Anti-PCSK9 inhibitor antibodies for treating hyperlipidemic patients undergoing lipoprotein apheresis

Abstract

To provide methods for reducing or eliminating a patient's need for lipoprotein apheresis therapy.SOLUTION: The methods comprise administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor. In certain embodiments, the PCSK9 inhibitor is an anti-PCSK9 antibody. The methods are useful in treating patients with hyperlipidemia and related conditions who are currently being treated with a therapeutic regimen comprising lipoprotein apheresis (e.g., LDL apheresis or Lp(a) apheresis).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of therapeutic treatment of diseases and disorders associated with high levels of lipids and lipoproteins. More specifically, the present invention relates to the use of PCSK9 inhibitors to treat patients with hyperlipidemia and related conditions who are currently being treated with therapeutic regimens that include lipoprotein apheresis (e.g., LDL apheresis or Lp(a) apheresis). [Background technology]

[0002] Hyperlipidemia is a general term that encompasses diseases and disorders characterized by or associated with elevated levels of lipids and / or lipoproteins in the blood. Hyperlipidemia includes hypercholesterolemia, hypertriglyceridemia, and the combination of hyperlipidemia and hyperlipoprotein a (Lp(a)). A particularly prevalent form of hyperlipidemia in many populations is hypercholesterolemia. A particular form of hyperlipidemia that is prevalent in many populations is hypercholesterolemia.

[0003] Hypercholesterolemia, particularly elevated low-density lipoprotein (LDL) cholesterol (LDL-C) levels, constitutes a major risk factor for the development of atherosclerosis and coronary heart disease (CHD) (Non-Patent Document 1). Low-density lipoprotein cholesterol has been identified as a primary target for cholesterol-lowering therapy and is generally accepted as a valid surrogate therapeutic endpoint. Numerous studies have demonstrated that lowering LDL-C levels reduces CHD risk, with a direct and strong relationship between LDL-C levels and CHD events: a 22% reduction in cardiovascular disease (CVD) mortality and morbidity for each 1 mmol / L (approximately 40 mg / dL) reduction in LDL-C. Greater reductions in LDL-C produce greater reductions in events, and intensive comparative data with standard statin therapy suggest that lower LDL-C levels result in greater benefit for patients at very high cardiovascular (CV) risk.

[0004] Familial hypercholesterolemia (FH) is an inborn error of lipid metabolism that predisposes individuals to cardiovascular disease (CVD) at an early age. Defects in at least three distinct genes encoding proteins involved in the hepatic clearance of low-density lipoprotein (LDL) cholesterol (LDL-C) can cause FH. Examples of such defects include mutations in the gene encoding the LDL receptor (LDLR), which removes LDL-C from the circulation, and the gene encoding apolipoprotein (Apo) B, the major protein in LDL particles. In some cases of FH, the gene encoding proprotein convertase subtilisin / kexin type 9 (PCSK9), an enzyme involved in the degradation of LDLR (gain of function mutation), is mutated. In all cases, FH is characterized by the accumulation of LDL-C in plasma from birth and the subsequent development of tendon xanthomas, xanthelasmas, atheromas, and CVD. FH can be classified as either heterozygous FH (heFH) or homozygous FH (hFH), depending on whether the individual has a genetic defect in one (heterozygous) or both (homozygous) copies of the relevant gene.

[0005] Current LDL-C-lowering medications include statins, cholesterol absorption inhibitors, fibrates, niacin, and bile acid sequestrants. Statins are a commonly prescribed treatment for lowering LDL-C. However, despite the availability of such lipid-lowering therapies, many high-risk patients do not achieve their guideline-targeted LDL-C levels. (Non-Patent Document 2). For patients who still cannot achieve guideline target levels of LDL-C despite available lipid-modifying therapies (LMT), mechanical removal of LDL-C by lipoprotein apheresis (e.g., LDL apheresis) is sometimes prescribed. Lipoprotein apheresis removes apoprotein B100-containing lipoproteins from the blood. It is generally regarded as a last resort for patients with advanced cardiovascular disease and persistently elevated LDL-C.

[0006] However, LDL apheresis is an invasive, burdensome, and expensive procedure for the patient. Apheresis generally involves mechanical removal of blood from the patient; the blood is centrifuged, filtered, or otherwise separated outside the body to remove undesirable components, and then reintroduced into the patient. Lipoprotein apheresis acutely reduces LDL-C concentrations by 50-75%, which translates to an average LDL-C reduction of approximately 30% over the time between apheresis sessions. The typical apheresis process is characterized by a transient decrease in serum lipoprotein concentrations, followed by a nearly linear return of lipoprotein levels to elevated "baseline" levels over the following period. This pattern of fluctuation in lipoprotein levels, characteristic of lipoprotein apheresis therapy, explains the need for periodic apheresis treatments throughout an individual's lifetime. Furthermore, because many apheresis facilities are geographically scattered, many patients must travel significant distances for this procedure, which typically requires administration over three hours and every week to four weeks, depending on the patient's LDL-C level and cardiovascular risk. Furthermore, this procedure requires the placement of a shunt for frequent vascular access. While generally well tolerated, low-density lipoprotein apheresis can result in hypotension, hypocalcemia, allergic reactions, and a rapid decline in serum protein levels. Compared with patients treated with lipid-lowering drugs alone, patients receiving apheresis in addition to lipid-lowering drugs have been reported to have lower quality of life (QoL) (QoL determined by questionnaire) (Non-Patent Document 3). Therefore, patients who are not achieving LDL-C goals despite optimized LMT therapy and who require apheresis to lower their LDL-C would greatly benefit from alternative LDL-C-lowering therapies that could reduce or eliminate the need for apheresis. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sharrett et al., 2001, Circulation 104:1108-1113 [Non-patent document 2] Gitt et al., 2010, Clin Res Cardiol 99(11):723-733 [Non-patent document 3] Schiel et al., 1995, Int J Artif Organs 18:786-793 Summary of the Invention [Means for solving the problem]

[0008] The present invention provides methods for treating hyperlipidemia in patients undergoing lipoprotein apheresis therapy. The treatment methods of the present invention result in a reduction in serum lipoprotein levels, thereby helping to eliminate or reduce the patient's need for lipoprotein apheresis. In certain embodiments, the frequency of apheresis treatments required by a patient to achieve a target lipoprotein level (e.g., a target LDL-C level) is reduced by administering the treatment methods of the present invention. In certain embodiments, administering the treatment methods of the present invention to a patient eliminates the need for apheresis to achieve a target lipoprotein level (e.g., a target LDL-C level).

[0009] According to one embodiment, the methods of the invention comprise administering one or more doses of a PCSK9 inhibitor to a patient who is being treated, or has been treated (within the last six months) with lipoprotein apheresis, wherein administration of the PCSK9 inhibitor to the patient results in a reduction in the level of at least one lipoprotein in the patient's serum, thereby reducing or eliminating the patient's need for lipoprotein apheresis treatment.

[0010] According to another aspect, the methods of the invention involve selecting a hypercholesterolemic patient who is or has been treated with an initial (pre-treatment) frequency of lipoprotein apheresis and administering one or more PCSK9 inhibitors, thereby reducing the level of at least one lipoprotein in the patient's serum and, as a result, reducing the frequency of lipoprotein apheresis required by the patient to achieve a target lipoprotein level.

[0011] Patients who may be treated or treatable by the methods of the invention include, for example, patients with hypercholesterolemia, including patients with familial hypercholesterolemia (FH). In certain embodiments, patients who may be treated or treatable by the methods of the invention are patients who have been diagnosed with (or are otherwise known to have) homozygous FH (hoFH) or heterozygous FH (heFH).

[0012] According to certain embodiments of the present invention, the PCSK9 inhibitor is administered to the patient as add-on therapy to the patient's existing lipid-lowering therapy (e.g., on top of the patient's background statin therapy).

[0013] The present invention also provides pharmaceutical compositions comprising a PCSK9 inhibitor for use to reduce or eliminate the need for lipoprotein apheresis treatment, or to reduce the frequency of lipoprotein apheresis treatment by a patient.

[0014] Representative PCSK9 inhibitors that can be used in accordance with the methods of the present invention include, for example, anti-PCSK9 antibodies, small molecule PCSK9 inhibitors, and scaffold-based PCSK9 binding molecules.

[0015] Other embodiments of the present invention will become apparent from a review of the detailed description that follows. [Brief explanation of the drawings]

[0016] [Figure 1]Figure 1 shows the overall design of the study described herein in Example 2. The study includes a 2-week screening period, an 18-week double-blind treatment period, and an optional 8-week follow-up / open-label extension period. [Figure 2] Figure 2 is a waterfall plot showing the percentage of individual patient declines in apheresis treatment from Week 7 to Week 18 in the placebo- and alirocumab-treated patient groups (ITT population). Only LDL-C values ​​from missed apheresis treatments at the point of care are counted as "no apheresis occurring." Missing apheresis treatment information (for any reason) from Week 7 to Week 18 is attributed to the apheresis treatment that "occurred" at that visit. [Figure 3] Figure 3 shows normalized apheresis rates from Weeks 7 to 18 in placebo- and alirocumab-treated patient groups. Apheresis rates are shown along the x-axis. The y-axis shows the percentage of patients exhibiting the corresponding apheresis rate range. [Figure 4] Figure 4 shows the proportion of placebo- and alirocumab-treated patients who underwent apheresis at various time points in the study, from Week 7 to Week 18. Patients shown in this table were receiving weekly (QW) apheresis at the start of the study. [Figure 5] Figure 5 shows the proportion of placebo- and alirocumab-treated patients who underwent apheresis at various time points in the study, from Week 7 to Week 18. Patients shown in this table were receiving apheresis every 2 weeks (Q2W) at the start of the study. [Figure 6] Figure 6 shows the percentage of placebo- and alirocumab-treated patients who underwent apheresis at various time points in the study, from Week 7 to Week 18. The patients shown in this table represent all patients enrolled in the study. [Figure 7]Figure 7 shows the calculated percent change in LDL-C from baseline in the placebo- and alirocumab-treated patient groups at various time points throughout the study design. The black arrow labeled "Apheresis Variation" indicates the time point at which apheresis frequency was advanced for individual patients based on their LDL-C levels. Prior to this time point, patients underwent apheresis at a consistent frequency based on the schedule established for each patient. [Figure 8] Figure 8 shows the calculated LDL-C levels (expressed as LS mean (+ / - SE) mg / dL) in the placebo- and alirocumab-treated patient groups at various time points during the study. The black arrow labeled "Apheresis Variation" indicates the time point at which apheresis frequency was advanced for individual patients based on their LDL-C levels. Prior to this time point, patients underwent apheresis at a consistent frequency based on the schedule established for each patient. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention Before the present invention is described, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is intended to be limited only by the appended claims.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. As used herein, the term "about," when used in connection with a specific recited numerical value, means that the value may vary by 1% or less from the recited value. For example, the expression "about 100" used herein includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0019] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are now described below. All publications mentioned herein are incorporated by reference in their entirety.

[0020] Methods for reducing or eliminating the need for lipoprotein apheresis The present invention generally relates to methods and compositions for reducing lipoprotein levels in patients who are undergoing or have undergone (e.g., within the last six months or more) lipoprotein apheresis to lower serum lipoprotein levels (e.g., LDL-C and / or Lp(a)). According to certain embodiments, the methods of the invention result in a reduction of lipoprotein levels in the serum of such patients, thereby reducing or eliminating the need for lipoprotein apheresis.

[0021] As used herein, the term "lipoprotein" refers to 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 and lipoprotein(a) (Lp(a)).

[0022] As used in the context of the present invention, "lipoprotein apheresis" refers to a therapeutic process involving the mechanical removal of blood from a patient, followed by 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 into the patient's bloodstream. For purposes of this disclosure, "LDL apheresis" and "Lp(a) apheresis" are considered types of apheresis and are therefore encompassed within the more general definition of "lipoprotein apheresis."

[0023] Specific types of lipoprotein apheresis encompassed within the scope of the present invention include, for example, double membrane filtration, immunoadsorption, heparin-induced LDL precipitation, direct lipid adsorption, dextran sulfate-cellulose adsorption (plasma or whole blood), heparin extracorporeal LDL precipitation (HELP) system, DFPP and thermofiltration plasma exchange, and hemoperfusion.

[0024] According to certain embodiments, the present invention includes methods for eliminating the need for lipoprotein apheresis therapy. As used herein, a particular patient's "need for lipoprotein apheresis therapy" is determined by a physician, physician's assistant, diagnostician, or medical professional based on the level of one or more lipoproteins (e.g., LDL-C and / or Lp(a)) measured or detected in the patient's serum. A patient's "need for lipoprotein apheresis therapy" may also be determined or influenced by other factors, such as the patient's family history, medical background, current medical therapy status, and generally accepted or prevalent target lipoproteins adopted by national medical associations and physician groups. For example, in certain situations, an LDL-C level equal to or greater than about 70 mg / dL indicates a patient's "need for lipoprotein apheresis therapy." In other situations, an LDL-C level equal to or greater than about 100 mg / dL indicates a patient's "need for lipoprotein apheresis therapy." In certain circumstances, an LDL-C level equal to or greater than about 150 mg / dL, 200 mg / dL, 250 mg / dL, 300 mg / dL, or 400 mg / dL indicates a patient's "need for lipoprotein apheresis therapy." Additionally, in other aspects, a relative comparison to a patient's LDL-C or Lp(a) level at a particular starting point, "baseline," may be used to determine whether a patient has a "need for lipoprotein apheresis therapy," regardless of whether the reduction in LDL-C or Lp(a) levels meets a particular percentage. For example, a reduction in LDL-C or Lp(a) of less than 50% from baseline (e.g., less than 40%, less than 35%, less than 30%, less than 25%, etc.) indicates a "need for lipoprotein apheresis therapy."

[0025] According to certain embodiments, the present invention includes a method for reducing the frequency of lipoprotein apheresis therapy in a patient. As will be appreciated by those skilled in the art, a patient may be treated with lipoprotein apheresis therapy at a specific frequency to achieve a specific lipoprotein target (e.g., an LDL-C level of less than 100 mg / dL or an LDL-C level of less than 70 mg / dL). The predetermined frequency is determined based on the number of apheresis treatments the patient needs in a specific period (e.g., weekly, monthly, etc.) to achieve and maintain the lipoprotein target. Exemplary lipoprotein apheresis frequencies include, for example, once weekly, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once per month, twice per month, once per two months, etc. The present invention includes a method 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 certain embodiments of the invention, the frequency of apheresis after administration of one or more PCSK9 inhibitors is reduced by at least 50% from the patient's pre-treatment apheresis frequency. For example, if the patient, prior to treatment with a PCSK9 inhibitor, had a lipoprotein-associated vasoconstriction (LPA) of 100 mg / kg, the patient's apheresis frequency is reduced by at least 50% from the patient's pre-treatment apheresis frequency. If a patient's lipoprotein apheresis treatment regimen is once weekly (QW) and the apheresis frequency for that patient is reduced to once every two weeks (Q2W) after treatment with a PCSK9 inhibitor, the patient is said to have achieved a 50% reduction in lipoprotein apheresis frequency after treatment. In certain embodiments, the frequency of apheresis after one or more doses of a PCSK9 inhibitor according to the present invention is reduced by 75% or 100% (i.e., eliminating the need for lipoprotein apheresis after treatment).

[0026] In the context of the present invention, lipoprotein apheresis frequency may be expressed in terms of pre-treatment frequency and post-treatment frequency. "Pre-treatment frequency" refers to the frequency of apheresis therapy required by a patient to achieve and / or maintain a specific target lipoprotein level before initiation of a therapeutic regimen including 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 initiation of a therapeutic regimen including administration of a PCSK9 inhibitor. The frequency of apheresis therapy required for a particular patient to achieve and / or maintain a specific target lipoprotein level is preferably determined by a qualified medical professional based on criteria universally accepted in the art, including the serum level of the lipoprotein desired to be reduced or controlled.

[0027] Thus, the present invention provides a method of treatment comprising administering one or more doses of a PCSK9 inhibitor to a patient, wherein the patient's post-treatment frequency of lipoprotein apheresis is less than the patient's pre-treatment frequency of lipoprotein apheresis. For example, the present invention includes a method of treatment comprising administering one or more doses of a PCSK9 inhibitor to a patient undergoing or having undergone lipoprotein apheresis therapy at a pre-treatment frequency, e.g., once per week, wherein, after receiving one or more doses of the PCSK9 inhibitor, the patient requires lipoprotein apheresis less frequently to achieve and / or maintain a specific target lipoprotein level (i.e., post-treatment frequency), e.g., once every two weeks, once every three weeks, or once every four weeks, or less frequently. In some examples, the patient's need for lipoprotein apheresis to achieve and / or maintain a specific target lipoprotein level is completely eliminated after administering one or more doses of a PCSK9 inhibitor.

[0028] According to certain embodiments, the present invention provides a method for reducing or eliminating the need for lipoprotein apheresis treatment, comprising administering one or more doses of a PCSK9 inhibitor to a patient with hyperlipidemia (e.g., hypercholesterolemia) who has been treated with lipoprotein apheresis for the last month, the last two months, the last three months, the last four months, the last five months, the last six months, or for a longer period of time. The method according to this aspect of the present invention reduces the level of at least one lipoprotein in the patient's serum, thereby reducing or eliminating the patient's need for lipoprotein apheresis therapy. For example, in certain embodiments of the present invention, after administration of one or more doses of a PCSK9 inhibitor, the patient's serum LDL-C level is reduced to below a predetermined level (e.g., below 100 mg / dL or below 70 mg / dL) such that the patient requires a reduced post-treatment frequency of lipoprotein apheresis therapy to achieve and / or maintain a specific target lipoprotein level, or such that it is concluded that lipoprotein apheresis is no longer necessary.

[0029] In certain embodiments, the rate (or frequency) of apheresis is expressed as the normalized rate of apheresis required for a patient to achieve and / or maintain a particular target lipoprotein level. As used herein, a particular patient's normalized rate of apheresis refers to the ratio of the actual number of apheresis treatments a patient receives over a defined period of time after initiation of an anti-PCSK9 treatment regimen to the number of apheresis treatments a patient receives over a comparable period of time prior to initiation of an anti-PCSK9 treatment regimen. The normalized apheresis ratio is defined as the number of apheresis treatments a patient has received divided by the number of apheresis treatments the patient has received. For example, if a patient receives eight apheresis treatments over an eight-week period (e.g., once per week) before starting a treatment regimen including administration of an anti-PCSK9 antibody, and the patient receives two apheresis treatments over an eight-week period (e.g., once every four weeks) after starting the anti-PCSK9 treatment regimen, the patient's normalized apheresis ratio is 2 / 8 = 0.25. The invention includes methods in which the patient's normalized apheresis ratio 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.

[0030] According to certain embodiments, patients treatable by the methods of the invention have hypercholesterolemia (e.g., a serum LDL-C level of 70 mg / dL or greater, or a serum LDL-C level of 100 mg / dL or greater). In certain embodiments, the patient's hypercholesterolemia is inadequately controlled with statin therapy. For example, the invention is a method of reducing or eliminating the frequency of lipoprotein apheresis therapy by a patient having hypercholesterolemia inadequately controlled with a daily dose of a statin selected from the group consisting of atorvastatin (including atorvastatin plus ezetimibe), rosuvastatin, cerivastatin, pitavastatin, fluvastatin, lovastatin, simvastatin (including simvastatin plus ezetimibe), pravastatin, and combinations thereof. The present invention also includes methods for reducing or eliminating the frequency of lipoprotein apheresis therapy by patients who have hypercholesterolemia and exhibit statin intolerance or who experience other or undesirable reactions to statin therapy (e.g., muscle skeletal pain, pain, weakness, or cramps (e.g., myalgia, myopathy, rhabdomyolysis, etc.)).

[0031] Treatment of lipid lesions The present invention further relates to methods and compositions for treating, reversing, or ameliorating physical symptoms of hypercholesterolemia. According to certain embodiments, the present invention provides a method for treating a patient with lipid lesions associated with hypercholesterolemia. For example, the present invention provides a method for treating a patient with one or more xanthelasmata. The methods and compositions according to this aspect of the invention comprise administering one or more doses of a PCSK9 inhibitor to a patient in need thereof, wherein lipid lesions present in the patient prior to treatment with the PCSK9 inhibitor are reduced, reversed, or eliminated after administering one or more doses of the PCSK9 inhibitor to the patient. According to certain embodiments, the present invention provides a method for treating a patient with lipid lesions associated with hypercholesterolemia, comprising selecting a patient with lipid lesions (e.g., xanthelasmas) and administering one or more doses of a PCSK9 inhibitor to the patient.

[0032] Patient selection The present invention includes methods and compositions useful for treating patients who are undergoing or have recently undergone lipoprotein apheresis (e.g., within the last 6 months, within the last 12 weeks, within the last 8 weeks, within the last 6 weeks, within the last 4 weeks, within the last 2 weeks, etc.). Patients treatable by the methods of the invention may also exhibit one or more additional selection criteria. For example, patients may be selected for treatment by the methods of the invention if they have been diagnosed or identified as at risk for developing a hypercholesterolemic condition, such as heterozygous familial hypercholesterolemia (heFH), homozygous familial hypercholesterolemia (HIFH), 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 mutations in LDLRAP1), and development of hypercholesterolemia distinct from familial hypercholesterolemia (non-FH). Diagnosis of familial hypercholesterolemia (e.g., heFH or hoFH) can be made by genotyping and / or clinical criteria. In patients whose genotypes have not been determined, the clinical diagnosis is unclear. The criteria can be based on either the Simon Broome criteria with definite FH criteria or the WHO / Dutch Lipid Network criteria with a score >8 points.

[0033] According to certain embodiments, patients may be selected based on having a medical history of coronary heart disease (CHD). As used herein, "medical history of CHD" (or "previous CHD history") includes one or more of the following: (i) acute myocardial infarction (MI), (ii) asymptomatic MI, (iii) unstable angina, (iv) coronary revascularization (e.g., percutaneous coronary intervention (PCI) or coronary artery bypass graft surgery (CABG)), and / or (v) clinically significant CHD diagnosed by invasive or non-invasive testing (e.g., coronary angiography, treadmill stress testing, stress echocardiography, or nuclear imaging).

[0034] According to certain embodiments, patients may be selected based on having non-coronary heart disease cardiovascular disease ("non-CHD CVD"), as used herein, including one or more of the following: (i) a previous history of ischemic stroke believed to be of atherothrombotic origin and with focal ischemic neurological deficit lasting more than 24 hours; (ii) peripheral artery disease; (iii) abdominal aortic aneurysm; (iv) atherosclerotic renal artery stenosis; and / or (v) carotid artery disease (transient ischemic attack or greater than 50% occlusion of a carotid artery).

[0035] According to certain embodiments, patients may be selected based on having one or more of the following additional risk factors, such as: (i) a history of moderate chronic kidney disease (CKD), as defined by an eGFR of 30≦eGFR<60 mL / mL / min / 1.73 m for 3 months or more; (ii) type 1 or type 2 diabetes mellitus, with or without target organ damage (e.g., retinopathy, nephropathy, microalbuminuria); or (iii) a calculated 10-year fatal CVD risk score of 5% or greater (ESC / EAS guidelines for the management of dyslipidemia, Conroy et al., 2003, EuHeart J. 24:987-1003).

[0036] In certain embodiments, patients may be selected based on having one or more additional risks selected from the group consisting of age (e.g., 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old), race, national origin, sex (male or female), exercise habits (e.g., regular exerciser, non-exerciser), other pre-existing medical conditions (e.g., type 2 diabetes, high blood pressure, etc.), and current medication status (e.g., current beta-blocker, niacin, ezetimibe, fibrates, omega-3 fatty acids, bile acid resins, etc.).

[0037] According to the present invention, patients may be selected based on a combination of one or more of the aforementioned selection criteria or treatment characteristics. For example, according to certain embodiments, patients suitable for treatment with the methods of the present invention are undergoing or have recently undergone lipoprotein apheresis (e.g., within the last 6 months) and also have (i) a history of CHD or (ii) a non-CHD condition. Patients may be selected based on having FH or non-FH in combination with CVD, and / or (iii) diabetes mellitus with target organ damage; such patients may also be selected based on having a serum LDL-C concentration of 70 mg / dL or greater.

[0038] According to certain other embodiments, patients suitable for treatment with the methods of the present invention may be selected based on having hypercholesterolemia that is not adequately controlled by a daily moderate-dose therapeutic statin regimen, as well as having FH or non-FH without CHD or non-CHD CVD, but either (i) a calculated 10-year fatal CVD risk SCORE of ≥ 5% or (ii) diabetes mellitus without target organ damage; such patients are also selected based on having a serum LDL-C concentration of 100 mg / dL or greater. That's fine.

[0039] PCSK9 inhibitors as add-on therapy The present invention includes methods of treatment in which a PCSK9 inhibitor is administered to a patient undergoing or who has recently undergone lipoprotein apheresis according to a specified dosage and frequency, and the PCSK9 inhibitor is administered as an add-on to the patient's already undergoing daily lipid-lowering therapy (if applicable), such as an add-on to a daily therapeutic statin regimen the patient is already undergoing.

[0040] For example, methods of the present invention include add-on treatment regimens in which a PCSK9 inhibitor is administered as add-on therapy to the same stable daily therapeutic statin regimen (i.e., the same statin dosage) that the patient was receiving before receiving the PCSK9 inhibitor. In other embodiments, the PCSK9 inhibitor is administered as add-on therapy to a therapeutic statin regimen that includes a higher or lower amount of statin than the statin dose the patient was receiving before administering the PCSK9 inhibitor. For example, after initiating a treatment regimen including a PCSK9 inhibitor administered at a particular dosing frequency and amount, the daily dose of statin administered or prescribed to the patient may (a) remain the same, (b) increase, or (c) decrease (e.g., uptitration or downtitration) compared to the daily statin dose the patient was taking before starting the PCSK9 inhibitor treatment regimen, depending on the patient's treatment needs.

[0041] Treatment effect The methods of the present invention result in a reduction in serum levels of one or more lipid components selected from the group consisting of LDL-C, ApoB100, non-HDL-C, total cholesterol, VLDL-C, triglycerides, Lp(a), and residual cholesterol. Thus, the lipoprotein-lowering effect of the therapeutic regimens of the present invention reduces the frequency or eliminates the need for lipoprotein apheresis to achieve patient target lipoprotein levels. For example, according to certain embodiments of the present invention, administration of a pharmaceutical composition comprising a PCSK9 inhibitor to patients undergoing or who have recently undergone lipoprotein apheresis can result in an average reduction from baseline in serum low-density lipoprotein cholesterol (LDL-C) of at least about 25%, 30%, 40%, 50%, 60%, or more; an average reduction from baseline in ApoB100 of at least about 25%, 30%, 40%, 50%, 60%, or more; an average reduction from baseline in non-HDL-C of at least about 25%, 30%, 40%, 50%, 60%, or more; or more, an average reduction from baseline in total cholesterol of at least about 10%, 15%, 20%, 25%, 30%, 35% or more, an average reduction from baseline in VLDL-C of at least about 5%, 10%, 15%, 20%, 25%, 30% or more, an average reduction from baseline in triglycerides of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or more, and / or an average reduction from baseline in Lp(a) of at least about 5%, 10%, 15%, 20%, 25% or more.

[0042] The present invention provides a method of treating a patient with hypercholesterolemia, the method comprising administering multiple doses of an anti-PCSK9 antibody to the patient at a dosage of about 75-150 mg per dose and at a dosing frequency of about once every two weeks (or a dosing regimen according to an uptitration dosing regimen as described elsewhere herein), wherein the patient is undergoing or has recently undergone lipoprotein apheresis, and about 12, 14, 16, 18, 20, 22, 24, or more weeks after treatment with the anti-PCSK9 antibody, the patient exhibits a reduction in LDL-C levels from baseline by at least 50%, thereby reducing the frequency of or eliminating the need for lipoprotein apheresis by the patient. In one embodiment, after one or more weeks after treatment with the anti-PCSK9 antibody, the patient exhibits a reduction in LDL-C levels from baseline by about 55%, 60%, 70%, or more. Indicates a decrease.

[0043] PCSK9 inhibitors The methods of the invention involve administering to a patient a therapeutic composition comprising a PCSK9 inhibitor. As used herein, a "PCSK9 inhibitor" is any agent that binds to or interacts with human PCSK9 and inhibits the normal biological function of PCSK9 in vitro or in vivo. Examples of classes of PCSK9 inhibitors include, but are not limited to, small molecule PCSK9 antagonists, nucleic acid-based inhibitors of PCSK9 expression or activity (e.g., siRNA or antisense), peptide-based molecules that specifically interact with PCSK9 (e.g., peptibodies), receptor molecules that specifically interact with PCSK9, proteins comprising the ligand-binding portion of the LDL receptor, PCSK9-binding scaffold molecules (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetraglycopeptide repeat proteins, fibronectin-based scaffold constructs, and other scaffolds based on naturally occurring repeat proteins (see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857 and references cited therein)), and anti-PCSK9 aptamers or portions thereof. According to certain embodiments, the PCSK9 inhibitors that can be used in accordance with the present invention are anti-PCSK9 antibodies or antigen-binding fragments of antibodies that specifically bind to human PCSK9.

[0044] As used herein, the term "human proprotein convertase subtilisin / kexin type 9" or "human PCSK9" or "hPCSK9" means PCSK9 having the nucleic acid sequence set forth in SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 198, or a biologically active fragment thereof.

[0045] As used herein, the term "antibody" is intended to refer to immunoglobulin molecules, and multimers thereof (e.g., IgM), comprising four polypeptide chains: two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or V H The heavy chain constant region is made up of three domains: C H 1. CH 2 and C H Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The V domain contains a V-chain constant region (CL1) and a V-chain constant region (CL2). H and V L The regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L Each of these CDRs is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present invention, the FRs of an anti-PCSK9 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence can be defined based on a side-by-side analysis of two or more CDRs.

[0046] The term "antibody," as used herein, also includes antigen-binding fragments of intact antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen and forms a complex. Antigen-binding fragments of antibodies may be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or may 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 the appropriate orientation, or to introduce codons, or to create cysteine ​​residues. The amino acids may be modified, added or deleted, etc.

[0047] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0048] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V associated with the domain H In an antigen-binding fragment having a domain, V H Domain and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region is a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a monomeric V H or V L It may also include a domain.

[0049] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Exemplary arrangements of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include, but are not limited to, the following: (i) VH -C H 1(ii)V H -C H 2(iii)V H -C H 3(iv)V H -C H 1-C H 2(v)V H -C H 1-C H 2-C H 3(vi)V H -C H 2-C H 3(vii)V H -C L (viii)V L -C H 1(ix)V L -C H 2(x)V L -C H 3(xi)V L -C H 1-C H 2(xii)V L -C H 1-C H 2-C H 3(xiii)V L -C H 2-C H 3(xiv)V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which provides a flexible or semi-flexible link between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may be non-covalently associated with each other and / or with one or more monomeric V H or V L The domains may be held together (eg, by disulfide bonds) to form homodimers or heterodimers (or other multimers) of any of the variable and constant domain arrangements listed above.

[0050] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted and used in the context of the antigen-binding fragments of antibodies of the present invention, using routine techniques available in the art.

[0051] The constant region of an antibody is important in the ability of the 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.

[0052] As used herein, the term "human antibody" refers to an antibody derived from human germline immunoglobulin sequences. The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions comprising the same or similar sequences. Nevertheless, the human antibodies of the invention may include amino acid residues, e.g., in the CDRs, and particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0053] As used herein, the term "recombinant human antibody" is intended to include all human antibodies that are prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (discussed further below), antibodies isolated from a recombinant combinatorial human antibody library (discussed further below), antibodies isolated from an animal (e.g., a mouse) transgenic with human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means involving the splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when transgenic animals of human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V and constant regions of the recombinant antibody. H and V L The amino acid sequence of the region is similar to that of the human germline V H and V L These are sequences that are derived from and related to sequences, but do not naturally occur within the human antibody germline repertoire in vivo.

[0054] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa, in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds, forming an approximately 75-80 kDa molecule composed of covalently linked light and heavy chains (half antibodies). These forms have been extremely difficult to separate, even after affinity purification.

[0055] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the antibody hinge region isotype. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to the level normally observed with a human IgG1 hinge. The present invention provides a method for the detection of the second form in the hinge, C H 2 or C H Antibodies with one or more mutations in the three regions may be included, which may be desirable, for example, to improve the yield of antibody in the desired form during production.

[0056] As used herein, an "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for purposes of the present invention. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0057] The term "specifically binds" or the like means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plate precipitation, and the like. For example, an antibody that "specifically binds" to PCSK9 as used in the context of the present invention is an antibody that binds to PCSK9 or a portion thereof and has a K measured by a surface plasmon resonance assay. Dless than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM. However, an isolated antibody that specifically binds to human PCSK9 may have cross-reactivity to other antigens, such as PCSK9 molecules from other (non-human) species.

[0058] Anti-PCSK9 antibodies useful in the methods of the present invention may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains, relative to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes methods involving the use of antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain are mutated back to the residue found in the germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody is derived). Furthermore, antibodies of the invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved or enhanced biological properties as an antagonist or agonist (as the case may be), reduced immunogenicity, etc. Uses of antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present invention.

[0059] The present invention also includes methods involving the use of anti-PCSK9 antibodies having one or more conservative substitutions and variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. For example, the present invention provides anti-PCSK9 antibodies having a conservative amino acid sequence. The present invention also includes the use of anti-PCSK9 antibodies having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0060] As used herein, the term "surface plasmon resonance" refers to real-time surface plasmon resonance (SPPR) measurements by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore® system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ). This refers to optical phenomena that allow us to analyze interactions in real time.

[0061] As used herein, the term "K D " refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0062] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, known as the paratope. A single antigen can have one or more epitopes. Thus, different antibodies may bind to different regions on the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are produced by the spatial juxtaposition of amino acids from different parts of a linear polypeptide chain. Linear epitopes are those produced by adjacent amino acid residues on a polypeptide chain. In certain circumstances, epitopes may include portions of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0063] According to certain embodiments, the anti-PCSK9 antibodies used in the methods of the present invention have pH-dependent binding properties. As used herein, the term "pH-dependent binding" means that the antibody or antigen-binding fragment thereof exhibits "reduced binding to PCSK9 at acidic pH compared to neutral pH" (for purposes of this disclosure, both terms may be used interchangeably). For example, antibodies with "pH-dependent binding properties" include antibodies and antigen-binding fragments thereof that bind to PCSK9 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibodies and antigen-binding fragments of the present invention bind to 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-fold or more higher affinity at neutral pH than at acidic pH.

[0064] According to this aspect of the invention, an anti-PCSK9 antibody with pH-dependent binding properties can have one or more amino acid mutations compared to a parent anti-PCSK9 antibody. For example, an anti-PCSK9 antibody with pH-dependent binding properties can contain one or more histidine substitutions or insertions in one or more CDRs of, for example, the parent anti-PCSK9 antibody. Thus, according to certain embodiments of the invention, methods are provided that include administering an anti-PCSK9 antibody that contains CDR amino acid sequences (e.g., heavy and light chain CDRs) that are identical to the CDR amino acid sequences of a parent anti-PCSK9 antibody, except that one or more amino acids in one or more CDRs of the parent antibody are substituted with histidine residues. An anti-PCSK9 antibody with pH-dependent binding can have, for example, one, two, three, four, five, six, seven, eight, nine, or more histidine substitutions, either within a single CDR of the parent antibody or distributed across multiple CDRs (e.g., two, three, four, five, or six) of the parent anti-PCSK9 antibody. For example, the invention includes the use of anti-PCSK9 antibodies that have pH-dependent binding and that comprise one or more histidine substitutions in HCDR1, one or more histidine substitutions in HCDR2, one or more histidine substitutions in HCDR3, one or more histidine substitutions in LCDR1, one or more histidine substitutions in LCDR2, and / or one or more histidine substitutions in LCDR3 of a parent anti-PCSK9 antibody.

[0065] As used herein, the term "acidic pH" refers to a pH of 6.0 or lower (e.g., less than about 6.0, less than about 5.5, less than about 5.0, etc.). The term "acidic pH" includes pH values ​​of about 6.0, 5.95, 5.90, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or lower. The term "neutral pH" refers to a pH of about 7.0 to about 7.4. The term "neutral pH" refers to a pH value of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, 7.11, 7.12, 7.13, 7.14, 7.15, 7.2, 7.25, 7.3, 7.45, 7.45, 7.55, 7.65, 7.75, 7.85, 7.95, 7.16, 7.17, 7.18, 7.19, 7.20, 7.21, 7.22, 7.23, 7.24, 7.25, 7.36, 7.26, 7.27, 7.28, 7.29, 7.39, 7.38, 7.39, 7.40, 7.41, Includes .35 and 7.4.

[0066] Examples of anti-PCSK9 antibodies that may be used in accordance with the present invention include, but are not limited to, alirocumab, evolocumab, bococizumab, roderucizumab, ralpancizumab, or antigen-binding portions of any of the foregoing antibodies.

[0067] Preparation of human antibodies Methods for preparing human antibodies in transgenic mice are known in the art, and any of these known methods may be used in accordance with the present invention to generate human antibodies that specifically bind to human PCSK9.

[0068] Using VELOCIMMUNE® technology (see, e.g., U.S. Patent No. 6,596,541, Regeneron Pharmaceuticals) or other known methods for generating monoclonal antibodies, we first isolated a chimeric antibody with a human variable region and a mouse constant region that has high affinity for PCSK9. This VELOCIMMUNE® technology involves the generation of a transgenic mouse whose genome contains human heavy and light chain variable regions operably linked to endogenous mouse constant region loci so that the mouse produces antibodies containing human variable regions and mouse constant regions in response to antigenic stimulation. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0069] Typically, VELOCIMMUNE® mice are sensitized with an antigen of interest, and lymphocytes (e.g., B cells) are collected from the mice that express antibodies. The lymphocytes are fused with a myeloma cell line to generate immortal hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions may be isolated and then linked to constant regions of the desired heavy and light chain isotype. Such antibody proteins may be produced in cells such as CHO cells. Alternatively, DNA encoding an antigen-specific chimeric antibody, or the light and heavy chain variable domains, may be isolated directly from antigen-specific lymphocytes.

[0070] First, high-affinity chimeric antibodies having human variable regions and murine constant regions are isolated. The antibodies are characterized and selected for desired properties, including affinity, selectivity, epitope, etc., using standard procedures known to those of skill in the art. The murine constant regions are replaced with the desired human constant regions to produce fully human antibodies of the invention, e.g., wild-type or denatured IgG1 or IgG4. While the constant region selected can vary depending on the specific application, high-affinity antigen binding and target specificity reside in the variable regions.

[0071] Generally, antibodies that can be used in the methods of the invention have high affinity, as described above, as measured by binding to either the antigen immobilized on a solid phase or the antigen in solution phase. The mouse constant regions are replaced with the desired human constant regions to produce fully human antibodies of the invention. While the constant region selected can vary depending on the specific application, high affinity antigen binding and target specificity reside in the variable regions.

[0072] Specific examples of human antibodies or antigen-binding fragments of antibodies that specifically bind to PCSK9 that can be used in accordance with the methods of the present invention include antibodies having three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR), and having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 11, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. Alternatively, specific examples of human antibodies or antigen-binding fragments of antibodies that specifically bind to PCSK9 that can be used in accordance with the methods of the present invention include any antibody or antigen-binding fragment having three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR), and having an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173, 181, and 189, or a sequence substantially similar to a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. The antibody or antigen-binding fragment thereof may comprise three light chain CDRs (LCVR1, LCVR2, LCVR3) comprised within a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 15, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. Alternatively, the antibody or antigen-binding fragment thereof may comprise three light chain CDRs (LCVR1, LCVR2, LCVR3) comprised within a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145, 153, 161, 169, 177, 185, and 193, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0073] The sequence identity between two amino acid sequences is determined over the entire length of the reference amino acid sequence, i.e., using the amino acid sequence identified by the SEQ ID NO:, using the best sequence alignment and / or over the region of best sequence alignment between the two amino acid sequences, where the best sequence alignment may be obtained using known tools, such as Align using standard settings, preferably EMBOSS: needle, matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0074] In certain embodiments of the invention, the antibody or antigen binding protein comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) from a heavy and light chain variable region amino acid sequence pair (HCVR / LCVR) selected from the group consisting of SEQ ID NOs: 1 / 6 and 11 / 15. Alternatively, in certain embodiments of the invention, the antibody or antigen binding protein comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) from a heavy and light chain variable region amino acid sequence pair (HCVR / LCVR) selected from the group consisting of SEQ ID NOs: 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.

[0075] In certain embodiments of the invention, anti-PCSK9 antibodies or antigen binding proteins that may be used in the methods of the invention have an HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 amino acid sequence selected from SEQ ID NOs: 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 Publication No. 2010 / 0166768), where SEQ ID NO: 16 includes a histidine for leucine substitution at amino acid residue 30 (L30H).

[0076] In certain embodiments of the invention, the antibody or antigen binding protein comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 1 / 6 and 11 / 15. In certain exemplary embodiments, the antibody or antigen binding protein comprises the HCVR amino acid sequence pair of SEQ ID NO: 1. and the LCVR amino acid sequence of SEQ ID NO: 6. In certain representative 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 certain 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 with a histidine substitution for leucine at amino acid residue 30 (L30H).

[0077] Pharmaceutical compositions and methods of administration The present invention relates to a method comprising administering a PCSK9 inhibitor to a patient, wherein the PCSK9 inhibitor is contained within a pharmaceutical composition. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide for appropriate transport, delivery, tolerability, etc. Many suitable formulations can be found in formularies known to all pharmaceutical chemists. Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) including vesicles (e.g., LIPOFECTIN®), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Powell et al., "Compendium of excipients for parenteral formulations” See also PDA (1998) J Pharm Sci Technol 52:238-311.

[0078] Exemplary pharmaceutical formulations comprising anti-PCSK9 antibodies that can be used in accordance with the present invention include any of the formulations described in U.S. Pat. No. 8,795,669 (which describes, inter alia, exemplary formulations comprising alirocumab), or WO 2013 / 166448, or WO 2012 / 168491.

[0079] Various delivery systems are known, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (e.g., Wu et al. 1987, J. Biol. Chem. 262:4429-4432). (See, e.g., Phys. Rev. 1999, 14:139-147.) Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example, by injection or bolus administration, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents.

[0080] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, pen delivery devices can be conveniently adapted to deliver the pharmaceutical compositions of the present invention subcutaneously. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Some disposable pen delivery devices do not have a replaceable cartridge. Rather, disposable pen delivery devices are sold pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of pharmaceutical composition, the entire device is discarded.

[0081] Many reusable pen and autoinjector delivery devices are adaptable for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN® (Owen Mumford, Inc., Woodsville, TN), to name just a few. stock, UK), DISETRONIC® pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), 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). Examples of disposable pen delivery devices suitable for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR® pen (sanofi-aventis), FLEXPEN® (Novo Nordisk), and KWIKPEN® (Eli Lilly), the SURECLICK® auto-injector (Amgen, Thousand Oaks, CA), PENLET® (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and the HUMIRA® pen (Abbott Labs, Abbott Park IL), to name just a few.

[0082] In certain circumstances, pharmaceutical compositions can be delivered in a controlled-release system. In one embodiment, a pump may be used (see Langer and Sefton, supra, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material may be used. See, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled-release system can be placed in proximity to the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, pp. 115-138). Other controlled-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0083] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations can be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injections. Examples of aqueous media for injection include adjuvants such as physiological saline and glucose-containing isotonic solutions, which may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), and the like. Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable preparations prepared in this manner are preferably filled into appropriate ampoules.

[0084] Conveniently, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in a dosage form of a unit dose suitable for the dose of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0085] Dosage PCSK9 inhibitors (e.g., anti-PCSK9 antibodies) administered to subjects according to the methods of the invention is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" refers to a dose of a PCSK9 inhibitor that results in a detectable decrease (at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more from baseline) in one or more parameters selected from the group consisting of LDL-C, ApoB100, non-HDL-C, total cholesterol, VLDL-C, triglycerides, Lp(a), and residual cholesterol, or an amount that reduces or eliminates the patient's need for lipoprotein apheresis, or an amount that reduces the patient's normalized apheresis rate (as defined elsewhere herein).

[0086] In the case of an anti-PCSK9 antibody, the therapeutically effective amount may be from about 0.05 mg to about 600 mg, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg , about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 3 The anti-PCSK9 antibody may be administered in an amount of 80 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg. According to certain exemplary embodiments of the present invention, the therapeutically effective amount of the anti-PCSK9 antibody is 75 mg, 150 mg, or 300 mg (e.g., in the case of alirocumab) or 140 mg or 420 mg (e.g., in the case of evolocumab). Other dosage amounts of the PCSK9 inhibitor will be apparent to those skilled in the art and are considered to be within the scope of the present invention.

[0087] The amount of anti-PCSK9 antibody contained in each dose can be expressed in milligrams of antibody per kilogram of patient body weight (i.e., mg / kg). For example, the anti-PCSK9 antibody can be administered to a patient at a dose of about 0.0001 to about 10 mg / kg of patient body weight.

[0088] Combination therapy As described elsewhere herein, the methods of the present invention may involve administering a PCSK9 inhibitor to a patient in combination with and in addition to a lipid-lowering therapy that the patient has previously been prescribed. For example, a PCSK9 inhibitor may be administered to a patient in combination with a stable daily statin treatment regimen, with a view to reducing or eliminating the need for lipoprotein apheresis. Representative daily statin treatment regimens that may be administered in conjunction with a PCSK9 inhibitor in accordance with 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 that may be administered in conjunction with a PCSK9 inhibitor in accordance with the present invention include, for example, (1) agents that inhibit cholesterol uptake and / or bile acid reabsorption (e.g., ezetimibe), (2) agents that increase lipoprotein catabolism (e.g., niacin), and / or (3) agents that play a role in cholesterol removal, such as 22-hydroxycholesterol. Activator of LXR transcription factors.

[0089] According to certain embodiments of the present invention, methods are provided that include administering to a patient a PCSK9 inhibitor (e.g., an anti-PCSK9 antibody such as alirocumab, evolocumab, bococizumab, roderucizumab, or ralpancizumab) in combination with an inhibitor of angiopoietin-like protein 3 (e.g., an anti-ANGPTL3 antibody such as REGN1500), an inhibitor of angiopoietin-like protein 4 (e.g., an anti-ANGPTL4 antibody such as the anti-ANGPTL4 antibody referred to as "H1H268P" or "H4H284P" in U.S. Pat. No. 9,120,851), or an inhibitor of angiopoietin-like protein 8 (e.g., an anti-ANGPTL8 antibody).

[0090] Within the method aspects of the present invention, an additional therapeutically active ingredient, such as any of the agents listed above or derivatives thereof, may be administered immediately prior to, simultaneously with, or immediately following administration of the PCSK9 inhibitor (for purposes of this disclosure, such administration regimens are considered to be administering the PCSK9 inhibitor "in combination with" the additional therapeutically active ingredient). The present invention includes pharmaceutical compositions and methods of use thereof in which a PCSK9 inhibitor is co-formulated with one or more additional therapeutically active ingredients, as described elsewhere herein.

[0091] Dosing regimen According to certain embodiments of the present invention, a subject may be administered multiple doses of a PCSK9 inhibitor (i.e., a pharmaceutical composition comprising a PCSK9 inhibitor) over a predetermined time period (e.g., in addition to a daily statin treatment regimen or other background lipid-lowering therapy). The method according to this aspect of the present invention comprises sequentially administering multiple doses of a PCSK9 inhibitor to a subject. As used herein, "sequentially administering" means administering each dose of the PCSK9 inhibitor to the subject at different times, e.g., on different days separated by predetermined intervals (e.g., hours, days, weeks, or months). The present invention includes methods comprising administering a single initial dose of a PCSK9 inhibitor to a patient, followed by one or more secondary doses of the PCSK9 inhibitor, and optionally, subsequently, one or more tertiary doses of the PCSK9 inhibitor.

[0092] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the chronological administration sequence of individual doses of a pharmaceutical composition comprising a PCSK9 inhibitor. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and then a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of PCSK9 inhibitor, but may generally differ from one another in terms of administration frequency. However, in certain embodiments, the amount of PCSK9 inhibitor contained in the initial, secondary, and / or tertiary doses differs from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of a treatment regimen as a "loading dose," followed by doses administered on a less frequent basis (e.g., "maintenance doses").

[0093] According to an exemplary embodiment of the present invention, each of the secondary and / or tertiary doses is administered 1 to 26 weeks (e.g., 1, 1 1 / 2 , 2, 2 1 / 2 , 3, 3 1 / 2 , 4, 4 1 / 2 , 5, 5 1 / 2 , 6, 6 1 / 2 , 7, 7 1 / 2 , 8, 8 1 / 2 , 9, 9 1 / 2 , 10, 10 1 / 2 , 11, 11 1 / 2 , 1 2, 12 1 / 2 , 13, 13 1 / 2 , 14, 14 1 / 2 , 15, 15 1 / 2 , 16, 16 1 / 2 , 17, 17 1 / 2 , 18, 18 1 / 2 , 19, 19 1 / 2 , 20, 20 1 / 2 , 21, 21 1 / 2 , 22, 22 1 / 2 , 23, 23 1 / 2 , 24, 24 1 / 2 , 25, 25 1 / 2 , 26, 26 1 / 2As used herein, the phrase "immediately preceding administered dose" refers to a dose of an antigen-binding molecule that is administered to a patient immediately prior to the administration of the next dose in a multiple administration sequence, without any intervening administrations.

[0094] The method according to this aspect of the invention can include administering any number of secondary and / or tertiary doses of a PCSK9 inhibitor to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0095] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2, 4, 6, 8, or more weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 1 to 2, 4, 6, 8, or more weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary throughout the course of the treatment regimen. The frequency of administration may be adjusted during the course of treatment by a physician depending on the needs of the individual patient after clinical testing.

[0096] The present invention includes dosing regimens that include an uptitration option (also referred to herein as "dose modification"). As used herein, "uptitration option" refers to a subsequent increase in the dose of a PCSK9 inhibitor if the patient does not achieve a specific reduction in one or more defined treatment parameters after receiving a specific number of doses of the PCSK9 inhibitor. For example, in a treatment regimen that includes administering a 75 mg dose of an anti-PCSK9 antibody to a patient every two weeks, if the patient has not achieved a serum LDL-C concentration of less than 70 mg / dL after eight weeks (i.e., five doses at weeks 0, 2, 4, 6, and 8), the dose of the anti-PCSK9 antibody is subsequently increased to, for example, 150 mg every two weeks (e.g., starting at week 10 or 12 or later).

[0097] In certain embodiments, the anti-PCSK9 antibody is administered to the subject at a dose of about 75 mg every two weeks, for example, for at least three doses (or over the course of a treatment regimen that may extend over days, weeks, months, or years).

[0098] In certain embodiments, the anti-PCSK9 antibody is administered to the subject at a dose of about 150 mg every two weeks, for example, for at least three doses (or over the course of a treatment regimen that can span days, weeks, months, or years).

[0099] In some embodiments, the antibody is administered to a subject at a dose of about 75 mg every two weeks for 12 weeks, and at week 8, if the subject's LDL-C level is less than 100 mg / dl and there has been a 30% reduction in LDL-C, the dose remains at about 75 mg every two weeks.

[0100] In another embodiment, the antibody is administered to a subject at a dose of about 75 mg every two weeks for 12 weeks, and if at week 8 the subject's LDL-C level is 100 mg / dl or greater, the dose is uptitrated to about 150 mg every two weeks.

[0101] In some embodiments, the antibody is administered to a subject at a dose of about 75 mg every two weeks for 12 weeks, and if at week 8 the subject's LDL-C level is less than 70 mg / dl and there is a 30% reduction in LDL-C, the dose of about 75 mg every two weeks remains.

[0102] In another embodiment, the antibody is administered to a subject at a dose of about 300 mg every four weeks.

[0103] In a further embodiment, the antibody is administered to a subject at a dose of about 300 mg every four weeks for a total of three doses, and if at week 8 the subject has not achieved the predetermined treatment goal or the subject's LDL-C has not had at least a 30% reduction from baseline, the dose is changed to about 150 mg every two weeks for an additional 36 weeks.

[0104] In certain embodiments, the anti-PCSK9 antibody is administered to a subject at a dose of about 150 mg every four weeks for at least three doses.

[0105] In some embodiments, the antibody is administered to a subject at a dose of about 150 mg every four weeks for 12 weeks, and at week 8, if the subject's LDL-C level is less than 100 mg / dl and there is a 30% reduction in LDL-C, the dose of about 150 mg every four weeks remains.

[0106] In another embodiment, the antibody is administered to a subject at a dose of about 150 mg every four weeks for 12 weeks, uptitrating to a dose of about 300 mg every two weeks if the subject's LDL-C level is equal to or greater than 100 mg / dl at week 8.

[0107] In some embodiments, the antibody is administered to a subject at a dose of about 150 mg every four weeks for 12 weeks, and if at week 8 the subject's LDL-C level is less than 70 mg / dl and there is a 30% reduction in LDL-C, the dose of about 150 mg every four weeks remains for an additional 12 weeks.

[0108] In another embodiment, the antibody is administered to a subject at a dose of about 300 mg every four weeks.

[0109] In a further embodiment, the antibody is administered to a subject at a dose of about 300 mg every four weeks for a total of three doses, and at week 8, if the subject has not achieved the predetermined treatment goal or the subject's LDL-C has not had at least a 30% reduction from baseline, the dose is changed to about 150 mg every two weeks for an additional 36 weeks. [Example]

[0110] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of the compositions, methods of making, and using the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0111] Example 1. Generation of human antibodies against human PCSK9 Human anti-PCSK9 antibodies were generated as described in U.S. Patent No. 8,062,640. The representative PCSK9 inhibitor used in the following examples 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 comprising SEQ ID NO:5 and a light chain comprising SEQ ID NO:9; a heavy chain variable region (HCVR) comprising SEQ ID NO:1 and a light chain variable region (LCVR) comprising SEQ ID NO:6; a heavy chain complementarity-determining region 1 (HCDR1) comprising SEQ ID NO:2; an HCDR2 comprising SEQ ID NO:3; an HCDR3 comprising SEQ ID NO:4; a light chain complementarity-determining region 1 (LCDR1) comprising SEQ ID NO:7; an LCDR2 comprising SEQ ID NO:8; and an LCDR3 comprising SEQ ID NO:10.

[0112] Example 2. A randomized, double-blind study to evaluate the efficacy and safety of alirocumab in patients with heterozygous familial hypercholesterolemia undergoing lipid apheresis therapy. Placebo-controlled parallel-group study Introduction The objective of this study was to evaluate the effect of alirocumab 150 mg administered once every 2 weeks (“Q2W”) compared with placebo on the frequency of LDL apheresis treatment in patients with HeFH receiving LDL apheresis therapy.

[0113] Adult men and women diagnosed with HeFH and receiving LDL apheresis therapy weekly or biweekly were enrolled in this study. Subjects maintained their background LMT treatment throughout the study. In such patients, the choice of placebo as a control was appropriate for the purpose of this study because it provided the least sensitive evaluation of the efficacy and safety of alirocumab.

[0114] Depending on the apheresis technique and duration, the average reduction in LDL-C levels ranges from 30% to 75% (Bambauer et al., Scientific World Journal, 2012, 2012:1-19). Weekly or biweekly treatment can reduce LDL-C levels by 40% to 50%. In this study, apheresis was not performed if LDL-C values ​​at the visit were 30% or more lower than baseline (pre-apheresis) LDL-C values. LDL-C levels rise toward baseline after each apheresis procedure but do not reach their original levels. Repeated weekly or biweekly apheresis continues to reduce baseline levels until a plateau is reached (Thompsen et al., Atherosclerosis, 2006, 189:31-38). Thus, determining the need for apheresis based on a 30% reduction in LDL-C levels is reasonable, because this reduction observed with alirocumab administration is consistent with the time course that achieves similar levels of LDL-C reduction as with the apheresis procedure.

[0115] Alirocumab Q2W (75 mg and 150 mg SC) and Q4W (150 mg and 300 mg SC) dosing regimens were evaluated in other Phase 3 studies of alirocumab. Based on the results of two dose-finding studies, the Q2W dosing regimen was selected, with maximal efficacy at week 12 provided by the 150 mg Q2W dosing, maintaining consistent LDL-C reductions throughout the dosing interval in all patients. Patients with heterozygous familial hypercholesterolemia generally have high baseline LDL-C levels. Given their lifetime exposure to high LDL-C, their target LDL-C is either 100 mg / dL or 70 mg / dL, depending on their history of CVD or other risk factors. To reach and maintain their target LDL-C, such patients were highly likely to require the most potent dose of alirocumab and the frequency required to maintain this effect at the end of the dosing interval. Therefore, a dose of 150 mg Q2W was selected for this study.

[0116] In the double-blind treatment period of this study, patients with HeFH who required apheresis every one or two weeks were administered alirocumab 150 mg Q2W for 18 weeks to evaluate its ability to lower LDL-C and reduce the frequency of apheresis from weeks 7 to 18 compared with the frequency during the 8 weeks before screening. Patients received alirocumab 150 mg Q2W through week 76 during the open-label treatment period.

[0117] Preliminary pharmacokinetic (PK) data from the Phase 2 studies, DFI11565, DFI11566, and R727 CL1003, showed that exposure to alirocumab decreased during the 8-week follow-up period following the double-blind treatment period, with total serum alirocumab concentrations still detectable, but at very low levels. Therefore, patients were followed up for the 8-week follow-up period (i.e., after the last dose) to establish serum alirocumab concentrations low enough to be ineffective. The patients were followed up for 10 weeks.

[0118] Research purpose The primary objective of the study was to evaluate the effect of alirocumab 150 mg Q2W compared with placebo on the frequency of LDL apheresis treatment in patients with HeFH receiving weekly or biweekly LDL apheresis therapy.

[0119] The secondary objectives of this study are: (a) to evaluate the effect of alirocumab 150 mg Q2W on LDL-C levels in HeFH patients undergoing LDL apheresis; (b) to evaluate the effect of alirocumab 150 mg Q2W on the following parameters: ApoB, non-HDL-C, total cholesterol, Lp(a), HDL-C, TG, and ApoA-1 in HeFH patients undergoing LDL apheresis during the study period; (c) to evaluate the safety and tolerability of alirocumab 150 mg Q2W in HeFH patients undergoing LDL apheresis; (d) to evaluate the PK of alirocumab 150 mg Q2W in HeFH patients undergoing LDL apheresis (QW vs. Q2W); (e) to evaluate the development of anti-alirocumab antibodies; (f) to evaluate PCSK9 levels in response to alirocumab therapy and before and after apheresis; and (g) to evaluate the quality of life (QOL) of these patients.

[0120] Study design This study was a randomized, double-blind, placebo-controlled, parallel-group trial in patients with HeFH receiving weekly or biweekly LDL apheresis therapy.

[0121] The study consisted of four periods: screening, double-blind treatment period, open-label treatment period, and follow-up. The double-blind treatment period included two intervals: Weeks 1 through 6 (scheduled administration of study medication on days 1, 15, and 29), during which apheresis frequency would be fixed to the individual patient's established schedule, and Weeks 7 through 18 (scheduled administration of study medication on days 43, 57, 71, 75, 99, and 113), during which apheresis frequency would be adjusted based on the patient's response to treatment.

[0122] Screening: Patients who had a stable apheresis schedule (every 7 or 14 days) at a stable apheresis facility for at least 4 weeks prior to the screening visit (week 2) and stable background medical LMT for at least 8 weeks (week 2) prior to the screening visit (week 2) entered the 2-week screening period.

[0123] Double-blind treatment: Patients who met all inclusion criteria and no exclusion criteria were randomly assigned in a 2:1 ratio to receive 18 weeks of treatment with alirocumab 150 mg SC Q2W or placebo in place of alirocumab SC Q2W.

[0124] Therapeutic injections during the double-blind treatment period were administered Q2W starting on the day of randomization (Week 0 (Day 1) / Visit 2). Patients were monitored at the clinical site for 30 minutes after the first injection. If LDL apheresis coincided with study drug administration, the study drug was administered immediately after completing the LDL apheresis procedure.

[0125] From week 1 to week 6, the frequency of apheresis was fixed to the schedule (QW or Q2W) established for each patient. Starting at week 7, LDL apheresis was administered based on the LDL-C value at that visit (determined by point-of-care testing). LDL apheresis was not administered if the LDL-C value at that visit was at least 30% lower than the baseline (day 1) LDL-C value before apheresis. The criterion of determining the need for apheresis based on a 30% reduction in LDL-C levels is reasonable. This is because the observed reductions observed with alirocumab are consistent with the time course that achieves similar levels of LDL-C reduction as the apheresis procedure. Investigators remained blinded to point-of-care LDL-C laboratory values ​​and were only alerted as to whether LDL apheresis should be administered.

[0126] Open-label Treatment: During the open-label treatment period, patients received alirocumab 150 mg SC Q2W. Treatment continued uninterrupted from the last dose of study drug in the double-blind treatment period (last dose at Week 16) through Week 18 (first dose of the open-label treatment period) and through Week 76.

[0127] The first injection during the open-label treatment period was administered at the 18-week visit (first visit of the open-label treatment period) as part of patient injection training. Subsequent injections were administered by the patient or designated caregiver (spouse, relative, etc.) at the patient's preferred location (e.g., home or work). Patients could also choose to return to the Q2W site to have injections administered by study staff.

[0128] Apheresis treatments were not required during the open-label treatment period of the study. At the investigator's discretion, patients were allowed to continue receiving apheresis treatments as needed. If a planned apheresis procedure coincided with a clinic visit or alirocumab dosing day, study assessments were performed before apheresis, and alirocumab was administered after apheresis.

[0129] Follow-up: Patients were seen at the end of the 86-week study visit.

[0130] Study General: All samples for clinical laboratory (lipids and lipid specialist team) and PK assessments were obtained immediately before and after the LDL apheresis procedure (if LDL apheresis was administered at that visit) and before study medication administration.

[0131] For patients not undergoing LDL apheresis at the visit, all samples for clinical laboratory evaluation were obtained before administration of study medication.

[0132] Overall safety was assessed at pre-determined time points by TEAEs, physical examination, vital signs (pulse rate and blood pressure), electrocardiogram (ECG), and clinical safety laboratory monitoring / assessment. The potential for development of anti-alirocumab antibodies was assessed. PCSK9 levels were assessed before and after apheresis.

[0133] The use of all medications and dietary supplements (including intake of red yeast products) known to alter serum lipids, including but not limited to statins, ezetimibe, fibrates, niacin, and bile acid resins, was permitted provided that treatment was stable for at least 8 weeks prior to the screening visit (week 2). Patients were instructed to continue their background medical LMT throughout the study, from the start of screening through the end-of-treatment visit.

[0134] Patients' diets remained stable throughout the study, starting from screening through the end-of-treatment visit.

[0135] Patients' exercise plans remained stable throughout the study, from screening through the end-of-treatment visit.

[0136] Patient selection The study protocol required the enrollment of approximately 63 patients at no more than 15 clinics in the United States and Germany. Patients were randomized as follows: One-third (approximately 21 people) received a placebo, and about two-thirds (approximately 42 people) of the enrolled patients received alirocumab.

[0137] The population consisted of adult men and women diagnosed with HeFH who were undergoing weekly or biweekly LDL apheresis therapy.

[0138] Randomization was stratified according to the following: apheresis frequency: QW vs. Q2W, and baseline Lp(a) level: normal (<30 mg / dL) vs. elevated (30 mg / dL). Because some patients with elevated LDL-C also had elevated Lp(a) levels, stratification ensured adequate representation in the placebo / treatment groups.

[0139] Inclusion Criteria: Patients enrolled in this study had to meet criteria 1-6 (below) to be eligible for inclusion in this study.

[0140] (1) Men and women aged 18 years or older at the time of the screening visit.

[0141] (2) Diagnosis of HeFH; (Note) The diagnosis of HeFH was made by genotyping or by clinical criteria. Clinical diagnosis in patients whose genotype was not identified was based on the Simon Broome criteria, which use the criteria for definitive FH, or the WHO / Dutch Lipid This could be based on a score >8 on the Network criteria.

[0142] (3) Prior to the screening visit (week 2), patients were currently receiving LDL apheresis therapy for at least 4 weeks QW or at least 8 weeks Q2W, and had initiated apheresis therapy at least 5 months prior to the screening visit (Note: Acceptable apheresis techniques include double membrane filtration, immunoadsorption, heparin-induced LDL precipitation, direct lipid adsorption, dextran sulfate adsorption (plasma), and dextran sulfate adsorption (whole blood)).

[0143] (4) Willingness and ability to comply with clinic visit and study-related procedures.

[0144] (5) provided signed informed consent, and

[0145] (6) Ability to understand and complete study-related questionnaires.

[0146] Exclusion criteria: Prospective patients who met any of the following criteria were excluded from the study.

[0147] (1) Homozygous FH;

[0148] (2) Background medical LMT (if applicable) had not been stable for at least 8 weeks prior to the screening visit (week 2).

[0149] (3) Patients receiving weekly apheresis had an unstable LDL apheresis schedule / settings for at least 4 weeks prior to the screening visit (week 2), and patients receiving biweekly apheresis had an unstable LDL apheresis schedule / settings for at least 8 weeks prior to the screening visit (week 2).

[0150] (4) LDL Affair Planning other than QW~Q2W.

[0151] (5) If you have started a new exercise program within 8 weeks prior to the screening visit (week 2) or have been exercising but are not stable.

[0152] (6) If you have started a new diet within 8 weeks prior to the screening visit (week 2) or if you have been on a diet but have not stabilized.

[0153] (7) Use of unstable doses or amounts of dietary supplements or over-the-counter medications known to adversely affect lipids prior to the screening visit (week 2) or at least 8 weeks between the screening visit and the randomization visit.

[0154] (8) presence of clinically significant uncontrolled endocrine disease known to affect serum lipids or lipoproteins;

[0155] (9) signs and symptoms of hypothyroidism (thyroid replacement therapy is possible);

[0156] (10) History of bariatric surgery within 12 months prior to the screening visit (week 2).

[0157] (11) Unstable weight (fluctuation > 5 kg) within 2 months prior to the screening visit (week 2)

[0158] (12) Newly diagnosed diabetes mellitus (within 3 months prior to the randomization visit (Day 1)) or uncontrolled diabetes (hemoglobin A1c [HbA1c] > 9%).

[0159] (13) Use of systemic corticosteroids, except when used in a stable regimen as replacement therapy for pituitary / adrenal disease for at least 6 weeks prior to randomization. Topical, intra-articular, intranasal, inhaled, and ophthalmic steroid therapy are not considered "systemic" and are permitted.

[0160] (14) If you are using estrogen or testosterone therapy even if your treatment regimen has not been stable for 6 weeks prior to the screening visit (week 2) and you do not plan to change your regimen during the study.

[0161] (15) Systolic blood pressure >160 mmHg or diastolic blood pressure >100 mmHg at the screening visit (Week 2) or randomization visit (Day 1) [Note: Blood pressure assessments for study eligibility may be obtained at any visit between the two screening or randomization visits for scheduled apheresis if the patient is not taking or is not scheduled to take the prescribed hypertension medication.

[0162] (16) History of myocardial infarction (MI), unstable angina leading to hospitalization, coronary artery bypass graft surgery (CABG), percutaneous coronary intervention (PCI), uncontrolled cardiac arrhythmia, carotid artery surgery or stent insertion, stroke, transient ischemia, or carotid artery revascularization within 3 months (week 2) prior to the screening visit, or endovascular procedure or surgical intervention for peripheral vascular disease within 1 month (week 2) prior to the screening visit.

[0163] (17) History of New York Heart Association stage III or IV heart failure within 12 months prior to the screening visit.

[0164] (18) History of hemorrhagic stroke

[0165] (19) History of cancer within the past 5 years, except for adequately treated basal cell skin cancer, squamous cell carcinoma, or in situ cervical cancer.

[0166] (20) a history of a positive test for human immunodeficiency virus;

[0167] (21) Any valid immunization within 1 month or 5 half-lives of screening, whichever is longer. Use of investigational drugs.

[0168] (22) Patients who have received at least one dose of alirocumab or any other anti-PCSK9 monoclonal antibody in any other clinical study;

[0169] (23) Conditions / circumstances, such as: (a) an abnormality identified at screening as clinically significant that, in the judgment of the Investigator or Co-Investigator, would preclude safe completion of the study or assessment of endpoints, e.g., major systemic disease, patients with short life expectancy; or (b) any reason that either the Investigator or Co-Investigator deems the patient unsuitable for the study, such as: (i) being deemed unable to meet the requirements of a particular protocol, such as scheduled visits; (ii) being deemed unable to tolerate injections by the patient or Investigator; (iii) the Investigator or Co-Investigator, pharmacist, study coordinator, other study staff, or anyone directly involved in the implementation of the protocol; or (iv) the existence of any other condition (e.g., geographic or social) that the Investigator feels may actually or anticipated constrain or limit the patient's participation for the duration of the study.

[0170] (24) Laboratory findings during the screening period (not including randomization testing): (a) positive test for hepatitis B surface antigen and / or hepatitis C antibody; (b) positive serum β-hCG or urine pregnancy test in women of childbearing potential; (c) TG >500 mg / dL >5.65 mmol / L (one repeat test allowed); (d) eGFR <15 mL / min / 1.73 m2 based on the 4-parameter Diet Modification Renal Disease Study Equation (calculated by the Central Lab); (e) alanine aminotransferase (ALT) or aspartate aminotransferase (AST) >3x upper limit of normal (ULN) (one repeat test allowed); (f) CPK >3x ULN (one repeat test allowed).

[0171] (25) Known hypersensitivity to the monoclonal antibody therapeutic drug or any component of the drug.

[0172] (26) Pregnant or breastfeeding women;

[0173] (27) Women of childbearing age who are not protected by a highly effective method of birth control (as defined in the Informed Consent Form [ICF] and / or local protocol addendum) and / or who are unwilling or unable to undergo pregnancy testing. Postmenopausal women must have been amenorrheic for at least 12 months.

[0174] research treatment Study treatment was a single 1 mL SC injection of a 150 mg dose of alirocumab or placebo in an autoinjector or prefilled syringe administered in the abdomen, thigh, or lateral upper arm region Q2W.

[0175] During the double-blind treatment period (Day 1-Week 18), eligible patients were randomized to receive alirocumab 150 mg SC Q2W or placebo in place of alirocumab SC Q2W.

[0176] During the open-label treatment period, patients received open-label alirocumab 150 mg SC Q2W starting at week 18 and the last dose at week 76.

[0177] Sterile alirocumab drug was supplied in auto-injectors or pre-filled syringes at a concentration of 150 mg / mL in histidine, pH 6.0, polysorbate 20, and sucrose.

[0178] Placebo-matched alirocumab was administered in the same formulation as alirocumab but without added protein. , supplied in an auto-injector or pre-filled syringe.

[0179] All patients and / or caregivers selected to inject study medication outside the hospital on the dosing day were trained by study staff before administering the injection. Patients / caregivers were instructed to administer study medication at the Week 18 visit, at which time they self-administered the first dose of the open-label treatment period. Subsequent injections were administered Q2W by the patient (self-injecting) or designated caregiver (spouse, relative, etc.) at the patient's preferred location (e.g., home or work).

[0180] Patients were also given the option to return to the site Q2W and have the injection administered by study personnel.

[0181] Treatment allocation method Patients were randomly assigned to receive placebo or alirocumab 150 mg Q2W in a 1:2 ratio using a permuted block design to ensure equal distribution of treatment assignments. Randomization was stratified according to frequency of apheresis treatment (every 7 or 14 days) and Lp(a) level (normal or elevated). Enrollment was limited to ensure that no more than two-thirds of patients underwent apheresis.

[0182] Regardless of assigned treatment during the double-blind treatment period, all patients received alirocumab 150 mg Q2W during the open-label treatment period.

[0183] Combination therapy Any treatment administered from the time of informed consent through the end of the follow-up period / final study visit was considered concomitant therapy. This included medications started before and administered during the study. Concomitant medications were kept to a minimum during the study. Concomitant medications (except those prohibited during the study) were allowed at the investigator's discretion at stable doses (if possible) if deemed necessary for the patient's welfare and unlikely to interfere with the investigational drug.

[0184] Prohibited Medications: Any background medical LMT that has not been stable for at least 8 weeks (week 2) prior to the screening visit is prohibited.

[0185] Allowed Medications: The use of all medications and dietary supplements (including intake of red yeast products) known to alter serum lipids, including but not limited to statins, ezetimibe, fibrates, niacin, and bile acid resins, is permitted as long as they have been on stable treatment for at least 8 weeks (week 2) prior to the screening visit. Patients were instructed to continue their background medical LMT throughout the entire study, from the start at screening through the end-of-treatment visit.

[0186] Patients' diets remained stable throughout the study, from the start at screening through the end-of-treatment visit.

[0187] Patients' exercise programs remained stable throughout the study, from initiation at screening to the end-of-treatment visit.

[0188] Study endpoints Baseline characteristics include standard demographics (eg, age, race, weight, height, etc.), disease characteristics including medical history, and medication history for each patient.

[0189] Primary Efficacy Endpoint: The primary efficacy endpoint was the sequential This is the rate of apheresis treatments over a 12-week period from week 7 to week 18, normalized by the number of apheresis treatments planned according to each patient's established schedule at the time of cleaning.

[0190] The normalized apheresis rate was defined for each patient as the number of actual apheresis treatments received from weeks 7 to 18 divided by the number of planned apheresis treatments per randomization stratum at baseline (6 for Q2W and 12 for QW).

[0191] If a patient withdrew before week 18, the actual number of apheresis procedures (weeks 7 through 18) was added to the imputed number of remaining planned apheresis treatments according to the numerator randomization strata.

[0192] The normalization effect for planned apheresis treatment allows for homogenization to the same percentage scale for patients who entered the study with different apheresis schedules (i.e., QW or Q2W apheresis), according to each patient's schedule established at baseline.

[0193] Secondary Efficacy Endpoints: For the following lipid endpoints, such as percent change from baseline to specific post-baseline attainment during the double-blind treatment period, two time points will be defined: before and after the apheresis procedure. For the primary efficacy endpoint, lipid parameters will be analyzed using assessments collected before each apheresis procedure. If apheresis is not performed at the protocol-specified visit, a single laboratory assessment planned for collection will be used for both time points (i.e., including the primary efficacy endpoint). Baseline values ​​will be defined as the last available value for each parameter before the first double-blind dose of study medication.

[0194] The secondary endpoints of this study were:

[0195] (1) Percent change in LDL-C (pre-apheresis) from baseline to week 6, regardless of adherence to treatment.

[0196] (2) Normalized rates of apheresis treatment during the 4-week period from Weeks 15 to 18, defined similarly to the primary efficacy endpoint.

[0197] (3) Percent change in ApoB (pre-apheresis) from baseline to week 6, regardless of adherence to treatment.

[0198] (4) Percent change from baseline to week 6 in non-HDL-C (pre-apheresis), regardless of adherence to treatment.

[0199] (5) Percent change in total cholesterol (pre-apheresis) from baseline to week 6, regardless of adherence to treatment.

[0200] (6) Percent change in ApoA-1 (pre-apheresis) from baseline to week 6, regardless of adherence to treatment.

[0201] (7) the proportion of patients with a ≥30% reduction in LDL-C (pre-apheresis) at week 6, regardless of adherence to treatment;

[0202] (8) Regardless of adherence to treatment, LDL-C (before apheresis) at week 6 the proportion of patients with a 50% or greater reduction;

[0203] (9) Percent change in LDL-C (pre-apheresis) from baseline to week 18, regardless of adherence to treatment.

[0204] (10) Percent change in ApoB (pre-apheresis) from baseline to week 18, regardless of adherence to treatment.

[0205] (11) Percent change from baseline to week 18 in non-HDL-C (pre-apheresis), regardless of adherence to treatment.

[0206] (12) Percent change in total cholesterol (pre-apheresis) from baseline to week 18, regardless of adherence to treatment.

[0207] (13) Percent change in ApoA-1 (pre-apheresis) from baseline to week 18, regardless of adherence to treatment.

[0208] (14) The proportion of patients who achieved a 30% or greater reduction in LDL-C (pre-apheresis) at week 18, regardless of adherence to treatment.

[0209] (15) The proportion of patients who achieved a 50% or greater reduction in LDL-C (pre-apheresis) at week 18, regardless of adherence to treatment.

[0210] (16) Change in W-BQ22 index score from baseline to week 18, regardless of adherence to treatment.

[0211] (17) Percent change in Lp(a) (pre-apheresis) from baseline to week 6, regardless of adherence to treatment.

[0212] (18) Percent change in HDL-C (pre-apheresis) from baseline to week 6, regardless of adherence to treatment.

[0213] (19) Percent change in TG levels (pre-apheresis) from baseline to week 6, regardless of adherence to treatment.

[0214] (20) Percent change in Lp(a) (pre-apheresis) from baseline to week 18, regardless of adherence to treatment.

[0215] (21) Percent change in HDL-C (pre-apheresis) from baseline to week 18, regardless of adherence to treatment.

[0216] (22) Percent change in TG levels (pre-apheresis) from baseline to week 18, regardless of adherence to treatment.

[0217] (23) Change in ApoB / ApoA-1 ratio (pre-apheresis) from baseline to week 6, regardless of adherence to treatment.

[0218] (24) Change in ApoB / ApoA-1 ratio (pre-apheresis) from baseline to week 18, regardless of adherence to treatment.

[0219] (25) The standardized rate of apheresis treatment during a 4-week period from week 7 to week 10 was therefore defined as the primary efficacy endpoint.

[0220] (26) The standardized rate of apheresis treatment during the 4-week period from week 11 to week 14 was therefore defined as the primary efficacy endpoint.

[0221] (27) Comprehensive assessment of apheresis frequency and LDL-C levels over a 12-week period from the beginning of week 7 to the end of week 18 (patients ranked according to LDL-C levels and apheresis rates; the mean rank for all patients was determined, and the difference from the mean rank for each treated patient's LDL-C level or apheresis rate for that variable was expressed as a percentage; the percentage differences for the two variables on a patient-by-patient basis were added to provide a cumulative percent difference. When analyzed in combination by treatment group, the resulting values ​​indicate lower LDL-C and / or reduced apheresis rates).

[0222] (28) Percentage 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 adherence to treatment.

[0223] (29) Percentage 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.

[0224] (30) Raw values, and percent and absolute changes in LDL-C and other lipids from baseline to both time points reached during the double-blind treatment period, regardless of adherence to treatment.

[0225] (31) Raw values, and percent and absolute changes in LDL-C and other lipids from baseline to each endpoint during the open-label treatment period, regardless of adherence to treatment.

[0226] (32) Safety parameters assessed throughout the study (AEs (including declared cardiovascular events), clinical laboratory data, vital signs, and ECG).

[0227] Other endpoints included: (1) anti-alirocumab antibodies assessed throughout the study; (2) percent change from baseline to weeks 18 and 6 in high-sensitivity C-reactive protein (hs-CRP) and HbA1c; (3) total alirocumab concentrations in serum pre- and post-apheresis; and (4) free or total PCSK9 levels pre- and post-apheresis.

[0228] Test Procedure All laboratory samples were collected before administration of any dose of study medication. Blood samples were collected for all lipid panel visits from patients in a fasting state (e.g., overnight (at least 10-hour fast), water only), if possible. Fasting, although preferred, was not a requirement for lipid panel samples. Alcohol consumption and smoking within 48 hours or strenuous exercise within 24 hours prior to blood collection were not permitted.

[0229] Total cholesterol, HDL-C, triglycerides (TG), apoB, apoA-1, and Lp(a) were measured directly at a central laboratory. LDL-C was calculated using the Friedewald formula. If triglycerides exceeded 400 mg / dL (4.52 mmol / L), LDL-C was not calculated, but was instead measured recursively (by beta-quantitation) at the central laboratory. Non-HDL-C was calculated by subtracting HDL-C from total cholesterol. The ApoB / ApoA-1 ratio was calculated.

[0230] Lipid Study Group (Fasting): Lipid study group blood samples (total-C, TG, HDL-C, and calculated LDL-C) were collected at pre-specified time points after fasting for at least 10 hours.

[0231] Lipid Study Group (Fasting): Blood samples for the lipid study group (ApoB, ApoA-1, and Lp[a]) were collected after fasting for at least 10 hours at pre-specified time points.

[0232] The Well-Being Questionnaire: W-BQ22 was used to assess the impact of hypercholesterolemia and treatment on well-being at specific time points.

[0233] Blood Pressure and Heart Rate: Blood pressure and heart rate were assessed at pre-specified time points. Blood pressure was preferably measured in a standard sitting position, on approximately the same day, in the same arm, and with the same device (after the patient had rested comfortably in a sitting position for at least 5 minutes). Blood pressure was measured in both arms at the initial screening visit. The arm with the highest diastolic blood pressure was determined at this visit, and blood pressure was measured in this arm throughout the study. This highest value was recorded on the electronic case report form (eCRF). Heart rate was measured at the time of blood pressure measurement.

[0234] Physical Examination: A full and complete physical examination was performed at pre-specified time points.

[0235] Weight and Height: Weight was obtained with the patient wearing underwear or very light clothing, no shoes, and with an empty bladder. It is preferable to use the same measuring device throughout the study. If possible, the use of a calibrated balance is recommended.

[0236] Electrocardiogram: Electrocardiograms were performed before blood collection during visits requiring blood sampling. Standard 12-lead ECGs were performed at pre-specified times. 12-lead ECGs were performed in the supine position after at least 10 minutes of rest. Each electrode was placed in the same location, whenever possible, for ECG recordings throughout the study. ECGs were interpreted by the investigator at each site. New and / or clinically significant changes in ECG parameters were immediately reviewed before any decision was made for the affected patient. Any clinically significant abnormalities were documented as AEs / SAEs, as appropriate. Each tracing was analyzed in comparison with the tracing recorded at screening. All ECG tracings were retained as source data. Heart rate was recorded from the ventricular rate, and PR, QRS, RR, and QT intervals were recorded.

[0237] Laboratory Tests: All laboratory samples (including PK and ADA samples) were collected after the assessments were performed and before administration of any dose of study medication at the visit corresponding to the dosing day.

[0238] Adjudicated cardiovascular events: Adjudicated cardiovascular events included all actively adjudicated cardiovascular AEs. Adjudicated categories were as follows: (1) CHD death, (2) non-fatal MI, (3) fatal and non-fatal ischemic stroke, (4) unstable angina requiring hospitalization, (5) congestive heart failure requiring hospitalization, and (6) ischemia-driven coronary artery bypass grafting (PCI, CABG).

[0239] statistical analysis With a sample size of 63 patients (42 alirocumab: 21 placebo), using a two-sided significance level and assuming a standard deviation of 40%, we estimated that there would be a 33% difference in mean apheresis rate with at least 85% power.

[0240] The primary efficacy analysis population was the primary intention-to-treat (ITT) population, defined as all patients randomized. The secondary efficacy analysis population consisted of patients who had at least one calculated pre-apheresis LDL-C value before the first dose of study drug (or who had not received any study drug). The randomized patients were based on those with at least one calculated LDL-C value in one of the pre-apheresis analytical windows (those who did not receive apheresis treatment were randomized) and those with at least one calculated LDL-C value in one of the pre-apheresis analytical windows through week 6. Statistical analysis was performed when the last patient completed all efficacy assessments at week 18. LDL-C data were analyzed post hoc using the Kroon formula to estimate the interval mean of LDL-C from weeks 6 to 18 (after potential discontinuation of apheresis therapy).

[0241] The standardized apheresis rate was calculated by dividing the number of treatments received over a 12-week period (weeks 7–18) by the number of scheduled treatments (6 for biweekly (Q2W) and 12 for weekly) and analyzed using a rank analysis of covariance (ANCOVA) model. Standardized apheresis rates ranged from 0 to 1, with 0 indicating that the patient missed all scheduled apheresis treatments between weeks 7 and 18 and 1 indicating that the patient received all scheduled treatments. A rate of 0.75 indicated that the patient received 75% of scheduled apheresis treatments (and missed 25% of scheduled treatments). Patient withdrawals were accounted for according to predefined criteria. Median treatment differences were determined using the Hodges-Lehmann estimate and 95% confidence intervals (CIs) using Moses-free confidence intervals.

[0242] A hierarchical inference approach was used to control for type I error (alpha). Because the primary endpoint analysis was significant at the 5% alpha level, key secondary efficacy endpoints were tested sequentially.

[0243] result Baseline characteristics The baseline characteristics of patients enrolled in the study are summarized in Tables 1–4.

[0244] [Table 1]

[0245] [Table 2]

[0246] [Table 3]

[0247] [Table 4]

[0248] Seventy-six patients with heFH who were undergoing regular weekly or Q2W lipoprotein apheresis provided consent to participate and were screened. As summarized in Tables 1-4, a total of 62 patients (mean ± SD age, 58.7 ± 9.7 years) were randomized. 58.1% of the randomized patients were male. At the screening visit, 28 patients were not receiving statins due to tolerability, and 34 patients indicated they were taking statins daily, of which 19 were taking the maximum daily dose. All patients had atherosclerosis (coronary and / or cerebrovascular and / or peripheral), and 56.5% All patients had a family history of coronary heart disease. The average patient had undergone regular lipoprotein apheresis for 7.6 ± 7.7 years (mean ± SD). The median (min:max) was 4.9 (0.5:32.9) years. Apheresis frequency was Q2W (56.5%) and QW (43.5%). 16.1% of patients had diabetes. 1.6% had chronic kidney disease. Baseline mean LDL-C was 4.7 mmol / L (180.7 mg / dL), with a median (min:max) of 4.7 (1.4:8.2) mmol / L (179.5 (53.0:316.0) mg / dL). Baseline mean Lp(a) was 43.9 mg / dL (median (min:max) was 19.0 (1.5:285.0) mg / dL).

[0249] Sixty patients completed the 6-week double-blind treatment period (40 [97.6%] in the alirocumab group and 20 [95.2%] in the placebo group), where the apheresis rate was determined by the patient's established schedule, and 57 completed the 18-week double-blind treatment period (37 [90.2%] and 20 [95.2%], respectively), where the apheresis schedule was determined by previously achieved LDL-C levels. Of the five patients (8.1%) who discontinued study treatment prematurely, one (4.8%) received placebo (discontinued due to an adverse event) and four (9.8%) received alirocumab (two due to an adverse event, one due to poor compliance, and one patient withdrew consent).

[0250] 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 (min-max) duration of apheresis treatment before the study started was 4.9 (0.5, 32.9) years.

[0251] Baseline characteristics were balanced between treatment groups. The calculated mean baseline LDL-C level (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). At baseline, 27 patients (43.5%) were on a weekly apheresis schedule, and 35 patients (56.5%) were on a Q2W schedule. The mean LDL-C level in patients receiving weekly apheresis was 3.9 ± 1.3 mmol / L (151.3 ± 51.3 mg / dL) compared with 5.3 ± 1.4 mmol / L (204.9 ± 55.7 mg / dL) in patients receiving 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 statins at screening, of which 19 (55.9%) were receiving the maximum daily dose. Baseline LDL-C levels were 4.0 ± 1.4 mmol / L (155.0 ± 54.6 mg / dL) in patients on statins compared with 5.4 ± 1.4 mmol / L (208.0 ± 53.2 mg / dL) in patients not taking statins. Thirty (48.4%) patients were from Germany, and 32 (51.6%) were from the United States.

[0252] The mean ± SD duration of injectable 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 placebo group.

[0253] Efficacy Results The efficacy results are summarized in Tables 5-8.

[0254] Table 5 shows the normalized rates of apheresis from weeks 7 to 18.

[0255] [Table 5]

[0256] [Table 6]

[0257] The primary efficacy endpoint achieved a statistically significant benefit for patients treated with alirocumab, yielding a Hodges-Lehmann median estimate of the treatment difference vs. placebo of 0.75 (95% confidence interval: 0.67-0.83). Thus, alirocumab-treated patients had an additional 0.75 (75%) reduction in the standardized rate of apheresis treatment compared with placebo-treated patients (P<0.0001). The median treatment difference in the standardized rate of apheresis treatment from weeks 7 through 18, when apheresis treatment was determined by previously achieved LDL-C concentrations, was 0.75 (95% CI: 0.67-0.83) for patients receiving weekly apheresis. 0.58-0.92), and 0.6 for alirocumab in patients on the Q2W plan. 7 (95% CI 0.50-1.00).

[0258] The median treatment difference in standardized rate of apheresis treatment over 4 weeks (weeks 15-18) was 0.50 (95% CI 0.50-1.00; P < 0.0001) for alirocumab, representing a 50% reduction in standardized rate of apheresis treatment vs. placebo.

[0259] 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 depiction of the percentage reduction in apheresis treatment rates for treated patients is shown in Figure 2. During this period, 63.4% of alirocumab patients did not undergo apheresis treatments, and 92.7% were able to forgo at least half of their treatments. That is, 26 patients in the alirocumab treatment group achieved a 100% reduction in apheresis treatment rates. In contrast, no patients in the placebo group achieved a 100% reduction in apheresis treatment rates. These results are also reflected in Table 6 and Figures 3-6, which show the apheresis treatment rates for patients in the placebo and alirocumab treatment groups from Weeks 7 to 18 of the study. The results demonstrate the impact of alirocumab on existing treatment regimens. Patients receiving the mab demonstrated a significant 75% reduction in the frequency of standardized apheresis treatments over 12 weeks compared with placebo (P<0.0001).

[0260] Table 7 and Figures 7 and 8 show the effect of alirocumab treatment compared to placebo with respect to changes in serum LDL-C levels throughout the study. Overall, alirocumab-treated patients showed a mean reduction in LDL-C from baseline of more than 50% at week 6, while patients in the placebo group did not show any significant degree of LDL-C reduction.

[0261] [Table 7]

[0262] Key secondary efficacy results for placebo- and alirocumab-treated patients are summarized in Table 8 Ta.

[0263] [Table 8]

[0264] [Table 9]

[0265] The mean pre-apheresis LDL-C level 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, compared with 5.0 mmol / L (192 mg / dL) and 4.8 mmol / L (185 mg / dL) for corresponding placebo patients (Figure 8). The LS mean ± SE (95% CI) percent change in pre-apheresis LDL-C levels from baseline at week 6 was -53.7 ± 2.3 (-58.2 to -49.2) in the alirocumab group and 1.6 ± 3.1 (-4.7 to 7.9) in the placebo group (LS mean ± SE percent change). By week 18, mean LDL-C in the alirocumab group had increased slightly to 2.9 mmol / L (110 mg / dL) compared with 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).

[0266] Cross-validation comparing point-of-care LDL-C values ​​with central laboratory values ​​showed that both measures were highly correlated (Pearson correlation 0.86).

[0267] The percent change from baseline in lipoprotein(a) by week 18 was -5.7% for alirocumab vs. -3.0% for placebo in patients with normal baseline values, and 4.9% vs. 7.6%, respectively, in patients with elevated baseline values. Between weeks 7 and 18, when apheresis treatment could be discontinued, alirocumab treatment was associated with lower mean ± SD time-averaged LDL-C levels (using the Kroon formula) over the entire (potential) apheresis interval: 2.4 ± 1.3 (92.7 ± 50.2 mg / dL) compared with 3.8 ± 1.7 mmol / L (146.7 ± 65.6 mg / dL) for placebo (P < 0.0001).

[0268] In post-hoc analyses, time-averaged LDL-C levels in alirocumab-treated patients were consistently lower than those in placebo-treated patients.

[0269] safety TEAEs were reported by 75.6% of patients in the alirocumab group and 76.2% of patients in the placebo group, but none were fatal. The rates of serious adverse events (9.8% for alirocumab and 9.5% for placebo) and events leading to treatment discontinuation (4.9% and 4.8%, respectively) were similar in both groups.

[0270] Three patients (7.3%) in the alirocumab group and none in the placebo group had LDL-C values ​​<0.7 mmol / L (25 mg / dL) on two consecutive pre-apheresis sessions. Two of these patients experienced at least one adverse event, including several serious adverse events (pneumonia, acute myocardial infarction, acute respiratory failure, congestive heart failure, sepsis, and aortic stenosis). Twenty-seven patients (23 [56.1%] in the alirocumab group and 4 [19.0%] in the placebo group) had two post-apheresis LDL-C values ​​<0.7 mmol / L; 15 (65.2%) and 2 (50.0%) of these patients experienced adverse events, respectively. None of the events were serious, but one patient (4.3%) discontinued alirocumab treatment.

[0271] Summary and Conclusions This study demonstrated that patients receiving alirocumab had a lower rate of apheresis treatments and fewer apheresis treatments over the course of the study compared with patients receiving placebo. Specifically, alirocumab treatment resulted in a median estimated reduction in apheresis treatments of 0.75 or 75% greater than placebo treatment (p<0.0001).

[0272] This study also showed that 63.4% of patients treated with alirocumab did not undergo apheresis, compared with 0% of patients treated with placebo (i.e., no patients in the placebo group were able to forgo apheresis at all, whereas 63.4% of patients in the alirocumab group were able to completely eliminate apheresis after alirocumab treatment). Additionally, 92.7% of patients treated with alirocumab experienced at least a 50% reduction in apheresis frequency, compared with 14.3% of placebo-treated patients. Furthermore, alirocumab reduced LDL-C by 55% (vs. PBO) from 175 mg / dL to 89.5 mg / dL at week 6. 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 treatment from 15 to 18 weeks.

[0273] Regarding safety, subcutaneous administration of alirocumab in patients with HeFH receiving weekly or biweekly LDL apheresis was generally safe and well tolerated. The number of patients reporting overall TEAEs and AESIs was similar between treatment groups.

[0274] All patients in this study were at high cardiovascular risk and had previously taken LLTs, including statins. At screening, only 54.8% of patients were taking statins, and 55.9% were at the maximum tolerated dose. The majority of the overall population had a history of downtitration of statin therapy due to tolerability issues (43.5%), and 62.9% had a history of switching to a different statin. Reasons for not taking statins or not taking the maximum daily dose ranged from muscle symptoms to concerns about side effects and local habits / local labeling, indicating that patients with heFH undergoing apheresis represent a diverse and challenging population with limited treatment options.

[0275] Patients in the United States exhibit different characteristics from those in Germany, with higher baseline LDL-C, more prevalent statin intolerance, and fewer apheresis regimens. In the United States, lipoprotein apheresis for heFH is often considered only for patients who have not had an adequate response to maximal tolerated medical therapy after 6 months and who have elevated LDL-C and other cardiovascular risk factors. In Germany, where apheresis centers are more common, apheresis is considered when dietary and LLT failures occur within 12 months and when the LDL-C threshold is lower; furthermore, weekly apheresis is preferred over Q2W. Furthermore, European recommendations for LDL-C reduction are based on a risk-stratified, goal-directed treatment approach, whereas U.S. guidelines advocate a dose-adapted approach. In view of the European guideline approach, German patients receiving alirocumab with a target LDL-C level of <1.8 mmol / L (70 mg / dL) and an LDL-C above this target would still meet the criteria for apheresis. Consequently, an alirocumab-containing LLT could prove complementary to lipoprotein apheresis in patients with very high LDL-C or who do not meet LDL-C targets.

[0276] In conclusion, this study achieved a significant reduction in the primary efficacy endpoint and demonstrated that PCSK9 inhibitors, such as alirocumab, are an effective treatment option to reduce or eliminate the need for lipoprotein apheresis treatment in patients, or to delay the need for such treatment.

[0277] The present invention is not limited in scope by the specific embodiments described herein. Indeed, 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. All such modifications are intended to fall within the scope of the appended claims.

Claims

1. A pharmaceutical for eliminating a patient's need for lipoprotein apheresis therapy or reducing the frequency of lipoprotein apheresis therapy required by a patient to achieve a target lipoprotein level, comprising a PCSK9 inhibitor, the patient has hypercholesterolemia and is being treated or has been treated with lipoprotein apheresis at an initial frequency, wherein the initial frequency is the frequency before treatment; administration of one or more doses of the PCSK9 inhibitor to a patient reduces the level of at least one lipoprotein in the patient's serum, thereby reducing the frequency of lipoprotein apheresis required by the patient to achieve a target lipoprotein level; The PCSK9 inhibitor is an antibody that specifically binds to PCSK9, and The antibody comprises HCVR / LCVR shown in SEQ ID NO: 1 / 6. The above medicine.

2. 2. The method of claim 1, wherein the initial frequency of apheresis is once a week or once every two weeks, wherein the initial frequency is the frequency before treatment.

3. 3. The method of claim 1, wherein the frequency of apheresis after administration of one or more doses of the PCSK9 inhibitor is once every three weeks.

4. 3. The method of claim 1, wherein the frequency of apheresis after administration of one or more doses of the PCSK9 inhibitor is once every four weeks.

5. 3. The method of claim 1, wherein the frequency of apheresis after administration of one or more doses of the PCSK9 inhibitor is once every five weeks.

6. 3. The method of claim 1, wherein the frequency of apheresis after administration of one or more doses of a PCSK9 inhibitor is less than once every five weeks.

7. 3. The method of claim 1, wherein after administration of one or more doses of the PCSK9 inhibitor, the patient no longer requires apheresis to maintain target lipoprotein levels.

8. 2. The pharmaceutical composition of claim 1, wherein the patient is diagnosed with heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH) and / or high lipoprotein(a) (Lp(a)) prior to treatment with one or more doses of the PCSK9 inhibitor.

9. 2. The pharmaceutical composition of claim 1, wherein the lipoprotein apheresis therapy is selected from the group consisting of cascade filtration, immunoadsorption, heparin-induced LDL precipitation, LDL-adsorbed (dextran sulfate) liposorber, LDL hemoperfusion, and LDL-hemoperfusion (liposorber D).

10. 2. The pharmaceutical composition of claim 1, wherein, prior to or at the time of treatment with one or more doses of the PCSK9 inhibitor, the patient is on a stable schedule of lipoprotein apheresis at an initial frequency for at least two weeks prior to administration of the first dose of the PCSK9 inhibitor, wherein the initial frequency is a pre-treatment frequency.

11. 2. The method of claim 1, wherein the patient is undergoing lipid-modifying therapy (LMT) on a stable background prior to administration of one or more doses of the PCSK9 inhibitor.

12. The method of claim 1, wherein the patient is receiving lipid-modifying therapy (LMT) in a stable background in parallel with administration of one or more doses of a PCSK9 inhibitor.

13. 13. The medicament of claim 11 or 12, wherein the stable background LMT is low, medium, or high dose statin therapy.

14. The pharmaceutical composition of claim 1, wherein the lipoprotein that is decreased in the patient's serum after administration of one or more doses of the PCSK9 inhibitor is one or more lipoproteins selected from the group consisting of LDL-C, ApoB, non-HDL-C, total cholesterol, and Lp(a).

15. The pharmaceutical composition of claim 1, wherein the target lipoprotein level is a serum LDL-C level of less than 200 mg / dL.

16. The pharmaceutical composition of claim 1, wherein the target lipoprotein level is a serum LDL-C level of less than 130 mg / dL.

17. The pharmaceutical composition of claim 1, wherein the target lipoprotein level is a serum LDL-C level of less than 100 mg / dL.

18. The method of claim 1, wherein the target lipoprotein level is a serum LDL-C level of less than 70 mg / dL.

19. 2. The pharmaceutical composition of claim 1, wherein the antibody that specifically binds to PCSK9 is administered to the patient at a dose of about 75 mg once every two weeks.

20. 2. The pharmaceutical composition of claim 1, wherein the antibody that specifically binds to PCSK9 is administered to the patient at a dose of about 150 mg once every two weeks.

21. 2. The pharmaceutical composition of claim 1, wherein the antibody that specifically binds to PCSK9 is administered to the patient at a dose of about 300 mg once every four weeks.

22. The pharmaceutical composition according to any one of claims 19 to 21, wherein the antibody is alirocumab.

23. The method of claim 1 , wherein the antibody comprises heavy and light chain CDR amino acid sequences having SEQ ID NOs: 2, 3, 4, 7, 8 and 10.

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