EA043821B1 - METHODS OF TREATING PATIENTS WITH HYPERLIPIDEMIA BY ADMINISTRATION OF A PCSK9 INHIBITOR IN COMBINATION WITH ANGPTL3 INHIBITOR - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the field of therapeutic treatment of diseases and disorders that are associated with elevated levels of lipids and lipoproteins. More specifically, the invention relates to the use of a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor in combination with an angiopoietin-like protein 3 (ANGPTL3) inhibitor for the treatment of patients with hypercholesterolemia and related conditions who do not respond to, achieve adequate control of, or are intolerant to standard lipid-modifying therapy. therapy. State of the art

Hyperlipidemia is a general term that covers diseases and disorders characterized by or associated with elevated levels of lipids and/or lipoproteins in the blood. Hyperlipidemias include hypercholesterolemia, hypertriglyceridemia, combined hyperlipidemia, and elevated lipoprotein(a) (Lp(a)). A particularly common form of hyperlipidemia in many populations is hypercholesterolemia.

Hypercholesterolemia, particularly elevated low-density lipoprotein cholesterol (LDL-C) levels, represents a major risk for the development of atherosclerosis and coronary heart disease (CHD) (Sharrett et al., 2001, Circulation 104:1108-1113). Low-density lipoprotein cholesterol has been identified as a primary target of cholesterol-lowering therapy and is accepted as a valid surrogate therapeutic endpoint. Numerous studies have shown that lowering LDL-C levels leads to a reduced risk of developing CHD, with a strong direct relationship between LDL-C levels and CHD;

Every 1 mmol/L (~40 mg/dL) reduction in LDL-C reduces mortality and cardiovascular disease (CVD) morbidity by 22%. Greater reductions in LDL-C levels lead to an even greater reduction in these events, and data comparing intensive versus standard statin treatment suggest that the lower the LDL-C levels, the greater the benefit for patients at very high risk of developing cardiovascular disease (CVD). ).

Familial hypercholesterolemia (FH) is an inherited lipid disorder that predisposes an individual to premature development of severe cardiovascular disease (CVD) (Kolansky et al. (2008), Am. J. Cardiology, 102 (11):1438-1443). FH can be either an autosomal dominant or an autosomal recessive disorder that results from mutations in the low-density lipoprotein receptor (LDLR) gene or at least three different genes that encode proteins involved in the hepatic clearance of 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 in the apolipoprotein (Apo) B gene, which is the major protein of the LDL particle. In some cases of FH mutation, the gene encoding proprotein convertase subtilisin/kexin type 9 (PCSK9), an enzyme involved in the degradation of LDLR (gain-of-function mutation), is affected. In all cases, FH is characterized by the accumulation of LDL-C in plasma from birth and the subsequent development of tendon xanthoma, xanthelasma, atheroma and cardiovascular disease. FH can be classified as heterozygous FH (heFH) or homozygous FH (hoFH) depending on whether the subject has a genetic defect in one (heterozygous) or both (homozygous) copies of the gene involved.

Currently available drugs that lower LDL-C levels include statins, cholesterol absorption inhibitors, fibrates, niacin, and bile acid sequestrants. Statin treatment is a commonly prescribed method for lowering LDL-C. However, despite the availability of these lipid-lowering therapies, many high-risk patients fail to achieve their recommended LDL-C target (Gitt et al., 2010, Clin. Res. Cardiol., 99(11):723-733). Patients who continue to fail to achieve their recommended LDL-C target despite available lipid-modifying therapy (LMT) are sometimes treated with mechanical removal of lipoproteins by apheresis (eg, LDL-C apheresis).

However, patients who do not achieve target LDL-C levels despite receiving an optimized LMT regimen would benefit significantly from alternative methods of lowering LDL-C or the use of a combination of therapeutic agents, such as the agents and regimens described here.

The essence of the invention

The present invention relates to methods, uses and compositions for treating hyperlipidemia in patients who are refractory to, inadequately controlled or intolerant to treatment with standard lipid-modifying therapy. The therapeutic methods of the present invention reduce serum lipoprotein levels to the normal and acceptable range and, as such, may reduce the risk of developing atherosclerosis or coronary heart disease.

In one aspect, the invention relates to a method of treating a patient suffering from hypercholesterolemia, where the patient is refractory to, does not achieve adequate control of, or does not tolerate treatment with standard lipid-modifying therapy, where the method includes treating the patient with a combination of a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor and an angiopoietin-like protein inhibitor 3 (ANGPTL3).

- 1 043821

In one embodiment, the invention relates to the administration of one or more doses of a PCSK9 inhibitor in combination with one or more doses of an ANGPTL3 inhibitor to a patient who is being treated or has been treated with standard lipid-modifying therapy but has proven refractory to such therapy. Administration of a combination of a PCSK9 inhibitor with an ANGPTL3 inhibitor to a patient results in a decrease in the level of at least one lipoprotein in the patient's blood serum and, therefore, reduces or eliminates the need for treatment with standard lipid-modifying therapy for the patient.

In a related aspect, the methods of the present invention include selecting a patient with hypercholesterolemia who is being treated or has been treated with standard lipid-modifying therapy and who is refractory to, does not achieve adequate control of, or does not tolerate such therapy, and administering one or more doses of an anti-PCSK9 antibody in combination with one or more more doses of anti-ANGPTL3 antibody to the patient, thereby reducing the level of at least one lipoprotein in the patient's blood serum and, therefore, replacing the use of standard lipid-modifying therapy with a combination treatment of anti-PCSK9 antibody plus anti-ANGPTL3 antibody to achieve target lipoprotein levels.

Patients who are treated or amenable to treatment by the methods of the present invention include, for example, patients with hypercholesterolemia, including patients with familial hypercholesterolemia (FH). In some embodiments, patients who are treated or amenable to treatment by the methods of the present invention are patients diagnosed with (or known to have) homozygous FH (hoFH) or heterozygous FH (heFH), or patients who undergo risk of developing abnormally high levels of lipids and/or lipoproteins associated with homozygous FH (hoFH) or heterozygous FH (heFH).

The present invention also provides pharmaceutical compositions containing a PCSK9 inhibitor and an ANGPTL3 inhibitor for use in the treatment of a patient who is refractory to, does not achieve adequate control of, or does not tolerate treatment with standard lipid-modifying therapy, such as statin therapy. The statin may be selected from the group consisting of atorvastatin (LIPITOR®), pitavastatin (LIVALO®), lovastatin (MEVACOR®), simvastatin (ZOCOR®), pravastatin (PRAVACHOL®), fluvastatin (LESCOL®) and rosuvastatin (CRESTOR®). Other standard lipid-modifying agents that may be used in patients suffering from hypercholesterolemia include, but are not limited to, fibrates, niacin, bile acid sequestrants, ezetimibe (ZETIA®), lomitapide (JUZTAPID™), phytosterols, orlistat (XENICAL®).

Exemplary PCSK9 inhibitors or ANGPTL3 inhibitors that may be used in the context of the methods of the present invention include, for example, anti-PCSK9 antibodies or anti-ANGPTL3 antibodies, small molecule inhibitors and scaffold-based inhibitors, i.e. PCSK9-binding molecules or ANGPTL3-binding molecules.

In some embodiments, it is contemplated that the use of a combination of a PCSK9 inhibitor with an ANGPTL3 inhibitor may be sufficiently effective in lowering serum lipid and/or lipoprotein levels such that the dose of standard lipid-modifying therapy may be reduced to eliminate any adverse effects, if at all. be cancelled.

In one embodiment, the methods provide treatment to a patient in need thereof with an antibody or antigen binding fragment thereof that specifically binds to PCSK9 in combination with an antibody or antigen binding fragment thereof that specifically binds, in particular, to ANGPTL3. In one embodiment, the anti-PCSK9 antibody is administered to the patient at a dose of approximately 75 mg at a frequency of once every two weeks. In one embodiment, the anti-PCSK9 antibody is administered to the patient at a dose of approximately 140 mg at a frequency of once every two weeks. In one embodiment, the anti-PCSK9 antibody is administered to the patient at a dose of approximately 150 mg at a frequency of once every two or four weeks. In one embodiment, the anti-PCSK9 antibody is administered to a patient at a dose of approximately 300 mg at a frequency of once every four weeks. In one embodiment, the anti-PCSK9 antibody is administered to a patient at a dose of approximately 420 mg at a frequency of once every four weeks.

In one embodiment, the anti-PCSK9 antibody is selected from the group consisting of alirocumab, evolocumab, bococizumab, lodelcizumab and ralpancizumab.

In one embodiment, the anti-PCSK9 antibody is alirocumab.

In one embodiment, an antibody or antigen binding fragment thereof that specifically binds to PCSK9 comprises a heavy chain variable region (HCVR) complementarity determining regions (CDRs) having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region (LCVR) CDRs with sequence SEQ ID NO: 17.

In one embodiment, an antibody or antigen binding fragment thereof that specifically binds PCSK9 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence of SEQ ID NO: 13, HCDR2 having the amino acid sequence of SEQ ID NO: ID NO:, 14, HCDR3, having the amino acid sequence SEQ ID NO: 15, light chain CDR1 (LCDR1) having the amino acid sequence SEQ ID NO: 18, LCDR2 having the amino acid sequence SEQ ID NO: 19, and LCDR3 having the amino acid sequence SEQ ID NO: 2043821 21.

In one embodiment, an antibody or antigen binding fragment thereof that specifically binds PCSK9 comprises HCVR having the amino acid sequence of SEQ

ID NO: 12, and LCVR having the amino acid sequence SEQ ID NO: 17.

In one embodiment, the anti-PCSK9 antibody is administered to the patient subcutaneously or intravenously.

In one embodiment, the anti-ANGPTL3 antibody is administered to a patient at a dose of approximately 150 mg at a frequency of once per week. In one embodiment, the anti-ANGPTL3 antibody is administered to a patient at a dose of approximately 300 mg at a frequency of once per week. In one embodiment, the anti-ANGPTL3 antibody is administered to a patient at a dose of approximately 450 mg at a frequency of once per week. In one embodiment, the anti-ANGPTL3 antibody is administered to a patient at a dose of approximately 300 mg at a frequency of once every two weeks. In one embodiment, the anti-ANGPTL3 antibody is administered to a patient at a dose of approximately 450 mg at a frequency of once every two weeks. In one embodiment, the anti-ANGPTL3 antibody is administered to a patient at a dose of approximately 20 mg/kg at a frequency of once every four weeks.

In one embodiment, the anti-ANGPTL3 antibody is evinacumab.

In one embodiment, an antibody or antigen binding fragment thereof that specifically binds to ANGPTL3 comprises a heavy chain variable region (HCVR) complementarity determining regions (CDRs) having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region (LCVR) CDRs with sequence SEQ ID NO: 3.

In one embodiment, an antibody or antigen binding fragment thereof that specifically binds to ANGTL3 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence of SEQ ID NO: 4, HCDR2 having the amino acid sequence of SEQ ID NO: ID NO: 5, HCDR3 having the amino acid sequence of SEQ ID NO: 6, light chain CDR1 (LCDR1) having the amino acid sequence of SEQ ID NO: 7, LCDR2 having the amino acid sequence of SEQ ID NO: 8, and LCDR3 having the amino acid sequence of SEQ ID NO: 9.

In one embodiment, an antibody or antigen binding fragment thereof that specifically binds to ANGPTL3 comprises an HCVR having the amino acid sequence of SEQ ID NO: 2 and an LCVR having the amino acid sequence of SEQ ID NO: 3.

In one embodiment, the anti-ANGPTL3 antibody is administered to a patient subcutaneously or intravenously.

In one embodiment, administration of an anti-PCSK9 antibody in combination with an anti-ANGPTL3 antibody results in an additive effect in reducing blood levels of LDL-C, non-LDL-C, and total cholesterol, but has no effect on blood HDL-C levels.

In one embodiment, administration of an anti-PCSK9 antibody in combination with an anti-ANGPTL3 antibody results in a synergistic effect in reducing blood levels of LDL-C, non-HDL-C, and total cholesterol, but has no effect on blood HDL-C levels.

In one embodiment, administration of an anti-PCSK9 antibody in combination with an anti-ANGPTL3 antibody results in a reduction in one or more of the following:

(a) reduction in the level of total cholesterol in the blood serum (TC);

(b) reduction in serum low-density lipoprotein cholesterol (LDL-C); or (c) reducing serum non-high-density lipoprotein cholesterol (non-HDL-C);

wherein the reduction in (a), (b) and/or (c) is determined relative to the serum TC level, serum LDL-C level and/or non-HDL-C serum level of the patient before or at the time of initiation of treatment a combination of a PCSK9 inhibitor and an ANGPTL3 inhibitor.

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

Brief description of the figures

In fig. Figure 1 shows the effect of H4H1276R and H1H316R on LDL-C levels in hyperlipidemic Ldlr-/+ mice when used alone or in combination. The mice were placed on a balanced diet and blood samples were collected five days before the start of the experiment.

In fig. Figure 2 shows the effect of H4H1276R and H1H316R on total cholesterol levels in hyperlipidemic Ldlr-/+ mice when used alone or in combination. The mice were placed on a balanced diet and blood samples were collected five days before the start of the experiment.

In fig. Figure 3 shows the effect of H4H1276R and H1H316R on HDL-C levels in hyperlipidemic Ldlr-/+ mice when used alone or in combination. The mice were placed on a balanced diet and blood samples were collected five days before the start of the experiment.

In fig. Figure 4 shows the effect of H4H1276P and H1H316P on non-HDL-C levels in hyperlipidemic Ldlr-/+ mice when used alone or in combination. The mice were placed on a balanced diet and blood samples were collected five days before the start of the experiment.

In fig. Figure 5 shows the effect of H4H1276P and H1H316P on LDL-C levels in hyperlipidemic Ldlr-/+ mice when used alone or in combination. Mice were placed on a Western diet with

- 3 043821 high fat diet for 3 weeks prior to treatment and maintained on this diet throughout the study.

In fig. 6 shows the effect of H4H1276R and H1H316R on total cholesterol levels in hyperlipidemic Ldlr-/+ mice when used alone or in combination. Mice were placed on a high-fat Western diet for 3 weeks before treatment and maintained on this diet throughout the study.

In fig. 7 shows the effect of H4H1276P and H1H316P on HDL-C levels in hyperlipidemic Ldlr-/+ mice when used alone or in combination. Mice were placed on a high-fat Western diet for 3 weeks before treatment and maintained on this diet throughout the study.

In fig. 8 shows the effect of H4H1276P and H1H316P on non-HDL-C levels in hyperlipidemic Ldlr-/+ mice when used alone or in combination. Mice were placed on a high-fat Western diet for 3 weeks before treatment and maintained on this diet throughout the study.

Detailed Description of the Invention

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

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. As used herein, the term approximately, when used in relation to a specific specified numerical value, means that the value may differ from the specified value by no more than 1%. For example, as used here, the expression approximately 100 includes 99 and 101 and all values in between (for example, 99.1, 99.2, 99.3, 99.4, etc.).

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

Methods for treating hyperlipidemia

The present invention generally relates to methods and compositions for lowering lipoprotein levels in patients suffering from hypercholesterolemia who do not respond to, achieve adequate control of, or are intolerant to standard lipid-modifying therapy (eg, statin therapy). In some embodiments, treatment with a PCSK9 inhibitor in combination with an ANGPTL3 inhibitor may serve to reduce lipoprotein levels in these patients to an acceptable range, thereby reducing the risk of developing atherosclerosis, stroke, and other cardiovascular diseases. In some embodiments, the described methods can be used to treat patients with hypercholesterolemia, including heterozygous familial hypercholesterolemia (heFH) and/or homozygous familial hypercholesterolemia (hoFH), in the event that these patients do not respond to, do not achieve adequate control of, or are intolerant to standard lipid-modifying therapy .

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

The present invention, in accordance with some embodiments, includes methods of treating patients who do not respond to, do not achieve adequate control of, or are intolerant to standard lipid-modifying therapy. As used herein, a specific patient who is refractory to, does not achieve adequate control of, or does not tolerate standard lipid-modifying therapy is determined by a physician, physician assistant, diagnostician, or other health care professional based on the level of one or more lipoproteins (eg, LDL-C and/or or non-HDL-C) measured or otherwise determined in the patient's serum after treatment with a standard lipid-modifying agent. A physician, physician assistant, diagnostician, or other health care professional may also determine how intolerant a patient is to standard lipid-modifying therapy based on the profile of standard lipid-modifying therapy side effects that the patient may experience, including, but not limited to, muscle pain, soreness, or weakness. (myalgia), headache, redness of the skin, trouble sleeping, abdominal cramps, flatulence, diarrhea, constipation, rash, nausea or vomiting. A patient who is refractory to, does not achieve adequate control of, or does not tolerate standard lipid-modifying therapy may also be identified or influenced by other factors, such as the patient's family history, general knowledge of the disease, current therapeutic treatment status, and conventional or dominant lipoprotein targets adopted by national medical associations and physician groups. For example, in certain cases, if a patient is being treated with a standard lipid-modifying agent and has an LDL-C level greater than or equal to approximately 70 mg/dL, then this indicates that the patient is refractory to, is not achieving adequate control of, or is intolerant of standard lipid-modifying therapy. and treatment using the therapeutic modalities described herein may be beneficial. In other cases, if a patient is undergoing therapy with a standard lipid-modifying agent and has an LDL-C level greater than or equal to approximately 100 mg/dL, then this indicates that the patient is refractory to, is not achieving adequate control of, or is intolerant of standard lipid-modifying therapy, and treatment using the therapeutic methods described here may have a positive effect. In some cases, if a patient is being treated with a standard lipid-modifying agent and has an LDL-C level greater than or equal to about 150 mg/dL, 200 mg/dL, 250 mg/dL, 300 mg/dL, 400 mg/dL or higher, then this indicates that the patient is refractory to, does not achieve adequate control of, or is intolerant of standard lipid-modifying therapy and may benefit from treatment using the therapeutic modalities described herein. In still other cases, whether or not a certain percentage reduction in LDL-C or non-HDL-C level has been achieved relative to the LDL-C level or non-HDL-C level in a patient at a certain baseline point (baseline period), these data can be used to determine the extent to which a patient has responded to standard lipid-modifying therapy or the extent to which the patient requires further treatment using the methods and agents of the present invention. For example, reducing LDL-C or non-HDL-C by less than 50% (e.g., less than 40%, less than 35%, less than 30%, less than 25%, etc.) from initial period may indicate a need for therapy using the methods and agents of the invention.

Therefore, the present invention includes methods of treatment comprising administering one or more doses of a PCSK9 inhibitor in combination with one or more doses of an ANGPTL3 inhibitor to a patient, whereby the levels of total cholesterol, LDL-C and/or non-HDL-C in the patient are significantly reduced. For example, the present invention includes therapeutic methods comprising administering one or more doses of a PCSK9 inhibitor and one or more doses of an ANGPTL3 inhibitor to a patient who is undergoing standard lipid-modifying therapy but is refractory to or intolerant of such therapy, where after administration of one or more doses of the inhibitor PCSK9 and one or more doses of an ANGPTL3 inhibitor, the patient can achieve normal total cholesterol, LDL-C, or non-LDL-C levels. In some cases, the patient may need to discontinue standard lipid-modifying therapy, or standard lipid-modifying therapy may be continued, but may be administered at lower doses, and may be used in combination with a PCSK9 inhibitor and an ANGPTL3 inhibitor, to achieve and/or maintain a specific target lipoprotein level. Alternatively, the patient may be given standard lipid-modifying therapy at the usual prescribed dose, but the frequency of administration of lipid-modifying agents may be reduced if the standard lipid-modifying therapy is given in combination with a combination of a PCSK9 inhibitor and an ANGPTL3 inhibitor. In some cases, the need for a patient to be treated with standard lipid-modifying therapy to achieve and/or maintain a specific target lipoprotein level may be completely eliminated after administration of one or more doses of a PCSK9 inhibitor in combination with an ANGPTL3 inhibitor.

In accordance with some embodiments, the present invention includes methods for reducing or eliminating the need for standard lipid-modifying therapy, wherein the methods include selecting a patient with hyperlipidemia (eg, hypercholesterolemia) who has been treated with lipid-modifying therapy within the past month, past 2 months, past 3 months , the last 4 months, the last 5 months, the last 6 months, or for a longer period of time, and administering one or more doses of a PCSK9 inhibitor in combination with an ANGPTL3 inhibitor to the patient. The methods of this aspect of the invention result in a decrease in the level of at least one lipoprotein in the patient's blood serum and, therefore, reduce or eliminate the need to treat the patient with standard lipid-modifying therapy. For example, in some embodiments of the present invention, after administration of one or more doses of a PCSK9 inhibitor in combination with an ANGPTL3 inhibitor, the patient's serum LDL-C level decreases below a certain level (for example, below 100 mg/dL or below 70 mg/dL), or the level total cholesterol is reduced to a certain level (for example, below 200 mg/dL or below 150 mg/dL).

In accordance with some embodiments, a patient who is amenable to treatment with the methods of the present invention has hypercholesterolemia (e.g., a serum LDL-C concentration greater than or equal to 70 mg/dL, or a serum LDL-C concentration greater than or equal to 100 mg/dL). dl). In some embodiments, the patient's hypercholesterolemia is not adequately controlled by standard lipid-modifying therapy, such as statin therapy. For example, the present invention includes methods of treating a patient who is refractory to, does not achieve adequate

- 5 043821 control or is intolerant to treatment with standard lipid-modifying therapy, such as statin therapy, or has hypercholesterolemia that is not adequately controlled by a daily dose of a selected statin selected from the group consisting of atorvastatin (including atorvastatin+ezetimibe), rosuvastatin, cerivastatin, pitavastatin , fluvastatin, lovastatin, simvastatin (including simvastatin + ezetimibe), pravastatin and combinations thereof. The present invention also includes methods for lowering cholesterol, LDL-C or non-LDL-C levels in a patient with hypercholesterolemia and who is intolerant to statins or who otherwise develops adverse or undesirable reaction(s) to statin therapy (e.g., musculoskeletal -muscle pain, headache, weakness or spasms (for example, myalgia, myopathy, rhabdomyolysis, etc.).

Patient selection

The present invention includes methods and compositions useful for treating patients suffering from hyperlipidemia who do not respond to, do not achieve adequate control of, or are intolerant to treatment with standard lipid-modifying therapy. Patients who are amenable to treatment with the methods of the present invention may also have one or more additional selection criteria. For example, a patient may be selected for treatment with the methods of the present invention if the patient is diagnosed or found to be at risk for developing a hypercholesterolemic condition, such as heterozygous familial hypercholesterolemia (heFH), homozygous familial hypercholesterolemia (hoFH), autosomal dominant hypercholesterolemia (ADH, eg ADH associated with one or more gain-of-function mutations in the PCSK9 gene), autosomal recessive hypercholesterolemia (ARH, eg ARH associated with mutations in LDLRAP1), and cases of hypercholesterolemia other than familial hypercholesterolemia (nonFH). Diagnosis of familial hypercholesterolemia (eg, heFH or hoFH) can be made using genotyping and/or clinical criteria. For patients who have not been genotyped, the clinical diagnosis can be based on the Simon Broome criteria or the criteria for defining FH, or the WHO/Dutch Clinic Network criteria for lipid disorders with a score >8.

In accordance with some embodiments, a patient may be selected based on a history of coronary artery disease (CHD). As used herein, the term CHD history (or recorded CHD history) includes one or more of:

(i) acute myocardial infarction (MI);

(ii) asymptomatic IM;

(iii) unstable angina;

(iv) coronary revascularization procedures (eg, percutaneous coronary intervention [PCI] or coronary artery bypass graft [CABG] procedures); and/or (v) clinically significant CHD diagnosed by invasive or noninvasive testing (eg, coronary angiography, treadmill stress test, stress echocardiography, or nuclear imaging).

In accordance with some embodiments, a patient may be selected based on the presence of cardiovascular disease other than coronary artery disease (non-CHDCVD). As used herein, the term non-CHD-CVD includes one or more of:

(i) documented previous ischemic stroke with focal ischemic neurological deficit that persists for more than 24 hours, believed to be of atherothrombotic origin;

(ii) peripheral arterial disease;

(iii) abdominal aortic aneurysm;

(iv) atherosclerotic stenosis of the renal arteries; and/or (v) carotid artery disease (transient ischemic attacks or >50% carotid artery occlusion).

In some embodiments, the patient may be selected based on the presence of one or more additional risk factors, such as (i) documented moderate chronic kidney disease (CKD), as defined by 30<eGFR<60 mL/min. 1.73 ^m2 for 3 months or more; (ii) diabetes mellitus type 1 or type 2 with or without end organ damage (eg, retinopathy, nephropathy, microalbuminuria); (iii) estimated 10-year risk of fatal outcome from CVD SCORE >5% (ESC/EAS Guidelines for management of dyslipidemias, Conroy et al., 2003, Eur. Heart J. 24:987-1003).

In some embodiments, the patient may be selected based on the presence of one or more additional risk factors selected from the group consisting of age (e.g., over 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old) , race, national origin, gender (male or female), exercise habits (eg, regular exercise, non-regular exercise), other pre-existing medical conditions (eg, type 2 diabetes, high blood pressure, etc.) and current treatment status (eg, currently taking beta blockers, niacin, ezetimibe, fibrates, omega-3

- 6 043821 fatty acids, bile acids, etc.).

In accordance with some embodiments of the present invention, a subject who is amenable to treatment with the methods of the invention has elevated levels of one or more inflammatory markers. Any marker of systemic inflammation can be used for the purposes of the present invention. Suitable inflammatory markers include, but are not limited to, C-reactive protein, cytokines (eg, IL-6, IL-8 and/or IL-17), and cell adhesion molecules (eg, ICAM-1, ICAM-3, BL-CAM , LFA-2, VCAM-1, NCAM and RESAM).

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

In some other embodiments, a patient eligible for treatment with the methods of the present invention, in addition to hypercholesterolemia that is not adequately controlled by a therapeutic regimen of daily moderate dose statins, may also be selected based on the presence of heFH or non-FH without CHD or cardiovascular disease a disease other than CVD but which has (i) an estimated risk of fatal outcome due to CVD over the next 10 years SCORE >5%; or (ii) diabetes mellitus without end organ damage; such patients may also be selected based on serum LDL-C concentrations greater than or equal to 100 mg/dL.

In accordance with some embodiments of the present invention, the subject who is treatable by the methods of the invention is a subject who has familial chylomicronemia syndrome (FCS, also known as lipoprotein lipase deficiency).

In accordance with some embodiments of the present invention, a subject who is amenable to treatment with the methods of the invention is a subject who is undergoing or has recently undergone lipoprotein apheresis (e.g., within the past six months, within the past 12 weeks, within the past 8 weeks, within last 6 weeks, during the last 4 weeks, during the last 2 weeks, etc.).

Administration of PCSK9 inhibitor plus ANGPTL3 inhibitor as adjunctive therapy

The present invention includes methods of treatment wherein a PCSK9 inhibitor plus an ANGPTL3 inhibitor is administered to a patient who is undergoing or has recently been exposed to standard lipid-modifying therapy (eg, statin therapy) in accordance with a specified amount and dosing frequency, and wherein a PCSK9 inhibitor and an ANGPTL3 inhibitor are administered to the patient in addition to previous lipid-modifying therapy (if applicable), that is, as an addition to a previous therapeutic regimen of daily statins.

For example, the methods of the present invention include additional therapeutic regimens wherein a PCSK9 inhibitor and an ANGPTL3 inhibitor are administered as adjunctive therapy to a similar unchanged daily statin therapeutic regimen (i.e., a similar statin dosage) that the patient was on prior to taking the PCSK9 inhibitors and ANGPTL3. In yet other embodiments, PCSK9 and ANGPTL3 inhibitors are administered as adjunctive therapy to a statin therapeutic regimen comprising a statin in an amount that is greater or less than the statin dose the patient was on prior to taking the PCSK9 and ANGPTL3 inhibitors. For example, following initiation of a therapeutic regimen that includes a PCSK9 inhibitor and an ANGPTL3 inhibitor, which are administered at a specified frequency and dosage, the daily dose of statin administered or prescribed to the patient may (a) remain unchanged, (b) increase, or (c) decrease (eg , up-titration or down-dose titration) compared to the daily dose of statins the patient was taking before starting a therapeutic regimen with PCSK9 and ANGPTL3 inhibitors, depending on the patient's therapeutic needs.

Therapeutic efficacy

The methods of the present invention can result in a decrease in serum levels of one or more lipid components selected from the group consisting of total cholesterol, LDL-C, non-HDL-C, ApoB100, VLDL-C, triglycerides, Lp(a) and residual cholesterol. For example, in some embodiments of the present invention, administration of a PCSK9 inhibitor in combination with an ANGPTL3 inhibitor to a suitable subject will result in an average percentage reduction from baseline in serum low-density lipoprotein cholesterol (LDL-C) levels of at least about 25%, 30%, 40%, 50%, 60% or more; an average percentage reduction from baseline in ApoB100 of at least about 25%, 30%, 40%, 50%, 60% or more; an average percentage reduction from non-HDL-C baseline of at least about 25%, 30%, 40%, 50%, 60% or more; average interest

- 7 043821 reduction compared to the initial level of total cholesterol by at least about 10%, 15%, 20%, 25%, 30%, 35% or more; an average percentage reduction from baseline in VLDL-C of at least about 5%, 10%, 15%, 20%, 25%, 30% or more; an average percentage reduction from baseline in triglyceride levels of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or more; and/or an average percentage reduction from baseline in Lp(a) of at least about 5%, 10%, 15%, 20%, 25% or more.

PCSK9 inhibitors and ANGPTL3 inhibitors

The methods of the present invention include administering to a patient a therapeutic composition containing a PCSK9 inhibitor and an ANGPTL3 inhibitor.

PCSK9 inhibitors

As used herein, the term 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. Non-limiting examples of groups of PCSK9 inhibitors include small molecule antagonists of PCSK9, nucleic acid-based inhibitors of PCSK9 expression or activity (e.g., siRNA or antisense molecules), peptide-based molecules that specifically interact with PCSK9 (e.g., peptibodies), receptor molecules that specifically interact with PCSK9, proteins containing the ligand-binding fragment of the LDL receptor, PCSK9-binding scaffolds (e.g., darpins, HEAT repeat proteins, ARM repeat proteins, tetratripeptide repeat proteins, fibronectin-based scaffold constructs, and other scaffolds based on those found in the nature of protein repeats, etc. [see, for example, Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol., 22: 849-857 and references therein]), and aptamers or fragments thereof against PCSK9. In accordance with some embodiments, PCSK9 inhibitors that can be used in the context of the present invention are anti-PCSK9 antibodies or antigen-binding antibody fragments that specifically bind to human PCSK9.

As used herein, the term human proprotein convertase subtilisin/kexin type 9 (PCSK9) or human PCSK9 or hPCSK9, refers to PCSK9 having the nucleic acid sequence shown in SEQ ID NO: 22 and the amino acid sequence of SEQ ID NO: 23, or its biological active fragment.

ANGPTL3 inhibitors

As used herein, the term ANGPTL3 inhibitor is any agent that binds to or interacts with human ANGPTL3 and inhibits the normal biological function of ANGPTL3 in vitro or in vivo. Non-limiting examples of groups of ANGPTL3 inhibitors include small molecule antagonists of ANGPTL3, nucleic acid-based inhibitors of ANGPTL3 expression or activity (e.g., siRNA or antisense molecules), peptide-based molecules that specifically interact with ANGPTL3 (e.g., peptibodies), receptor molecules that specifically interact with ANGPTL3, ANGPTL3-binding scaffolds (e.g. Darpins, HEAT repeat proteins, ARM repeat proteins, tetratripeptide repeat proteins, fibronectin-based scaffold constructs and other naturally occurring protein repeat-based scaffolds, etc. [see, for example, Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol., 22: 849-857 and references cited there]) and aptamers or fragments thereof against ANGPTL3. In some embodiments, ANGPTL3 inhibitors that can be used in the context of the present invention are anti-ANGPTL3 antibodies or antigen-binding antibody fragments that specifically bind to human ANGPTL3.

As used herein, the term human angiopoietin-like protein-3 or human ANGPTL3 or hANGPTL3 refers to ANGPTL3 having the amino acid sequence SEQ ID NO: 1 (see also the sequence under accession number in NCBI NP 055310), or a biologically active fragment thereof.

As used herein, the term antibody is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: _CH 1, _CH 2 and _CH 3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain (C _L 1). The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), between which lie more conserved regions called framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In various embodiments, the FR of the anti-PCSK9 antibody (or antigen binding fragment thereof) may be identical to human germline sequences or may be

- 8 043821 modified naturally or artificially. A consensus amino acid sequence can be determined based on parallel analysis of two or more CDRs.

As used herein, the term antibody also includes antigen-binding fragments of complete antibody molecules. As used herein, the terms antigen binding region of an antibody, antigen binding fragment of an antibody, and the like include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

Antigen-binding antibody fragments can be prepared, for example, from complete antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and optionally constant regions of the antibody. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and processed chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant regions into a suitable configuration or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen binding fragments include:

(i) Fab fragments;

(ii) F (ab') ₂ -fragments;

(iii) Fd fragments;

(iv) Fv fragments;

(v) single chain Fv molecules (scFv);

(vi) dAb fragments and (vii) minimal recognition elements consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR) such as the CDR3 peptide) or a conformationally restricted peptide FR3-CDR3 -FR4.

The term antigen binding fragment as used herein also includes other genetically engineered molecules such as domain specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, tribodies, tetrabodies and minibodies, nanobodies (eg , monovalent nanobodies, bivalent nanobodies, etc.), small molecule modular protein (SMIP) immunotherapies, and shark IgNAR variable domains as used herein.

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

In some embodiments, an antigen binding fragment of an antibody may comprise at least one variable region covalently linked to at least one constant region. Non-limiting exemplary configurations of variable and constant regions that may be found in the antigen binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) V _H -C _H 1-C _H 2-C _H 3; (vi) V _H Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (xiii) V _l -C _h 2-C _h 3; and (xiv) V _L -C _L . In any configuration of variable and constant regions, including any of the above illustrative configurations, the variable and constant regions may be linked directly to each other or may be linked through a complete or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in flexible or semi-flexible linkage between adjacent variable and/or constant regions in a single polypeptide. In addition, the antigen binding fragment of an antibody of the present invention may include a homodimer or heterodimer (or other multimer) of any of the variable and constant region configurations noted above in non-covalent association with each other and/or with one or more monomeric V _H or V _L regions (eg disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments can be monospecific or polyspecific (eg, bispecific).

A polyspecific antigen-binding antibody fragment will typically contain at least two different variable regions, where each variable region is capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any polyspecific antibody format, including the exemplary bispecific antibody formats described herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using conventional techniques available in the art.

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

As used herein, the term human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may, however, include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), for example, in the CDR and, in particular, CDR3 . However, as used herein, the term human antibody is not intended to include antibodies in which CDR sequences derived from the germ line of other mammalian species, such as mouse, have been grafted onto human framework sequences. The term includes antibodies produced recombinantly in a non-human mammal or in cells of a non-human mammal. This term is not intended to include antibodies isolated or produced in the body of a human subject.

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

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

The frequency of occurrence of the second form in different intact IgG isotypes is due to, but is not limited to, structural differences associated with the isotype of the antibody hinge region. Substitution of one amino acid in the hinge region of human IgG4 can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology, 30: 105) to levels typically observed using the hinge region of human IgG1. The present invention covers antibodies having one or more mutations in the hinge region, C _H 2 or C _H 3, which may be desirable, for example, in production to increase the yield of the desired form of the antibody.

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

The term specifically binds or the like means that the antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining how specifically an antibody binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that specifically binds to PCSK9 or that specifically binds to ANGPTL3, as used in the context of the present invention, includes antibodies that bind to PCSK9 or ANGPTL3, or fragments thereof, with a K _D below about 1000 nM, below about 500 nM, below about 300 nM, below about 200 nM, below about 100 nM, below about 90 nM, below about 80 nM, below about 70 nM, below about 60 nM, below about 50 nM, below about 40 nM, below about 30 nM, below about 20 nM, below about 10 nM, below about 5 nM, below about 4 nM, below about 3 nM, below about 2 nM, below about 1 nM, or below about 0.5 nM, as measured by surface plasmon resonance . However, an isolated antibody that specifically binds to human PCSK9 or human ANGPTL3 is cross-reactive to other antigens, such as PCSK9 molecules or ANGPTL3 molecules from other (non-human) species.

The anti-PCSK9 antibody and anti-ANGPTL3 antibody useful in the methods of the present invention may contain one or more amino acid substitutions, insertions and/or deletions in the framework region and/or CDR regions of the heavy and light chain variable regions compared to the corresponding germline sequences from which antibodies are derived. Such mutations can be easily determined by comparing the amino acid sequences described here with germline sequences available, for example, in publicly available antibody sequence databases. The present invention includes methods involving the use of antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions have been mutated to the corresponding germline sequence residue(s). line from which the antibody is derived, or into the corresponding residue(s) of another human germline sequence, or subjected to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as germline mutations). One of ordinary skill in the art, starting with the heavy and light chain variable region sequences described herein, can readily produce numerous antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In some embodiments, all residues of the framework region and/or CDR regions in the VH and/or _VL regions are reverse mutated to residues found in the original germline sequence from which the antibody is derived. In still other embodiments, only certain residues are backmutated into the original germline sequence, for example, only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In yet other embodiments, one or more framework residues and/or CDR regions are mutated to the corresponding residue(s) of another germline sequence (ie, a germline sequence that is different from the germline sequence from which the antibody is originally derived). In addition, the antibodies of the present invention may contain any combination of two or more germline mutations in the framework region and/or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of a certain germline sequence, while certain other residues that differ from the original germline sequence, are retained, or are mutated to the corresponding residue of another germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be readily tested for one or more desirable properties, such as increased binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate) , reduced immunogenicity, etc. The use of antibodies and antigen binding fragments obtained by this general method is covered by the present invention.

The present invention also includes methods comprising the use of an anti-PCSK9 antibody and an anti-ANGPTL3 antibody containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences described herein with one or more conservative substitutions. For example, the present invention includes the use of an anti-PCSK9 antibody and an anti-ANGPTL3 antibody having the amino acid sequences HCVR, LCVR and/or CDRs, for example, with 10 or less, 8 or less, 6 or less, 4 or less, etc. d. conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

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

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

- 11 043821

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

In accordance with some embodiments, the anti-PCSK9 antibody and anti-ANGPTL3 antibody used in the methods of the present invention are antibodies with pH-dependent binding characteristics. As used herein, the expression pH-dependent binding characteristics means that the antibody or antigen-binding fragment thereof exhibits reduced binding to PCSK9 at acidic pH compared to neutral pH (for the purposes of this description, both expressions can be used interchangeably) or that the antibody or antigen-binding fragment thereof exhibit reduced binding to ANGPTL3 at acidic pH compared to neutral pH (for the purposes of this description, both expressions can be used interchangeably). For example, antibodies with pH-dependent binding characteristics include antibodies and antigen binding fragments thereof that bind to PCSK9 or ANGPTL3 with higher affinity at neutral pH than at acidic pH. In some embodiments, the antibodies and antigen binding fragments of the present invention bind to PCSK9 or ANGPTL3 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 with higher affinity at neutral pH than at acidic pH.

In accordance with this aspect of the invention, an anti-PCSK9 antibody and an anti-ANGPTL3 antibody with pH-dependent binding characteristics may have one or more amino acid changes relative to the parent anti-PCSK9 antibody or the parent anti-ANGPTL3 antibody. For example, an anti-PCSK9 antibody or an anti-ANGPTL3 antibody with pH-dependent binding characteristics may contain one or more histidine substitutions or insertions, for example, in one or more CDRs of the parent anti-PCSK9 antibody or the parent anti-ANGPTL3 antibody. Thus, in accordance with some embodiments of the present invention, methods are provided including administering an anti-PCSK9 antibody and an anti-ANGPTL3 antibody that contain CDR amino acid sequences (e.g., heavy and light chain CDRs) that are identical to the CDR amino acid sequences of the parent anti -PCSK9 antibody or the parent anti-ANGPTL3 antibody, except for the replacement of one or more amino acids of one or more CDRs of the parent antibody with a histidine residue. Anti-PCSK9 antibodies or anti-ANGPTL3 pH-dependent binding antibodies may contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more histidine substitutions within a single CDR or distributed in multiple ( for example, 2, 3, 4, 5 or 6) CDR of the original anti-PCSK9 antibody or the original anti-ANGPTL3 antibody. For example, the present invention includes the use of anti-PCSK9 antibodies and anti-ANGPTL3 antibodies with pH-dependent binding comprising one or more histidine substitutions in HCDR1, one or more histidine substitutions in HCDR2, one or more histidine substitutions in HCDR3, one or more histidine substitutions in LCDR1, one or more histidine substitutions in LCDR2 and/or one or more histidine substitutions in LCDR3 of the original anti-PCSK9 antibody or the original anti-ANGPTL3 antibody.

As used herein, the expression acidic pH means a pH value of 6.0 or lower (eg, below about 6.0, below about 5.5, below about 5.0, etc.). The expression acidic pH value includes pH values of approximately 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. As used herein, the term neutral pH means a pH of about 7.0 to about 7.4. The expression neutral pH value includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

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

A non-limiting example of an anti-ANGPTL3 antibody that can be used in the context of the present invention includes evinacumab.

Obtaining human antibodies

Anti-PCSK9 antibodies and anti-ANGPTL3 antibodies can be prepared according to any antibody production/isolation method known in the art. For example, antibodies for use in the methods of the present invention can be produced by hybridoma technologies, phage display, yeast display, etc. Antibodies for use in the methods of the present invention may be, for example, chimeric antibodies, humanized antibodies, or fully human antibodies.

- 12 043821

Methods for producing human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to obtain human antibodies that specifically bind to PCSK9 or ANGPTL3.

For example, using VELOCIMMUNE™ technology (see, for example, US Patent No. 6594541, Regeneron Pharmaceuticals) or any other known method for producing monoclonal antibodies, high-affinity chimeric antibodies to PCSK9 or ANGPTL3 with a human variable region and a mouse constant region are initially isolated. VELOCIMMUNE® technology involves producing a transgenic mouse having a genome containing human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody containing the human variable region and the mouse constant region in response to antigenic stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, a VELOCIMMUNE® mouse is stimulated with the antigen of interest and lymphatic cells (such as B cells) from the mice that express antibodies are isolated. Lymphatic cells can be fused with cells of a myeloma lineage to generate immortal hybridoma cell lines, and such hybridoma cell lines are processed and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the desired isotype heavy chain and light chain constant regions. Such an antibody protein can be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

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

In general, antibodies that can be used in the methods of the present invention have high affinity, as described above, when measured by binding to antigen immobilized on a solid phase or in a solution phase. The mouse constant regions are replaced with the desired human constant regions to produce fully human antibodies of the invention. Although the selected constant region may vary depending on the particular application, the high affinity antigen binding characteristics and target binding specificity are in the variable region.

Specific examples of human antibodies or antigen-binding antibody fragments that specifically bind to PCSK9 that can be used in the context of the methods of the present invention include antibodies or antigen-binding proteins containing six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of the amino acid pair heavy and light chain variable region (HCVR/LCVR) sequences comprising SEQ ID NO: 12/17.

In some embodiments of the present invention, an anti-PCSK9 antibody or antigen binding fragment thereof that can be used in the methods of the present invention comprises heavy and light chain complementarity determining regions (HCDR1-HCDR2HCDR3/LCDR1-LCDR2-LCDR3) comprising the amino acid sequences of SEQ ID NO: 13, 14, 15, 18, 19 and 21.

In some embodiments of the present invention, an anti-PCSK9 antibody or antigen binding fragment thereof that can be used in the methods of the present invention comprises an HCVR having the amino acid sequence of SEQ ID NO: 12 and an LCVR having the amino acid sequence of SEQ ID NO: 17.

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

In some embodiments of the present invention, an anti-ANGPTL3 antibody or antigen binding fragment thereof that can be used in the methods of the present invention comprises heavy and light chain complementarity determining regions (HCDR1-HCDR2-13043821

HCDR3/LCDR1-LCDR2-LCDR3), including amino acid sequences SEQ ID NO: 4, 5, 6,

7, 8 and 9.

In some embodiments of the present invention, an anti-ANGPTL3 antibody or antigen binding fragment thereof that can be used in the methods of the present invention comprises an HCVR having the amino acid sequence of SEQ ID NO: 2 and an LCVR having the amino acid sequence of SEQ ID NO: 3.

Pharmaceutical compositions and methods of administration

The present invention includes methods that include administering a PCSK9 inhibitor to a patient in combination with an ANGPTL3 inhibitor, wherein the PCSK9 inhibitor and the ANGPTL3 inhibitor are contained in the same or different pharmaceutical compositions. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients and other agents that provide suitable transport, delivery, tolerability and the like. Numerous suitable formulations can be found in a reference book familiar to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions and emulsions water-in-oil, emulsions based on carbowasc (polyethylene glycols with different molecular weights), semi-solid gels and semi-solid mixtures containing carbowasc. See also Powell et al. Compendium of excipients for parenteral formulations, PDA (1998) J Pharm Sci Technol 52:238–311.

Examples of pharmaceutical compositions containing anti-PCSK9 antibodies and/or anti-ANGPTL3 antibodies that can be used in the context of the present invention include any of the compositions described in US Pat. No. 8,795,669 (which describes, among other things, exemplary compositions containing alirocumab) or in WO2013/166448 or WO 2012/168491.

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

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a conventional needle or syringe. Moreover, with regard to subcutaneous delivery, a pen-type device is readily applicable for delivery of the pharmaceutical composition of the present invention. Such a pen-type device may be a reusable or disposable device. The reusable pen-type device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition in the cassette has been administered and the cassette has been emptied, the empty cassette can be easily removed and replaced with a new cassette that contains the pharmaceutical composition. The pen-shaped device can then be reused. The disposable pen-shaped device does not have a replaceable cartridge. However, the disposable pen-type device comes pre-filled with a pharmaceutical composition contained in a reservoir in the device. Once the pharmaceutical composition reservoir is empty, the entire device is discarded.

Numerous pen or refillable auto-injector delivery systems are useful for subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ 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), to name a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park IL), to name a few of them.

In some situations, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer,

- 14 043821 see Sefton, 1987, CRC Crit. Ref. Biomed, pp. 14: 201). In another embodiment, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system may be located close to the target of the composition, resulting in only a portion of the systemic dose being required (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115- 138). Other controlled release systems are discussed in Langer, 1990, Science, 249:1527-1533.

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

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in unit dosage form providing a dose of the active ingredients. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

Dosage

The amount of a PCSK9 inhibitor (eg, anti-PCSK9 antibody) or ANGPTL3 inhibitor (eg, anti-ANGPTL3 antibody) administered to a subject in accordance with the methods of the present invention is generally a therapeutically effective amount. As used herein, the expression therapeutically effective amount of a PCSK9 inhibitor means a dose of a PCSK9 inhibitor, when administered in combination with an ANGPTL3 inhibitor, resulting in a detectable reduction (at least about 5%, 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to the baseline period) of one or more indicators selected from the group consisting of total cholesterol, LDL- C, ApoB100, non-HDL-C, VLDL-C, triglycerides, Lp(a) and residual cholesterol, or an amount that reduces or eliminates the patient's need for other therapeutic interventions, such as lipoprotein apheresis, or that reduces the patient's normal level apheresis.

For an anti-PCSK9 antibody, a therapeutically effective amount may be from about 0.05 to about 600 mg, such as 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 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, 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 mg or about 600 mg anti-PCSK9 antibody. In some exemplary embodiments of the present invention, the therapeutically effective amount of the anti-PCSK9 antibody is 75 mg, 150 mg, or 300 mg (eg, alirocumab) or 140 mg or 420 mg (eg, evolocumab). Other dosages of PCSK9 inhibitors will be apparent to those skilled in the art and are contemplated within the scope of the present invention.

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

The amount of ANGPTL3 inhibitor (eg, anti-ANGPTL3 antibody) administered to a subject in accordance with the methods of the present invention will generally be a therapeutically effective amount. As used herein, the term therapeutically effective amount of an ANGPTL3 inhibitor means a dose of an ANGPTL3 inhibitor, in combination with a PCSK9 inhibitor, resulting in a detectable reduction (at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,

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45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to the baseline period) of one or more indicators selected from the group consisting of total cholesterol, LDL-C, ApoB100, non- HDL-C

VLDL-C, triglycerides, Lp(a) and residual cholesterol, or an amount that prevents or attenuates atherosclerosis in a subject (as described elsewhere herein).

For an anti-ANGPTL3 antibody, a therapeutically effective amount may be from about 0.05 to about 600 mg, such as 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 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, 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, or about 600 mg anti-ANGPTL3 antibody. Other dosages of ANGPTL3 inhibitors will be apparent to those skilled in the art and are contemplated within the scope of the present invention.

The amount of anti-ANGPTL3 antibody contained in individual doses may be expressed in units of milligrams of antibody per kilogram of patient body weight (ie, mg/kg). For example, an anti-ANGPTL3 antibody may be administered to a patient at a dose of from about 0.0001 to about 10 mg/kg of the patient's body weight.

Combined treatment

As described elsewhere herein, the methods of the present invention may involve administering a PCSK9 inhibitor in combination with an ANGPTL3 inhibitor to a patient who is refractory to, does not achieve adequate control of, or is intolerant to standard lipid-lowering therapy. In some embodiments, the need for further administration of lipid-lowering therapy may be completely eliminated. In some embodiments, the combination use of a PCSK9 inhibitor with an ANGPTL3 inhibitor can be used in combination with (in addition to) the patient's previously prescribed lipid-lowering therapy. For example, in the context of reducing at least one parameter of lipid/lipoprotein metabolism in a patient suffering from hyperlipidemia (eg, hypercholesterolemia), where the patient is refractory to, does not achieve adequate control of, or does not tolerate standard lipid-lowering therapy, the combination of a PCSK9 inhibitor with an ANGPTL3 inhibitor may be used. administered to the patient in combination with an unchanged therapeutic regimen of daily statins. Exemplary daily statin therapeutic regimens with which a PCSK9 inhibitor plus an ANGPTL3 inhibitor may be administered in the context of the present invention include, for example, atorvastatin (10, 20, 40, or 80 mg daily) (atorvastatin/ezetimibe 10/10 or 40/10 mg every day), rosuvastatin (5, 10 or 20 mg every day), cerivastatin (0.4 or 0.8 mg every day), pitavastatin (1, 2 or 4 mg every day), fluvastatin (20, 40 or 80 mg every day), simvastatin (5, 10, 20, 40 or 80 mg every day), simvastatin/ezetimibe (10/10, 20/10, 40/10 or 80/10 mg every day), lovastatin (10, 20, 40 or 80 mg every day), pravastatin (10, 20, 40 or 80 mg every day) and combinations thereof. Other lipid-modifying therapeutic regimens with which a PCSK9 inhibitor plus an ANGPTL3 inhibitor can be administered in the context of the present invention include, for example, (1) an agent that inhibits cholesterol uptake and/or bile acid reabsorption (eg, ezetimibe); (2) an agent that enhances lipoprotein catabolism (such as niacin); and/or (3) activators of the transcription factor LXR, which plays a role in the elimination of cholesterol, such as 22-hydroxycholesterol.

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

A non-limiting example of an anti-ANGPTL3 antibody for use in the context of the present invention includes evinacumab.

Administration modes

In accordance with some embodiments of the present invention, a subject may be administered multiple doses of a PCSK9 inhibitor (e.g., a pharmaceutical composition containing a PCSK9 inhibitor) and an ANGPTL3 inhibitor (i.e., a pharmaceutical composition containing an ANGPTL3 inhibitor) over a period of time (e.g., in addition to to a therapeutic regimen of daily statins or other background lipid-modifying therapy). Methods in accordance with this aspect of the invention involve sequentially administering multiple doses of a PCSK9 inhibitor and an ANGPTL3 inhibitor to a subject. As used herein, the term sequential administration means that each dose of PCSK9 inhibitor and ANGPTL3 inhibitor is administered to a subject at a different time point, eg, on different days separated by a predetermined interval (eg, hours, days, weeks or months). The present invention includes methods that include sequentially administering to a patient one initial dose of a PCSK9 inhibitor and an ANGPTL3 inhibitor, followed by one or more secondary doses of a PCSK9 inhibitor and an ANGPTL3 inhibitor, and optionally followed by one or more tertiary doses of a PCSK9 inhibitor and an ANGPTL3 inhibitor.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration of individual doses of a pharmaceutical composition containing a PCSK9 inhibitor and an ANGPTL3 inhibitor. Thus, the starting dose is the dose that is administered at the beginning of the treatment regimen (also called the initial dose); secondary doses are doses that are administered after the initial dose; and tertiary doses represent doses that are administered after secondary doses. Initial, secondary and tertiary doses may contain the same amount of PCSK9 inhibitor and ANGPTL3 inhibitor, but may generally differ in frequency of administration. However, in some embodiments, the amounts of PCSK9 inhibitor and ANGPTL3 inhibitor contained in the initial, secondary, and/or tertiary doses are different from each other (eg, they may be adjusted up or down as needed) over the course of treatment. In some embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses, followed by doses that are administered less frequently (eg, maintenance doses).

In accordance with illustrative embodiments of the present invention, each secondary and/or tertiary dose is administered from 1 to 26 (eg, 1, 1, 2, 2, 2, 3, 3, 4, 4, 5, 5, 6, 6 ^{1/ 2} , 7, 7 ^1/2 , 8, 8 ^1/2 , 9, 9 ^1/2 , 10, 10 ^1/2 , 11, 11 ^1/2 , 12, 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 ^1/2 or more) weeks after the immediately preceding dose. As used herein, the expression immediately preceding dose means, in a multiple dosing sequence, the dose of antigen binding molecule that is administered to a patient prior to administration of the immediately next dose in the sequence, with no intervening doses.

Methods in accordance with this aspect of the invention may include administering to a patient any number of secondary and/or tertiary doses of a PCSK9 inhibitor and an ANGPTL3 inhibitor. For example, in some embodiments, only one secondary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses. Likewise, in some embodiments, only one tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dose can 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 immediately after the previous dose. Likewise, in embodiments involving multiple tertiary doses, each tertiary dose can 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 immediately after the previous dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to the patient may vary during the course of treatment. During treatment, the physician will also adjust the frequency of administration depending on the needs of the individual patient after a clinical examination.

Examples

The following examples are provided to provide those skilled in the art with a complete disclosure and description of methods for making and using the methods and compositions of the invention, and are not intended to limit the scope of what applicants consider to be the invention. Attempts have been made to ensure the accuracy of the numbers used (eg quantities, temperatures, etc.), however some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1. Preparation of human antibodies to human PCSK9.

Human anti-PCSK9 antibodies were prepared as described in US Pat. No. 8,062,640. An exemplary PCSK9 inhibitor used in the following example is a human anti-PCSK9 antibody designated H1H316P, also known as alirocumab or PRAULENT®. H1H316P has the following amino acid sequence characteristics: a heavy chain containing SEQ ID NO: 16 and a light chain containing SEQ ID NO: 20; a heavy chain variable region (HCVR) comprising SEQ ID NO: 12 and a light chain variable region (LCVR) comprising SEQ ID NO: 17; heavy chain complementarity determining region 1 (HCDR1) containing SEQ ID NO: 13, HCDR2 containing SEQ ID NO: 14, HCDR3 containing SEQ ID NO: 15, light chain complementarity determining region 1 (LCDR1) containing SEQ ID NO: 18, LCDR2 containing SEQ ID

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Claims (23)

N0: 19 and LCDR3 containing SEQ ID NO: 21. Example 2. Preparation of human antibodies to human ANGPTL3. Human anti-ANGPTL3 antibodies were prepared as described in US Pat. No. 9,018,356. An exemplary ANGPTL3 inhibitor used in the following example is a human anti-ANGPTL3 antibody designated H4H276S, also known as evinacumab. H4H276S has the following amino acid sequence characteristics: a heavy chain containing SEQ ID NO: 10 and a light chain containing SEQ ID NO: 11; a heavy chain variable region (HCVR) comprising SEQ ID NO: 2 and a light chain variable region (LCVR) comprising SEQ ID NO: 3; heavy chain complementarity determining region 1 (HCDR1) containing SEQ ID NO: 4, HCDR2 containing SEQ ID NO: 5, HCDR3 containing SEQ ID NO: 6, light chain complementarity determining region 1 (LCDR1) containing SEQ ID NO: 7, LCDR2 containing SEQ ID NO: 8 and LCDR3 containing SEQ ID NO: 9. Example 3. Effect of in vivo treatment with a combination of anti-hANGPTL3 antibody and anti-PCSK9 antibody on circulating lipid levels in hyperlipidemic Ldlr-/+ mice. The effects of one anti-hANGPTL3 antibody H4H1276 (evinacumab), one anti-PCSK9 antibody H1H316P (alirocumab) and both antibodies in combination on serum lipid levels were determined in LDLR-/+ mice. These mice have hyperlipidemia primarily with circulating cholesterol in the form of LDL due to partial deficiency of LDLR, the major LDL-C uptake receptor. In the first study, blood samples were pre-collected from male LDLR-/+ mice on a balanced diet 5 days before the start of the experiment, and the mice were divided into groups of 5 mice each. Antibodies H4H1276P, H1H316P, their combination and an isotype-matched control (hIgG4) with irrelevant specificity were administered in each case at a dose of 10 mg/kg by subcutaneous injection on day 0 of the experiment. Blood samples were collected from mice after 4 hours of fasting on consecutive days after antibody administration, and serum lipid levels (total cholesterol, LDL-C, non-HDL-C, and HDL-C) were determined on an ADVIA® 1800 Chemistry System (Siemens ). Mean values were calculated for each group at each time point. The results, expressed as mean ± SEM, serum lipid concentrations are shown in FIG. 1, 2, 3 and 4. In the second experiment, male LDLR−/+ mice were placed on a high-fat Western diet for 3 weeks before antibody injection, and the mice were fed this diet throughout the study period. The rest of the experiment was carried out according to a protocol similar to that described for the first experiment. The results, expressed as mean ± SEM, serum lipid concentrations are shown in FIG. 5, 6, 7 and 8. Levels of circulating antibodies (serum Ab) in both experiments were determined using a standard ELISA assay. Briefly, plates were coated with goat anti-human Fc antibody (Sigma-Aldrich) to capture serum Abs. Serum was then added to the plates, and captured antibodies were detected by chemiluminescence using horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Sigma-Aldrich). The results, expressed as mean ± SEM, are shown in Tables 1A and 1B (first experiment) and Tables 2A and 2B (second experiment). Control: mice treated with an isotype-matched control Ab. Summary of results Administration of the combination of H1H316R and H4H1276R as single subcutaneous doses to LDLR-/+ mice fed a balanced diet and a high-fat Western diet resulted in significant reductions in total cholesterol, LDL-C, and non-HDL-C and had an additive effect on lipid levels in blood serum compared with the corresponding separate administration of each antibody. CLAIM 1. A method of treating a patient suffering from hypercholesterolemia, where the patient is refractory to, does not achieve adequate control with, or cannot tolerate treatment with a statin, fibrates, niacin, bile acid sequestrants, ezetimibe, lomitapide, phytosterols, or orlistat, wherein the method includes treating the patient with a combination of a proprotein convertase inhibitor subtilisin /kexin type 9 (PCSK9) and angiopoietin-like protein 3 (ANGPTL3) inhibitor, where the PSCK9 inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to PCSK9, and the ANGPTL3 inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to ANGPTL3, and anti The -PCSK9 antibody is selected from the group consisting of alirocumab, evolocumab, bococizumab, lodelcizumab and ralpancizumab, and wherein the antibody or antigen binding fragment thereof that specifically binds to ANGPTL3 contains complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) having an amino acid sequence SEQ ID NO: 2, and the light chain variable region (LCVR) CDR with - 18 043821 consequently SEQ ID NO: 3. 2. The method according to claim 1, wherein the hypercholesterolemia is heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH). 3. The method of claim 1, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 75 mg at a frequency of once every two weeks. 4. The method of claim 1, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 140 mg at a frequency of once every two weeks. 5. The method of claim 1, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 150 mg at a frequency of once every two weeks or once every four weeks. 6. The method of claim 1, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 300 mg at a frequency of once every four weeks. 7. The method of claim 1, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 420 mg at a frequency of once every four weeks. 8. The method according to any one of claims 1 to 7, wherein the antibody or antigen binding fragment thereof that specifically binds to PCSK9 comprises heavy chain variable region (HCVR) complementarity determining regions (CDRs) having the amino acid sequence of SEQ ID NO: 12, and Light chain variable region (LCVR) CDR with sequence SEQ ID NO: 17. 9. The method according to any one of claims 1 to 8, wherein the antibody or antigen binding fragment thereof that specifically binds to PCSK9 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence of SEQ ID NO: 13, HCDR2 having the amino acid sequence of SEQ ID NO: : 14, HCDR3 having the amino acid sequence SEQ ID NO: 15, light chain CDR1 (LCDR1) having the amino acid sequence SEQ ID NO: 18, LCDR2 having the amino acid sequence SEQ ID NO: 19, and LCDR3 having the amino acid sequence SEQ ID NO: : 21. 10. The method according to any one of claims 1 to 9, wherein the antibody or antigen binding fragment thereof that specifically binds to PCSK9 comprises an HCVR having the amino acid sequence of SEQ ID NO: 12 and an LCVR having the amino acid sequence of SEQ ID NO: 17. 11. The method according to any one of claims 1 to 10, wherein the anti-PCSK9 antibody is alirocumab. 12. The method according to claim 1, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is administered to the patient subcutaneously or intravenously. 13. The method of claim 1, wherein the anti-ANGPTL3 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 150 mg once a week. 14. The method of claim 1, wherein the anti-ANGPTL3 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 300 mg once a week. 15. The method of claim 1, wherein the anti-ANGPTL3 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 450 mg at a frequency of once per week. 16. The method of claim 1, wherein the anti-ANGPTL3 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 300 mg at a frequency of once every two weeks. 17. The method of claim 1, wherein the anti-ANGPTL3 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 450 mg at a frequency of once every two weeks. 18. The method of claim 1, wherein the anti-ANGPTL3 antibody or antigen-binding fragment thereof is administered to the patient at a dose of approximately 20 mg/kg at a frequency of once every four weeks. 19. The method according to any one of claims 1 to 18, wherein the anti-ANGPTL3 antibody or antigen binding fragment thereof comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence of SEQ ID NO: 4, HCDR2 having the amino acid sequence of SEQ ID NO: 5, HCDR3 having the amino acid sequence of SEQ ID NO: 6, light chain CDR1 (LCDR1) having the amino acid sequence of SEQ ID NO: 7, LCDR2 having the amino acid sequence of SEQ ID NO: 8, and LCDR3 having the amino acid sequence of SEQ ID NO: 9. 20. The method according to any one of claims 1 to 19, wherein the anti-ANGPTL3 antibody or antigen binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 2, and an LCVR having the amino acid sequence of SEQ ID NO: 3. 21. The method according to any one of claims 1 to 20, wherein the anti-ANGPTL3 antibody is evinacumab. 22. The method according to claim 1, wherein the anti-ANGPTL3 antibody or antigen-binding fragment thereof is administered to the patient subcutaneously or intravenously. 23. The method according to any one of claims 1 to 22, where the combination of a PCSK9 inhibitor and an ANGPTL3 inhibitor is designed to reduce one or more of the following indicators: (a) reduction in the level of total cholesterol in the blood serum (TC); (b) reduction in serum low-density lipoprotein cholesterol (LDL-C); or (c) reducing serum non-high-density lipoprotein cholesterol (non-HDL-C); where the decrease in indicators (a), (b) and/or (c) is determined relative to the level of TC in blood serum