EA041556B1 - INHIBITORY ANTIBODIES AGAINST PCSK9 FOR THE TREATMENT OF PATIENTS WITH HYPERLIPIDEMIA UNDER LIPOPROTEIN APHERESIS - Google Patents

Description

Detailed description

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

Unless otherwise defined, all technical and scientific terms used in this specification have the meaning generally understood by one of ordinary skill in the art to whom this invention belongs. As used herein, the term about, when used with reference to a specific recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, an expression near 100 includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

While any methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, preferred methods and materials will now be described. All publications cited in this document are incorporated herein by reference in their entirety for description.

Ways to reduce or eliminate the need for lipoprotein apheresis.

The present invention relates generally to methods and compositions for lowering lipoprotein levels in patients who are undergoing or have undergone (e.g., within the past six months or more) lipoprotein apheresis to lower serum levels of lipoproteins (e.g., LDL-C and/or Lp( a)). In some embodiments, the methods of the present invention result in a decrease in serum lipoprotein levels in such patients such that the need for lipoprotein apheresis is reduced or eliminated.

As used in this description, the term lipoprotein means a biomolecular particle containing both protein and lipid. Examples of lipoproteins include, for example, low density lipoprotein (LDL), very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and lipoprotein (a) (Lp(a)).

As used in the context of the present invention, lipoprotein apheresis means a therapeutic process involving the mechanical removal of blood from a patient, followed by the removal of lipoproteins (e.g., LDL-C and/or Lp(a)) from the patient's blood through processes such as filtration, adsorption , precipitation, etc. and finally, reintroducing the treated blood back into the patient's circulation. For the purposes of this disclosure, LDL apheresis and Lp(a) apheresis are considered to be types of apheresis, and are therefore covered by the definition of the more general definition of lipoprotein apheresis.

Specific types of lipoprotein apheresis that are included within the context of the present invention include, for example, double membrane filtration, immunoadsorption, heparin-induced LDL precipitation, direct lipid adsorption, dextran-cellulose sulfate (plasma or whole blood) adsorption, extracorporeal heparin LDL precipitation (HELP), DFPP and thermofiltration plasmapheresis and hemoperfusion.

The present invention, in some embodiments, includes methods for eliminating the need for lipoprotein apheresis therapy. As used herein, in a particular patient, the need for lipoprotein apheresis therapy is determined by a physician, paramedic, diagnostician, or other healthcare professional based on the level of one or more lipoproteins (e.g., LDL-C and/or Lp(a)), measured or otherwise found in the patient's serum. The need for lipoprotein apheresis therapy in a patient may also be determined or influenced by other factors such as the patient's family history, general medical history, treatment status, and generally accepted or prevailing reference levels of lipoproteins accepted by national medical associations and physician groups. . For example, in some contexts, an LDL-C level greater than or equal to about 70 mg/dl indicates a need for lipoprotein apheresis therapy in a patient. In other contexts, an LDL-C level greater than or equal to about 100 mg/dL indicates a need for lipoprotein apheresis therapy in a patient. In some contexts, an LDL-C level greater than or equal to about 150, 200, 250, 300, 400 mg/dl or greater indicates a need for lipoprotein apheresis therapy in the patient. In yet other contexts, whether or not a specific percentage reduction in LDL-C or Lp(a) is achieved, the relative level of LDL-C or Lp(a) in a patient at a particular baseline (at baseline) can be used to determine whether the patient has a need for lipoprotein apheresis therapy. For example, a decrease in LDL-C or Lp(a) less than 50% (eg, less than 40, less than 35, less than 30, less than 25%, etc.) from baseline may indicate a need for lipoprotein apheresis therapy.

The present invention, in some embodiments, includes methods for reducing the frequency of lipoprotein apheresis therapy in a patient. As will be appreciated by those of ordinary skill in the art, a patient may be treated with lipoprotein apheresis at a specific frequency to achieve a specific goal of lowering lipoprotein levels (e.g., LDL-C level less than 100 mg/dL, LDL-C level less than 70 mg/dL, and etc.). The prescribed frequency is determined based on the number of apheresis treatments required by the patient during a particular period of time (eg, weekly, monthly, etc.) to reach and maintain the target lipoprotein level for the patient. Exemplary lipoprotein apheresis rates include, for example, once a week, once every two weeks, once every 3 weeks, once every four weeks, once every five weeks, once a month, twice a month, once at two months, etc. The present invention includes methods for reducing the frequency of lipoprotein apheresis therapy in a patient by administering one or more doses of a PCSK9 inhibitor to the patient. According to some embodiments of the present invention, the frequency of apheresis following the administration of one or more doses of a PCSK9 inhibitor is reduced by at least 50% of the frequency of apheresis prior to treatment of the patient. For example, if a patient prior to treatment with a PCSK9 inhibitor is on a once-weekly (QW) lipoprotein apheresis regimen, and after treatment with a PCSK9 inhibitor, the patient's apheresis rate is reduced to once every 2 weeks (Q2W), then it is stated that the patient achieved a 50% reduction in the frequency of lipoprotein apheresis after treatment. In some embodiments, the frequency of apheresis after administration of one or more doses of a PCSK9 inhibitor of the present invention is reduced by 75% or 100% (ie, after treatment, the need for lipoprotein apheresis is eliminated).

The frequency of lipoprotein apheresis, in the context of the present invention, can be expressed in terms of frequency before treatment and frequency after treatment. Pre-Treatment Frequency refers to the frequency of apheresis therapy required by a patient to achieve and/or maintain a specific target lipoprotein level prior to starting a treatment regimen comprising administration of a PCSK9 inhibitor. Post-treatment frequency refers to the frequency of apheresis therapy required by a patient to achieve and/or maintain a specific target lipoprotein level after starting a treatment regimen comprising administration of a PCSK9 inhibitor. The frequency of apheresis therapy required to achieve and/or maintain a particular target lipoprotein level for a particular patient is preferably determined by a qualified healthcare professional based on generally accepted criteria in the art, including the serum lipoprotein level that is being attempted to be reduced or otherwise controlled.

The present invention accordingly includes methods of treatment comprising administering one or more doses of a PCSK9 inhibitor to a patient, whereby the rate of lipoprotein apheresis on the patient after treatment is less than the rate of lipoprotein apheresis on the patient prior to treatment. For example, the present invention includes therapeutic methods comprising administering one or more doses of a PCSK9 inhibitor to a patient who is receiving or has received lipoprotein apheresis therapy at a pre-treatment frequency of, for example, once a week, where, after receiving one or more doses of a PCSK9 inhibitor , the frequency of lipoprotein apheresis required by a patient to achieve and/or maintain a specific target lipoprotein level (i.e., post-treatment frequency) is, for example, once every two weeks, once every three weeks, one once every four weeks or less often. In some cases, the need for lipoprotein apheresis required by a patient to achieve and/or maintain a particular target lipoprotein level is eliminated entirely after administration of one or more doses of a PCSK9 inhibitor.

In some embodiments, the present invention includes methods of reducing or eliminating the need for lipoprotein apheresis therapy, wherein the methods include selecting a patient with hyperlipidemia (e.g., hypercholesterolemia) who received lipoprotein apheresis treatment within the last month, last 2 months, the last 3 months, the last 4 months, the last 5 months, the last 6 months, or longer, and administering one or more doses of a PCSK9 inhibitor to the patient. The methods according to this aspect of the invention result in a decrease in the level of at least one lipoprotein in the patient's serum, and therefore reduce or eliminate the need for lipoprotein apheresis therapy in the patient. For example, in some embodiments of the present invention, following the administration of one or more doses of a PCSK9 inhibitor, the patient's serum LDL-C level falls below a predetermined level (e.g., less than 100 or less than 70 mg/dL), such that the frequency of apheresis therapy lipoprotein after treatment required by the patient to achieve and/or maintain a specific target lipoprotein level is reduced, or it is concluded that lipoprotein apheresis is no longer required.

In some embodiments, the apheresis rate (or rate) is expressed as a normalized apheresis rate required by a patient to achieve and/or maintain a particular target lipoprotein level. As used herein, a normalized apheresis rate for a particular patient is defined as the number of valid apheresis treatments a patient receives during a given time period after initiation of an anti-PCSK9 antibody treatment regimen divided by the number of apheresis treatments a patient receives during an equivalent time period before initiation of an anti-PCSK9 antibody treatment regimen.

For example, if prior to initiation of a PCSK9 antibody regimen, a patient received 8 apheresis treatments over an 8-week period (e.g., once a week), and after initiation of a PCSK9 antibody regimen, the patient received 2 apheresis treatments per over an 8-week period (eg, once every four weeks), then the patient's normalized apheresis rate is 2/8=0.25. The present invention includes methods whereby the normalized apheresis rate for a patient after administration of a PCSK9 inhibitor is, for example, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 or less than 0.1.

In some embodiments, the patient who may be treated with the methods of the present invention has hypercholesterolemia (eg, a serum LDL-C concentration greater than or equal to 70 mg/dL, or a serum LDL-C concentration greater than or equal to 100 mg/dL). In some embodiments, the patient's hypercholesterolemia is not adequately controlled by statin therapy. For example, the present invention includes methods for reducing or eliminating the frequency of lipoprotein apheresis therapy in a patient who has hypercholesterolemia that is not adequately controlled by a daily dose of a statin selected from the group consisting of atorvastatin (including atorvastatin+ezetimibe), rosuvastatin, cerivastatin, pitavastatin, fluvastatin , lovastatin, simvastatin (including simvastatin+ezetimibe), pravastatin, and combinations thereof. The present invention also includes methods for reducing or eliminating the frequency of lipoprotein apheresis therapy in a patient who has hypercholesterolemia and who exhibits statin intolerance or who otherwise experiences adverse or unwanted reaction(s) to statin therapy (e.g., skeletal muscle pain, aches, weakness or spasms (eg, myalgia, myopathy, rhabdomyolysis, etc.)).

Methods for the treatment of lipid damage.

The present invention further relates to methods and compositions for the treatment, management or resolution of physical manifestations of hypercholesterolemia. In some embodiments, the present invention provides methods for treating a patient having lipid damage associated with hypercholesterolemia. For example, the present invention provides a method for treating a patient having one or more xanthelasmas. Methods and compositions according to this aspect of the invention include administering one or more doses of a PCSK9 inhibitor to a patient in need thereof, wherein the lipid lesions that existed in the patient prior to treatment with the PCSK9 inhibitor are reduced, resolved, or eliminated upon administration of one or more doses of the PCSK9 inhibitor to the patient. In some embodiments, the present invention includes methods for treating a patient having lipid damage associated with hypercholesterolemia, wherein the methods include selecting a patient with lipid damage (eg, xanthelasma) and administering one or more doses of a PCSK9 inhibitor to the patient.

- 5 041556

Selection of patients.

The present invention includes methods and compositions useful in the treatment of patients undergoing or recently undergoing lipoprotein apheresis (e.g., within the last six months, within the last 12 weeks, within the last 8 weeks, within the last 6 weeks, within the last 4 weeks, within the last 2 weeks, etc.). Patients who may be treated with the methods of the present invention may also exhibit one or more additional selection criteria. For example, a patient may be selected for treatment using the methods of the present invention if the patient is diagnosed or identified as being at risk for developing a hypercholesterolemia condition such as, for example, heterozygous familial hypercholesterolemia (heFH), homozygous familial hypercholesterolemia (hoFH), autosomal dominant hypercholesterolemia (ADH , e.g., ADH associated with one or more gain-of-function mutations in the PCSK9 gene), autosomal recessive hypercholesterolemia (ARH, e.g., ARH associated with LDLRAP1 mutations), and cases of hypercholesterolemia other than familial hypercholesterolemia (nonFH). The diagnosis of familial hypercholesterolemia (eg, heFH or hoFH) can be made based on genotyping and/or clinical criteria. For patients who are not genotyped, the clinical diagnosis can be based on either the Simon Broom criteria with criteria for specific FH, or the WHO/Dutch Lipid Network criteria with a score of >8 points.

In some embodiments, a patient may be selected based on having a medical history of coronary heart disease (CHD). As used herein, a history of CHD (or documented history of CHD) includes one or more of: (i) acute myocardial infarction (MI); (ii) asymptomatic MI; (iii) unstable angina; (iv) coronary revascularization procedures (eg, percutaneous coronary intervention [PCI] or coronary artery bypass grafting [CABG]); and/or (v) clinically significant CHD diagnosed by invasive or non-invasive testing (such as coronary angiography, treadmill exercise testing, stress echocardiography, or radionuclide imaging).

In some embodiments, a patient may be selected on the basis that they have a cardiovascular disease that is non-coronary heart disease (non-CHD CVD). As used herein, non-CHD CVD includes one or more of: (i) documented prior ischemic stroke with focal ischemic neurological deficit that lasted more than 24 hours, considered to be atherothrombotic in origin; (ii) peripheral arterial disease; (iii) abdominal aortic aneurysms; (iv) atherosclerotic renal artery stenosis; and/or (v) carotid disease (transient ischemic attacks or >50% carotid obstruction).

In some embodiments, a patient may be selected based on having one or more additional risk factors, such as, for example, (i) documented moderate chronic kidney disease (CKD) as measured by 30<eGFR<60 ml/min /1.73 m ² for 3 months or more; (ii) type 1 or type 2 diabetes mellitus with or without target organ damage (eg, retinopathy, nephropathy, microalbuminuria); (iii) a calculated risk score of 10-year fatal CVD >5% (ESC/EAS Guidelines for management of dyslipidemias, Conroy et al., 2003, Eur. Heart J. 24:987-1003).

In some embodiments, a patient may be selected based on having one or more additional risk factors selected from a group consisting of age (e.g., older than 40, 45, 50, 55, 60, 65, 70, 75, or age 80), race, national origin, gender (male or female), exercise habits (eg, regular exerciser, non-exerciser), other pre-existing medical conditions (eg, type II diabetes, high blood pressure, and etc.), and the current status of drug treatment (eg currently taking beta-blockers, niacin, ezetimibe, fibrates, omega-3 fatty acids, bile acid sequestrants, etc.). According to the present invention, patients may be selected based on a combination of one or more of the above selection criteria or therapeutic characteristics. For example, in some embodiments, a patient eligible for treatment using the methods of the present invention, in addition to past or recent past (eg, within the past six months, lipoprotein apheresis, may further be selected based on having heFH or non-FH in combination with: (i) a history of documented CHD, (ii) non-CHD CVD, and/or (iii) diabetes mellitus without target organ damage; such patients may also be selected based on their serum concentration LDL-C greater than or equal to 70 mg/dl.

In some other embodiments, a patient suitable for treatment using the methods of the present invention, in addition to having hypercholesterolemia that is not adequately controlled by a daily moderate dose in a statin therapeutic regimen, may further be selected based on that he has heFH or non-FH without CHD, or non-CHD CVD, but has either (i) an estimated risk score for 10-year fatal CVD >5%; or (ii) diabetes mellitus without target organ damage; such patients may also be selected on the basis that they have a serum LDL-C concentration greater than or equal to 100 mg/dL.

Administration of a PCSK9 inhibitor as adjunctive therapy.

The present invention includes methods of treatment whereby a PCSK9 inhibitor is administered to a patient undergoing or recently undergoing lipoprotein apheresis according to a specific amount and frequency of dosing, and wherein the PCSK9 inhibitor is administered as an adjunct to the patient's pre-existing lipid-lowering therapy (if applicable). ), such as in addition to the patient's previously existing daily statin therapy regimen.

For example, the methods of the present invention include complementary treatment regimens where the PCSK9 inhibitor is administered as add-on therapy to the same stable daily statin therapeutic regimen (i.e., the same statin dosage amount) that the patient followed prior to taking the PCSK9 inhibitor. In other embodiments, the PCSK9 inhibitor is administered as add-on therapy to a statin therapeutic regimen comprising a statin in an amount that is greater than or less than the dose of statin that the patient was taking prior to taking the PCSK9 inhibitor. For example, upon initiation of a treatment regimen comprising a PCSK9 inhibitor administered at a specific dosage frequency and amount, the daily dose of a statin administered or administered to a patient may (a) remain the same, (b) increase, or (c) decrease (e.g., at dose increase or dose reduction) compared to the daily dose of the statin that the patient was taking before starting the PCSK9 inhibitor regimen, depending on the patient's therapeutic needs.

Therapeutic efficacy.

The methods of the present invention result in a decrease in serum levels of one or more lipid components selected from the group consisting of LDL-C, ApoB100, non-HDL-C, total cholesterol, VLDL-C, triglycerides, Lp(a) and remnant cholesterol. The lipoprotein-lowering effects of the treatment regimen of the present invention therefore result in a reduced frequency of conduction, or the elimination of the need for lipoprotein apheresis in order to achieve a patient's target lipoprotein level. For example, in some embodiments of the present invention, administration of a pharmaceutical composition containing a PCSK9 inhibitor to a patient undergoing or recently undergoing lipoprotein apheresis will result in an average percentage reduction in serum low-density lipoprotein (LDL-C) cholesterol from baseline of at least least about 25, 30, 40, 50, 60% or more; an average percentage reduction in ApoB100 from baseline of at least about 25%, 30%, 40%, 50%, 60% or more; a mean percentage reduction in non-HDL-C from baseline of at least about 25%, 30%, 40%, 50%, 60% or greater; an average percentage reduction in total cholesterol from baseline of at least about 10%, 15%, 20%, 25%, 30%, 35% or more; an average percentage reduction in VLDL-C from baseline of at least about 5%, 10%, 15%, 20%, 25%, 30% or more; an average percentage reduction in triglycerides from baseline of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or more; and/or an average percentage reduction in Lp(a) from baseline of at least about 5%, 10%, 15%, 20%, 25% or more.

The present invention includes methods of treating a patient with hypercholesterolemia, methods comprising administering multiple doses of an anti-PCSK9 antibody to a patient at a dosage amount of about 75 to 150 mg per dose, and a dosing frequency of about once every two weeks, (or on a dosing schedule in accordance with escalation dosing regimen as described elsewhere in this document) where the patient is undergoing or has recently undergone lipoprotein apheresis, and where, at about 12, 14, 16, 18, 20, 22, 24 or more weeks treatment with an anti-PCSK9 antibody, the patient exhibits a decrease in LDL-C from baseline of at least 50%, and therefore results in a reduced frequency or elimination of the need for lipoprotein apheresis in the patient. In some embodiments, after one or more weeks of treatment with an anti-PCSK9 antibody, the patient exhibits a decrease in LDL-C from baseline of about 55%, 60%, 70%, or more.

PCSK9 inhibitors.

The methods of the present invention include administering to a patient a therapeutic composition containing 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 categories of PCSK9 inhibitors include small molecule PCSK9 antagonists, nucleic acid-based (e.g., siRNA or antisense strand) inhibitors of PCSK9 expression or activity, peptide-based molecules that specifically interact with PCSK9 (e.g., peptibodies), receptor molecules that specifically interact with PCSK9, proteins containing the ligand-binding portion of the LDL receptor, molecules with a PCSK9-binding scaffold (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, fibronectin-based scaffolds, and other scaffolds based on natural proteins with repeats, etc. (see e.g. Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein]) and anti-PCSK9 aptamers or parts thereof.In some embodiments, the PCSK9 inhibitors that can be used in the context of the present invention are antibodies against anti-PCSK9 or antigen-binding antibody fragments that specifically bind to human PCSK9.

The term subtilisin/kexin type 9 proprotein convertase or human PCSK9 or hPCSK9, as used herein, 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 its biological active fragment.

The term antibody, as used herein, is intended to refer to immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains, and two light (L) chains linked together by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, C _H 2 and C _H 3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or V _L ) and a light chain constant region. The light chain constant region contains one domain (CL1). The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with regions that are more conserved called framework regions (FRs). Each VH and V _L is composed of three CDRs and four FRs arranged in the amino to carboxy direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, an anti-PCSK9 FR antibody (or an antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally occurring or artificially modified.

The amino acid consensus sequence can be determined based on parallel analysis of two or more CDRs.

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

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

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

- 8 041556 VH or V _L monomeric domain.

In some embodiments, the antigen-binding antibody fragment may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, illustrative variable and constant domain configurations that can be found in an antigen-binding fragment of an antibody of the present invention include: (i) Vh-Ch1; (ii) Vh-Ch2; (in) Vh-Ch3; (iv) Vh-Ch1-Ch2; (v) Vh-Ch1-Ch2-Ch3; (vi) Vh-Ch2Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (xiii) VlC _h 2-C _h 3; and (xiv) V _l -C _l . In any configuration of variable and constant domains, including any of the exemplary configurations above, the variable and constant domains may either be directly linked to each other or may be linked through a complete or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in flexible or semi-flexible binding between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) with any of the variable and constant domain configurations above in non-covalent association with each other and/or with one or more VH or V _L monomeric domains ( for example, via disulfide bond(s)).

By analogy with full antibody molecules, antigen binding fragments can be monospecific or multispecific (eg, bispecific).

A multispecific antigen binding antibody fragment will typically comprise at least two different variable domains, where each variable domain is capable of specifically binding to a separate antigen or to different epitopes 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 established techniques available in the art.

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

The term human antibody, as used herein, is intended to include antibodies having variable and constant regions derived from human fetal immunoglobulin sequences. Human antibodies of the invention may, however, include amino acid residues not encoded by human germ line immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation), for example, in CDRs and in particular , CDR3. However, the term human antibody, as used herein, is not intended to include antibodies in which the CDR sequences are derived from the embryonic line of one other mammalian species, such as a mouse, and have been grafted onto human framework sequences.

The term recombinant human antibody, as used herein, is intended to include all human antibodies that are produced, expressed, generated, 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 combinatorial library of recombinant human antibodies (described further below), antibodies isolated from an animal (e.g., mouse) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies produced, expressed, engineered, or isolated by any other means that involve splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human embryonic line immunoglobulin sequences. In some embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, somatic mutagenesis in vivo) and thus the amino acid sequences of the VH and V _L regions of the recombinant antibody are sequences , which, while derived from and related to human fetal VH and V _L sequences, cannot naturally exist in the fetal human antibody array in vivo.

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

The frequency of appearance of the second form in different intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region of the antibody isotype. A single amino acid substitution at the human IgG4 hinge region can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels normally seen with the human IgG1 hinge. The present invention encompasses antibodies having one or more mutations in the hinge, C _H 2 or C _H 3 region, which may be desirable, for example, upon preparation, in order to improve the yield of the desired antibody form.

An isolated antibody, as used herein, 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 isolated 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 the purposes of the present invention. An isolated antibody also includes an antibody in situ in a recombinant cell. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. In some embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

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

Anti-PCSK9 antibodies useful for the methods of the present invention may contain one or more amino acid substitutions, insertions and/or deletions in the framework regions and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. . Such mutations can be readily confirmed by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. The present invention includes methods involving the use of antibodies and antigen-binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework and/or CDR regions are mutated at the appropriate residue ( residues) of the germline sequence from which the antibody is derived, or at the corresponding residue(s) of another human germline sequence, or a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as germline mutations). One of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can readily generate numerous antibodies and antigen binding fragments that contain one or more individual germline mutations, or combinations thereof. In some embodiments, all framework and/or CDR residues in the VH and/or V _L domains are back-mutated to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only some residues are backmutated back to the original germline sequence, such as only mutated residues found in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated to the corresponding residue(s) from a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived) . In addition, the antibodies of the present invention may contain any combination of two or more germline mutations in the framework and/or CDR regions, for example, where some individual residues are mutated to the corresponding residue of a particular germline sequence, while some other residues, which are different from the original

- 10 041556 germline sequences are retained or mutated to the corresponding residue from a different germline sequence. Once produced, antibodies and antigen-binding fragments that contain one or more germline mutations can be readily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (in depending on the situation), reduced immunogenicity, etc. The use of antibodies and antigen-binding fragments obtained by this general method is covered by the present invention.

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

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

The term K _D as used herein is intended to mean the equilibrium dissociation constant for a particular antibody-antigen interaction.

The term epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes can be either conformational or linear. A conformational epitope is produced by spatially contiguous amino acids from different segments of the linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. Under some circumstances, an epitope may include fragments of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

In some embodiments, the PCSK9 antibody used in the methods of the present invention is an antibody with pH dependent binding characteristics. As used herein, the expression pH-dependent binding means that the antibody or antigen-binding fragment thereof exhibits reduced binding to PCSK9 at acidic pH as compared to neutral pH (for the purposes of this disclosure, both terms can be used interchangeably). For example, antibodies with pH-dependent binding characteristics include antibodies and antigen-binding fragments thereof that bind to PCSK9 with higher affinity at neutral pH than at acidic pH. In some embodiments, antibodies and antigen-binding fragments of the present invention bind to PCSK9 with at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100 or more times higher affinity at neutral pH than at acidic pH.

According to this aspect of the invention, anti-PCSK9 antibodies with pH dependent binding characteristics may have one or more amino acid variations relative to the parent anti-PCSK9 antibody. For example, an anti-PCSK9 antibody with pH dependent binding characteristics may contain one or more histidine substitutions or insertions, for example, in one or more CDRs of the parent anti-PCSK9 antibody. Thus, according to some embodiments of the present invention, methods are provided comprising administering an anti-PCSK9 antibody that contains amino acid sequences of CDRs (e.g., heavy and light chain CDRs) that are identical to the amino acid sequences of the CDRs of the parent anti-PCSK9 antibody, except for replacing one or more amino acids in one or more CDRs of the parent antibody per histidine residue. Anti-PCSK9 antibodies with pH-dependent binding may have, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more histidine substitutions, either in a single CDR of the parent antibody or distributed over a plurality (e.g., 2, 3, 4, 5, or 6) CDR of the parent anti-PCSK9 antibody. For example, the present invention includes the use of anti-PCSK9 antibodies with pH-dependent binding, comprising one or more histidine substitutions in HCDR1, one or more histidine substitutions in HCDR2, one or more histidine substitutions in HCDR3, one or more histidine substitutions in LCDR1, one or more more histidine substitutions in LCDR2 and/or one or more histidine substitutions in LCDR3 of the parent anti-PCSK9 antibody.

As used herein, the term 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 term acidic pH includes pH values of about 6.0, 5.95, 5.90, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5 .5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. As used herein, the expression neutral pH

- 11 041556 means a pH of about 7.0 to about 7.4. The expression neutral pH includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

Non-limiting examples of PCSK9 antibodies that can be used in the context of the present invention include, for example, alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, or antigen binding portions of any of the above antibodies.

Obtaining human antibodies.

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

Using VELOCIMMUNE™ technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals) or any other known method for generating monoclonal antibodies, high affinity anti-PCSK9 chimeric antibodies having a human variable region and a mouse constant region are first isolated. The VELOCIMMUNE® technology involves generating a transgenic mouse having a genome containing 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 heavy and light chain variable regions of the antibody is isolated and operably linked to the DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, a VELOCIMMUNE® mouse is sensitized with an antigen of interest and lymphatic cells (such as B cells) that express antibodies are recovered from the mice. Lymph cells can be hybridized with a myeloma cell line to obtain immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. The DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the desired isotypic heavy chain and light chain constant regions. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

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

Typically, antibodies that can be used in the methods of the present invention have high affinities as described above when measured by binding to an antigen, either immobilized on a solid phase or in a liquid phase. Mouse constant regions are replaced with the desired human constant regions to generate fully human antibodies of the invention. While the constant region chosen may vary according to the particular application, antigen binding characteristics with high affinity and target specificity are maintained in the variable region.

Specific examples of human antibodies or antigen-binding antibody fragments that specifically bind to PCSK9 that can be used in the context of the methods of the present invention include any antibody or antigen-binding fragment that contains three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within the variable region. heavy chain (HCVR) having an amino acid sequence selected from the group consisting of ID NO:1 and 11, or a sequence substantially similar to it, having at least 90, at least 95, at least 98, or at least 99 % sequence identity. Alternatively, specific examples of human antibodies or antigen-binding fragments of antibodies that specifically bind to PCSK9 that can be used in the context of the methods of the present invention include any antibody or antigen-binding fragment that contains three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of ID NOs: 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 149 , 157, 165, 173, 181, and 189, or a substantially similar sequence having at least 90, at least 95,

- 12 041556 at least 98 or at least 99% sequence identity. An antibody or antigen-binding fragment may comprise three light chain CDRs (LCVR1, LCVR2, LCVR3) contained within a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of ID NOs: 6 and 15, or substantially similar to it. a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. Alternatively, the antibody or antigen binding fragment may comprise three light chain CDRs (LCVR1, LCVR2, LCVR3) contained within a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of ID NOs: 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145, 153, 161, 169, 177, 185, and 193, or an essentially similar sequence having at least 90, at least at least 95, at least 98, or at least 99% sequence identity.

Sequence identity between two amino acid sequences is determined over the full length of the reference amino acid sequence, i. amino acid sequence identified with SEQ ID NO using the best sequence alignment and/or by the region of best sequence alignment between two amino acid sequences, where the best sequence alignment can be obtained using software tools known in the art, e.g. Align using standard settings, preferably EMBOSS ::needle, Matrix: Blosum62, 10.0 gap opening, 0.5 gap continuation.

In some embodiments of the present invention, the antibody or antigen-binding protein comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) from heavy and light chain variable region (HCVR/LCVR) amino acid sequence pairs selected from the group consisting of ID NO:1/6 and 11/15. Alternatively, in some embodiments, the antibody or antigen-binding protein comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) from heavy and light chain variable region (HCVR/LCVR) amino acid sequence pairs selected from the group consisting of from 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.

In some embodiments of the present invention, the PCSK9 antibody or antigen binding protein that can be used in the methods of the present invention have the amino acid sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 selected from SEQ ID NO:2/3/4/ 7/8/10 (mAb316P [also referred to as REGN727 or alirocumab]) and 12/13/14/16/17/18 (mAb300N) (See U.S. Patent Application Published No. 2010/0166768) and 12/13/14/16 /17/18, where SEQ ID NO:16 contains the replacement of histidine with leucine at amino acid residue 30 (L30H).

In some embodiments, the implementation of the present invention, the antibody or antigen-binding protein contains pairs of amino acid sequences HCVR/LCVR selected from the group consisting of ID NO:1/6 and 11/15. In some exemplary embodiments, 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 some exemplary embodiments, 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 some exemplary embodiments, the antibody or antigen-binding protein comprises the HCVR amino acid sequence of SEQ ID NO:11 and the HCVR amino acid sequence of SEQ ID NO:15 containing a histidine to leucine substitution at amino acid residue 30 (L30H).

Pharmaceutical compositions and methods of administration.

The present invention includes methods that include administering a PCSK9 inhibitor to a patient, wherein the PCSK9 inhibitor is contained in a pharmaceutical composition. Pharmaceutical compositions according to the invention are formulated with suitable carriers, excipients, and other means that provide suitable transfer, delivery, tolerability, and the like. Many suitable dosage forms can be found in the pharmacological reference book known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These dosage forms include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in emulsions -water and water-in-oil, emulsions with carbovax (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbovax. See also Powell et al. Compendium of excipients for parenteral formulations PDA (1998) J Pharm Sci Technol 52:238-311.

Illustrative pharmaceutical dosage forms containing antibodies against PCSK9 that can be used in the context of the present invention include any of the dosage forms described in US 8,795,669 (describes, among other things, illustrative dosage forms containing alirocumab), or in WO 2013/166448 or WO 2012/168491.

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

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition to subcutaneous delivery, a pen-shaped delivery device can be easily used in delivering the pharmaceutical composition of the present invention. Such a pen-style delivery device may be in the form of a reusable or disposable device. A reusable pen-style delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been injected and the cartridge becomes empty, the empty cartridge can be easily removed and replaced with a new cartridge that contains the pharmaceutical composition. The pen-style delivery device can then be reused. In the device in the form of a single-use delivery pen, there is no replaceable cartridge. Most commonly, a disposable pen delivery device comes pre-filled with a pharmaceutical composition contained in a container within the device. Once the container is emptied of the pharmaceutical composition, the whole device is discarded.

Numerous reusable pen and autoinjector delivery devices have applications in subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ handle (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ handle, HUMALOG™ handle, HUMALIN 70 handle /30™ (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (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), among others. Examples of reusable delivery pen devices having applications in subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), SURECLICK ™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and HUMIRA™ Pen (Abbott Labs, Abbott Park IL), among others.

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

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations can be obtained by known methods. For example, injectable preparations may be prepared, for example, by dissolving, suspending or emulsifying an antibody or salt thereof, as described above, in a sterile aqueous or oily medium conventionally used for injection. Aqueous injection media are, for example, saline, isotonic saline containing glucose, and other auxiliaries, etc., which can be used in combination with an appropriate solubilizer such as alcohol (eg ethanol), polyalcohol (eg propylene glycol , polyethylene glycol), a non-ionic surfactant (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizer such as benzyl benzoate, benzyl alcohol, etc. The injection form thus obtained is preferably used to fill an appropriate ampoule.

Preferably, the pharmaceutical compositions for oral or parenteral administration described above are prepared in unit dose dosage forms adjusted to match the dosage of the active ingredients. Such unit dosage forms include, for example, tablets, pills, capsules, injection forms (ampoules), suppositories, and the like.

Dosage.

The amount of a PCSK9 inhibitor (eg, 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 the dose of a PCSK9 inhibitor that results in a detectable reduction (at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75% or more of baseline) on one or more parameters selected from the group consisting of LDL-C, ApoB100, non-HDL-C, total cholesterol, VLDL-C, triglycerides, Lp(a) and remnant cholesterol, or an amount that reduces or eliminates the patient's need for lipoprotein apheresis, or that reduces the patient's normalized rate of apheresis (as described elsewhere herein).

In the case of an anti-PCSK9 antibody, the therapeutically effective amount may be from about 0.05 mg to about 600 mg, such as about 0.05 mg, about 0.1, about 1.0, about 1.5, about 2.0, about 10 , about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, or about 600 mg of anti-PCSK9 antibody. According to some exemplary embodiments of the present invention, a therapeutically effective amount of an anti-PCSK9 antibody is 75, 150, or 300 (eg, in the case of alirocumab), or 140 or 420 mg (eg, in the case of evolocumab). Other dosage amounts of PCSK9 inhibitors will be understood by those of ordinary skill in the art and are contemplated within the scope of the present invention.

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

Combined therapies.

As described elsewhere in this document, the methods of the present invention may include administering a PCSK9 inhibitor to a patient in combination with (along with) the patient's prior lipid-lowering therapy. For example, in the context of reducing or eliminating the need for lipoprotein apheresis, a PCSK9 inhibitor may be administered to a patient in combination with a stable daily statin therapeutic regimen. Exemplary daily therapeutic regimens for statins in combination with which a PCSK9 inhibitor may be administered in the context of the present invention include, for example, atorvastatin (10, 20, 40 or 80 mg daily), (atorvastatin/ezetimibe 10/10 or 40/10 mg daily ), rosuvastatin (5, 10, or 20 mg daily), cerivastatin (0.4 or 0.8 mg daily), pitavastatin (1, 2, or 4 mg daily), fluvastatin (20, 40, or 80 mg daily), simvastatin ( 5, 10, 20, 40, or 80 mg daily), simvastatin/ezetimibe (10/10, 20/10, 40/10, or 80/10 mg daily), lovastatin (10, 20, 40, or 80 mg daily), pravastatin (10, 20, 40 or 80 mg daily) and combinations thereof. Other lipid-lowering therapies in combination with which a PCSK9 inhibitor may be administered in the context of the present invention include, for example, (1) an agent that inhibits cholesterol uptake and/or bile acid reabsorption (eg, ezetimibe); (2) an agent that enhances lipoprotein catabolism (such as niacin); and/or (3) LXR transcription factor activators that play a role in cholesterol elimination, such as 22-hydroxycholesterol.

According to some embodiments of the present invention, methods are provided comprising administering a PCSK9 inhibitor (e.g., an anti-PCSK9 antibody such as alirocumab, evolocumab, bococizumab, lodelcizumab, or ralpancizumab) to a patient in combination with an angiopoietin-like protein 3 inhibitor (e.g., an anti-ANGPTL3 antibody, such as REGN1500), an angiopoietin-like protein 4 inhibitor (for example, an anti-ANGPTL4 antibody, such as an anti-ANGPTL4 antibody referred to in US Pat. No. 9,120,851 as H1H268P or H4H284P), or an angiopoietin-like protein 8 inhibitor (for example, an anti-ANGPTL8 antibody).

In the context of the methods of the present invention, additional therapeutically active component (s), for example, any of the agents above, or derivatives thereof, can be administered immediately before, simultaneously with, or shortly after administration of the PCSK9 inhibitor; (For the purposes of this disclosure, such administration regimens are considered to be the administration of a PCSK9 inhibitor in combination with an additional therapeutically active ingredient). The present invention includes pharmaceutical compositions and methods of using them, in which the PCSK9 inhibitor is combined with one or more additional therapeutically active component (components), as described elsewhere in this document.

Introduction modes.

According to some embodiments of the present invention, multiple doses of a PCSK9 inhibitor (i.e., a pharmaceutical composition comprising a PCSK9 inhibitor) may be administered to a subject over a predetermined period of action (e.g., along with a daily statin therapeutic regimen or other initial lipid-lowering therapy). ). Methods according to this aspect of the invention include sequentially administering multiple doses of a PCSK9 inhibitor to a subject. As used herein, sequential administration means that each dose of a PCSK9 inhibitor is administered to a subject at a different time point, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention includes methods that include sequentially administering to a patient a single initial dose of a PCSK9 inhibitor, followed by one or more secondary doses of a PCSK9 inhibitor, and optionally followed by one or more tertiary doses of a PCSK9 inhibitor.

The terms initial dose, secondary doses, and tertiary doses refer to the time sequence of administration of individual doses of a pharmaceutical composition containing a PCSK9 inhibitor. Thus, the starting dose is the dose that is administered at the start of a treatment regimen (also referred to as the baseline dose); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. Initial, secondary and tertiary doses may all contain the same amount of PCSK9 inhibitor, but generally may differ from each other in terms of frequency of administration. In some embodiments, however, the amount of PCSK9 inhibitor contained in the initial, secondary, and/or tertiary doses is different (eg, adjusted up or down as appropriate) during the course of treatment. In some embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the start of a treatment regimen as loading doses followed by successive doses administered on a less frequent basis (eg, maintenance doses).

According to illustrative embodiments of the present invention, each secondary and/or tertiary dose is administered through 1 to 26 (for example, 1, 11/2, ₂ , 21/2, 3, 31/2, ₄ , 41/2, 5, 51 /2, 6, 61/2, 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2 , 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2, 22 , 221/2, 23, 231/2, 24, 241/2, 25, 251/2, 26, 261/2, or more) weeks after the immediately preceding dose. The phrase immediately preceding dose, as used herein, means, in a sequence of multiple administrations, the dose of an antigen-binding molecule that is administered to a patient prior to the next same dose in the sequence without any intervening doses.

Methods according to this aspect of the invention may include administering to a patient any number of secondary and/or tertiary doses of a PCSK9 inhibitor. For example, in some embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in some embodiments, only 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 involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to a patient 1 to 2, 4, 6, 8 or more weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to a patient 1-2, 4, 6, 8 or more weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to a patient may vary during the course of a treatment regimen. The frequency of administration can also be adjusted during the course of treatment by the attending physician, depending on the needs of the individual patient after clinical examination.

The present invention includes administration regimens including a dose escalation variant (also referred to herein as dose modification). As used herein, a dose escalation option means that, after receiving a specific number of doses of a PCSK9 inhibitor, if the patient has not achieved an established reduction in one or more of the target therapeutic parameters, the dose of the PCSK9 inhibitor is further increased. For example, in the case of a treatment regimen administering doses of 75 mg of anti-PCSK9 antibody to a patient at a biweekly frequency, if after 8 weeks (i.e. 5 doses administered at Week 0, Week 2 and Week 4, Week 6 and Week 8), the patient does not achieve a serum LDL-C concentration of less than 70 mg/dL, then the dose of anti-PCSK9 antibody is increased to, for example, 150 mg administered every two weeks thereafter (eg, starting at Week 10 or Week 12 or later).

In some embodiments, the PCSK9 antibody is administered to the subject at a dose of about 75 mg once every two weeks, for example, in a volume of at least three doses (or for a treatment regimen spanning days, weeks, months, or years).

In some embodiments, the PCSK9 antibody is administered to the subject at a dose of about 150 mg once every two weeks, for example, in a volume of at least three doses (or for a treatment regimen spanning days, weeks, months, or years).

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In several embodiments, the antibody is administered to the subject at a dose of about 75 mg biweekly for 12 weeks, and the dose remains at 75 mg biweekly if, at week 8, the subject's LDL-C is less than 100 mg /dl and there will be a 30% reduction in LDL-C.

In other embodiments, the antibody is administered to a subject at a dose of about 75 mg once every two weeks for 12 weeks, and the dose is increased to about 150 mg once every two weeks if, at week 8, the subject's LDL-C value is greater than or equal to 100 mg/dl.

In several embodiments, the antibody is administered to the subject at a dose of about 75 mg biweekly for 12 weeks, and the dose remains at 75 mg biweekly if, at week 8, the subject's LDL-C is less than 70 mg /dl and there will be a 30% reduction in LDL-C.

In one other embodiment, the antibody is administered to the subject at a dose of about 300 mg once every four weeks.

In a further embodiment, the antibody is administered to the subject at a dose of about 300 mg once every four weeks in a volume of three doses, and the dose is changed to 150 mg every other week for another 36 weeks if, at week 8, the subject has not reached the predetermined treatment goal, or the subject does not have at least a 30% reduction in LDL-C from baseline.

In some embodiments, the PCSK9 antibody is administered to the subject at a dose of about 150 mg once every four weeks for at least three doses.

In several embodiments, the antibody is administered to the subject at a dose of about 150 mg once every four weeks for 12 weeks, and the dose remains at 150 mg once every four weeks if, at week 8, the subject's LDL-C value is less than 100 mg /dl and there will be a 30% reduction in LDL-C.

In other embodiments, the antibody is administered to the subject at a dose of about 150 mg once every four weeks for 12 weeks, and the dose is increased to about 300 mg once every two weeks if, at week 8, the subject's LDL-C value is greater than or equals 100 mg/dl.

In several embodiments, the antibody is administered to the subject at a dose of about 150 mg once every four weeks for 12 weeks, and the dose remains at 150 mg once every four weeks for another 12 weeks if, at week 8, the LDL-C value the subject will be less than 70 mg/dL and will have a 30% reduction in LDL-C.

In one other embodiment, the antibody is administered to the subject at a dose of about 300 mg once every four weeks.

In a further embodiment, the antibody is administered to the subject at a dose of about 300 mg once every four weeks for three doses, and the dose is changed to 150 mg every other week for another 36 weeks if, at week 8, the subject has not previously reached defined treatment goal, or the subject does not have at least a 30% reduction in LDL-C from baseline.

Examples

The following examples are provided 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 what the inventors consider their invention. Efforts have been made to ensure accuracy with respect to the numerical values used (eg for amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric.

Example 1 Generation of human anti-human PCSK9 antibodies.

Human anti-PCSK9 antibodies were generated as shown in US Pat. No. 8,062,640. An exemplary PCSK9 inhibitor used in the following Example is a human anti-PCSK9 antibody designated mAb316P, also known as REGN727 or alirocumab. mAb316P has the following amino acid sequence characteristics: a heavy chain containing SEQ ID NO:5 and a light chain containing SEQ ID NO:9; a heavy chain variable region (HCVR) comprising SEQ ID NO:1 and a light chain variable domain (LCVR) comprising SEQ ID NO:6; complementarity determining heavy chain region 1 (HCDR1) containing SEQ ID NO:2, HCDR2 containing SEQ ID NO:3, HCDR3 containing SEQ ID NO:4, complementarity determining light chain region 1 (LCDR1) containing SEQ ID NO:7, LCDR2 containing SEQ ID NO:8 and LCDR3 containing SEQ ID NO:10.

Example 2 Randomized, double anonymous, placebo-controlled, parallel group study to evaluate the efficacy and safety of alirocumab in patients with heterozygous familial hypercholesterolemia undergoing lipid apheresis therapy.

Introduction.

The aim of this study was to evaluate the effect of alirocumab 150 mg administered once every two weeks (Q2W) versus placebo on the frequency of LDL apheresis treatments in HeFH patients undergoing LDL apheresis therapy.

This study included adult men and women diagnosed with HeFH who were treated with LDL apheresis at a frequency of once a week or once every two

- 17 041556 weeks. Baseline treatment of subjects with LMT was maintained throughout the study. In these patients, the choice of placebo as a control is appropriate for the purposes of this study, as it provides a more reliable assessment of the efficacy and safety of alirocumab.

The average reduction in LDL-C ranges from 30% to 75% depending on the method and duration of apheresis (Bambauer et al., ScientificWorldJoumal, 2012, 2012:1-19). When treated weekly or twice a week, LDL-C levels can be reduced by 40-50%. In the present study, apheresis was not performed when the LDL-C value at that visit was >30% lower than the baseline (pre-apheresis) LDL-C value. LDL-C levels rise to baseline after each apheresis procedure but do not reach baseline; with repeated apheresis weekly or twice a week, baseline levels continue to fall until a plateau is reached (Thompsen et al., Atherosclerosis, 2006, 189:31-38). As such, a criterion for determining the need for apheresis based on a 30% reduction in LDL-C levels is warranted, as this reduction, if observed with alirocumab, will be constant over time, reaching approximately the same level of LDL-C reduction as and during the apheresis procedure.

Alirocumab dosing regimens Q2W (75 and 150 mg SC) and Q4W (150 and 300 mg SC) were evaluated in another phase 3 trial of alirocumab. Based on the results of 2 dosing studies, the Q2W dosing regimen was chosen to maintain a constant reduction in LDL-C throughout the dosing interval in all patients, with maximum efficacy at 12 weeks provided by a dosing of 150 mg Q2W. Patients with heterozygous familial hypercholesterolemia usually have high baseline levels of LDL-C. Given that these patients have had lifetime exposure to elevated LDL-C, their LDL-C target is either 100 or 70 mg/dL, depending on their history of CVD or other risk factors. In order to reach and maintain their LDL-C target, these patients were expected to most likely require the most potent doses of alirocumab and at the frequency of administration needed to maintain this exposure at the end of the dosing interval. Therefore, a dose of 150 mg Q2W was chosen for this study.

During the double anonymous treatment period of this study, 150 mg alirocumab Q2W administered for 18 weeks to HeFH patients requiring apheresis every 1 or 2 weeks to lower LDL-C was evaluated for the ability to reduce the frequency of apheresis from week 7 up to week 18 compared with the frequency during the 8 weeks before screening. Patients received 150 mg alirocumab Q2W until week 76 during the open-label treatment period.

Preliminary pharmacokinetic (PK) data from the Phase 2 studies, DFI11565, DFI11566, and R727-CL-1003, showed that alirocumab exposure fell over the 8-week prospective period that followed treatment during the double-anonymity period, with still detectable total serum concentrations of alirocumab, but at very low levels. Therefore, to confirm sufficiently low, ineffective serum concentrations of alirocumab, patients were followed up for a prospective period of 8 weeks (i.e., 10 weeks after the last dose).

Research objectives.

The primary study objective was to evaluate the effect of alirocumab Q2W 150 mg versus placebo on the frequency of LDL apheresis therapy in HeFH patients treated with weekly or twice weekly LDL apheresis.

The secondary objectives of the study were to: (a) Evaluate the effect of 150 mg alirocumab Q2W on LDL-C levels in HeFH patients treated with LDL apheresis; (b) To evaluate the effect of 150 mg alirocumab Q2W on the following lipid parameters: ApoB, non-HDL-C, total cholesterol, Lp(a), HDL-C, TG, and ApoA-1 in HeFH patients treated with apheresis LDL during the study; (c) To evaluate the safety and tolerability of 150 mg alirocumab Q2W in HeFH patients undergoing LDL apheresis therapy; (d) Assess the PK of 150 mg alirocumab Q2W in HeFH patients treated with LDL apheresis (QW vs. Q2W); (e) Assess the development of antibody production against alirocumab; (f) Assess PCSK9 levels in response to alirocumab therapy and before and after apheresis; and (g) Assess quality of life (QOL) in these patients.

Clinical study plan.

The present study was a randomized, double-anonymous, placebo-controlled, parallel group study of HeFH patients treated with LDL apheresis once a week or every 2 weeks.

The study consisted of 4 periods: a screening period, a dual-anonymity treatment period, an open-label treatment period, and a prospective period. The treatment period under double anonymity included 2 intervals: from day 1 to week 6 (with planned administration of study drug on days 1, 15 and 29), when the frequency of apheresis will be fixed according to the established schedule for the individual patient, and from week 7 to week 18 (with planned study drug administration on days 43, 57, 71, 85, 99, and 113), when the frequency of apheresis will be adjusted based on the patient's response to treatment.

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Screening: Patients who were on a stable apheresis schedule (every 7 or 14 days), with stable apheresis settings for at least 4 weeks prior to the Screening visit (week -2) and stable baseline medical LMT for at least 8 weeks prior to screening visits (week -2) were included in the 2-week screening period.

Treatment under double anonymity: Patients who met all inclusion criteria and none of the exclusion criteria were randomly divided in a 2:1 ratio to receive 18 weeks of treatment: 150 mg alirocumab SC Q2W, or placebo for alirocumab SC Q2W.

Therapeutic injections during the treatment period under conditions of double anonymity were administered Q2W, starting from the day of randomization (week 0 [day 1]/visit 2). Patients were monitored at the clinical site for 30 minutes after the first dose. If LDL apheresis coincided with study drug administration, study drug was administered immediately after completion of the LDL apheresis procedure.

From day 1 to week 6, the frequency of apheresis was recorded according to the established schedule for the individual patient (QW or Q2W). Starting at week 7, LDL apheresis was performed based on the LDL-C value at that visit (determined at the testing site). LDL apheresis was NOT performed when the LDL-C value at this visit was at least 30% lower than the LDL-C value at baseline before apheresis (Day 1). The criterion for determining the need for apheresis, based on a 30% reduction in LDL-C levels, is justified, as this reduction, if observed with alirocumab, will be constant over time, reaching approximately the same level of LDL-C reduction as with the procedure. apheresis. Investigators remained anonymous with respect to laboratory LDL-C values at the site and only received a warning signal as to whether or not LDL apheresis should be performed.

Open-label treatment: During open-label treatment, patients received 150 mg alirocumab SC Q2W. Treatment continued without interruption from the last dose of study drug during the double-anonymity treatment period (last dose at week 16) to week 18 (first during open-label treatment), until week 76.

The first injection during the open treatment period was administered at the week 18 visit (the first visit of the open treatment period) as part of patient education on injection administration. Subsequent injections were administered by the patient or an authorized guardian (spouse, relative, etc.) at the patient's preferred location (eg, home or workplace). Patients were also allowed to choose to return to the Q2W site to receive an injection administered by study staff.

Apheresis treatment is not required during the treatment period of an open-label study. At the investigator's discretion, patients were allowed to continue with apheresis procedures if necessary. In the event that the planned apheresis procedure coincided with a clinical visit or alirocumab dosing day, study evaluations were performed before apheresis and alirocumab was administered after apheresis.

Prospective Period: Patients were followed up at the end of the study visit at week 86.

Studies throughout the trial period: All clinical laboratory specimens (for lipid analysis and in particular lipidograms) and PK assessments were obtained immediately before and immediately after LDL apheresis (if LDL apheresis is performed at that visit) and before administration of study drug.

For those patients who did not have LDL apheresis at the visit, all specimens for clinical laboratory evaluation were obtained prior to study drug administration.

An overall safety assessment was performed through monitoring/assessment of TEAE, exercise, vital signs (pulse rate and blood pressure), electrocardiograms (ECGs), and clinical safety laboratory tests at predetermined timestamps. The potential appearance of antibodies against alirocumab was assessed. PCSK9 levels were assessed before and after apheresis.

The use of all medications and dietary supplements (including consumption of red fermented foods) known to alter serum lipids, including (but not limited to) statins, ezetimibe, fibrates, niacin, and bile acid sequestrants, was permitted provided that the therapy was stable for at least 8 weeks prior to the screening visit (week -2). Patients were instructed to continue receiving their original medical LMT throughout the study from screening through to the end of the treatment visit.

Patients' diets remained stable throughout the study from screening until the end of the treatment visit.

Exercise regimens remained stable throughout the study from screening through to the end of the treatment visit.

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Selection of patients.

The study protocol required enrollment of approximately 63 patients from at least 15 study centers in the US and Germany. Patients were randomized as follows: approximately one third of the included patients (~21) received placebo; and approximately two-thirds of the included patients (~42) received alirocumab.

The study population consisted of adult males and females diagnosed with HeFH who were treated with LDL apheresis every 1 or 2 weeks.

Randomization was divided according to: apheresis rate: QW versus Q2W; and baseline Lp(a) levels: normal (<30 mg/dl) versus elevated (>30 mg/dl). Since some patients with elevated LDL-C also had elevated Lp(a) levels, the separation provided an appropriate representation in the placebo/treatment groups.

Inclusion Criteria: Patients included in this study were required to meet conditions 1-6 (below) in order to be eligible for enrollment in the study:

(1) men and women aged >18 at the time of the screening visit;

(2) diagnosis of HeFH; (Note: HeFH was diagnosed based on either genotyping or clinical criteria. For those patients who were not genotyped, a clinical diagnosis can be made based on the Simon-Broom criteria with criteria for a specific FH, or WHO/Dutch Lipid Network criteria with a score >8 points);

(3) at the time in question they were treated with LDL QW apheresis for at least 4 weeks or Q2W for at least 8 weeks before the screening visit (week -2) and started apheresis therapy for at least 5 months before; (Note: Acceptable apheresis methods are: double membrane filtration, immunoadsorption, heparin-induced LDL precipitation, direct lipid adsorption, dextran sulfate adsorption (plasma), dextran sulfate adsorption (whole blood));

(4) willingness and ability to follow the schedule of clinical visits and procedures related to the study;

(5) providing signed informed consent; and (6) the ability to understand and complete survey-related questionnaires.

Exclusion Criteria: Prospective patients who met any of the following criteria were excluded from the study:

(1) homozygous FH;

(2) baseline medical LMT (if applicable) that was not stable for at least 8 weeks prior to the screening visit (week -2);

(3) schedule for LDL apheresis/apheresis units that were not stable for at least 4 weeks prior to the screening visit (week -2) for patients who have weekly apheresis and at least 8 weeks prior to the screening visit (week -2). 2) for patients who undergo apheresis twice a week;

(4) LDL apheresis schedule other than QW - Q2W;

(5) initiation of a new exercise program or exercise that did not remain stable in the 8 weeks prior to the screening visit (week -2);

(6) initiation of a new diet or a diet that was not stable in the 8 weeks prior to the screening visit (week -2);

(7) use of nutraceuticals or over-the-counter therapies known to affect lipids at a dose/amount that has not been stable for at least 8 weeks prior to the screening visit (week -2), or between screening and the randomization visit;

(8) the presence of any clinically significant uncontrolled endocrine disorder known to affect serum lipids or lipoproteins;

(9) signs and symptoms of hypothyroidism (thyroid replacement therapy is allowed);

(10) history of bariatric surgery within 12 months prior to screening visit (week -2);

(11) unstable body weight (variation >5 kg) in the interval of 2 months prior to the screening visit (week -2);

(12) newly diagnosed (within 3 months prior to randomization visit (Day 1)) diabetes mellitus or poorly controlled (hemoglobin A1c [HbA1c] >9%) diabetes;

(13) use of systemic corticosteroids unless used as replacement therapy for pituitary/adrenal disease on a stable regimen for at least 6 weeks prior to randomization; topical, intra-articular, nasal, inhaled and ophthalmic steroid therapies are not considered systemic and are permitted;

(14) use of estrogen or testosterone therapy if the regimen has not been stable in the past 6 weeks prior to the screening visit (week -2) and there are no plans to change the regimen during the study;

(15) systolic blood pressure >160 mm Hg or diastolic blood pressure >100 mm Hg at the screening visit (week -2) or at the time of randomization (day 1) (observation: evaluation

- 20 041556 study eligibility blood pressure may be obtained at a visit taking place between these 2 visits if the patient did not take, or did not plan to take, the prescribed hypertensive medications at the screening visit or randomization visit due to the apheresis schedule );

(16) history of myocardial infarction (MI), unstable angina leading to hospitalization, coronary artery bypass grafting (CABG), percutaneous coronary intervention (PCI), uncontrolled cardiac arrhythmia, carotid stenting or surgery, stroke, transient ischemic attack, or revascularization carotid arteries within 3 months prior to the screening visit (week -2), or an endovascular procedure or surgery for peripheral vascular disease within 1 month prior to the screening visit (week -2);

(17) history of New York Heart Association class III or IV heart failure within 12 months prior to screening visit;

(18) known history of hemorrhagic stroke;

(19) history of malignancy within the past 5 years, excluding appropriately treated basal cell skin cancer, squamous cell skin cancer, or cervical cancer in situ;

(20) known history of positive human immunodeficiency virus testing;

(21) use of any active investigational medicinal products within 1 month or 5 half-lives prior to screening, whichever is longer;

(22) patients who have been treated with at least 1 dose of alirocumab or any other anti-PCSK9 monoclonal antibody in any other clinical study;

(23) conditions/situations such as: (a) Any clinically significant pathology identified at screening that, in the opinion of the Investigator or any Associate Investigator, might interfere with the safe completion of the study or limit the evaluation of endpoints; eg major systemic diseases, patients with short life expectancy; or (b) Considered by the Investigator or any Associate Investigator to be unsuitable for this study for any reason, such as: (i) deemed unable to meet specific protocol requirements, such as scheduled visits; (ii) deemed incapable of tolerating injections, as from the patient or investigator; (iii) the investigator or any junior investigator, pharmacist, study coordinator, other or related research personnel directly involved in the implementation of the protocol, etc.; (iv) the existence of any other conditions (eg, geographic or social), either actual or perceived, which, in the opinion of the investigator, might limit or limit the patient's participation during the study period;

(24) laboratory results during the screening period (not including tests at randomization): (a) a positive test result for hepatitis B surface antigen and/or hepatitis C antibody; (b) a positive serum beta-hCG test or urine pregnancy test in women of childbearing potential; (c) TG>500 mg/dL (>5.65 mmol/L) (1 repeat test is allowed); (d) eGFR<15 ml/min/1.73 m ² according to a 4-variable equation for studying dietary modification in kidney disease (calculated at the Central Laboratory); (e) alanine aminotransferase (ALT) or aspartate aminotransferase (AST) > 3χ upper limit of normal (ULN) (1 retest is allowed); (f) CPK>3xULN (1 reanalysis allowed);

(25) known hypersensitivity to monoclonal antibody therapeutics or to any component of the medicinal product;

(26) pregnant or lactating women;

(27) Women of reproductive potential who are not protected by highly effective pregnancy prevention method(s) (as defined in the Informed Consent Form [ICF] and/or local protocol supplement) and/or who are unwilling or unable to undergo pregnancy testing . Postmenopausal women must be amenorrheic for at least 12 months.

Study treatment.

Treatment within the study was a single SC injection of 1 ml for a 150 mg dose of alirocumab or placebo provided in an auto-injector or pre-filled syringe injected into the abdomen, thigh, or Q2W injected into the outer area of the upper arm.

When treated during the double-anonymity period (day 1 to week 18), eligible patients were randomized to receive: 150 mg alirocumab SC Q2W, or Placebo for alirocumab SC Q2W.

For open-label treatment, patients received open-label alirocumab 150 mg SC Q2W starting at week 18, with the last dose administered at week 76.

The sterile drug alirocumab was delivered at a concentration of 150 mg/ml in histidine, pH 6.0, polysorbate 20 and sucrose in an auto-injector or pre-filled syringe.

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The placebo drug corresponding to alirocumab was delivered in the same dosage form as alirocumab, without added protein, in an auto-injector or pre-filled syringe.

All patients and/or caregivers who opted for out-of-clinic injection of study drug on dose days were trained by study staff prior to injection. Patients/carers were instructed to administer study drug at the week 18 visit and self-administered the first dose during the open-label treatment period at the week 18 visit. etc.) in a location preferred by the patient (e.g. at home or workplace).

Patients were also allowed to return to the Q2W site to receive injections administered by study staff.

Distribution method for treatment groups.

Patients were randomly assigned to receive either placebo or 150 mg alirocumab Q2W at a ratio of 1:2 using a block randomization design to ensure even distribution across treatment groups. Randomization was stratified according to the frequency of the apheresis procedure (every 7 or 14 days) and Lp(a) levels (normal or elevated). Inclusion in the study was limited such that apheresis was performed in no more than two-thirds of the patients.

All patients, regardless of allocation to treatment groups during the treatment period under double anonymity, received 150 mg alirocumab Q2W during the treatment period in open mode.

Concomitant medications.

Any treatment administered from the time of informed consent to the end of the prospective period/final study visit was considered concomitant treatment. This treatment includes medications started before the study and continued during the study. Concomitant medications were kept to a minimum during the study. If it was deemed necessary for the patient's well-being and it was unlikely that patients would interfere with the action of the investigational medicinal product, then concomitant medicinal products (other than those prohibited during the study) were allowed at the discretion of the investigator, at a stable dose (when possible).

Prohibited Medications: Any baseline medical LMT that was not stable for at least 8 weeks prior to the Screening visit (week 2) was prohibited.

Approved Medications: All medications and dietary supplements (including consumption of red fermented foods) known to alter serum lipids, including but not limited to statins, ezetimibe, fibrates, niacin, and bile acid sequestrants, were permitted provided that this therapy was stable for at least 8 weeks prior to the screening visit (week 2). Patients were instructed to continue taking their baseline medical LMT throughout the study, from screening through to the final treatment visit.

Patients' diets remained stable throughout the study, from screening through to the final treatment visit.

The patients' exercise programs remained stable throughout the study, from screening through to the final treatment visit.

Study endpoints.

Baseline characteristics will include standard demographics (eg, age, race, weight, height, etc.), disease characteristics, including history and drug history for each patient.

Primary efficacy endpoints: The primary efficacy endpoint is the rate of apheresis treatments during the 12-week period from week 7 to week 18, normalized by the number of planned apheresis treatments according to the established schedule for each patient at screening, from week -10 to weeks -2.

The normalized apheresis rate is defined for each patient as: The number of actual apheresis treatments obtained from week 7 to week 18 divided by the number of planned apheresis treatments per randomization stratum at baseline (6 for Q2W or 12 for QW).

In the event of a patient dropping out before week 18, the number of valid apheresis treatments (week 7 to week 18) will be added to the estimated number of remaining scheduled apheresis treatments according to the randomization stratum for the numerator.

The effect of normalization on planned apheresis treatments, according to the established schedule for each patient at baseline, ensures uniformity with respect to the same score scale for patients entering the study with different apheresis schedules (i.e., QW apheresis or Q2W).

Secondary efficacy endpoints: For the following lipid endpoints, such as percentage change from baseline to a specific visit after baseline during

- 22 041556 treatment in the period under conditions of double anonymity, 2 time stamps are set: before and after the apheresis procedure. For key efficacy endpoints, lipid parameters will be analyzed using scores collected prior to each apheresis procedure. In the event that apheresis is not performed at the protocol visit, the only laboratory assessment scheduled for collection is used for both time stamps (i.e., including key efficacy endpoints). The baseline value is defined as the last available value for each of the parameters prior to the first double-blind dose of study drug.

The secondary endpoints of the present study were:

(1) percent change in LDL-C from baseline (pre-apheresis) to week 6, regardless of adherence to treatment;

(2) a standardized rate of apheresis treatment during the 4-week period from week 15 to week 18, defined similarly as for the primary efficacy endpoints;

(3) percent change in ApoB from baseline (pre-apheresis) to week 6, regardless of adherence to treatment;

(4) percent change in non-HDL-C from baseline (pre-apheresis) to week 6, regardless of adherence to treatment;

(5) percentage change in total cholesterol from baseline (pre-apheresis) to week 6, regardless of adherence to treatment;

(6) percent change in ApoA-1 from baseline (pre-apheresis) to week 6, regardless of adherence to treatment;

(7) proportion of patients with >30% decrease in LDL-C (before apheresis) at week 6, regardless of treatment adherence;

(8) proportion of patients with >50% decrease in LDL-C (before apheresis) at week 6, regardless of treatment adherence;

(9) percent change in LDL-C from baseline (pre-apheresis) to week 18, regardless of adherence to treatment;

(10) percent change in ApoB from baseline (pre-apheresis) to week 18, regardless of adherence to treatment;

(11) percent change in non-HDL-C from baseline (pre-apheresis) to week 18, regardless of treatment adherence;

(12) percent change in total cholesterol from baseline (pre-apheresis) to week 18, regardless of adherence to treatment;

(13) percent change in ApoA-1 from baseline (pre-apheresis) to week 18, regardless of adherence to treatment;

(14) proportion of patients with >30% decrease in LDL-C (before apheresis) at week 18, regardless of treatment adherence;

(15) proportion of patients with >50% decrease in LDL-C (before apheresis) at week 18, regardless of treatment adherence;

(16) change in W-BQ22 score from baseline to week 18, regardless of treatment adherence;

(17) percentage change in Lp(a) from baseline (pre-apheresis) to week 6, regardless of adherence to treatment;

(18) percent change in HDL-C from baseline (pre-apheresis) to week 6, regardless of adherence to treatment;

(19) percent change in TG levels from baseline (pre-apheresis) to week 6, regardless of adherence to treatment;

(20) percent change in Lp(a) from baseline (pre-apheresis) to week 18, regardless of adherence to treatment;

(21) percent change in HDL-C from baseline (pre-apheresis) to week 18, regardless of treatment adherence;

(22) percent change in TG levels from baseline (pre-apheresis) to week 18, regardless of adherence to treatment;

(23) change in the ApoB/ApoA-1 ratio (before apheresis) from baseline to week 6, regardless of treatment adherence;

(24) change in ApoB/ApoA-1 ratio (before apheresis) from baseline to week 18, regardless of adherence to treatment;

(25) standardized scores for apheresis treatments during the 4-week period from week 7 to week 10, respectively, as defined for the primary efficacy endpoints;

(26) standardized scores for apheresis treatment procedures during the 4-week period from week 11 to week 14, defined respectively as primary efficacy endpoints;

- 23 041556 (27) a comprehensive assessment of the frequency of apheresis and LDL-C values for a 12-week period from the beginning of week 7 to the end of week 18 (patients were ranked according to their LDL-C values and apheresis score); determine the average rank for all patients and the difference in the level of LDL-C for each of the treated patients or the apheresis score from the average rank for this variable is expressed as a percentage; the percentage difference for the 2 variables based on the data for each patient is added to provide the percentage difference from the sum of the factors. When combined and analyzed for the treatment group, the resulting number will indicate a low LDL-C value and/or a reduced apheresis rate);

(28) proportion of patients achieving LDL-C levels <200 mg/dl, <130 mg/dl, <100 mg/dl, and <70 mg/dl before apheresis at week 6, regardless of treatment adherence;

(29) proportion of patients achieving LDL-C levels <200 mg/dl, <130 mg/dl, <100 mg/dl, and <70 mg/dl before apheresis at week 18, regardless of adherence to treatment;

(30) baseline and percentage and absolute change in LDL-C and other lipids from baseline to both time points at each visit, regardless of treatment adherence, when treated in a period of double anonymity;

(31) baseline values and percentage and absolute change in LDL-C and other lipids from baseline to each visit, regardless of adherence to treatment, during open-label treatment;

(32) safety parameters (AE [including confirmed cardiovascular events], laboratory data, vital signs, and ECG) assessed throughout the study.

Other endpoints:

(1) anti-alirocumab antibodies assessed throughout the study;

(2) percent change in highly sensitive C-reactive protein (hs-CRP) and HbA1c from baseline to weeks 18 and 6;

(3) serum concentrations of total alirocumab before and after apheresis;

(4) free and total PCSK9 levels before and after apheresis.

research procedures.

All laboratory samples were collected prior to dosing study drug. Blood samples were collected from patients on an empty stomach, if possible (for example, during (at least 10 hours of fasting), water only) for all clinical visits to obtain lipidograms. Fasting was preferred, but optional, for lipid profile samples. Alcohol consumption and smoking within the 48-hour interval or vigorous exercise within the 24-hour interval prior to blood sampling were not permitted.

Total-C, HDL-C, TG, ApoB, ApoA-1 and Lp(a) were directly measured at the central laboratory. LDL-C was calculated using the Friedewald formula. If TG values exceeded 400 mg/dL (4.52 mmol/L), then the central laboratory measured (by beta quantification method) LDL-C automatically instead of calculating it. He-HDL-C was calculated by subtracting HDLC from total-C. The ApoB/ApoA-1 ratio was calculated.

Lipidogram (fasting): Blood samples for lipidogram (total-C, TG, HDL-C and calculated LDL-C) were collected after at least a 10-hour fasting period at predetermined time points.

Special Lipidogram (Fasting): Blood samples for a special lipid profile (ApoB, ApoA-1 and Lp[a]) were collected after at least a 10 hour fasting period at predetermined time points.

Patient Feeling Questionnaire: W-BQ22 was used to assess the effect of hypercholesterolemia and treatment on well-being at set time points.

Blood pressure and heart rate: Blood pressure and heart rate were assessed at predetermined time points. Blood pressure was preferably measured in a sitting position under standardized conditions, at approximately the same time of day, on the same arm, using the same instrument (after the patient has been comfortably resting in the sitting position for at least 5 minutes) . At the first screening visit, blood pressure was measured on both arms. The arm with the highest diastolic pressure was determined at this visit, and blood pressure was measured on that arm throughout the study. This highest value was recorded in the electronic individual registration card (eCRF). Heart rate was measured during blood pressure measurement.

Medical examination: A thorough and complete medical examination was performed at predetermined time points.

Body weight and height: The body weight value was obtained for the patient dressed in underwear or very light clothing and without shoes and with an empty bladder. The same scales were preferably used throughout the study. It was recommended, if possible, to use a calibrated scale with a reference scale.

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Electrocardiogram: Electrocardiograms were taken prior to blood sampling during visits requiring blood sampling. A standard 12-lead ECG was performed at predetermined time points. A 12-lead ECG was performed after at least 10 minutes of rest and in the supine position. The electrodes were placed in the same place, as far as possible, for each ECG recording throughout the study. The ECG was interpreted on site by the investigator. Any new and/or clinically significant changes in ECG parameters were immediately rechecked for confirmation before making any decision for this patient. Any clinically significant anomaly was documented as AE/SAE, as appropriate for the specific situation. Each entry was analyzed in comparison with the entry recorded during the screening. All ECG records were saved as primary data. Heart rate was recorded by ventricular rhythm, and PR, QRS, RR and QT intervals were to be recorded.

Laboratory Testing: All laboratory samples (including PK and ADA samples) were collected after evaluations and before dosing study drug at visits that corresponded to the day of dosing.

Confirmation of cardiovascular events.

Confirmed cardiovascular events included all positively confirmed cardiovascular AEs. The categories of confirmation were as follows:

(1) death from CHD;

(2) non-fatal MI;

(3) fatal and non-fatal ischemic stroke;

(4) unstable angina requiring hospitalization;

(5) chronic heart failure requiring hospitalization;

(6) ischemia-guided coronary revascularization procedure (PCI, CABG).

Statistical analysis.

It is estimated that a sample size of 63 patients (alirocumab 42: placebo 21) could have at least 85% study power to establish a 33% difference in mean apheresis rates using a two-tailed significance level and a standard deviation tolerance of 40%.

The population for the primary efficacy analysis was the primary set of all randomized patients according to the intended treatment (ITT population), defined as all randomized patients. The population for secondary efficacy analysis was based on randomized patients who had at least one pre-apheresis calculated LDL-C value before the first dose of study drug (or before randomization if the patient did not receive any study drug), and at least at least one calculated LDL-C value in one of the analysis windows prior to apheresis up to week 6. Statistical analysis was performed when the last patient completed all efficacy evaluations at week 18. LDL-C data were analyzed post hoc using Kroon's formula to estimate the range of mean LDL values -C at weeks 6-18 (after potential withdrawal of apheresis therapy).

The standardized rate of apheresis treatments was analyzed by the treatment rate obtained during the 12-week period (week 7 to week 18) divided by the number of scheduled treatments (6 for Q2W and 12 for weekly) using an analysis-ranked analysis of covariance (ANCOVA) model. . The standardized apheresis score could range from 0 to 1, with 0 indicating that the patient missed all scheduled apheresis treatments between week 7 and week 18, and 1 indicating that the patient received all scheduled treatments. A score of 0.75 indicated that the patient received 75% of scheduled apheresis treatments (and missed 25% of scheduled treatments). Exclusions of patients from the study were taken into account according to predetermined criteria. Mean treatment difference was determined using the Hodges-Lehman score and 95% confidence intervals (CI) using the confidence interval of the Moses distribution.

A hierarchical deductive approach was used to control for Type I errors. Since the primary endpoint analysis was significant at 5% alpha, key secondary efficacy endpoints were tested sequentially.

Results.

Characteristics of the initial level.

Baseline characteristics of patients included in the study are summarized in Table 1. 1-4.

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Table 1 Patient characteristics at baseline

Characteristic Placebo (N=21) Alirocumab 150 mg Q2W (Ν=41) All (Ν=62) Age Medium (SD) 57.0 (10.5) 59.5 (9.2) 58.7 (9.7) Min: Max 27:73 41:79 27:79 Age group (years) [n(%)] <65 16 (76.2%) 25 (61.0%) 41 (66.1%) >=65 5 (23.8%) 16 (39.0%) 21 (33.9%) Gender [η(%)] Male 10 (47.6%) 26 (63.4%) 36 (58.1%) female 11 (52.4%) 15 (36.6%) 26 (41.9%) Race [n(%)] White 21 (100%) 39 (95.1%) 60 (96.8%) black or African American 0 2 (4.9%) 2 (3.2%) Asian 0 0 0 Others, mixed 0 0 0 Country [n(%)] Germany 10 (47.6%) 20 (48.8%) 30 (48.4%) USA 11 (52.4%) 21 (51.2%) 32 (51.6%) Weight (kg) Medium (SD) 86.9 (21.3) 90.0 (16.1) 88.9 (17.9) Min: Max 56:124 60:124 56:124 ΒΜΙ group (kg/m ² ) [n(%)] >=30 8 (38.1%) 23 (56.1%) 31 (50%) Table 2. LM LMT source Levels Taking statins Taking the maximum daily dose statins* T initial ur placebo (N=21) 13 (61.9%) 8 (38.1%) Aries with randomiza Alirocumab 150 mg Q2W (Ν=41) 21 (51.2%) 11 (26.8%) tions All (Ν=62) 34 (54.8%) 19 (30.6%) Any LMT other than statins 16 (76.2%) 26 (63.4%) 42 (67.7%)

*reasons for patients not taking statins or the maximum daily dose of statins: muscle symptoms, increased CPK, elevated LFTs, concomitant medications, advanced age, low BMI, impaired glucose homeostasis, regional practice, potential for cognitive impairment.

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Table 3. Treatment history with apheresis

Placebo Alirocumab 150 (N=21) mg Q2W (N=41) All (N=62) The frequency at which the subject is apheresis QW 9 (42.9%) 18 (43.9%) 27 (43.5%) Q2W 12 (57.1%) 23 (56.1%) 35 (56.5%) Time since first known apheresis treatment (years) Average (SD) 8.32 (8.11) 7.22(7.60) 7.59 (7.73) median 5, 32 4.54 4.89 Min: Max 0.5:27.3 0.5:32.9 0.5:32.9

Table 4. Lipid parameters at baseline

Lipid Parameter Placebo (N=21) Alirocumab 150 mg Q2W (N=41) All (N=62) Calculated LDL-C (mg/dl) Medium (SD) median QI:Q3 Min: Max He-HDL-C (mg/dl) 191.6 (68.9) 180.0 140.0: 240.0 81:316 175.1 (54.6) 176.0 129.0: 219.0 53:275 180.7 (59.7) 179.5 133.0: 227.0 53:316 Medium (SD) 224.5 (68.0) 210.3 (62.8) 215.1 (64.4) Min: Max 90:313 56:265 56:313 Total-C (mg/dl) Medium (SD) 272.3 (73.6) 256.9 (60.6) 262.1 (65.1) Min: Max 147:399 140:377 140:399 HDL-C (mg/dl) Medium (SD) 47.8 (17.3) 46.6 (16.2) 47.0 (16.4) Min: Max 24:98 24:108 24:108 TG (mg/dl) Medium (SD) 164.4 (61.6) 176.0 (87.2) 172.1 (79.0) Min: Max 81:308 59:393 59:393 Lipoprotein-(a) (mg/dl) Medium (SD) 45.7 (49.5) 43.0 (54.5) 43.9 (52.4) Min: Max 7:202 1.5:285 21.5:285

Seventy-six patients with heFH who underwent regular weekly or Q2W lipoprotein apheresis agreed to participate and were screened. As summarized in Table. 1-4, a total of 62 patients (age: mean ± SD age, 58.7 ± 9.7 years) were randomized. 58.1% of randomized patients were men. At the screening visit, 28 patients did not receive any statins due to tolerability, and 34 patients indicated daily statin use, with 19 of them taking the maximum daily dose. All patients had atherosclerotic disease (coronary and/or cerebrovascular and/or peripheral) and 56.5% had a family history of coronary heart disease. On average, patients underwent regular lipoprotein apheresis at

- 27 041556 course (mean ± SD) 7.6 ± 7.7 years; median (Min:Max): 4.9 (0.5:32.9) years. The frequency of apheresis was

Q2W (56.5%) and QW (43.5%). 16.1% of patients had diabetes. 1.6% of patients had chronic kidney disease. Mean baseline LDL-C was 4.7 mmol/L (180.7 mg/dL); median (Min:Max): 4.7 (1.4:8.2) mmol/L [179.5 (53.0:316.0) mg/dL]. The mean Lp(a) at baseline was

43.9 mg/dL (median [Min:Max]: 19.0 [1.5:285.0] mg/dL).

Sixty patients completed a 6-week treatment period under double anonymity (40 [97.6%] in the alirocumab group and 20 [95.2%] in the placebo group) (i.e. when the apheresis score was determined according to the established schedule patient), and 57 completed an 18-week treatment period under double anonymity (37 [90.2%] and 20 [95.2%], respectively) when the apheresis schedule was determined by previously achieved LDL-C. Of the 5 (8.1%) patients who discontinued treatment prematurely in the study, 1 (4.8%) received placebo (removed due to adverse events) and 4 (9.8%) received alirocumab (2 were removed due to adverse events). events and 1 due to poor adherence to instructions, 1 patient did not provide consent).

The mean ± standard deviation (SD) age of the population was 58.7 ± 9.7 years, 36 (58.1%) patients were male, and 60 (96.8%) were Caucasian. The median (minimum, maximum) duration of apheresis procedures before entering the study was 4.9 (0.5, 32.9) years.

Baseline characteristics were balanced between treatment groups. The mean calculated baseline LDL-C (at study entry) was 4.5 ± 1.4 mmol/L (175.1 mg/dL) in the alirocumab group and 5.0 ± 1.8 mmol/L (191, 6 mg/dl) in the placebo group (P=0.35). Twenty-seven (43.5%) patients followed the weekly apheresis schedule at baseline, and 35 (56.5%) patients followed the Q2W schedule. Mean LDL-C in patients undergoing weekly apheresis was 3.9 ± 1.3 mmol/L (151.3 ± 51.3 mg/dL) versus 5.3 ± 1.4 mmol/L ( 204.9±55.7 mg/dl) in patients undergoing Q2W apheresis. Thirty-eight (61.3%) patients had normal baseline lipoprotein (a) levels (<30 mg/dL), and 24 (38.7%) had elevated levels. Thirty-four (54.8%) patients were taking a statin at screening, 19 (55.9%) of whom received the maximum daily dose. Baseline LDL-C values were 4.0 ± 1.4 mmol/L (155.0 ± 54.6 mg/dL) among statin-treated patients compared with 5.4 ± 1.4 mmol/L (208, 0±53.2 mg/dl) in patients not taking a statin. Thirty patients (48.4%) were from Germany and 32 (51.6%) from the United States.

The mean ± SD duration of injection exposure was 17.4 ± 2.3 weeks (8.6 ± 1.3 injections) in the alirocumab group and 17.5 ± 3.1 weeks (8.4 ± 1.7 injections) in the with placebo.

Efficiency results.

The results of efficiency are summarized in table. 5-8.

Tab. 5 shows the standardized apheresis score from week 7 to week 18.

Table 5 Primary Efficacy Analysis: Standardized Score of Apheresis Treatment Procedures from Week 7 to Week 18 (ITT)

Standardized rate of procedures apheresis treatment from Weeks 7 to Week 18 Mean (SD) Median Min:Max placebo (N=21) 0.806 (0.191) 0.833 0.42:1.00 Alirocumab 150 mg Q2W (N=41) 0.128 (0.242) 0.000 0.00:1.00 Hodges-Lehman score of median difference in treatment — 0.750 95% free Moses distribution CI (0.667-0.833) p-value in comp. placebo With <,0001

Only when there was a justified omission of apheresis treatment at the treatment site, the LDLC value was considered a case where apheresis was not performed. Missing information on apheresis treatment (for any reason) from week 7 to week 18 was considered a result of the apheresis treatment performed at the visit. The standardized rate of apheresis treatments is defined as: Number of actual apheresis treatments/planned apheresis treatments (6 for Q2W or 12 for QW).

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Table 6. Standardized score for apheresis treatments from week 7 to week 18 (ITT)

Standardized Score of Apheresis Treatment Procedures Placebo (N=21) Alirocumab 150 mg Q2W (N=41) 0 0 26 (63.4%) > 0 and <= 0.25 0 7 (17.1%) > 0.25 and <= 0.5 3 (14.3%) 5 (12.2%) > 0.5 and <= 0.75 5 (23.8%) 1 (2.4%) > 0.75 and < 1 7 (33.3%) 1 (2.4%) 1 6 (28.6%) 1 (2.4%)

Only when there was a justified omission of apheresis treatment at the treatment site, the LDLC value was considered a case where apheresis was not performed. Missing information on apheresis treatment (for any reason) from week 7 to week 18 was considered a result of the apheresis treatment performed at the visit. Two patients (1 with placebo and 1 with alirocumab) discontinued study treatments before week 6, and standardized apheresis scores were administered as 1.

The primary efficacy endpoints achieved a statistically significant benefit in favor of patients treated with alirocumab, with a Hodges-Lehman estimate of the median difference in treatment versus placebo of 0.75 (95% confidence interval: 0.67-0.83).

Therefore, alirocumab-treated patients had a 0.75 (75%) additional reduction in the standardized rate of apheresis treatments compared to placebo-treated patients (P<0.0001). The median difference in treatment for a standardized score of apheresis treatments from week 7 to week 18, when apheresis treatment outcome was determined by previously achieved LDL-C concentration, was 0.75 (95% CI 0.58-0.92) for patients who performed apheresis weekly, and 0.67 (95% CI 0.50-1.00) for patients on a Q2W schedule, in favor of alirocumab.

The median difference in treatment for the standardized score of apheresis treatments over a 4-week period (week 15 to week 18) was 0.50 (95% CI 0.50 to 1.00; P<0.0001) in favor of alirocumab, indicating a 50% reduction in the standardized rate of apheresis treatments compared to placebo.

As shown in Table. 5, the mean rate of apheresis treatments in patients treated with alirocumab was 0.128, while the mean rate of apheresis treatments in the placebo group was 0.806. A graphical representation of the percentage reduction in apheresis treatment rate for individual patients treated with alirocumab compared to individual patients in the placebo group over a 12-week period is shown in FIG. 2. During this period, 63.4% of alirocumab-treated patients did not undergo apheresis procedures and 92.7% avoided at least half of the procedures. This means that 26 patients in the alirocumab treatment group achieved a 100% reduction in the rate of apheresis treatments. In contrast, none of the patients in the placebo group achieved a 100% reduction in the rate of apheresis treatments. These results are also reflected in table. 6 and in FIG. 3-6 illustrating the rate of apheresis treatments for patients in the placebo and alirocumab treatment groups from week 7 to week 18 of the study. The results show that for patients in whom alirocumab was added to their existing regimen, it was possible to significantly reduce the frequency of their standardized 12-week apheresis therapy by 75% compared with placebo (P<0.0001).

Tab. 7 and FIG. 7 and 8 show the effects of alirocumab treatment versus placebo in relation to changes in serum LDL-C levels during the course of the study. Overall, patients treated with alirocumab showed a mean decrease in LDL-C from baseline over 50% at week 6, while patients in the placebo group did not show any significant reduction in LDL-C.

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Table 7 Analysis of LDL-C percentage change from baseline at week 6 (before apheresis): MMRM - ITT

Percent change from baseline in calculated LDL cholesterol Placebo (N=21) Alirocumab 150 mg Q2W (N=41) Baseline (mg/dl) Medium (SD) 191.6 (68.9) 175.1 (54.6) median 180.0 176.0 Min: Max 81:316 53:275 Percent change from baseline at week 6 (%) Average LS (SE) 1.6 (3.1) -53.7 (2.3) Mean Difference LS (SE) Versus Placebo -55.3 (3.9) 95%CI (-63.1 to -47.5) p-value vs. placebo <,0001

Note: Least squares (LS) means that the standard errors (SE) and p-value are taken from the results of the MMRM (repeated measures mixed effects model) analysis. The model includes fixed categorical effects of treatment group, randomization stratum as per IVRS, time point, treatment at time point, and relationship with time stratum, and continuous fixed covariates of calculated baseline LDL-C values and calculated baseline LDL-C values at relationship with points in time. The time points in the model include timestamps twice a week from week 2 (before apheresis) to week 18 (before apheresis).

Key secondary efficacy outcomes for patients treated with placebo and patients treated with alirocumab are summarized in Table 1. 8.

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Table 8. Analysis of Key Performance Outcomes

Endpoints Result placebo Result Alirocumab Comparison R- meaning Percentage change calculated LDL-C from baseline to week 6 (Before apheresis) LS avg.: 16% LS avg.: -53.7% Diff: -55.3% < 0.0001 Rate of apheresis treatments from week 15 to week 18 Median: 1,000 Median: 0,000 Diff*: 0.500 < 0.0001 Percent change in Apo B from baseline to week 6 (Before apheresis) LS avg.: 1.2% LS Mean: 42.8% Diff: -44.0% < 0.0001 Percentage non-HDL-C change from baseline to week 6 (Before apheresis) LS avg.: 2.8% LS Mean: 47.1% Diff: -50.0% < 0.0001 Percent Change in Total-C from Baseline to Week 6 (Before Apheresis) LS avg.: 3.1% LS avg:36.4% Diff: -39.4% < 0.0001 Haro A-1 Percent change from LS avg.: 0.0% LS avg.: 4.2% Diff: 4.2% 0.3012

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baseline to week 6 (Before apheresis) > 30% reduction in LDL- WITH from baseline to Weeks 6 (Before apheresis) Share: 4.8% LS avg.: 95.1% Rel. risks 144.4 < 0.0001 > 50% reduction in LDL- From baseline to Week 6 (Before apheresis) Share: 0 LS avg.: 63.4% Rel. risks: 48.1 0.0006 Percent change in calculated LDL-C from baseline to Week 18 (Before apheresis) LS avg.: 3.9% LS avg.: -42.5% Diff: -46.4% < 0.0001 Percentage Apo B change from baseline to Week 18 (Before apheresis) LS avg.: 1.8% LS average: 33.5% Diff: -35.2% < 0.0001 Percentage non-HDL-C change from baseline to Week 18 (Before apheresis) LS avg.: 4.7% LS avg:45.8% Diff: -40.4% < 0.0001 Percent Change in Total-C from Baseline to Week 18 (Before Apheresis) LS avg.: 4.6% LS Mean: 27.3% Diff: -31.9% < 0.0001 Percentage LS avg.: LS avg.: Diff: 0.0404

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change in Aro A-1 from baseline to Week 18 (Before apheresis) -0.7% 8.1% 8.8% > 30% reduction in LDL- C from original up to Weeks 18 (Before apheresis) Share: 0 LS avg.: 65.9% Rel. risks: 39.8 < 0.0001 > 50% reduction in LDL- C from original up to Weeks 18 (Before apheresis) Share: 0 LS avg.: 43.9% Rel. risks: 18.8 < 0.0001 W-BQ22 (Feeling Score) Change from Baseline to Week 18 LS avg.: -0.34 LS avg.: -0.86 Diff: -0.52 0.6887 Percentage change in Lp(a) from baseline to Week 6 (Before apheresis) LS avg.: -4.0% LS Mean: 18.0% Diff: -14.1% 0.0262 Percentage HDL-C change from baseline to week 6 (Before apheresis) LS Mean: 4.0% LS avg.: 9.2% Diff: 5.2% 0.2168 Percentage change in TG from baseline to week 6 (Before apheresis) LS avg.: thirty% LS avg.: -12.9% Diff: -15.8% 0.0204 Percentage LS avg.: LS avg.: Diff: 0.6805 change in Lp(a) from baseline to week 18 (Before apheresis) -0.9% -50% -4.1% Percentage HDL-C change from baseline to week 18 (Before apheresis) LS avg.: 2.5% LS avg.: 10.5% Diff: 8.0% 0.2022 Percentage change in TG from baseline to week 18 (Before apheresis) LS avg.: 4.2% LS avg.: -1.4% Diff: -5.6% 0.5770

Mean pre-apheresis LDL-C decreased from 4.5 mmol/L (175 mg/dL) at baseline to 2.3 mmol/L (90 mg/dL) at week 6 in the alirocumab group; relevant data for patients in

- 33 041556 placebo group were 5.0 mmol/l (192 mg/dl) and 4.8 mmol/l (185 mg/dl) (Fig. 2). LS mean±SE (95% CI) percent change in pre-apheresis LDL-C from baseline at week 6 was -53.7±2.3 (-58.2 to -49.2) in the alirocumab and 1.6 ± 3.1 (-4.7 to 7.9) in the placebo group (LS mean ± SE percentage difference -55.3 ± 3.9, 95% CI -63.1 to -47, 5; P<0.0001). By week 18, mean LDL-C in the alirocumab group increased slightly to 2.9 mmol/L (110 mg/dL) compared to 4.9 mmol/L (191 mg/dL) in the placebo group ( LS mean ± SE percent difference -46.4 ± 7.9, 95% CI -62.3 to -30.5; P<0.0001).

Cross-validation, comparing LDL-C values at the treatment site with values from the central laboratory, showed that both measurements had a high degree of correlation (Pearson's correlation 0.86).

The percentage change in lipoprotein (a) from baseline to week 18 was -5.7% for alirocumab versus -3.0% for placebo among patients with normal baseline values, and 4.9% versus 7.6% , respectively, in patients with elevated baseline values. During weeks 7-18, when apheresis treatment could be stopped, alirocumab treatment was associated with a lower ± SD time-averaged LDL-C value (using Kroon's 18 formula) over a (potential) apheresis interval of 2.4 ± 1.3 mmol/L (92.7±50.2 mg/dL) vs 3.8±1.7 mmol/L (146.7±65.6 mg/dL) for placebo (P<0, 0001).

In post hoc analysis, time-averaged LDL-C values in alirocumab-treated patients were consistently lower than those in placebo-treated patients.

Safety.

75.6% of patients in the alirocumab group and 76.2% of patients in the placebo group reported TEAE, none of which was fatal. The rates of serious adverse events (9.8% for alirocumab and 9.5% for placebo) and discontinuation events (4.9% and 4.8%, respectively) were also similar in both groups.

Three patients (7.3%) in the alirocumab and group had two consecutive pre-apheresis LDL-C values <0.7 mmol/L (25 mg/dL), and no patient in the placebo group had such values. Two of these patients had at least one adverse event, one of which had multiple serious adverse events (pneumonia, acute myocardial infarction, acute respiratory failure, congestive heart failure, sepsis, and aortic valve stenosis). Twenty-seven patients (23 [56.1%] treated with alirocumab and 4 [19.0%] treated with placebo) had two consecutive LDL-C values <0.7 mmol/L after apheresis, of which 15 (65.2 %) and 2 (50.0%), respectively, had an adverse event. None of the events were serious, but one patient (4.3%) stopped treatment with alirocumab.

Generalized results and conclusions.

The present study shows that patients treated with alirocumab exhibited a reduced rate of apheresis treatments and a reduced number of apheresis treatments during the study, compared with patients treated with placebo. In particular, treatment with alirocumab resulted in a higher reduction in the rate of apheresis treatments compared with placebo, with a median estimate of 0.75 or 75% (p<0.0001).

The present study also demonstrated that 63.4% of patients treated with alirocumab did not undergo apheresis compared with 0% of patients treated with placebo (i.e., none of the patients in the placebo group could completely avoid apheresis treatments, in while for 63.4% of patients in the alirocumab treatment group it was possible to exclude the apheresis treatment procedure along with subsequent alirocumab treatment). In addition, 92.7% of patients treated with alirocumab had at least a 50% reduction in apheresis rates compared with 14.3% for patients treated with placebo. Moreover, alirocumab reduced LDL-C by 55% (compared to RVO) at week 6 from 175 to 89.5 mg/dl. Significant reductions in apoB, non-HDL-C, and TC were also observed in patients treated with alirocumab. Alirocumab treatment reduced the standardized rate of apheresis treatments from week 15 to week 18.

From a safety standpoint, subcutaneous alirocumab in HeFH patients treated with LDL apheresis once every 1 or 2 weeks was generally safe and well tolerated. The number of patients reporting overall TEAE and AESI was comparable across the treatment groups.

All patients in this study were at high risk for cardiovascular disease and had previously received LLT, including statins. At screening, only 54.8% of patients were taking statins, 55.9% of whom were receiving the maximum tolerated dose. A large proportion of the total population reported dose reduction with a history of statin treatment due to tolerability problems (43.5%), and 62.9% reported switching between different statins. Various reasons have been given for not taking statins or not taking them at the maximum daily dose, from muscle symptoms to concerns about side effects, and regional practices/local labeling, indicating that heFH patients receiving apheresis present a diverse and intractable population with limited therapeutic options.

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Patients in the US showed characteristics different from those in Germany, with higher baseline LDL-C, higher incidence of statin intolerance, and less frequent apheresis regimen. In the US, lipoprotein apheresis in the case of heFH is often considered acceptable only for those patients who at 6 months do not have an adequate response to maximally tolerated drug therapy and have elevated LDL-C with the addition of other cardiovascular risk factors. In Germany, where apheresis centers are more common, apheresis is considered necessary when there is no successful outcome from diet and LLT within 12 months, and a lower LDL-C threshold, and a weekly regimen is preferred over Q2W apheresis. In addition, recommendations for lowering LDL-C from Europe are based on a target-adjusted, patient-at-risk approach, while guidelines in the US advocate a dose-adapted approach. Given the approach recognized in the European guidelines, with an LDL-C target of <1.8 mmol/L (70 mg/dL), German patients receiving alirocumab with an LDL-C above this target would still be eligible. for apheresis. Therefore, LLT with alirocumab may be an adjunct to lipoprotein apheresis in patients with very high LDL-C or patients who cannot meet the LDL-C target.

In conclusion, this study achieved a significant reduction in the primary efficacy endpoints and demonstrated that PCSK9 inhibitors such as alirocumab are an effective therapeutic option to reduce or eliminate the patient's need for lipoprotein apheresis therapy or to delay the requirement for such therapies.

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

Claims (16)

1. A method of eliminating a patient's need for lipoprotein apheresis therapy or reducing the frequency of lipoprotein apheresis therapy required for a patient to achieve a target lipoprotein level, wherein the method includes selecting a patient with hypercholesterolemia who is or has been treated with lipoprotein apheresis from an initial (for preliminary treatment) frequency, and administering one or more doses of a PCSK9 inhibitor to a patient, thereby lowering at least one serum lipoprotein level in the patient and eliminating the patient's need for lipoprotein apheresis therapy or reducing the frequency of lipoprotein apheresis required by the patient to achieve a target lipoprotein level, wherein the target lipoprotein level is a serum LDL-C level of less than 200 mg/dl, less than 130 mg/dl, less than 100 mg/dl, or less than 70 mg/dl, wherein the PCSK9 inhibitor is an antibody or antigen-binding fragment thereof that specifically connection are identified with PCSK9, wherein said antibody or antigen-binding fragment thereof comprises heavy and light chain variable heavy/light chain variable region (HCVR/LCVR) complementarity determining regions (CDRs) from an amino acid sequence pair containing SEQ ID NO: 1/6. 2. The method according to claim 1, where the initial (for pre-treatment) frequency of apheresis is once a week or once every two weeks. 3. The method of claim 1 or 2, wherein the frequency of apheresis following administration of one or more doses of the PCSK9 inhibitor is once every three weeks, once every four weeks, once every five weeks, or less than once every five weeks . 4. The method of claim 1 or 2, wherein after administration of one or more doses of the PCSK9 inhibitor, the patient no longer requires apheresis to maintain the target lipoprotein level. 5. The method of claim 1, wherein the lipoprotein apheresis therapy is selected from the group consisting of: cascade filtration, immunoadsorption, heparin-induced LDL precipitation, LDL adsorption (dextran sulfate) by liposorber, LDL hemoperfusion, and LDL hemoperfusion (liposorber D). 6. The method of claim 1, wherein the patient, prior to or during treatment with one or more doses of the PCSK9 inhibitor, undergoes lipoprotein apheresis on a stable schedule at an initial (pre-treatment) frequency for at least 2 weeks prior to the first dose of the inhibitor PCSK9. 7. The method of claim 1 wherein the patient is receiving baseline stable lipid modifying therapy (LMT) prior to administration of one or more doses of a PCSK9 inhibitor. 8. The method of claim 1, wherein the patient is receiving stable baseline lipid-modifying therapy (LMT) coinciding with administration of one or more doses of a PCSK9 inhibitor. 9. The method of claim 7 or 8, wherein the baseline stable LMT is low, moderate, or high dose statin therapy. 10. The method according to claim 1, where the lipoprotein, which is reduced in the patient's serum after administration of one - 35 041556 or more doses of a PCSK9 inhibitor is one or more lipoproteins selected from the group consisting of LDL-C, ApoB, non-HDL-C, total cholesterol and Lp(a). 11. The method of claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds to PCSK9 is administered to the patient at a dose of about 75 mg once every two weeks. 12. The method of claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds to PCSK9 is administered to the patient at a dose of about 150 mg once every two weeks. 13. The method of claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds to PCSK9 is administered to the patient at a dose of about 300 mg once every four weeks. 14. The method of any one of claims 11-13, wherein the antibody or antigen-binding fragment thereof is alirocumab. 15. The method of claim 1, wherein the antibody or antigen-binding fragment thereof comprises heavy and light chain CDR amino acid sequences having SEQ ID NOS: 2, 3, 4, 7, 8 and 10. 16. The method of claim 15, wherein the antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO:1 and an LCVR having the amino acid sequence of SEQ ID NO:6.