CA2848201C

Methods for reducing lipoprotein(a) levels by administering an inhibitor of proprotein convertase subtilisin kexin-9 (pcsk9)

Abstract

The present invention provides methods for reducing lipoprotein(a) (Lp(a)) in patients. The methods of the present invention comprise selecting a patient who exhibits elevated serum Lp(a), and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor. In certain embodiments, the PCSK9 inhibitor is an anti-PCSK9 antibody such as the exemplary antibody referred to herein as mAb316P.

Term

6 yearsleft in the term

Expires 12 September 2032.

20 claims: 1 independent, 19 dependent

1
A pharmaceutical composition for use in reducing lipoprotein(a) (Lp(a)) levels, in a patient who exhibits a serum Lp(a) level above 30 mg/dL, wherein the pharmaceutical composition comprises a PCSK.9 inhibitor and a carrier, wherein the PCSK9 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds PCSK.9, and wherein the antibody or antigen binding fragment thereof comprises heavy and light chain CDRs of the HCVR and LCVR as set forth in the amino acid sequences of SEQ ID NOs:90 and 92, respectively.

339 paragraphs in 74 sections, as filed

METHODS FOR REDUCING LIPOPROTEN(a) LEVELS BY ADMINISTERING AN INHIBITOR OF PROPROTEIN CONVERTASE SUBT1LISIN KEXIN-9 (PCSK9)

FIELD OF THE INVENTION

[0001] The present invention relates to the field of therapeutic treatments of diseases and disorders which are associated with elevated levels of lipoproteins. More specifically, the invention relates to the administration of PCSK9 inhibitors to reduce the levels of serum Lp(a) in a patient.

BACKGROUND

[0002] Lipoprotein^) (Lpfa)) is a low-density lipoprotein-like particle formed by the association of apolipoprotein(a) (Apo(a)) with apoplipoprotein B100 (ApoBIOQ). The Apo(a) component is covalently linked to the ApoB100 component in the assembled Lp(a) particle via a disuifide bond. Elevated serum Lp(a) has been shown in several studies to correlate with a variety of atherosclerotic and thrombotic disorders. (See. e.g., Marcovina and Koschlnsky (1998), Am. J. Cardiol 82.57U-66U; ignatescu eta/. (1998), Tfoomb. Haemost. 80:231-232: Lippi and Guidi (2000), O. J. Med. 93:75-84; Bennet et al. (2008), Arch. Intern. Med. f68:598-608; The Emerging Risk Factors Collaboration (2009), J. Am. Med. Assoc. 302:412-423; and LamonFava et al. (2011 ), J. Lipid Res. 52:1181 -1187). Thus, therapeutic reduction of serum Lp(a) levels has been suggested as a means for treating or reducing the risk of cardiovascular disorders. There are few available therapeutic options for lowering serum Lp(a) levels.

Examples of treatments that have been tested and/or proposed for lowering serum Lp(a) include administration of acetylsalicylic acid, L-camitine, niacin oranacetcapib, or LDL apheresis. (See. e.g., Parhofer (2011), Cutr. Pbarm Des 17:871-876). No currently available treatments, however, provide adequate and practical therapy for elevated Lp(a).

[0003) Proprotein convertase subtiiisin/kexin type 9 (PCSK9) is a proprotein convertase belonging to the proteinase K subfamily of die secretory subtilase family. The use of PCSK9 inhibitors (anfi-PCSK9 antibodies) to reduce serum total cholesterol, LDL cholesterol and serum triglycerides is described in US Patent Appl. Publ. No. 2010/0166768. Nonetheless, heretofore, PCSK9 inhibitors have not been shown to lower Lp(a) levels in patients. Thus, there remains a need in the art for therapeutic methods of lowering serum Lp(a) levels.

BRIEF SUMMARY OF THE INVENTION

[0004) The present invention addresses the aforementioned need in the art by providing methods for towering serum Lp(a) levels in an individual. The methods of the invention comprise selecting a patient who exhibits elevated serum Lp(a). and administering a pharmaceutical composition comprising a PCSK9 inhibitor to foe patient. The patient is selected on the basis of having an elevated serum Lp(a) level that is indicative of enhanced risk

-1for cardiovascular and/or thrombotic occlusive diseases and disorders. The patient may also be selected on the basis of exhibiting additional risk factors for such diseases and disorders in which a reduction in Lp(a) levels would be beneficial or risk-lowering. For example, patients with hypercholesterolemia (e.g., heFH, nonFH, etc.) may be good candidates for treatment with the therapeutic methods of the present invention.

[0005] PCSK9 inhibitors which may be administered in accordance with the methods of the present invention include, eg., anti-PCSK9 antibodies or antigen-binding fragments thereof. Specific exemplary anti-PCSK9 antibodies which may be used in the practice of the methods of the present invention include any antibodies or antigen-binding fragments as set forth in US Patent Appl Publ. No. 2010/0166768, and/or disclosed herein.

[0006] The PCSK9 inhibitor may be administered to a subject subcutaneously or intravenously. Furthermore, the PCSK9 inhibitor may be administered to a patient who is on a therapeutic statin regimen at the time of therapeutic intervention.

[0007] Other embodiments of the present invention will become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION

[0008] Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0009] 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, when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression about 100 includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc ).

[0010] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are now described.

Elevated Serum Lp(a) Levels

[0011] The present invention provides methods for reducing serum Lp(a) levels in a patient. The methods of the invention comprise selecting a patient who exhibits elevated serum Lp(a), and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor. As used herein, the expressions Lp(a) or lipoprotein(a) refer to a low-density lipoprotein-like

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PCT/US2012/054756 particle formed by the association of apolipoprotein(a) (apo(a)) with apolipoprotein B100 (apo B100).

[0012] In fee context of fee present invention, elevated serum Lp(a)” means a serum Lp(a) ievel greater than about 14 mg/dL. In certain embodiments, a patient is considered to exhibit elevated serum Lp(a) if the level of serum Lp(a) measured in the patient Is greater than about 15 mg/dL, 20 mg/dL, 25 mg/dL, 30 mg/dL, 35 mg/dL, 40 mg/dL, 45 mg/dL, 50 mg/dL, 60 mg/dL, 70 mg/dL, 80 mg/dL, 90 mg/dL, 100 mg/dL, 120 mg/dL. 140 mg/dL, 160 mg/dL, 180 mg/dL. or 200 mg/dL. The serum Lp(a) level can be measured in a patient post-prandial, in some embodiments, the Lp(a) level is measured after a period of time of fasting (e.g., after fasting for 6 hrs. 8 hrs, 10 hrs. 12 hrs or more). An exemplary method for measuring serum Lp(a) in a patient is by rate immune-nephelometry, although any clinically acceptable diagnostic method can be used in the context of the present invention.

Patient Population

[0013] The methods of fee present invention are useful for reducing serum Lp(a) levels in human subjects that exhibit an elevated level of serum Lpfa), In some instances the patient is otherwise healthy except for exhibiting elevated serum Lp(a). For example, the patient may not exhibit any other risk factor of cardiovascular, thrombotic or other diseases or disorders at the time of treatment. In other instances, however, the patient is selected on the basis of being diagnosed witfi, or at risk of developing, a disease or disorder that is caused by or correlated wife elevated serum Lp(a). For example, at the time of, or prior to administration of the pharmaceutical composition of the present invention, the patient may be diagnosed with or identified as being at risk of developing a cardiovascular disease or disorder, such as, e g,, coronary artery disease, acute myocardial infarction, asymptomatic carotid atherosclerosis, stroke, peripheral artery occlusive disease, etc. The cardiovascular disease or disorder, in some instances, is hypercholesterolemia. For example, a patient may be selected for treatment wife toe methods of toe present invention if the patient is diagnosed with or identified as being at risk of developing a hypercholesterolemia condition such as. e.g., heterozygous Familial Hypercholesterolemia (heFH), homozygous Familial Hypercholesterolemia (hoFH), as well as incidences of hypercholesterolemia that are distinct from Familial Hypercholesterolemia (nonFH).

[0014] In other instances, at the time of, or prior to administration of the pharmaceutical composition of the present invention, the patient may be diagnosed with or identified as being at risk of developing a thrombotic occlusive disease or disorder, such as, e.g., pulmonary embolism, centrai retinal vein occlusion, etc. In certain embodiments, the patient is selected on the basis of being diagnosed with or at risk of developing a combination of two or more of the abovementioned diseases or disorders. For example, at the time of. or prior to administration of the pharmaceutical composition of toe present invention, the patient may be diagnosed with or

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PCT/US2012/054756 identified as being at risk of developing coronary artery disease and pulmonary embolism. Other diagnostic combinations (e.g., atherosclerosis and central retinal vein occlusion, heFH and stroke, etc.) are also included in the definition of the patient populations that are treatable by Sie methods of the present invention.

(00151 In yet other instances, the patient who is to be treated with the methods of the present invention is selected on the basts of one or more factors selected from the group consisting of age (e.g., older than 40,45,50,55,60,65,70.75, or 60 years), race, gender {male or female), exercise habits (e g., regular exerciser, non-exerciser), other preexisting medical conditions (e.g., type-ll diabetes, high blood pressure, etc.), and current medication status (e.g., currently taking statins [e.g.cerivastatm, atorvastatin, simvastatin, pitavastatm, rosuvastatin, fiuvastatin. lovastatin, pravastatin, etc.], beta blockers, niacin, etc.). The present invention also includes methods for reducing serum Lp(a) levels in patients who are intolerant of, non-responsive to. or inadequately responsive to conventional statin therapy. Potential patients can be seiected/screened on the basis of one or more of these factors (e g., by questionnaire, diagnostic evaluation, etc.) before being treated with the methods of the present invention.

(0016] The present invention also includes methods for increasing transirrtestinal cholesterol excretion (TICE) in a subject by administering a PCSK9 inhibitor to the subject. For example, the present invention provides methods for increasing TICE in a subject by administering to the subject an anti-PCSK9 antibody, e.g., the antibody referred to herein as ”mAb3i6P“. According to certain embodiments, the present invention includes methods comprising identifying a subject for which enhanced TICE would be beneficial, or identifying a subject that exhibits impaired TICE, and administering a PCSK9 inhibitor (e.g, mAb316P) to the subject.

PCSK9 Inhibitors (0017] The methods of tie present invention comprise administering to a patient a therapeutic composition comprising a PCSK9 inhibitor. As used herein, a PCSK9 inhibitor is any agent which binds to or interacts with human PCSK9 and inhibits the normal biological function of PCSK9 in vitro or in vivo. Non-limiting examples of categories of PCSK9 inhibitors include small molecule PCSK9 antagonists, peptide-based PCSK9 antagonists (e.g, peptibody molecules), and antibodies or antigen-binding fragments of antibodies that specifically bind human PCSK9. (0018] The term “human proprotein convertase subtilisin/kexin type 9” or human PCSK9 or hPCSK9, as used herein, refers to PCSK9 having the nucleic acid sequence shown in SEQ ID NO:754 and the amino acid sequence of SEQ ID NO:755, or a biologically active fragment thereof.

(0019] The term “antibody, as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g, IgM). Each heavy chain

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PCT/US2012/054756 comprises a heavy chain variable region (abbreviated herein as HCVR or V«) and a heavy chain constant region. The heavy chain constant region comprises three domains. ΟΗ1, Ch2 and Ch3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V0 and a tight chain constant region. The light chain constant region comprises one domain (Ct.l ). The V« and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs). interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the foilowing order: FR1. CDR1. FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the antiPCSK9 antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

(0020] The term ‘antibody/ as used herein, also includes antigen-binding fragments of full antibody molecuies. The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources. DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. Trie DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

(0021] Non-limiting examples of antigen-binding fragments indude: (i) Fab fragments; (ii) F(ab')2 fragments; (ill) Fd fragments: (iv) Fv fragments: (v) single-chain Fv (scFv) moiecuies, (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (COR) such as a CDR3 peptide), or a constrained FR3-COR3-FR4 peptide Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domaindeleted antibodies, chimeric antibodies. CDR-grafted antibodies, diabodies, triabcdies, tetrabodies, minibodies, nanobodies (e,g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs). and shark variable IgNAR domains, are also encompassed within the expression antigen-binding fragment. as used herein.

(0022] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally

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PCT/US2012/054756 comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the Vh and Vl domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain Vh-Vh, V^V. or V;,-V;. dimers Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V« or VL domain.

[0023] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigenbinding fragment of an antibody of the present invention include: (i) Vh-C>(1; (ii) Vh-C«2; (iii) V>tCh3; (iv) V(!-Cp1-Cti2; (v) 7=,-0,,1-C(j2-Cn3; (vi) Vh-Ci<2-C,3: (vii) (viii) Vt-CM1 : (ix) VL-C(<2: (x) Vl-Ch3; (xi) Vl-Cb1-Ch2; (xil) Vl-Ch1-Ch2-Ch3; (xiii) Vl-C^-C^; and (xiv) Vt-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a frill or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5,10,1S, 20.40,60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.

Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or Vt. domain (e.g., by disulfide bond(s)).

[0024] As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

[0025] The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody may be selected on the basts of whether it is desirable for the antibody to mediate cytotoxicity.

[0026] 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 invention may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or sitespecific mutagenesis in vitro or by somatic mutation in vivo}, for example in the CDRs and in particular CDR3. However, the term human antibody’, as used herein, is not intended to

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PCT/US2012/054756 include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0027] The term recombinant human antibody', as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.

Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to In vitro mutagenesis (or, when an animal transgenic for human lg sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and ty, regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0028] Human antibodies can exist in two forms that are associated with hinge heterogeneity, in one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.

[0029] 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. A single amino acid substitution in the hinge region of the human lgG4 hinge can significantly reduce the appearance of the second form (Angai et al. (1993) Molecuiar Immunology 30:105) to levels typically observed using a human IgGi hinge. The instant invention encompasses antibodies having one or more mutations in the hinge, C^2 or Ck3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.

[0030] An isolated antibody. as used herein, means an anybody 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 naturally exists or is naturally produced, is an isolated antibody for purposes of the present invention. An isolated antibody also

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PCT/US2012/054756 includes an antibody in situ within a recombinant call. Isoiated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.

(0031j The term specifically binds, or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that specifically binds PCSK9, as used in the context of the present invention, includes antibodies that bind PCSK9 or portion thereof with a Ko of less than about 1000 nM. iess than about 500 nM. less than about 300 nM, less than about 200 nM, iess than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, iess than about 60 nM. less than about 50 nM, less than about 40 nM. less than about 30 nM, iess than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, iess than about 2 nM. less than about 1 nM or less than about 0.5 nM, as measured in a surface plasmon resonance assay. An isolated antibody that specifically binds human PCSK9, however, have cross-reactivity to other antigens, such as PCSK9 molecules from other (non-human) species.

[0032] The anti-PCSK9 antibodies useful for the methods of the present invention may comprise one or more amino add substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino add sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention indudes methods involving toe use of antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disdosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino add substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as germline mutations). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germiine mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues with-n the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g..

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PCT/US2012/054756 only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residuets) are mutated to the corresponding residue(s) of a different germline sequence (/. e„ a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. The use of antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[0033} The present invention also includes methods involving the use of anti-PCSKS 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 includes the use of anti-PCSK9 antibodies having HCVR, LCVR. and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer. 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.

[0034J The term surface plasmon resonance, as used herein, refers to an optical phenomenon that aliows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0035] The term Ko ”, as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0036] The term epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or iinear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear poiypeptide chain. A iinear epitope is one produced by adjacent amino acid residues in a poiypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or suifonyi groups on the antigen.

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Preparation of Human Antibodies (00371 Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to human PCSK9.

(0038] Using VELOCIMMUNE™ technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals) or any other known method for generating monoclonal antibodies, high affinity cnimenc antibodies to PCSK9 are initially isolated having a human variable region and a mouse constant region. The VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.

(0039] Generally, a VELOCIMMUNE® mouse is challenged vwth the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identity hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

(0040] Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc, using standard procedures known to those skilled in the art. The mouse constant regions are replaced with a desired human constant region to generate the folly human antibody of the invention, for example wild-type or modified lgG1 or lgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region, [0041] In general, the antibodies that can be used in the methods of the present invention possess high affinities, as described above, when measured by binding to antigen either immobilized on solid phase or in solution phase. The mouse constant regions are replaced with desired human constant regions to generate the fully human antibodies of the invention. While the constent region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

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[0042] Specific examples of human antibodies or antigen-binding fragments of antibodies that specifically bind PCSK9 which can be used in the context of the methods of the present invention include any antibody or antigen-binding fragment which comprises the 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 SEQ ID NOs: 2. 18. 22. 26,42.. 46. 50. 66,70. 74. 90, 94, 98.114, 118,122.138. 142, 146.162, 166,170.186, 190.194, 210,214. 218, 234, 238,242,258, 262, 266, 282, 286, 290, 306, 310, 314, 330, 334, 338. 354, 358, 362. 378, 382. 386. 402,406.410. 426.430. 434, 450, 454. 458, 474,478.482, 498. 502, 506, 522, 526, 530, 546, 550, 554, 570, 574, 578, 594, 598, 602,618, 622, 626,642. 646.650,666,670,674,690,694, 698, 714,718, 722, 738 and 742, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. The antibody or antigen-binding fragment may comprise the three light chain CDRs (LCVR1, LCVR2, LCVR3) contained within a tight chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 20,24, 34,44,48, 58,68, 72,82, 92,96,106,116, 120,130,140, 144,154, 164,168,178, 188, 192,202, 212, 216,226,236. 240,250.260,264.274.284, 288, 298. 308,312, 322. 332.336, 346. 356,360, 370,380,384. 394,404,408,418,428.432, 442,452.456,466,476.480,490, 500. 504,514, 524, 528. 538, 548, 552, 562, 572, 576, 586, 596, 600. 610,620, 624, 634,644, 648. 658,668, 672,682, 692.696,706,716,720,730,740 and 744. or a substantially similar sequence thereof having ai ieast 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0043] In certain embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises the six CDRs (HCDR1, HCDR2. HCDR3, LCDR1. LCDR2 and LCDR3) from the heavy and light chain variable region amino acid sequence pairs (HCVR/LCVR) selected from the group consisting of SEQ ID NOs: 2/10,18/20,22/24,26/34, 42/44, 46/48, 50/58,66/68, 70/72, 74/82, 90/92, 94/96, 98/106.114/116.118/120, 122/130, 138/140,142/144,146/154,162/164, 166/168,170/178,186/188,190/192,194/202, 210/212, 214/216, 218/226,234/236,238/240, 242/250, 258/260, 262/264, 266/274,282/284, 286/288. 290/298,306/308,310/312,314/322, 330/332.334/336,338/346,354/356,358/360. 362/370, 378/380, 382/384, 386/394.402/404, 406/408,410/418,426/428,430/432,434/442. 450/452. 454/456,458/466.474/476,478/480,482/490,498/500,502/504,506/514. 522/524. 526/528. 530/538, 546/548. 550/552. 554/562, 570/572, 574/576, 578/586, 594/596, 598/600. 602/610. 618/620, 622/624,626/634, 642/644,646/648,650/658,666/668,670/672,674/682, 690/692, 694/696, 698/706, 714/716. 718/720, 722/730, 738/740 and 742/744.

(0044] In certain embodiments of the present invention, the anti-PCSK9 antibody, or antigenbinding fragment thereof, that can be used in the methods of the present invention has HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 amino acid sequences selected from SEQ ID

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NOs: 76/78/80/84/86/88 (mAb316P) and 220/222/224/228/230/232 (mAb300N) (See US Patent App. Putt No. 2010/0166768).

(0045j In certain embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises HCVR/LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NOs: 2/10, 18/20, 22/24,26/34,42/44,46/48, 50/58, 66/68,70/72,74/82, 90/92, 94/96, 98/106,114/116,118/120,122'130, 138/140,142'144,146/154, 162/164, 166/168,170/178,186/188,190/192,194/202,210/212, 214/216,218/226,234/236.238/240, 242/250,258/260,262/264. 266/274, 282/284,286/288,290/298, 306/308, 310/312, 314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370,378/380, 382/384, 386/394. 402/404, 406/408, 410/418.426/428. 430/432,434/442.450/452,454/456.458/466.474/476. 478/480, 482/490,498/500, 502/504. 506/514, 522/524, 526/528,530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600, 602/610,618/620,622/624,626/634, 642/644, 646/648, 650/658,666/668, 670/672. 674/682, 690/692,694/696,698/706,714/716, 718/720, 722/730. 738/740 and 742/744.

Pharmaceutical Compositions and Methods of Administration (0O46J The present invention includes methods which comprise administering a PCSK9 inhibitor to a patient, 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 suitable transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to ai! pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et ai. Compendium of excipients for parenteral formulations PDA (1998) J Pharm Sci Technol 52:238-311.

(0047J Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, micropartides, microcapsufes, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.

(00481 A pharmaceutical composition of the present invention can be delivered

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PCT/US2012/054756 subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications tn delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0049] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVO PEN™ I, II and Hi (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen. Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt. Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen. Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMlRArM Pen (Abbott Labs, Abbott Park IL), to name only a few.

[0050] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see. Medical Applications of Controlled Release, Langer and Wise (eds.), 1974. CRC Pres., Boca Raton. Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, eg., Goodson, 1984, in Medical Applications of Coniroiled Release, supra, vol. 2. pp. 115-138). Other controlled release systems are discussed in the review by Langer. 1990, Science 249:1527-1533.

[0051] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be

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PCT/US2012/054756 prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oft, soybean oil. etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

(0052} Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose Include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

Dosage (0053} The amount of PCSK9 inhibitor (e.g.. anti-PCSK9 antibody) administered to a subject according to the methods of the present invention is, generally, a therapeutically effective amount. As used herein, the phrase therapeutically effective amount means a dose of PCSK9 inhibitor that results in a detectable reduction in serum Lp(a). For exampie, therapeutically effective amount of a PCSK9 inhibitor indudes, e.g., an amount of PCSK9 inhibitor that causes a reduction of at least 2%. 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more in Lp(a) levels when administered to a human patient, e.g., as illustrated in Example 2. herein Alternatively, animat models can be used to establish whether a particular amount of a candidate PCSK9 inhibitor is a therapeutically effective amount.

(0054} In the case of an anti-PCSK9 antibody, a therapeutically effective amount can be from about 0.05 mg to about 600 mg, e.g., about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg. about 110 mg, about 120 mg, about 130 mg. about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240mg, about 250 mg, about 260 mg, about 270 mg. about 280 mg, about 290 mg, about 300mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360mg, about 370 mg, about 38C mg, about 390 mg, about 400 mg, about 410 mg, about 420mg, about 430 mg. about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg. about 590 mg. or about 600 mg. of the anti-PCSK9 antibody.

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[0055] The amount of anti-PCSK9 antibody contained within the individual doses may be expressed in terms of milligrams of antibody per kilogram of patient body weight (i.e., mg/kg). For example, (he anti-PCSKS antibody may be administered to a patient ata dose of about 0.0001 to about 10 mg/kg of patient body weight.

Combination Therapies

[0056] The methods of the present invention, according to certain embodiments, may comprise administering a pharmaceutical composition comprising an anti-PCSK9 antibody to a patient who is on a therapeutic regimen for the treatment of hyperchoiesteroiemia at the time of, or just prior to, administration of the pharmaceutics! composition of the invention. For example, a patient who has previously been diagnosed with hyperchoiesteroiemia may have been prescribed and is taking a stable therapeutic regimen of another drug prior to and/or concurrent with administration of a pharmaceutical composition comprising an anti-PCSKS antibody. The prior or concurrent therapeutic regimen may comprise, e.g., (1) an agent which induces a cellular depletion of cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)coenzyme A (CoA) reductase, such as a statin (e.g., cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvasiatin, lovastatin, pravastatin, etc.); (2) an agent which inhibits cholesterol uptake and or bile acid re-absorption; (3) an agent which increase lipoprotein catabolism (such as niacin); and/or (4) activators of the LXR transcription factor that plays a role in cholesterol elimination such as 22-hydroxychoiesteroi. In certain embodiments, the patient, prior to or concurrent with administration of an anti-PCSK9 antibody is on a fixed combination of therapeutic agents such as ezetimibe plus simvastatin; a statin with a bile resin (e.g., cholestyramine, colestipol, colesevelam); niacin plus a statin (e.g.. niacin with lovastatin); or with other lipid lowering agents such as omega-3-fatty acid ethyl esters (for example, omacor).

Administration Regimens

[0057] According to certain embodiments of the present invention, multiple doses of a PCSK9 inhibitor may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multipie doses of a PCSK9 inhibitor. As used herein, sequentially administering” means that each dose of PCSK9 inhibitor is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of a PCSK9 inhibitor, followed by one or more secondary doses of the PCSK9 inhibitor, and optionally foilowed by one or more tertiary doses of the PCSK9 inhibitor.

[0058] The terms initial dose, secondary doses, and tertiary doses,” refer to the temporal sequence of administration of the PCSK9 inhibitor. Thus, the initial dose is the dose which is administered at the beginning of the treatment regimen (also referred to as the baseline dose’);

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PCT/US2012/054756 the secondary doses are the doses which are administered after the initial dose; and the tertiary doses are the doses which are administered after the secondary doses. The initiai, secondary, and tertiary doses may all contain the same amount of PCSK9 inhibitor, but will generally differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of PCSK9 inhibitor contained in the initial, secondary and/or tertiary doses will vary from one another (e.g., adjusted up or down as appropriate) during the course of treatment.

(0059] In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1 to 30 (e.g., 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 after the immediately preceding dose. The phrase the immediately preceding dose. as used herein, means, in a sequence of multiple administrations, the dose of PCSK9 inhibitor which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[0060] The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of a PCSK9 inhibitor. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e g., 2,3,4, 5,6,7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient, in other embodiments, two or more (e.g,, 2,3,4.5.6,7,8, or more) tertiary doses are administered to the patient.

(oosi] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 29 days after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary dosas, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 1 to 60 days after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

EXAMPLES (0062] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be

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PCT/US2012/054756 accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1. Generation of Human Antibodies to Human PCSK9

[0063} Human anti-PCSKS antibodies were generated as described in US Patent App. Publ No. 2010/0166768. The exemplary PCSK9 inhibitor used in the following Example is the human anti-PCSK9 antibody designated mAb316P. mAb316P has the following amino acid sequence characteristics: heavy chain variable region (HCVR) comprising SEQ ID NO:90; light chain variable domain (LCVR) comprising SEQ ID NO:92; heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO:76; HCDR2 comprising SEQ ID NO:78: HCDR3 comprising SEQ ID NO:80; light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO:84; LCDR2 comprising SEQ ID NO:86; and LCDR3 comprising SEQ ID NO:88.

Example 2: Clinical Trials of An Anti-PCSK9 Monoclonal Antibody, As Mono- or Add-On Therapy in Heterozygous Familial and Non-Familial Hypercholesterolemia

Introduction

[0064] This example describes the results from two clinical trials in which single doses of the anti-PCSK9 monoclonal antibody mAb316P were administered intravenously (IV) or subcutaneously (SC) to healthy volunteers, and a multiple-dose clinical trial of mAb316P in subjects with either Familial Hypercholesterolemia (FH) or nonFH, receiving stable doses of atorvastatin or dietary intervention only.

Methods

A. Study Design

[0065] Three clinical trials were conducted using mAb316P administered to human patients. Two of the trials were single dose placebo-controlled studies of mAb3l6P administered IV or SC, respectively. The third trial was a phase 1b, double-blind, randomized, placebo-controlled, ascending multiple-dose evaluation of mAb316P administered SC to subjects with FH or nonFH. The third trial was conducted in two parts: Part A and Part B. In Part A of the third trial, each subject received a total of 3 administrations of mAb316P (50 mg, 100 mg or 150 mg) or matching placebo. In Part B of the third trial, each subject received 2 administrations of either mAb3l6P (200 mg) or matching placebo. In both parts of the multiple SC dose trial, the first dose was administered in an inpatient unit where subjects were confined and observed for 48 hours post-dose Subsequent doses were administered in an outpatient setting with at least 2 hours of post-dose observation.

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B. Dose and Dose Escalation

[0066] The single dose IV study included 5 sequential cohorts (0.3,1.0, 3.0,6.0, or 12.0 mg/kg of mAb316P), and the single SC dose trial included 4 sequential dose cohorts (50.100, 150, and 250 mg of mAb316P). Based on initial results from these trials, the third triai evaluated SC mAb316P doses of 50 mg, 100 mg, 150 mg, or placebo administered on days 1, 29 and 43 to 7 groups of subjects with FH or nonFH (Part A), and 200 mg or placebo administered on days 1 and 29 to an eighth group of subjects with FH or nonFH (Part B). The dosing regimens and patient compositions of the three trials are summarized in Table 1.

Table 1

<td> mAb 316 Dose</td><td> Patient Group</td><td> Total No. Patients (mAb316:Pbo)</td><td> | i Screening Patient Type | LDL-C</td><td> Atorvastatin Dose</td>

<td colspan="5"> Single IV Dose</td>

<td colspan="5"> Single SC Dose</td>

<td> 50 mg</td><td> 1</td><td rowspan="4"> 8 (6:2)</td><td rowspan="4"> I I > 100 Volunteers । * I 1</td><td rowspan="4"> None</td>

<td colspan="5"> Multiple SC Dose</td>

<td> 2</td><td> 10 (8:2)</td><td> nonFH I</td>

<td> 4</td><td> 10(8:2)</td><td> , i > luo mg/dL nonFH i</td>

<td> 6</td><td> 10 (8:2)</td><td> nonFH |</td>

<td> 7</td><td> 10 (8:2)</td><td> nonFH | >130 mg/dL</td><td> None</td>

<td colspan="5"> PartB</td>

<td> 200 mg</td><td> 8</td><td> 10 (8:2)</td><td> FH& nonFH | >100 mg/dL</td><td> 10-40 mg OD</td>

[0067] In the multiple SC dose trial, each dose level commenced with a sentinel group of 3 (FH, non-FH, or both) subjects, with at least 1. but not more than 2, subjects receiving mAb316P. Enrollment of additional subjects at a given dose level only proceeded after the initial 3 subjects had safeiy completed their day 3 post-treatment assessments. Enrollment in

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PCT/US2012/054756 the next higher dose level was opened once 10 subjects had completed the day 15 safety assessments. Enrollment once started at each dose level, continued independent of the dose escalation decision and day 15 safety review.

C. Subjects {0058] Subjects in foe single-dose studies were healthy men and women (18-65 years of age 50-95 kg in body weight, body mass index [BMI] » 18-30 kg/m2) with serum LDL-C >100 mg/dL (2.59 mmol/L). The use of ail non-study agents to alter lipids was prohibited throughout these studies.

(0069] Subjects in the muitiple-dose study had heterozygous FH or nonFH (18-65 years of age, BMI=18.0-35.0 kg/m2) and were on stable atorvastatin therapy (10 to 40 mg per day) with LDL cholesterol >100 mg/dL (2.59 mmol/L), or nonFH and were on diet only with LDL cholesterol >130 mg/dL (3.36 mmol/L) (Table 1). All subjects reviewed and signed an informed consent previously approved by an institutional review board prior to any study related procedures. Subjects in the FH group met Simon Broome criteria for definite or possible FH (Simon Broome Register Group (1991), BMJ 303:893-896; Neil et a/., (2000), BMJ 321:148) while nonFH subjects did not Those taking atorvastatin were on a stable dose. 10 to 40 mg daily, for at least 28 days prior to baseline, and remained on the same dose throughout the study. NonFH patients on diet only could not have received lipid-lowering therapy for at least 28 days prior to baseline, and remained off such treatment throughout the study. Potentially eligible patients not on atorvastatin could enter a 4 week run-in period during which were switched to a dose of atorvastatin (10 to 40 mg per day) that was likely to maintain LDL cholesterol near their pre-study level and >100 mg/dL (2.59 mmol/L). All subjects had triglycerides £300 mg/dL and blood pressure controlled on fewer than 3 antihypertensive medications. Patients with a history of cerebrovascular, cardiovascular, or peripheral vascular atherosclerotic disease, diabetes, or disorders known to produce secondary elevations of LDL cholesterol were excluded as were those with hepatic transaminases (ALT or AST) >1.2 times the upper limit of normal (x ULN), >2+ proteinuria or creatine kinase (CK) >3 x ULN, unless clearly exercise related in which case they were required to have repeat testing with CK <3 x ULN.

D. Laboratory Measurements and Methods (0070] In the multi-dose study, serum lipid (total-, HDL-, LDL-, non-HOL-, and VLDL cholesterol) and apolipoprotein (Apo) B, A1 and Lp(a), hs-CRP and safety laboratory tests were performed after 12 hour overnight fasts (water only) on screening (day -21 to -3), day -2 (for those on atorvastatin), day-1, days 1,2,3,8,15, 29,43, 57, 71,85, 99,120, and theend-ofstudy (day 148). All laboratory measurements were performed by a central laboratory which maintained Part til certification by CDC Lipid Standardization Program and accreditation by the College of American Pathologists. Triglycerides and cholesterol were measured with enzymatic

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PCT/US2012/054756 colonmetric tests (Olympus AU2700 or AU5400 Analyzer, Olympus, Center Valley, Pennsylvania, USA) with calibration directly traceable to CDC reference procedures. Apo B containing lipoproteins were precipitated with dextran sulphate and HDL cholesterol was measured on the supernatant. (Warnick et al., (1978), J. Lipid Res. 19:65-76). LDL- and VLDL cholesterol were measured after preparative ultracentrifugation (beta-quantification). Apo A1, B, Lp(a) and hs-CRP were measured with rate immune-nephelometry (Dade Behring BNH nephelometer, Siemens Healthcare Diagnostics, Deerfield, Illinois, USA). Ali lipid, apolipoprotein, and hs-CRP values were blinded to the investigators, study staff, and patients from after day -2 for the atorvastatin treated groups or day -1 for diet only group.

E. Statistical Plan (00711 Effects of mAb316P on lipid parameters in the single-dose studies were assessed using analysis of covariance (ANCOVA) models. Least squares means of differences between treatment groups and the pooled placebo group. 95% confidence intervals, and p-values for comparison between treatment groups versus placebo by visit were obtained within the framework of ANCOVA. Significance for all tests was set at 0.05.

(0072} In toe multi-dose study, ali subjects who received placebo and atorvastatin were pooled into two placebo groups of either FH or nonFH subjects. The 6 subjects in both the FH and nonFH (atorvastatin treated) placebo groups, and 5 and 8 subjects in each of toe respective FH and nonFH mAb3l6P-treated dose groups provided at least 80% power to detect a treatment difference of 30% (SD=15%) versus placebo in mean percentage change from baseline of LDL cholesterol at each study visit when comparing each dose with placebo with a 2-sided test at the 5% significance level. No adjustments were made for multiple comparisons. Group 7 results were summarized separately by treatment and placebo and the two groups were compared individually. ANCOVA with treatment arm as the fixed effect and frie relevant baseline value as a covariate was used to analyze the continuous variables. Missing values were imputed by the Last Observation Carried Forward (LOCF) method. All p-values, except for triglycerides and Lp(a), which were derived from the Rank-based analysis of covariates, were drawn from the ANCOVA model.

(0073} To summarize the lipid and lipoprotein effects, results were selected from study day 57 on which the effects of two subsequent 2-week dosing periods could be observed. The study was not powered for a direct statistical comparison of the response of FH subjects to nonFH subjects.

Results

A. Study Population

[0074] A total of 40 subjects were randomized in the single dose IV study and 32 were randomized in the single dose SC study. For Part A of the multiple-dose SC study, a total of 97

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PCT/US2012/054756 subjects were screened and 62 were randomized {21 FH and 41 nonFH). A total of 10 subjects were included in Part B of the multi-dose SC study (4 FH and 6 nonFH). Baseline characteristics of the subjects in the singie dose studies and Part A of the multi-dose study are shown in Table 2A. Baseline characteristics of the subjects in Part B of the multi-dose study are shown in Table 2B.

Table 2A

<td rowspan="2"></td><td rowspan="2"> Single Dose i IV</td><td rowspan="2"> Single Dose SC</td><td colspan="3"> Multiple Dose SC - Part A</td>

<td> FH</td><td> nonFH</td><td> nonFH Diet Alone (no atorvastatin)</td>

<td> No. Patients</td><td> 40 |</td><td> 32</td><td> 21</td><td> 30</td><td> 10</td>

<td> Median Age</td><td> 36 I</td><td> 34</td><td> 40</td><td> 52</td><td> 52</td>

<td colspan="6"> Gender</td>

<td> Female</td><td> 35% j</td><td> 26%</td><td> 19%</td><td> 41%</td><td> 44%</td>

<td> Median BMI (kg/m2)</td><td> 26 Î</td><td> 25</td><td> 27</td><td> 27</td><td> 27</td>

<td> White</td><td> 55% |</td><td> 44%</td><td> 86%</td><td> 93%</td><td> 100%</td>

<td> Black/African American</td><td> 37.5% I I</td><td> 50%</td><td> 14%</td><td> 7%</td><td> 0%</td>

<td> American Indian/ Alaskan Native</td><td> 7.5% J</td><td> 6%</td><td> 0%</td><td> 0%</td><td> 0%</td>

<td colspan="6"> Atorvastatin Dose</td>

<td colspan="6"> Baseline Value</td>

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Table 2B

<td></td><td colspan="2"> Multiple</td><td> Dose SC -</td><td colspan="3"> Part B (200 mg dose)</td>

<td></td><td colspan="2"> FH and NonFH Combined</td><td colspan="2"> FH</td><td colspan="2"> NonFH</td>

<td> No. Patients</td><td> 2</td><td> 8</td><td> 1</td><td> 3</td><td> 1</td><td> 5</td>

<td> Median Age</td><td> 35</td><td> 45.5</td><td> 27</td><td> 50</td><td> 43</td><td> 41</td>

<td colspan="7"> Gender</td>

<td> Female</td><td> 50%</td><td> 62.5%</td><td> 0%</td><td> 33.3%</td><td> 100%</td><td> 80%</td>

<td> Median BMI (kg/m''‘)</td><td> 29.39</td><td> 26.02</td><td> 28.26</td><td> 25.43</td><td> 30.52</td><td> 26.61</td>

<td> White</td><td> 100%</td><td> 100%</td><td> 100%</td><td> 100%</td><td> 100%</td><td> 100%</td>

<td colspan="7"> Atorvastatin Dose</td>

<td colspan="7"> Baseline Value</td>

B. Lipid and Lipoprotein Response

[0075] Administration of a single IV or SC dose of mAb3l6P to healthy volunteers produced similar mean maximum percent reductions in LDL cholesterol of 55-60%. The degree and duration of LDL cholesterol lowering was dose dependent and LDL cholesterol reductions were sustained at least until 29 and 22 days after administration of higher dose levels of IV or SC mAb316P, respectively. Similarly, in the multiple-dose study, the mean percent reduction from baseline in LDL cholesterol was dose dependent and exceeded 50% two weeks after dosing with 150 mg in the FH and nonFH on atorvastatin and the nonFH diet alone populations . All but 1 subject in each population that received 150 mg in the multiple-dose study experienced a reduction of at feast 40% from basefine.

[0076] Lipid and apolipoprotein baseline and day 57 levels for all treatment groups in Part A of the multiple-dose study are shown in Tables 3A (atorvastatin-treaied subjects) and 38 (diet only - no atorvastatin treatment).

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Table 3A

<td colspan="2" rowspan="3"></td><td colspan="4"> Atorvastatin-Treated Subjects</td>

<td rowspan="2"> Placebo (Pbo)</td><td colspan="3"> mAb316P Dose</td>

<td colspan="2"> Parameter</td><td> N=12</td><td> N=13</td><td> N=13</td><td> N=13</td>

<td> LDL</td><td> Baseline</td><td> 125(19)</td><td> 114(15)</td><td> 121 (31)</td><td> 123(27)</td>

<td> Cholesterol</td><td> day 57</td><td> 195(17)</td><td> 148(29)</td><td> 130 (16)</td><td> 127(26)</td>

<td> mean (SD)</td><td> % change vs Pbo</td><td></td><td> -39.2</td><td> -53.7</td><td> -61.0</td>

<td> mg/dL</td><td> p value vs Pbo</td><td></td><td> <0.0001</td><td> <0.0001</td><td> <0.0001</td>

<td> Total</td><td> Baseline</td><td> 193(20)</td><td> 191 (24)</td><td> 192 (27)</td><td> 197 (32)</td>

<td> Cholesterol</td><td> day 57</td><td> 195 (17)</td><td> 148(29)</td><td> 130(16)</td><td> 127(26)</td>

<td> mean (SD)</td><td> % change vs Pbo</td><td></td><td> -24.6</td><td> -33.2</td><td> -36.4</td>

<td> mg/dL</td><td> p value vs Pbo</td><td></td><td> <0.0001</td><td> <0.0001</td><td> <0.0001</td>

<td> HDL</td><td> Baseline</td><td> 43 (10)</td><td> 52 (16)</td><td> 48 (10)</td><td> 48(11)</td>

<td> Cholesterol</td><td> day 57</td><td> 42(10)</td><td> 55(14)</td><td> 51 (10)</td><td> 54(12)</td>

<td> mean (SD)</td><td> % change vs Pbo</td><td></td><td> 13.2</td><td> 11.3</td><td> 18.2</td>

<td> mg/dL</td><td> p value vs Pbo</td><td></td><td> 0.0153</td><td> 0.0336</td><td> 0.0009</td>

<td rowspan="2"> LDL Cholesterol</td><td> Baseline</td><td> 125(20)</td><td> 115(18)</td><td> 119(30)</td><td> 123(29)</td>

<td> mean (SD)</td><td> % change vs Pbo</td><td></td><td> -41.7</td><td> -55.8</td><td> -62.4</td>

<td> mg/dL</td><td> p value vs Pbo</td><td></td><td> <0.0001</td><td> <0.0001</td><td> <0.0001</td>

<td rowspan="2"> Triglycerides median (min:max)</td><td> Baseline</td><td> 113 (60:244)</td><td> 118(43:171)</td><td> 135 (46:210)</td><td> 117(65:278)</td>

<td> mg/dL</td><td> % change vs Pbo</td><td></td><td> -16.3</td><td> -7.5</td><td> -11.7</td>

<td></td><td> p value vs Pbo</td><td></td><td> 0.0142</td><td> 0.2515</td><td> 0.0609</td>

<td rowspan="2"> Apolipoprotein B</td><td> Baseline</td><td> 107(12)</td><td> 104(15)</td><td> 100(21)</td><td> 106(22)</td>

<td> mean (SD)</td><td> % change vs Pbo</td><td></td><td> -31.5</td><td> -42.0</td><td> -46.4</td>

<td> mg/dL</td><td> p value vs Pbo</td><td></td><td> <0.0001</td><td> <0.0001</td><td> <0.0001</td>

<td rowspan="2"> Apolipoprotein At</td><td> Baseline</td><td> 132(19)</td><td> 155(36)</td><td> 144 (23)</td><td> 145(23)</td>

<td> mean (SD)</td><td> % change vs Pbo</td><td></td><td> 9.9</td><td> 6.9</td><td> 13.5</td>

<td> mg/dL</td><td> p value vs Pbo</td><td></td><td> 0.019</td><td> 0.086</td><td> 0.013</td>

<td> Lipoprotein</td><td> Baseline</td><td> 22 (2:121)</td><td> 46 (5:151)</td><td> 50 (7:142)</td><td> 61 (5:154)</td>

<td rowspan="3"> (a) median (min.max) mg/dL</td><td> day 57</td><td> 13 (2:119)</td><td> 42 (4:145)</td><td> 26 (3.95)</td><td> 47(5:119)</td>

<td> % change vs Pbo</td><td></td><td> -15.5</td><td> -24.1</td><td> -18.3</td>

<td> p value vs Pbo</td><td></td><td> 0.1109</td><td> 0.0015</td><td> 0.1226</td>

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Table 3B

<td colspan="2" rowspan="2"></td><td colspan="2"> Diet Only - No Atorvastatin Treatment |</td>

<td> Placebo (Pbo)</td><td> 150mgmAb316P |</td>

<td colspan="2"> Parameter</td><td> N=2</td><td> N=8 |</td>

<td rowspan="4"> LDL Cholesterol mean (SO) mg/dL</td><td> Baseline</td><td> 152 (16)</td><td> 179(49)</td>

<td> % change vs Pbo</td><td></td><td> -57.0</td>

<td> p value vs Pbo</td><td> <0.0001</td><td> 0.0023 I</td>

<td rowspan="4"> Total Cholesterol mean (SD) mg/dL</td><td> Baseline</td><td> 228 (6)</td><td> 257 (58) |</td>

<td> % change vs Pbo</td><td></td><td> -43.3 |</td>

<td> p value vs Pbo</td><td></td><td> 0.0025 |</td>

<td rowspan="4"> HDL Cholesterol mean (SD) mg/dL</td><td> Baseline</td><td> 54(14</td><td> 50 (8)</td>

<td> % change vs Pbo</td><td></td><td> -18.9 |</td>

<td> p value vs Pbo</td><td></td><td> 017 j</td>

<td rowspan="4"> LDL Cholesterol mean (SD) mg/dL</td><td> Baseiine</td><td> 152(12)</td><td> 177(49) |</td>

<td> % change vs Pbo</td><td></td><td> -58.4</td>

<td> p value vs Pbo</td><td></td><td> <0.0025 |</td>

<td rowspan="2"> Triglycerides median</td><td> Baseiine</td><td> 116 (86:146)</td><td> 127(82:268) |</td>

<td> (minimax)</td><td> % change vs Pbo</td><td></td><td> -16.86</td>

<td> mg/dL</td><td> p value vs Pbo</td><td></td><td> 0.4820 |</td>

<td rowspan="4"> Apolipoprotein B mean (SD) mg/dL</td><td> Baseline</td><td> 118 (23)</td><td> 138(37)</td>

<td> % change vs Pbo</td><td></td><td> -45.0</td>

<td> p value vs Pbo</td><td></td><td> 0.003 |</td>

<td rowspan="2"> Apolipoprotein A1</td><td> Baseline</td><td> 138(6)</td><td> 151 (15)</td>

<td rowspan="2"> mean (SD) mg/dL</td><td> % change vs Pbo</td><td></td><td> -19.2</td>

<td> p value vs Pbo</td><td></td><td> 0.073 |</td>

<td rowspan="2"> Lipoprotein (a) median</td><td> Baseline</td><td> 47 (30:63)</td><td> 34(5:111) |</td>

<td> (minimax)</td><td> % change vs Pbo</td><td></td><td> -43.8</td>

<td> mg/dL</td><td> p value vs Pbo</td><td></td><td> 0.0415 |</td>

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PCT/US2012/054756 (0077} The LDL cholesterol response at day 57 was similar in aii subjects irrespective of FH or nonFH, atorvastatin treated or on diet alone. Corresponding changes were also observed in total- and ποπ-HDL cholesterol and Apo B (Tables 3A and 38). Importantly, reductions were also observed in Lp(a). Additionally, in patients taking atorvastatin favorable changes were seen In both HDL cholesterol and apoAl.

[0078J Similar improvements were observed in the mAb316P-treated patients in Part 8 of the multiple-dose study. That is, subcutaneous administration of 200 mg of mAb316P induced rapid, substantial, and sustained reductions in LDL-C, total cholesterol. non-HDL-C, apoB, and in the ratio of apoB/apoA. Patients who received 200 mg of rrAb3l6P SC also appeared to demonstrate a trend toward higher levels of HDL-C and ApoAl.

Discussion

[00791 This Example describes human trials of the anti-PCSK9 antibody referred to herein as mAb3l6P, starting with two single ascending dose studies in healthy volunteers and extending into a large multiple-dose proof of concept trial in patients with both FH and nonFH treated with either a statin or on diet atone. These trials confirm the potential to bring about rapid and significant reductions in LDL cholesterol with PCSK9 inhibition in both familial and nonfamilial hypercholesterolemia, whether on a statin or on diet alone. The populations tested encompass the large majority of patients with hypercholesterolemia.

[0080) The robust and reproducible effect on LDL cholesterol of mAb316P from single IV dosing, through single SC dosing, to multiple SC doses is seen within an unexpectedly short time after administration, reaching a maximum at approximately 2 weeks. This rapidity exceeds that for all other therapeutic modalities for hypercholesterolemia other than removal of LDL cholesterol via apheresis, and is more rapid than for statins. That large reductions of LDL choiesterol can be achieved with mAb316P in patients already on relatively high doses of atorvastatin known to produce 40% to 50% LDL cholesterol reductions clearly distinguishes the potential of mAb316P from other investigational therapies that have been added to statins, such as ezetimibe. bile acid séquestrants, and squalene synthase inhibitors. The LDL cholesterol lowering efficacy of mAb316P, combined with apparent toferability and safety, highlights the surprising therapeutic advantages of mAb316P over several other Apo B/LDL-C lowering agents in development such as Apo B antisense, MTP inhibitors, and thyroid hormone analogues. In fact, the achievement in many of the subjects in these studies of low LDL cholesterol levels of 50 mg per deciliter (1.29 mmol/L) or less without any increase in hepatic transaminases is in marked contrast to results obtained with agents that inhibit hepatic VLDL and LDL formation. Epidemiology studies demonstrating that genetic under-expression or even the absence of PCSK9 and life-long low levels of LDL cholesterol appear not to be associated with unexpected morbidity or mortality, provide a level of comfort with regard to the therapeutic potential of drug inhibition of PCSK9.

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[00811 The anti-PCSK9 antibodies of the present invention such as mAb316P also provide patients unable to tolerate statins (now estimated to be approximately 5% to 10% of all statin treated patients) a significant opportunity to achieve large (at feast 50%) reductions in LDL cholesterol. For many of the estimated 10 million patients worldwide with FH, the anti~PCSK9 antibodies of the invention, including mAb316P, offer the real possibility that their use along with statins may finally achieve optimal LDL cholesterol control and potentially further reduce their substantial risk of the early and recurrent cardiovascular disease. The currently disclosed therapeutic methodologies may also offer many patients the potential to discontinue LDL apheresis. an invasive, time consuming, expensive every 2-waak procedure that provides only short-term LDL cholesterol reduction.

[0C82[ Additional unexpected beneficial effects on other lipids were seen in HDL cholesterol, Apo A1 and, most surprisingly, Lp(a) which trended to, or reached, statistical significance, including in patients treated with atorvastatin. importantly, before now, no therapeutic biologic agents (e.g., anti-PCSK9 antibodies) have been shown clinically to reduce Lp(a), which has been thought not to be cleared via the LDL receptor. Thus, the current experiments are the first demonstration of the ability of a PCSK9 inhibitor to reduce serum Lp(a) levels in patients.

[0083] Regarding safety. mAb3i6P injection site reactions were minimal. mAb3i 6P was also generally safe and well tolerated with no trend in drug-related adverse events and no evidence of hepato- or myo-toxicity. The few CK elevations that were seen were exercise related.

[0084] In summary, this Example shows that mAb316P inhibition of PCSK9, besides effectively lowering LDL cnolestero! in human patients, surprisingly reduced Lp(a) levels as well.

Example 3: A Randomized, Double-Blind, Placebo Controlled, 12 Week Study of the Safety and Efficacy of An Anti-PCSK9 Monoclonal Antibody in Patients with Heterozygous Familial Hypercholesterolemia

[0085] A clinical trial was conducted to assess the efficacy of varying subcutaneous (SC) doses and dosing regimens of mAb316P on serum low-density lipoprotein cholesterol (LDL-C) and other lipids/apolipoproteins (e.g.. total cholesterol, high-density lipoprotein cholesterol, triglycerides, apo B, Apo A1, and Lp[a]) in patients with heterozygous familial hypercholesterolemia (heFH).

Patient Population

[0086} The patient population for this study included men and women, ages 18-75 years old, who were diagnosed with heFH, exhibited LDL-C levels of 100 mg/dL or greater, and who were on a stable daily statin cose (either with or without ezetimibe) at the start of the study. Patients were excluded if they were taking any additional lipid lowering compounds such as fibrates, niacin, omega-3 fatty acids, bile acid resins, plant stands (e g, Benecoi, flaxseed oil, psyllium), or red yeast rice. A total of 77 patients completed the study.

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Drug Formulation

[0087] The drug formulation comprised 150 mg/mL of mAb316P, 10 mM histidine, 0.2% polysorbate 20,10% sucrose, and had a pH of 6.0.

Method of Administration

[00881 The drug formulation (or piacebo) was administered to the patients by subcutaneous injection into the abdomen. Each treatment included two subcutaneous injections of 1 mL each.

Administration Regimens

[0089] The patients were divided into five groups. Group 1 included 15 patients who received placebo once every two weeks; Group 2 included 16 patients who received 150 mg of mAb3l6P once every two weeks: Group 3 included 15 patients who received 150 mg of mAb316P once every four weeks: Group 4 included 16 patients who received 200 mg of mAb316P once every four weeks: and Group 5 included 15 patients who received 300 mg of mAb316P once every four weeks. The study duration was 12 weeks.

Efficacy Assessment

[0090] Blood samples were collected from the patients throughout the study, and during an 8 week follow-up period. The samples were collected after at least a 12 hour fast (in the morning before any drug intake). Measurements of the following parameters were determined from the blood samples. (1) Total Cholesterol; (2) LDL-C; (3) HDL-C; (4) Triglycerides (TO): (5) Apo B;

(6) Apo A1 ; and (7) Lp(a). The median percent changes in these parameters compared to placebo at the end of the study (week 12) are summarized in Table 4.

Table 4

<td> I Group</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td> I No. of Patients</td><td> 15</td><td> 16</td><td> 15</td><td> 16</td><td> 15</td>

<td> | Treatment</td><td> Placebo</td><td> mAb316P</td><td> mAb316P</td><td> mAb316P</td><td> mAb316P</td>

<td> I Dosing Frequency</td><td> Q2W</td><td> Q2W</td><td> Q4W</td><td> Q4W</td><td> Q4W</td>

<td> i Total Choiesteroi</td><td> -6.52</td><td> -38.27</td><td> -18.89</td><td> -15.31</td><td> -28.84</td>

<td> | Triglycerides</td><td> -10.55</td><td> -16.23</td><td> -16.73</td><td> -9.87</td><td> -4.92</td>

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Example 4: A Randomized, Double-Blind, Parallei-Group, Placebo Controlled, 12 Week Study of the Safety and Efficacy of An Anti-PCSK9 Monoclonal Antibody in Patients with Primary Hypercholesterolemia on Stable Atorvastatin Therapy

[0091J A clinical trial was conducted to assess the efficacy of five doses and two dose regimens of mAb316P over 12 weeks in patients with primary hypercholesterolemia and LDL-C levels greater than 100 mg/dL on ongoing stable atorvastatin therapy. Efficacy was assessed based on changes in serum low-density lipoprotein cholesterol (LDL-C) and other lipids/apolipoprotems (e g., total cholesterol, high-density lipoprotein cholesterol, triglycerides, apo B, Apo A1, and Lp[a]) over the course of the study.

Patient Population

[0092] The patient population for trits study included men and women, ages 18-75 years old, who were diagnosed with primary hypercholesterolemia, exhibited LDL-C levels of 100 mg/dL or greater, and who were on stable atorvastatin therapy of 10,20 or 40 mg for at least six weeks prior to the start of the study. Patients were excluded if they were taking any additional lipid lowering compounds such as fibrates, niacin, omega-3 fatty acids, bile acid resins, plant stanols (e.g., Benecoi, flaxseed oil, psyllium), or red yeast rice. A total of 118 patients completed the study.

Drug Formulation

[0093J The drug formulation comprised 150 mg/mL of mAb3l6P, 10 mM histidine, 0.2% polysorbate 20,10% sucrose, and had a pH of 6.0.

Method of Administration

[0094] The drug formulation (or placebo) was administered to the patients by subcutaneous injection into the abdomen. Each treatment included two subcutaneous injections of 1 mL each.

Administration Regimens

[0093] The patients were divided into six groups. Group 1 included 20 patients who received placebo once every two weeks; Group 2 included 19 patients who received 50 mg of mAb316P once every two weeks; Group 3 included 20 patients who received 100 mg of mAb316P once every two weeks; Group 4 included 18 patients who received 150 mg of mAb3l6P once every two weeks; Group 5 included 20 patients who received 200 mg of mAb3l6P once every four weeks; and Group 6 included 21 patients who received 300 mg of mAb3l6P once every four weeks. The study duration was 12 weeks.

Efficacy Assessment

[0096] Blood samples were collected from the patients throughout the study, and during an 8 week follow-up period. The samples were collected after at least a 12 hour fast (in the morning

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PCT/US2012/054756 before any drug intake). Measurements of the following parameters were determined from the blood samples: (1) Total Cholesterol; (2) LDL-C; (3) HDL-C; (4) Triglycerides (TG); (5) Apo B;

(6) Apo A1 ; and (7) Lp(a). The median percent changes in these parameters compared to placebo at the end of the study (week 12) are summarized in Table 5.

Table 5

<td> Group</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td>

<td> No. of Patients</td><td> 20</td><td> 19</td><td> 20</td><td> 18</td><td> 20</td><td> 21</td>

<td> Treatment</td><td> Placebo</td><td> mAb316P</td><td> rnAb316P</td><td> mAb316P</td><td> mAb316P</td><td> mAb316P</td>

<td> Dosing Frequency</td><td> Q2W</td><td> Q2W</td><td> Q2W</td><td> Q2W</td><td> Q4W</td><td> Q4W</td>

<td> Total Cholesterol</td><td> -3.73</td><td> -23.34</td><td> -40.21</td><td> -45.03</td><td> -29.51</td><td> -33.48</td>

<td> Triglycerides</td><td> +22.42</td><td> -7.85</td><td> -11.41</td><td> -22.93</td><td> +1.27</td><td> -9.17</td>

Example 5: A Randomized, Double-Blind, Parallel-Group, Placebo Controlled, Fixed Dose Study of the Safety and Efficacy of An Anti-PCSK9 Monoclonal Antibody CoAdministered with 80 mg Atorvastatin in Patients with Primary Hypercholesterolemia

[0097] A clinical trial was conducted to assess the efficacy of mAb316P when co-administered with 80 mg of atorvastatin over 8 weeks In patients with primary hypercholesterolemia and LDLC levels greater than 100 mg/dL. Efficacy was assessed based on changes in serum lowdensity lipoprotein cholesterol (LDL-C) and other liplds/apolipoproteins (e.g., total cholesterol, high-density lipoprotein cholesterol, triglycerides, apo B, Apo A1, and Lp[a]) over the course of the study.

Patient Population

[0098) The patient population for this study included men and women, ages 13-75 years old, who were diagnosed with primary hypercholesterolemia, exhibited LDL-C levels of 100 mg/dL or greater, and who were on stable atorvastatin therapy of 10 mg for at least six weeks prior to the start of the study. Patients were excluded if they were taking any additional lipid lowering compounds such as fibrates, niacin, omega-3 fatty acids, bile acid resins, plant stanols (e.g., Benecol, flaxseed o3, psyHium), or red yeast rice. A total of 88 patients completed the study.

Drug Formulation

[0099] The drug formulation comprised 150 mg/mL of mAb316P, 10 mM histidine, 0.2% polysorbate 20,10% sucrose, and had a pH of 6.0.

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Method of Administration (oiooj The drug formulation (or placebo) was administered to the patients by subcutaneous injection into toe abdomen. Each treatment included one subcutaneous injection of 1 mt.

Administration Regimens

[oioi] The patients were divided into three groups. Group 1 included 29 patients who received placebo once every two weeks plus 80 mg atorvastatin daily: Group 2 included 30 patients who received 150 mg of mAb316P once every two weeks plus 80 mg atorvastatin daily; and Group 3 included 29 patients who received 150 mg of mAb316P once every two weeks plus 10 mg atorvastatin daily. The study duration was 8 weeks.

Efficacy Assessment

[0102] Biood samples were collected from the patients throughout the study, and during an 8 week follow-up period The samples were collected after at least a 12 hour fast (in the morning before any drug intake). Measurements of the following parameters were determined from the blood samples; (1) Total Cholesterol; (2) LDL-C; (3) HDL-C; (4) Triglycerides (TG); (5) Apo B;

(6) Apo A1 ; (7) Apo B/Apo A1 ratio; and (8) Lp(a). The percent changes in these parameters compared to placebo at the end of the study are summarized In Table 6.

Table 6

<td> Group</td><td colspan="2"> 1</td><td colspan="2"> 2</td><td colspan="3"> 3</td>

<td> No. of Patients</td><td> 29</td><td></td><td> 30</td><td></td><td colspan="3"> 29</td>

<td> Treatment</td><td> Placebo</td><td></td><td> mAb316P</td><td></td><td colspan="3"> mAb316P</td>

<td> Dosina Frequency</td><td> Q2W</td><td> !</td><td> Q2W</td><td></td><td colspan="3"> Q2W</td>

<td> Daily Atorvastatin</td><td> 80 mg</td><td> t</td><td> 80 mg</td><td></td><td colspan="3"> 10 mg</td>

<td> Total Cholesterol</td><td> -16.59</td><td> I</td><td> -47.21</td><td></td><td colspan="3"> -40.45</td>

<td> Triglycerides</td><td> -11.89</td><td> •</td><td> -24.67</td><td></td><td colspan="3"> -3.98</td>

[0103] Taken together, the results from these studies (Examples 3-5) confirm the ability of mAB316P to effectively lower LDL cholesterol and beneficially affect other lipid/apolipoprotein parameters in patients when administered at various doses and dosing regimens. These clinical Examples also confirm the unexpected finding that mAB3i6P is able to significantly reduce Lp(a) levels in patients under various circumstances.

[0104] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described

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PCT/US2012/054756 herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Contents74

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Document	Office	Kind	Date
201161535392	United States of America	P	2011-09-16
201161535392	United States of America	P	2011-09-16
61535392	United States of America	–	2011-09-16
201161559162	United States of America	P	2011-11-14
201161559162	United States of America	P	2011-11-14
61559162	United States of America	–	2011-11-14
201261641321	United States of America	P	2012-05-02
201261641321	United States of America	P	2012-05-02
61641321	United States of America	–	2012-05-02
2012054756	United States of America	W	2012-09-12
2012054756	United States of America	W	2012-09-12
61535392	–	–	–
61559162	–	–	–
61641321	–	–	–
PCTUS2012054756	–	–	–
US201161535392P	–	–	–
US201161559162P	–	–	–
US201261641321P	–	–	–
WO2012US54756	–	–	–

Members56

Family members, oldest first
Document	Office	Kind
CA2848201A1	Canada	A1	2013-03-21
WO2013039969A1	World Intellectual Property Organization (WIPO)	A1	2013-03-21
TW201329099A	Taiwan Province of China	A	2013-07-16
US2013243784A1	United States of America	A1	2013-09-19
AU2012308797A1	Australia	A1	2014-03-13
CO6910163A2	Colombia	A2	2014-03-31
IL231011A0	Israel	A0	2014-03-31
IL231011D0	Israel	D0	2014-03-31
PH12014500342A1	Philippines	A1	2014-03-31
AR087881A1	Argentina	A1	2014-04-23
MX2014002158A	Mexico	A	2014-05-07
KR20140075728A	Republic of Korea	A	2014-06-19
SG2014010524A	Singapore	A	2014-06-27
EA201490641A1	Eurasian Patent Organization (EAPO)	A1	2014-06-30
CN103930444A	China	A	2014-07-16
EP2756004A1	European Patent Office (EPO)	A1	2014-07-23
CL2014000605A1	Chile	A1	2014-08-22
MA35430B1	Morocco	B1	2014-09-01
JP2014527967A	Japan	A	2014-10-23
ZA201401114B	South Africa	B	2015-04-29
NZ621135A	New Zealand	A	2016-10-28
BR112014005522A2	Brazil	A2	2017-03-21
AU2012308797B2	Australia	B2	2017-06-01
JP6158813B2	Japan	B2	2017-07-05
TWI601740B	Taiwan Province of China	B	2017-10-11
EA028278B1	Eurasian Patent Organization (EAPO)	B1	2017-10-31
JP2017206520A	Japan	A	2017-11-24
US10076571B2	United States of America	B2	2018-09-18
US2018333490A1	United States of America	A1	2018-11-22
MX363642B	Mexico	B	2019-03-28
KR101964031B1	Republic of Korea	B1	2019-04-02
PH12014500342B1	Philippines	B1	2019-05-22
PH12014500342B1	Philippines	B1	2019-06-14
MY170089A	Malaysia	A	2019-07-04
EP3536712A1	European Patent Office (EPO)	A1	2019-09-11
EP2756004B1	European Patent Office (EPO)	B1	2019-12-25
IL231011A	Israel	A	2020-01-30
IL231011B	Israel	B	2020-01-30
ES2773111T3	Spain	T3	2020-07-09
CN103930444B	China	B	2020-08-04
CN111789944A	China	A	2020-10-20
CA2848201CThis record	Canada	C	2020-10-27
US11116839B2	United States of America	B2	2021-09-14
EP3536712B1	European Patent Office (EPO)	B1	2023-05-31
EP4252857A2	European Patent Office (EPO)	A2	2023-10-04
ES2953713T3	Spain	T3	2023-11-15
EP4252857A3	European Patent Office (EPO)	A3	2023-12-06
EP4252857B1	European Patent Office (EPO)	B1	2024-09-25
EP4252857C0	European Patent Office (EPO)	C0	2024-09-25
EP4461373A2	European Patent Office (EPO)	A2	2024-11-13
ES2992345T3	Spain	T3	2024-12-11
HRP20241450T1	Croatia	T1	2025-01-03
EP4461373A3	European Patent Office (EPO)	A3	2025-01-22
RS66342B1	Serbia	B1	2025-01-31
HUE069234T2	Hungary	T2	2025-02-28
PL4252857T3	Poland	T3	2025-03-03

4 legal events, as the office reported them to INPADOC

Over the term

Point at a mark for the event

Events

Legal events, newest first
	Event	Code
2024-08-26	Maintenance fee for patent paidMPN	MPN
2024-08-26	Fee paidST27 STATUS EVENT CODE: A-4-4-U10-U00-U101 (AS PROVIDED BY THE NATIONAL OFFICE); EVENT TEXT: MAINTENANCE REQUEST RECEIVEDU00	U00
2024-08-26	Full renewal or maintenance fee paidST27 STATUS EVENT CODE: A-4-4-U10-U11-U102 (AS PROVIDED BY THE NATIONAL OFFICE); EVENT TEXT: MAINTENANCE FEE PAYMENT DETERMINED COMPLIANTU11	U11
2017-09-19	Examination requestEEER	EEER