RU2772712C2 - Methods for treatment of hyperlipidemia in patients with diabetes by injection of pcsk9 inhibitor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to the use of recombinant antibodies that specifically bind human proprotein convertase of subtilisin/kexin type 9 (PCSK9); it can be used in medicine for the treatment of patients with a high cardiovascular risk, suffering from hypercholesterinemia and diabetes mellitus type 1 or 2, receiving a therapeutic agent based on insulin.

EFFECT: invention allows for safe and effective reduction in the LDL-C level in patients with diabetes mellitus type 1 or 2 that have a high cardiovascular risk and a high LDL-C level despite the treatment with the maximum tolerated dose of statins.

19 cl, 5 dwg, 16 tbl, 3 ex

Description

RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application No. 62/517672, filed June 9, 2017, U.S. Provisional Application No. 62/532,162, filed July 13, 2017, and European Patent Application No. 18305565.6, filed May 4, 2018. The contents of each of these related applications are incorporated herein by reference in their entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The present invention relates to the field of therapeutic effects on diseases and disorders that are associated with elevated levels of lipids and lipoproteins. More specifically, the present invention relates to the use of PCSK9 inhibitors for the treatment of diabetic patients suffering from hyperlipidemia, including hypercholesterolemia.

BACKGROUND OF THE INVENTION

[0003] Hyperlipidemia is a general term that encompasses diseases and disorders characterized by or associated with elevated levels of lipids and/or lipoproteins in the blood. Types of hyperlipidemia include hypercholesterolemia, hypertriglyceridemia, combined hyperlipidemia, and elevated lipoprotein A (Lp(a)). A particularly common form of hyperlipidemia in many populations is hypercholesterolemia.

[0004] Hypercholesterolemia, in particular, increased levels of low-density lipoprotein (LDL) cholesterol (LDL-C), is 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 is identified as the main target of cholesterol-lowering therapy and is accepted as a suitable surrogate endpoint of therapy. Numerous studies have demonstrated that lowering LDL-C levels reduces the risk of CHD, and there is a strong positive relationship between LDL-C levels and CHD events; For every 1 mmol/L (~40 mg/dL) LDL-C level decrease, CVD mortality and CVD incidence are reduced by 22%. Larger reductions in LDL-C lead to greater reductions in CHD events, and comparative data from intensive versus standard statin treatment indicate that the lower the LDL-C level, the greater the benefit for patients at very high risk of CVD. ) diseases.

[0005] Cardiovascular disease (CVD) is a major cause of morbidity and mortality in patients with type 1 (T1) or type 2 (T2) diabetes mellitus (DM), and in patients with diabetes treated with insulin, the risk of CV is still above. In addition, the presence of comorbid DM among patients with atherosclerotic CVD (ASCVD) significantly increases the risk of CV events. Several studies and meta-analyses have shown that lowering LDL-C levels with statin use leads to a significant reduction in CV events in patients with DM, with a further reduction in CV risk associated with additional lowering of LDL-C levels with the use of ezetimibe as a concomitant drug. However, even with currently available therapies, many patients with DM remain permanently impaired in lipid metabolism and therefore are at residual risk of CV events.

[0006] Current LDL-C lowering drugs include subtilisin/kexin type 9 (PCSK9) proprotein convertase inhibitors, such as anti-PCSK9 antibodies. Although PCSK9 antibodies have been the subject of extensive clinical trials, the efficacy and safety of alirocumab in the diabetic population has not been fully established. Thus, there is a need in the art to identify anti-PCSK9 antibody regimens that provide optimal efficacy and safety in the treatment of hypercholesterolemia in patients with diabetes receiving an insulin-based therapy who are at high risk for CV.

BRIEF DESCRIPTION OF THE INVENTION

[0007] The present invention provides methods for treating hypercholesterolemia in a patient with diabetes mellitus (DM) receiving an insulin-based therapy. In some embodiments, the methods involve administering one or more doses of an antibody or antigen-binding fragment thereof that specifically binds human PCSK9 to a patient with hypercholesterolemia and diabetes. In certain embodiments, the patient is at high cardiovascular risk. In certain embodiments, the patient is receiving a concomitant antidiabetic therapy in addition to the insulin based therapy.

[0008] According to one aspect, the methods provide a method for treating hypercholesterolemia in a patient with type 1 diabetes mellitus (T1DM), the method comprising

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has (i) T1DM and (ii) hypercholesterolemia not adequately controlled by a maximum tolerated dose statin-based therapy; and

(b) administering to the patient 75 mg, 150 mg, or 300 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase, wherein the patient receives concomitant insulin therapy.

[0009] In certain embodiments, the patient is administered 75 mg of the antibody or antigen-binding fragment every two weeks. In other embodiments, the patient is administered 150 mg of the antibody or antigen-binding fragment every two weeks. In other embodiments, the patient is administered 300 mg of the antibody or antigen-binding fragment every four weeks.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs from the HCVR/LCVR amino acid sequence pair comprising SEQ ID NO: 1/6. In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs listed under SEQ ID NOs: 2, 3, and 4 and three light chain CDRs listed under SEQ ID NOs: 7, 8, and 10. In some embodiments, the antibody or its antigen-binding fragment comprise a heavy chain variable region (HCVR) with the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (LCVR) with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to an antibody or antigen-binding fragment thereof that comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope on PCSK9 that and an antibody containing HCVR with the amino acid sequence of SEQ ID NO: 1 and LCV R with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope on PCSK9 that overlaps with an epitope of an antibody comprising HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequences of the heavy and light chain CDRs having SEQ ID NOs: 86, 87, 88, 90, 91, and 92. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR with an amino acid sequence at least 90%, 95% or 99% identical to the amino acid sequence shown under SEQ ID NO:85, and LCVR with an amino acid sequence identical to at least 90%, 95% or 99% of the amino acid sequence given under SEQ ID NO:89.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, and LY3015014. In some embodiments, the antibody or antigen-binding fragment thereof is alirocumab.

[0013] In certain embodiments, the method disclosed herein further comprises (c) administering to the patient one or more subsequent doses of 75 mg of the antibody or antigen-binding fragment approximately every two weeks if, for example, after 8 weeks, the level The patient's LDL-C is below the threshold, or one or more subsequent doses of 150 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if, for example, after 8 weeks, the patient's LDL-C level equals or exceeds the threshold . In some embodiments, the threshold level is 70 mg/dl.

[0014] In certain embodiments, the method disclosed herein further comprises (c) administering to the patient one or more subsequent doses of 300 mg of the antibody or antigen-binding fragment approximately every four weeks if, for example, after 8 weeks, the level The patient's LDL-C is below the threshold, or one or more subsequent doses of 150 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if, for example, after 8 weeks, the patient's LDL-C level equals or exceeds the threshold . In some embodiments, the threshold level is 70 mg/dL.

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof is administered subcutaneously.

[0016] In certain embodiments, the patient additionally receives a concomitant lipid corrective therapy (LMT). In some embodiments, the LMT is selected from the group consisting of a statin, a cholesterol absorption inhibitor, a fibrate, a niacin, an omega-3 fatty acid, and a bile acid sequestrant. In certain embodiments, the LMT is a statin-based therapy. In some embodiments, the statin is selected from the group consisting of atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin, and cerivastatin. In certain embodiments, the statin therapy is a maximum tolerated statin therapy. In some embodiments, the cholesterol absorption inhibitor is ezetimibe. In some embodiments, the implementation of the patient suffers from intolerance to a statin.

[0017] In some embodiments, the insulin therapy is selected from the group consisting of human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degludec, insulin aspart, and basal insulin. In some embodiments, the patient additionally receives an additional concomitant antidiabetic therapy in addition to the insulin based therapy. In some embodiments, the additional antidiabetic agent is selected from the group consisting of a glucagon-like peptide 1 (GLP-1) therapy, a gastrointestinal peptide, a glucagon receptor agonist or antagonist, a glucose-dependent insulinotropic polypeptide (GIP) receptor agonist or antagonist, an antagonist or inverse agonist ghrelin, xenin, xenin analog, biguanide, sulfonylurea, meglitinide, thiazolidinedione, DPP-4 inhibitor, alpha-glucosidase inhibitor, sodium-dependent glucose transporter 2 (SGLT-2) inhibitor, SGLT-1 inhibitor, agonist or receptor modulator peroxisome proliferator-activated (PPAR-) (alpha, gamma, or alpha/gamma), amylin, amylin analog, G protein-coupled receptor 119 (GPR119) agonist, GPR40 agonist, GPR120 agonist, GPR142 agonist, systemic or poorly absorbed TGR5 agonist , an immunotherapeutic agent used in diabetes, anti-inflammatory drugs for metabolic syndrome and diabetes, adenosine monophosphate-activated protein kinase (AMPK) stimulator, 11-beta-hydroxysteroid dehydrogenase 1 inhibitor, glucokinase activator, diacylglycerol-O-acyltransferase (DGAT) inhibitor, glucose transporter-4 modulator, somatostatin receptor 3 agonist, lipid-lowering agent, and their combinations.

[0018] In some embodiments, the implementation of the antibody or its antigennegative fragment reduces the level of LDL-C in the patient, for example, at least 30%, 35%, 40% or 45%. In some embodiments, the antibody or antigen-binding fragment thereof reduces non-HDL-C cholesterol levels in a patient, eg, by at least 25%, 30%, 35%, or 40%. In some embodiments, the antibody or antigen-binding fragment thereof reduces apolipoprotein C3 (ApoC3) levels in a patient (e.g., by at least about 6.0%, about 6.5%, about 7.0%, or about 7.5% after 12 or 24 weeks of treatment). In some embodiments, the antibody or antigen-binding fragment thereof reduces the number of lipoprotein particles in a patient (eg, by at least about 20%, about 30%, about 40%, or about 50% after 12 or 24 weeks of treatment). In other embodiments, the antibody or antigen-binding fragment thereof reduces the size of lipoprotein particles in a patient (eg, by at least about 1.5%, about 2%, about 2.5%, or about 3% after 12 or 24 weeks of treatment).

[0019] In some embodiments, the antibody or antigen-binding fragment thereof (a) does not affect the patient's hemoglobin A1c (HbA1c) level and/or (b) does not affect the patient's fasting plasma glucose (FPG) level.

[0020] According to another aspect, the methods provide a method for treating hypercholesterolemia in a patient with type 1 diabetes mellitus (T1DM), the method comprising

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has (i) T1DM and (ii) hypercholesterolemia not adequately controlled by a maximum tolerated dose statin-based therapy;

(b) administering to the patient every two weeks 75 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase;

(c) administering to the patient one or more subsequent doses of 75 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if, for example, after 8 weeks, the patient's LDL-C level is lower than 70 mg/dL, or administering one or several subsequent doses of 150 mg of the antibody or antigen-binding fragment approximately every two weeks if, for example, after 8 weeks, the patient's LDL-C level is 70 mg/dL or greater, where the antibody or antigen-binding fragment contains HCVR with the amino acid sequence under SEQ ID NO: 1 and LCVR with the amino acid sequence under SEQ ID NO: 6, and where the patient receives concomitant insulin therapy.

[0021] In accordance with another aspect, the methods provide a method of treating hypercholesterolemia in a patient with type 1 diabetes mellitus (T1DM), the method comprising

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has (i) T1DM and (ii) hypercholesterolemia not adequately controlled by a maximum tolerated dose statin-based therapy;

(b) administering to the patient every four weeks 300 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase; and

(c) administering to the patient one or more subsequent doses of 300 mg of the antibody or antigen-binding fragment thereof approximately every four weeks if, after 8 weeks, the patient's LDL-C level is above a threshold level, or administering one or more subsequent doses of 150 mg of an antibody or antigen-binding fragment approximately every two weeks if, after 8 weeks, the patient's LDL-C level is equal to or less than the cut-off level,

wherein the antibody or antigen-binding fragment thereof comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6, and wherein the patient is receiving concomitant insulin therapy. In one embodiment, the threshold level is 15 mg/dl. In another embodiment, the threshold level is 25 mg/dl.

[0022] According to another aspect, the methods provide a method of treating hypercholesterolemia in a patient with type 2 diabetes mellitus (T2DM), the method comprising

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has (i) T2DM and (ii) hypercholesterolemia not adequately controlled by a statin-based therapy at the maximum tolerated dose; and

(b) administering to the patient 75 mg, 150 mg, or 300 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase, wherein the patient receives concomitant insulin therapy.

[0023] In certain embodiments, the patient is administered 75 mg of the antibody or antigen-binding fragment every two weeks. In other embodiments, the patient is administered 150 mg of the antibody or antigen-binding fragment every two weeks. In other embodiments, the patient is administered 300 mg of the antibody or antigen-binding fragment every four weeks.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs from the HCVR/LCVR amino acid sequence pair comprising SEQ ID NO: 1/6. In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs listed under SEQ ID NOs: 2, 3, and 4 and three light chain CDRs listed under SEQ ID NOs: 7, 8, and 10. In some embodiments, the antibody or its antigen-binding fragment comprise a heavy chain variable region (HCVR) with the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (LCVR) with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to an antibody or antigen-binding fragment thereof that comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope on PCSK9 that and an antibody containing HCVR with the amino acid sequence of SEQ ID NO: 1 and LCV R with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope on PCSK9 that overlaps with an epitope of an antibody comprising HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) complementarity determining regions (CDRs) comprising the amino acid sequences shown under SEQ ID NOS: 85 and 89, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequences of the heavy and light chain CDRs having SEQ ID NOs: 86, 87, 88, 90, 91, and 92. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR with the amino acid sequence, at least 90%, 95% or 99% identical to the amino acid sequence listed under SEQ ID NO:85, and LCVR with an amino acid sequence identical to at least 90%, 95% or 99% of the amino acid sequence listed under SEQ ID NO:89.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, and LY3015014. In some embodiments, the antibody or antigen-binding fragment thereof is alirocumab.

[0027] In certain embodiments, the method disclosed herein further comprises (c) administering to the patient one or more subsequent doses of 75 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if the patient's LDL-C level is below a threshold level, or administration of one or more subsequent doses of 150 mg of the antibody or antigen-binding fragment approximately every two weeks if the patient's LDL-C level equals or exceeds the threshold level. In some embodiments, the threshold level is 70 mg/dL.

[0028] In certain embodiments, the method disclosed herein further comprises (c) administering to the patient one or more subsequent doses of 300 mg of the antibody or antigen-binding fragment approximately every four weeks if, for example, after 8 weeks, the level The patient's LDL-C is below the threshold, or one or more subsequent doses of 150 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if, for example, after 8 weeks, the patient's LDL-C level equals or exceeds the threshold . In some embodiments, the threshold level is 70 mg/dL.

[0029] In certain embodiments, the antibody or antigen-binding fragment thereof is administered subcutaneously.

[0030] In certain embodiments, the patient additionally receives a concomitant lipid corrective therapy (LMT). In some embodiments, the LMT is selected from the group consisting of a statin, a cholesterol absorption inhibitor, a fibrate, a niacin, an omega-3 fatty acid, and a bile acid sequestrant. In certain embodiments, the LMT is a statin-based therapy. In some embodiments, the statin is selected from the group consisting of atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin, and cerivastatin. In certain embodiments, the statin therapy is a maximum tolerated statin therapy. In certain embodiments, the cholesterol absorption inhibitor is ezetimibe.

[0031] In certain embodiments, the patient suffers from statin intolerance.

[0032] In some embodiments, the insulin therapy is selected from the group consisting of human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degludec, insulin aspart, and basal insulin.

[0033] In certain embodiments, the patient additionally receives a concomitant antidiabetic therapy in addition to the insulin based therapy. In some embodiments, the additional antidiabetic agent is selected from the group consisting of a glucagon-like peptide 1 (GLP-1) therapy, a gastrointestinal peptide, a glucagon receptor agonist or antagonist, a glucose-dependent insulinotropic polypeptide (GIP) receptor agonist or antagonist, an antagonist or inverse agonist ghrelin, xenin, xenin analog, biguanide, sulfonylurea, meglitinide, thiazolidinedione, DPP-4 inhibitor, alpha-glucosidase inhibitor, sodium-dependent glucose transporter 2 (SGLT-2) inhibitor, SGLT-1 inhibitor, agonist or receptor modulator peroxisome proliferator-activated (PPAR-) (alpha, gamma, or alpha/gamma), amylin, amylin analog, G protein-coupled receptor 119 (GPR119) agonist, GPR40 agonist, GPR120 agonist, GPR142 agonist, systemic or poorly absorbed TGR5 agonist , an immunotherapeutic agent used in diabetes, anti-inflammatory drugs for metabolic syndrome and diabetes, adenosine monophosphate-activated protein kinase (AMPK) stimulator, 11-beta-hydroxysteroid dehydrogenase 1 inhibitor, glucokinase activator, diacylglycerol-O-acyltransferase (DGAT) inhibitor, glucose transporter-4 modulator, somatostatin receptor 3 agonist, lipid-lowering agent, and their combinations.

[0034] In some embodiments, the implementation of the antibody or its antigennegative fragment reduces the level of LDL-C in the patient, for example, at least 30%, 35%, 40% or 45%. In some embodiments, the antibody or antigen-binding fragment thereof reduces non-HDL-C cholesterol levels in a patient, eg, by at least 20%, 25%, 30%, or 35%. In some embodiments, the antibody or antigen-binding fragment thereof reduces apolipoprotein C3 (ApoC3) levels in a patient (e.g., by at least about 6.0%, about 6.5%, about 7.0%, or about 7.5% after 12 or 24 weeks of treatment). In some embodiments, the antibody or antigen-binding fragment thereof reduces the number of lipoprotein particles in a patient (eg, by at least about 20%, about 30%, about 40%, or about 50% after 12 or 24 weeks of treatment). In other embodiments, the antibody or antigen-binding fragment thereof reduces the size of lipoprotein particles in a patient (eg, by at least about 1.5%, about 2%, about 2.5%, or about 3% after 12 or 24 weeks of treatment).

[0035] In some embodiments, the antibody or antigen-binding fragment thereof (a) does not affect the patient's hemoglobin A1c (HbA1c) level and/or (b) does not affect the patient's fasting plasma glucose (FPG) level.

[0036] According to another aspect, the methods provide a method of treating hypercholesterolemia in a patient with type 2 diabetes mellitus (T2DM), the method comprising

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has (i) T2DM and (ii) hypercholesterolemia not adequately controlled by a maximum tolerated dose statin-based therapy;

(b) administering to the patient every two weeks 75 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase;

(c) administering to the patient one or more subsequent doses of 75 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if, after 8 weeks, the patient's LDL-C level is below 70 mg/dl, or administering one or more subsequent doses, 150 mg of the antibody or antigen-binding fragment thereof approximately every two weeks, if, for example, after 8 weeks, the patient's LDL-C level is 70 mg/dL or more,

wherein the antibody or antigen-binding fragment thereof comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6, and wherein the patient is receiving concomitant insulin therapy.

[0037] According to another aspect, the methods provide a method for treating hypercholesterolemia in a patient with type 2 diabetes mellitus (T2DM), the method comprising

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has (i) T2DM and (ii) hypercholesterolemia not adequately controlled by a maximum tolerated dose statin-based therapy;

(b) administering to the patient every four weeks 300 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase; and

(c) administering to the patient one or more subsequent doses of 300 mg of the antibody or antigen-binding fragment thereof approximately every four weeks if, after 8 weeks, the patient's LDL-C level is above a threshold level, or administering one or more subsequent doses of 150 mg of an antibody or antigen-binding fragment approximately every two weeks if, after 8 weeks, the patient's LDL-C level is equal to or less than the cut-off level,

wherein the antibody or antigen-binding fragment thereof comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6, and wherein the patient is receiving concomitant insulin therapy. In one embodiment, the threshold level is 15 mg/dl. In another embodiment, the threshold level is 25 mg/dL.

[0038] According to another aspect, the methods provide a method for treating hypercholesterolemia in a patient with T2DM and atherosclerotic cardiovascular disease (ASCVD), the method comprising

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has (i) T2DM, (ii) ASCVD, and (iii) hypercholesterolemia not adequately controlled by a maximum tolerated dose statin-based therapy; and

(b) administering to the patient 75 mg, 150 mg, or 300 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase, wherein the patient receives concomitant insulin therapy.

[0039] In some embodiments, ASCVD is defined as coronary heart disease (CHD), ischemic stroke, or peripheral arterial disease. In some embodiments, CHD includes acute myocardial infarction, asymptomatic myocardial infarction, and unstable angina.

[0040] In certain embodiments, the patient is administered 75 mg of the antibody or antigen-binding fragment every two weeks. In certain embodiments, the patient is administered 150 mg of the antibody or antigen-binding fragment every two weeks. In certain embodiments, the patient is administered 300 mg of the antibody or antigen-binding fragment every four weeks.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs from the HCVR/LCVR amino acid sequence pair comprising SEQ ID NO: 1/6. In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs listed under SEQ ID NOs: 2, 3, and 4 and three light chain CDRs listed under SEQ ID NOs: 7, 8, and 10. In some embodiments, the antibody or its antigen-binding fragment comprise a heavy chain variable region (HCVR) with the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (LCVR) with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope on PCSK9 as an antibody comprising HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope on PCSK9 that overlaps with an epitope of an antibody containing HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with am foreign acid sequence under SEQ ID NO: 6.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) complementarity determining region (CDR) containing the amino acid sequences shown under SEQ ID NOS: 85 and 89, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequences of the heavy and light chain CDRs having SEQ ID NOs: 86, 87, 88, 90, 91, and 92. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR with the amino acid sequence, at least 90%, 95% or 99% identical to the amino acid sequence listed under SEQ ID NO:85, and LCVR with an amino acid sequence identical to at least 90%, 95% or 99% of the amino acid sequence listed under SEQ ID NO:89.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, and LY3015014. In some embodiments, the antibody or antigen-binding fragment thereof is alirocumab.

[0044] In certain embodiments, the method further comprises

(c) administering to the patient one or more subsequent 75 mg doses of the antibody or an antigen-binding fragment thereof approximately every two weeks if the patient's LDL-C level is below a threshold level, or administering one or more subsequent 150 mg doses of the antibody, or its antigen-binding fragment approximately every two weeks if the patient's LDL-C level equals or exceeds the threshold level.

[0045] In certain embodiments, the method further comprises

(c) administering to the patient one or more subsequent 300 mg doses of the antibody or an antigen-binding fragment thereof approximately every four weeks if the patient's LDL-C level is below a threshold level, or administering one or more subsequent 150 mg doses of the antibody, or its antigen-binding fragment approximately every two weeks if the patient's LDL-C level equals or exceeds the threshold level.

[0046] In some embodiments, the threshold level is 70 mg/dL.

[0047] In certain embodiments, the antibody or antigen-binding fragment thereof is administered subcutaneously.

[0048] In certain embodiments, the patient additionally receives a concomitant lipid corrective therapy (LMT). In some embodiments, the LMT is selected from the group consisting of a statin, a cholesterol absorption inhibitor, a fibrate, a niacin, an omega-3 fatty acid, and a bile acid sequestrant. In certain embodiments, the LMT is a statin-based therapy. In some embodiments, the statin is selected from the group consisting of atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin, and cerivastatin. In certain embodiments, the statin therapy is a maximum tolerated statin therapy. In certain embodiments, the cholesterol absorption inhibitor is ezetimibe.

[0049] In certain embodiments, the patient suffers from statin intolerance.

[0050] In some embodiments, the insulin therapy is selected from the group consisting of human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degludec, insulin aspart, and basal insulin. In certain embodiments, the patient is receiving a concomitant antidiabetic therapy in addition to the insulin based therapy. In some embodiments, the additional antidiabetic agent is selected from the group consisting of a glucagon-like peptide 1 (GLP-1) therapy, a gastrointestinal peptide, a glucagon receptor agonist or antagonist, a glucose-dependent insulinotropic polypeptide (GIP) receptor agonist or antagonist, an antagonist or inverse agonist ghrelin, xenin, xenin analog, biguanide, sulfonylurea, meglitinide, thiazolidinedione, DPP-4 inhibitor, alpha-glucosidase inhibitor, sodium-dependent glucose transporter 2 (SGLT-2) inhibitor, SGLT-1 inhibitor, agonist or receptor modulator peroxisome proliferator-activated (PPAR-) (alpha, gamma, or alpha/gamma), amylin, amylin analog, G protein-coupled receptor 119 (GPR119) agonist, GPR40 agonist, GPR120 agonist, GPR142 agonist, systemic or poorly absorbed TGR5 agonist , an immunotherapeutic agent used in diabetes, anti-inflammatory drugs for metabolic syndrome and diabetes, adenosine monophosphate-activated protein kinase (AMPK) stimulator, 11-beta-hydroxysteroid dehydrogenase 1 inhibitor, glucokinase activator, diacylglycerol-O-acyltransferase (DGAT) inhibitor, glucose transporter-4 modulator, somatostatin receptor 3 agonist, lipid-lowering agent, and their combinations.

[0051] In some embodiments, the implementation of the antibody or its antigennegative fragment reduces the level of LDL-C in the patient, for example, at least 30%, 35%, 40% or 45%. In some embodiments, the antibody or antigen-binding fragment thereof reduces non-HDL-C cholesterol levels in a patient, eg, by at least 20%, 25%, 30%, 35%. In some embodiments, the implementation of the antibody or its antigennegative fragment reduces the level of ApoC3 in the patient. In some embodiments, the antibody or antigen-binding fragment thereof reduces the number and/or size of lipoprotein particles in a patient. In some embodiments, the antibody or antigen-binding fragment thereof

(a) do not affect the patient's hemoglobin A1c (HbA1c) level and/or

(b) do not affect the patient's fasting plasma glucose (FPG) level.

[0052] According to another aspect, the methods provide a method for treating hypercholesterolemia in a patient with T2DM and ASCVD, the method comprising

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has (i) T2DM, (ii) ASCVD, and (iii) hypercholesterolemia not adequately controlled by a maximum tolerated dose statin-based therapy;

(b) administering to the patient every two weeks 75 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase; and

(c) administering to the patient one or more subsequent doses of 75 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if the patient's LDL-C level is below 70 mg/dL, or administering one or more subsequent doses of 150 mg, antibody or antigen-binding fragment approximately every two weeks if the patient's LDL-C level is 70 mg/dl or greater,

wherein the antibody or antigen-binding fragment thereof comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6, and wherein the patient is receiving concomitant insulin therapy.

[0053] Other embodiments will be apparent from the review of the following detailed description.

BRIEF DESCRIPTION OF GRAPHICS

[0054] Figure 1 is a diagram illustrating the general outline of the main phase of the study described in Example 2 herein. The study includes a screening period, a double-blind treatment period, and a safety data observation period.

[0055] Figure 2 is a graph showing mean LS (+/-SE) percent change in calculated LDL-C levels from baseline in a population of ITT patients with type 1 diabetes according to IVRS. Least squares means and standard errors (SE) were obtained using MMRM (repeated measures mixed effects model) analysis. The model includes fixed categorical effects of treatment group, IVRS randomization strata, time point, and treatment group-time point, stratum-time point, treatment group-stratum, treatment group-stratum-time point relationships, and fixed covariates of baseline LDL-value. C and baseline value-point-in-time relationship. The MMRM model was applied to all patients in the ITT population (i.e. type 1 and type 2 diabetic patients).

[0056] Figure 3 is a graph showing mean LS (+/-SE) percent change in calculated LDL-C levels from baseline in a population of ITT patients with type 2 diabetes according to IVRS. Least squares means and standard errors (SE) were obtained using MMRM (repeated measures mixed effects model) analysis. The model includes fixed categorical effects of treatment group, IVRS randomization strata, time point, and treatment group-time point, stratum-time point, treatment group-stratum, treatment group-stratum-time point relationships, and fixed covariates of baseline LDL-value. C and baseline value-point-in-time relationship. The MMRM model was applied to all patients in the ITT population (i.e. patients with type 1 and type 2 diabetes).

[0057] Figure 4 is a graph showing the percentage change from baseline to week 24 for cholesterol levels other than HDL-C, LDL-C, ApoB, and LDL-PN in the ITT Type 2 Diabetes and ASCVD population.

[0058] Figure 5 is a graph showing the percentage of individuals achieving non-HDL-C cholesterol <100 mg/dl, LDL-C <70 mg/dl and ApoB <80 mg/dl at week 24 in the population ITT with type 2 diabetes and ASCVD.

DETAILED DESCRIPTION

[0059] The methods are not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not meant to be limiting, as the scope of the methods presented will only be limited by the appended claims.

[0060] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning, which is usually understood by a person skilled in the art. As used herein, the term "approximately", when used with reference to a specific numerical value being described, means that the value may differ from the described value by no more than 1%. For example, as used herein, "about 100" includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

[0061] While any methods and materials similar or equivalent to those described herein can be used, preferred methods and materials are described below. All publications cited in this document are incorporated herein by reference in their entirety.

Methods for treating a patient with hypercholesterolemia and diabetes receiving an insulin-based therapy

[0062] Methods and compositions are provided for the treatment of diabetic patients suffering from hypercholesterolemia receiving an insulin-based therapy. In accordance with some variants of implementation of these methods lead to a decrease in the levels of lipoproteins (eg, LDL-C and/or Lp(a)) in the blood serum of such patients.

[0063] The present invention also relates to a PCSK9 inhibitor (e.g., an antibody or antigen-binding fragment thereof that specifically binds to PCSK9 (e.g., human PCSK9) or a composition containing a PCSK9 inhibitor for use in the treatment of hypercholesterolemia diabetic patients receiving the agent Insulin Based Therapy In some embodiments, the PCSK9 inhibitor or composition is useful in lowering the serum levels of lipoproteins (eg, LDL-C and/or Lp(a)) in such patients.

[0064] As used herein, the term "lipoprotein" means a biomolecular particle containing both a protein and a lipid. Examples of lipoproteins include, for example, low density lipoprotein (LDL), very low density lipoprotein (VLDL), medium density lipoprotein (IDL), and lipoprotein (a) (Lp(a)).

[0065] Diabetes mellitus, often referred to simply as diabetes, is a group of metabolic diseases in which an individual has high blood sugar levels, either because the body does not produce enough insulin or because the cells do not respond to the insulin produced. The most common types of diabetes are (1) type 1 diabetes, in which the body is unable to produce insulin; (2) type 2 diabetes, in which the body is unable to use insulin properly, coupled with increasing insulin deficiency over time, and (3) gestational diabetes, in which a woman develops diabetes as a result of pregnancy. All forms of diabetes increase the risk of long-term complications, which usually develop over many years. The basis of most of these long-term complications is damage to blood vessels, and they can be divided into two categories: "macrovascular" disease resulting from atherosclerosis of large blood vessels, and "microvascular" disease resulting from damage to small blood vessels. Examples of macrovascular diseases are coronary heart disease, myocardial infarction, stroke, and peripheral vascular disease. Examples of microvascular diseases are diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy.

[0066] In some embodiments, the patient to be treated has type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) and is receiving an insulin-based therapy. In some embodiments, the patient was diagnosed with T1DM or T2DM at least one year ago. In some embodiments, the patient was diagnosed with T1DM before the age of 30 years. In some embodiments, the T1DM patient has a C-peptide level below 0.2 pmol/mL. In some embodiments, the patient has a glycated hemoglobin (HbA1c) level of less than 10%.

[0067] In accordance with some embodiments, the patient to be treated suffers from hypercholesterolemia not adequately controlled by a lipid corrective therapy (LMT) agent. Hypercholesterolemia is considered to be insufficiently controlled by LMT if the patient's serum LDL-C concentrations are not reduced to an established, medically acceptable level, such as less than 70 mg/dL (taking into account the patient's relative risk of coronary heart disease) after at least 4 weeks of treatment with LMT. In certain embodiments, the LMT is a maximum tolerated dose statin therapy. As used herein, "maximum tolerated dose statin therapy" means the highest dose of a statin that can be administered to a patient without unacceptable unwanted side effects in that patient. For example, the methods disclosed herein provide for the treatment of a T1DM or T2DM patient suffering from hypercholesterolemia not adequately controlled by a daily dose of a statin selected from the group consisting of atorvastatin (including atorvastatin+ezetimibe), rosuvastatin, cerivastatin, pitavastatin, fluvastatin , lovastatin, simvastatin (including simvastatin + ezetimibe), pravastatin and combinations thereof. In certain embodiments, the patient is not receiving concomitant statin therapy if the patient is intolerant to such therapy. Patients with statin intolerance may have, for example, skeletal muscle-related symptoms other than those due to strain or injury, such as pain, aches, weakness, or spasms that occur or worsen while taking a statin-based therapy and disappear when the statin-based therapy is discontinued.

Patient Selection

[0068] The methods and compositions of the present invention are useful in the treatment of patients having hypercholesterolemia and diabetes and receiving an insulin based therapy. The patient to be treated may also demonstrate one or more additional selection criteria. For example, a patient may be selected for treatment if the patient's calculated LDL-C level is 70 mg/dl, 100 mg/dl, or 130 mg/dl or greater. The patient may be treated with the maximum tolerated dose of the statin, optionally in combination with at least one other lipid-adjusting therapy (LMT) for at least 4 weeks, or the patient may be treated with the optimal dose of at least one non-statin LMT for at least 4 weeks if the patient is statin intolerant. The maximum tolerated dose of a statin can be defined, for example, as the dose prescribed in accordance with regional practice or local guidelines, or is the dose that is maximally tolerated due to the occurrence of adverse effects at higher doses, as indicated in the local package insert for pediatric patients. Statin intolerance can be defined, for example, as the inability to tolerate at least 2 statins: one statin at the lowest daily starting dose and the other statin at any dose due to skeletal muscle related symptoms other than symptoms arising from strain or injuries such as pain, aches, weakness, or spasms that come on or get worse while taking the statin-based therapy and disappear when the statin-based therapy is stopped. Patients not receiving a statin in accordance with the daily dosing regimen (for example, 1 to 3 times a week) are also considered unable to tolerate the daily dose.

[0069] In addition, a patient may be selected for treatment if the patient has a high risk of developing cardiovascular disease (CV). In some embodiments, the high-risk CV patient has a documented history of cardiovascular disease (CVD) and/or at least one additional CV risk factor. CVD includes, without limitation, coronary artery disease (CHD) and equivalent risks of CHD. CHD includes, without limitation, acute myocardial infarction (MI), asymptomatic MI, unstable angina, coronary revascularization procedure (eg, percutaneous coronary angioplasty (PCI) or coronary artery bypass surgery (CABG)) and clinically significant CHD (eg, diagnosed with invasive or non-invasive examinations such as coronary angiography, treadmill exercise electrocardiogram, stress echocardiography, or radionuclide imaging). Equivalent risks of CHD include, but are not limited to, peripheral arterial disease (eg, as described in the inclusion criteria in Example 2) and prior ischemic stroke with focal ischemic neurological deficit that persists for more than 24 hours of atherothrombotic origin. Risk factors for CV include, without limitation, hypertension, current cigarette smoking, age ≥45 years for men and ≥55 years for women, history of micro/macroalbuminuria, history of diabetic retinopathy, family history of premature CHD (father or brother under 55 years of age). ; mother or sister under age 65), low HDL-C (<40 mg/dL [1.0 mmol/L] in men and <50 mg/dL [1.3 mmol/L] in women) and documented chronic kidney disease (CKD) (eg, as defined in the inclusion criteria in Example 2).

[0070] In some embodiments, the high-risk CV patient has atherosclerotic cardiovascular disease (ASCVD). In some embodiments, ASCVD is defined as coronary heart disease (CHD), ischemic stroke, or peripheral arterial disease. In some embodiments, CHD includes acute myocardial infarction, asymptomatic myocardial infarction, and unstable angina. In some embodiments, CHD is defined as acute myocardial infarction, asymptomatic myocardial infarction, or unstable angina.

Insulin-based therapy

[0071] As described herein, patients with diabetes selected for treatment with the methods of the present invention have received and continue to receive an insulin-based therapy, including insulin or its derivatives. The types of insulin on the market differ in the origin of the insulin (eg bovine, porcine, human insulin) as well as in their composition, which can affect the profile of action (onset of action and duration of action). By combining different insulin-based products, a wide range of action profiles can be obtained and blood sugar levels can be set as close to physiological as possible. Exemplary insulin-based therapies may include natural insulin such as human insulin as well as extended-acting modified insulins such as insulin glargine (Gly(A21)-Arg(B31)-Arg(B32) human insulin, e.g., ^Lantus® ). Insulin glargine is administered as an acidic clear solution, and due to the properties of its solution in the physiological pH range of the subcutaneous tissue, it precipitates as a stable hexamer associate. Insulin glargine is administered once daily and differs from other long-acting insulins in its flat serum profile and associated reduction in the risk of nocturnal hypoglycemia (Schubert-Zsilavecz et al., 2:125-130 (2001)). Insulin glargine can be administered at concentrations above 100 U/ml, for example 270-330 U/ml insulin glargine or 300 U/ml insulin glargine (as described in EP 2387989). Other exemplary insulin based therapies include insulin glulisine (eg ^Apidra® ), insulin detemir (eg Levemir®), insulin lispro (eg ^Humalog® , ^Liprolog® ), insulin degludec (eg ^{DegludecPlus®} , IdegLira ( ^NN9068 ) ), insulin aspart and insulin aspart formulations (eg NovoLog ^® ), basal insulin and analogues (eg LY2605541, LY2963016, NN1436), pegylated insulin lispro (eg LY-275585), long-acting insulins (eg NN1436, Insumera (PE0139), AB-101, AB-102, Sensulin LLC), intermediate acting insulins (eg Humulin ^® N, Novolin ^® N), rapid acting and short acting insulins (eg Humulin ^® R, Novolin ^® R , Linjeta ^® (VIAject ^® ), insulin PH20, NN1218, HinsBet ^® ), premixed insulins, SuliXen ^® , NN1045, insulin plus Symlin ^® , PE-0139, hydrogel-packed insulin ACP-002 and insulin types for oral, inhaled , transdermal and buccal or sublingual (eg Exubera®, ^Nasulin® , ^Afrezza® , ^tregopil insulin, TPM-02 insulin, ^Capsulin® , Oral- ^lyn® , ^Cobalamin® oral insulin, ORMD-0801, Oshadi oral insulin, NN1953 , NN1954, NN1956, ^VIAtab® ). Also suitable are those insulin derivatives which are linked to albumin or other protein by a bifunctional linker.

PCSK9 inhibitors

[0072] The methods involve administering to a patient a therapeutic composition comprising a PCSK9 inhibitor. As used herein, "PCSK9 inhibitor" means any agent that binds or interacts with human PCSK9 and inhibits the normal biological function of PCSK9 in vitro or in vivo. Non-limiting examples of categories of PCSK9 inhibitors include small molecule PCSK9 antagonists, nucleic acid-based (e.g., siRNA or antisense) inhibitors of PCSK9 expression or activity, peptide-based molecules that specifically interact with PCSK9 (e.g., peptibodies), receptor molecules that specifically interact with PCSK9, proteins containing the ligand-binding portion of the LDL receptor, scaffold molecules that bind PCSK9 (e.g., engineered ankyrin repeat proteins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, fibronectin-based scaffolds, and other scaffolds based on naturally occurring repeat proteins, etc. [see e.g. Boersma and Pluckthun, 2011, Curr Opin Biotechnol 22: 849-857 and references cited therein]), and aptamers to PCSK9 or parts thereof. In some embodiments, PCSK9 inhibitors useful in the context of the methods of the present invention are anti-PCSK9 antibodies or antigen-binding antibody fragments that specifically bind human PCSK9.

[0073] The term "human subtilisin/kexin type 9 proprotein convertase" or "human PCSK9" or "hPCSK9" as used herein refers to PCSK9 with the nucleic acid sequence shown under SEQ ID NO:197 and the amino acid sequence under SEQ ID NO:198 or its biologically active fragment.

[0074] The term "antibody" as used herein is intended to refer to immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains, and two light (L) chains linked together by disulfide bonds, as well as their multimers (for example , IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V _H ) and a heavy chain constant region. The heavy chain constant region contains three domains: C _H 1, C _H 2 and C _H 3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or V _L ) and a light chain constant region. The light chain constant region contains one domain (C _L 1). The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with regions that are more conserved called framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, the FR of an anti-PCSK9 antibody (or antigen-binding site thereof) may be identical to human germline immunoglobulin sequences, or may be altered naturally or artificially. The amino acid consensus sequence can be determined based on a side-by-side analysis of two or more CDRs.

[0075] The term "antibody" as used herein also includes antigen-binding fragments of whole antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to complex formation. Antigen-binding fragments of an antibody can be obtained, for example, from whole antibody molecules using any suitable standard techniques, such as proteolytic cleavage or recombinant genetic engineering techniques, including the manipulation of DNA encoding variable and optionally constant domains of antibodies, and its expression. 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. DNA can be sequenced and chemically manipulated or manipulated using molecular biology techniques, for example, to arrange one or more variable and/or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, modify, add or remove amino acids, and etc.

[0076] Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic an antibody hypervariable region (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide) or a FR3-CDR3- FR4. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, nanobodies (e.g., monovalent antibodies, bivalent antibodies, etc.) , small molecule modular protein (SMIP) immunotherapies, and shark IgNAR variable domains are also included in the expression "antigen binding fragment" as used herein.

[0077] An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and will typically contain at least one CDR that is adjacent to or in a reading frame with one or more framework sequences. In antigen-binding fragments having a V _H domain associated with a V _L domain, the V _H and V _L domains may be located relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain V _H -V _H , V _H -V _L or V _L -V _L dimers. Alternatively, the antigen-binding fragment of an antibody may comprise a V _H or V _L monomeric domain.

[0078] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting illustrative variable and constant domain configurations that can be identified in an antigen-binding fragment of an antibody include (i) V _{H -CH} 1; (ii) V _H -C _H 2; (iii) V _H -C _H 3; (iv) V _H -C _H 1 -C _H 2; (v) V _H -C _H 1 -C _H 2 -C _H 3; (vi) V _H -C _H 2 -C _H 3; (vii) V _H -C _L ; (viii) V _L -C _H 1; (ix) V _{L -CH} 2; (x) V _L -C _H 3; (xi) V _L -C _H 1 -C _H 2; (xii) V _L -C _H 1 -C _H 2 -C _H 3; (xiii) V _L -C _H 2 -C _H 3 and (xiv) V _L -C _L . In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may either be directly linked to each other, or may be linked through an entire or incomplete hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible bond between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment of an antibody may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations set forth above in non-covalent association with each other and/or with one or more V _H or r V _L monomeric domains (e.g. , via disulfide bond(s)).

[0079] As with whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody will typically contain at least two distinct variable domains, where each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen-binding antibody fragment according to the methods of the present invention using standard techniques available in the art.

[0080] The constant region of an antibody is important in terms of the ability of the antibody to bind complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype can be selected based on whether the antibody is required to mediate cytotoxicity.

[0081] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation), such as in CDRs, and in particular CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0082] The term "recombinant human antibody" as used herein is intended to include all human antibodies that are produced, expressed, generated, or isolated by recombinant methods, such as antibodies expressed with a recombinant expression vector transfected into a host cell ( described in more detail below), antibodies isolated from a combinatorial library of recombinant human antibodies (described in more detail below), antibodies isolated from an animal (e.g., mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992 ) Nucl. Acids Res. 20:6287-6295), or antibodies produced, expressed, generated, or isolated by any other means, which involve joining human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, in the case of an animal transgenic for human Ig sequences, in vivo somatic mutagenesis) and thus the amino acid sequences of the V _H and V _L regions of the recombinant antibodies are sequences which, although derived from and related to the human germline V _H and V _L sequences, may not occur naturally in an in vivo human germline antibody array.

[0083] Human antibodies can occur in two forms due to the heterogeneity of the hinge regions. In one form, the immunoglobulin molecule contains a stable four-chain construct of about 150-160 kDa in which the dimers are held together by a disulfide bond that links the heavy chains. In the second form, the dimers are not connected by chain-linking disulfide bonds, and a molecule 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 affine purification.

[0084] The frequency of appearance of the second form in different isotypes of intact IgG is due, without limitation, to structural differences associated with the isotype of the hinge region of the antibody. A single amino acid substitution in the human IgG4 hinge region can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels normally seen with the human IgG1 hinge. The methods of the present invention encompass antibodies with one or more mutations in the hinge region, C _H 2 or _CH 3 region, which may be preferred, for example, upon preparation, to increase the yield of the preferred form of the antibody.

[0085] The term "isolated antibody" as used herein means an antibody that has been identified and isolated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or recovered from/from at least one component of an organism or tissue or cell in which the antibody is originally present or naturally produced is an "isolated antibody" for the purposes of the methods of the present invention. An isolated antibody also includes an antibody in situ in a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In certain embodiments, the isolated antibody may be substantially free of other cellular material and/or other chemical compounds.

[0086] The term "specifically binds" and the like means that the antibody or antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining the presence of specific binding of an antibody to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds" PCSK9, as used in the context of the methods of the present invention, includes antibodies that bind PCSK9, or a portion thereof, with a K _D of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM as measured in a surface plate assay zmono resonance. However, an isolated antibody that specifically binds to human PCSK9 is cross-reactive with other antigens, such as PCSK9 molecules from other (non-human) species.

[0087] PCSK9 antibodies useful in the methods of the present invention may contain one or more amino acid substitutions, insertions and/or deletions in the framework regions and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences, from which antibodies were obtained. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The methods involve 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 are mutated with respect to the corresponding germline sequence residue(s). from which the antibody was derived, or to the corresponding sequence residue(s) of another human germline, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations") . One of ordinary skill in the art can readily generate, from the heavy and light chain variable region sequences disclosed herein, a plurality of antibodies and antigen-binding fragments that contain one or more individual germline mutations, or combinations thereof. In certain embodiments, all framework and/or CDR residues in the V _H and/or V _L domains are back-mutated to residues occurring in the original germline sequence from which the antibody originated. In other embodiments, only certain residues are back-mutated to residues of the original germline sequence, e.g., only residues located in the first 8 amino acids of FR1 or the last 8 amino acids of FR4 are mutated, or only residues located in CDR1, CDR2, or CDR3 are mutated. . In other embodiments, one or more framework and/or CDR residues are mutated into the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody originally originated). In addition, antibodies may contain any combination of two or more germline mutations in the framework regions and/or CDR regions, e.g., where certain individual residues are mutated into the corresponding residue of a certain germline sequence, while certain other residues that differ from the sequence of the original germline, are retained or mutated into the corresponding residue of the sequence of another germline. Once produced, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more of the desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced biological antagonistic or agonistic properties (in the case of necessary), reduced immunogenicity, and the like. The use of antibodies and antigen-binding fragments produced by this general method is encompassed by the methods of the present invention.

[0088] The methods involve the use of PCSK9 antibodies containing variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the methods of the present invention include the use of PCSK9 antibodies with the amino acid sequences HCVR, LCVR, and/or CDR with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

[0089] The term "Surface Plasmon Resonance" as used herein refers to an optical phenomenon that enables real-time analysis of interactions by detecting changes in protein concentrations in a biosensor array, such as with the BIAcore™ system (Biacore Life Sciences Division at GE Healthcare, Piscataway, New Jersey).

[0090] The term "K _D ", as used herein, refers to the equilibrium dissociation constant of the interaction of a particular antibody and antigen.

[0091] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. One antigen may have more than one epitope. Thus, different antibodies may bind to different sites on the antigen and may have different biological effects. Epitopes may be conformational or linear. A conformational epitope is formed by spatially contiguous amino acids from different segments of a linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. Under certain conditions, the epitope may include fragments of saccharides, phosphoryl groups or sulfonyl groups of the antigen.

[0092] In certain embodiments, the PCSK9 antibody used in the methods is an antibody with pH dependent binding characteristics. As used herein, "pH dependent binding" means that an antibody or antigen binding fragment thereof exhibits "reduced binding to PCSK9 at acidic pH compared to neutral pH" (both expressions may be used interchangeably for the purposes of this disclosure). For example, antibodies "with pH-dependent binding characteristics" include antibodies and antigen-binding fragments thereof that bind to PCSK9 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibodies and antigen-binding fragments bind PCSK9 to at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 100 times or higher affinity at neutral pH than at acidic pH.

[0093] In this aspect, anti-PCSK9 antibodies with pH dependent binding characteristics may have one or more amino acid variations relative to the parent anti-PCSK9 antibody. For example, an anti-PCSK9 antibody with pH-dependent binding characteristics may contain one or more histidine substitutions or histine insertions, for example, in one or more CDRs of the parent anti-PCSK9 antibody. Thus, in certain embodiments, methods are provided comprising administering an anti-PCSK9 antibody that contains amino acid sequences of CDRs (e.g., heavy and light chain CDRs) that are identical to the amino acid sequences of the CDRs of the parent anti-PCSK9 antibody, except for substitution of one or more amino acids in one or several CDRs of the parent antibody per histidine residue. Anti-PCSK9 antibodies with pH-dependent binding may have, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more histidine substitutions, either within a single CDR of the parent antibody, or distributed over several (e.g. , 2, 3, 4, 5, or 6) CDR of the parent anti-PCSK9 antibody. For example, the methods of the present invention include the use of anti-PCSK9 antibodies with pH-dependent binding comprising one or more histidine substitutions in HCDR1, one or more histidine substitutions in HCDR2, one or more histidine substitutions in HCDR3, one or more histidine substitutions in HCDR3, one or more histidine in LCDR1, one or more histidine substitutions in LCDR2, and/or one or more histidine substitutions in LCDR3 of the parent anti-PCSK9 antibody.

[0094] As used herein, the expression "acidic pH" means a pH of 6.0 or less (eg, less than about 6.0, less than about 5.5, less than about 5.0, etc.). The expression "acidic pH" 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 less. Used in this document, the expression "neutral pH" means a pH value of from about 7.0 to about 7.4. The expression "neutral pH" includes pH values of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0095] Non-limiting examples of PCSK9 antibodies that can be used in the context of the methods of the present invention include, for example, alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, LY3015014, or antigen binding sites of any of the above antibodies.

Obtaining human antibodies

[0096] 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 methods of the present invention to generate human antibodies that specifically bind to human PCSK9.

[0097] Using VELOCIMMUNE™ technology (see, for example, US 6596541, Regeneron Pharmaceuticals) or any other known method for producing monoclonal antibodies, high affinity chimeric anti-PCSK9 antibodies with a human variable region and a mouse constant region are initially isolated. The ^{VELOCIMMUNE®} technology involves generating a transgenic mouse with a genome containing human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigen challenge. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding the constant regions of the human heavy and light chains. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[0098] Typically, a ^{VELOCIMMUNE®} -derived mouse is exposed to an antigen of interest and lymphatic cells (such as B cells) are recovered from mice that express antibodies. Lymph cells can be fused with a myeloma cell line to form immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the constant regions of the desired heavy chain and light chain isotypes. 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.

[0099] High affinity chimeric antibodies with a human variable region and a mouse constant region are initially isolated. Antibodies are characterized and selected for the desired characteristics, including affinity, selectivity, epitope, etc., using conventional procedures known to those skilled in the art. The murine constant regions are replaced with the desired human constant region to produce a fully human antibody, such as wild-type IgG1 or IgG4 or modified IgG1 or IgG4. While the constant region chosen may vary depending on the particular application, the variable region retains the characteristics of high affinity antigen binding and target specificity.

[00100] In general, antibodies that can be used have high affinity as described above when measuring antigen binding both when immobilized on the solid phase and in the liquid phase. Mouse constant regions are replaced with the desired human constant regions to obtain fully human antibodies. While the constant region chosen may vary depending on the particular application, the variable region retains the characteristics of high affinity antigen binding and target specificity.

[00101] Specific examples of human antibodies or antigen-binding fragments of antibodies that specifically bind to PCSK9 that can be used in the context of the methods of the present invention include any antibody or antigen-binding fragment that contains three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in the heavy chain variable region (HCVR), with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 11, or a substantially similar sequence, which is characterized by at least 90%, at least 95%, at least 98 % or at least 99% sequence identity. Alternatively, specific examples of human antibodies or antigen-binding antibody fragments that specifically bind to PCSK9 that can be used in the context of the methods of the present invention include any antibody or antigen-binding fragment that contains the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in the variable heavy chain region (HCVR), with an amino acid sequence selected from the group consisting of SEQ ID NOs 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173, 181, and 189, or a substantially similar sequence that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. An antibody or antigen-binding fragment may comprise three light chain CDRs (LCVR1, LCVR2 and LCVR3) contained in a heavy chain variable region (LCVR) with an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 15, or substantially similar to it. a sequence that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. Alternatively, the antibody or antigen-binding fragment may comprise three light chain CDRs (LCVR1, LCVR2 and LCVR3) contained within a light chain variable region (LCVR) with an amino acid sequence selected from the group consisting of SEQ ID NOs 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145, 153, 161, 169, 177, 185, and 193, or a substantially similar sequence that is characterized by at least 90%, at least at least 95%, at least 98%, or at least 99% sequence identity.

[00102] Sequence identity between two amino acid sequences is determined over the entire length of the reference amino acid sequence, i.e., the amino acid sequence defined under SEQ ID NO, by the best sequence alignment, and/or the area of best sequence alignment between two amino acid sequences, where the best sequence alignment can be obtained by methods known in the art, for example, Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[00103] In certain embodiments, the antibody or antigen-binding protein comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) from heavy and light chain variable region (HCVR/LCVR) amino acid sequence pairs selected from the group consisting of SEQ ID NO:1/6 and 11/15. Alternatively, in certain embodiments, the antibody or antigen-binding protein comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) from heavy and light chain variable region (HCVR/LCVR) amino acid sequence pairs selected from the group consisting of SEQ ID NOs :37/41, 45/49, 53/57, 61/65, 69/73, 77/81, 85/89, 93/97, 101/105, 109/113, 117/121, 125/129, 133 /137, 141/145, 149/153, 157/161, 165/169, 173/177, 181/185, and 189/193.

[00104] In certain embodiments, an anti-PCSK9 antibody or antigen-binding protein useful in the methods of the present invention has the amino acid sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 selected from SEQ ID NO: 2/3/4/ 7/8/10 (mAb316P [also referred to as "REGN727" or "alirocumab"]) and 12/13/14/16/17/18 (mAb300N) (see US Patent Application Publication No. 2010/0166768) and 12 /13/14/16/17/18, where SEQ ID NO:16 contains the replacement of histidine with leucine at amino acid residue 30 (L30H).

[00105] In certain embodiments, the antibody or antigen-binding protein thereof comprises HCVR/LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NOs: 1/6 and 11/15. In certain exemplary embodiments, the antibody or antigen-binding protein comprises the amino acid sequence of HCVR of SEQ ID NO: 1 and the amino acid sequence of LCVR of SEQ ID NO: 6. In certain exemplary embodiments, the antibody or antigen-binding protein comprises the amino acid sequence of HCVR of SEQ ID NO: 11 and the LCVR amino acid sequence of SEQ ID NO: 15. In certain exemplary embodiments, the antibody or antigen-binding protein comprises the HCVR amino acid sequence of SEQ ID NO: 11 and the LCVR amino acid sequence of SEQ ID NO: 15 containing a histidine to leucine substitution at amino acid residue 30 ( L30H).

Pharmaceutical compositions and routes of administration

[00106] The methods of the present invention provide for the administration of a PCSK9 inhibitor to a patient, wherein the PCSK9 inhibitor is contained in a pharmaceutical composition. Pharmaceutical compositions are formulated with suitable carriers, excipients, and other means that provide suitable transfer, delivery, tolerability, and the like. Many suitable formulations can be found in the reference book known to pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing (cationic or anionic) lipids (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water emulsions and water-in-oil, carbovax emulsions (polyethylene glycols of various molecular weights), semi-liquid gels and semi-liquid mixtures containing carbovax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[00107] Exemplary pharmaceutical formulations containing PCSK9 antibodies that can be used in the context of the methods of the present invention include any of the formulations set forth in US Pat. , or WO2012/168491.

[00108] Various delivery systems are known and can be used to administer a pharmaceutical composition, e.g. encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see e.g. Wu et al., 1987, J Biol Chem 262:4429-4432). Routes of administration include, without limitation, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucosal surfaces (e.g. oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active means.

[00109] The pharmaceutical composition can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, a delivery device in the form of a pen is widely used in the delivery of a pharmaceutical composition. Such a delivery device in the form of a pen can be reusable and disposable. A refillable pen delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. After all of the pharmaceutical composition has been administered from the cartridge, and after the cartridge has been emptied, the empty cartridge can be easily disposed of and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. The disposable pen delivery device does not have a replaceable cartridge. Instead, the disposable pen delivery device is delivered pre-filled with the pharmaceutical composition contained in the device's reservoir. After the reservoir is emptied of the pharmaceutical composition, the device is completely disposed of.

[00110] Numerous reusable pens and autoinjector delivery devices are used in the subcutaneous delivery of a pharmaceutical composition. Examples include, without limitation, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, syringe - HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Indiana), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), syringe pen BD™ (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), just to name a few. Examples of refillable pen delivery devices used in the subcutaneous administration of a pharmaceutical composition according to the methods of the present invention include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly ), SURECLICK ^TM autoinjector (Amgen, Thousand Oaks, CA), PENLET ^TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA ^TM pen (Abbott Labs, Abbott Park, IL), while only a few of them are mentioned.

[00111] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed near the target of the composition, requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115- 138). Other controlled release systems are reviewed by Langer, 1990, Science 249:1527-1533.

[00112] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectable preparations can be obtained using known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying an antibody or salt thereof, as described above, in a sterile aqueous or oily medium commonly used for injection. The aqueous injection medium is, for example, physiological saline, an isotonic solution containing glucose and other excipients, etc., which can be used in conjunction with a suitable solubilizing agent such as an alcohol (for example, ethanol), a polyhydric alcohol (for example, propylene glycol, polyethylene glycol), non-ionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil, and the like, which can be used in conjunction with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injectable composition thus obtained is preferably filled into a suitable ampoule.

[00113] Advantageously, the oral or parenteral pharmaceutical compositions described above are formulated into unit dosage forms suitable for dose adjustment of the active ingredients. Such unit dosage forms include, for example, tablets, pills, capsules, injectables (ampoules), suppositories, etc.

Dose

[00114] The amount of a PCSK9 inhibitor (eg, anti-PCSK9 antibody) administered to a patient is typically a therapeutically effective amount. As used herein, "therapeutically effective amount" means a dose of a PCSK9 inhibitor that results in a detectable reduction (at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40 %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more of baseline) of one or more parameters selected from the group consisting of LDL-C, ApoB, ApoB100, cholesterol, other than HDL-C, total cholesterol, VLDL-C, triglycerides, ApoC3, TRL particles, Lp(a) and residual cholesterol.

[00115] In the case of an anti-PCSK9 antibody, a therapeutically effective amount can be from about 0.05 mg to about 600 mg, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 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, approximately 250 mg, approximately 260 mg, approximately 270 mg, approximately 280 mg, approximately 290 mg, approximately 300 mg, approximately 310 mg, approximately 320 mg, approx. approximately 330 mg, approximately 340 mg, approximately 350 mg, approximately 360 mg, approximately 370 mg, approximately 380 mg, approximately 390 mg, approximately 400 mg, approximately 410 mg, approximately 420 mg, approximately 430 mg, approximately 440 mg, approximately 450 mg, approximately 460 mg, approximately 470 mg, approximately 480 mg, approximately 490 mg, approximately 500 mg, approximately 510 mg, approximately 520 mg, approximately 530 mg, approximately 540 mg, approximately 550 mg, approximately 560 mg, approximately 570 mg, about 580 mg, about 590 mg, or about 600 mg of anti-PCSK9 antibody. In some exemplary embodiments, a therapeutically effective amount of an anti-PCSK9 antibody is 30 mg, 40 mg, or 75 mg (e.g., in the case of alirocumab for patients weighing less than 50 kg and/or 17 years of age or younger), 50 mg , 75 mg or 150 mg (for example, in the case of alirocumab for patients with a body weight of 50 kg or more and / or aged 17 years or younger), or 140 mg or 420 mg (for example, in the case of evolocumab). Other dosage amounts of PCSK9 inhibitors will be apparent to those skilled in the art.

[00116] The amount of PCSK9 antibody contained in individual doses can be expressed as milligrams of antibody per kilogram of patient body weight (ie, mg/kg). For example, the PCSK9 antibody may be administered to a patient at a dose of about 0.0001 to about 10 mg/kg of body weight.

Introduction schemes

[00117] In certain embodiments, multiple doses of a PCSK9 inhibitor (i.e., a pharmaceutical composition containing a PCSK9 inhibitor) may be administered to the subject over a period of time (e.g., along with a daily statin or other background LMT treatment regimen). The methods of this aspect include sequentially administering multiple doses of a PCSK9 inhibitor to a subject. As used herein, the term "sequential administration" means that each dose of a PCSK9 inhibitor is administered to a subject at different time points, such as different days separated by a predetermined interval (eg, hours, days, weeks, or months). The methods of the present invention sequentially administer to the patient one initial dose of a PCSK9 inhibitor, followed by one or more secondary doses of a PCSK9 inhibitor, and then optionally one or more tertiary doses of a PCSK9 inhibitor.

[00118] The terms "initial dose", "secondary doses" and "tertiary doses" refer to the time sequence of administration of individual doses of a pharmaceutical composition containing a PCSK9 inhibitor. Thus, "starting dose" is the dose administered at the start of a treatment regimen (also referred to as "starting dose"); "secondary doses" are doses that are administered after the initial dose; and "tertiary doses" are doses that are administered after secondary doses. All of the initial, secondary, and tertiary doses may contain the same amount of PCSK9 inhibitor, but, as a rule, they may differ from each other in the frequency of administration. However, in certain embodiments, the amounts of PCSK9 inhibitor contained in the initial, secondary, and/or tertiary doses differ from each other (eg, adjusted up or down as necessary) over the course of treatment. In certain embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses", then subsequent doses are administered at a lesser frequency (eg, "maintenance doses").

[00119] According to exemplary embodiments, each secondary and/or tertiary dose is administered 1-26 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks after the immediately preceding dose. The term "immediately preceding dose" as used herein means the dose of an antigen-binding molecule in a multi-dose sequence that is administered to a patient prior to the next next dose in the sequence, with no intermediate doses.

[00120] The methods of this aspect may include administering any number of secondary and/or tertiary doses of a PCSK9 inhibitor to a patient. For example, in certain embodiments, only one secondary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in certain 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.

[00121] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to a patient 1-2, 4, 6, 8 or more weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the remaining tertiary doses. For example, each tertiary dose may be administered to a patient 1-2, 4, 6, 8 or more weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to a patient may vary over the course of a treatment regimen. The frequency of administration can be adjusted by the physician during the course of treatment, depending on the needs of the individual patient after clinical examination.

[00122] The methods of the present invention include escalation regimens (also referred to herein as "dose escalation"). As used herein, the term "dose escalation" means that after receiving a certain number of doses of a PCSK9 inhibitor, if the patient has not achieved a certain reduction in one or more certain therapeutic parameters, the dose of the PCSK9 inhibitor is subsequently increased. For example, in the case of a treatment regimen that involves the administration of anti-PCSK9 antibody in doses of 75 mg to a patient every two weeks, if after 8 weeks (i.e. after 5 doses administered at week 0, 2 weeks, 4 weeks , 6 weeks and 8 weeks) the patient has not achieved a serum LDL-C concentration of less than 70 mg/dL, then the dose of anti-PCSK9 antibody is increased, for example, to 150 mg, which is administered thereafter once every two weeks (for example, starting from 10 weeks or 12 weeks or later).

[00123] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds PCSK9 is administered to a patient at a dose of approximately 75 mg once every two weeks. In certain embodiments, a dose of approximately 75 mg is continued if the patient's LDL-C level measured after one or more, two or more, three or more, four or more, or five or more doses is <70 mg/dL . In certain embodiments, a dose of approximately 75 mg is discontinued if the patient's LDL-C level measured after one or more, two or more, three or more, four or more, or five or more doses remains ≥70 mg /dl, and while the antibody or antigennegative fragment that specifically binds PCSK9, subsequently administered to the patient at a dose of approximately 150 mg, with a frequency of once every two weeks.

[00124] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds PCSK9 is administered to a patient at a dose of approximately 300 mg once every four weeks. In certain embodiments, a dose of approximately 300 mg is continued if the patient's LDL-C level measured after one or more, two or more, three or more, four or more, or five or more doses is <70 mg/dL . In certain embodiments, a dose of approximately 300 mg is discontinued if the patient's LDL-C level measured after one or more, two or more, three or more, four or more, or five or more doses remains ≥70 mg /dl, and while the antibody or antigennegative fragment that specifically binds PCSK9, subsequently administered to the patient at a dose of approximately 150 mg, with a frequency of once every two weeks.

[00125] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds PCSK9 is administered to a patient at a dose of approximately 150 mg once every two weeks.

[00126] In some embodiments, if an antibody or antigen-binding fragment thereof that specifically binds PCSK9 is administered to a patient at a dose of about 150 mg once every two weeks, the dose of about 150 mg is stopped if the patient has an LDL-C level measured after at least one dose, or at least two, three, four, or five consecutive doses, is <10, 15, 20, or 25 mg/dL, and an antibody or antigen-binding fragment thereof that specifically binds PCSK9 is administered sequentially, to the patient at a dose of approximately 75 mg once every two weeks. Without wishing to be bound by any theory, it is believed that very low levels of LDL-C (eg, <10, 15, 20, or 25 mg/dl) may exacerbate diabetes. In certain embodiments, a dose of approximately 150 mg is administered to the patient as a constant dose. In some embodiments, a dose of about 150 mg is administered to the patient after dose adjustments as described herein (eg, from about 75 mg every two weeks or from about 300 mg every four weeks).

Means of combination therapy

[00127] As described elsewhere herein, the methods may involve administering a PCSK9 inhibitor to a patient in combination with ("alongside") a previously administered lipid management therapy (LMT) to the patient. LMT agents include, but are not limited to, statins, fibrates, niacin (e.g., nicotinic acid and its derivatives), bile acid sequestrants, ezetimibe, lomitapide, phytosterols, orlistat, etc. For example, a PCSK9 inhibitor can be administered to a patient in combination with a constant daily therapeutic regimen taking a statin. Exemplary daily statin therapeutic regimens with which a PCSK9 inhibitor can be administered in combination in the context of the methods of the present invention include, for example, atorvastatin (10, 20, 40, or 80 mg per day), (atorvastatin/ezetimibe 10/10 or 40/ 10 mg per day), rosuvastatin (5, 10 or 20 mg per day), cerivastatin (0.4 or 0.8 mg per day), pitavastatin (1, 2 or 4 mg per day), fluvastatin (20, 40 or 80 mg per day), simvastatin (5, 10, 20, 40, or 80 mg per day), simvastatin/ezetimibe (10/10, 20/10, 40/10 or 80/10 mg per day), lovastatin (10, 20, 40 or 80 mg per day), pravastatin (10, 20, 40 or 80 mg per day) and combinations thereof. In certain embodiments, the statin therapy is a statin therapy at the maximum tolerated dose for the patient. Other LMT agents that may be administered in combination with a PCSK9 inhibitor within the context of the methods of the present invention include, for example, (1) an agent that inhibits cholesterol absorption and/or bile acid reabsorption (eg, ezetimibe); (2) an agent that increases lipoprotein catabolism (such as niacin); and/or (3) activators of the transcription factor LXR, which plays a role in the excretion of cholesterol, such as 22-hydroxycholesterol.

[00128] According to some embodiments, methods are provided comprising administering a PCSK9 inhibitor (e.g., an anti-PCSK9 antibody such as alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, or LY3015014) to a patient in combination with an angiopoietin-like protein 3 inhibitor (e.g., an anti-ANGPTL3 antibody, such as REGN1500), an angiopoietin-like protein 4 inhibitor (e.g., an anti-ANGPTL4 antibody, such as an anti-ANGPTL4 antibody, referred to in US Pat. (for example, antibodies to ANGPTL8).

[00129] According to some embodiments, methods are provided comprising administering to a patient a PCSK9 inhibitor (e.g., an anti-PCSK9 antibody such as alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, or LY3015014), in combination with an additional antidiabetic therapy agent in addition to the agent insulin therapy. Illustrative types of complementary antidiabetic therapies include, without limitation

(a) all medicines mentioned in the Rote Liste 2016 (for example, all antidiabetic drugs mentioned in chapter 12 of the Rote Liste 2014), all weight loss or appetite suppressants mentioned in chapter 06 of the Rote Liste 2016, all lipid-lowering agents mentioned in Rote Liste 2016 chapter 58, all antihypertensives mentioned in Rote Liste 2016 chapter 17, all nephroprotective drugs mentioned in Rote Liste or all diuretics mentioned in Rote Liste 2016 chapter 36;

(b) glucagon-like peptide 1 (GLP-1) therapies, including GLP-1, GLP-1 analogs, and GLP-1 receptor agonists, e.g., GLP-1(7-37), GLP-1(7 -36)amide, lixisenatide (eg Lyxumia ^® ), exenatide (eg exendin-4, r-exendin-4, Byetta ^® , Bydureon ^® , exenatide NexP), exenatide-LAR, liraglutide (eg Victoza ^® ), semaglutide , taspoglutide, albiglutide, dulaglutide, albugon, oxyntomodulin, geniproside, ACP-003, CJC-1131, CJC-1134-PC, GSK-2374697, PB-1023, TTP-054, langlenatide (HM-11260C), CM-3, GLP-1 eligen, AB-201, ORMD-0901, NN9924, NN9926, NN9927, nodexen, viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, ZP-3022, CAM-2036, DA-3091 , DA-15864, ARI-2651, ARI-2255, exenatide-XTEN (VRS-859), exenatide-XTEN+glucagon-XTEN (VRS-859+AMX-808) and polymer-bound GLP-1 and GLP-1 analogs ;

(c) GLP-1/GIP dual agonists (eg, RG-7697 (MAR-701), MAR-709, BHM081, BHM089, BHM098); dual GLP-1/glucagon receptor agonists (eg, BHM-034, OAP-189 (PF-05212389, TKS-1225), TT-401/402, ZP2929, LAPS-HMOXM25, MOD-6030);

(d) GLP-1/gastrin dual agonists (eg ZP-3022).

(e) gastrointestinal peptides such as peptide YY 3-36 (PYY3-36) or analogues thereof and pancreatic polypeptide (PP) or analogues thereof;

(f) glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, ghrelin antagonists or inverse agonists, xenin and analogues thereof;

(g) dipeptidyl peptidase-IV (DPP-4) inhibitors, eg alogliptin (eg Nesina ^® , Kazano ^® ), linagliptin (eg Ondero ^® , Trajenta ^® , Tradjenta ^® , Trayenta ^® ), saxagliptin (eg Onglyza ^{® ,} Komboglyze XR ^® ), sitagliptin (eg Januvia ^® , Xelevia ^® , Tesavel ^® , Janumet ^® , Velmetia ^® , Juvisync ^® , Janumet XR ^® ), anagliptin, teneligliptin (eg Tenelia ^® ), trelagliptin, vildagliptin (eg Galvus ^® , ^Galvumet® ), gemigliptin, omarigliptin, evogliptin, dutogliptin, DA-1229, MK-3102, KM-223, KRP-104, PBL-1427, pinoxacin hydrochloride and Ari-2243;

(h) sodium dependent glucose transporter 2 (SGLT-2) inhibitors, eg canagliflozin, dapagliflozin, remogliflozin, remogliflozin etabonate, sergliflozin, empagliflozin, ipragliflozin, tofogliflozin, luseogliflozin, ertugliflozin, EGT-0001442, LIK-066, SBM-TFC- 039 and KGA-3235 (DSP-3235);

(i) dual SGLT-2 and SGLT-1 inhibitors (eg LX-4211, LIK066);

(j) SGLT-1 inhibitors (eg LX-2761, KGA-3235) or SGLT-1 inhibitors in combination with anti-obesity drugs such as ileal bile acid transport (IBAT) inhibitors (eg GSK-1614235+ GSK-2330672);

(k) biguanides (eg metformin, buformin, phenformin);

(l) thiazolidinediones (eg pioglitazone, rosiglitazone), glitazone analogs (eg lobeglitazone);

(m) peroxisome proliferator-activated receptor (PPAR-) agonists or modulators (alpha, gamma, or alpha/gamma) (eg, saroglitazar (eg, ^Lipaglyn® ), GFT-505) or PPAR gamma partial agonists (eg, Int-131 );

(n) sulfonylureas (eg tolbutamide, glibenclamide, glimepiride, ^Amaryl® , glipizide) and meglitinides (eg nateglinide, repaglinide, mitiglinide);

(o) alpha-glucosidase inhibitors (eg acarbose, miglitol, voglibose);

(q) amylin and amylin analogs (eg pramlintide, ^Symlin® );

(p) G protein coupled receptor 119 (GPR119) agonists (e.g. GSK-1292263, PSN-821, MBX-2982, APD-597, ARRY-981, ZYG-19, DS-8500, HM-47000, YH -Chem1);

(q) GPR40 agonists (eg, TUG-424, P-1736, P-11187, JTT-851, GW9508, CNX-011-67, AM-1638, AM-5262);

(r) GPR120 agonists and GPR142 agonists;

(s) systemic or poorly absorbed TGR5 (GPBAR1=G-protein coupled bile acid receptor 1) agonists (eg, INT-777, XL-475, SB756050);

(t) immunotherapeutic agents used in diabetes, e.g., oral C-C chemokine receptor type 2 (CCR-2) antagonists (e.g., CCX-140, JNJ-41443532), interleukin 1 beta (IL-1ß) antagonists (e.g., AC-201) or oral monoclonal antibodies (MoA) (eg, metalosamide, VVP808, PAZ-320, P-1736, PF-05175157, PF-04937319);

(v) anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, eg nuclear factor kappa B inhibitors (eg ^Triolex® );

(w) adenosine monophosphate-activated protein kinase (AMPK) stimulants, eg, imeglimin (PXL-008), Debio-0930 (MT-63-78), R-118;

(x) 11-beta-hydroxysteroid dehydrogenase 1 (11-beta-HSD-1) inhibitors (eg, LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329, HSD-016, BI-135585);

(y) glucokinase activators (eg, PF-04991532, TTP-399 (GK1-399), GKM-001 (ADV-1002401), ARRY-403 (AMG-151), TAK-329, TMG-123, ZYGK1);

(z) diacylglycerol O-acyltransferase (DGAT) inhibitors (eg pradigast (LCQ-908)), protein tyrosine phosphatase 1 inhibitors (eg troduskvemin), glucose-6-phosphatase inhibitors, fructose-1,6-bisphosphatase inhibitors, glycogen phosphorylase inhibitors, phosphoenolpyruvate carboxykinase inhibitors, glycogen synthase kinase inhibitors, pyruvate dehydrogenase kinase inhibitors;

(aa) glucose-4 transporter modulators, somatostatin receptor 3 agonists (eg, MK-4256);

(bb) one or more lipid-correcting agents are also suitable as combination partners, ^e.g. Inegy ^® , Simcor ^® ), atorvastatin (eg Sortis ^® , Caduet ^® ), rosuvastatin (eg Crestor ^® ), pravastatin (eg Lipostat ^® , Selipran ^® ), fluvastatin (eg Lescol ^® ), pitavastatin (eg Livazo ^® , Livalo ^® ), lovastatin (eg Mevacor ^® , Advicor ^® ), mevastatin (eg Compactin ^® ), rivastatin, cerivastatin (Lipobay ^® ), fibrates such as bezafibrate (eg Cedur ^® retard), ciprofibrate (eg Hyperlipen ^® ), fenofibrate (eg Antara ^® , Lipofen ^® , Lipanthyl ^® ), gemfibrozil (eg Lopid ^® , Gevilon ^® ), etofibrate, simfibrate, ronifibrate, clinofibrate, clofibride, nicotinic acid and its derivatives (eg niacin, in including sustained release formulations based on niacin), ago nicotinic acid 1 receptor nists (eg, GSK-256073), PPAR delta agonists, acetyl-CoA acetyltransferase (ACAT) inhibitors (eg, avasimib), cholesterol uptake inhibitors (eg, ezetimibe, Ezetrol ^® , Zetia ^® , Liptruzet ^® , ^Vytorin® , S-556971), bile acid binders (eg, cholestyramine, colesevelam), ileal bile acid transport (IBAT) inhibitors (eg, GSK-2330672, LUM-002), microsomal triglyceride transport protein inhibitors (MTP) (eg, lomitapide (AEGR-733), SLx-4090, granotapide), subtilisin/kexin type 9 proprotein convertase 9 (PCSK9) modulators (eg, alirocumab (REGN727/SAR236553), AMG-145, LGT-209, PF -04950615, MPSK3169A, LY3015014, ALD-306, ALN-PCS, BMS-962476, SPC5001, ISIS-394814, 1B20, LGT-210, 1D05, BMS-PCSK9Rx-2, SX-PCK9, RG7652), LDL receptor positive regulators , for example, liver-selective thyroid hormone receptor beta-agonists (eg, eprotir (KB-2115), M B07811, sobetirome (QRX-431), VIA-3196, ZYT1), HDL-enhancing compounds such as cholesterol ester transfer protein (CETP) inhibitors (eg, anacetrapib (MK0859), dalcetrapib, evacetrapib, JTT-302, DRL -17822, TA-8995, R-1658, LY-2484595, DS-1442), or dual CETP/PCSK9 inhibitors (eg, K-312), ATP-binding cassette regulators (ABC1), lipid metabolism modulators (eg, BMS -823778, TAP-301, DRL-21994, DRL-21995), phospholipase A2 (PLA2) inhibitors (eg darapladib, ^Tyrisa® , varespladib, rilapladib), ApoA-I enhancers (eg RVX-208, CER-001, MDCO-216, CSL-112), cholesterol synthesis inhibitors (eg, ETC-1002), lipid metabolism modulators (eg, BMS-823778, TAP-301, DRL-21994, DRL-21995), and omega-3 fatty acids and their derivatives (eg icosapent ethyl (AMR101), ^Epanova® , AKR-063, NKPL-66, PRC-4016, CAT-2003);

(cc) bromocriptine (eg Cycloset ^® , Parlodel ^® ), phentermine and phentermine formulations or combinations (eg Adipex-P, Ionamine, Qsymia ^® ), benzphetamine (eg Didrex ^® ), diethylpropion (eg Tenuate ^® ) , phendimetrazine (eg Adipost ^® , Bontril ^® ), bupropion and combinations (eg Zyban ^® , Wellbutrin XL ^® , Contrave ^® , Empatic ^® ), sibutramine (eg Reductil ^® , Meridia ^® ), topiramate (eg Topamax ^® ) , zonisamide (eg Zonegran ^® ), tesofensine, opioid antagonists such as naltrexone (eg Naltrexin ^® , naltrexone + bupropion), cannabinoid receptor 1 (CB1) antagonists (eg TM-38837), melanin-concentrating hormone (MCH-1) antagonists ) (e.g., BMS-830216, ALB-127158(a)), MC4 receptor agonists and partial agonists (e.g., AZD-2820, RM-493), neuropeptide Y5 (NPY5) or NPY2 antagonists (e.g., wellneperite, S-234462 ), NPY4 agonists (eg, PP-1420), beta-3-adrenergic agonists, leptin or leptin mimetics, agonist 5-hydroxytryptamine 2c receptor (5HT2c) (eg, lorcaserin, Belviq ^® ), pramlintide/metreleptin, lipase inhibitors such as cetilistat (eg, Cametor ^® ), orlistat (eg, Xenical ^® , Calobalin ^® ), angiogenesis inhibitors (eg , ALS-L1023), beta-histidine and histamine H3 antagonists (eg HPP-404), AgRP (agouti-like protein) inhibitors (eg TTP-435), serotonin reuptake inhibitors such as fluoxetine (eg Fluctine ^® ), duloxetine (eg, Cymbalta ^® ), dual or triple monoamine uptake inhibitors (reuptake of dopamine, norepinephrine, and serotonin) such as sertraline (eg, Zoloft ^® ), tesofensine, methionine aminopeptidase 2 (MetAP2) inhibitors (eg, beloranib), and antisense oligonucleotides against the production of fibroblast growth factor receptor 4 (FGFR4) (eg, ISIS-FGFR4Rx) or prohibitin-targeting peptide-1 (eg, ^Adipotide® ); and

(dd) nitric oxide donors, AT1 antagonists or angiotensin II receptor (AT2) antagonists such as telmisartan (eg ^Kinzal® , ^Micardis® ), candesartan (eg ^Atacand® , ^Blopress® ), valsartan (eg ^Diovan® , Co -Diovan ^® ), losartan (eg Cosaar ^® ), eprosartan (eg Teveten ^® ), irbesartan (eg Aprovel ^® , CoAprovel ^® ), olmesartan (eg Votum ^® , Olmetec ^® ), tazosartan, azilsartan (eg Edarbi ^® ), dual angiotensin receptor blockers (dual ARBs), angiotensin converting enzyme (ACE) inhibitors, ACE-2 activators, renin inhibitors, prorenin inhibitors, endothelin converting enzyme (ECE) inhibitors, endothelin receptor blockers (ET1/ETA), endothelin antagonists, diuretics , aldosterone antagonists, aldosterone synthase inhibitors, alpha-blockers, alpha-2-adrenergic antagonists, beta-blockers, mixed alpha/beta blockers, calcium antagonists, calcium channel blockers (CCB), nasal formulations n based on the calcium channel blocker diltiazem (eg, CP-404), dual mineralocorticoids/CCBs, centrally acting antihypertensives, neutral endopeptidase inhibitors, aminopeptidase-A inhibitors, vasopeptide inhibitors, dual vasopeptide inhibitors such as neprilysin-ACE inhibitors or neprilysin-A inhibitors ECE, dual-acting angiotensin (AT) and neprilysin receptor inhibitors, dual AT1/ETA antagonists, advanced glycation end-product (AGE) cleavers, recombinant renalase, high blood pressure vaccines such as RAAS (renin-angiotensin-aldosterone system) vaccines , AT1 or AT2 vaccines, drugs based on the pharmacogenomics of high blood pressure, such as modulators of gene polymorphisms with antihypertensive effect, inhibitors of platelet aggregation, and others; as well as

(ee) their combinations, which are suitable.

[00130] In some embodiments, the additional antidiabetic therapy is a GLP-1 therapy (eg, lixisenatide). In some embodiments, the GLP-1 therapy is formulated with methionine (eg, L-methionine or D-methionine). In some embodiments, polysorbate (e.g., polysorbate 20, polysorbate 80), poloxamer (e.g., poloxamer 188), benzalkonium chloride, histidine, lysine, and/or EDTA are absent or substantially absent from the GLP-1 therapy formulation. In some embodiments, the GLP-1 therapy formulation is free or substantially free of surfactants such as polyols (e.g., polypropylene glycols, polyethylene glycols, poloxamers, pluronics, tetronics), partial esters or fatty acid esters, and ethers. polyhydric alcohols such as glycerol and sorbitol (eg Span.RTM., Tween.RTM., Myrj.RTM., Brij.RTM., Cremophor.RTM). The GLP-1 therapy formulation may contain a suitable preservative (eg, phenol, m-cresol, benzyl alcohol, and/or p-hydroxybenzoate esters) and suitable tonicity modifiers (eg, glycerol, dextrose, lactose, sorbitol, mannitol, glucose , NaCl, calcium or magnesium compounds such as CaCl ₂ ). The concentrations of glycerol, dextrose, lactose, sorbitol, mannitol and glucose are usually in the range of 100-250 mm, NaCl in concentrations up to 150 mm.

[00131] In some embodiments, an insulin-based therapy that the patient is receiving is combined with an additional anti-diabetic therapy (eg, any of the aforementioned anti-diabetic therapies that are not insulin-based therapies). For example, in certain embodiments, the anti-diabetic therapy includes a combination of an insulin-based therapy (eg, insulin glargine) and a GLP-1 therapy (eg, lixisenatide). Such therapies may be provided either separately or as a single pharmaceutical composition. For example, insulin glargine and lixisenatide can be formulated into one pharmaceutical composition (eg ^Soliqua® 100/33) for daily injection.

[00132] In the context of these methods, an additional therapeutically active component(s), for example, any of the above agents or derivatives thereof, can be administered immediately before, simultaneously with, or immediately after administration of the PCSK9 inhibitor; (For the purposes of the present invention, such administration regimens are considered to be the administration of a PCSK9 inhibitor "in combination with" an additional therapeutically active component). The methods of the present invention include pharmaceutical compositions and methods of using them, in which the PCSK9 inhibitor is formulated together with one or more additional therapeutically active components, as described elsewhere in this document.

Administration of a PCSK9 inhibitor as add-on therapy

[00133] Methods of the present invention for treatment comprising treating a patient with hypercholesterolemia and diabetes with a PCSK9 inhibitor, such as an antibody or antigen-binding fragment thereof, that specifically binds PCKS9, wherein the PCSK9 inhibitor can be administered as an adjunct to the patient's initial insulin-based therapy and/or to LMT (if applicable), for example, as an adjunct to the patient's initial daily therapeutic regimen with insulin and/or a statin.

[00134] For example, the methods include additional therapeutic regimens wherein the PCSK9 inhibitor is administered as adjunctive therapy to the same constant multiple daily insulin regimen and/or daily statin regimen (i.e., the same dosage amount of the statin) that the patient received prior to receiving the PCSK9 inhibitor. In other embodiments, the PCSK9 inhibitor is administered as adjunctive therapy to an insulin and/or statin therapeutic regimen comprising insulin and/or statin in an amount greater than or less than the dose of insulin or statin that the patient was receiving prior to receiving the PCSK9 inhibitor. For example, after starting a therapeutic regimen that includes a PCSK9 inhibitor administered at a certain frequency and in a certain amount, the daily dose of insulin and/or statin administered or administered to a patient may (a) remain the same, (b) increase, or (c) decrease (e.g., dose up or down) compared to the daily dose of the statin that the patient was taking prior to starting a PCSK9 inhibitor therapeutic regimen, based on the patient's therapeutic needs.

Therapeutic efficacy

[00135] The methods result in a decrease in serum levels of one or more lipid components selected from the group consisting of LDL-C, ApoB, ApoB100, non-HDL-C cholesterol, total cholesterol, VLDL-C, triglycerides, Lp( a), HDL-C, LDL particle number, LDL particle size, ApoC3, ApoA-1, triglyceride-rich lipoprotein cholesterol (TRL-C) and residual cholesterol. In some embodiments, administration of a pharmaceutical composition containing a PCSK9 inhibitor to a patient will result in an average percentage reduction from baseline in serum low-density lipoprotein (LDL-C) cholesterol levels of at least about 25%, 30%, 35 %, 40%, 45%, 50%, 55%, 60%, or more; an average percent reduction from baseline in ApoB levels of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more; an average percent reduction from baseline in ApoB100 levels of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more; an average percentage reduction from baseline in non-HDL-C cholesterol of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more; an average percentage reduction from baseline in total cholesterol levels of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, or more; an average percent reduction from baseline in VLDL-C levels 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; an average percentage reduction from baseline in the number of LDL particles of at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more; an average percentage reduction from baseline in LDL particle size of at least about 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% or more; average percent reduction from baseline in apolipoprotein C3 (ApoC3) levels of at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0 %, 9.0%, 10% or more; an average percentage increase from baseline in HDL-C levels of at least about 1%, 2%, 3%, 4%, 5%, or more; an average percentage increase from baseline in ApoA-1 levels of at least about 1%, 2%, 3%, 4%, 5%, or more; an average percentage reduction from baseline in TRL-C levels of at least about 5%, 10%, 15%, 20%, 25%, 30%, or more; and/or an average percentage reduction from baseline in Lp(a) levels of at least about 5%, 10%, 15%, 20%, 25%, or more.

[00136] The methods of the present invention provide for the treatment of a patient with hypercholesterolemia and T1DM who is receiving an insulin-based therapy, the methods comprising administering multiple doses of an anti-PCSK9 antibody or antigen-binding fragment thereof to the patient at a dose of about 75 to 150 mg per dose, and a dosing frequency of approximately once every two weeks or every four weeks, or a dosing regimen in accordance with a dosing regimen with increasing doses, as disclosed herein. After approximately 8, 10, 12, 14, 16, 18, 20, 22, 24 or more weeks of treatment with an anti-PCSK9 antibody, a patient may experience a decrease in LDL-C levels from baseline of at least 35%, 50 % or 60%. In some embodiments, after one or more weeks of treatment with an anti-PCSK9 antibody, the patient experiences a decrease in LDL-C levels from baseline of about 35%, 50%, or 60% or more.

[00137] The methods of the present invention also provide for the treatment of a patient with hypercholesterolemia and T2DM who is receiving an insulin-based therapy, the methods comprising administering multiple doses of an anti-PCSK9 antibody or antigen-binding fragment thereof to the patient at a dose of about 75 to 150 mg per dose, and a dosing frequency of approximately once every two weeks or every four weeks, or a dosing regimen in accordance with a dosage escalation regimen as disclosed herein. After approximately 8, 10, 12, 14, 16, 18, 20, 22, 24 or more weeks of treatment with an anti-PCSK9 antibody, a patient may experience a decrease in LDL-C levels from baseline of at least 40%, 48 % or 54%. In some embodiments, after one or more weeks of treatment with an anti-PCSK9 antibody, the patient experiences a decrease in LDL-C levels from baseline of about 40%, 48%, or 54% or more.

[00138] As disclosed herein, the methods of the present invention do not alter a patient's diabetic parameters. For example, in certain embodiments, the method has no effect (e.g., no change greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) the patient's hemoglobin A1c (HbA1c) level. In certain embodiments, the method has no effect (e.g., no change greater than 2%, 4%, 6%, 8%, 10%, 12%, 15%, 18%, or 20%) of plasma glucose levels fasting (FPG) in a patient.

[00139] In other embodiments, the invention relates to the use of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase for the treatment of hypercholesterolemia in a patient with type 1 diabetes mellitus (T1DM).

[00140] In other additional embodiments, the present invention relates to methods for treating hypercholesterolemia in a patient with type 1 diabetes mellitus (T1DM).

[00141] In one embodiment, said use and/or method comprises the steps of:

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has

(i) T1DM and

(ii) hypercholesterolemia not adequately controlled with a maximum tolerated dose statin therapy; and

(b) administering to the patient 75 mg, 150 mg, or 300 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase, wherein the patient receives concomitant insulin therapy.

[00142] In one embodiment of said use and/or method, 75 mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

[00143] In one embodiment of said use and/or method, 150 mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

[00144] In one embodiment of said use and/or method, 300 mg of the antibody or antigen-binding fragment is administered to the patient every four weeks.

[00145] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs listed under SEQ ID NOs: 2, 3 and 4 and three light chain CDRs listed under SEQ ID NOs: 7, 8 and 10.

[00146] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) with the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (LCVR) with the amino acid sequence of SEQ ID NO: : 6.

[00147] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof is selected from the group consisting of alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, and LY3015014.

[00148] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof is alirocumab.

[00149] In one embodiment, said use and/or method further comprises the steps of:

(c) administering to the patient one or more subsequent 75 mg doses of the antibody or an antigen-binding fragment thereof approximately every two weeks if the patient's LDL-C level is below a threshold level, or administering one or more subsequent 150 mg doses of the antibody, or its antigen-binding fragment approximately every two weeks if the patient's LDL-C level equals or exceeds the threshold level.

[00150] In one embodiment, said use and/or method further comprises the step of:

(c) administering to the patient one or more subsequent 300 mg doses of the antibody or an antigen-binding fragment thereof approximately every four weeks if the patient's LDL-C level is below a threshold level, or administering one or more subsequent 150 mg doses of the antibody, or its antigen-binding fragment approximately every two weeks if the patient's LDL-C level equals or exceeds the threshold level.

[00151] In one embodiment of said application and/or method, the threshold level is 70 mg/dL.

[00152] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof is administered subcutaneously.

[00153] In one embodiment of said use and/or method, the patient is additionally receiving a lipid-correcting therapy (LMT) concomitant agent.

[00154] In one embodiment of said use and/or method, the LMT is selected from the group consisting of a statin, cholesterol absorption inhibitor, fibrate, niacin, an omega-3 fatty acid, and a bile acid sequestrant.

[00155] In one embodiment of said use and/or method, the LMT is a statin-based therapy.

[00156] In one embodiment of said use and/or method, the statin is selected from the group consisting of atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin, and cerivastatin.

[00157] In one embodiment of said use and/or method, the LMT is a maximum tolerated dose statin therapy.

[00158] In one embodiment of said use and/or method, the cholesterol absorption inhibitor is ezetimibe.

[00159] In one embodiment of said use and/or method, the patient suffers from statin intolerance.

[00160] In one embodiment of said use and/or method, the insulin based therapy is selected from the group consisting of human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degludec, insulin aspart, and basal insulin.

[00161] In one embodiment of said use and/or method, the patient is receiving a concomitant antidiabetic therapy in addition to an insulin based therapy.

[00162] In one embodiment of said use and/or method, the additional concomitant antidiabetic therapy is selected from the group consisting of a glucagon-like peptide 1 (GLP-1) therapy, a gastrointestinal peptide, a glucagon receptor agonist or antagonist, an agonist, or glucose-dependent insulinotropic polypeptide (GIP) receptor antagonist, ghrelin antagonist or inverse agonist, xenin, xenin analog, biguanide, sulfonylurea, meglitinide, thiazolidinedione, DPP-4 inhibitor, alpha-glucosidase inhibitor, sodium-dependent glucose transporter 2 (SGLT-2) inhibitor , SGLT-1 inhibitor, peroxisome proliferator-activated receptor (PPAR-) agonist or modulator (alpha, gamma, or alpha/gamma), amylin, amylin analog, G protein-coupled receptor 119 (GPR119) agonist, GPR40 agonist, agonist GPR120, GPR142 agonist, systemic or poorly absorbed TGR5 agonist, immunotherapeutic agent, we use anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, adenosine monophosphate-activated protein kinase (AMPK) stimulant, 11-beta-hydroxysteroid dehydrogenase 1 inhibitor, glucokinase activator, diacylglycerol-O-acyltransferase (DGAT) inhibitor, glucose transporter-4 modulator, somatostatin agonist receptor 3, a lipid-lowering agent, and combinations thereof.

[00163] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces LDL-C levels in a patient by at least 30%, 35%, 40%, or 45%.

[00164] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces non-HDL-C cholesterol levels in a patient by at least 25%, 30%, 35%, or 40%.

[00165] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces apolipoprotein C3 (ApoC3) levels in a patient.

[00166] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces the number and/or size of lipoprotein particles in a patient.

[00167] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof

(a) do not affect the patient's hemoglobin A1c (HbA1c) level and/or

(b) do not affect the patient's fasting plasma glucose (FPG) level.

[00168] In additional embodiments, the present invention relates to uses and/or methods for treating hypercholesterolemia in a patient with type 1 diabetes mellitus (T1DM), the method comprising the steps of:

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has

(i) T1DM and

(ii) hypercholesterolemia not adequately controlled with a maximum tolerated dose statin therapy;

(b) administering to the patient every two weeks 75 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase; and

(c) administering to the patient one or more subsequent doses of 75 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if the patient's LDL-C level is below 70 mg/dl, or administering one or more subsequent doses of 150 mg, antibody or antigen-binding fragment approximately every two weeks if the patient's LDL-C level is 70 mg/dl or greater,

wherein the antibody or antigen-binding fragment thereof comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6, and wherein the patient is receiving concomitant insulin therapy.

[00169] In additional embodiments, the present invention relates to uses and/or methods for treating hypercholesterolemia in a patient with type 2 diabetes mellitus (T2DM), the method comprising the steps of:

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has

(i) T2DM and

(ii) hypercholesterolemia not adequately controlled with a maximum tolerated dose statin therapy; and

(b) administering to the patient 75 mg, 150 mg, or 300 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase, wherein the patient receives concomitant insulin therapy.

[00170] In one embodiment of said use and/or method, 75 mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

[00171] In one embodiment of said use and/or method, 150 mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

[00172] In one embodiment of said use and/or method, 300 mg of the antibody or antigen-binding fragment is administered to the patient every four weeks.

[00173] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs listed under SEQ ID NOs: 2, 3 and 4 and three light chain CDRs listed under SEQ ID NOs: 7, 8 and 10.

[00174] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) with the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (LCVR) with the amino acid sequence of SEQ ID NO: : 6.

[00175] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof is selected from the group consisting of alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, and LY3015014.

[00176] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof is alirocumab.

[00177] In one embodiment, said use and/or method further comprises the steps of:

(c) administering to the patient one or more subsequent 75 mg doses of the antibody or an antigen-binding fragment thereof approximately every two weeks if the patient's LDL-C level is below a threshold level, or administering one or more subsequent 150 mg doses of the antibody, or its antigen-binding fragment approximately every two weeks if the patient's LDL-C level equals or exceeds the threshold level.

[00178] In one embodiment, said use and/or method further comprises the steps of:

(c) administering to the patient one or more subsequent 300 mg doses of the antibody or an antigen-binding fragment thereof approximately every four weeks if the patient's LDL-C level is below a threshold level, or administering one or more subsequent 150 mg doses of the antibody, or its antigen-binding fragment approximately every two weeks if the patient's LDL-C level equals or exceeds the threshold level.

[00179] In one embodiment of said application and/or method, the threshold level is 70 mg/dL.

[00180] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof is administered subcutaneously.

[00181] In one embodiment of said use and/or method, the patient is additionally receiving a lipid corrective therapy (LMT) concomitant agent.

[00182] In one embodiment of said use and/or method, the LMT is selected from the group consisting of a statin, cholesterol absorption inhibitor, fibrate, niacin, an omega-3 fatty acid, and a bile acid sequestrant.

[00183] In one embodiment of said use and/or method, the LMT is a statin-based therapy.

[00184] In one embodiment of said use and/or method, the statin is selected from the group consisting of atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin, and cerivastatin.

[00185] In one embodiment of said use and/or method, the statin therapy is a maximum tolerated dose statin therapy.

[00186] In one embodiment of said use and/or method, the cholesterol absorption inhibitor is ezetimibe.

[00187] In one embodiment of said use and/or method, the patient suffers from statin intolerance.

[00188] In one embodiment of said use and/or method, the insulin based therapy is selected from the group consisting of human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degludec, insulin aspart, and basal insulin.

[00189] In one embodiment of said use and/or method, the patient is receiving a concomitant antidiabetic therapy in addition to an insulin based therapy.

[00190] In one embodiment of said use and/or method, the additional antidiabetic therapy is selected from the group consisting of a glucagon-like peptide 1 (GLP-1) therapy, a gastrointestinal peptide, a glucagon receptor agonist or antagonist, an agonist or an antagonist. glucose-dependent insulinotropic polypeptide (GIP) receptor, ghrelin antagonist or inverse agonist, xenin, xenin analog, biguanide, sulfonylurea, meglitinide, thiazolidinedione, DPP-4 inhibitor, alpha-glucosidase inhibitor, sodium-dependent glucose transporter 2 (SGLT-2) inhibitor, SGLT-1 inhibitor, peroxisome proliferator-activated receptor (PPAR-) agonist or modulator (alpha, gamma, or alpha/gamma), amylin, amylin analog, G protein-coupled receptor 119 (GPR119) agonist, GPR40 agonist, GPR120 agonist , GPR142 agonist, systemic or poorly absorbed TGR5 agonist, immunotherapy used in diabetes e, anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, adenosine monophosphate-activated protein kinase (AMPK) stimulator, 11-beta-hydroxysteroid dehydrogenase 1 inhibitor, glucokinase activator, diacylglycerol-O-acyltransferase (DGAT) inhibitor, glucose transporter-4 modulator, somatostatin receptor 3 agonist , lipid-lowering agent and combinations thereof.

[00191] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces LDL-C levels in a patient by at least 30%, 35%, 40%, or 45%.

[00192] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces non-HDL-C cholesterol levels in a patient by at least 20%, 25%, 30%, or 35%.

[00193] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces ApoC3 levels in a patient.

[00194] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces the number and/or size of lipoprotein particles in a patient.

[00195] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof

(a) do not affect the patient's hemoglobin A1c (HbA1c) level and/or

(b) do not affect the patient's fasting plasma glucose (FPG) level.

[00196] In additional embodiments, the present invention relates to uses and/or methods for treating hypercholesterolemia in a patient with type 2 diabetes mellitus (T2DM), the method comprising the steps of:

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has

(i) T2DM and

(ii) hypercholesterolemia not adequately controlled with a maximum tolerated dose statin therapy;

(b) administering to the patient every two weeks 75 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase; and

(c) administering to the patient one or more subsequent doses of 75 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if the patient's LDL-C level is lower than 70 mg/dL, or administering one or more subsequent doses of 150 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if the patient's LDL-C level is 70 mg/dL or greater, wherein the antibody or antigen-binding fragment thereof comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6, and where the patient is receiving concomitant insulin therapy.

[00197] In other embodiments, the invention relates to the use of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase for the treatment of hypercholesterolemia in a patient with type 2 diabetes mellitus (T2DM) and atherosclerotic heart disease. vascular disease (ASCVD).

[00198] In other additional embodiments, the present invention relates to methods for treating hypercholesterolemia in a patient with T2DM and ASCVD.

[00199] In one embodiment, said use and/or method comprises the steps of:

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has

(i) T2DM,

(ii) ASCVD and

(iii) hypercholesterolemia not adequately controlled with a maximum tolerated dose statin-based therapy; and

(b) administering to the patient 75 mg, 150 mg, or 300 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase, wherein the patient receives concomitant insulin therapy.

[00200] In one embodiment of said use and/or method, ASCVD is defined as coronary heart disease (CHD), ischemic stroke, or peripheral arterial disease.

[00201] In one embodiment of said use and/or method, CHD includes acute myocardial infarction, asymptomatic myocardial infarction, and unstable angina.

[00202] In one embodiment of said use and/or method, 75 mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

[00203] In one embodiment of said use and/or method, 150 mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

[00204] In one embodiment of said use and/or method, 300 mg of the antibody or antigen-binding fragment is administered to the patient every four weeks.

[00205] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs listed under SEQ ID NOs: 2, 3 and 4 and three light chain CDRs listed under SEQ ID NOs: 7, 8 and 10.

[00206] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) with the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (LCVR) with the amino acid sequence of SEQ ID NO: : 6.

[00207] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof is selected from the group consisting of alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, and LY3015014.

[00208] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof is alirocumab.

[00209] In one embodiment, said use and/or method further comprises the steps of:

(c) administering to the patient one or more subsequent 75 mg doses of the antibody or an antigen-binding fragment thereof approximately every two weeks if the patient's LDL-C level is below a threshold level, or administering one or more subsequent 150 mg doses of the antibody, or its antigen-binding fragment approximately every two weeks if the patient's LDL-C level equals or exceeds the threshold level.

[00210] In one embodiment, said use and/or method further comprises the steps of:

(c) administering to the patient one or more subsequent 300 mg doses of the antibody or an antigen-binding fragment thereof approximately every four weeks if the patient's LDL-C level is below a threshold level, or administering one or more subsequent 150 mg doses of the antibody, or its antigen-binding fragment approximately every two weeks if the patient's LDL-C level equals or exceeds the threshold level.

[00211] In one embodiment of said application and/or method, the threshold level is 70 mg/dl.

[00212] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof is administered subcutaneously.

[00213] In one embodiment of said use and/or method, the patient is additionally receiving a lipid-correcting therapy (LMT) concomitant agent.

[00214] In one embodiment of said use and/or method, the LMT is selected from the group consisting of a statin, a cholesterol absorption inhibitor, a fibrate, a niacin, an omega-3 fatty acid, and a bile acid sequestrant.

[00215] In one embodiment of said use and/or method, the LMT is a statin-based therapy.

[00216] In one embodiment of said use and/or method, the statin is selected from the group consisting of atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin, and cerivastatin.

[00217] In one embodiment of said use and/or method, the statin therapy is a maximum tolerated dose statin therapy.

[00218] In one embodiment of said use and/or method, the cholesterol absorption inhibitor is ezetimibe.

[00219] In one embodiment of said use and/or method, the patient suffers from statin intolerance.

[00220] In one embodiment of said use and/or method, the insulin based therapy is selected from the group consisting of human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degludec, insulin aspart, and basal insulin.

[00221] In one embodiment of said use and/or method, the patient is receiving a concomitant antidiabetic therapy in addition to an insulin based therapy.

[00222] In one embodiment of said use and/or method, the additional antidiabetic therapy is selected from the group consisting of a glucagon-like peptide 1 (GLP-1) therapy, a gastrointestinal peptide, a glucagon receptor agonist or antagonist, an agonist or an antagonist. glucose-dependent insulinotropic polypeptide (GIP) receptor, ghrelin antagonist or inverse agonist, xenin, xenin analog, biguanide, sulfonylurea, meglitinide, thiazolidinedione, DPP-4 inhibitor, alpha-glucosidase inhibitor, sodium-dependent glucose transporter 2 (SGLT-2) inhibitor, SGLT-1 inhibitor, peroxisome proliferator-activated receptor (PPAR-) agonist or modulator (alpha, gamma, or alpha/gamma), amylin, amylin analog, G protein-coupled receptor 119 (GPR119) agonist, GPR40 agonist, GPR120 agonist , GPR142 agonist, systemic or poorly absorbed TGR5 agonist, immunotherapy used in diabetes e, anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, adenosine monophosphate-activated protein kinase (AMPK) stimulator, 11-beta-hydroxysteroid dehydrogenase 1 inhibitor, glucokinase activator, diacylglycerol-O-acyltransferase (DGAT) inhibitor, glucose transporter-4 modulator, somatostatin receptor 3 agonist , lipid-lowering agent and combinations thereof.

[00223] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces LDL-C levels in a patient by at least 30%, 35%, 40%, or 45%.

[00224] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces non-HDL-C cholesterol levels in a patient by at least 20%, 25%, 30%, or 35%.

[00225] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces ApoC3 levels in a patient.

[00226] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof reduces the number and/or size of lipoprotein particles in a patient.

[00227] In one embodiment of said use and/or method, the antibody or antigen-binding fragment thereof

(a) do not affect the patient's hemoglobin A1c (HbA1c) level and/or

(b) do not affect the patient's fasting plasma glucose (FPG) level.

[00228] In additional embodiments, the present invention relates to routes of administration and/or methods for treating hypercholesterolemia in a patient with T2DM and ASCVD, the method comprising the steps of:

(a) selecting a patient at high cardiovascular risk receiving an insulin-based therapy that has

(i) T2DM,

(ii) ASCVD and

(iii) hypercholesterolemia not adequately controlled with a maximum tolerated dose statin-based therapy;

(b) administering to the patient every two weeks 75 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase; and

(c) administering to the patient one or more subsequent doses of 75 mg of the antibody or antigen-binding fragment thereof approximately every two weeks if the patient's LDL-C level is below 70 mg/dL, or administering one or more subsequent doses of 150 mg, antibody or antigen-binding fragment approximately every two weeks if the patient's LDL-C level is 70 mg/dl or greater,

wherein the antibody or antigen-binding fragment thereof comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6, and wherein the patient is receiving concomitant insulin therapy.

EXAMPLES

[00229] The following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and prepare and use the compositions of the present invention, and are not intended to limit the scope of what the present inventors contemplate. as his invention. Efforts have been made to ensure accuracy with respect to the numbers used (eg amounts, temperatures, etc.), however, some experimental errors and deviations must be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric.

Example 1 Preparation of Human Anti-Human PCSK9 Antibodies

[00230] Human anti-PCSK9 antibodies were prepared as described above in US Pat. No. 8,062,640. An exemplary PCSK9 inhibitor used in the following example is a human anti-PCSK9 antibody referred to as "mAb316P", also known as "REGN727" or "alirocumab". mAb316P has the following amino acid sequence characteristics: a heavy chain containing SEQ ID NO:5 and a light chain containing SEQ ID NO:9; a heavy chain variable region (HCVR) containing SEQ ID NO:1 and a light chain variable domain (LCVR) containing SEQ ID NO:6; heavy chain complementarity determining region 1 (HCDR1) containing SEQ ID NO:2, HCDR2 containing SEQ ID NO:3, HCDR3 containing SEQ ID NO:4, light chain complementarity determining region 1 (LCDR1) containing SEQ ID NO:7, LCDR2 containing SEQ ID NO:8, and LCDR3 containing SEQ ID NO:10.

Example 2 A randomized, double-blind, placebo-controlled, parallel-group study to evaluate the efficacy and safety of alirocumab in insulin-treated patients with type 1 or type 2 diabetes and hypercholesterolemia at high cardiovascular risk not adequately controlled by LDL- C-corrective therapy at the maximum tolerated dose

INTRODUCTION

[00231] More than 380 million people worldwide have diabetes, most of whom will die due to cardiovascular disease (CVD). Compared to people without diabetes, those with diabetes are at higher risk of developing CVD, develop associated clinical complications at an earlier age, and have a reduced life expectancy of approximately 6-7 years. In addition to the significant consequences for humanity due to the disease, CVD contributes greatly to the overall health care costs of these patients.

[00232] This study, called Odyssey DM-Insulin, included adult patients with type 1 or type 2 diabetes mellitus receiving an insulin-based therapy with hypercholesterolemia at high risk of cardiovascular (CV) disease, not adequately controlled by maximally tolerated doses of a statin, with or without another lipid-correcting therapy (LMT).

RESEARCH OBJECTIVES

[00233] The primary objectives of the present study were to: (a) evaluate the efficacy of alirocumab compared with placebo in reducing calculated low-density lipoprotein (LDL-C) cholesterol levels after 24 weeks of treatment in high CV risk diabetic patients treated with with insulin, and with hypercholesterolemia not adequately controlled with LDL-C corrective therapy at the maximum tolerated dose; and (b) evaluate the safety and tolerability of alirocumab in diabetic patients treated with insulin.

[00234] Secondary study objectives were to evaluate the efficacy of alirocumab versus placebo on other lipid parameters at 12 and 24 weeks (e.g. measured levels of LDL-C, non-HDL-C cholesterol ), apolipoprotein B (Apo B), total cholesterol (TC), lipoprotein A (Lp(a)), high-density lipoprotein cholesterol (HDL-C), triglyceride levels (TG), triglyceride-rich lipoprotein (TGRL), apolipoprotein A- 1 (Apo A-1), apolipoprotein C3 (ApoC3) and the number and size of LDL particles.

RESEARCH PLAN

[00235] This was a randomized, double-blind, placebo-controlled, multinational, multicenter, phase 3b study to evaluate the efficacy and safety of alirocumab given by subcutaneous (SC) injection in insulin-treated patients at high CV risk in type 1 diabetes mellitus or type 2 and with hypercholesterolemia not adequately controlled with LDL-C corrective therapy at the maximum tolerated dose. The study consisted of a screening period of up to 3 weeks, a double-blind treatment period of 24 weeks, and a safety data observation period of 8 weeks after the end of the double-blind treatment period.

[00236] Patients, provided that they do not suffer from statin intolerance, took a constant, maximum tolerated dose of a statin-based therapy with or without another lipid-correcting therapy (LMT). The dose and dosing regimen of the statin, as well as the dosing regimen and doses of other lipid-correcting treatments (if applicable), were stable throughout the duration of the study, including 4 weeks before the screening period, during the screening period, and from screening to randomization. Patients were on a stable diet for glucose and lipid control throughout the duration of the study from screening to the 24 week visit. Patients were treated for diabetes in accordance with local/regional standards of care.

[00237] Patients were stratified by type of diabetes (i.e., type 1 diabetes versus type 2 diabetes). The recruitment of patients with type 2 diabetes was completed after stratification of approximately 400 patients. The recruitment of patients with type 1 diabetes was completed at the end of the target recruitment period.

[00238] Alirocumab was administered subcutaneously with an initial dose of 75 mg Q2W for 12 weeks with a blind escalation of alirocumab to 150 mg Q2W at week 12 if LDL-C at the week 8 visit was ≥70 mg/dL (1.81 mmol/l). Patients with LDL-C <70 mg/dL (1.81 mmol/L) at the week 8 visit continued with alirocumab 75 mg Q2W until the end of the treatment period.

[00239] Data on lipid parameters from blood samples were hidden after randomization. No attempt was made by the investigator or the patient to independently evaluate the patient's lipid profile after randomization prior to the week 24 visit, except for patient safety, at the discretion of the investigator.

[00240] Patients visited the research center at 3, 0, 8, 12, 20 and 24 weeks, undergoing laboratory tests at each visit. In addition, phone call visits were made at 4 and 32 weeks.

[00241] Adverse events (AE) that occurred within 70 days after the last dose of investigational drug product (IMP) were documented. Patients with a serious adverse event (SAE) or an adverse event of special interest (AESI) were followed up until resolution, stabilization, or death.

PATIENT SELECTION

[00242] The study included 517 patients, including 76 T1DM patients and 441 T2DM patients.

Inclusion Criteria

[00243] Patients included in this study met all of the following criteria:

[00244] (1) Patient with type 1 or type 2 diabetes treated with insulin and characterized by LDL-C levels ≥70 mg/dL (1.81 mmol/L) not adequately controlled by a constant maximum dose/regimen a statin that was tolerated by the patient at least 4 weeks before the screening visit (week -3) with or without another LMT. The maximum statin dose/regimen tolerated by the patient was the highest recorded dose/regimen tolerated by the patient, based on Investigator judgment or consideration. Some examples of acceptable reasons for a patient taking a lower dose of a statin include, but are not limited to, adverse effects at high doses, older age, low body weight index (BMI), regional practices, local package insert, or concomitant medications. Patients could take a dose of the statin every other day if the dose is taken continuously (eg, a dose every Monday, Wednesday, Friday, etc.). Concomitant treatment with more than 1 statin was not allowed. Patients who had documented statin intolerance as judged by the investigator and who as a result stopped receiving a statin-based therapy were also eligible to participate in the study. The reason(s) for the violation of the maximum dose/statin regimen (including statin intolerance) were documented in the individual registration card.

[00245] (2) Patients ≥18 years of age or of legal age at the screening visit, whichever is greater.

[00246] (3) Patients diagnosed with type 1 or type 2 diabetes at least one year before the screening visit (week -3). Patients diagnosed with type 1 diabetes must meet all of the following criteria:

(a) diagnosed before age 30;

(b) treatment with a multiple daily injection/basal prandial insulin regimen or an insulin pump regimen within 6 months of diagnosis; and

(c) C-peptide level <0.2 pmol/ml at the screening visit.

[00247] (4) Glycosylated hemoglobin (HbA1c) <10% at screening visit (week -3). Patients with elevated HbA1c levels (up to 10%) were eligible, provided there was no plan to target lower HbA1c levels during the study, as assessed by the investigator.

[00248] (5) Patients with a documented history of CVD (including CHD and/or CHD equivalent risks) and/or at least one additional CV risk factor.

[00249] History of CHD included at least one of the following:

(a) acute myocardial infarction (MI);

(b) asymptomatic MI;

(c) unstable angina;

(d) a coronary revascularization procedure (eg, percutaneous coronary intervention (PCI) or coronary artery bypass surgery (CABG)); and

(e) clinically significant CHD diagnosed by invasive or non-invasive testing (such as coronary angiography, treadmill exercise electrocardiogram, stress echocardiography, or radionuclide imaging).

[00250] Equivalent risks of CHD included at least one of the following:

(a) documented peripheral arterial disease that meets one of the following criteria:

(i) current intermittent claudication (muscle discomfort in the lower limb that is both repetitive and aggravated by exercise and relieved by rest for 10 minutes) of suspected atherosclerotic origin together with an ankle-brachial index < 0.90 in each leg at a state of rest;

(ii) a history of intermittent claudication (muscle discomfort in the lower limb that is both repetitive and aggravated by exercise and relieved by rest for 10 minutes) together with an endovascular procedure or surgery in one or both legs for a reason atherosclerotic disease, and

(iii) history of critical limb ischemia with thrombolysis, endovascular procedure, or surgery to one or both legs due to atherosclerotic disease; and

(b) a documented ischemic stroke with focal ischemic neurological deficit that lasted more than 24 hours, thought to be atherothrombotic in origin. Computed tomography or magnetic radioimaging should have been performed to exclude hemorrhage and non-ischemic neurological disease.

[00251] Cardiovascular risk factors included at least one of the following:

(a) hypertension (established while taking medications for hypertension);

(b) current cigarette smoking;

(c) age ≥45 years for men and ≥55 years for women;

(d) history of micro/macroalbuminuria;

(e) a history of diabetic retinopathy (preproliferative or proliferative);

(f) family history of premature CHD (father or brother under age 55; mother or sister under age 65);

(g) low HDL-C (<40 mg/dL (1.0 mmol/L) in men and <50 mg/dL (1.3 mmol/L) in women); and

(h) documented moderate chronic kidney disease (CKD) characterized by 15 ≤ eGFR <60 ml/min/1.73 m ² for 3 months or more, including screening visit).

[00252] (6) Written informed consent signatories.

Exclusion Criteria

[00253] Patients who met all of the above inclusion criteria were screened for the following exclusion criteria:

[00254] (1) Exclusion criteria related to study methodology:

(a) it is planned to start a new LMT during the course of the study or to change the dose of the current LMT;

(b) not taking a constant dose of LMT (including a statin or other LMT) for at least 4 weeks prior to the screening visit (week -3) or from screening to randomization, unless statin intolerance occurred, in which case the statin was discontinued for 4 weeks before the screening visit/during the screening period;

(c) the use of nutraceutical products or over-the-counter therapeutics that may affect lipid levels and the dose of which has not been constant for at least 4 weeks prior to the Screening Visit (week -3) or between the Screening Visit and the Randomization Visit.

(d) consumption of red fermented rice products within 4 weeks prior to the screening visit (week -3) or between the screening visit and the randomization visit.

(e) use of systemic corticosteroids, except when used as replacement therapy for pituitary/adrenal disease, on a continuous regimen for at least 6 weeks prior to randomization. Steroid preparations for topical, intra-articular, nasal, inhalation and ophthalmic use are not considered "systemic" and are permitted;

(f) the use of continuous hormone replacement therapy, unless the regimen was constant during the last 6 weeks prior to the screening visit (week -3) and no change in regimen is planned during the study.

(g) recent (within 3 months prior to screening visit (week -3) or between screening visit and randomization visit) MI, unstable angina leading to hospitalization, uncontrolled cardiac arrhythmia, CABG, PCI, carotid surgery or stenting, stroke, transient ischemic attack (TIA), endovascular procedure or surgery for peripheral vascular disease;

(h) planned planned PCI, CABG, carotid or peripheral revascularization during the course of the study;

(i) a history of class III or IV heart failure (see Table 1) according to the New York Heart Association (NYHA) within the past 12 months;

(j) systolic blood pressure >180 mm Hg. Art. or diastolic blood pressure >110 mm Hg. Art. during a screening or randomization visit;

(k) a patient who received plasmapheresis treatment within 2 months prior to the Screening Visit (week -3) or between the Screening Visit and the Randomization Visit, or is about to undergo it during the course of the study;

(l) a history of hemorrhagic stroke;

(m) a history of loss of PCSK9 function (ie, a genetic mutation or sequence change) or a history of homozygous familial hypercholesterolemia;

(n) Newly diagnosed or actively progressing cancer within the past 5 years, unless basal cell skin cancer, squamous cell skin cancer, or cervical cancer in situ has been cured, to the extent possible;

(o) a history of a positive HIV test result;

(p) a patient who has taken any active investigational medicinal products for 1 month or 5 half-lives, whichever is longer.

(q) patients not previously instructed on a cholesterol-lowering diet prior to the screening visit (week -3).

(r) a patient who withdrew consent at the time of screening (starting with a signed ICF);

(s) unstable weight, defined as a change of >5 kg within 2 months prior to the screening visit, as judged by the investigator.

(t) BMI >45 kg/m2 or plans to undergo bariatric surgery, weight loss programs, or plans to start weight loss medications during the study;

(u) recent initiation of weight loss medications (i.e., within 3 months prior to the screening visit or between screening and randomization) or recent bariatric surgery (within the last 6 months) and an active phase of weight loss, as assessed by the investigator;

(v) Patients who have not been treated with insulin for at least 6 months prior to the Screening Visit, or who are not on a consistent insulin regimen (i.e. there has been a change in insulin type, total injection time/frequency, route or location) administration, such as exclusively basal (type 2 diabetes), basal-prandial, etc.) within at least 3 months prior to the screening visit, or a potential need to change the type/frequency of insulin administration or route of administration during the study period;

(w) not taking a steady dose of insulin for at least 3 months prior to screening (i.e. more than a 30% change in total daily insulin dose as estimated by the Investigator) or the likelihood of needing to increase the insulin/hypoglycemic regimen during the course of the study, as judged by the investigator (eg, addition of a new agent, plans to increase insulin dose, etc.);

(x) other hypoglycemic drugs administered to the patient have not been constant for at least

3 months prior to screening visit;

(y) a history of recent decompensation of diabetes within 2 months prior to the screening visit (ie, diabetic ketoacidosis or hyperosmolar hyperglycemic status (HHS));

(z) receiving or planning to receive renal replacement therapy during the study (eg, hemodialysis, kidney transplant, etc.);

(aa) the presence of any clinically significant uncontrolled endocrine disorder known to affect serum lipids or lipoproteins. Patients on thyroid hormone replacement therapy may be included if thyroxine dosage has been constant for at least 3 months prior to screening and the patient's thyroid stimulating hormone (s-TSH) sensitivity levels are within ±10% from the normal range of the laboratory value at the screening visit;

(bb) laboratory data during the screening period (not including laboratory tests at randomization other than a pregnancy test):

(i) serum TG level >400 mg/dL (>4.52 mmol/L) (1 repeat lab test allowed);

(ii) a positive serum or urine test for pregnancy in women of reproductive age;

(iii) a positive test for hepatitis B surface antigen or hepatitis C virus antibody;

(iv) (eGFR) <15 ml/min/1.73 m2 according to the 4-variable kidney disease diet modification equation

(MDRD);

(v) ALT or AST >3 x ULN (1 repeat lab test allowed); or

(vi) creatine phosphokinase (CPK) level >3 x ULN (1 reassessment allowed); or

(cc) states/situations such as

(i) patients with short life expectancy;

(ii) the need for concomitant treatment that may affect the initial assessment;

(iii) the inability to meet certain protocol requirements (eg, the need for hospitalization, the ability to attend educational institutions, etc.);

(iv) the patient was the investigator or any junior investigator, research assistant, pharmacist, study coordinator, other staff member or relative directly involved in the implementation of the protocol.

(v) non-contact or any condition that could render the patient non-compliant with study procedures;

(vi) any technical/administrative reason preventing randomization of a patient in the study; or

(vii) any clinically significant abnormality identified at screening that, in the opinion of the Investigator or any Associate Investigator, would prevent safe completion of the study or would limit the assessment of endpoints, such as major systemic diseases, patients with short life expectancy.

Table 1 New York Heart Association (NYHA) functional classification of heart failure

Class Patient symptoms Class I (Norm) No limitation of physical activity. Ordinary physical activity does not cause excessive fatigue, heart palpitations, or shortness of breath (shortness of breath). Class II (lungs) Slight restrictions on physical activity. Nothing disturbs at rest, but ordinary physical activity leads to fatigue, heart palpitations or shortness of breath. Class III (moderate) Severe limitation of physical activity. Nothing disturbs at rest, but less than normal activity leads to fatigue, heart palpitations, or shortness of breath. Class IV (severe) Inability to perform any physical activity without discomfort. Symptoms of heart failure at rest. In the case of any physical activity, the discomfort increases.

[00255] (2) Exclusion criteria that apply to active comparators and/or mandatory background therapies: all contraindications to background therapies or warnings/precautions for use (if applicable) presented on the appropriate national product labeling.

[00256] (3) Exclusion criteria related to current state of knowledge regarding alirocumab:

(a) hypersensitivity to alirocumab or to any of the ingredients of alirocumab;

(b) a pregnant or lactating woman;

(c) women of reproductive age who are not protected by highly effective contraceptive method(s) (as defined by the ICF and/or by regional protocol in the case of a specific regional requirement) and/or who are unwilling or unable to undergo a pregnancy test. Women of reproductive age must have a confirmed negative pregnancy test result at the screening and inclusion visits. They must use an effective method of contraception for the duration of study treatment and for at least 10 weeks after the last IMP injection. The methods of contraception used had to meet the criteria for a highly effective method of birth control according to the "International Conference on Harmonization of Technical Requirements for Registration of Pharmaceutical Products for Human Use. M3(R2): Guidelines for Non-Clinical Safety Studies for Human Clinical Trials and Marketing Authorization of Pharmaceutical Products .ICH.2009 Jun: 1-25." In postmenopausal women, menstruation should be absent for at least 12 months.

INVESTIGATIONAL TREATMENTS

Investigational medicinal product

[00257] A sterile drug product based on alirocumab was supplied at a concentration of 75 ml/ml and 150 mg/ml in aqueous buffer, pH 6.0, with sucrose, histidine and polysorbate 20, in a volume of 1 ml, in an autoinjector (also known as a pre-filled pen). Sterile placebo for alirocumab was given in the same formulation as alirocumab without added protein in a 1 ml volume in a pre-filled pen for patients undergoing injection training and for those in the placebo treatment group . During the screening period, the patient (or other designated person) was required to perform a placebo self-injection exercise using a pre-filled pen prior to the first IMP administration.

[00258] For patients randomized to receive alirocumab, the starting dose was 75 mg administered subcutaneously once Q2W. The dose was increased blindly to 150 mg Q2W at week 12 for patients randomized to receive alirocumab if the LDL-C value at week 8 is ≥70 mg/dL (1.81 mmol/L). Patients randomized to placebo received a subcutaneous injection of Q2W for the entire 24-week treatment period.

Way and method of administration

[00259] A manual for the pre-filled pen (auto-injector manual) was provided to research centers, and instructions for use (auto-injector for use) were provided to the patient. Each IMP administration was a 1 ml subcutaneous injection into the abdomen, thigh, or outer shoulder (i.e., deltoid region). If another concomitant drug was administered at the same site scheduled for the IMP injection, the patient was advised to use the alternate site for the IMP injection.

[00260] The IMP may be administered by self-injection or with the help of another designated person (eg, spouse, relative, etc.). In the event that a designated person was to administer alirocumab to a patient during the study, it was ensured that the person was properly trained prior to the injection. All who planned to administer the IMP were trained by the study staff.

[00261] Instructions were provided to the patient (or other designated person (eg, spouse, relative, etc.) who will administer the injections) during training and as needed during the course of the study. Careful observation was made at the first visit and at other visits and feedback was provided as needed.

[00262] The used pre-filled pen was discarded in a sharps container provided to patients. It has been recommended that subcutaneous IMP injections be alternated within the anatomical region (eg, right thigh, then left thigh or right abdomen, then left abdomen). Patients also had the opportunity to receive injections in another anatomical region (eg, thigh, then abdomen or outer shoulder, etc.) during the study.

[00263] Patients were asked to store the IMP in a refrigerator. Prior to administration, the IMP should be removed and left in a safe place at room temperature for approximately 30-40 minutes. Thereafter, IMP should be administered as soon as possible.

Timing of introduction

[00264] During the screening period, patients or another designated person were required to perform a placebo self-injection exercise using a pre-filled pen prior to the first administration of the IMP.

[00265] At the randomization visit, the patient or other designee (such as spouse, relative, etc.) received the first injection of IMP at the study site under the direct supervision of a staff member of the study center. Patients were observed at the study site for at least 30 minutes after such a first injection of this study. If the assigned person changed during the course of the study, the new assigned person was trained using a placebo.

[00266] Then subcutaneous injections of IMP were performed outside the clinic, Q2W until the last injection. If the injection was scheduled on the same date as the study site visit, then the IMP was administered after the completion of blood sampling. Exceptionally, if the patient preferred to receive an injection at a study site, and arrangements were in place for injections to be given at a study center, this was also permitted.

[00267] IMP should be administered subcutaneously with Q2W, ideally around the same time of day. However, an interval period of ±3 days was acceptable. The time of day was chosen based on the preferences of the patients.

[00268] If, by mistake or due to other circumstances, the injection was delayed for more than 7 days after the missed date or missed completely, then the patient was asked to return to the original IMP schedule without administering the delayed injections. If, by mistake or due to other circumstances, the injection was delayed less than 7 days after the missed date, the patient was asked to administer the delayed injection and then resume the original IMP schedule.

UNINVESTIGATIONAL DRUGS

[00269] The following drug classes have been identified as non-IMPs because the drug is either background therapy or a potential rescue agent:

(a) statins;

(b) cholesterol absorption inhibitors (ezetimibe);

(c) bile acid sequestrants (eg cholestyramine, colestipol, colesevelam);

(d) nicotinic acid;

(e) fibrates (eg fenofibrate);

(f) omega-3 fatty acids (≥ 1000 mg daily); and

(g) types of insulin.

[00270] For background LMT, including those based on statins, research centers followed national product labeling for safety monitoring and patient management. Patients were on a consistent, patient-tolerated maximum dose/schedule of statin-based therapy with or without other LMT during the study. Lipid profile values in samples obtained after randomization were masked. However, for safety reasons, research centers have been made aware of dangerous levels of TG in order to make decisions about background LMT for the patient.

[00271] From the screening visit (week -3) until the visit at week 24, the background LMT was not changed. During this time, there was no dose adjustment, discontinuation or initiation of other statins or other LMT, except in exceptional circumstances in which particular concerns (including, without limitation, dangerous levels of TG reported by the central laboratory) justified such changes, at the discretion of the investigator. In the case of dangerous levels of TG, which were confirmed by repeated analyzes, the researcher conducted research, assisted the patient and added another LMT in his / her physician's opinion.

[00272] All fibrates were resolved on admission if the patient tolerated the medication and remained on a constant dose. If a patient required fibrate during the course of the study (i.e., as an emergency treatment in response to dangerous TG levels), only fenofibrate was allowed to be added. Background LMT and insulin were provided by the sponsor. Patients received these drugs in accordance with regional regulations.

[00273] STUDY BLINDING PROCEDURES

[00274] Alirocumab and placebo for alirocumab were provided in identically matched pre-filled pens and packaged identically, with labeling included to protect the blinding system. Each treatment kit was labeled with a number that was generated by a computer program received from the sponsor. Treatment kit numbers were obtained by the Investigator at the time of patient randomization, and follow-up patient visits were scheduled using IVRS/IWRS, which were available 24 hours a day, 7 days a week.

[00275] Under a double-blind design, study patients, investigators, and study site staff remained uninformed about study treatment and did not have access to randomization (treatment codes), except as described below.

Adverse event

[00276] The treatment code was open to reporting to a health authority any Suspected Unanticipated Serious Adverse Reaction (SUSAR), i.e., any serious adverse event that is both unexpected (based on the Investigator's Brochure) and reasonably associated with the use of an IMP in according to the opinion of one of the investigator and/or sponsor.

Lipid parameters

[00277] Lipid parameter values from blood samples obtained after the randomization visit conducted by the central laboratory were not shared with the research centers, so they could not draw conclusions about the treatment group of their patients based on the level of LDL-C achieved. The sponsor's steering group did not have access to lipid parameters associated with patient identification until after the final lock of the database occurred. For safety reasons, alerts about dangerous TG levels at TG values ≥500 mg/dL at any time after randomization were sent to the investigator.

[00278] At the end of the double-blind treatment period (week 24 visit), the investigator continued to monitor the patient's lipid levels in accordance with standard practice. Any lipid values after randomization were edited in the original documents and not shared with the sponsor.

Antibodies to alirocumab

[00279] The results of the study of antibodies to alirocumab of patients were not transmitted to research centers during the study. The sponsor's steering group did not have access to the results of the anti-alirocumab antibody study associated with the patient ID number until after the final blocking of the database. The technicians involved in the determination of patient anti-alirocumab antibody titers were removed from the operating group and a method was established to prevent potential disclosure.

Unlocking the randomization code throughout the study

[00280] In the case of AE, the code was revealed when knowledge of the IMP was necessary to treat the patient. If possible, the sponsor's monitoring team/medical expert should have been contacted prior to revealing the code. All calls were properly documented by the monitoring team with the date and time of the call, the name of the person contacted by the monitoring team, the patient ID, documentation of the request, and the decision to remove blinding or not.

[00281] Code discovery can be performed at any time using the appropriate Interactive Voice Response System (IVRS)/Interactive Web Response System (IWRS) module, depending on which system was used at the research center, and/or by calling any other telephone number provided by the sponsor for this purpose. However, it would be preferable to contact the study physician to discuss the case before lifting the blind. If blinding was lifted, the investigator was asked to document the date, time of day, and reason for the release of the code, and report this information on the appropriate e-CRF page. When documenting the reason for blinding cancellation, the investigator did not provide any details regarding the nature of the IMP. The investigator did not disclose details of the IMP to either the sponsor's representative or staff until the database was closed. In addition, when completing forms (eg, AE, SAE), the study treatment was not disclosed on the forms.

[00282] The code breaking materials were also stored in a form serving for the "24-Hour Ready System"; but this system should only be used in the most exceptional cases (i.e., when the IVR/IWR system is unavailable or the investigator and/or site staff cannot be contacted). However, the preferred option was de-blinding with IVRS. The clinical monitoring group informed the investigators about the availability of materials for the disclosure of the region code. A patient card, including the corresponding telephone number of the “24-hour system”, was provided to each patient who will take part in the study. Sponsor has also been allowed to perform deblinding on some SAEs in order to comply with regulatory reporting requirements (i.e. for some SAEs that are both related and unexpected).

[00283] If the code was disclosed, the patient permanently stopped taking the IMP.

METHOD OF DISTRIBUTION OF PATIENTS IN THE TREATMENT GROUP

[00284] A randomized list of treatment kit numbers was centrally compiled by the sponsor. IMPs (alirocumab 75 or 150 mg kits or placebo kits) were packaged according to this listing.

[00285] The trial supply operations manager provided a randomized list of treatment kit numbers, and the study biostatistician provided the randomization scheme to the provider of the centralized treatment distribution system. The said centralized treatment distribution system provider then generated a patient randomization list according to which it distributed the treatments to the patients.

[00286] Patients were randomized to receive either placebo or alirocumab during the treatment period in a double-blind study. The randomization ratio of alirocumab:placebo was 2:1. For each randomized patient, there were several matching treatment kit numbers (restocking visits) that were allocated through a centralized treatment distribution system. Randomization was stratified by type of diabetes (i.e., type 1 versus type 2).

[00287] Treatment kit numbers were allocated using a centralized treatment distribution system at the randomization visit (day 1, week 0) and then at week 12 as restocking visits and at unscheduled visits if needed.

[00288] For patients in the alirocumab treatment group, the treatment set allocated at week 12 was based on their LDL-C level at week 8, according to dose escalation rules. A regular exchange of data was planned between the central laboratory and the provider of the centralized treatment distribution system in order to blindly pass to the research centers and the sponsor.

[00289] Prior to randomizing a patient, the investigator or their designee had to contact the centralized treatment distribution system.

[00290] A randomized patient was defined as a patient who was enrolled and who received a treatment kit number from a centralized treatment distribution system, as documented in the enrollment log. The patient could not be randomized more than once per study. If a treatment was used without recourse to a centralized treatment distribution system, the patient was considered non-randomized and was excluded from the study.

[00291] Depending on the choice of research center, two types of centralized treatment distribution systems were used: IVRS and IWRS.

PACKAGING AND LABELING

[00292] During the double-blind treatment period, each double-blind treatment kit, either alirocumab or alirocumab placebo, was formulated to contain 6 pre-filled pens in a child-safe package. To protect the blinding system, all boxes for the double-blind injection kit had the same appearance and were therefore marked with a double-blind marking.

[00293] In addition to the double-blind injection sets, a training set containing 1 placebo for alirocumab in a pre-filled pen was prepared to instruct patients on the injection to be performed prior to randomization at the Screening Visit (week - 3, visit 1). If deemed necessary, a second placebo injection session for alirocumab was performed prior to randomization using an additional training set. Placebo injection training was performed and recorded in the CRF, including if the designated person who administered the IMP to the patient changed during the course of the study.

[00294] The packaging corresponded to the mode of administration. The content of the label was in accordance with local regulatory specifications and requirements.

STORAGE CONDITIONS AND SHELF LIFE

[00295] Researchers or other authorized persons (eg, pharmacists) were responsible for storing the IMP in a secure and secure location in accordance with local regulations, labeling specifications, policies, and procedures. Control of IMP storage conditions, in particular temperature control (eg, refrigeration), and information regarding stability in use, and instructions for handling IMPs should be in accordance with sponsor's guidelines.

[00296] The IMPs were stored in a refrigerator at +2°C to +8°C (36 to 46°F) at the research center. The temperature of the refrigerator at the research center was checked daily and recorded in a log. The IMP that was stored at the study center was stored in an appropriate locked room, under the responsibility of the investigator or a designee or other authorized person, in accordance with the storage conditions indicated on the label.

[00297] After restocking IMP kits for patients during visits to the study site, appropriate arrangements were made to transport the IMP kits from the study center to the patient's refrigerator.

STUDY ENDPOINTS

[00298] Baseline characteristics included standard demographics (eg, age, race, weight, height, etc.), disease characteristics, including medical and drug history for each patient.

Primary efficacy endpoint

[00299] The primary efficacy endpoint was the percentage change in LDL-C from baseline to week 24 in the Intent-to-Treat (ITT) population using all LDL-C values regardless of adherence (estimated ITT value) . The percentage change was defined as 100x (calculated LDL-C at 24 weeks - calculated LDL-C at baseline)/calculated LDL-C at baseline.

[00300] The initial LDL-C level calculated was the last LDL-C level obtained prior to the first IMP injection in the double-blind study. The calculated LDL-C level at week 24 was the LDL-C level obtained during the analysis interval at week 24. All calculated LDL-C levels (scheduled or unscheduled, fasted or non-fasted) between weeks 8-24 can be used to derive a value for the primary efficacy endpoint, if appropriate, as defined above.

Primary efficacy endpoints

[00301] Safety parameters (AE, laboratory parameters, vital signs) were assessed throughout the study. The security data observation was as follows:

(a) the period to treatment was defined as from signed informed consent to the first dose of double-blind IMP;

(b) Treatment-Emerged Adverse Event Period (TEAE), defined as the time from the first dose of double-blind IMP to the last dose of IMP+70 days (10 weeks), since a residual treatment effect is expected up to 10 weeks after the double-blind IMP study is stopped. ; and

(c) the post-treatment period was defined as the time starting the day after the end of the TEAE period until resolution/stabilization of all SAEs and AESIs, whichever comes last.

[00302] AE was any adverse medical event in a patient or a patient participating in a clinical study who was administered a pharmaceutical product, and which should not necessarily have a causal relationship with this treatment.

[00303] SAE is any adverse medical event that, at any dose,

(a) resulted in death;

(b) was life threatening. The term "life-threatening" in the definition of "serious" referred to an event in which the patient was at risk of death at the time of the event; however, he did not refer to a phenomenon that could hypothetically cause death if it were more severe;

(c) required hospitalization in a hospital or an extension of an ongoing hospitalization;

(d) resulted in permanent or significant disability/disability;

(e) was a congenital anomaly/congenital malformation; or

(f) was an important medical event.

[00304] Medical and scientific opinion should be taken into account when deciding whether rapid reporting is appropriate in other situations, such as important medical events that may not be directly life-threatening or result in death or hospitalization, but may put the patient at risk or may require medical or surgical intervention (i.e. special measures or corrective therapy) to prevent one of the other outcomes listed in the definition above.

[00305] The following list of medically significant events is intended to serve as a guide for determining which condition should be considered a medically important event. The list is not intended to be exhaustive:

(a) intensive care in the emergency room or at home due to allergic bronchospasm, abnormal blood composition (i.e. agranulocytosis, aplastic anemia, bone marrow aplasia, myelodysplasia, pancytopenia, etc.) or seizures (convulsions, epilepsy , epileptic seizure, short-term loss of consciousness, etc.);

(b) development of drug dependence or drug abuse;

(c) ALT >3 x ULN + total bilirubin >2 x ULN or asymptomatic elevation of ALT >10 x ULN;

(d) suicide attempt or any occurrence indicating suicidal tendencies;

(e) fainting, loss of consciousness (except in documented cases as a result of taking a blood sample);

(f) bullous skin eruptions;

(g) cancers diagnosed during the study or aggravated during the study;

(h) chronic neurodegenerative diseases (newly diagnosed) or aggravated during the study; and

(i) suspected transmission of the infectious agent if there is suspicion of transmission of the infectious agent through the medicinal product (for example, contamination of the product).

[00306] An Adverse Event of Special Interest (AESI) is an AE (serious or non-serious) that needs to be tracked, documented, and controlled in a predetermined manner. For this study, AESIs were

(a) ALT elevation: ALT ≥3 x ULN (if baseline ALT<ULN) or ALT ≥2 times baseline (if baseline ALT ≥ULN);

(b) about allergic events: allergic reactions to the drug and/or local reactions at the injection site, which the investigator considers allergic (or have an allergic component), which require consultation with another physician for further evaluation of hypersensitivity/allergy in accordance with the medical opinion of the investigator must be reported as AESI;

(c) pregnancy: pregnancy occurring in a female patient or the patient's male partner (if permitted by the female partner and local regulatory authorities) during the study or within 70 days of the last dose of study drug. Pregnancy was recorded as AESI in all cases. A pregnancy was qualified as SAE only if it met one or more of the SAE criteria. In case of pregnancy of a female patient included in the study, the administration of the study product was stopped. Follow-up of the pregnancy was mandatory until the outcome of the pregnancy was determined.

(d) overdose of IMP with clinical manifestations. An overdose (accidental or intentional) was an event suspected by the investigator or spontaneously reported by the patient (not based on systematic injection counts) and defined as taking a dose of at least twice the intended dose during the intended therapeutic interval (i.e., 2 or more injections were administered within <7 calendar days), which should be reported using the term “symptomatic overdose (accidental or intentional)”, with an indication of the circumstances in parentheses (e.g., “symptomatic overdose (accidental)” or “symptomatic overdose ( intentional)"). The patient was under observation and appropriate symptomatic treatment was prescribed. The circumstances of the overdose were explicitly stated in specific words, and symptoms, if any, were recorded on separate AE/SAE forms. Asymptomatic overdose was suggested to be reported as standard AE;

(e) Neurological events: Neurological events that require additional investigations/procedures and/or referral were suggested to be reported as AESI. If the event did not require additional investigations/procedures and/or referral, it was suggested that it be reported as a standard AE; and

(f) Neurocognitive events: All neurocognitive events were considered as AESI.

Secondary endpoints of effectiveness.

[00307] The key secondary endpoints of the present invention were as follows:

(a) percent change in calculated LDL-C level from baseline to week 24, using all LDL-C levels during the treatment period to evaluate efficacy (treatment estimate);

(b) percentage change in measured LDL-C from baseline to week 24 (estimated ITT value);

(c) percent change in calculated LDL-C level from baseline to week 12 (estimated ITT value);

(d) percentage change in measured LDL-C from baseline to week 12 (estimated ITT value);

(e) percent change in non-HDL-C cholesterol from baseline to week 24 (estimated ITT value);

(f) percentage change in Apo B level from baseline to week 24 (estimated ITT value);

(g) percentage change in total cholesterol from baseline to week 24 (estimated ITT value);

(h) proportion of patients achieving LDL-C levels <70 mg/dl before week 24 (estimated value on treatment);

(i) proportion of patients achieving LDL-C levels <50 mg/dl by week 24 (estimated value on treatment);

(j) the proportion of patients achieving non-HDL-C cholesterol levels <100 mg/dl before week 24 (treatment estimate);

(k) the proportion of patients achieving non-HDL-C cholesterol <80 mg/dl by week 24 (treatment estimate);

(l) percentage change in Lp(a) from baseline to week 24 (estimated ITT value);

(m) percent change in HDL-C from baseline to week 24 (estimated ITT);

(n) percentage change in TG level from baseline to week 24 (estimated ITT value);

(o) percent change in LDL-C particle number from baseline to week 24 (estimated ITT value); and

(p) percent change in LDL-C particle size from baseline to week 24 (estimated ITT value).

[00308] The following diabetes-related endpoints were also measured in this study:

(a) absolute change in HbA1c from baseline to 12 and 24 weeks (estimated by ITT and estimated by treatment);

(b) absolute change in FPG level from baseline to 12 and 24 weeks (estimated by ITT and estimated by treatment);

(c) absolute change in total daily insulin dose from baseline to weeks 12 and 24 (estimated ITT and estimated treatment values); and

(d) absolute change in the number of glucose-lowering treatments from baseline to 12, and

24 weeks (estimated ITT and estimated values on treatment).

[00309] Other efficacy endpoints in this study included

(a) percent change in calculated LDL-C level from baseline to week 12 (estimated value on treatment);

(b) percentage change in measured LDL-C from baseline to 12 and 24 weeks (estimated value on treatment);

(c) percent change in non-HDL, Apo B, total cholesterol, Lp(a), HDL-C, and TG from baseline to week 12 (estimated by ITT and estimated by treatment) and 24 weeks (estimated by treatment) ;

(d) the proportion of patients achieving calculated LDL-C <50 and <70 mg/dL by week 12 (estimated by ITT and estimated by treatment) and 24 (estimated by ITT);

(e) the proportion of patients with a decrease of 50% or more from baseline in calculated LDL-C at 12 and 24 weeks (estimated by ITT);

(f) the proportion of patients achieving non-HDL-C cholesterol <80 mg/dl and <100 mg/dl at 12 weeks (estimated by ITT and estimated by treatment) and 24 weeks (estimated by ITT);

(g) proportion of patients achieving Apo B <80 mg/dL at 12 and 24 weeks (estimated ITT and estimated values on treatment);

(h) percentage change in the number and size of LDL-C particles from baseline to week 12 (estimated by ITT and estimated by treatment) and 24 weeks (estimated by treatment);

(i) percentage change in TGRL, Apo A-1 and Apo C-III levels from baseline to 12 and 24 weeks (estimated by ITT and estimated by treatment);

(j) absolute change in the ratio of Apo B/Apo A-1 and TC/HDL-C from baseline to 12 and 24 weeks (estimated by ITT and estimated by treatment);

(k) proportion of patients achieving calculated LDL-C <70 and <50 mg/dL at 12 and 24 weeks according to baseline A1c <8% or ≥8% (estimated ITT and estimated values on treatment); and

(l) Proportion of patients achieving calculated LDL-C <70 mg/dL and <50 mg/dL at 12 and 24 weeks according to baseline A1c <median A1c or ≥median A1c (estimated ITT and estimated values on treatment) .

RESEARCH PROCEDURES

[00310] The interval period for week 0 was +3 days. The interval period for 8, 12 and 24 weeks was ±3 days. The interval period for 4, 20 and 32 weeks was ±7 days. For all visits after Day 1/Inclusion Visit, if the date of one visit was changed, then the next visit followed the original pattern as outlined in Figure 1.

Collection of blood samples

[00311] All blood sampling, including blood sampling for lipid parameters (e.g., TC, LDL-C, HDL-C, TG, non-HDL-C cholesterol, Apo A, Apo B, Apo C-III , Lp(a), LDL particle size and number), as well as plasma glucose levels, were performed in the morning, fasted (i.e. overnight, at least 10-12 hours of fasting and abstinence from smoking) , and prior to IMP injection for all site visits throughout the study. Drinking alcohol within 48 hours and intense physical activity within 24 hours prior to blood sampling were not recommended. If the patient was not fasted, no blood samples were taken and the patient was given a new appointment the next day (or as close to that date as possible) with instructions to fast (see conditions above).

Laboratory research

[00312] Laboratory data were collected in accordance with the study schedule shown in Figure 1 and the following guidelines:

(a) hematology: all visits except weeks 4 and 20; may be performed at week 0, depending on the circumstances and based on the clinical discretion of the investigator;

(b) chemical analyses: all visits except visits 3 and 6; may be performed at week 0, as appropriate and based on the clinical discretion of the investigator, with the exception of plasma glucose testing, which must be performed at week 0 for all patients;

(c) HbA1c: at screening and at 0, 12 and 24 weeks;

(d) lipid panel: at screening and at 0, 8, 12, 20 and 24 weeks;

(e) LDL-C measured by beta-cholesterol quantitation: 0, 12 and 24 weeks;

(f) other lipid scores (Apo B, Apo A-1, Apo C-III, LDL particle size and number, Lp[a]): 0, 12 and 24 weeks;

(g) liver test: all visits except visits 3 and 6; may be performed at week 0, depending on the circumstances and based on the clinical discretion of the investigator. In the event that total bilirubin levels are above the normal range, differentiation into conjugated and unconjugated bilirubin will occur automatically;

(h) creatine phosphokinase (CPK) level: all visits except visits 3 and 6; may be performed at week 0, depending on the circumstances and based on the clinical discretion of the investigator;

(i) hepatitis B surface antigen: only at the screening stage;

(j) hepatitis C virus antibody: at screening and week 24; if ALT levels increase during the study, antibodies to hepatitis C virus should be determined. If the result of the analysis of antibodies to hepatitis C virus was positive during the study, a second test was performed;

(k) pregnancy testing (only in women of childbearing potential): serum pregnancy test at screening only, urine pregnancy test at 0 and 24 weeks;

(l) thyroid stimulating hormone: screening only for patients who are receiving thyroid hormone replacement therapy;

(m) C-peptide: only at the screening stage;

(n) PCSK9 levels at week 0 only; and

(o) Anti-alirocumab antibody levels (week 0, week 12, and week 24).

Collection of urine samples

[00313] A urinalysis was performed at screening and at the week 24 visit. pH, specific gravity, and the presence of blood, protein, glucose, ketones, nitrates, leukocyte esterase, urobilinogen, and bilirubin were assessed using a test strip. If the test strip results were abnormal, then standard microscopy was performed. Microscopy was used to evaluate for the presence of red blood cells (RBCs), RBC aggregations, leukocytes (WBCs), WBC aggregations, epithelial cells (transitional, renal tubular, and squamous), casts (hyaline, epithelial, WBC, RBC, granular, adipose, cellular, broad granular , waxy), crystals (triple phosphate, calcium oxalate, calcium phosphate, calcium carbonate, uric acid, amorphous, ammonium biurate, bilirubin, leucine, tyrosine, cystine), bacteria, budding yeast, yeast hyphae, Trichomonas, oval fat bodies, fat, mucus and sperm.

[00314] A single urine sample was tested for albumin and creatinine at the screening step and at the week 24 visit to determine the albumin:creatinine ratio. Any clinically significant abnormality was immediately rechecked for confirmation before any decision was made for the respective patient.

Physical examination

[00315] A general physical examination was performed. If a new clinically significant anomaly or worsening from baseline was detected after inclusion, an AE was reported and the patient was referred for further clinical investigations and/or specialist advice according to the investigator's medical judgment.

Blood pressure and heart rate

[00316] Blood pressure (BP) was measured in a sitting position under standard conditions, at about the same time of day, on the same arm, with the same apparatus (after the patient rested quietly in a sitting position for at least 5 minutes). Values were recorded in e-CRF; both systolic and diastolic BP were recorded. At the first screening visit, BP was measured on both arms. At this visit, the arm with the higher diastolic pressure was determined and BP was measured on that arm throughout the study. This is the highest value recorded in the e-CRF.

[00317] Heart rate was measured during BP measurement.

Body weight and height

[00318] Body weight was determined with the patient wearing underwear or very light clothing and no shoes, with an empty bladder. The same scales were used throughout the study.

[00319] Height was measured because self-assessment of growth was not acceptable.

iTAQ

[00320] The ITAQ was a patient-reported outcome measure (PRO) to assess treatment acceptability over a 4-week period prior to completing the questionnaire. The patient was asked to fill it out at visits at 8 and 24 weeks.

Insulin Log

[00321] Patients were instructed to complete an insulin log to record his/her daily insulin dose (for basal insulin and for prandial insulin, as appropriate) at least 7 days prior to each visit, and were asked to bring this information to the next research visit. The patient was able to record the daily insulin dose for more than 7 days prior to the study visits, however, only the last 7 days prior to each visit were entered into the CRF.

RESULTS

[00322] A total of 76 T1DM patients and 441 T2DM patients were enrolled.

[00323] All of the 76 randomized T1DM patients were treated and therefore included in the safety population. The two randomized patients with T1DM (both in the alirocumab arm) were not included in the "Treatment Based" (ITT) population.

[00324] Of the 441 patients with T2DM, 3 were untreated (1 in the alirocumab group and 2 in the placebo group) and therefore were not included in the safety population. Twelve randomized patients with T2DM (7 in the alirocumab group and 5 in the placebo group) were not included in the ITT population.

[00325] Patients were not included in the ITT population if there was no calculated LDL-C value available at baseline or in one of the analysis periods up to week 24.

STUDY PATIENTS

[00326] Six (7.9%) patients with T1DM discontinued study treatment prematurely (3 [5.9%] in the alirocumab group (2 patients discontinued due to AE) and 3 [12.0%] in the placebo group (2 All 3 patients in the alirocumab group also did not complete the study period, while in the placebo group 2 patients also did not complete the study period and 1 patient remained in the study until the end of the study period.

Table 2 Distribution of patients with T1DM according to IVRS

Placebo (N=25) Alirocumab (N=51) Total (N=76) Randomized but untreated 0 0 0 Randomized and treated 25 (100) 51 (100) 76 (100) Completed study treatment period 22 (88.0) 48 (94.1) 70 (92.1) Did not complete study treatment period 3 (12.0) 3 (5.9) 6 (7.9) Completed the study period 22 (88.0) 49 (96.1) 71 (93.4) Did not complete study period 3 (12.0) 2 (3.9) 5 (6.6) Reason for stopping treatment Adverse event 2 (8.0) 2 (3.9) 4 (5.3) Subject declined to continue 1 (4.0) 1 (2.0) 2 (2.6) Reason for termination of the study Subject declined to continue 3 (12.0) 2 (3.9) 5 (6.6) Status at the time of last contact during the study live 25 (100) 51 (100) 76 (100) Dead 0 0 0

Percentages are calculated using the number of randomized patients as the denominator.

[00327] Thirty-nine (8.8%) patients with T2DM discontinued study treatment prematurely (29 (9.9%) in the alirocumab group and 10 (6.8%) in the placebo group). Of the 29 patients in the alirocumab arm, 18 patients also did not complete the study, and 11 patients remained in the study until the end of the study period. In the placebo group, out of 10 patients, 7 patients also did not complete the study period, and 3 patients remained in the study until the end of the study period.

[00328]

[00329] Table 3. Distribution of patients with T2DM according to IVRS

Placebo (N=147) Alirocumab (N=294) Total (N=441) Randomized but untreated 2 (1.4) 1 (0.3) 3 (0.7) Reason for no treatment Other 2 (1.4) 1 (0.3) 3 (0.7) Randomized and treated 145 (98.6) 293 (99.7) 438 (99.3) Completed study treatment period 135 (91.8) 264 (89.8) 399 (90.5) Did not complete study treatment period 10 (6.8) 29 (9.9) 39 (8.8) Completed the study period 138 (93.9) 275 (93.5) 413 (93.7) Did not complete study period 7 (4.8) 18 (6.1) 25 (5.7) Reason for stopping treatment Adverse event 2 (1.4) 15 (5.1) 17 (3.9) Subject declined to continue 3 (2.0) 8 (2.7) 11 (2.5) Insufficient adherence to study protocol 2 (1.4) 0 2 (0.5) Fatal outcome 1 (0.7) 0 1 (0.2) Other 2 (1.4) 6 (2.0) 8 (1.8) Reason for termination of the study Adverse event 0 1 (0.3) 1 (0.2) Fatal outcome 1 (0.7) 0 1 (0.2) Subject declined to continue 4 (2.7) 13 (4.4) 17 (3.9) Lost to follow up 0 1 (0.3) 1 (0.2) Insufficient adherence to study protocol 2 (1.4) 1 (0.3) 3 (0.7) Other 0 2 (0.7) 2 (0.5) Status at the time of last contact during the study live 146 (99.3) 294 (100) 440 (99.8) Dead 1 (0.7) 0 1 (0.2)

Percentages are calculated using the number of randomized patients as the denominator.

Demographics and characteristics at baseline

[00330] Baseline characteristics were generally similar between the alirocumab and placebo groups. 60.5% of randomized T1DM patients were male, while T2DM randomized male patients were 54.2%. T1DM patients were younger with a mean age of 56.1 (SD=9.5) than T2DM patients with a mean age of 64.0 (SD=9.1). The mean BMI for T1DM patients was 30.0 kg/m2 (SD=5.9) and the mean BMI of 32.6 kg/m2 (SD=5.06) was observed for T2DM patients.

Table 4 Patient Demographics and Characteristics at Baseline - Patients with Type 1 Diabetes According to IVRS.

Placebo (N=25) Alirocumab (N=51) Total (N=76) Age (years) Number Mean value (SD) 58.5 (7.8) 54.9 (10.1) 56.1 (9.5) Age group (years) [n(%)] <65 19 (76.0) 42 (82.4) 61 (80.3) >=65 to <75 6 (24.0) 8 (15.7) 14 (18.4) >=75 0 1 (2.0) 1 (1.3) Gender [n (%)] Number Male 17 (68.0) 29 (56.9) 46 (60.5) female 8 (32.0) 22 (43.1) 30 (39.5) Weight, kg) Mean value (SD) 86.98 (18.41) 87.93 (18.47) 87.62 (18.33) BMI (kg/m²) Mean value (SD) 28.71 (4.82) 30.59 (6.25) 29.97 (5.85) Systolic blood pressure (mmHg) Mean value (SD) 126.5 (15.5) 129.9 (14.5) 128.8 (14.8) Diastolic blood pressure (mmHg) Mean value (SD) 69.5 (8.5) 74.7 (9.8) 73.0 (9.7) Heart rate (beats per minute) Mean value (SD) 68.4 (9.3) 73.0 (10.9) 71.5 (10.6) BMI: body weight index

Table 5. Patient Demographics and Characteristics at Baseline - Patients with Type 2 Diabetes According to IVRS.

Placebo (N=147) Alirocumab (N=294) Total (N=441) Age (years) Number 147 294 441 Mean value (SD) 64.0 (9.4) 63.9 (8.9) 64.0 (9.1) Age group (years) [n(%)] <65 73 (49.7) 143 (48.6) 216 (49.0) >=65 to <75 55 (37.4) 126 (42.9) 181 (41.0) >=75 19 (12.9) 25 (8.5) 44 (10.0) Gender [n (%)] Number 147 294 441 Male 78 (53.1) 161 (54.8) 239 (54.2) female 69 (46.9) 133 (45.2) 202 (45.8) Weight, kg) Mean value (SD) 92.58 (20.80) 92.45 (18.38) 92.49 (19.20) BMI (kg/m²) Mean value (SD) 32.69 (5.50) 32.55 (4.84) 32.60 (5.06) Systolic blood pressure (mmHg) Mean value (SD) 132.4 (15.5) 131.7 (16.1) 131.9 (15.9) Diastolic blood pressure (mmHg) Mean value (SD) 75.1 (8.2) 76.0 (9.2) 75.7 (8.8) Heart rate (beats per minute) Mean value (SD) 72.5 (10.9) 72.8 (10.6) 72.7 (10.7) BMI: body weight index

[00331] Estimated LDL-C at baseline was higher in T1DM patients (mean=121.0 mg/dl, SD=51.2) than in T2DM patients (mean=110.4 mg/dl , SD=37.3). Triglyceride levels at baseline were lower in T1DM patients with median (Q1:Q3) = 102.0 mg/dL (76.5:135.0) than in T2DM patients with median (Q1:Q3) = 147. 0 mg/dl (105.0 : 212.0 ).

Table 6. Lipid parameters at baseline - pooled results of quantification in standard units - patients with type 1 diabetes according to IVRS.

Placebo (N=25) Alirocumab (N=51) Total (N=76) Estimated LDL-C (mg/dL) Mean value (SD) 110.2 (31.2) 126.4 (58.2) 121.0 (51.2) Measured LDL-C (mg/dL) Mean value (SD) 109.8 (31.4) 127.7 (58.1) 121.7 (51.1) Non-HDL-C cholesterol (mg/dl) Mean value (SD) 130.7 (34.2) 150.2 (62.9) 143.8 (55.6) Total cholesterol level (mg/dl) Mean value (SD) 195.2 (36.0) 205.1 (65.1) 201.9 (57.1) HDL-C level (mg/dl) Mean value (SD) 64.4 (17.4) 54.9 (13.7) 58.1 (15.6) Triglycerides (mg/dl) Mean value (SD) 100.0 (38.1) 119.1 (58.4) 112.8 (53.1) Triglyceride-rich lipoprotein (mg/dL) Mean value (SD) 20.9 (7.6) 23.7 (11.4) 22.8 (10.4) Lipoprotein-(a) level (mg/dl) Mean value (SD) 24.8 (29.2) 22.8 (23.0) 23.5 (25.1) Apolipoprotein B level (mg/dl) Mean value (SD) 87.0 (21.0) 99.7 (35.6) 95.4 (31.8) Apolipoprotein A1 (mg/dl) Mean value (SD) 166.3 (23.8) 152.3 (26.4) 157.0 (26.2)

In terms of other lipid parameters, patients with T1DM showed a percentage reduction from baseline in the number of LDL-C particles (mean LS) of 40.7% at week 12 and 44.4% at week 24 and a percentage decrease in LDL particle size -C by 2.3% at week 12 and 2.3% at week 24. ApoC3 levels were reduced in these patients by 6.9% at week 12 and 7.5% at week 24.

Table 7. Lipid parameters at baseline - pooled results of quantitative assessment in standard units - patients with type 2 diabetes according to IVRS.

Placebo (N=147) Alirocumab (N=294) Total (N=441) Estimated LDL-C (mg/dL) Mean value (SD) 109.6 (39.1) 110.8 (36.5) 110.4 (37.3) Measured LDL-C (mg/dL) Mean value (SD) 110.5 (37.4) 112.1 (34.3) 111.6 (35.3) Non-HDL-C cholesterol (mg/dl) Mean value (SD) 144.9 (48.5) 144.7 (42.6) 144.8 (44.6) Total cholesterol level (mg/dl) Mean value (SD) 189.9 (47.6) 190.2 (42.4) 190.1 (44.1) HDL-C level (mg/dl) Mean value (SD) 44.9 (13.2) 45.5 (12.5) 45.3 (12.7) Triglycerides (mg/dl) Mean value (SD) 189.4 (148.2) 174.6 (110.1) 179.5 (124.2) Triglyceride-rich lipoprotein (mg/dL) Mean value (SD) 34.6 (27.4) 33.2 (20.5) 33.7 (23.0) Lipoprotein-(a) level (mg/dl) Mean value (SD) 29.7 (37.6) 38.9 (49.6) 35.8 (46.1) Apolipoprotein B level (mg/dL) Mean value (SD) 96.2 (26.8) 97.0 (24.7) 96.7 (25.4) Apolipoprotein A1 (mg/dL) Mean value (SD) 141.7 (23.8) 141.6 (22.8) 141.6 (23.1)

[00332] With respect to other lipid parameters, T2DM patients showed a percentage decrease from baseline in LDL-C particle number (mean LS) of 37.6% at week 12 and 38.3% at week 24 and a percentage decrease particle size LDL-C by 2.6% at week 12 and 2.8% at week 24. ApoC3 levels were reduced in these patients by 6.3% at week 12 and 5.8% at week 24.

[00333] In patients with T1DM, the mean duration of diabetes and insulin use were similar in the treatment groups. The mean duration of diabetes was 34.92 years (SD=12.67) and the mean duration of insulin use was 34.81 years (SD=12.77). In patients with T2DM, mean duration of diabetes and insulin use were similar across the treatment groups. The mean duration of diabetes was 16.75 years (SD=8.13) and the mean duration of insulin use was 8.01 years (SD=6.90) in patients with T2DM.

[00334] the Duration of hypercholesterolemia was generally similar in the treatment groups and in patients with T1DM and T2DM.

[00335] The proportion of patients with statin intolerance reported by the investigator was 31.6% in T1DM patients and 23.8% in T2DM patients.

[00336] The proportion of patients receiving fibrates at randomization was 2.6% in T1DM patients and 8.8% in T2DM patients.

[00337] The proportion of patients taking cholesterol absorption inhibitors (including ezetimibe) at randomization was higher in the alirocumab group (13.6%) than in the placebo group (7.6%), in particular in patients with T2DM: 45 patients (15.3%) versus 10 patients (6.8%).

[00338] History of cardiovascular disorders and risk factors were generally similar in the treatment groups. The following differences were observed between patients with T1DM and T2DM:

(1) ASCVD was more common in T2DM patients than in T1DM patients (40.1% vs. 21.1%) with more frequent coronary heart disease (34.7% vs. 15.8%) and stroke (8, 2% vs. 2.6%) and rarer PAD (4.3% vs. 9.2%) in T2DM patients relative to T1DM patients.

(2) Among non-ASCVD patients, 56.7% of T1DM patients had target organ damage (microalbuminuria, macroalbuminuria) and/or CKD and/or retinopathy compared with 39.4% of T2DM patients. However, among patients without ASCVD, the following additional cardiovascular risk factors have been observed:

(a) more common in T1DM patients than in T2DM patients: current smoker (20.0% vs. 14.0%), preproliferative diabetic retinopathy (36.7% vs. 12.9%), and proliferative diabetic retinopathy (20.0% vs. 5.7%).

(b) Less common in T1DM patients than in T2DM patients: hypertension (55.0% vs. 84.8%), microalbuminuria (10.0% vs. 19.7%), low HDL-C (16, 7% versus 28.0%).

(3) The presence of 3 or more additional cardiovascular risk factors in patients without ASCVD was observed in 45% of patients with T1DM and 55.7% of patients with T2DM.

[00339] In general, patients with T1DM and T2DM were treated with high and medium intensity statins in both treatment groups, with a higher proportion of patients treated with medium intensity statins (58.9%). Overall, 59.0% of patients with T1DM and T2DM were treated with a statin alone.

Table 8. Means of background lipid-correcting therapy during randomization

Placebo (N=172) Alirocumab (N=345) Total (N=517) Any statin [n (%)] 129 (75.0) 258 (74.8) 387 (74.9) Statin intensity ^{a, c} [n (%)] Number 127 257 384 High 38 (29.5) 102 (39.5) 140 (36.2) Medium 85 (65.9) 143 (55.4) 228 (58.9) Low 4 (3.1) 12 (4.7) 16 (4.1) Statin only [n (%)] 100 (58.1) 205 (59.4) 305 (59.0) Any statin in addition to another LMT [n (%)] 29 (16.9) 53 (15.4) 82 (15.9) Other LMT only (no statin) [n (%)] 11 (6.4) 32 (9.3) 43 (8.3) Any non-statin LMT ^b [n (%)] 40 (23.3) 85 (24.6) 125 (24.2) Fibrates 15 (8.7) 26 (7.5) 41 (7.9) Bile acid sequestrant 0 1 (0.3) 1 (0.2) cholesterol absorption inhibitor 13 (7.6) 47 (13.6) 60 (11.6) Nicotinic acid and derivatives 2 (1.2) 3 (0.9) 5 (1.0) Omega-3 fatty acids >= 1000 mg/day 17 (9.9) 16 (4.6) 33 (6.4) PCSK9 inhibitor 0 0 0 Nutraceuticals affecting lipids/other 4 (2.3) 10 (2.9) 14 (2.7) No LMT (neither statins nor other LMT) [n (%)] 32 (18.6) 55 (15.9) 87 (16.8) Note: ^a Only for patients currently taking a statin. ^b with or without statins. ^c High-intensity statin corresponds to atorvastatin 40–80 mg daily, rosuvastatin 20–40 mg daily, or simvastatin 80 mg daily. Moderate intensity statin corresponds to 10-20 mg of atorvastatin daily or 5-10 mg of rosuvastatin daily, or 20-40 mg of simvastatin daily, or 40-80 mg of pravastatin daily, or 40 mg of lovastatin daily, or 80 mg of fluvastatin daily, or 2- 4 mg pitavastatin daily. Low-intensity statins correspond to simvastatin 10 mg daily, or pravastatin 10-20 mg daily, or lovastatin 20 mg daily, or fluvastatin 20-40 mg daily, or pitavastatin 1 mg daily. Patients receiving a statin at more than one intensity level are counted as taking the highest intensity statin. % is calculated using the number of patients randomized as the denominator, excluding each statin intensity, where % is calculated using the number of patients taking statins as the denominator, and for each daily dose category, where % is calculated using the number of patients taking that specific statin, as the denominator.

[00340] The impact of the investigational medicinal product on the safety assessment population is summarized in Table 9.

Table 9. Exposure to Investigational Medicinal Product - Injection

Placebo (N=170) Alirocumab (N=344) Duration of exposure to IMP injection (weeks) Mean value (SD) 23.31 (3.92) 23.16 (3.90) Duration of exposure to IMP injection by category [n (%)] >=1 to <4 weeks 0 2 (0.6) >=4 weeks to <8 weeks 5 (2.9) 6 (1.7) >=8 weeks to <12 weeks 1 (0.6) 6 (1.7) >=12 weeks to <16 weeks 3 (1.8) 7 (2.0) >=16 weeks to <24 weeks 13 (7.6) 34 (9.9) >=24 weeks 148 (87.1) 289 (84.0) Number of IMP injections Mean value (SD) 11.3 (2.0) 11.3 (2.1) Dose change [n (%)] Patients with dose escalation ^a But. 77/326 (23.6) Note: ^a Patients with post-exposure dose escalation via IVRS at week 12 to at least one injection of alirocumab 150 mg. The denominator corresponds to patients who received at least one injection after exposure via IVRS on W12. Patients were considered to be in the treatment group they were actually assigned to. The duration of IMP exposure in weeks is defined as: (date of last IMP injection + 14 days - date of first IMP injection)/7, regardless of interruptions.

EFFICIENCY

Primary efficacy endpoint

[00341] Alirocumab was superior to placebo in percent change in calculated LDL-C from baseline to week 24 in the T1DM and T2DM ITT patient population (as shown in Tables 10 and 11 and Figures 2 and 3). Among patients with T1DM, the proportion of individuals achieving LDL-C <70 mg/dl (<1.8 mmol/l) was 70.2% in the alirocumab group and 5.1% in the placebo group (P<0.0001) and the proportion of individuals achieving LDL-C <50 mg/dl (1.3 mmol/l) was 55.1% in the alirocumab group and 0% in the placebo group (P-value not calculated). Among patients with T2DM, the proportion of individuals achieving LDL-C <70 mg/dl (<1.8 mmol/l) was 76.4% in the alirocumab group and 7.4% in the placebo group (P<0.0001) and the proportion of individuals achieving LDL-C <50 mg/dl (1.3 mmol/l) was 50.7% in the alirocumab group and 2.4% in the placebo group (P<0.0001). Sensitivity analyzes for the primary efficacy endpoint showed similar results in both populations (data not shown).

Table 10. Percent Change in Calculated LDL-C from Baseline to Week 24: MMRM - Patients with Type 1 Diabetes According to IVRS

Estimated LDL cholesterol levels Placebo (N=25) Alirocumab (N=49) Initial level (mmol/l) Number 25 49 Mean value (SD) 2.9 (0.8) 3.2 (1.2) Median value 2.6 3.0 Min : Max 2:5 1:7 Baseline (mg/dL) Number 25 49 Mean value (SD) 110.2 (31.2) 122.5 (47.8) Median value 100.0 114.0 Min : Max 71:180 48:273 Percent change from baseline at week 24 (%) Least Squares Mean (SE) -3.9 (5.3) -51.8 (3.7) Difference for least squares mean (SE) versus placebo -47.8 (6.5) 95%CI (-60.7 to -35.0) P-value versus placebo <0.0001* Note: least squares means, standard errors (SE), and p-value derived from MMRM-based analysis (Mixed-Effects Models for Repeated Measures). The model includes fixed categorical effects of treatment group, IVRS randomization strata, time point and treatment group-time, stratum-time, treatment-stratum, treatment-stratum-time-point relationships, and fixed covariates of baseline LDL-value. C and baseline value-point-in-time relationship. The MMRM model is applied to all patients in the ITT population (i.e. patients with type 1 and type 2 diabetes). The MMRM model and baseline description were performed on patients with a baseline value and a postbaseline value in at least one of the analysis periods used in the model. The p-value is followed by '*' if it is statistically significant according to the fixed hierarchical approach applied to provide a tight control of the total Type I error rate of 0.05.

Table 11. Percent Change in Calculated LDL-C from Baseline to Week 24: MMRM - Patients with Type 2 Diabetes According to IVRS

Estimated LDL cholesterol levels Placebo (N=142) Alirocumab (N=297) Initial level (mmol/l) Number 142 287 Mean value (SD) 2.8 (1.0) 2.9 (0.9) Median value 2.6 2.7 Min : Max eighteen 1:7 Baseline (mg/dL) Number 142 287 Mean value (SD) 109.5 (38.7) 110.3 (35.9) Median value 102.0 104.0 Min : Max 43:309 45:279 Percent change from baseline at week 24 (%) Least Squares Mean (SE) 0.8 (2.2) -48.2 (1.6) Difference for least squares mean (SE) versus placebo -49.0 (2.7) 95%CI (from -54.4 to -43.6) P-value versus placebo <0.0001* Note: least squares means, standard errors (SE), and p-value derived from MMRM-based analysis (Mixed-Effects Models for Repeated Measures). The model includes fixed categorical effects of treatment group, IVRS randomization strata, time point and treatment group-time, stratum-time, treatment-stratum, treatment-stratum-time-point relationships, and fixed covariates of baseline LDL-value. C and baseline value-point-in-time relationship. The MMRM model is applied to all patients in the ITT population (i.e. patients with type 1 and type 2 diabetes). The MMRM model and baseline description were performed on patients with a baseline value and a postbaseline value in at least one of the analysis periods used in the model. The p-value is followed by '*' if it is statistically significant according to the fixed hierarchical approach applied to provide a tight control of the total Type I error rate of 0.05.

Secondary efficacy endpoints

[00342] Alirocumab resulted in a significant reduction from baseline to week 24 (compared to placebo) in non-HDL-C, ApoB, total cholesterol, and Lp(a) levels, as well as an increase in HDL-C in the ITT population patients with T1DM and T2DM (as shown in tables 12 and 13, respectively).

Table 12. Percent Change in Key Secondary Efficacy Endpoints from Baseline - Patients with Type 1 Diabetes

Selected Key Secondary Effectiveness Endpoints
% change from baseline, mean (SD) Placebo (N=25) Alirocumab (N=49) Difference vs placebo, % [95% CI], P-value Calculated LDL-C (week 12) -4.5 (5.0) -49.4 (3.5) -44.8 (6.1) [-56.9 to -32.8], p<0.0001. Non-HDL-C cholesterol (week 24) -3.2 (4.8) -45.9 (3.3) -42.7 (5.8) [-54.2 to -31.3], p<0.0001. Apo B level (week 24) -0.4 (4.3) -39.4 (3.0) -39.0 (5.3) [-49.4 to -28.7], p<0.0001. Total cholesterol (week 24) -0.7 (3.6) -29.9 (2.5) -29.2 (4.3) [-37.8 to -20.7], p<0.0001. Lp(a) (week 24) -4.3 (5.3) -23.0 (3.8) -18.7 (6.5) [-31.4 to -6.0], p<0.0001. HDL-C Level (Week 24)† 7.3 (3.5) 11.2 (2.4) 3.9 (4.1) [-4.2 to 12.0], p=0.343‡ Triglycerides (Week 24)† 1.9 (6.7) -13.6 (4.7) -15.5 (8.1) [-31.4 to 0.4], p=0.056‡ Number of LDL particles (week 24) -4.4 (4.6) -44.4 (3.2) -40.0 (5.6) [-51.0 to -28.9], p<0.0001‡ LDL particle size (week 24) 0.8 (0.5) -2.3 (0.3) -3.0 (0.6) [-4.2 to -1.9], p<0.0001‡ ApoC3 level (week 24, ITT) 1.6 (5.8) -7.4 (4.0) -9.0 [22.7 to 4.8] ApoA-I level (week 24, ITT) 7.9 (2.6) 10.2 (1.8) 2.3 [3.8 to 8.4] TRL-C level (week 24, ITT) -8.4 (8.1) -24.7 (5.6) -16.3 [-35.4 to 2.8] TRL-C level (week 24, on treatment) -4.2 (8.2) -24.3 (5.8) -20.1 [39.5 to 0.6] ^† Hierarchical testing stopped at the triglyceride endpoint in T2D participants and at the HDL-C endpoint in T1D participants, so all subsequent statistical comparisons were not considered statistically significant. ‡P-values are provided for illustrative purposes only. Apo - apolipoprotein; ITT - depending on the prescribed treatment; LDL-C - low density lipoprotein cholesterol; Lp (a) - lipoprotein (a); LS - least squares method; non-HDL-C cholesterol - other than low-density lipoprotein cholesterol; TRL-C, triglyceride-rich lipoprotein cholesterol; SE - standard error; SD - standard deviation.

Table 13. Percent Change in Key Secondary Efficacy Endpoints from Baseline - Patients with Type 2 Diabetes

Selected Key Secondary Effectiveness Endpoints % change from baseline, mean (SD) Placebo (N=142) Alirocumab (N=287) Difference vs placebo, % [95% CI], P-value Calculated LDL-C (week 12) 1.4 (2.1) -48.8 (1.4) -50.2 (2.5) [55.2 to -45.3], p<0.0001. Non-HDL-C cholesterol (week 24) 0.7 (2.0) -37.9 (1.4) -38.7 (2.4) [43.4 to -33.9], p<0.0001. Apo B level (week 24) 3.3 (1.7) -33.4 (1.3) -36.7 (2.1) [40.9 to -32.5], p<0.0001. Total cholesterol (week 24) 0.8 (1.5) -26.8 (1.0) -27.6 (1.8) [31.2 to -24.1], p<0.0001. Lp(a) (week 24) -0.5 (2.2) -19.0 (1.6) -18.4 (2.7) [23.7 to -13.2], p<0.0001. HDL-C Level (Week 24)† 3.7 (1.4) 8.1 (1.0) 4.4 (1.7) [1.1 to 7.7]
P<0.01 Triglycerides (Week 24)† 0.0 (2.7) -5.7 (2.0) -5.7 (3.4) [12.3 to 0.9], p=0.0902‡ Number of LDL particles (week 24) 1.9 (1.9) -38.3 (1.3) -40.2 (2.3) [44.7 to -35.6], p<0.0001‡ LDL particle size (week 24) -0.3 (0.2) -2.8 (0.1) -2.5 (0.2) [2.9 to 2.0], p<0.0001‡ ApoC3 level (week 24, ITT) 4.2 (2.3) -5.8 (1.7) -10.0 [15.6 to 4.4] ApoA-I level (week 24, ITT) 4.5 (1.0) 7.4 (0.7) 2.9 [0.5 to 5.4] TRL-C level (week 24, ITT) 1.4 (3.3) -15.7 (2.3) -17.2 (4.0) [25.1 to -9.3] TRL-C level (week 24, on treatment) 2.2 (3.4) -16.4 (2.4) -18.6 [from 26.6 to 10.5] ^† Hierarchical testing stopped at the triglyceride endpoint in T2D participants and at the HDL-C endpoint in T1D participants, so all subsequent statistical comparisons were not considered statistically significant. ‡P-values are provided for illustrative purposes only. Apo - apolipoprotein; ITT - depending on the prescribed treatment; LDL-C - low density lipoprotein cholesterol; Lp (a) - lipoprotein (a); LS - least squares method; non-HDL-C cholesterol - other than low-density lipoprotein cholesterol; TRL-C, triglyceride-rich lipoprotein cholesterol; SE - standard error; SD - standard deviation.

Diabetes related endpoints

[00343] In general, treatment with FPG and HbA1c, as well as glucose lowering, remained constant over time in patients with T1DM and T2DM in both treatment groups.

[00344] With regard to HbA1c, in the T1DM cohort, in the alirocumab group, the mean HbA1c% was 7.84% (SD=0.94) at baseline with a mean absolute change=-0.03% (0.6), in while in the placebo group, the mean HbA1c was 7.68% (0.78) at baseline, with a mean absolute change of -0.23% (0.36). In the T2DM cohort, the alirocumab group had a mean HbA1c of 7.52% (0.96) at baseline with a mean absolute change=0.18% (0.74), while the placebo group had a mean HbA1c of 7 .54% (1.02) at baseline, with mean absolute change=0.06% (0.66).

[00345] Regarding FPG, in the T1DM cohort, in the alirocumab group, the mean FPG was 173 mg/dL (SD=70.6) at baseline with a mean absolute change=-0.03 mg/dL (0.6), while in the placebo group, the mean FPG was 166.5 mg/dl (75.6) at baseline, with a mean absolute change=14.6 mg/dl (75.9). In the T2DM cohort, the alirocumab group had a mean FPG of 154.1 mg/dl (50.1) at baseline with a mean absolute change of 9.5 mg/dl (61.8), while the placebo group had a mean the FPG value was 153.5 mg/dl (52.5) at baseline, with a mean absolute change=10.0 mg/dl (47.0).

Safety

[00346] A total of 344 patients (51 with T1DM and 293 with T2DM) were treated with alirocumab, and 170 patients (25 with T1DM and 145 with T2DM) were treated with placebo.

[00347] In general, the rates of patients with any emerging treatment adverse events (TEAE) were similar in the treatment groups in the population to assess the safety of patients with T1DM or T2DM (see table 14).

Table 14 Adverse Event Profile Overview: Adverse Events Occurring During Treatment

Placebo (N=170) n (%) Alirocumab (N=344) n (%) Total (N=514) n (%) Patients with any TEAE 109 (64.1) 222 (64.5) 331 (64.4) Patients with any SAE occurring during treatment 14 (8.2) 25 (7.3) 39 (7.6) Patients with any TEAE resulting in death 1 (0.6) 0 1 (0.2) Patients with any TEAE that resulted in definitive discontinuation of treatment 4 (2.4) 17 (4.9) 21 (4.1) Note: TEAE: Post-Treatment Adverse Event, SAE: Serious Adverse Event. n (%) = number and percentage of patients with at least one TEAE.

[00348] TEAEs were more common (≥ 10%) in the following system organ classes (SOCs):

(a) infections and infestations (21.8% in the alirocumab group versus 21.8% in the placebo group);

(b) gastrointestinal disorders (13.1% in the alirocumab group versus 12.4% in the placebo group);

(c) musculoskeletal and connective tissue disorders (21.5% in the alirocumab group versus 15.9% in the placebo group); and

(d) General and injection site disorders (11.0% in the alirocumab group vs. 8.8% in the placebo group)

[00349] At the PT level, the most frequently reported TEAEs (≥ 2%) in the alirocumab group and with a difference of ≥ 0.5% in the placebo group were observed in descending order of frequency in the alirocumab group: myalgia (4.4% vs. 1.8%) arthralgia (2.9% vs 1.8%), bronchitis (2.6% vs 0.6%), dizziness (2.6% vs 1.2%) and peripheral edema (2.0% vs 0.6 %). In contrast, the most commonly reported TEAEs (≥ 2%) in the placebo group and with a difference of ≥ 0.5% in the alirocumab group were: influenza (2.3% vs. 2.9%), pain in the extremities (1.7% vs. 2 .9%) hypoglycemia (1.7% vs. 2.4%), cough (1.5% vs. 2.9%), musculoskeletal pain (1.2% vs. 2.4%), upper respiratory tract infection (0.9% vs. 2.4%), hyperglycemia (0.9% vs. 2.4%) and pneumonia (0.6% vs. 2.4%).

[00350] Overall, treatment-related SAEs were reported in 25 patients (7.3%) in the alirocumab group and in 14 patients (8.2%) in the placebo group. SAEs (at PT level) reported in more than 1 patient in both treatment groups were pneumonia (1 patient (0.3%) in the alirocumab group vs. 2 patients (1.2%) in the placebo group), vertebro - foraminal stenosis (in 2 patients (0.6%) in the alirocumab group compared with no patients in the placebo group) and urinary tract infections (in 2 patients (0.6%) compared with no patients in the placebo group). One death from myocardial infarction was reported in a patient with T2DM in the placebo group 1 month after the first dose of IMP (Visit 3). Overall, 17 patients (4.9%) in the alirocumab group and 4 patients (2.4%) in the placebo group experienced TEAE resulting in permanent treatment discontinuation. At the PT level, the proportion of patients with TEAE that led to permanent discontinuation of treatment, more than one patient in the treatment group had headache (2 patients (0.6%) in the alirocumab group and no patient in the placebo group), cognitive impairment (2 patients (0.6%) versus no patients), allergic dermatitis (2 patients (0.6%) versus no patients), and myalgia (3 patients (0.9%) versus 2 patients (1.2%) ).

[00351] For Adverse Events of Special Interest (AESI), an increase in ALT meeting AESI criteria was defined as ALT ≥3 x ULN (if baseline ALT <ULN) or ALT ≥2 times baseline (if baseline ALT≥ULN). These events were reported in 2 patients (0.6%) in the alirocumab group versus 1 patient (0.6%) in the placebo group.

[00352] Allergic reactions to a drug that meet the AESI criteria have been defined as allergic events that require consultation with another physician for further evaluation. These events were reported in 5 patients (1.5%) in the alirocumab group versus 4 patients (2.4%) in the placebo group. These reactions were mainly related to skin and subcutaneous tissue disorders, which were reported in 3 patients (0.9%) in the alirocumab group (1 allergic dermatitis, 1 eczema, and 1 photosensitivity reaction) and in 2 patients (1.2%) in the alirocumab group. placebo group (1 dermatitis, 1 drug rash). Two other AESI allergic drug reactions in the alirocumab group were drug hypersensitivity and eosinophilia.

[00353] Neurological events that meet the AESI criteria have been defined as neurological events that require additional examinations/procedures and/or referral to a specialist. These events were reported in 1 patient (0.3%) in the alirocumab (parasthesia) group versus 1 patient (0.6%) in the placebo group. Both events have been reported in patients with T2DM.

[00354] All neurocognitive phenomena were considered as AESI. Neurocognitive events according to sponsor or FDA group were reported in 4 patients (1.2%) in the alirocumab group compared with no patients in the placebo group. All events were reported in patients with T2DM: cognitive impairment was reported in 2 patients (0.6%), and memory impairment and amnesia in 1 patient (0.3%) in each case. It should be noted that 2 cases of cognitive impairment also led to the final discontinuation of treatment.

[00355] Local injection site reactions meeting the AESI criteria were defined as injection site reactions that were allergic and required consultation with another physician, or injection site reactions that were not allergic that were clinically significant (e.g., edema or erythema reaction with a diameter >2.5 cm; a reaction that interferes with activity). LISR confirmed by the investigator as being associated with IMP ('in eCRF') was reported in 6 patients (1.7%) in the alirocumab group versus 8 patients (4.7%) in the placebo group (placebo injection site reaction for alirocumab) . There are no reports of local injection site reactions (LISR) meeting the AESI criteria, defined as reactions that require consultation with another physician for further evaluation.

[00356] There were no reports of symptomatic overdose or pregnancy.

[00357] Analyzes of liver function tests (ALT, AST, ALP, total bilirubin), CPK, and renal function tests (creatinine, eGFR, BUN) showed no difference between treatment groups in changes over time for any of the studied parameters. PCSA analysis did not reveal an increase in PCSA ALT in any of the treatment groups during the study. In patients with normal baseline CPK values, an increase of >3 ULN (and ≤10 ULN) was reported in 7 patients (2.1%) in the alirocumab group compared to 1 patient (0.6%) in the placebo group. All patients suffered from T2DM. An increase in CPK > 10 ULN has not been reported.

[00358] Numerical, non-significant differences were observed in the proportions of patients with mild, moderate or severe reduction in glomerular filtration rate (GFR) during the treatment period, regardless of baseline: mild, moderate and severe reduction in GFR in 49.7%, 28.1 % and 3.8% of patients, respectively, in the alirocumab group and 50.6%, 24.4%, and 3.6% of patients, respectively, in the placebo group. Similarly, an increase in blood creatinine (≥30% and <100%) was measured in 13 (3.8%) patients in the alirocumab group versus 5 patients (3.0%) in the placebo group. No patient had an increase in blood creatinine >= 100%. There were no significant differences in the assessment of renal function.

[00359] No significant differences in vital signs were observed between treatment groups.

Example 3 Analysis of Individuals with Type 2 Diabetes Mellitus and ASCVD from Odyssey DM-Insulin Clinical Trials

[00360] Individuals with diabetes often have high levels of atherogenic lipoproteins and cholesterol, reflected in elevated levels of low-density lipoprotein (LDL-C) cholesterol, non-high-density lipoprotein cholesterol (non-HDL-C cholesterol), apolipoprotein B (ApoB) and low density lipoprotein particle number (LDL-PN). The presence of atherosclerotic cardiovascular disease (ASCVD) increases the risk of future cardiovascular events.

[00361] In this analysis, the present inventors evaluated the efficacy and safety of alirocumab in subjects with T2DM, high LDL-C or non-HDL-C cholesterol, and set the ASCVD to obtain the maximum tolerated dose of a statin in the DM-Insulin study. Participants in the DM-Insulin study with ASCVD and T1DM were not included in this analysis due to the low number of individuals in this group (alirocumab: n=11; placebo: n=5). As used in this example, ASCVD was defined as ischemic heart disease (CHD; acute and asymptomatic myocardial infarction (MI) and unstable angina), ischemic stroke, or peripheral arterial disease.

[00362] Baseline and efficacy data were analyzed according to studies. Efficacy analyzes included the percentage reduction from baseline in non-HDL-C, LDL-C, ApoB, and LDL-PN cholesterol levels at week 24, as well as the percentage of individuals achieving non-HDL-C levels <100 mg/dL ( <2.59 mmol/L), LDL-C <70 mg/dL (<1.81 mmol/L), and ApoB <80 mg/dL at week 24. Intention to Treatment (ITT) analysis included all randomized individuals with baseline LDL-C value and at least one LDL-C value up to week 24.

[00363] This analysis included 177 DM-Insulin individuals with established ASCVD and T2DM (Table 15).

Table 15 Baseline Characteristics (Randomized Population)

Alirocumab n=119) Placebo (n=58) Age, years, mean (SD) 66.2 (8.7) 64.9 (8.9) Gender, male, n (%) 79 (66.4) 32 (55.2) BMI, kg/m ² , mean (SD) 32.6 (4.5) 33.4 (5.8) CHD, n (%) 102 (85.7) 51 (87.9) Acute MI 59 (49.6) 18 (31.0) Asymptomatic MI 4 (3.4) 4 (6.9) Unstable angina 15 (12.6) 4 (6.9) Coronary revascularization procedure 80 (67.2) 37 (63.8) Other clinically significant CHD ^† 31 (26.1) 15 (25.9) Ischemic stroke, n (%) 27 (22.7) 9 (15.5) PAD, n (%) 13 (10.9) 6 (10.3) HTN, ^‡ n (%) 105 (88.2) 53 (91.4) CKD, ^§ n (%) 37 (31.1) 13 (22.4) Cases of diabetes with target organ damage, ^ǁ n (%) 60 (50.4) 28 (48.3) Statin, n (%) 88 (73.9) 39 (67.2) Any non-statin LLT, n (%) 34 (28.6) 11 (19.0) HbA1c, %, mean value (SD) 7.5 (0.9) 7.4 (1.0) FPG, mg/dL [mmol/L], mean value (SD) 162.6 (52.5) [9.0 (2.9)] 146.7 (45.2) [8.1 (2.5)] Insulin, n (%) 119 (100) 57 (98.3) ^¶ Other than insulin GLT, n (%) biguanides 57 (47.9) 33 (56.9) Sulfonylureas 11 (9.2) 7 (12.1) DPP-4 inhibitor 21 (17.6) 7 (12.1) GLP-1 receptor agonist 11 (9.2) 8 (13.8) SGLT2 inhibitor 10 (8.4) 11 (19.0) Lipids, mg/dL [mmol/L], mean (SD) Non-HDL-C cholesterol levels 142.8 (41.5) 147.0 (54.9) [3.70 (1.08)] [3.81 (1.42)] LDL-C level 107.2 (35.1) 111.9 (46.4) [2.78 (0.91)] [2.90 (1.20)] ApoB level 96.4 (25.1) 98.7 (32.0) LDL-PN, nmol/l, mean value (SD) 1339.5 (408.5) 1425.0 (467.9) ^† Diagnosed by invasive or non-invasive testing. ^‡ Includes patients with established HTN taking anti-HTN drugs. ^§ Defined as an estimated glomerular filtration rate of 15-60 ml/min/1.73 ^m2 . ^ǁ Defined as microalbuminuria, macroalbuminuria, retinopathy and/or CKD. ^¶ One individual in the placebo group did not receive insulin at the time of randomization and remained without insulin treatment throughout the study.
BMI - body weight index; DPP4 - dipeptidyl peptidase 4; GLP-1, glucagon-like peptide 1; GLT is a glucose lowering treatment; FPG - fasting plasma glucose level; HbA1c - glycated hemoglobin; HTN - hypertension; SGLT2 is sodium-glucose cotransporter type 2.

[00364] Regardless of the distribution of treatment, 89.3% of the surveyed individuals had a history of hypertension, and 28.2% had chronic kidney disease (CKD) in addition to ASCVD. Overall, 20.3% had a history of ischemic stroke and 10.7% had peripheral arterial disease (PAD). At baseline, mean (standard deviation [SD]) non-HDL-C cholesterol was 144.2 (46.2) mg/dL [3.73 (1.20) mmol/L]; mean LDL-C was 108.7 (39.1) mg/dL [2.82 (1.01) mmol/L], regardless of treatment distribution.

Efficiency

[00365] Alirocumab reduced non-HDL-C, ApoB, LDL-PN, and LDL-C cholesterol from baseline at week 24 compared to control (Figure 4). At week 24, a significantly higher proportion of people achieved non-HDL-C cholesterol <100 mg/dL (<2.59 mmol/L), LDL-C <70 mg/dL (<1.81 mmol/L) and ApoB level <80 mg/dL compared to control (all P<0.0001; FIG. 5).

Safety

[00366] Safety analyzes were performed in a pooled population of individuals with T2DM, high LDL-C or non-HDL-C cholesterol, and established ASCVD receiving the maximum tolerated dose of a statin in the DM-Insulin study, and individuals with T1DM or T2DM, high levels of LDL-C or non-HDL-C cholesterol and established by ASCVD receiving maximum tolerated doses of a statin in the DM-Dyslipidemia study (see Chan et al. (2017) Ann Transl Med. 5 (23): 477, which is included to this document by reference in its entirety). A total of 66.4% (alirocumab) and 67.0% (control) of patients reported treatment-related adverse events (TEAE; table 16). The pattern of adverse events was similar in both groups. Mean (SD) HbA1c levels were similar in each treatment group at baseline (alirocumab: 7.3 [0.9]%; control: 7.3 [0.9]%) and week 24 (alirocumab: 7.6 [0.9]% 1.2]%; control: 7.5 [1.2]%; safety analysis). FPG levels were also similar regardless of treatment distribution at baseline (alirocumab: 154.2 [47.9] mg/dL, 8.6 [2.7] mmol/L; control: 149.5 [43.7] mg /dl, 8.3 [2.4] mmol/l) and at week 24 (alirocumab: 164.7 [54.9] mg/dl, 9.1 [3.0] mmol/l; control: 159, 4 [48.4] mg/dL, 8.9 [2.7] mmol/L; safety analysis).

Table 16. Security summary

n(%) Alirocumab (n=213) Control (n=104) TEAE 142 (66.7) 70 (67.3) Occurred during the treatment of SAE 28 (13.1) 10 (9.6) TEAE leading to death 1 (0.5) 1 (1.0) TEAE leading to definitive treatment discontinuation 13 (6.1) 2 (2.3) TEAE occurring in >2% of people, according to the preferred term urinary tract infection 8 (3.8) 6 (5.8) Diarrhea 8 (3.8) 6 (5.8) Nasopharyngitis 6 (2.8) 5 (4.8) Flu 7 (3.3) 4 (3.8) hypertension 8 (3.8) 4 (3.8) Headache 7(3.3) 1 (1.0) Musculoskeletal pain 7(3.3) 3 (2.9) Arthralgia 3 (1.4) 3(2.9) Bronchitis 3 (1.4) 3 (2.9) Cough 1 (0.5) 3 (2.9) Fatigue 5 (2.3) 3 (2.9) hyperglycemia 0 (0.0) 3 (2.9) hypotension 2 (0.9) 3 (2.9) Nausea 4 (1.9) 3 (2.9) Pain in the limb 4 (1.9) 3 (2.9) Backache 6 (2.8) 2 (1.9) Dizziness 6 (2.8) 3 (2.9) Cataract 5 (2.3) 1 (1.0) Myalgia 5 (2.3) 1 (1.0) SAE, Serious Adverse Event.

conclusions

Among individuals with T2DM and ASCVD who had high levels of LDL-C despite taking the maximum tolerated dose of statins, alirocumab significantly reduced atherogenic cholesterol and LDL-PN compared to controls.

--->

SEQUENCE LIST

<110> Sanofi Biotechnology

<120> METHODS FOR TREATMENT OF HYPERLIPIDEMIA IN DIABETES PATIENTS WITH PCSK9 INHIBITOR ADMINISTRATION

<130> 602044:SA9-208

<150> US 62/517,672

<151> 2017-06-09

<150> US 62/532,162

<151> 2017-07-13

<150>EP 18305565.6

<151> 2018-05-04

<160> 198

<170> PatentIn version 3.5

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fifteen

<210> 32

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 32

Ile Asn Gln Asp Gly Ser Glu Lys

fifteen

<210> 33

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 33

Ala Arg Asp Ile Val Leu Met Val Tyr His Met Asp Tyr Tyr Tyr Tyr

1 5 10 15

Gly Met Asp Val

twenty

<210> 34

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 34

Gln Ser Leu His His Ser Asn Gly Asn Asn Tyr

1 5 10

<210> 35

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 35

Leu Gly Ser

one

<210> 36

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 36

Met Gln Thr Leu Gln Thr Pro Leu Thr

fifteen

<210> 37

<211> 131

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide VH; m2CX1D05

<400> 37

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Ser His

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Tyr Glu Ile Gln Ile Gly Arg Tyr Gly Met Asn Val Tyr

100 105 110

Tyr Leu Met Tyr Arg Phe Ala Ser Trp Gly Gln Gly Thr Leu Val Thr

115 120 125

Val Ser Ser

130

<210> 38

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR1 VH; m2CX1D05

<400> 38

Gly Gly Thr Phe Asn Ser His Ala Ile Ser

1 5 10

<210> 39

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR2 VH; m2CX1D05

<400> 39

Trp Met Gly Gly Ile Asn Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln

1 5 10 15

Lys Phe Gln Gly

twenty

<210> 40

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR3 VH; m2CX1D05

<400> 40

His Tyr Glu Ile Gln Ile Gly Arg Tyr Gly Met Asn Val Tyr Tyr Leu

1 5 10 15

Met Tyr Arg Phe Ala Ser

twenty

<210> 41

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide LC; m2CX1D05

<400> 41

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Ser Ala

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Gly Asp Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Ala

210

<210> 42

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR1 VL; m2CX1D05

<400> 42

Arg Ala Ser Gln Gly Ile Arg Ser Ala Leu Asn

1 5 10

<210> 43

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR2 VL; m2CX1D05

<400> 43

Leu Leu Ile Tyr Asn Gly Ser Thr Leu Gln Ser

1 5 10

<210> 44

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR3 VL; m2CX1D05

<400> 44

Gln Gln Phe Asp Gly Asp Pro

fifteen

<210> 45

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide VH; 1B20

<400> 45

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Trp Tyr Lys Pro Leu Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 46

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR1 VH; 1B20

<400> 46

Gly Tyr Ser Phe Thr Asn Tyr Trp Ile Ser

1 5 10

<210> 47

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR2 VH; 1B20

<400> 47

Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro

1 5 10 15

Ser Phe Gln Gly

twenty

<210> 48

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR3 VH; 1B20

<400> 48

Asp Tyr Trp Tyr Lys Pro Leu Phe Asp Ile

1 5 10

<210> 49

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide LC; 1B20

<400> 49

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Ser Ser Phe Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ala

210 215 220

<210> 50

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR1 VL; 1B20

<400> 50

Arg Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 51

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR2 VL; 1B20

<400> 51

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser

1 5 10

<210> 52

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR3 VL; 1B20

<400> 52

Gln Gln Tyr Ser Ser Phe Pro Ile

fifteen

<210> 53

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic antibody heavy chain variable region polypeptide

<400> 53

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Gly Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Gly Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Arg Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser

115 120

<210> 54

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide of the CDR1 region of the heavy chain of the antibody AX132

<400> 54

Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Gly Met Tyr Trp Val Arg

1 5 10 15

<210> 55

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide of the CDR2 region of the heavy chain of the antibody AX132

<400> 55

Trp Ile Gly Trp Ile Asp Pro Gly Ser Gly Gly Thr Lys Tyr Asn Glu

1 5 10 15

Lys Phe Lys Gly Lys Ala Thr

twenty

<210> 56

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide of the CDR3 region of the heavy chain of the antibody AX132

<400> 56

Cys Ala Arg Glu Arg Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln

1 5 10 15

<210> 57

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic antibody light chain variable region polypeptide

<400> 57

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Val Trp Asp Ser Ser Pro Pro

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 58

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide of the light chain CDR1 region of antibodies AX213 and AX132

<400> 58

Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly Ser Tyr Leu Asn Trp Tyr

1 5 10 15

Gln

<210> 59

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide of the light chain CDR2 region of antibodies AX213 and AX132

<400> 59

Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro

1 5 10

<210> 60

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide of the light chain CDR3 region of antibodies AX132 and AX213

<400> 60

Tyr Tyr Cys Gln Val Trp Asp Ser Ser Pro Pro Val Val Phe Gly Gly

1 5 10 15

<210> 61

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic antibody heavy chain variable region polypeptide

<400> 61

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Gly Asn Gly Gly Thr Arg Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asn Asp Gly Tyr Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser

115 120

<210> 62

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide of the CDR1 region of the heavy chain of the antibody AX213

<400> 62

Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr Gly Ile Asn Trp Val Arg

1 5 10 15

<210> 63

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide of the CDR2 region of the heavy chain of the antibody AX213

<400> 63

Trp Ile Gly Arg Ile Asp Pro Gly Asn Gly Gly Thr Arg Tyr Asn Glu

1 5 10 15

Lys Phe Lys Gly Lys Ala Thr

twenty

<210> 64

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide of the CDR3 region of the heavy chain of the antibody AX213

<400> 64

Cys Ala Arg Ala Asn Asp Gly Tyr Ser Phe Asp Tyr Trp Gly Gln

1 5 10 15

<210> 65

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic antibody light chain variable region polypeptide

<400> 65

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Val Trp Asp Ser Ser Pro Pro

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 66

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide of the light chain CDR1 region of antibodies AX213 and AX132

<400> 66

Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly Ser Tyr Leu Asn Trp Tyr

1 5 10 15

Gln

<210> 67

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide of the light chain CDR2 region of antibodies AX213 and AX132

<400> 67

Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro

1 5 10

<210> 68

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide of the light chain CDR3 region of antibodies AX132 and AX213

<400> 68

Tyr Tyr Cys Gln Val Trp Asp Ser Ser Pro Pro Val Val Phe Gly Gly

1 5 10 15

<210> 69

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic VH polypeptide sequence of antibody AX1

<400> 69

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Asp Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Arg Leu Ser Trp Asp Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 70

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide CDR1 VH AX1 antibody sequence

<400> 70

Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg

1 5 10 15

<210> 71

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic peptide CDR2 VH AX1 peptide sequence

<400> 71

Trp Ile Gly Arg Ile Asn Pro Asp Ser Gly Ser Thr Lys Tyr Asn Glu

1 5 10 15

Lys Phe Lys Gly Arg Ala Thr

twenty

<210> 72

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide CDR3 VH AX1 peptide sequence

<400> 72

Cys Ala Arg Gly Gly Arg Leu Ser Trp Asp Phe Asp Val Trp Gly Gln

1 5 10 15

<210> 73

<211> 109

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Antibody AX synthetic VL polypeptide sequence

<400> 73

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Ala Tyr Asp Tyr Ser Leu Gly

85 90 95

Gly Tyr Val Phe Gly Asp Gly Thr Lys Val Glu Ile Lys

100 105

<210> 74

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide CDR1 VL sequence of antibody AX1

<400> 74

Arg Ala Ser Gln Asp Ile Ser Arg Tyr Leu Ala

1 5 10

<210> 75

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide CDR2 VL sequence of antibodies AX1, AX9, AX189

<400> 75

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 76

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide CDR3 VL sequence of antibody AX1

<400> 76

Ala Ala Tyr Asp Tyr Ser Leu Gly Gly Tyr Val

1 5 10

<210> 77

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic VH polypeptide sequence of antibodies AX9, AX189

<400> 77

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Tyr Asn Gly Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Gly Tyr Tyr Leu Gly Ser Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 78

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide CDR1 VH sequence of antibodies AX9, AX189

<400> 78

Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg

1 5 10 15

<210> 79

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic CDR2 peptide sequence of VH antibodies AX9, AX189

<400> 79

Trp Ile Gly Arg Ile Asp Pro Tyr Asn Gly Gly Thr Lys Tyr Asn Glu

1 5 10 15

Lys Phe Lys Gly Lys Ala Thr

twenty

<210> 80

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic CDR3 peptide sequence of VH antibodies AX9, AX189

<400> 80

Cys Ala Arg Tyr Gly Tyr Tyr Leu Gly Ser Tyr Ala Met Asp Tyr Trp

1 5 10 15

Gly Gln

<210> 81

<211> 109

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic VL polypeptide sequence of antibody AX189

<400> 81

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Arg Tyr

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Ala Tyr Asp Tyr Ser Leu Ser

85 90 95

Gly Tyr Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 82

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide CDR1 VL sequence of antibody AX189

<400> 82

Arg Ala Ser Gln Asp Val Ser Arg Tyr Leu Thr

1 5 10

<210> 83

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide CDR2 VL sequence of antibodies AX1, AX9, AX189

<400> 83

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 84

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide CDR3 VL sequence of antibody AX189

<400> 84

Gln Ala Tyr Asp Tyr Ser Leu Ser Gly Tyr Val

1 5 10

<210> 85

<211> 115

<212> PROTEIN

<213> Homo sapiens

<400> 85

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 86

<211> 5

<212> PROTEIN

<213> Homo sapiens

<400> 86

Ser Tyr Gly Ile Ser

fifteen

<210> 87

<211> 17

<212> PROTEIN

<213> Homo sapiens

<400> 87

Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln

1 5 10 15

gly

<210> 88

<211> 6

<212> PROTEIN

<213> Homo sapiens

<400> 88

Gly Tyr Gly Met Asp Val

fifteen

<210> 89

<211> 109

<212> PROTEIN

<213> Homo sapiens

<400> 89

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr Ser Thr

85 90 95

Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 90

<211> 14

<212> PROTEIN

<213> Homo sapiens

<400> 90

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser

1 5 10

<210> 91

<211> 7

<212> PROTEIN

<213> Homo sapiens

<400> 91

Glu Val Ser Asn Arg Pro Ser

fifteen

<210> 92

<211> 9

<212> PROTEIN

<213> Homo sapiens

<400> 92

Asn Ser Tyr Thr Ser Thr Ser Met Val

fifteen

<210> 93

<211> 123

<212> PROTEIN

<213> Homo sapiens

<400> 93

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asp Tyr Asp Phe Trp Ser Ala Tyr Tyr Asp Ala Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 94

<211> 10

<212> PROTEIN

<213> Homo sapiens

<400> 94

Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn

1 5 10

<210> 95

<211> 17

<212> PROTEIN

<213> Homo sapiens

<400> 95

Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Ser Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 96

<211> 14

<212> PROTEIN

<213> Homo sapiens

<400> 96

Asp Tyr Asp Phe Trp Ser Ala Tyr Tyr Asp Ala Phe Asp Val

1 5 10

<210> 97

<211> 111

<212> PROTEIN

<213> Homo sapiens

<400> 97

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Ser Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser

85 90 95

Leu Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 98

<211> 14

<212> PROTEIN

<213> Homo sapiens

<400> 98

Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10

<210> 99

<211> 7

<212> PROTEIN

<213> Homo sapiens

<400> 99

Gly Asn Ser Asn Arg Pro Ser

fifteen

<210> 100

<211> 11

<212> PROTEIN

<213> Homo sapiens

<400> 100

Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Val

1 5 10

<210> 101

<211> 114

<212> PROTEIN

<213> Homo sapiens

<400> 101

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Ala Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Tyr Tyr Asp Gly Ile Asn Lys His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Leu Asp Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 102

<211> 10

<212> PROTEIN

<213> Homo sapiens

<400> 102

Gly Phe Thr Phe Ser Ser Tyr Gly Met His

1 5 10

<210> 103

<211> 17

<212> PROTEIN

<213> Homo sapiens

<400> 103

Val Ile Tyr Tyr Asp Gly Ile Asn Lys His Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 104

<211> 5

<212> PROTEIN

<213> Homo sapiens

<400> 104

Asp Arg Gly Leu Asp

fifteen

<210> 105

<211> 113

<212> PROTEIN

<213> Homo sapiens

<400> 105

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Ser Lys Asn Tyr Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 106

<211> 17

<212> PROTEIN

<213> Homo sapiens

<400> 106

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Ser Lys Asn Tyr Leu

1 5 10 15

Val

<210> 107

<211> 7

<212> PROTEIN

<213> Homo sapiens

<400> 107

Trp Ala Ser Thr Arg Glu Ser

fifteen

<210> 108

<211> 9

<212> PROTEIN

<213> Homo sapiens

<400> 108

Gln Gln Tyr Tyr Ser Thr Pro Trp Thr

fifteen

<210> 109

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic variable region polypeptide (FR1-FR4) Vh heavy chain of anti-PCSK9 monoclonal antibody pJG04 (clones LGT-209 and LGT-210)

<400> 109

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Thr Met

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Glu His Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Tyr Tyr Tyr Tyr Asn Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 110

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Anti-PCSK9 monoclonal antibody heavy chain synthetic peptide CDR1 of clones LGT-209, LGT-210 and LGT-211

<400> 110

Thr Met Tyr Met Ser

fifteen

<210> 111

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Anti-PCSK9 monoclonal antibody heavy chain synthetic peptide CDR2 of clones LGT-209, LGT-210 and LGT-211

<400> 111

Arg Ile Asp Pro Ala Asn Glu His Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 112

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide complementarity determining region 3 (CDR3) of Vh heavy chain of anti-PCSK9 monoclonal antibody pJG04 (clones LGT-209 and LGT-210).

<400> 112

Ser Tyr Tyr Tyr Tyr Asn Met Asp Tyr

fifteen

<210> 113

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic variable region polypeptide (FR1-FR4) Vk light chain of anti-PCSK9 monoclonal antibody pJG04 (clones LGT-209 and LGT-211)

<400> 113

Gln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Gly Val Phe Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Gly Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Leu Gln Trp Ser Ser Asp Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 114

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Anti-PCSK9 monoclonal antibody CDR1 synthetic light chain peptide of clones LGT-209, LGT-210 and LGT-211

<400> 114

Arg Ala Ser Gln Ser Val Ser Tyr Met His

1 5 10

<210> 115

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Anti-PCSK9 monoclonal antibody CDR1 synthetic light chain peptide of clones LGT-209, LGT-210 and LGT-211

<400> 115

Gly Val Phe Arg Arg Ala Thr

fifteen

<210> 116

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic light chain CDR3 peptide of mouse anti-PCSK9 monoclonal antibody LFU720 and anti-PCSK9 monoclonal antibody of clones LGT-209, LGT-210 and LGT-211

<400> 116

Leu Gln Trp Ser Ser Asp Pro Pro Thr

fifteen

<210> 117

<211> 118

<212> PROTEIN

<213> Homo sapiens

<400> 117

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Glu Ile Ser Pro Phe Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Arg Pro Leu Tyr Ala Ser Asp Leu Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 118

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 118

Gly Tyr Thr Phe Thr Ser Tyr

fifteen

<210> 119

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 119

Ser Pro Phe Gly Gly Arg

fifteen

<210> 120

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic heavy chain variable region CDR peptide

<400> 120

Glu Arg Pro Leu Tyr Ala Ser Asp Leu

fifteen

<210> 121

<211> 107

<212> PROTEIN

<213> Homo sapiens

<400> 121

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Arg Tyr Ser Leu Trp Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 122

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic light chain variable region CDR peptide

<400> 122

Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala

1 5 10

<210> 123

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic light chain variable region CDR peptide

<400> 123

Ser Ala Ser Tyr Arg Tyr Thr

fifteen

<210> 124

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic light chain variable region CDR peptide

<400> 124

Gln Gln Arg Tyr Ser Leu Trp Arg Thr

fifteen

<210> 125

<211> 118

<212> PROTEIN

<213> Homo sapiens

<400> 125

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Glu Ile His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Arg Pro Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 126

<211> 10

<212> PROTEIN

<213> Homo sapiens

<400> 126

Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His

1 5 10

<210> 127

<211> 17

<212> PROTEIN

<213> Homo sapiens

<400> 127

Glu Ile His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ser

<210> 128

<211> 9

<212> PROTEIN

<213> Homo sapiens

<400> 128

Glu Arg Pro Leu Tyr Ala Met Asp Tyr

fifteen

<210> 129

<211> 107

<212> PROTEIN

<213> Homo sapiens

<400> 129

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr His Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Arg Tyr Ser Leu Trp Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 130

<211> 11

<212> PROTEIN

<213> Homo sapiens

<400> 130

Lys Ala Ser Gln Asp Val His Thr Ala Val Ala

1 5 10

<210> 131

<211> 7

<212> PROTEIN

<213> Homo sapiens

<400> 131

His Ala Ser Tyr Arg Tyr Thr

fifteen

<210> 132

<211> 9

<212> PROTEIN

<213> Homo sapiens

<400> 132

Gln Gln Arg Tyr Ser Leu Trp Arg Thr

fifteen

<210> 133

<211> 118

<212> PROTEIN

<213> Homo sapiens

<400> 133

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Glu Ile His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Arg Pro Leu Tyr Ala Ser Asp Leu Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 134

<211> 10

<212> PROTEIN

<213> Homo sapiens

<400> 134

Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His

1 5 10

<210> 135

<211> 17

<212> PROTEIN

<213> Homo sapiens

<400> 135

Glu Ile His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ser

<210> 136

<211> 9

<212> PROTEIN

<213> Homo sapiens

<400> 136

Glu Arg Pro Leu Tyr Ala Ser Asp Leu

fifteen

<210> 137

<211> 107

<212> PROTEIN

<213> Homo sapiens

<400> 137

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr His Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Arg Tyr Ser Leu Trp Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 138

<211> 11

<212> PROTEIN

<213> Homo sapiens

<400> 138

Lys Ala Ser Gln Asp Val His Thr Ala Val Ala

1 5 10

<210> 139

<211> 7

<212> PROTEIN

<213> Homo sapiens

<400> 139

His Ala Ser Tyr Arg Tyr Thr

fifteen

<210> 140

<211> 9

<212> PROTEIN

<213> Homo sapiens

<400> 140

Gln Gln Arg Tyr Ser Leu Trp Arg Thr

fifteen

<210> 141

<211> 118

<212> PROTEIN

<213> Mus musculus

<400> 141

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Arg Pro Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 142

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 142

Gly Tyr Thr Phe Thr Ser Tyr

fifteen

<210> 143

<211> 6

<212> PROTEIN

<213> Mus musculus

<400> 143

Asn Pro Ser Asn Gly Arg

fifteen

<210> 144

<211> 9

<212> PROTEIN

<213> Mus musculus

<400> 144

Glu Arg Pro Leu Tyr Ala Met Asp Tyr

fifteen

<210> 145

<211> 108

<212> PROTEIN

<213> Mus musculus

<400> 145

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Arg Tyr Ser Thr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 146

<211> 11

<212> PROTEIN

<213> Mus musculus

<400> 146

Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 147

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic light chain variable region CDR peptide

<400> 147

Ser Ala Ser Tyr Arg Tyr Thr

fifteen

<210> 148

<211> 9

<212> PROTEIN

<213> Mus musculus

<400> 148

Gln Gln Arg Tyr Ser Thr Pro Arg Thr

fifteen

<210> 149

<211> 115

<212> PROTEIN

<213> Mus musculus

<400> 149

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Tyr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Leu Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 150

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 150

Gly Tyr Thr Phe Thr Asp Tyr

fifteen

<210> 151

<211> 6

<212> PROTEIN

<213> Mus musculus

<400> 151

Asn Pro Asn Asn Gly Gly

fifteen

<210> 152

<211> 6

<212> PROTEIN

<213> Mus musculus

<400> 152

Trp Leu Leu Phe Ala Tyr

fifteen

<210> 153

<211> 108

<212> PROTEIN

<213> Mus musculus

<400> 153

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Leu Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Phe Tyr Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 154

<211> 11

<212> PROTEIN

<213> Mus musculus

<400> 154

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala

1 5 10

<210> 155

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 155

Ser Ala Ser Tyr Arg Tyr Ser

fifteen

<210> 156

<211> 9

<212> PROTEIN

<213> Mus musculus

<400> 156

Gln Gln Phe Tyr Ser Tyr Pro Tyr Thr

fifteen

<210> 157

<211> 123

<212> PROTEIN

<213> Mus musculus

<400> 157

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gly Gly Ile Tyr Tyr Arg Tyr Asp Arg Asn Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 158

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 158

Gly Tyr Thr Phe Thr Asp Tyr

fifteen

<210> 159

<211> 6

<212> PROTEIN

<213> Mus musculus

<400> 159

Asn Pro Asn Asn Gly Gly

fifteen

<210> 160

<211> 14

<212> PROTEIN

<213> Mus musculus

<400> 160

Gly Gly Ile Tyr Tyr Arg Tyr Asp Arg Asn Tyr Phe Asp Tyr

1 5 10

<210> 161

<211> 107

<212> PROTEIN

<213> Mus musculus

<400> 161

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 162

<211> 11

<212> PROTEIN

<213> Mus musculus

<400> 162

Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 163

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 163

Tyr Thr Ser Ser Leu His Ser

fifteen

<210> 164

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 164

Gln Gln Tyr Ser Lys Leu Pro Phe Thr

fifteen

<210> 165

<211> 117

<212> PROTEIN

<213> Mus musculus

<400> 165

Glu Val Lys Leu Val Glu Ser Glu Gly Gly Leu Val Gln Pro Gly Ser

1 5 10 15

Ser Met Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Val Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Asn Tyr Asp Gly Ser Asn Thr Ser Tyr Leu Asp Ser Leu

50 55 60

Lys Ser Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Glu Lys Phe Ala Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 166

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 166

Gly Phe Thr Phe Ser Asp Tyr

fifteen

<210> 167

<211> 6

<212> PROTEIN

<213> Mus musculus

<400> 167

Asn Tyr Asp Gly Ser Asn

fifteen

<210> 168

<211> 8

<212> PROTEIN

<213> Mus musculus

<400> 168

Glu Lys Phe Ala Ala Met Asp Tyr

fifteen

<210> 169

<211> 108

<212> PROTEIN

<213> Mus musculus

<400> 169

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Phe Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Asn Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly His Ser Pro Lys Leu Leu Ile

35 40 45

Phe Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 170

<211> 11

<212> PROTEIN

<213> Mus musculus

<400> 170

Lys Ala Ser Gln Asp Val Ser Asn Ala Leu Ala

1 5 10

<210> 171

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic light chain variable region CDR peptide

<400> 171

Ser Ala Ser Tyr Arg Tyr Thr

fifteen

<210> 172

<211> 9

<212> PROTEIN

<213> Mus musculus

<400> 172

Gln Gln His Tyr Ser Thr Pro Trp Thr

fifteen

<210> 173

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic polypeptide

<400> 173

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg His

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ala Arg Ile Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 174

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 174

Gly Phe Thr Phe Thr Arg His Thr Ile His

1 5 10

<210> 175

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 175

Arg Ile Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 176

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 176

Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr

1 5 10

<210> 177

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic polypeptide

<400> 177

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Arg Ile Gln Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 178

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 178

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 179

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 179

Ser Ala Ser Phe Leu Tyr Ser

fifteen

<210> 180

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 180

Gln Gln Ser Tyr Arg Ile Gln Pro Thr

fifteen

<210> 181

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic polypeptide

<400> 181

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Thr

20 25 30

Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 182

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 182

Gly Phe Thr Phe Ser Ser Thr Ala Ile His

1 5 10

<210> 183

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 183

Arg Ile Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 184

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 184

Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr

1 5 10

<210> 185

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic polypeptide

<400> 185

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Pro Ala Leu His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 186

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 186

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 187

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 187

Ser Ala Ser Phe Leu Tyr Ser

fifteen

<210> 188

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 188

Gln Gln Ser Tyr Pro Ala Leu His Thr

fifteen

<210> 189

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic polypeptide

<400> 189

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Lys Leu

20 25 30

Gly Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Thr Ile Ser Ser Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Ile Ser Phe Gln Gly Gly Thr Tyr Thr Tyr Val Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 190

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 190

Gly Phe Pro Phe Ser Lys Leu Gly Met Val

1 5 10

<210> 191

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 191

Thr Ile Ser Ser Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

gly

<210> 192

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 192

Glu Gly Ile Ser Phe Gln Gly Gly Thr Tyr Thr Tyr Val Met Asp Tyr

1 5 10 15

<210> 193

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic polypeptide

<400> 193

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Arg

20 25 30

Asn Gly Ile Thr Tyr Ser Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Leu Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Tyr Gln Asn

85 90 95

Leu Glu Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 194

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 194

Arg Ser Ser Lys Ser Leu Leu His Arg Asn Gly Ile Thr Tyr Ser Tyr

1 5 10 15

<210> 195

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 195

Gln Leu Ser Asn Leu Ala Ser

fifteen

<210> 196

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic peptide

<400> 196

Tyr Gln Asn Leu Glu Leu Pro Leu Thr

fifteen

<210> 197

<211> 2076

<212> DNA

<213> Homo sapiens

<400> 197

atgggcaccg tcagctccag gcggtcctgg tggccgctgc cactgctgct gctgctgctg 60

ctgctcctgg gtcccgcggg cgcccgtgcg caggaggacg aggacggcga ctacgaggag 120

ctggtgctag ccttgcgttc cgaggaggac ggcctggccg aagcacccga gcacggaacc 180

acagccacct tccaccgctg cgccaaggat ccgtggaggt tgcctggcac ctacgtggtg 240

gtgctgaagg aggagaccca cctctcgcag tcagagcgca ctgcccgccg cctgcaggcc 300

caggctgccc gccggggata cctcaccaag atcctgcatg tcttccatgg ccttcttcct 360

ggcttcctgg tgaagatgag tggcgacctg ctggagctgg ccttgaagtt gccccatgtc 420

gactacatcg aggaggactc ctctgtcttt gccgagca tcccgtggaa cctggagcgg 480

attacccctc cacggtaccg ggcggatgaa taccagcccc ccgacgggagg cagcctggtg 540

gaggtgtatc tcctagacac cagcatacag agtgaccacc gggaaatcga gggcagggtc 600

atggtcaccg acttcgagaa tgtgcccgag gaggacggga cccgcttcca cagacaggcc 660

agcaagtgtg acagtcatgg cacccacctg gcaggggtgg tcagcggccg ggatgccggc 720

gtggccaagg gtgccagcat gcgcagcctg cgcgtgctca actgccaagg gaagggcacg 780

gttagcggca ccctcatagg cctggagttt attcggaaaa gccagctggt ccagcctgtg 840

gggccactgg tggtgctgct gcccctggcg ggtgggtaca gccgcgtcct caacgccgcc 900

tgccagcgcc tggcgagggc tggggtcgtg ctggtcaccg ctgccggcaa cttccgggac 960

gatgcctgcc tctactcccc agcctcagct cccgaggtca tcacagttgg ggccaccaat 1020

gcccaagacc agccggtgac cctggggact ttggggacca actttggccg ctgtgtggac 1080

ctctttgccc caggggagga catcattggt gcctccagcg actgcagcac ctgctttgtg 1140

tcacagagtg ggacatcaca ggctgctgcc cacgtggctg gcattgcagc catgatgctg 1200

tctgccgagc cggagctcac cctggccgag ttgaggcaga gactgatcca cttctctgcc 1260

aaagatgtca tcaatgaggc ctggttccct gaggaccagc gggtactgac ccccaacctg 1320

gtggccgccc tgccccccag cacccatggg gcaggttggc agctgttttg caggactgta 1380

tggtcagcac actcggggcc tacacggatg gccacagccg tcgcccgctg cgccccagat 1440

gaggagctgc tgagctgctc cagtttctcc aggagtggga agcggcgggg cgagcgcatg 1500

gaggcccaag ggggcaagct ggtctgccgg gcccacaacg cttttggggg tgagggtgtc 1560

tacgccattg ccaggtgctg cctgctaccc caggccaact gcagcgtcca cacagctcca 1620

ccagctgagg ccagcatgggg gacccgtgtc cactgccacc aacagggcca cgtcctcaca 1680

ggctgcagct cccactggga ggtggaggac cttggcaccc acaagccgcc tgtgctgagg 1740

ccacgaggtc agcccaacca gtgcgtggggc cacagggagg ccagcatcca cgcttcctgc 1800

tgccatgccc caggtctgga atgcaaagtc aaggagcatg gaatcccggc ccctcaggag 1860

caggtgaccg tggcctgcga ggagggctgg accctgactg gctgcagtgc cctccctggg 1920

acctcccacg tcctgggggc ctacgccgta gacaacacgt gtgtagtcag gagccgggac 1980

gtcagcacta caggcagcac cagcgaaggg gccgtgacag ccgttgccat ctgctgccgg 2040

agccggcacc tggcgcaggc ctcccaggag ctccag 2076

<210> 198

<211> 692

<212> PROTEIN

<213> Homo sapiens

<400> 198

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln

690

<---

Claims (42)

1. A method for the treatment of hypercholesterolemia, involving administering to a patient in need thereof 75 mg, 150 mg, or 300 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase every two weeks or every four weeks, where the antibody or antigen-binding fragment contains three heavy chain CDRs listed under SEQ ID NOS: 2, 3 and 4, and three light chain CDRs listed under SEQ ID NOS: 7, 8 and 10, and wherein the patient is a patient at high cardiovascular risk receiving an insulin-based therapy that has (i) type 1 diabetes mellitus (T1DM) and (ii) hypercholesterolemia not sufficiently controlled by a statin-based therapy at the maximum tolerated dose. 2. A method for the treatment of hypercholesterolemia, involving administering to a patient in need thereof 75 mg, 150 mg, or 300 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase, where the antibody or antigen-binding fragment contains three heavy chain CDRs listed under SEQ ID NOS: 2, 3 and 4, and three light chain CDRs listed under SEQ ID NOS: 7, 8 and 10, and wherein the patient is a patient at high cardiovascular risk receiving an insulin based therapy that has (i) type 2 diabetes mellitus (T2DM) and (ii) hypercholesterolemia not adequately controlled by a statin based therapy at the maximum tolerated dose. 3. The method of claim 2, wherein the patient additionally has atherosclerotic cardiovascular disease (ASCVD). 4. The method of claim 3 wherein the ASCVD includes ischemic heart disease (CHD), ischemic stroke, or peripheral arterial disease. 5. The method of claim. 4, where CHD includes acute myocardial infarction, asymptomatic myocardial infarction and unstable angina. 6. The method according to any one of paragraphs. 1-5, where (a) 75 mg of the antibody or antigen-binding fragment is administered to the patient every two weeks; (b) 150 mg of the antibody or antigen binding fragment is administered to the patient every two weeks, or (c) 300 mg of the antibody or antigen binding fragment is administered to the patient every four weeks. 7. The method according to any one of paragraphs. 1-6, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) with the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (LCVR) with the amino acid sequence of SEQ ID NO: 6. 8. The method according to any one of paragraphs. 1-7, additionally providing administration to the patient of one or more subsequent doses of 75 mg of the specified antibody or antigen-binding fragment every two weeks if the patient's LDL-C level is below the threshold level, or administration of one or more subsequent doses of 150 mg of the specified antibody or antigen-binding fragment thereof fragment every two weeks if the patient's LDL-C level is at or above the cut-off level. 9. The method according to any one of paragraphs. 1-7, additionally providing administering one or more subsequent doses of 300 mg of said antibody or antigen-binding fragment thereof to the patient every four weeks if the patient's LDL-C level is below a threshold level, or administering one or more subsequent doses of 150 mg of said antibody or antigen-binding fragment thereof fragment approximately every two weeks if the patient's LDL-C level is at or above the cut-off level. 10. The method according to claim 8 or claim 9, wherein the threshold level is 70 mg/dl. 11. The method according to any one of paragraphs. 1-10, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously. 12. The method according to any one of paragraphs. 1-11, where the patient additionally receives a concomitant lipid corrective therapy (LMT). 13. The method of claim 12, wherein the LMT is selected from the group consisting of a statin, cholesterol absorption inhibitor, fibrate, niacin, an omega-3 fatty acid, and a bile acid sequestrant. 14. The method according to p. 13, where LMT is a statin-based therapy, (a) wherein the statin is selected from the group consisting of atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin and cerivastatin; and/or (b) the statin therapy is a maximum tolerated dose statin therapy. 15. The method according to any one of paragraphs. 1-13, where the patient suffers from statin intolerance. 16. The method according to any one of paragraphs. 1-15, wherein the insulin based therapy is selected from the group consisting of human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degludec, insulin aspart, and basal insulin. 17. The method according to any one of paragraphs. 1-16 wherein the patient is receiving a concomitant antidiabetic therapy in addition to an insulin based therapy, wherein the additional antidiabetic therapy is selected from the group consisting of glucagon like peptide 1 (GLP-1) therapy, gastrointestinal peptide, agonist or glucagon receptor antagonist, glucose-dependent insulinotropic polypeptide (GIP) receptor agonist or antagonist, ghrelin antagonist or inverse agonist, xenin, xenin analog, biguanide, sulfonylurea, meglitinide, thiazolidinedione, DPP-4 inhibitor, alpha-glucosidase inhibitor, sodium-dependent transporter inhibitor glucose 2 (SGLT-2), SGLT-1 inhibitor, peroxisome proliferator-activated receptor (PPAR-) agonist or modulator (alpha, gamma, or alpha/gamma), amylin, amylin analog, G protein-coupled 119 receptor agonist ( GPR119), GPR40 agonist, GPR120 agonist, GPR142 agonist, systemic or poorly absorbed T agonist GR5, diabetes immunotherapy, anti-inflammatory agents for metabolic syndrome and diabetes, adenosine monophosphate-activated protein kinase (AMPK) stimulator, 11-beta-hydroxysteroid dehydrogenase 1 inhibitor, glucokinase activator, diacylglycerol-O-acyltransferase (DGAT) inhibitor, glucose transporter modulator -4, a somatostatin receptor 3 agonist, a lipid-lowering agent, and combinations thereof. 18. The method according to any one of paragraphs. 1-17, where the antibody or antigen-binding fragment (a) reduce the level of LDL-C in the patient by at least 40%; (b) reduce the level of non-HDL-C cholesterol in the patient by at least 35%; (c) reduce the level of ApoC3 in the patient; (d) reduce the number and/or size of lipoprotein particles in a patient; (e) do not affect the patient's hemoglobin A1c (HbA1c) level and/or (f) do not affect the patient's fasting plasma glucose (FPG) level. 19. A method for the treatment of hypercholesterolemia, involving administering every two weeks to a patient in need thereof 75 mg of an antibody or antigen-binding fragment thereof that specifically binds human subtilisin/kexin type 9 (PCSK9) proprotein convertase; and administering one or more subsequent 75 mg doses of the antibody or antigen-binding fragment thereof to the patient every two weeks if the patient's LDL-C level is below 70 mg/dL, or administering one or more subsequent 150 mg doses of the antibody or antigen-binding fragment thereof fragment every two weeks if the patient's LDL-C level is 70 mg/dl or greater, wherein the antibody or antigen-binding fragment thereof comprises HCVR with the amino acid sequence of SEQ ID NO: 1 and LCVR with the amino acid sequence of SEQ ID NO: 6, and wherein the patient is receiving concomitant insulin therapy, and wherein the patient is a patient at high cardiovascular risk receiving an insulin-based therapy that has (i) T1DM or T2DM and (ii) hypercholesterolemia not adequately controlled by a statin-based therapy at the maximum tolerated dose.

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