JP2019108353A - Methods for inhibiting atherosclerosis by administering inhibitor of pcsk9 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a therapeutic method and a composition for inhibiting atherosclerotic plaque formation. An antibody or antigen that specifically binds to PCSK9 for use in inhibiting an increase in the number, total area, severity, or instability of atherosclerotic lesions in a subject with atherosclerosis. A pharmaceutical composition comprising a binding protein, wherein the antibody or antigen binding protein comprises heavy and light chain CDR amino acid sequences having SEQ ID NOs: 2, 3, 4, 7, 8, and 10. [Selection diagram] None

Description

RELATED APPLICATIONS This application is related to US Provisional Application No. 61 / 832,459 filed on Jun. 7, 2013; European Patent Application No. 13305762.0 filed on Jun. 7, 2013; October 2013 U.S. Provisional Application No. 61 / 892,215, filed on 17th; European Patent Application No. 13306436.0, filed on October 18, 2013; U.S. Provisional Application No .: filed on February 26, 2014 And claim the benefit of US Provisional Application No. 62 / 002,508 filed May 23, 2014. The contents of each of the above applications are incorporated herein in their entirety by reference.

  The present invention relates to the field of therapeutic treatments for atherosclerosis. More specifically, the present invention relates to the administration of a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor that inhibits atherosclerotic plaque formation in a subject.

  Atherosclerosis represents a major cause of death and cardiovascular morbidity in the western world. Atherosclerosis risk factors include high low density lipoprotein (LDL) cholesterol levels, low high density lipoprotein (HDL) cholesterol levels, hypertension, diabetes mellitus, family history, male gender, tobacco smoke, And high serum cholesterol.

  Proprotein Convertase Subtilisin / Kexin Type 9 (PCSK9) is a proprotein convertase that belongs to the proteinase K subfamily of the secretory subtilase family. Protein Convertase Subtilisin / Kexin Type 9 (PCSK9) is a serine protease involved in LDL metabolism and is mainly expressed in liver, kidney and intestine. Evidence suggests that PCSK9 increases plasma LDL cholesterol by promoting the degradation of the LDL receptor, which mediates LDL endocytosis in the liver, which is the main pathway of LDL clearance from the circulation .

  The use of PCSK9 inhibitors (anti-PCSK9 antibodies) to reduce serum total cholesterol, LDL cholesterol and serum triglycerides is described in US Pat. Nevertheless, PCSK9 inhibitors have not been reported to reduce or inhibit the progression of atherosclerotic plaque formation in subjects.

U.S. Patent No. 8,062,640 U.S. Patent No. 8,357,371 U.S. Patent Application Publication No. 2013/0064834

  There remains a need in the art for therapeutic methods of inhibiting atherosclerotic plaque formation.

The present invention addresses the need in the art for therapeutic methods by providing methods and compositions for inhibiting atherosclerotic plaque formation in a subject. In certain embodiments, the methods and compositions of the present invention comprise selecting a subject having or at risk of developing atherosclerosis, and proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor (eg, anti- Generally, administering a pharmaceutical composition comprising a PCSK9 antibody or an antigen binding protein). In another aspect, the invention provides a pharmaceutical composition comprising a PCSK9 inhibitor for use in the inhibition of atherosclerotic plaque formation in a subject. In yet another aspect, the invention provides a method of inhibiting atherosclerotic plaque formation in a subject by administering to the subject a pharmaceutical composition comprising a PCSK9 inhibitor.

  In one embodiment, the subject is non-hyperlipidemic. In another embodiment, the subject is apparently healthy.

  In another embodiment, the present invention is a method of treating atherosclerosis or inhibiting its progression in a subject comprising: (a) selecting a subject suffering from stroke or myocardial infarction; and (b 2.) A method comprising administering to a subject a pharmaceutical composition comprising a PCSK9 inhibitor, thereby treating atherosclerosis or inhibiting its progression. In one embodiment, the subject is non-hyperlipidemic.

  In another aspect, the invention provides a pharmaceutical composition comprising a PCSK9 inhibitor for use in the treatment of atherosclerosis or inhibition of the progression thereof in a subject suffering from stroke or myocardial infarction.

  In another aspect, the invention is a method of treating atherosclerosis or inhibiting its progression in a non-hyperlipidemic subject: (a) having or developing atherosclerosis Selecting a non-hyperlipidemic subject known to be at risk; and (b) administering to the subject a pharmaceutical composition comprising a PCSK 9 inhibitor, thereby treating atherosclerosis. Or providing a method comprising inhibiting the progression thereof. In one embodiment, the subject is apparently healthy. In another embodiment, the subject is non-hypercholesterolemic. In another embodiment, the subject is non-hypertriglyceridemic.

  In another aspect, the invention relates to the treatment of atherosclerosis or inhibition of its progression in a non-hyperlipidemic subject known to have or be at risk of developing atherosclerosis. Pharmaceutical compositions comprising a PCSK9 inhibitor for use in

  In certain embodiments, the subject has a disease or disorder selected from the group consisting of type I diabetes mellitus, type II diabetes mellitus, Kawasaki disease, chronic inflammatory disease, and hypertension. In another embodiment, the subject has elevated levels of an inflammatory marker. In a further embodiment, the inflammatory marker is a C-reactive protein. In another further embodiment, the inflammatory marker is a proinflammatory cytokine.

In one exemplary embodiment, the PCSK9 inhibitor is an antibody or antigen binding protein that specifically binds PCSK9. In other embodiments, the antibody comprises heavy and light chain CDR amino acid sequences having SEQ ID NOs: 12, 13, 14, 16, 17, and 18. In one embodiment, the antibody or antigen binding protein comprises HCVR having the amino acid sequence of SEQ ID NO: 11 and LCVR having the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the antibody or antigen binding protein comprises heavy and light chain CDR amino acid sequences having SEQ ID NOs: 2, 3, 4, 7, 8, and 10. In one embodiment, the antibody or antigen binding protein comprises HCVR having the amino acid sequence of SEQ ID NO: 1 and LCVR having the amino acid sequence of SEQ ID NO: 6. In one embodiment, the antibody or antigen binding protein binds to the same epitope on PCSK9 as an antibody comprising heavy and light chain variable domain amino acid sequences as set forth in SEQ ID NOs: 1 and 6; or 11 and 15, respectively. In some embodiments, the antibody or antigen binding protein competes with PCSK9 for binding to an antibody comprising heavy and light chain variable domain amino acid sequences as set forth in SEQ ID NOs: 1 and 6; or 11 and 15, respectively. .

  In another embodiment, the PCSK9 inhibitor reduces atherosclerotic plaque formation in a subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

  In some embodiments, the subject has heterozygous familial hypercholesterolemia (heFH). In another embodiment, the subject has a form of hypercholesterolemia (nonFH) that is not familial hypercholesterolemia.

  In certain embodiments, the subject receives another lipid-modifying agent prior to and / or during administration of the antibody or antigen binding protein. Therapeutic lipid modifiers include statins, cholesterol uptake inhibitors, ezetimibe, fibrates, niacin, omega-3 fatty acids, and bile acid resins. Statins include cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin and pravastatin.

  In some embodiments, the antibody or antigen binding protein is administered subcutaneously.

  The invention also provides a unit dosage form comprising a pharmaceutical composition of a PCSK9 inhibitor. In certain embodiments, the PCSK9 inhibitor is an antibody or antigen binding fragment and the unit dosage form comprises 75 mg, 150 mg, 200 mg, or 300 mg antibody or antigen binding fragment. The unit dosage form may be a prefilled syringe, a prefilled autoinjector, a vial, a cartridge, a reusable syringe, or a reusable autoinjector; the unit dosage form may be sealed to further indicate the dose.

  The invention also provides products or kits comprising pharmaceutical compositions and containers of PCSK9 inhibitors, and in some embodiments, instructions for use.

  The invention also provides (a) inhibition of atherosclerotic plaque formation in the subject; (b) treatment of atherosclerosis or inhibition of its progression in a subject suffering from stroke or myocardial infarction; or (c) atherosclerosis PCSK9 inhibitors in the manufacture of a medicament for the treatment of atherosclerosis or inhibition of its progression in non-hyperlipidemic subjects known to have or be at risk of developing arteriosclerosis Providing the use of the composition.

1A-D are a series of graphs showing the effect of mAb 316P, atorvastatin, and their combinations on several parameters after a 18 week treatment period. FIG. 1A shows the effect on mean plasma total cholesterol; FIG. 1B shows the effect on triglyceride levels. FIGS. 1C and 1D show that after 12 weeks of treatment, FPLC lipoproteins were separated and lipoproteins on cholesterol as assessed by testing the effect of mAb 316P alone (FIG. 1C) and the combination with atorvastatin (FIG. 1D) It is a figure which shows a profile. *** P <0.0045 compared to control; † P <0.0045 compared to atorvastatin; ‡‡‡ P <0.0045 comparing 3 mg / kg mAb 316 P and 10 mg / kg mAb 316 P N = 15 per group). FIG. 2A is a graph of the effect of mAb 316P, atorvastatin, and their combinations on liver low density lipoprotein receptor protein levels. *** P <0.0045 compared to control; PP <0.05 compared to atorvastatin バ ス タ チ ン P <0.01; † P <0.0045; 3 mg / kg mAb 316 P and 10 mg / ‡ P <0.05 (n = 8 per group) comparing kg mAb 316P. FIG. 2B is a graph showing the effect of mAb 316P, atorvastatin, and their combinations on non-HDL cholesterol levels. *** P <0.001 compared to control; #P <0.05 compared to atorvastatin; #### <0.01; #### P <0.001. 3A-F are a series of photographs showing the effect of mAb 316P, atorvastatin, and their combinations on plaque morphology. Control (FIG. 3A), 3 mg / kg mAb 316P (FIG. 3B), 10 mg / kg mAb 316 P (FIG. 3C), atorvastatin (FIG. 3D), 3 mg / kg mAb 316 P + atorvastatin (FIG. 3E) and 10 mg / kg respectively after 18 weeks of treatment Representative images of hematoxylin-phloxine-saffron-stained atherosclerotic lesions at the cross-section of the aortic root area for the kg mAb 316P + atorvastatin (FIG. 3F) group. 4A-D are a series of graphs showing the effect of mAb 316P, atorvastatin, and combinations thereof on atherosclerosis development at the aortic root and aortic arch. After 18 weeks of treatment, total lesion area per cross section (FIG. 4A) and number of lesions (FIG. 4B) were evaluated. Lesion severity was assessed and classified as no lesion, mild (type I-III) and severe (type IV-V) lesions (FIG. 4C). Total plaque mass in the aortic arch (FIG. 4D) was analyzed after oil-red O staining. Data are expressed as percentage of stained area. * P <0.05 compared to the control *** P <0.0045; PP <0.05 compared to atorvastatin PP <0.01; † P <0.0045; ‡ P <0.05; ‡‡ P <0.01; ‡‡‡ P <0.0045 (n = 8 per group in the area of origin and aortic arch compared to 3 mg / kg mAb 316 P and 10 mg / kg mAb 316 P N = 6 to 7). FIG. 5 is a graph showing the correlation between mean plasma total cholesterol and atherosclerotic lesion area. The square root of the lesion area was plotted against the mean total cholesterol. Linear regression analysis was performed. FIG. 6 is a series of photographs showing the effect of mAb 316P, atorvastatin, and combinations thereof on lesion composition. Representative immunostaining of macrophages with Mac-3 for control and 18 weeks after treatment with mAb316P alone and in combination with atorvastatin, followed by representative of Sirius red stain of collagen and alpha actin in smooth muscle cells (SMC) image. 7A-C are a series of graphs showing the effect of mAb 316P, atorvastatin, and their combinations on lesion composition. The amount of macrophages (FIG. 7A left) and the amount of necrosis (FIG. 7A right), including cholesterol cleft as a destabilizing agent and SMC (FIG. 7B left) and collagen (FIG. 7B right) as a stabilizing factor It was determined in severe (type IV-V) lesions after correction for lesion size. * P <0.05, ** P <0.01, *** P <0.001 in comparison to the control; #P <0.05, ## P <0.01, # in comparison to atorvastatin ## P <0.001. The plaque stability index was calculated as the ratio of stabilizer to destabilizer (FIG. 7C). * P <0.05, *** P <0.0045 in comparison with the control; <P <0.05, † P <0.0045 in comparison with atorvastatin 10 mg / kg of 3 mg / kg mAb 316 P ‡ P <0.05 (n = 15 per group) compared to mAb 316P. The continuation of Figure 7-1. 8A-C are a series of graphs showing the effect of mAb 316P, atorvastatin, and their combinations on markers of vascular inflammation. The number of monocytes adhering to the endothelium (FIG. 8A) and the number of T cells in the aortic root area (FIG. 8B) were determined per cross section. In addition, cell adhesion molecule 1 (ICAM-1) was determined as a percentage of stained area (FIG. 8C). The representative image was included. * P <0.05, ** P <0.01, *** P <0.0045 compared to the control; PP <0.05, † P <0.0045 compared to atorvastatin.

  It is understood that the present invention is not limited to the particular methods and experimental conditions described, as the methods and conditions can vary before the invention is described. Also, the terminology used herein is for the purpose of describing particular embodiments only, and is intended to be limiting as the scope of the present invention is limited only by the scope of the appended claims. It is understood that it is not.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" means that when used with reference to the numerical values specifically recited, the value can vary from the stated value to 1% or less. For example, the expression "about 100" as used herein includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

  Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, exemplary methods and materials are described below. All publications mentioned herein and the Sequence Listing filed simultaneously herein are incorporated by reference in their entirety.

Methods for Inhibiting Atherosclerosis The methods of the present invention select subjects that have or are at risk of developing atherosclerosis, and administer a pharmaceutical composition comprising a PCSK9 inhibitor to these subjects Including.

  Atherosclerosis risk factors are well known to those skilled in the art and include high low density lipoprotein (LDL) cholesterol levels, low high density lipoprotein (HDL) cholesterol levels, hypertension, diabetes mellitus, family history, male Gender, tobacco smoke, and high serum cholesterol, including but not limited to. Methods for assessing these risk factors for a given subject are also well known to those skilled in the art.

In certain embodiments, the selected subject is non-hyperlipidemic. "Non-hyperlipidemic" is a subject that is non-hypercholesterolemic and / or non-hypertriglyceridemic subject. "Non-hypercholesterolemic" is a subject that does not meet current criteria established for hypercholesterolemic subjects. "Non-hypertriglyceridemia" is a subject that does not meet current criteria established for hypertriglyceridemia subjects (e.g., Harrison's Principles of Experimental Medicine, 13th Edition, McGraw-Hill, Inc., See N. Y). Hypercholesterolemic subjects have an LDL level of> 160 mg / dL, or> 130 mg / dL and male gender, a family history of premature coronary heart disease, cigarette smoking (more than 10 per day), hypertension, Low HDL (<35 mg / dL), diabetes mellitus,
It has at least two risk factors selected from the group consisting of hyperinsulinemia, abdominal obesity, high lipoproteins (a), and a personal history of cerebrovascular disease or obstructive peripheral vascular disease. Hypertriglyceridemia subjects have triglyceride (TG) levels of> 250 mg / dL. Thus, non-hyperlipidemic subjects are defined as having cholesterol and triglyceride levels below the limits set as described above for both hypercholesterolemia and hypertriglyceridemia subjects. In certain embodiments, the selected subject has not been treated for non-hyperlipidemic or hyperlipidemia.

  In one embodiment, the subject selected is apparently healthy. As used herein, “apparently healthy” refers to an individual who has not previously had an acute and adverse cardiovascular event such as myocardial infarction (ie, a secondary adverse event due to a primary adverse cardiovascular event). Individual who is not at increased risk of adverse cardiovascular events. An apparently healthy individual also shows no other symptoms of the disease.

  In certain embodiments, the selected subject has previously suffered from an acute adverse cardiovascular event, such as myocardial infarction, stroke, angina and / or peripheral arteriovascular disease. In one embodiment, the selected subject has previously suffered from an acute adverse cardiovascular event, such as myocardial infarction, stroke, angina and / or peripheral arteriovascular disease, but is not hyperlipidemic. It is symptomatic. In one embodiment, the selected subject has previously suffered from an acute adverse cardiovascular event, such as myocardial infarction, stroke, angina and / or peripheral arteriovascular disease, but is not hyperlipidemic. Neither symptomatic nor hyperlipidemic.

  In certain embodiments, the selected subject has a disease or disorder selected from the group consisting of type I diabetes mellitus, type II diabetes mellitus, Kawasaki disease, chronic inflammatory disease, and hypertension. In one embodiment, the selected subject has a disease or disorder selected from the group consisting of type I diabetes mellitus, type II diabetes mellitus, Kawasaki disease, chronic inflammatory disease, and hypertension, but non-hyperlipidemic It is symptomatic. In one embodiment, the selected subject has a disease or disorder selected from the group consisting of type I diabetes mellitus, type II diabetes mellitus, Kawasaki disease, chronic inflammatory disease, and hypertension, but non-hyperlipidemic Neither symptomatic nor hyperlipidemic.

  In certain embodiments, the selected subject has elevated levels of an inflammatory marker. In one embodiment, the selected subject is non-hyperlipidemic, although levels of inflammatory markers are elevated. Any marker of systemic inflammation may be utilized for the purposes of the present invention. Suitable inflammatory markers include C-reactive protein (eg, incorporated in their entirety by reference, see US Pat. No. 7,964,614), cytokines (eg, Il-6, IL-8, And / or IL-17), and cell adhesion molecules (eg, ICAM-1, ICAM-3, BL-CAM, LFA-2, VCAM-1, NCAM, and PECAM), but is not limited thereto.

  Levels of inflammatory markers can be obtained by any of the art recognized assays. Typically, levels are measured by measuring the level of markers in body fluids, such as blood, lymph, saliva, urine and the like. Levels can be determined by ELISA to determine the presence of the marker, or by immunoassays or other conventional techniques. The level of the marker measured in the subject can be compared to a suitable control (eg, a predetermined value and / or a value obtained from a fitted healthy subject) to determine if the level of the inflammatory marker is elevated.

  In one embodiment, the present invention provides a pharmaceutical composition comprising a PCSK9 inhibitor for use in the inhibition of atherosclerotic plaque formation in a subject, as well as in the treatment or progression of atherosclerosis in a subject. .

  The present invention is also directed to the manufacture of a medicament for any of the methods described herein, including the inhibition of atherosclerotic plaque formation, and the treatment of atherosclerosis in the subject or the inhibition thereof. The use of a pharmaceutical composition comprising a PCSK9 inhibitor is provided.

PCSK9 Inhibitors In certain embodiments, the methods of the present invention comprise administering to a subject a therapeutic composition comprising a PCSK9 inhibitor.

  As used herein, a "PCSK9 inhibitor" is any agent that binds or interacts with human PCSK9 and inhibits the normal biological function of PCSK9 in vitro or in vivo. Non-limiting examples of the category of PCSK9 inhibitors specifically bind to small molecule PCSK9 antagonists, antagonistic nucleic acid molecules (eg, RNAi molecules), peptide-based PCSK9 antagonists (eg, "peptibody" molecules), and human PCSK9 Antibody or antigen binding fragment of the antibody.

  As used herein, the terms "proprotein convertase subtilisin / kexin type 9" or "PCSK9" refers to PCSK9 having the nucleic acid sequence shown in SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 198, or biologically active thereof Say a fragment.

  In certain embodiments, administration of a PCSK 9 inhibitor results in at least 10% (eg, 10% 20%) atherosclerotic plaque formation in a subject (eg, a human subject) relative to atherosclerotic plaque formation in an untreated subject. Decrease by 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).

In certain embodiments, the PCSK9 inhibitor is an antibody or antigen binding protein that specifically binds PCSK9. As used herein, the term "antibody" is an immunoglobulin molecule comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, and large quantities thereof. Refers to the body (eg, IgM). Each heavy chain comprises a heavy chain variable region (abbreviated as HCVR or V _H herein) and a heavy chain constant region. The heavy chain constant region comprises three _domains, a C H 1, _{C H} 2 and _{C H} 3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or _VL ) and a light chain constant region. The light chain constant region is comprised of one domain _{(C C} L 1). The V _H and V _L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), dispersed in more conserved regions, termed framework regions (FR). Each V _H and V _L is composed of the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, 3 CDRs arranged from amino to carboxy terminus in FR4 and 4 FRs. In different embodiments of the invention, the FRs of the anti-PCSK9 antibody (or antigen binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. Amino acid consensus sequences may be defined based on side-by-side analysis of two or more CDRs.

The term "antibody" as used herein also includes whole antibody molecules and antigen binding fragments of antigen binding proteins. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, "antigen-binding protein" and the like are any that specifically bind an antigen to form a complex. It includes naturally occurring, enzymatically obtained, synthetic or genetically engineered polypeptides or glycoproteins. Antigen binding fragments of antibodies may, for example, be prepared using any suitable standard techniques, such as proteolysis, or recombinant genetic engineering involving the manipulation and expression of DNA encoding the variable and optionally constant domains of antibodies. For example, they can be derived from whole antibody molecules. Such DNA is known and / or available from, for example, commercial sources, DNA libraries (eg, including phage antibody libraries) or can be synthesized. The DNA may, for example, place one or more variable and / or constant domains in a suitable configuration, or introduce codons, create cysteine residues, modify, add or delete amino acids, etc. Sequences and manipulations are carried out chemically or by using molecular biological techniques to eliminate them.

  Non-limiting examples of antigen binding fragments or antigen binding proteins are: (i) Fab fragments; (ii) F (ab ') 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv ( (vi) dAb fragments; and (vii) antibody hypervariable regions (eg, isolated complementarity determining regions (CDRs) such as CDR3 peptides), or restricted FR3-CDR3-FR4 peptides It contains a minimal recognition unit consisting of amino acid residues that mimic. Domain specific antibody, single domain antibody, domain deleted antibody, chimeric antibody, CDR grafted antibody, diabody, triabody, tetrabody, minibody, nanobody (eg, monovalent nanobody, bivalent nanobody, etc.), small molecule immunopharmaceutical Other modifying molecules such as (SMIP) and shark variable IgNAR domains are also encompassed within the expressions "antigen binding fragment" and "antigen binding protein" as used herein.

Antigen binding fragments of antibodies typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition, and generally comprises at least one CDR adjacent to or in frame with one or more framework sequences. In antigen binding fragments having a V _H domain associated with a V _L domain, the V _H and V _L domains are positioned relative to one another in any suitable arrangement. For example, the variable region may be a dimer, containing VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen binding fragment of the antibody may contain monomeric VH or VL domains.

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 found within an antigen-binding fragment of an antibody of the invention are: (i) 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 -CL; (viii) VL-C H 1; (ix) VL-C H 2; (x) VL-C H 3, (xi) VL-C H 1-C H _2; and a (xiv) VL-CL; ( xii) VL-C H 1-C H 2-C H 3; (xiii) VL-C H 2-C H 3. In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains are directly linked to each other or linked by a complete or partial hinge or linker region Be done. The hinge region is at least two (eg, 5, 6, 7, 8, 9, 10, 11, 12) providing flexible or semi-flexible linkages between adjacent variable and / or constant domains in a single polypeptide molecule. , 13, 14, 15, 20, 25, 30, 35, 40, 50, 60 or more) amino acids. Moreover, the antigen binding fragments of the antibodies of the invention are listed above in non-covalent association (e.g. by disulfide bonding) with each other and / or with one or more monomeric VH or VL domains. Homodimers or heterodimers (or other multimers) of any variable and constant domain configuration may be included.

As with whole antibody molecules, antigen binding fragments may be monospecific or multispecific (eg bispecific). Multispecific antigen binding fragments of antibodies typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a different antigen or to a different epitope of the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody format disclosed herein, may be used in the context of antigen binding fragments of antibodies of the invention using routine techniques available in the art. It is adapted to use.

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

  The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention still have amino acid residues not encoded by human germline immunoglobulin sequences (eg, in CDRs and particularly CDR3) (eg, by random or site directed mutagenesis in vitro, or in vivo (Mutation introduced by somatic mutation) may be included. However, as used herein, the term "human antibody" includes antibodies wherein CDR sequences derived from the germline of another mammalian species, such as mouse, are grafted onto human framework sequences. Not intended.

  As used herein, the term "recombinant human antibody" refers to an antibody (described further below) expressed using a recombinant expression vector that is transfected into host cells, a recombinant combinatorial human antibody library Antibodies (described further below), antibodies isolated from animals which are transgenic for human immunoglobulin genes (eg, mice) (eg, Taylor et al. (1992) Nucl. Acids Res. 20. Recombinant means, such as antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences) or Includes all human antibodies that are prepared, expressed, produced or isolated by It is intended to be. 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 in vivo somatic mutagenesis, if animal transgenic for human Ig sequences is used). The amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but may not naturally occur in human antibody germline repertoire in vivo.

  Human antibodies may exist in two forms associated with hinge heterogeneity. In the first form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by heavy chain disulfide bonds. In the second form, the dimers are formed by light and heavy chains (half antibody) covalently linked to a molecule of about 75-80 kDa, which is not linked by an inter-chain heavy chain disulfide bond. These forms are extremely difficult to separate, even after affinity purification.

The frequency of appearance 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. Single amino acid substitutions in the hinge region of human IgG4 hinges can significantly reduce the appearance of the second form to levels typically observed with human IgG1 hinges (eg, Angal et al. (1993). See Molecular Immunology 30: 105). The present invention includes antibodies having one or more mutations in the hinge, CH2 or CH3 region, which is desirable, for example, to improve the yield of the desired antibody form in production.

  As used herein, an "isolated antibody" means 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 the antibody is naturally present or produced naturally is an "isolated antibody" for the purpose of the present invention is there. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is one that has been subjected to at least one purification or isolation step. According to one embodiment, the isolated antibody is substantially free of other cellular substances and / or chemicals.

The term "specifically binds" etc. means that the antibody or its antigen binding protein forms a complex with the antigen which 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 PCSK9 used in the context of the present invention may be 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 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 about 0 with a _{K D} that is measured in .5nM less in a surface plasmon resonance assay include antibodies that bind to PCSK9 or portion thereof. However, isolated antibodies that specifically bind human PCSK9 may have cross-reactivity with other antigens, such as PCSK9 molecules from other (non-human) species.

  Exemplary non-limiting PCSK9 inhibitors are described herein, for example, US Patent Application Publications US20130115223, US20130071405, US20130064834, US20130064825, US20120122954, US Patent Application Publications US20130115223, US20130071834, US20130064952, US20120122954, each being incorporated herein by reference in its entirety. It includes the inhibitors described in US20120015435, US201110313024, US20110015252, US201102302542, US20110009628, and US Serial Number 61 / 682,349. In some embodiments, the PCSK9 inhibitor is an antibody having the VH / VL sequence of Ab "LGT209" as described in WO 2011/072263; Ab "L1 L3" as described in WO 2010/029513. Antibody having the VH / VL sequence of: antibody having the VH / VL sequence of Ab “5L1721H23_6L3” described in WO 2011/111007; VH of the Ab “5L1721H23_6L3H3” described in WO 2011/111007 / Antibody having the VH sequence; antibody having the VH / VL sequence of Ab "31H4" described in WO 2009/026558; VH of Ab "1 B20" described in US Patent Application Publication No. US 2009/0232795 / VL Antibody having the column; antibody having the VH / VL sequence of Ab "21B12" described in US Patent Application Publication No. US 2009/0142352; Ab "508. 20. antibody described in US Patent Application Publication No. US 2012/0195910". Or an antibody having the VH / VL sequence of Ab "508.20.33" described in U.S. Patent Application Publication No. US 2012/0195910.

Anti-PCSK9 antibodies useful in the methods and compositions of the present invention may comprise one or more of the heavy chain and light chain variable domain framework and / or CDR regions as compared to the corresponding germline sequence from which the antibody is derived. These amino acid substitutions, insertions and / or deletions may be included. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to, for example, germline sequences available from public antibody sequence databases. The present invention provides an antibody derived from any amino acid sequence disclosed herein,
And methods involving the use of antigen-binding fragments thereof, wherein one or more amino acids within one or more framework and / or CDR regions correspond to the germline sequence from which the antibody is derived Or a corresponding residue of another human germline sequence, or a conservative amino acid substitution of the corresponding germline residue (such sequence changes are referred to collectively herein). (Called “germline mutations”). One of skill in the art, including the heavy and light chain variable region sequences disclosed herein, facilitates numerous antibodies and antigen binding fragments, including one or more individual germline mutations or combinations thereof. Can be produced. In certain embodiments, all of the framework and / or CDR residues within the VH and / or _VL domains are backmutated to residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues, for example, only variant residues found within the first eight amino acids of FR1 or the last eight amino acids of FR4, or only variant residues found within CDR1, CDR2 or CDR3 Is backmutated to the original germline sequence. In other embodiments, one or more frameworks and / or one or more CDR residues are different germline sequences (ie, germline sequences that differ from the germline sequences from which the antibody was originally derived). Is mutated to the corresponding residue of Furthermore, the antibodies of the invention may comprise any combination of two or more germline mutations within the framework and / or CDR regions, eg individual residues which are here Some other residues that are mutated to the corresponding residues of the germline sequence, while differing from the original germline sequence, are maintained or mutated to the corresponding residues of the different germline sequence. Once obtained, antibodies and antigen-binding fragments thereof that contain one or more germline mutations can improve binding specificity, increase binding affinity, improve or enhance antagonist or agonist biological properties (as Depending on one or more desired properties such as immunogenicity reduction etc. can be easily tested. The use of antibodies and antigen binding fragments obtained in this general manner is encompassed within the present invention.

  The invention also includes methods involving the use of anti-PCSK9 antibodies comprising 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 provides, for example, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less for any HCVR, LCVR and / or CDR amino acid sequence disclosed herein. Use of an anti-PCSK9 antibody having HCVR; LCVR; CDRs; HCVR and LCVR; HCVR and CDRs; LCVR and CDRs; or HCVR, LCVR and CDR amino acid sequences with conserved amino acid substitutions of 3 or less, 2 or less or 1 or less including. In certain embodiments, the substitution is in a CDR amino acid sequence, eg, CDR1, CDR2 and / or CDR3. In other embodiments, other embodiments, the substitution is in the CDR3 amino acid sequence.

  The term "surface plasmon resonance" as used herein refers, for example, to detection of changes in protein concentration within a biosensor matrix using the BIACORETM system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ) , Optical phenomena that allow analysis of real-time interactions.

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

The term "epitope" refers to an antigenic determinant that binds to a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions of an antigen and may have different biological effects. The epitopes may be conformational or linear. Conformational epitopes are produced by amino acids spatially aligned from different segments of the linear polypeptide chain. Linear epitopes are those produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may comprise a portion of a sugar, a phosphoryl group, or a sulfonyl group in an antigen.

  According to one embodiment, the anti-PCSK9 antibody used in the method of the invention is an antibody having pH dependent binding properties. As used herein, the phrase "pH dependent binding" means that the antibody or antigen binding protein exhibits "a reduction in binding to PCSK9 at acidic pH as compared to neutral pH". (Both representations may be used interchangeably for the purpose of the present disclosure). For example, antibodies having "pH dependent binding properties" include antibodies and antigen binding fragments thereof that bind PCSK9 with higher affinity at neutral pH than acidic pH. In certain embodiments, the antibodies and antigen binding fragments of the invention are at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 at neutral pH than acidic pH. 65, 70, 75, 80, 85, 90, 95, 100-fold or more higher binding to PCSK9.

  According to this aspect of the invention, an anti-PCSK9 antibody with pH dependent binding properties may have one or more amino acid variations relative to a parent anti-PCSK9 antibody. For example, an anti-PCSK9 antibody with pH dependent binding properties may contain, for example, one or more histidine substitutions or insertions in one or more CDRs of the parent anti-PCSK9 antibody. Thus, according to one embodiment of the present invention, identical to the CDR amino acid sequence of the parent anti-PCSK9 antibody except for the substitution of one or more amino acids of one or more of the CDRs of the parent antibody with a histidine residue. There is provided a method comprising administering an anti-PCSK9 antibody comprising CDR amino acid sequences (eg, heavy and light chain CDRs) Anti-PCSK9 antibodies with pH dependent binding are distributed within a single CDR of the parent antibody or across multiple (eg 2, 3, 4, 5, or 6) CDRs of the parent anti-PCSK9 antibody, eg, 1, 2 , 3, 4, 5, 6, 7, 8, 9 or more histidine substitutions may be present. For example, the invention relates to one or more histidine substitutions in HCDR1, one or more histidine substitutions in HCDR2, one or more histidine substitutions in HCDR3 of the parent anti-PCSK9 antibody, LCDR1. Anti-PCSK9 antibodies with pH dependent binding, including one or more histidine substitutions, one or more histidine substitutions in LCDR2, and / or one or more histidine substitutions in LCDR3 Including use.

  As used herein, the expression "acidic pH" means a pH of 6.0 or less (eg, less than about 6.0, less than about 5.5, less than about 5.0, etc.). The expression "acidic pH" is 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, including pH values of 5.0 or less. As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. The expression "neutral pH" comprises pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4. .

Preparation of Human Antibodies Methods of producing 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 human PCSK9.

High affinity chimeric antibodies to PCSK9, having human variable regions and mouse constant regions, are first isolated using the VELOCIMMUNETM technology or any other known method of generating monoclonal antibodies. With VELOCIMMUNE® technology,
A genome comprising human heavy and light chain variable regions operably linked to an endogenous mouse constant region locus such that the mouse produces antibodies comprising human variable regions and mouse constant regions in response to antigenic stimulation It involves generation of transgenic mice. DNA encoding the variable regions of the antibody heavy and light chains is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

  Generally, VELOCIMMUNE® mice are challenged with the antigen of interest, and lymphoid cells (eg, B cells) are recovered from mice expressing the antibody. A hybridoma cell line capable of preparing an immortalized hybridoma cell line by fusing lymphoid cells with a myeloma cell line, screening and selecting such a hybridoma cell line to produce an antibody specific to an antigen of interest Identify DNA encoding the heavy and light chain variable regions can be isolated and ligated to the heavy chain and light chain desired isotype constant regions. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, antigen-specific chimeric antibodies or DNA encoding light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

  First, high affinity chimeric antibodies having human variable regions and mouse constant regions are isolated. Antibodies are characterized and selected for desirable properties, including affinity, selectivity, epitopes etc. using standard procedures known to those skilled in the art. The mouse constant region is replaced with the desired human constant region to generate a fully human antibody of the invention, such as wild type IgG1 or IgG4, or modified IgG1 or IgG4. While the constant region chosen may vary according to the particular application, high affinity antigen binding and target specificity properties are present in the variable region.

  In general, antibodies that can be used in the methods of the invention have high affinity, as described above, when immobilized on a solid phase or measured by binding to an antigen in a liquid phase. The mouse constant region is replaced with the desired human constant region to generate a fully human antibody of the invention. While the constant region chosen may vary according to the particular application, high affinity antigen binding and target specificity properties are present in the variable region.

Specific examples of antigen binding fragments of human antibodies or antibodies that specifically bind to PCSK9 that can be used in the context of the method of the invention include SEQ ID NOs: 1 and 11 or at least 90%, at least 95%, at least 98 Three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of substantially similar sequences having 90% or at least 99% sequence identity Including any antibody or antigen binding fragment. Alternatively, specific examples of antigen binding fragments of human antibodies or antibodies that specifically bind to PCSK9 that can be used in the context of the method of the invention include SEQ ID NOs: 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173, 181 and 189 or at least 90%, at least 95%, at least 98% or at least 99% sequence identity Any antibody or antigen binding comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of It contains fragments. The antibody or antigen binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 15 or substantially similar sequences having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. The three light chain CDRs (LCVR1, LCVR2, LCVR3) contained in the light chain variable region (LCVR) may be included. Alternatively, the antibody or antigen binding fragment may be one of SEQ ID NOs: 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 1
29, 137, 145, 153, 161, 169, 177, 185, and 193 or at least 90%, at least 95%, at least 98% or at least 99% substantially identical sequences having similar identity selected from the group The three light chain CDRs (LCVR1, LCVR2, LCVR3) contained within the light chain variable region (LCVR) having the amino acid sequence

  Sequence identity between two amino acid sequences is determined using optimal sequence alignment over the entire length of the reference amino acid sequence (ie the amino acid sequence identified by SEQ ID NO) and / or between the two amino acid sequences. Preferably, EMBOSS :: needle, Matrix: Blosum 62, Gap Open 10.0, Gap Extend 0, where optimal sequence alignment is determined using known tools such as Align, standard settings. .5 can be obtained.

  In one embodiment of the invention, the antibody or antigen binding protein is selected from the heavy and light chain variable region amino acid sequence pair (HCVR / LCVR) selected from the group consisting of SEQ ID NOs: 1/6 and 11/15. CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) are included. Alternatively, in one embodiment of the present invention, the antibody or antigen binding protein is SEQ ID NO: 37/41, 45/49, 53/57, 61/65, 69/73, 77/81, 85/89, 93/97. 101/105, 109/113, 117/121, 125/129, 133/137, 141/145, 149/153, 157/161, 165/169, 173/177, 181/185, and 189/193. And 6 CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) from heavy and light chain variable region amino acid sequence pairs (HCVR / LCVR) selected from the group consisting of

  In one embodiment of the invention, anti-PCSK9 antibodies or antigen binding proteins that can be used in the methods of the invention are SEQ ID NOs: 2/3/4/7/8/10 (mAb 316 P) and 12/13/14 / HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 selected from 16/17/18 (mAb 300 N) (see US Patent Application Publication No. 2010/0166768) and 12/13/14/16/17/18 It has an amino acid sequence, wherein SEQ ID NO: 16 comprises a substitution of histidine for leucine at amino acid residue 30 (L30H).

  In one embodiment of the invention, the antibody or antigen binding protein comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 1/6 and 11/15. In one exemplary embodiment, the antibody or antigen binding protein comprises the HCVR amino acid sequence of SEQ ID NO: 1 and the LCVR amino acid sequence of SEQ ID NO: 6. In one exemplary embodiment, the antibody or antigen binding protein comprises the HCVR amino acid sequence of SEQ ID NO: 11 and the LCVR amino acid sequence of SEQ ID NO: 15. In one exemplary embodiment, the antibody or antigen binding protein is the HCVR amino acid sequence of SEQ ID NO: 11 and the LCVR amino acid sequence of SEQ ID NO: 15, comprising a histidine substitution (L30H) for leucine at amino acid residue 30. Contains the amino acid sequence.

In certain embodiments, the antibody or antigen binding protein is APOE * 3 Leiden. When administered by subcutaneous injection weekly to CETP mice, it exhibits one or more of the following characteristics:
a. Reduce total cholesterol levels by about 37% relative to untreated control group when administered at 3 mg / kg;
b. When administered at 10 mg / kg, reduce total cholesterol levels by about 46% relative to untreated control group;
c. When administered at 3 mg / kg in combination with 3.6 mg / kg / day atorvastatin, reduce total cholesterol levels by about 48% relative to untreated control group;
d. When administered at 10 mg / kg in combination with 3.6 mg / kg / day atorvastatin, reduce total cholesterol levels by about 58% relative to untreated control group;
e. When administered at 3 mg / kg in combination with 3.6 mg / kg / day atorvastatin, reduce total cholesterol levels by approximately 36% relative to treatment with 3.6 mg / kg / day atorvastatin alone;
f. When 10 mg / kg is administered in combination with 3.6 mg / kg / day atorvastatin, the total cholesterol level is reduced by about 48% relative to treatment with 3.6 mg / kg / day atorvastatin alone;
g. Decreases about 33% triglyceride levels relative to untreated control group when administered at 3 mg / kg;
h. Decreases about 36% triglyceride levels relative to untreated control group when administered at 10 mg / kg;
i. When administered at 3 mg / kg in combination with 3.6 mg / kg / day atorvastatin, reduce triglyceride levels by approximately 40% relative to treatment with 3.6 mg / kg / day atorvastatin;
j. When administered at 10 mg / kg in combination with 3.6 mg / kg / day atorvastatin alone, it reduces triglyceride levels by approximately 51% relative to treatment with 3.6 mg / kg / day atorvastatin;
k. Increase hepatic LDLR expression relative to untreated control group when administered at 3 mg / kg;
l. Increases hepatic LDLR expression by about 178% relative to untreated control group when administered at 10 mg / kg;
m. When administered at 3 mg / kg in combination with 3.6 mg / kg / day atorvastatin, approximately 71% liver LDLR expression is increased relative to treatment with 3.6 mg / kg / day atorvastatin alone;
n. When administered at 10 mg / kg in combination with 3.6 mg / kg / day atorvastatin, treatment with only 3.6 mg / kg / day atorvastatin increases hepatic LDLR expression by about 140%;
o. Reduces atherosclerotic lesion size by about 70% relative to untreated control group when administered at 3 mg / kg;
p. Reduces atherosclerotic lesion size by about 87% relative to untreated control group when administered at 10 mg / kg;
q. Reduce atherosclerotic lesion size by about 88% relative to untreated control group when administering 3 mg / kg in combination with 3.6 mg / kg / day atorvastatin;
r. When administered at 10 mg / kg in combination with 3.6 mg / kg / day atorvastatin, reduce atherosclerotic lesion size by about 98% relative to untreated control group;
s. When administered at 3 mg / kg in combination with 3.6 mg / kg / day atorvastatin, the relative atherosclerotic lesion size is reduced by about 82% relative to treatment with 3.6 mg / kg / day atorvastatin alone;
t. When administered at 10 mg / kg in combination with 3.6 mg / kg / day atorvastatin, the relative atherosclerotic lesion size is reduced by about 97% relative to treatment with 3.6 mg / kg / day atorvastatin alone;
u. When administered at 3 mg / kg, reduces triglyceride levels by approximately 72% relative to treatment with 3.6 mg / kg / day atorvastatin alone;
v. When administered at 10 mg / kg, reduces triglyceride levels by about 79% relative to treatment with 3.6 mg / kg / day atorvastatin alone;
w. When administered at 3 mg / kg, reduce total cholesterol levels by approximately 22% relative to treatment with 3.6 mg / kg / day atorvastatin alone;
x. When administered at 10 mg / kg, reduce total cholesterol levels by about 34% relative to treatment with 3.6 mg / kg / day atorvastatin alone;
y. When administered at 3 mg / kg, increase the percentage of about 236% undiseased aortic segment relative to untreated control group;
z. When administered at 10 mg / kg, increase the percentage of about 549% undiseased aortic segment relative to untreated control group;
aa. When administered at 3 mg / kg in combination with 3.6 mg / kg / day atorvastatin, increase the percentage of about 607% undiseased aortic segment over control; and bb. When administering 3 mg / kg in combination with 3.6 mg / kg / day atorvastatin, increase the percentage of approximately 1118% undiseased aortic segment over control.

Pharmaceutical Compositions and Methods of Administration The present invention includes methods comprising administering a PCSK9 inhibitor to a subject, wherein the PCSK9 inhibitor is contained within a pharmaceutical composition. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients and other agents that provide for appropriate transfer, delivery, tolerability and the like. A large number of suitable formulations can be found in formulations known to all drug chemists (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA). These preparations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (such as LIPOFECTINTM), DNA conjugates Water-in-oil and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowaxes. Powell et al. "Compendium of
"excipients for parental formulations"
See also 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, encapsulation in liposomes, capable of expressing microparticles, microcapsules, mutant viruses Recombinant cells, receptor-mediated endocytosis (see, eg, 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, inhalation and oral routes. The compositions may be administered by any conventional route, for example by infusion and bolus injection, by absorption through epithelial or mucosal skin linings (eg, oral mucosa, rectal and intestinal mucosa etc), and other biological Administered in conjunction with active factors.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, pen delivery devices and auto-injector delivery devices have applications readily for delivering the pharmaceutical compositions of the present invention. Such delivery devices may be reusable or disposable, and may be adapted to administer variable or fixed doses. Reusable delivery devices generally utilize replaceable cartridges 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 delivery device can then be reused. In disposable delivery devices, there is no replaceable cartridge. Rather, the disposable delivery device comes pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is emptied in the reservoir, the entire device is discarded. Delivery devices adapted for variable dosing may include a mechanism for setting the dose within the range of dose volume (in some cases limiting the range to values less than the total volume contained in the cartridge) Can) The delivery device adapted for fixed dosing may include a mechanism for delivering the total volume of the cartridge when the delivery device is activated. In such cases, the cartridge may be a prefilled syringe.

A number of delivery devices have use in the delivery of the pharmaceutical compositions of the present invention. Only a few examples include AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG
MIX 75/25 ¹ m pen, HUMALOGTM pen, HUMALIN 70 / 30TM pen (Eli Lilly and Co., Indianapolis, IN), NOVOPENTM I, II and III (Novo Nordisk, Copenhagen, Denmark) ), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENTM, OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIK (Trademark) (sanofi-aventis, Frankfurt, Germany) are included, Not a constant. Only a few examples of disposable pen delivery devices with application for subcutaneous delivery of the pharmaceutical composition of the invention include SOLOSTARTM Pen (sanofi-aventis), FLEXPENTM (Novo Nordisk), and KWIKPENTM ) (Eli Lilly), SURECLICKTM automatic injector (Amgen, Thousand Oaks, CA), PENLETTM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRATM pen ( Includes, but is not limited to Abbott Labs, Abbott Park IL). In some embodiments, the reusable pen or auto-injector delivers a fixed dose. In other embodiments, the reusable pen or auto-injector can deliver a variable dose. In different embodiments, the reusable pen or auto-injector can deliver a single dose or multiple doses.

In one embodiment, the pharmaceutical composition is delivered in a controlled release system. In one embodiment, a pump may be used, including a micropump (Langer, supra;
Sefton, 1987, CRC Crit. Ref. Biomed. Eng.
14: 201). In another embodiment, polymeric materials can be used; Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres. See Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed close to the target of the composition, thus requiring only a small systemic dose (e.g., 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: 1525-1533.

Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, infusion and the like. These injectable formulations may be prepared by known methods. For example, the injectable preparation may be prepared, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or a salt thereof in an aqueous medium or oily medium usually used for injection. As an aqueous medium for injection, for example, physiological saline, an isotonic solution containing glucose, and alcohol (for example, ethanol), polyhydric alcohol (for example, propylene glycol, polyethylene glycol), nonionic surfactant [ For example, there are other adjuvants which may be used in combination with a suitable solubilizing agent such as polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil) and the like. As an oily medium, for example, sesame oil, soybean oil and the like which can be used in combination with a solubilizing agent such as benzyl benzoate and benzyl alcohol are used. The injection so prepared is filled into suitable ampoules.

  Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in unit dose forms suitable for adapted doses of the active ingredient. As used herein, a "unit dosage form" is one in which each unit is calculated to produce the desired therapeutic effect in association with a pharmaceutical diluent, carrier or vehicle (ie, a PCSK9 inhibitor) Refers to physically discrete units suitable as a single dosage form for human and / or animal subjects, containing a predetermined amount. In some embodiments, the PCSK9 inhibitor is an antibody or antigen binding protein and the unit dosage form comprises 75 mg, 150 mg, 200 mg, or 300 mg of antibody or antigen binding protein. Examples of suitable unit dosage forms for liquid pharmaceutical compositions include applicators, eg, syringes, including vials, prefilled syringes and reusable syringes, automatic syringes, including prefilled automatic syringes and reusable automatic syringes Other suitable unit dosage forms include tablets, pills, capsules, suppositories, wafers, any of the divided plurality mentioned above, and as described herein. Or generally include other forms known in the art. The unit dosage form may be sealed. The amount of PCSK9 inhibitor may be indicated to the applicator for a unit dosage form containing the pharmaceutical composition in the applicator.

  The invention includes (a) one or more unit dosage forms comprising a pharmaceutical composition of the invention, and (b) a product or kit comprising a container or package. The product or kit may further comprise (c) a package attachment with a label or instructions for use. In some embodiments, the product can further comprise (d) one or more unit dosage forms of a lipid modification therapy (eg, a blister of a tablet comprising HMG-CoA reductase inhibitor as an active ingredient).

Dosage and Administration Regimens The amount of PCSK9 inhibitor (eg, anti-PCSK9 antibody) administered to a subject in accordance with the methods and compositions of the present invention is generally a therapeutically effective amount. The phrase "therapeutically effective amount" as used herein means a dose of PCSK9 inhibitor that results in a detectable decrease in atherosclerotic lesions. For example, a "therapeutically effective amount" of a PCSK9 inhibitor, for example, when administered to a human patient as shown in the examples herein, is at least 2 for example in the sclerosing lesion area or lesion severity. %, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, an amount of PCSK9 inhibitor that causes a reduction of 90% or more. Alternatively, animal models can be used to establish whether a particular amount of candidate PCSK9 inhibitor is a therapeutically effective amount.

In the case of anti-PCSK9 antibodies, the therapeutically effective amount is from about 0.05 mg to about 600 mg of the anti-PCSK9 antibody, eg, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2. 0 mg, about 3 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, About 130 mg, about 140 mg, about 150 mg, about 160 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, 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 43
0 mg, about 440 mg, about 450 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 590 mg, about 590 mg Or about 600 mg.

  In one embodiment, the anti-PCSK9 antibody is administered to the subject at a dose of 75 mg. In one embodiment, the anti-PCSK9 antibody is administered to the subject at a dose of 150 mg. In one embodiment, the anti-PCSK9 antibody is administered to a subject at a dose of 300 mg.

  The amount of anti-PCSK9 antibody contained within an individual dose may be expressed in the form of milligrams (ie, mg / kg) of antibody per kilogram of patient weight. For example, the anti-PCSK9 antibody may be administered to the patient at a dose of about 0.0001 to about 10 mg / kg of patient weight.

  According to one embodiment of the present invention, multiple doses of PCSK9 inhibitor may be administered to a subject over a defined time course. The method according to this aspect of the invention comprises sequentially administering multiple doses of PCSK9 inhibitor to the subject. As used herein, “sequentially administering” means that each dose of PCSK9 inhibitor is separated by different time points, eg, different intervals (eg, hours, days, weeks or months) on different days. Means administered to a subject. The present invention sequentially serializes a patient with a single initial dose of PCSK9 inhibitor, followed by one or more secondary doses of PCSK9 inhibitor, optionally followed by one or more tertiary doses of PCSK9 inhibitor. Including methods comprising administering.

  The terms "initial dose," "second dose," and "tertiary dose" refer to the temporal sequence of administration of PCSK9 inhibitor. Thus, the "initial dose" is the dose administered first of the treatment regimen (also referred to as the "baseline dose"); the "secondary dose" is the dose administered after the "initial dose"; A dose is the dose administered after the "second dose." However, in certain embodiments, the amount of PCSK9 inhibitor contained in the initial, secondary and / or tertiary doses varies from one another (eg, adjusted up and down as needed) during the course of treatment. The initial, secondary and tertiary doses may all contain the same amount of PCSK9 inhibitor.

  In certain embodiments, the secondary and / or tertiary doses are each 1-30 days of the immediately preceding dose (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more days). As used herein, the phrase "immediate dose" refers to the dose of PCSK9 inhibitor administered to a patient prior to the administration of the next dose in the sequence, without an intervening dose in a multiple dose sequence. means.

  In certain embodiments, the method comprises administering to the patient any number of secondary and / or tertiary doses of a PCSK9 inhibitor. For example, in one embodiment, only a single secondary dose is administered to a 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 certain embodiments, only a single 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 where multiple secondary doses are involved, 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 to 29 days after the immediately preceding dose. Similarly, in embodiments in which multiple tertiary doses are involved, each tertiary dose may be administered as frequently as other tertiary doses. For example, each tertiary dose may be administered to the patient 1 to 60 days after the immediately preceding dose. Alternatively, the frequency at which the secondary and / or tertiary doses are administered to the patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by the physician, depending on the individual patient's needs after consultation.

  In one embodiment, the anti-PCSK9 antibody is administered to the subject every other week with an initial dose of about 75 mg.

  In a further embodiment, the antibody is administered to the subject every other week with an initial dose of about 150 mg.

  In one embodiment, the initial dose is administered during the initial dose period, after which the subject's LDL-C value is monitored and the LDL-C value is measured to be at or below the target LDL-C value. By determining whether it is necessary to change the initial dose to a secondary dose. For example, a subject may be started with treatment with an initial dose of 75 mg of antibody at a target LDL-C value of 100 milligrams per deciliter (mg / dL). If the subject's LDL-C reaches the target LDL-C value at the end of the initial dose period, the subject continues to be treated with the initial dose, but is changed to a secondary dose if the target value is not reached. Ru. In one embodiment, the initial dose is approximately 75 mg administered biweekly, the target LDL-C value is 100 mg / dL, and if necessary, the second dose is administered biweekly at 150 mg. For example, subjects can be monitored initially about 8 weeks after treatment, and if their LDL-C values exceed 100 mg / dL, they can be dosed at a secondary dose of 150 mg, biweekly dosing . Monitoring may continue over the treatment period. In another embodiment, the initial dose is biweekly for 75 mg of antibody, the target LDL-C value is 70 mg / dL, and the secondary dose is 150 mg for biweekly if the target LDL-C value is not reached.

Conventional Therapy and Combination Therapy According to one embodiment, the method of the invention is a treatment for the treatment of hypercholesterolemia and / or atherosclerosis, at or just prior to the time of administration of the pharmaceutical composition of the invention. A subject in a regimen comprises administering a pharmaceutical composition comprising an anti-PCSK9 antibody. For example, a patient who has previously been diagnosed with hypercholesterolemia and / or atherosclerosis may have a stable therapeutic regimen of another lipid modification therapy before and / or simultaneously with the administration of the pharmaceutical composition comprising the anti-PCSK9 antibody. May be prescribed and ingested. In other embodiments, the subject has not been previously treated with lipid modification therapy.

  According to an embodiment, the method of the invention comprises administering to the subject, as a monotherapy, a pharmaceutical composition comprising an anti-PCSK9 antibody in the absence of simultaneous lipid modification therapy.

  According to one embodiment, the method of the invention also comprises administering to the subject a pharmaceutical composition comprising an anti-PCSK9 inhibitor in combination with another lipid modification therapy.

The lipid modification therapy used herein is, for example, 3-hydroxy-3-methylglutaryl such as (1) statins (eg, cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin etc) (HMG) -an agent which induces cellular depletion of cholesterol synthesis by inhibiting coenzyme A (CoA) reductase; (2) an agent which inhibits cholesterol uptake and / or bile acid reabsorption (eg ezetimibe); 3) agents that increase lipoprotein catabolism (eg, niacin); and / or (4) activators of LXR transcription factors that play a role in cholesterol removal, such as 22-hydroxycholesterol. Lipid modification therapies also include ezetimibe plus simvastatin; statins and bile resins (eg cholestyramine, colestipol, colesevelam); fixed combinations of therapeutic agents such as niacin plus statins (eg niacin and lovastatin); or omega-3 fatty acid ethyls Includes fixed combinations with other lipid lowering agents such as esters (eg, Omacor).

  The following examples are proposed to provide the person skilled in the art with a complete disclosure and explanation of how to make and use the methods and compositions of the present invention, limiting the scope of what the inventors regard as their invention. It is not intended to be.

Generation of Human Antibodies to Human PCSK9 Human anti-PCSK9 antibodies were generated as described in US Pat. No. 8,062,640. An exemplary PCSK9 inhibitor used in the following examples is a human anti-PCSK9 antibody designated as "mAb 316P", also known herein as "alilocumab". mAb 316 P has the following amino acid sequence characteristics: heavy chain variable region (HCVR) comprising SEQ ID NO: 1; light chain variable domain (LCVR) comprising SEQ ID NO: 6; heavy chain complementarity determining region 1 comprising SEQ ID NO: 2 HCDR1); HCDR2 comprising SEQ ID NO: 3 HCDR3 comprising SEQ ID NO: 4; light chain complementarity determining region 1 comprising SEQ ID NO: 7 (LCDR1); LCDR2 comprising SEQ ID NO: 8; and LCDR3 comprising SEQ ID NO: 10.

Achieving low density lipoprotein cholesterol targets in patients with high cardiovascular risk: Results from a controlled care trial population Background: US guidelines support LDL-C targets based on patients' cardiovascular (CV) risk profile, however Little is known about the real-world patterns of achieving LDL-C goals in specific high CV risk conditions.
Methods: Patients were identified from the OptumInsight IMPACT database (large US multi-peer claims database) with LDL-C measurements between July 2011 and June 2012 and high CV risk conditions. The most recent LDL-C measurement was defined as the index day and high CV risk states were identified in a hierarchy between pre-index periods as follows: Recent Acute Coronary Syndrome (ACS, June Pre) Within indicator days), coronary events (myocardial infarction, hospitalization for unstable angina, coronary revascularization), stroke, and peripheral vascular disease (PVD).
Results: In total, 110, 739 patients met the inclusion criteria. Median (IQR) age was 59 (53-65) years, 53.7% male, and median (IQR) LDL-C was 116 (92-143) mg / dL. As of the index day, 2.7% recently had ACS, but 42.1%, 9.2% and 46.0% have evidence of coronary events, stroke and PVD respectively It was The following table (Table 1) represents the general distribution of patients by LDL-C levels at the index date for different high CV risk conditions.

  Conclusions: In the large peer cohort of high CV risk patients, slightly achieved <70 mg / dL (optional goal for very high CV risk) or <100 mg / dL LDL-C target. The LDL-C target achieved a slight improvement in patients with conditions representing higher CV risk and recently achieved the highest in patients with ACS, but the majority of these patients are still above the LDL-C target there were. These data indicate that there is a need for continuous effort to address gaps in LDL-C goal achievement and improve CV outcomes in high risk patients.

Achieving Low Density Lipoprotein Cholesterol Targets and Lipid Reduction Therapy in High Cardiovascular Risk Management Care Study Population Background: US guidelines support statins as first-line therapy to reduce LDL-C, but statins and other lipids Little is known about the real-world patterns of achieving LDL-C goals with reduction therapy (LLT).
Methods: LDL-C measurements between July 2011 and June 2012 and high CV risk conditions (coronary events (myocardial infarction, myocardial infarction, unstable angina) from the OptumInsight IMPACT database (large US multi-peer claims database) The patients were identified on admission for coronary artery revascularization, stroke, and peripheral vascular disease). The LLT formulation was evaluated at the time of the latest LDL-C measurement (index day) and high potency statin (atorvastatin 40/80 mg, rosuvastatin 20/40 mg, simvastatin 80 mg), standard potency statin (other statin), non- It was classified as statin LLT (ezetimibe, niacin, fibrate, bile acid scavenging factor), and no LLT.
Results: In total, 110, 739 patients met the inclusion criteria. Median (IQR) age was 59 (53-65) years, 53.7% male, and median (IQR) LDL-C was 116 (92-143) mg / dL. As of the index day, 10.8% was high-intensity statin, 26.9% was standard-intensity statin, 5.3% was non-statin LLT, and 57.0% was no LLT . The following table (Table 2) represents the general distribution of patients by LDL-C levels at the index date for the different LLT types.

  Conclusions: In this large CV group of patients with high CV risk, only a few achieved <70 mg / dL (optional goal for very high CV risk) or <100 mg / dL LDL-C targets. Furthermore, despite receiving high potency statins, many other patients did not reach the LDL-C goal. These data indicate that there is a continuing effort to improve adhesion to LLT and the need for new therapies to improve goal achievement and CV outcomes.

Monoclonal antibody MAb 316 P against PCSK-9 is described in APOE * 3 Leiden. Background to dose-dependently reduce atherosclerosis, induce a more stable plaque phenotype and potentiate the effects of atorvastatin in CETP transgenic mice The aim of this study is APOE * 3 Leiden. It was to investigate the effects of the two forms of mAb 316P, alone and in combination with atorvastatin, on plasma lipids, atherosclerosis development, and lesion composition of CETP mice. It is characterized by the accumulation of residual lipoproteins (remnant lipoproteins) and increased ultra-LDL (VLDL) cholesterol and LDL-C ratio, all characteristics of familial dysbetalipoproteinemia (FD) in humans It is an established model for hyperlipidemia and atherosclerosis associated with it. In contrast to normal wild-type mice, where APOE * 3 Leiden mice have very rapid clearance of apoB-containing lipoproteins, (V) LDL clearance is impaired and TG levels are increased, thereby being recognized in humans Mimic the slow clearance APOE * 3 Leiden. Lipoprotein profiles in CETP mice reflect that of FD patients, with similar responses to lipid modification therapies, including statins, fibrates, niacin, and cholesteryl ester transfer protein (CETP) inhibitors. This is shown by the comparable reduction of cholesterol in all apoB-containing lipoprotein subfractions with statin treatment. It was hypothesized that mAb 316P alone could reduce the progression of atherosclerosis and add to the atheroprotective effect of atorvastatin. APOE * 3 Leiden. Inhibition of atherosclerosis by atorvastatin in CETP mice has been previously observed.

Method i) Animals 90 female APOE * 3 Leiden. Expressing human CETP under the control of its native flanking region. CETP transgenic mice (9-13 weeks old) were used.
During the study, mice were housed under standard conditions with a 12 hour light and dark cycle and had free access to food and water. Animal experiments were approved by the Institutional Animal Care and Use Committee at the Netherlands Organization for Applied Research facility.

ii) Experimental design Mice were fed a semi-synthetic cholesterol-enriched diet containing 15% (w / w) cocoa butter and 0.15% cholesterol (Western-type diet [WTD]; Hope Farms, Woerden, The Netherlands). , Given a 3-week induction period, increased plasma total cholesterol (TC) levels to -15 mmol / L. Body weight (BW) and food intake were monitored regularly during the study. After matching based on BW, TC, plasma TG, and age, mice (n = 15 / group) are given WTD alone or mAb 316P (3 or 10 mg / kg) alone or atorvastatin (3.6 mg / kg) Treatment with the two dosage forms in combination with / d) was given for 18 weeks and also added the atorvastatin alone group. MAb 316P was administered weekly via subcutaneous injection and atorvastatin was added to the diet. The dose of atorvastatin was calculated to attempt a TC reduction of about 20% to 30%. At the end of the treatment period, all animals were sacrificed by CO ₂ inhalation. The liver and heart were isolated to assess liver LDLR protein levels, lipid levels, atherosclerosis development, and plaque composition.

iii) Measurement of plasma lipids, lipoprotein analysis, and MAb 316P levels Plasma was isolated from blood collected in ethylenediaminetetraacetic acid (EDTA) coated cups via tail vein hemorrhage after every 2 to 4 weeks and after fasting for 4 hours. It was released. Plasma TC and TG were determined using an enzymatic kit according to the manufacturer's protocol (Catalog No. 1458216 and Catalog No. 1488872, respectively; Roche / Hitachi) and mean plasma TC and TG levels were calculated. Lipoprotein profiles for TC were measured after lipoprotein separation by fast protein liquid chromatography (FPLC) after 4, 12 and 18 weeks of treatment. MAb 316 P levels were measured by human Fc enzyme linked immunosorbent assay.

iv) Liver LDLR protein level Liver tissue was dissolved in lysis buffer (50 mM Tris-HCL [pH = 7.4], 150 mM
NaCl, 0.25% deoxycholic acid, 1% NP-40 [Igepal], 1 mM
Homogenize in EDTA, protease inhibitor cocktail [complete, Roche], 1 mM PMSF, 1 mM Na3VO4), and then centrifuge at 6500 rpm, 4 ° C. for 30 minutes. Protein concentration in cell lysates was determined by bicinchoninic acid protein assay (Thermo Scientific) according to the manufacturer's instructions. 50 μg of protein lysate was separated by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Millipore). Blot the goat anti-mouse LDLR from R & D Systems, rabbit anti-goat horseradish peroxidase (HRP) from AbD Serotec or mouse anti-α-tubulin from Sigma and horse anti-mouse HRP from Cell Signaling Technologies (manufacturer's instructions Blots were developed with West Femto Super Signal ECL (Thermo Scientific) and processed with Chemi-Doc-it imaging system. The intensity of the protein band was quantified using Image J software.

v) Histological evaluation of atherosclerosis Hearts were isolated, fixed in formalin and embedded in paraffin. Cross sections (5 μm each) of the entire aortic root area were stained with hematoxylin-phloxine-saffron. For each mouse, four sections at 50 μm intervals were used for quantitative and qualitative evaluation of atherosclerotic lesions. In order to determine atherosclerotic lesion size and severity, the lesions are classified into five categories I) early fatty streaks according to American Heart Association classification, II) regular fatty streaks, III) mild plaques, IV) It was classified into moderate plaque, and V) serious plaque. The total lesion area and number of lesions per cross section and the undiseased segment percentage were calculated. To assess lesion severity as a percentage of all lesions, Type I-III lesions were classified as mild lesions and Type IV-V lesions were classified as severe lesions. In order to determine the total plaque mass in the thoracic aorta, the perfusion-fixed aorta (from aortic origin to the diaphragm) is flushed with extravascular fat, opened longitudinally and held in front, oil red for lipids Formerly in Verschuren et al. It was stained as described by (Arterioscler Thromb Vasc Biol. 2005; 25: 161-167). Data were normalized to the surface analyzed and expressed as a percentage of stained area. Photographs / images were taken with an Olympus BX51 microscope and lesion areas were measured using Cell D imaging software (Olympus Soft Imaging Solutions).

  At the aortic root, smooth with mouse anti-human alpha actin (1: 800; Monosan, Uden, The Netherlands) before determining lesion composition as a percentage of lesion area for severe lesions (type IV-V) Macrophages were immunostained with muscle cells (SMC) and rat anti-mouse Mac-3 (1:25; BD Pharmingen, the Netherlands) followed by collagen staining with Sirius red stain. Necrosis and cholesterol clefts, monocyte adhesion to the endothelium, T cell abundance in the aortic root area and plaque stability index (macrophage as a destabilizing agent and collagen and SMC as a stabilization factor to the necrotic area (Previously defined by Kuhnast et al.). (J Hypertens. 2012; 30: 107-116), Stary et al. (Arterioscler Thromb Vasc Biol. 1995; 15: 1512-1531) and Kuhnast et al. It was determined as described by (PLoS One. 2013; 8: e66467). The rat anti-mouse CD54 antibody GTX 76543 (GeneTex, Inc., San Antonio, TX, USA) was used for immunostaining of cell adhesion molecule 1 (ICAM-1). Photographs / images of lesions were taken on an Olympus BX40 microscope with a Nuance 2 multispectral imaging system, and stained areas were quantified using Image J software.

vi) Liver lipid analysis and fecal excretion of bile acids and neutral sterols Liver tissue samples were homogenized in phosphate buffered saline and the protein content was determined. Lipids were previously described in Post et al. As described by (Hepatology. 1999; 30: 491-500), they were extracted, separated by high-speed thin layer chromatography on silica gel plates and analyzed with TINA 2.09 software (Raytest Isotopen Messgerate Straubenhardt, Germany).

  Mice were housed 5 mice per cage and feces were collected during two consecutive periods of 72 hours and 48 hours respectively. Aliquots of lyophilised feces have been previously described by Post et al. Used for the determination of neutral and acid sterols content by gas liquid chromatography as described by (Arterioscler Thromb Vasc Biol. 2003; 23: 892-897).

vii) Flow cytometric analysis After 8 weeks of treatment, peripheral blood mononuclear cells (PBMC) are isolated from fresh blood samples and GR-1 + (neutrophils / granules using flow cytometry (FACS) analysis Spheres), GR-1- (lymphocytes / monocytes), CD3 + (T-cells), CD19 + (B-cells) and CD11b + / Ly6C ^low and CD11b + / Ly6C ^hi (monocytes) cells. The following conjugated monoclonal antibodies were used from Becton Dickinson: GR-1 FITC, CD3 PerCpCy5-5, CD19 V450, CD11b APC and Ly6C PE-Cy7.

viii) Statistical analysis Significance of differences between groups was calculated nonparametrically using the Kruskal-Wallis test for independent samples followed by the Mann-Whitney U-test for independent samples. Linear regression was used to assess the correlation between variables. As atherosclerotic lesion areas showed a secondary dependency on plasma cholesterol exposure, they were transformed using a square root transformation. IBM SPSS Statistics 20 for Windows® (SPSS, Chicago, USA) was used for statistical analysis. All groups were compared to the control and atorvastatin groups, and 3 mg / kg mAb 316P was compared to 10 mg / kg mAb 316P with or without atorvastatin. Values were expressed as mean ± SD. P-values <0.05 were considered statistically significant for single comparisons. The Bonferroni method was used to determine significance levels in the case of multiple comparisons. In the figure, significant effects after correction for multiple comparisons are shown as *** compared to the control group, shown as † compared to the atorvastatin group, with 3 mg / kg mAb 316 P and 10 mg / kg mAb 316 P It is shown by comparison and ‡‡‡.

Results i) MAb 316P and Atorvastatin alone treatment and their combinations are described in APOE * 3 Leiden. Lower plasma total cholesterol and triglycerides in CETP mice Circulating mAb316P levels are detected in all groups receiving mAb316P and during the 18-week trial, 5-12 μg / mL (3 mg / kg dose) and 12-30 μg / mL It fluctuated between (10 mg / kg dose). APOE * 3 Leiden. In cholesterol-containing WTD (control group). CETP mice reached mean plasma TC and 16.2 ± 1.8 mmol / L and 2.9 ± 0.6 mmol / L, respectively (FIGS. 1A and 1B). MAb 316 P had a mean plasma TC (-37%, P <0.001; -46%, P <0.001) and TG (-33%, P <0.001; -39%) compared to controls. P <0.001) was reduced, and TC (-48%, P <0.001; -58%, P <0.001) was further reduced in combination with atorvastatin. Compared to atorvastatin, both combination treatments have TC (-36%, P <0.001; -48%, P <0.001) and TG (-40%, P) to a greater extent than atorvastatin alone. <0.001; -51%, P <0.001). mAb 316P alone (-14%, P <0.01; 3 mg
The reduction in TC after mAb316P vs. 10 mg mAb316P) and in combination with atorvastatin (-19%, P <0.001; 3 mg mAb316P + atorvastatin vs 10 mg mAb316P + atorvastatin) is dose-dependent and persistent across trials The TC reduction after mAb 316P (FIG. 1C), atorvastatin, and their combination (FIG. 1D) is limited to apoB-containing lipoproteins. No effect on BW (gain) and food intake was noted in any treatment group compared to controls.

ii) MAb 316P (with or without atorvastatin) lowers plasma lipids by reducing low density lipoprotein receptor degradation and reduces non-HDL cholesterol Measurement of hepatic LDLR protein levels, PCSK9 inhibition by mAb 316P It was verified whether plasma lipids were reduced by rescue of degradation (FIG. 2A). Liver LDLR protein levels were combined with mAb316P treatment alone (+ 80%, P <0.01; + 133%, P <0.01) and atorvastatin (+ 98%, P <0.001; + 178%, P <0. 05) It increased later. Both combination treatments increased LDLR protein levels to a greater extent (+ 71%, P <0.0045; + 140%, P <0.01) compared to atorvastatin alone. The inverse correlation between LDLR protein levels and plasma TC confirmed the involvement of LDLR in the reduction of TC by mAb 316P (R2 = 0.50, P <0.001).

  Non-HDL cholesterol levels were also measured to see if PCSK9 inhibition by mAb 316P reduced such levels by upregulation of LDLR (FIG. 2B).

iii) MAb 316P does not affect hepatic lipid and fecal bile acid and neutral sterol excretion To evaluate the consequences of mAb 316 P induced changes in hepatic lipid metabolism and lipoprotein metabolism in fecal excretion, fecal liver lipids and The excretion of bile acids and neutral sterols was determined. MAb 316 P has no effect on liver weight or liver and cholesterol content of TG, while atorvastatin and combination treatments have liver weight (-15%, P = 0.067, respectively) compared to control group; -17%, P <0.05, N.S. after correction for multiple comparisons; -20%, P <0.0045) and liver cholesteryl ester (-48%, P <0.0045 respectively; -41% , P <0.0045 and-44%, P <0.05, corrected for N.S. multiple comparisons) but with no change in liver triglycerides (Table 3). Fecal output of bile acids and neutral sterols did not change with treatment (Table 4). These data indicate that hepatic cholesterol homeostasis is maintained during mAb 316 P and statin treatment, despite the larger influx of cholesterol from the plasma compartment.

iv) MAb 316 P dose-dependently reduce atherosclerotic development and enhance the atheroprotective effect of atorvastatin In the absence and presence of atorvastatin, the effect of mAb 316 P on the development of atherosclerosis, 18 Weeks later were evaluated at the aortic root and aortic arch. Representative images of atherosclerotic lesions show that mAb 316P, atorvastatin, and their combinations reduce lesion progression, as shown in FIG. To confirm the reduction in atherosclerotic development, the number of lesion areas and lesions per cross section (Figures 4A and 4B) was evaluated along with the lesion severity (Figure 4C). For the control group, the total lesion area was 278 ± 89 × 10 ³ μm ² per cross section, consisting of 4.0 ± 0.7 lesions per cross section. MAb 316 P dose-dependently reduced atherosclerotic lesion size compared to controls (-71%, P <0.001; -88%, P <0.001), and the effect of atorvastatin was dose-dependent (-89%, P <0.001; -98%, P <0.001). In addition, mAb 316P (with or without atorvastatin) also reduced the number of lesions (-17%, P <0.05, respectively, after correction for NS multiple comparisons; -30%, P <0.0045, respectively) And -41%, P <0.001; -77%, P <0.001). Mice treated with mAb 316P (alone and in combination with atorvastatin) had sections without lesions and fewer severe (type IV-V) lesions compared to controls. Atorvastatin alone reduces lesion size (-35%, P <0.05, corrected for N.S. multiple comparisons) and reduces severity to a lesser extent without affecting the number of lesions or undiseased segments I did. The combination had a lesion size (-82%, P <0.001; -97%, P <0.001) and a number of lesions (-38%, P <0.001; -76) as compared to atorvastatin monotherapy. %, P <0.001), and increased the amount of undiseased segments.

To evaluate the effect of mAb 316P treatment on lesion development at another spot along the aorta that is prone to develop atherosclerosis, the plaque surface in the aortic arch was measured (FIG. 4D). At this site, lesion development is delayed compared to the aortic root. 10 mg / kg mAb 316P alone (-67%, P <0.05, after correction for N.S. multiple comparisons) and both doses combined with atorvastatin (-73%), consistent with an atherogenic effect on aortic origin , P <0.0045; -73%, P <0.0045) reduced the total plaque area.

  The antiatherogenic effect of mAb 316P and atorvastatin was evaluated (Figure 5) and a strong correlation between plasma TC levels at the aortic root and atherosclerotic lesion area was observed (R2 = 0.84, P <0.001 ; Figure 5), which illustrates the important role of cholesterol in the development of atherosclerosis.

v) MAb 316 P reduces monocyte and T cell recruitment and improves the lesion stability index The number of monocytes adhering to activated endothelium as a functional marker of vascular wall inflammation (FIG. 8A) and aortic genesis The number of T cells in the beginning region (FIG. 8B) was counted per cross section and calculated (FIG. 7A). In the control group, there were 5.7 ± 4.2 adherent monocytes and 16.7 ± 7.7 T cells. When administered alone and in combination with atorvastatin, high doses of mAb 316 P (10 mg / kg) were obtained using adherent monocytes (-57%, P <0.01, after correction for NS multiple comparisons, and -75 %, P <0.001) and T cell abundance (-37%, P <0.05, N.S. after correction for multiple comparisons, and -62%, P <0.001) were reduced . Endothelial ICAM-1 expression was assessed by immunohistochemistry to clearly show the mechanism by which mAb 316P decreases monocyte adhesion (FIG. 8C). For controls, 39% of endothelium are positive for ICAM-1, 19% (P <0.001) after 10 mg / kg mAb 316P treatment alone and 16% (P <0.001) when given in combination with atorvastatin Compared with). Thus, the decrease in monocyte adhesion was reinforced by the decrease in adhesion molecule expression in endothelial cells after mAb 316P treatment alone and in combination with atorvastatin.

  After investigating the lesion morphology, the treatment effect on the plaque composition was analyzed in severe lesions (type IV-V) considered to be the most vulnerable lesions as shown by the representative images in FIG. To illustrate that plaque stability is not necessarily dependent on the size of the lesion, representative images of similar sized lesions for the control and mAb 316P groups were included. Lesion macrophage area (Fig. 7A left) + lesion necrotic core area (including cholesterol cleft) (Fig. 7A right) is quantified as a destabilizing factor, while SMC in the fibrous cap (Fig. 7B left) and collagen The area (FIG. 7B right) was quantified as a stabilization factor. All were expressed as a percentage of total lesion area. Lesions in the control group consisted of 10.3% macrophages, 4.8% necrotic cores and cholesterol clefts, SMC in a 3.1% cap and 48.4% collagen. The lesion stability index (FIG. 7C) for the control group was 3.5 ± 0.8. When administered in combination with atorvastatin, low doses of mAb 316P (3 mg / kg) reduce the destabilizing agent (-26%, P <0.05, after correction for multiple comparisons) and stabilizes it (+ 19%, P <0.05, corrected for N.S. multiple comparisons) was increased, while high dose (10 mg / kg) alone and in combination with atorvastatin resulted in destabilizing factor (-37) %, P <0.001; + 73%, P <0.001), and the stabilization factor (+ 19%, P <0.05, N.S. after correction for multiple comparisons; + 29%, P <0) .001) increased. Thus, MAb 316P is the lesion stability index alone (+ 24%, NS; + 113%, P <0.0045) and in combination with atorvastatin (+ 116%, P <0.05, NS multiplex). Lesion stability was improved as indicated by the increase in comparisons; 556%, P <0.001).

vi) MAb 316P Tends to Decrease Circulating Monocytes The effect of mAb 316P alone and in combination with atorvastatin on white blood cell counts was assessed by flow cytometry. Interestingly, granulocytes / neutrophils (-20%, P <0.05, N. S. after correction for multiple comparisons), as mAb 316 P alone and in combination with atorvastatin, as a percentage of the PBMC population; 34%, P <0.001) and monocytes (-28%, P <0.05, N.S. after correction for multiple comparisons; -39%, P <0.001) were reduced. More specifically, mAb 316P alone and in combination with atorvastatin reduce the proinflammatory Ly6C ^hi (-8%, P = 0.061; -19%, P <0.001), and anti-inflammatory Ly6C ^low ( + 12%, P = 0.089; + 35%, P <0.001) There is a tendency to increase monocytes. Thus, the effect of mAb 316P on vascular recruitment and monocyte adhesion is increased by the reduction in circulating monocytes.

SUMMARY This study was designed to investigate the effects of mAb 316P itself and in combination with atorvastatin on the development of atherosclerosis. Taken together, these data show that mAb 316P dose-dependently reduces plasma cholesterol, atherosclerosis progression and plaque fragility, APOE * 3 Leiden. To demonstrate that it potentiates the beneficial effects of atorvastatin in CETP mice. This test is the first test to show that antibodies against PCSK9 reduce the development of atherosclerosis.

Claims (39)

  A method of inhibiting atherosclerotic plaque formation in a subject comprising administering to the subject a pharmaceutical composition comprising a PCSK9 inhibitor.   A pharmaceutical composition comprising a PCSK9 inhibitor for use in the inhibition of atherosclerotic plaque formation in a subject.   The method or pharmaceutical composition according to claim 1 or 2, wherein the subject is non-hyperlipidemic.   The method or pharmaceutical composition according to claim 1 or 2, wherein the subject is apparently healthy. A method of treating or inhibiting the progression of atherosclerosis in a subject:
Selecting the subject suffering from stroke or myocardial infarction; and administering to the subject a pharmaceutical composition comprising a PCSK9 inhibitor, thereby treating atherosclerosis or inhibiting its progression.   A pharmaceutical composition comprising a PCSK9 inhibitor for use in the treatment of atherosclerosis or the inhibition of the progression thereof in a subject suffering from stroke or myocardial infarction.   7. The method or pharmaceutical composition according to claim 5 or 6, wherein the subject is non-hyperlipidemic. A method of treating atherosclerosis or inhibiting its progression in a non-hyperlipidemic subject:
Selecting a non-hyperlipidemic subject known to be at risk for developing atherosclerosis, and administering to the subject a pharmaceutical composition comprising a PCSK9 inhibitor, The method comprising treating atherosclerotic disease or inhibiting its progression.   PCSK9 inhibitors for use in the treatment of atherosclerosis or inhibition of its progression in non-hyperlipidemic subjects known to have or be at risk of developing atherosclerosis Pharmaceutical composition containing.   The method or pharmaceutical composition according to claim 8 or 9, wherein the subject is apparently healthy.   11. The method or pharmaceutical composition of any one of claims 1 to 10, wherein the subject is non-hypercholesterolemic.   12. The method or pharmaceutical composition of any one of claims 1-11, wherein the subject is non-hypertriglyceridemic.   13. The subject according to any one of claims 1 to 12, wherein the subject has a disease or disorder selected from the group consisting of type I diabetes mellitus, type II diabetes mellitus, Kawasaki disease, chronic inflammatory disease, and hypertension. A method or pharmaceutical composition.   14. The method or pharmaceutical composition of any one of claims 1-13, wherein the subject has an elevated level of an inflammatory marker. The method or pharmaceutical composition according to claim 14, wherein the inflammation marker is C-reactive protein.   15. The method or pharmaceutical composition according to claim 14, wherein the inflammatory marker is an inflammatory cytokine.   17. The method or pharmaceutical composition of any one of claims 1-16, wherein the PCSK9 inhibitor is an antibody or antigen binding protein that specifically binds PCSK9.   18. The method or pharmaceutical composition of claim 17, wherein the antibody or antigen binding protein comprises heavy and light chain CDR amino acid sequences having SEQ ID NOs: 12, 13, 14, 16, 17, and 18.   18. The method or pharmaceutical composition according to claim 17, wherein the antibody or antigen binding protein comprises HCVR having the amino acid sequence of SEQ ID NO: 11 and LCVR having the amino acid sequence of SEQ ID NO: 15.   18. The method or pharmaceutical composition of claim 17, wherein the antibody or antigen binding protein comprises heavy and light chain CDR amino acid sequences having SEQ ID NOs: 2, 3, 4, 7, 8, and 10.   21. The method or pharmaceutical composition according to claim 20, wherein the antibody or antigen binding protein comprises HCVR having the amino acid sequence of SEQ ID NO: 1 and LCVR having the amino acid sequence of SEQ ID NO: 6.   22. Any of the claims 17-21, wherein the antibody or antigen binding protein binds to the same epitope on PCSK9 as an antibody comprising heavy and light chain variable domain amino acid sequences as set forth in SEQ ID NOs: 1 and 6; or 11 and 15 respectively. The method or pharmaceutical composition according to any one of the preceding claims.   The antibody or antigen binding protein competes with PCSK9 for binding to an antibody comprising the heavy and light chain variable domain amino acid sequences as set forth in SEQ ID NOs: 1 and 6; or 11 and 15, respectively. The method or pharmaceutical composition according to any one of the preceding claims.   25. Any of claims 1-23, wherein the PCSK9 inhibitor reduces atherosclerotic plaque formation in the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. The method or pharmaceutical composition according to any one of the preceding claims.   25. The method or pharmaceutical composition of any one of claims 1-24, wherein the subject has heterozygous familial hypercholesterolemia (heFH).   26. The method or pharmaceutical composition of any one of claims 1-25, wherein the subject has a form of hypercholesterolemia (non FH) that is not familial hypercholesterolemia.   27. The method or pharmaceutical composition according to any one of the preceding claims, wherein the subject receives another lipid modifying agent before and / or during the administration of the antibody or antigen binding protein.   28. The method or pharmaceutical composition of claim 27, wherein the therapeutic lipid modifier is selected from the group consisting of statins, ezetimibe, fibrates, niacin, omega-3 fatty acids, and bile acid resins. 29. The method or pharmaceutical composition according to claim 28, wherein the statin is selected from the group consisting of cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin and pravastatin.   30. The method or pharmaceutical composition of any one of claims 1-29, wherein the subject has not received another lipid modifying agent before and / or during administration of the antibody or antigen binding protein.   31. The method or pharmaceutical composition of any one of claims 1 to 30, wherein the antibody or antigen binding protein is administered subcutaneously.   A unit dosage form comprising the pharmaceutical composition of any one of claims 17-23.   33. The unit dosage form of claim 32, wherein the PCSK9 inhibitor is an antibody or antigen binding fragment and the unit dosage form comprises 75 mg, 150 mg, 200 mg, or 300 mg antibody or antigen binding fragment.   34. The unit dosage form of claim 33, selected from the group consisting of prefilled syringes, prefilled autoinjectors, vials, cartridges, reusable syringes, and reusable autoinjectors.   35. The unit dosage form of claim 34, wherein the unit dosage form is sealed.   36. The unit dosage form of any one of claims 34-35, wherein the amount of antibody or antigen binding fragment is indicated in a sealed dosage form.   An article of manufacture comprising a pharmaceutical composition and a container according to any one of claims 17-23.   39. A product according to claim 37, comprising one or more unit dosage forms according to claims 32-36 and instructions for use. (A) inhibition of atherosclerotic plaque formation in the subject;
(B) treatment of atherosclerosis or inhibition of its progression in a subject suffering from stroke or myocardial infarction; or (c) known to be at risk for developing atherosclerosis. 39. Use of a pharmaceutical composition according to any one of claims 1 to 38 in the manufacture of a medicament for the treatment of atherosclerosis or the inhibition of the progression thereof in a non-hyperlipidemic subject.

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