CN110913889A

CN110913889A - Methods of treating hyperlipidemia in diabetic patients by administering PCSK9 inhibitors

Info

Publication number: CN110913889A
Application number: CN201880037888.2A
Authority: CN
Inventors: M·布加斯-博巴诺维奇
Original assignee: Sanofi Biotechnology SAS
Current assignee: Sanofi Biotechnology SAS
Priority date: 2017-06-09
Filing date: 2018-06-09
Publication date: 2020-03-24
Also published as: EP3634469A1; MX2019014831A; RU2019144346A; JP2020522544A; JP2023123842A; KR20200026826A; AU2018280567A1; TW202310872A; IL271212A; TW201904608A; RU2019144346A3; CA3066317A1

Abstract

Methods for treating patients with high cardiovascular risk who are suffering from hypercholesterolemia and type 1 or type 2 diabetes who are receiving insulin treatment are provided. These methods generally include administering to a subject a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds hPCSK9 in combination with insulin therapy.

Description

Methods of treating hyperlipidemia in diabetic patients by administering PCSK9 inhibitors

RELATED APPLICATIONS

This application claims priority from U.S. provisional patent application No. 62/517,672 filed on 9.6.2017, U.S. provisional patent application No. 62/532,162 filed on 13.7.2017, and european patent application No. 18305565.6 filed on 4.5.2018. The contents of each of these related applications are hereby incorporated by reference in their entirety.

Technical Field

The present invention relates to the field of therapeutic treatment of diseases or disorders associated with elevated levels of lipids or lipoproteins. More specifically, the present invention relates to the treatment of diabetic patients suffering from hyperlipidemia, including hypercholesterolemia, with PCSK9 inhibitors.

Background

Hyperlipidemia is a general term that encompasses diseases and conditions characterized by or associated with elevated levels of lipids and/or lipoproteins in the blood. Hyperlipidemia includes hypercholesterolemia, hypertriglyceridemia, combined hyperlipidemia and elevated lipoprotein a (lp (a)). A particularly prevalent form of hyperlipidemia in many populations is hypercholesterolemia.

Hypercholesterolemia, in particular an increased level of low-density lipoprotein (LDL) cholesterol (LDL-C), constitutes a major risk for the development of atherosclerosis and Coronary Heart Disease (CHD) (Sharrett et al, 2001, Circulation 104: 1108-. Low density lipoprotein cholesterol was identified as the primary target for cholesterol lowering therapy and accepted as an effective surrogate therapeutic endpoint. Numerous studies have demonstrated that lowering LDL-C levels reduces the risk of CHD, with a strong direct relationship between LDL-C levels and CHD events; for every 1mmol/L (-40 mg/dL) decrease in LDL-C, cardiovascular disease (CVD) mortality and morbidity decreases by 22%. More reduction in LDL-C leads to more reduction in CHD events and enhanced comparative data with standard statin treatment indicate that the lower the LDL-C level, the greater the benefit for patients with very high Cardiovascular (CV) risk.

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in patients with type 1 (T1) or type 2 (T2) Diabetes (DM), and insulin-treated diabetic patients have an even higher CV risk. Furthermore, the presence of comorbid DM in patients with atherosclerotic cvd (ascvd) significantly increases the risk of CV events. Several studies and meta-analyses showed that lowering LDL-C with statins resulted in a significant reduction in CV events in DM patients, and the use of concomitant ezetimibe (ezetimibe) resulted in a further reduction in CV risk associated with additional LDL-C lowering. However, even with currently available treatments, many patients with DM continue to have persistent lipid abnormalities and are therefore exposed to a residual risk of CV events.

Current LDL-C-lowering drugs include proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, such as anti-PCSK 9 antibodies. Although anti-PCSK 9 antibodies have been extensively studied clinically, the efficacy and safety of aleuromab (alirocumab) in the diabetic population is not fully understood. Therefore, there is a need in the art to identify a therapeutic regimen for an anti-PCSK 9 antibody that provides optimal efficacy and safety in treating hypercholesterolemia in insulin-treated diabetic patients at high CV risk.

Disclosure of Invention

The present disclosure provides methods for treating hypercholesterolemia in a patient suffering from Diabetes (DM) receiving insulin therapy. In certain embodiments, the methods comprise administering one or more doses of an antibody or antigen-binding fragment thereof that specifically binds to human PCSK9 to a patient with hypercholesterolemia and diabetes. In certain embodiments, the patient has a high cardiovascular risk. In certain embodiments, the patient receives concomitant anti-diabetic therapy in addition to insulin therapy.

According to one aspect, the method comprises a method for treating hypercholesterolemia in a patient having type 1 diabetes (T1DM), the method comprising: (a) selecting a high cardiovascular risk patient receiving insulin treatment with (i) T1DM, and (ii) hypercholesterolemia not adequately controlled by maximally tolerated statin treatment; and (b) administering 75mg, 150mg, or 300mg of an antibody or antigen-binding fragment thereof that specifically binds human proprotein convertase subtilisin/kexin type 9 (PCSK9) to the patient, wherein the patient receives concomitant insulin therapy.

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

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs of an HCVR/LCVR amino acid sequence pair comprising SEQ ID NO: 1/6. In certain embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs shown as

SEQ ID NOs

2, 3, and 4, and three light chain CDRs shown as

SEQ ID NOs

7, 8, and 10. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having the amino acid sequence of SEQ ID NO:1 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the antibody or antigen-binding fragment thereof competes for binding with an antibody or antigen-binding fragment thereof comprising a HCVR having the amino acid sequence of SEQ ID No. 1 and a LCVR having the amino acid sequence of SEQ ID No. 6. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope of PCSK9 as an antibody comprising a HCVR having the amino acid sequence of SEQ ID No. 1 and a LCVR having the amino acid sequence of SEQ ID No. 6. In certain embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of PCSK9 that overlaps with an epitope of an antibody comprising a HCVR having the amino acid sequence of SEQ ID No. 1 and a LCVR having the amino acid sequence of SEQ ID No. 6.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises heavy and light chain CDR amino acid sequences having SEQ ID NOs 86, 87, 88, 90, 91, and 92. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence at least 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 85, and a LCVR having an amino acid sequence at least 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 89.

In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: alirocumab (alirocumab), efuzumab (evolocumab), bococizumab, lodelcizumab (lodelcizumab), ralpancizumab and LY 3015014. In certain embodiments, the antibody or antigen-binding fragment thereof is aleucizumab.

In certain embodiments, the methods disclosed herein further comprise: (c) administering about every two weeks one or more of the following doses of the antibody or antigen-binding fragment thereof to the patient if the level of LDL-C in the patient is below a threshold level, e.g., after 8 weeks, or about every two weeks one or more of the following doses of 150mg of the antibody or antigen-binding fragment thereof if the level of LDL-C in the patient is greater than or equal to the threshold level, e.g., after 8 weeks. In certain embodiments, the threshold level is 70 mg/dL.

In certain embodiments, the methods disclosed herein further comprise: (c) administering to the patient one or more of the following doses of 300mg of the antibody or antigen-binding fragment thereof about every four weeks if, e.g., after 8 weeks, the LDL-C level in the patient is below a threshold level, or about every two weeks of one or more of the following doses of 150mg of the antibody or antigen-binding fragment thereof if, e.g., after 8 weeks, the LDL-C level in the patient is greater than or equal to a threshold level. In certain embodiments, the threshold level is 70 mg/dL.

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

In certain embodiments, the patient further receives concomitant Lipid Modification Therapy (LMT). In certain embodiments, the LMT is selected from the group consisting of: statins, cholesterol absorption inhibitors, fibrates, nicotinic acids, omega-3 fatty acids, and bile acid sequestrants. In certain embodiments, the LMT is a statin therapy. In certain embodiments, the statin is selected from the group consisting of: atorvastatin (atorvastatin), rosuvastatin (rosuvastatin), simvastatin (simvastatin), pravastatin (pravastatin), lovastatin (lovastatin), fluvastatin (fluvastatin), pitavastatin (pitavastatin), and cerivastatin (cerivastatin). In certain embodiments, the statin treatment is a maximum tolerated dose statin treatment. In certain embodiments, the cholesterol absorption inhibitor is ezetimibe. In certain embodiments, the patient is intolerant to statins.

In certain embodiments, the subject further receives additional concomitant antidiabetic therapy in addition to insulin therapy, in certain embodiments, the additional concomitant antidiabetic therapy is selected from the group consisting of glucagon-like peptide 1(GLP-1) therapy, gastrointestinal peptides, glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, growth hormone releasing hormone antagonists or inverse agonists, xenin, xenin analogs, biguanides, sulfonylureas, meglitinides (meglitinides), thiazolidinediones, DPP-4 inhibitors, α -glucosidase inhibitors, sodium-dependent glucose transporter 2(SGLT-2) inhibitors, SGLT-1 inhibitors, peroxisome proliferator activated receptors (PPAR 2, gamma α, or gamma 539) agonists, GPR-3 agonists, GPR119, GPR 120-G54 agonists, GPR142, GPR 3 agonists, GPR142 agonists, GPR 3 agonists, GPR-4 agonists, and GPR 3 agonists.

In certain embodiments, the antibody or antigen-binding fragment thereof reduces the LDL-C level of the patient, e.g., by at least 30%, 35%, 40% or 45%. In certain embodiments, the antibody or antigen-binding fragment thereof reduces non-HDL-C levels in the patient, e.g., by at least 25%, 30%, 35%, or 40%. In certain embodiments, the antibody or antigen-binding fragment thereof reduces the apolipoprotein C3(ApoC3) level of the 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 certain embodiments, the antibody or antigen-binding fragment thereof reduces the number of lipoprotein particles in the patient (e.g., 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 the patient (e.g., by at least about 1.5%, about 2%, about 2.5%, or about 3% after 12 or 24 weeks of treatment).

In certain 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 fasting blood glucose (FPG) levels in the patient.

According to another aspect, the method comprises a method for treating hypercholesterolemia in a patient having type 1 diabetes (T1DM), the method comprising:

(a) selecting a high cardiovascular risk patient receiving insulin treatment with (i) T1DM, and (ii) hypercholesterolemia not adequately controlled by maximally tolerated statin treatment; and

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

(c) administering to the patient one or more of the following doses of 75mg of the antibody or antigen-binding fragment thereof about every two weeks if, e.g., after 8 weeks, the LDL-C level in the patient is less than 70mg/dL, or 150mg of the antibody or antigen-binding fragment thereof about every two weeks if, e.g., after 8 weeks, the LDL-C level in the patient is greater than or equal to 70mg/dL, wherein the antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO. 1 and an LCVR having the amino acid sequence of SEQ ID NO. 6, and wherein the patient receives concomitant insulin therapy.

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

(c) administering to the patient one or more than one dose of 300mg of the antibody or antigen-binding fragment thereof about every four weeks if, e.g., after 8 weeks, the LDL-C level in the patient is below a threshold level, or one or more than one dose of 150mg of the antibody or antigen-binding fragment thereof about every two weeks if, e.g., after 8 weeks, the LDL-C level in the patient is greater than or equal to the threshold level,

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

According to another aspect, the method comprises a method for treating hypercholesterolemia in a patient having type 2 diabetes (T2DM), the method comprising:

(a) selecting a high cardiovascular risk patient receiving insulin treatment with (i) T2DM, and (ii) hypercholesterolemia not adequately controlled by maximally tolerated statin treatment; and

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

SEQ ID NOs

2, 3, and 4, and three light chain CDRs shown as

SEQ ID NOs

7, 8, and 10. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having the amino acid sequence of SEQ ID NO:1 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the antibody or antigen-binding fragment thereof competes for binding with an antibody or antigen-binding fragment thereof comprising a HCVR having the amino acid sequence of SEQ ID No. 1 and a LCVR having the amino acid sequence of SEQ ID No. 6. In certain embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of PCSK9 that is the same as the epitope of an antibody comprising a HCVR having the amino acid sequence of SEQ ID No. 1 and a LCVR having the amino acid sequence of SEQ ID No. 6. In certain embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of PCSK9 that overlaps with an epitope of an antibody comprising a HCVR having the amino acid sequence of SEQ ID No. 1 and a LCVR having the amino acid sequence of SEQ ID No. 6.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises Complementarity Determining Regions (CDRs) of a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR) comprising the amino acid sequences set forth in SEQ ID NOS: 85 and 89, respectively. In certain embodiments, the antibody or antigen-binding fragment thereof comprises heavy and light chain CDR amino acid sequences having SEQ ID NOs 86, 87, 88, 90, 91, and 92. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence at least 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 85, and a LCVR having an amino acid sequence at least 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID No. 89.

In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: alikumab, efuzumab, bococizumab, ludwizumab, ralpancizumab and LY 3015014. In certain embodiments, the antibody or antigen-binding fragment thereof is aleucizumab.

In certain embodiments, the methods disclosed herein further comprise: (c) administering one or more of the following doses of the antibody or antigen-binding fragment thereof to the patient about every two weeks if the level of LDL-C in the patient is below a threshold level, or about every two weeks if the level of LDL-C in the patient is greater than or equal to the threshold level, 150mg of the antibody or antigen-binding fragment thereof about every two weeks. In certain embodiments, the threshold level is 70 mg/dL.

In certain embodiments, the methods disclosed herein further comprise: (c) administering to the patient one or more of the following doses of 300mg of the antibody or antigen-binding fragment thereof about every four weeks if, e.g., after 8 weeks, the LDL-C level in the patient is below a threshold level, or about every two weeks of one or more of the following doses of 150mg of the antibody or antigen-binding fragment thereof if, e.g., after 8 weeks, the LDL-C level in the patient is greater than or equal to the threshold level. In certain embodiments, the threshold level is 70 mg/dL.

In certain embodiments, the patient further receives concomitant Lipid Modification Therapy (LMT). In certain embodiments, the LMT is selected from the group consisting of: statins, cholesterol absorption inhibitors, fibrates, nicotinic acids, omega-3 fatty acids, and bile acid sequestrants. In certain embodiments, the LMT is a statin therapy. In certain embodiments, the statin is selected from the group consisting of: atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin and cerivastatin. In certain embodiments, the statin treatment is a maximally tolerated dose statin treatment. In certain embodiments, the cholesterol absorption inhibitor is ezetimibe.

In certain embodiments, the patient is intolerant to statins.

In certain embodiments, the insulin treatment is selected from the group consisting of: human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degluvium, insulin aspart and basal insulin.

In certain embodiments, the additional antidiabetic therapy is selected from the group consisting of glucagon-like peptide 1(GLP-1) therapy, gastrointestinal peptides, glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, ghrelin antagonists or inverse agonists, xenin analogs, biguanides, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, α -glucosidase inhibitors, sodium-dependent glucose transporter 2(SGLT-2) inhibitors, SGLT-1 inhibitors, peroxisome proliferator-activated receptor (PPAR-) (α, γ or α/γ) agonists or modulators, amylin analogs, G protein coupled receptor monophosphoryl 119 (119) agonists, GPR40 agonists, GPR120 agonists, GPR142 agonists, systemic or low absorption agonists, agonists for the treatment of glucose-mediated diseases, glucose-mediated kinase (GDR 57) and glucose-mediated kinase inhibitors for the treatment of glucose-dependent glucose-releasing hormone, glucose-releasing hormone antagonists or inverse agonists, oxmin, meglumine analogs, thiazolidinediones, DPP-4 inhibitors, GPR-1 inhibitors, GPR-54 agonists, GPR-120 agonists, GPR142 agonists, systemic or low absorption agonists, GPR 57 agonists, and anti-kinase inhibitors for the treatment of glucose-mediated diseases.

In certain embodiments, the antibody or antigen-binding fragment thereof reduces the LDL-C level of the patient, e.g., by at least 30%, 35%, 40% or 45%. In certain embodiments, the antibody or antigen-binding fragment thereof reduces non-HDL-C levels in the patient, e.g., by at least 20%, 25%, 30%, or 35%. In certain embodiments, the antibody or antigen-binding fragment thereof reduces the apolipoprotein C3(ApoC3) level of the 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 certain embodiments, the antibody or antigen-binding fragment thereof reduces the number of lipoprotein particles in the patient (e.g., 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 the patient (e.g., by at least about 1.5%, about 2%, about 2.5%, or about 3% after 12 or 24 weeks of treatment).

(a) selecting a high cardiovascular risk patient receiving insulin treatment with (i) T2DM, and (ii) hypercholesterolemia not adequately controlled by maximally tolerated statin treatment;

(c) administering to the patient one or more of the following doses of the antibody or antigen-binding fragment thereof about every two weeks if, e.g., after 8 weeks, the LDL-C level in the patient is less than 70mg/dL, or one or more of the following doses of 150mg of the antibody or antigen-binding fragment thereof about every two weeks if, e.g., after 8 weeks, the LDL-C level in the patient is greater than or equal to 70mg/dL,

wherein the antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO. 1 and an LCVR having the amino acid sequence of SEQ ID NO. 6, and wherein the patient receives concomitant insulin treatment.

(c) administering to the patient one or more than one dose of 300mg of the antibody or antigen-binding fragment thereof about every four weeks if, e.g., after 8 weeks, the LDL-C level in the patient is above a threshold level, or one or more than one dose of 150mg of the antibody or antigen-binding fragment thereof about every two weeks if, e.g., after 8 weeks, the LDL-C level in the patient is below or equal to a threshold level,

According to another aspect, the method comprises a method for treating hypercholesterolemia in a patient having T2DM and atherosclerotic cardiovascular disease (ASCVD), the method comprising:

(a) selecting a high cardiovascular risk patient receiving insulin treatment with (i) T2DM, (ii) ASCVD, and (iii) hypercholesterolemia not adequately controlled by maximally tolerated statin treatment; and

In certain embodiments, the ASCVD is defined as Coronary Heart Disease (CHD), ischemic stroke, or peripheral arterial disease. In certain embodiments, the CHD comprises acute myocardial infarction, asymptomatic myocardial infarction, and unstable angina.

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

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the heavy and light chain CDRs of an HCVR/LCVR amino acid sequence pair comprising SEQ ID NO: 1/6. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs shown in

SEQ ID NOs

2, 3, and 4, and the three light chain CDRs shown in

SEQ ID NOs

7, 8, and 10. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having the amino acid sequence of SEQ ID NO:1 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope of PCSK9 as an antibody comprising a HCVR having the amino acid sequence of SEQ ID No. 1 and a LCVR having the amino acid sequence of SEQ ID No. 6. In certain embodiments, the antibody or antigen-binding fragment thereof binds to an epitope of PCSK9 that overlaps with an epitope of an antibody comprising a HCVR having the amino acid sequence of SEQ ID No. 1 and a LCVR having the amino acid sequence of SEQ ID No. 6.

In certain embodiments, the method further comprises: (c) administering one or more of the following doses of the antibody or antigen-binding fragment thereof to the patient about every two weeks if the level of LDL-C in the patient is below a threshold level, or about every two weeks if the level of LDL-C in the patient is greater than or equal to the threshold level, 150mg of the antibody or antigen-binding fragment thereof about every two weeks.

In certain embodiments, the method further comprises: (c) administering to the patient one or more of the following doses of 300mg of the antibody or antigen-binding fragment thereof about every four weeks if the level of LDL-C in the patient is below a threshold level, or about every two weeks of one or more of the following doses of 150mg of the antibody or antigen-binding fragment thereof if the level of LDL-C in the patient is greater than or equal to the threshold level.

In certain embodiments, the threshold level is 70 mg/dL.

In certain embodiments, the patient is intolerant to statins.

In certain embodiments, the patient is further receiving concomitant antidiabetic therapy in addition to insulin therapy, in certain embodiments, the additional antidiabetic therapy is selected from the group consisting of glucagon-like peptide 1(GLP-1) therapy, gastrointestinal peptides, glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, growth hormone releasing hormone antagonists or inverse agonists, xenin analogs, biguanides, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, α -glucosidase inhibitors, sodium-dependent glucose transporter 2(SGLT 2) inhibitors, SGLT-1 inhibitors, peroxisome proliferator activated receptors (PPAR-) (α, gamma or gamma/gamma) agonists or modulators, anti-inflammatory agonists or agonists for the glucose transporter 67120, agonists, GPR kinase inhibitors, GPR142 agonists, GPR-3 agonists, GPR142 inhibitors, GPR-3 agonists, GPR142 agonists, GPR4 agonists, GPR receptor antagonists, GPR4 agonists, GPR receptor agonists, GPR 3 agonists, GPR4 agonists, GPR receptor agonists, GPR 3 agonists, GPR4 agonists, and GPR receptor agonists, GPR receptor antagonists, and combinations thereof for the treatment of diabetes mellitus.

In certain embodiments, the antibody or antigen-binding fragment thereof reduces the LDL-C level of the patient, e.g., by at least 30%, 35%, 40% or 45%. In certain embodiments, the antibody or antigen-binding fragment thereof reduces non-HDL-C levels in the patient, e.g., by at least 20%, 25%, 30%, 35%. In certain embodiments, the antibody or antigen-binding fragment thereof reduces ApoC3 levels in the patient. In certain embodiments, the antibody or antigen-binding fragment thereof reduces the number and/or size of lipoprotein particles in the patient. In certain 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 fasting blood glucose (FPG) levels in the patient.

According to another aspect, the method comprises a method for treating hypercholesterolemia in a patient having T2DM and ASCVD, the method comprising:

(a) selecting a high cardiovascular risk patient receiving insulin treatment with (i) T2DM, (ii) ASCVD, and (iii) hypercholesterolemia not adequately controlled by maximally tolerated statin treatment;

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

(c) administering to the patient one or more of the following doses of the antibody or antigen-binding fragment thereof about every two weeks if the LDL-C level in the patient is less than 70mg/dL, or about every two weeks if the LDL-C level in the patient is greater than or equal to 70mg/dL, 150mg of the antibody or antigen-binding fragment thereof about every two weeks,

Other embodiments will become apparent from a review of the detailed description that follows.

Drawings

Fig. 1 is a diagram illustrating the overall design of the main stages of the study described in example 2 herein. The study included a screening phase, a double-blind treatment phase, and a safety observation phase.

FIG. 2 is a graph showing LS mean (+/-SE) as a percentage change from baseline for calculated LDL-C levels in the ITT population of patients with type 1 diabetes mellitus according to IVRS. Least Squares (LS) mean and Standard Error (SE) were obtained from mixed effects model (MMRM) analysis of replicate measurements. The model includes fixed classification effects for treatment groups, randomized layers by IVRS, time points, treatment group interaction with time points, layer (strata) interaction with time points, treatment group interaction with layer and layer interaction with time points, and continuous fixed covariates of baseline calculated LDL-C values and baseline values interaction with time points. The MMRM model was performed on all patients in the ITT population (i.e., type 1 and type 2 diabetic patients).

FIG. 3 is a graph showing the LS mean (+/-SE) as a percentage change from baseline for calculated LDL-C levels in the ITT population of patients with type 2 diabetes mellitus according to IVRS. Least Squares (LS) mean and Standard Error (SE) were obtained from mixed effects model (MMRM) analysis of replicate measurements. The model includes fixed classification effects for treatment groups, randomized layers by IVRS, time points, treatment group interaction with time points, layer interaction with time points, treatment group interaction with layer and layer interaction with time points, and continuous fixed covariates of baseline calculated LDL-C values and baseline value interaction with time points. The MMRM model was performed on all patients in the ITT population (i.e., type 1 and type 2 diabetic patients).

FIG. 4 is a graph showing the percent change from baseline to week 24 of non-HDL-C, LDL-C, ApoB and LDL-PN in the ITT population with type 2 diabetes and ASCVD.

FIG. 5 is a graph showing the percentage of individuals who reached non-HDL-C <100mg/dL, LDL-C <70mg/dL, and ApoB <80mg/dL at week 24 in an ITT population with type 2 diabetes and ASCVD.

Detailed description of the preferred embodiments

The methods are not limited to the specific methods and experimental conditions described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present method will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. As used herein, the term "about," when used in reference to a specifically recited value, means that the value may differ from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

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

Method for treating patients with hypercholesterolemia and diabetes during insulin therapy

Methods and compositions for treating diabetic patients with hypercholesterolemia during insulin therapy are provided. According to certain embodiments, the methods result in a reduction in lipoprotein levels (e.g., LDL-C and/or lp (a)) in the serum of the patients.

The present disclosure also provides PCSK9 inhibitors (e.g., antibodies or antigen-binding fragments thereof that specifically bind PCSK9 (e.g., human PCSK9) or compositions comprising PCSK9 inhibitors for use in treating diabetic patients with hypercholesterolemia during insulin therapy in certain embodiments, PCSK9 inhibitors or compositions are useful in reducing the levels of lipoproteins (e.g., LDL-C and/or lp (a)) in the serum of such patients.

As used herein, the term "lipoprotein" means a biomolecule particle that contains both proteins and lipids. Examples of lipoproteins include, for example, Low Density Lipoprotein (LDL), Very Low Density Lipoprotein (VLDL), Intermediate Density Lipoprotein (IDL) and lipoprotein (a) (lp (a)).

Diabetes (Diabetes mellitus), often referred to simply as Diabetes (Diabetes), is a group of metabolic diseases in which a person has high blood glucose levels 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 fails to utilize insulin properly, and is accompanied by an increase in insulin deficiency over time; (3) gestational diabetes, where a woman suffers from diabetes due to her pregnancy. All forms of diabetes increase the risk of long-term complications, which often occur many years later. Most of these long-term complications are based on damage to blood vessels and can be divided into two categories: "macrovascular" disease caused by atherosclerosis of large blood vessels and "microvascular" disease caused by damage to small blood vessels. Examples of macrovascular disease conditions are ischemic heart disease, myocardial infarction, stroke and peripheral vascular disease. Examples of microvascular diseases are diabetic retinopathy, diabetic nephropathy and diabetic neuropathy.

According to certain embodiments, the patient to be treated has type 1 diabetes (T1DM) or type 2 diabetes (T2DM) and is receiving insulin therapy. In certain embodiments, the patient has been diagnosed with T1DM or T2DM for at least one year. In certain embodiments, the patient was diagnosed with T1DM before the age of 30. In certain embodiments, a T1DM patient has a C peptide level of less than 0.2 pmol/mL. In certain embodiments, the patient has a glycosylated hemoglobin (HbA1c) level of less than 10%.

According to certain embodiments, the patient to be treated has hypercholesterolemia that is not adequately controlled by Lipid Modification Therapy (LMT). If the patient's serum LDL-C concentration does not fall to a recognized medically acceptable level, e.g., below 70mg/dL, at least 4 weeks after LMT, then the hypercholesterolemia is considered to be insufficiently controlled by LMT (given the relative risk of coronary heart disease in the patient). In certain embodiments, the LMT is the most tolerated statin therapy. As used herein, "maximally tolerated statin therapy" means the highest dose of statin that can be administered to a patient without causing unacceptable adverse side effects in the patient. For example, the methods disclosed herein include treating patients with hypercholesterolemia that is not adequately controlled by a daily dose of a statin selected from the group consisting of: atorvastatin (including atorvastatin + ezetimibe), rosuvastatin, cerivastatin, pitavastatin, fluvastatin, lovastatin, simvastatin (including simvastatin + ezetimibe), pravastatin, and combinations thereof. In certain embodiments, if the patient is intolerant to the treatment, the patient does not receive concomitant statin therapy. For example, a statin intolerant patient may have skeletal muscle-related symptoms such as pain, weakness, or spasticity that begin or increase during statin treatment and also stop when statin treatment stops, unlike symptoms due to a sprain or trauma.

Patient selection

The methods and compositions of the present invention are useful for treating patients with hypercholesterolemia and diabetes and undergoing insulin therapy. The patient to be treated may also exhibit one or more additional selection criteria. For example, a patient may be selected for treatment if the patient has a calculated LDL-C level greater than or equal to 70mg/dL, 100mg/dL, or 130 mg/dL. The patient may be treated with a maximum tolerated dose of a statin, optionally in combination with at least one other Lipid Modification Therapy (LMT), for at least 4 weeks, or wherein if the patient is statin intolerant, the patient has been treated with an optimal dose of at least one non-statin LMT for at least 4 weeks. A maximally tolerated dose of a statin may be defined, for example, as a dose prescribed based on regional practice or local guidelines, or as a dose that is maximally tolerated due to adverse reactions to higher doses prescribed in local prescription information for pediatric patients. Statin intolerance may be defined, for example, as the inability to tolerate at least 2 statins due to skeletal muscle-related symptoms that begin or increase during statin treatment and also stop when statin treatment stops, as opposed to symptoms due to sprains or trauma: the lowest daily starting dose of one statin, and any dose of the other statin. Patients who do not receive a daily regimen of statins (e.g., 1 to 3 times per week) are also considered to be unable to tolerate daily doses.

In addition, a patient may be selected for treatment if the patient has a high risk of Cardiovascular (CV). In certain embodiments, a high CV risk patient has a recorded history of cardiovascular disease (CVD) and/or at least one additional CV risk factor. CVD includes, but is not limited to, Coronary Heart Disease (CHD) and risk of CHD. CHD includes, but is not limited to, acute Myocardial Infarction (MI), asymptomatic MI, unstable angina, coronary revascularization procedure (e.g., Percutaneous Coronary Intervention (PCI) or coronary artery bypass graft surgery (CABG)), and clinically significant CHD (e.g., diagnosed by invasive or non-invasive tests such as coronary angiography, pressure testing using a treadmill, stress echocardiogram, or nuclear imaging). CHD risk-equivalent conditions include, but are not limited to, peripheral arterial disease (e.g., as described in the inclusion criteria of example 2) and prior ischemic stroke with atherosclerotic thrombotic origin, with focal ischemic neurological deficits lasting more than 24 hours. CV risk factors include, but are not limited to, hypertension, recent smoking, male age ≧ 45 years, female age ≧ 55 years, history of microalbuminuria, history of diabetic retinopathy, family history of early-onset CHD (father or brother before age 55; mother or sister before age 65), low HDL-C (male <40mg/dL [1.0mmol/L ], and female <50mg/dL [1.3mmol/L ]), and recorded Chronic Kidney Disease (CKD) (e.g., as defined in the inclusion criteria of example 2).

In certain embodiments, a high CV risk patient has atherosclerotic cardiovascular disease (ASCVD). In certain embodiments, ASCVD is defined as Coronary Heart Disease (CHD), ischemic stroke, or peripheral artery disease. In certain embodiments, CHD includes acute myocardial infarction, asymptomatic myocardial infarction, and unstable angina. In certain embodiments, CHD is defined as acute myocardial infarction, asymptomatic myocardial infarction, or unstable angina.

Insulin treatment

As shown herein, the diabetic patient selected for treatment by the methods of the invention has been subjected to and followed byFollowed by insulin therapy comprising insulin or a derivative thereof. Insulin on the market differs in the origin of the insulin (e.g. bovine, porcine, human insulin) and also in their composition, whereby the characteristics of the action (onset of action and duration of action) can be influenced. By combining different insulin products, it is possible to obtain a variety of action profiles and to establish blood glucose levels as close to physiological as possible. Exemplary insulin treatments may include naturally occurring insulins, such as human insulin, as well as modified insulins with extended duration of action, such as insulin glargine (Gly (a21) -Arg (B31) -Arg (B32) human insulin, e.g.,

). Insulin glargine is injected as an acidic clear solution and, due to its solution properties in the physiological pH range of the subcutaneous tissue, precipitates as a stable hexameric associate. Insulin glargine is injected once daily and is more noticeable than other long-acting insulins due to its flat serum profile and the corresponding reduced risk of nocturnal hypoglycemia (Schubert-Zsilavecz et al, 2:125-130 (2001)). Insulin glargine may be administered at a concentration above 100U/mL, for example, 270-330U/mL or 300U/mL insulin glargine (as disclosed in EP 2387989), other exemplary insulin treatments including: insulin glulisine (e.g. insulin glulisine)

) Insulin detemir (e.g. insulin detemir)

) Insulin lispro (e.g. insulin lispro)

) Insulin deglutamide (e.g. insulin deglutamide)

IdegLira (NN9068)), insulin aspart, and preparations of aspart (e.g., insulin aspart

) Basal insulins and analogs (e.g., LY2605541, LY2963016, NN1436), pegylated insulin lispro (e.g., LY-275585), long-acting insulins (e.g., NN1436, Insumera (PE0139), AB-101, AB-102, Sensulin LLC), intermediate-acting insulins (e.g.

) Rapid acting and short acting insulins (e.g. insulin

PH20 insulin, NN1218,

) Pre-mixing insulin,

NN1045, insulin plus

PE-0139, ACP-002 hydrogel insulin, and oral, inhalable, transdermal, and oral or sublingual insulin (e.g., insulin

Insulin tregpil, TPM-02 insulin,

Oral insulin, ORMD-0801, Oshadi oral insulin, NN1953, NN1954, NN1956,

). Also suitable are those insulin derivatives which are bound to albumin or another protein via a bifunctional linker.

PCSK9 inhibitors

The method comprises administering to the patient a therapeutic composition comprising a PCSK9 inhibitor. As used herein, a "PCSK 9 inhibitor" is any agent that binds to or interacts with human PCSK9 and inhibits the normal biological function of PCSK9 in vitro or in vivo. Non-limiting examples of PCSK9 inhibitor classes include small molecule PCSK9 antagonists, nucleic acid-based inhibitors of PCSK9 expression or activity (e.g., sirnas or antisense), peptide-based molecules that specifically interact with PCSK9 (e.g., peptibodies), receptor molecules that specifically interact with PCSK9, proteins comprising ligand-binding portions of LDL receptors, PCSK9 binding scaffold molecules (e.g., darpins, HEAT repeat proteins, ARM repeat proteins, triangular tetrapeptide repeat proteins, fibronectin-based scaffold constructs, and other scaffolds based on naturally occurring repeat proteins, etc.), [ see, e.g., Boersma Pluckthun,2011, curr. opin.biotechnol.22:849-857, and references cited therein ], and anti-PCSK 9 aptamers or portions thereof. According to certain embodiments, PCSK9 inhibitors that may be used in the context of the methods of the invention are anti-PCSK 9 antibodies or antigen-binding fragments of antibodies that specifically bind to human PCSK 9.

As used herein, the term "human proprotein convertase subtilisin/kexin type 9" or "human PCSK 9" or "hPCSK 9" refers to a polypeptide having the amino acid sequence of SEQ ID NO: 197 and the nucleic acid sequence set forth in SEQ ID NO: 198, or a biologically active fragment thereof.

As used herein, the term "antibody" is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V)_H) And a heavy chain constant region. The heavy chain constant region comprises three domains, C_H1、C _H2 and C _H3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V)_L) And a light chain constant region. The light chain constant region comprises a domain (C)_L1)。V_HAnd V_LThe regions may be further subdivided into hypervariable regions, known as Complementarity Determining Regions (CDRs), interspersed with more conserved regions known as Framework Regions (FRs). Each V_HAnd V_LConsists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2,CDR2, FR3, CDR3, FR 4. In various embodiments, the FR of the anti-PCSK 9 antibody (or antigen-binding portion thereof) may be identical to human germline sequences, or may be natural or artificially modified. Amino acid consensus sequences can be defined based on parallel analysis of two or more CDRs.

As used herein, the term "antibody" also includes antigen-binding fragments of intact antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from whole antibody molecules, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or can be readily obtained, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthetic. 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 into the appropriate configuration, or to introduce codons, generate cysteine residues, modify, add or delete amino acids, and the like.

Non-limiting examples of antigen-binding fragments include: (i) a Fab fragment; (ii) a F (ab')2 fragment; (iii) (ii) a fragment of Fd; (iv) (iv) an Fv fragment; (v) single chain fv (scFv) molecules; (vi) a dAb fragment; and (vii) a minimal recognition unit (e.g., an isolated Complementarity Determining Region (CDR), such as a CDR3 peptide) consisting of the amino acid residues that mimic a hypervariable region of an antibody, or a constrained (constrained) FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.), Small Modular Immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also included within the expression "antigen-binding fragments" as used herein.

An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition, and will typically comprise at least one CDR that is adjacent to or in frame with one or more framework sequences. In a region having a sum of V_LDomain linked V_HIn antigen-binding fragments of domains, V_HAnd V_LThe domains may be placed in any suitable arrangement relative to each other. For example, the variable region may be dimeric and contain V_H-V_H、V_H-V_LOr V_L-V_LA dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomeric V_HOr V_LA domain.

In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary structures of variable and constant domains that can be found in antigen-binding fragments of antibodies include: (i) v_H-C_H1；(ii)V_H-C _H2；(iii)V_H-C _H3；(iv)V_H-C_H1-C _H2；(v)V_H-C_H1-C_H2-C _H3；(vi)V_H-C_H2-C _H3；(vii)V_H-C_L；(viii)V_L-C_H1；(ix)V_L-C _H2；(x)V_L-C _H3；(xi)V_L-C_H1-C _H2；(xii)V_L-C_H1-C_H2-C _H3；(xiii)V_L-C_H2-C _H3; and (xiv) V_L-C_L. In any of the variable and constant domains, including any of the exemplary structures shown above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region can be comprised of at least 2 (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, which results in flexible or semi-flexible connections between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, antigen-binding fragments of antibodies mayTo include non-covalent linkage to each other and/or to one or more monomers V_HOr V_LA homodimer or heterodimer (or other multimer) of any of the variable and constant domains shown above in association with the domains (e.g., by disulfide bonds).

As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding a separate antigen or 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 antigen-binding fragments of antibodies of the methods of the invention using conventional techniques available in the art.

The constant regions of antibodies are important in the ability of the antibody to fix complement and mediate cell-dependent cellular cytotoxicity. Thus, the isotype of an antibody may be selected based on whether antibody-mediated cytotoxicity is desired.

As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, human antibodies may comprise amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, particularly in CDR 3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.

As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies isolated from recombinants, combinatorial human antibody libraries (described further below), isolated from animals transgenic for human immunoglobulin genes (e.g., mice)(iv) see, e.g., Taylor et al (1992) Nucl. acids Res.20:6287-6295) or by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, to in vivo somatic mutagenesis), and thus the V of the recombinant antibody_HAnd V_LThe amino acid sequence of the region is such that: when it is derived from human germline V_HAnd V_LSequences and sequences related thereto may not naturally exist in human antibody germline repertoires in vivo.

Human antibodies can exist in two forms associated with hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of about 150-160kDa, wherein the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds and form a molecule of about 75-80kDa, consisting of covalently coupled light and heavy chains (half-antibodies). Even after affinity purification, these forms are extremely difficult to isolate.

The frequency of occurrence of the second form in the various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. Single amino acid substitutions in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al (1993) Molecular Immunology 30:105) to levels typically observed with the human IgG1 hinge. The method of the invention comprises at the hinge, C _H2 or C _H3 region has one or more mutations of the antibody, which can be for example in production of the desired to improve the desired antibody form yield.

As used herein, "isolated antibody" 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 isolated or removed from at least one component of an organism or from a tissue or cell in which the antibody naturally occurs or naturally occurs is an "isolated antibody" for purposes of the methods of the invention. Isolated antibodies also include in situ antibodies within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

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

anti-PCSK 9 antibodies useful in the present methods may comprise one or more amino acid substitutions, insertions, and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The methods include the use of antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue of the germline sequence from which the antibody is derived, or to the corresponding residue of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residue (such sequence changes are herein generally used forReferred to as "germline mutations"). One of ordinary skill in the art can readily generate a number of antibodies and antigen-binding fragments that comprise one or more individual germline mutations (individual germline mutations) or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V_HAnd/or V_LAll framework and/or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., mutated residues found only within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or mutated residues found only in CDR1, CDR2, or CDR 3. In other embodiments, one or more framework and/or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, an antibody may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residues of a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced biological properties of antagonism or agonism (as the case may be), reduced immunogenicity, and the like. The use of antibodies and antigen-binding fragments obtained in this general manner is included in the present methods.

The methods comprise the use of an anti-PCSK 9 antibody comprising a variant of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the methods of the invention include the use of anti-PCSK 9 antibodies having HCVR, LCVR and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows, for example, the use of BIAcore^TMThe system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ) analyzes real-time interactions by detecting changes in protein concentration within the biosensor matrix (matrix).

As used herein, the term "K_D"is intended to mean the equilibrium dissociation constant for a particular antibody-antigen interaction.

The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, termed the paratope of an antigen. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a portion of a sugar, phosphoryl, or sulfonyl group on an antigen.

According to certain embodiments, the anti-PCSK 9 antibody used in the method is an antibody with pH-dependent binding characteristics. As used herein, the expression "pH-dependent binding" means that the antibody or antigen-binding fragment thereof exhibits "reduced binding to PCSK9 at acidic pH as compared to neutral pH" (for the purposes of this disclosure, the two expressions may be used interchangeably). For example, "an antibody having pH-dependent binding characteristics" includes antibodies and antigen-binding fragments thereof that bind PCSK9 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibodies and antigen-binding fragments bind PCSK9 with at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more times higher affinity at neutral pH than at acidic pH.

According to this aspect, an anti-PCSK 9 antibody having pH-dependent binding characteristics may have one or more amino acid variations relative to a parent anti-PCSK 9 antibody. For example, an anti-PCSK 9 antibody with pH-dependent binding characteristics may contain one or more histidine substitutions or insertions, e.g., in one or more CDRs of a parent anti-PCSK 9 antibody. Thus, according to certain embodiments, there is provided a method comprising administering an anti-PCSK 9 antibody comprising CDR amino acid sequences (e.g., heavy and light chain CDRs) identical to those of a parent anti-PCSK 9 antibody except for the replacement of one or more amino acids of one or more CDRs of the parent antibody with a histidine residue. An anti-PCSK 9 antibody with pH-dependent binding can 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 interspersed among multiple (e.g., 2, 3, 4, 5, or 6) CDRs of the parent anti-PCSK 9 antibody. For example, the methods of the invention comprise the use of an anti-PCSK 9 antibody with pH-dependent binding comprising one or more histidine substitutions in HCDR1, one or more histidine substitutions in HCDR2, one or more histidine substitutions in HCDR3, one or more histidine substitutions in LCDR1, one or more histidine substitutions in LCDR2, and/or one or more histidine substitutions in LCDR3 of the parent anti-PCSK 9 antibody.

As used herein, the expression "acidic pH" means a pH of 6.0 or less (e.g., less than about 6.0, less than about 5.5, less than about 5.0, etc.). The expression "acidic pH" includes pH values of about 6.0, 5.95, 5.90, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. As used herein, the expression "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

Non-limiting examples of anti-PCSK 9 antibodies that can be used in the context of the methods of the invention include, for example, alikumab, efuzumab, bococizumab, ludwizumab, ralpancizumab, LY3015014, or an antigen-binding portion of any of the foregoing antibodies.

Preparation of human antibodies

Methods for making human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the methods of the present invention to make human antibodies that specifically bind to human PCSK 9.

Use of VELOCIMMUNE^TMThe high affinity chimeric antibodies against PCSK9 with human variable and mouse constant regions were initially isolated by techniques (see, e.g., US 6,596,541, regeneron pharmaceuticals) or any other known method for generating monoclonal antibodies.

The technology involves producing a transgenic mouse having a genome of human heavy and light chain variable regions operably linked to an endogenous mouse constant region locus, such that the mouse produces antibodies comprising the human variable regions and a mouse constant region in response to antigen stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

Generally, challenge with an antigen of interest

Mice, and lymphocytes (such as B cells) are recovered from the antibody-expressing mice. Lymphocytes can be fused with myeloma cell lines to prepare immortal hybridoma cell lines, and such hybridoma cell lines screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. The DNA encoding the heavy and light chain variable regions can be isolated and ligated to the desired isotype constant regions of the heavy and light chains. Such antibody proteins may be produced in cells (such as CHO cells). Alternatively, DNA encoding the antigen-specific chimeric antibody or the light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

Initially, high affinity chimeric antibodies with human variable regions and mouse constant regions were isolated. The desired characteristics of the antibody, including affinity, selectivity, epitope, etc., are characterized and selected using standard methods known to those skilled in the art. The mouse constant region is replaced with the desired human constant region to generate fully human antibodies, e.g., wild-type or modified IgG1 or IgG 4. While the constant region selected may vary depending on the particular use, high affinity antigen binding and target-specific characteristics are present in the variable region.

In general, antibodies that can be used have high affinity when measured by binding to an antigen immobilized on a solid phase or in a solution phase, as described above. The mouse constant region was replaced with the desired human constant region to generate fully human antibodies. While the constant region selected may vary depending on the particular use, high affinity antigen binding and target-specific characteristics are present in the variable region.

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

Sequence identity between two amino acid sequences is determined over the entire length of a reference amino acid sequence (i.e., using the amino acid sequence identified by SEQ ID NO) and/or over the region of optimal sequence alignment between the two amino acid sequences using optimal sequence alignment, which can be determined using tools known in the art (e.g., Align), using standard settings, preferably EMBOSS:: needle, Matrix: blosum62, Gap Open 10.0, Gap extended 0.5.

In certain embodiments, the antibody or antigen binding protein comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 1/6 and 11/15 (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR 3). Alternatively, in certain embodiments, the antibody or antigen binding protein comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) from a heavy and light chain variable region amino acid sequence pair (HCVR/LCVR) selected from the group consisting of: 37/41, 45/49, 53/57, 61/65, 69/73, 77/81, 85/89, 93/97, 101/105, 109/113, 117/121, 125/129, 133/137, 141/145, 149/153, 157/161, 165/169, 173/177, 181/185 and 189/193.

In certain embodiments, anti-PCSK 9 antibodies or antigen-binding proteins useful in the methods have an HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 amino acid sequence selected from SEQ ID NOs: 2/3/4/7/8/10(mAb316P [ also referred to as "REGN 727" or "aleucirumab" ]) and 12/13/14/16/17/18(mAb300N) (see U.S. patent application publication No. 2010/0166768) and 12/13/14/16/17/18, wherein SEQ ID NO: 16 contains a histidine substitution at amino acid residue 30 for leucine (L30H).

In certain embodiments, the antibody or antigen binding protein comprises a sequence selected from the group consisting of SEQ ID NOs: 1/6 and 11/15 in a HCVR/LCVR amino acid sequence pair. In certain exemplary embodiments, the antibody or antigen binding protein comprises SEQ ID NO:1 and the HCVR amino acid sequence of SEQ ID NO: 6. In certain exemplary embodiments, the antibody or antigen binding protein comprises SEQ ID NO: 11 and the HCVR amino acid sequence of SEQ ID NO:15, LCVR amino acid sequence. In certain exemplary embodiments, the antibody or antigen binding protein comprises SEQ ID NO: 11 and the HCVR amino acid sequence of SEQ ID NO:15, said SEQ ID NO:15 comprising a histidine substituted for leucine at amino acid residue 30 (L30H).

Pharmaceutical compositions and methods of administration

The methods comprise administering a PCSK9 inhibitor to the patient, wherein the PCSK9 inhibitor is comprised in a pharmaceutical composition. Pharmaceutical compositions are formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. Many suitable formulations can be found in formulations known to all medicinal chemists: remington's pharmaceutical sciences, Mack Publishing Company, Easton, Pa. These include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicle-containing lipids (cationic or anionic), such as LIPOFECTIN^TM) DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al, "Complex of excipients for formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

Exemplary pharmaceutical formulations comprising anti-PCSK 9 antibodies that may be used in the context of the methods of the invention include US 8,795,669 (describing, inter alia, exemplary formulations comprising aleucirumab) or any of the formulations described in WO2013/166448 or WO 2012/168491.

A variety of delivery systems are known and may be used to administer pharmaceutical compositions, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing a mutant virus, receptor-mediated endocytosis (see, e.g., Wu et al, 1987, J.biol.chem.262: 4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example by infusion or bolus injection (bolus injection), absorbed through epithelial or mucocutaneous linings (e.g. oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other bioactive agents.

The pharmaceutical composition may be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with regard to subcutaneous delivery, the pen delivery device is readily applied to deliver pharmaceutical compositions. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges (cartridges) containing pharmaceutical compositions. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device may then be reused. In a disposable pen delivery device, there is no replaceable cartridge. In contrast, disposable pen delivery devices are pre-filled with a pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

Many reusable pen and autoinjector delivery devices have application for subcutaneous delivery of pharmaceutical compositions. Examples include, but are not limited to, AUTOPEN^TM(Owen Mumford,Inc.,Woodstock,UK)，DISETRONIC^TMPen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25^TMPen, HUMALOG^TMPen, HUMALIN 70/30^TMPen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN^TMI. II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR^TM(Novo Nordisk,Copenhagen,Denmark)，BD^TMPen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN^TM，OPTIPEN PRO^TM，OPTIPEN STARLET^TMAnd OPTICLIK^TM(sanofi-aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices having applications for subcutaneous delivery of the pharmaceutical compositions of the methods of the present invention include, but are not limited to, SOLOSTAR^TMPen (sanofi-aventis) and FLEXPEN^TM(Novo Nordisk) and KWIKPEN^TM(EliLilly)、SURECLICK^TMAuto-injectors (Amgen, Thousand Oaks, Calif.), PENLET^TM(Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA^TMPens (Abbott Labs, Abbott Park IL), to name a few.

In some cases, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton,1987, CRC Crit. Ref. biomed. Eng.14: 201). In another embodiment, a polymeric material may be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.),1974, CRC Pres, Boca Raton, Florida. In yet another embodiment, a Controlled Release system may be placed in the vicinity of the target of the composition, thus requiring only a small fraction of the systemic dose (see, e.g., Goodson,1984, Medical Applications of Controlled Release, supra, Vol.2, p.115-138). Other controlled release systems are discussed in reviews by Langer,1990, Science 249: 1527-.

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

Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms suitable for the unit dose with which a dose of active ingredient is to be fitted. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like.

Dosage form

The amount of PCSK9 inhibitor (e.g., an anti-PCSK 9 antibody) administered to the patient is typically a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" means a dose of a PCSK9 inhibitor that results in a detectable decrease (of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more from baseline) in one or more parameters selected from the group consisting of: LDL-C, ApoB100, non-HDL-C, total cholesterol, VLDL-C, triglycerides, ApoC3, TRL particles, lp (a), and residual cholesterol.

In the case of anti-PCSK 9 antibodies, a therapeutically effective amount may be about 0.05mg to about 600mg, e.g., about 0.05mg, about 0.1mg, about 1.0mg, about 1.5mg, about 2.0mg, about 10mg, about 20mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 75mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 400mg, about 440mg, about 450mg, about 500mg, about 390mg, about 500mg, about, About 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, or about 600mg of an anti-PCSK 9 antibody. According to certain exemplary embodiments, the therapeutically effective amount of the anti-PCSK 9 antibody is 30mg, 40mg, or 75mg (e.g., where aliskirumab is for a patient weighing less than 50kg, and/or less than or equal to 17 years old), 50mg, 75mg, or 150mg (e.g., where aliskirumab is for a patient weighing greater than or equal to 50kg, and/or less than or equal to 17 years old), or 140mg or 420mg (e.g., where efuzumab is for a patient weighing greater than or equal to 50kg, and/or less than or equal to 17 years old). Other doses of PCSK9 inhibitors will be apparent to one of ordinary skill in the art.

The amount of anti-PCSK 9 antibody contained in a single dose may be expressed in milligrams of antibody per kilogram of patient body weight (i.e., mg/kg). For example, the anti-PCSK 9 antibody can be administered to the patient at a dose of about 0.0001 to about 10mg/kg body weight.

Administration regimen

According to certain embodiments, multiple doses of a PCSK9 inhibitor (i.e., a pharmaceutical composition comprising a PCSK9 inhibitor) may be administered to a subject over a defined time course (e.g., in addition to a daily therapeutic statin regimen or other background LMT). The method according to this aspect comprises sequentially administering to the subject multiple doses of a PCSK9 inhibitor. As used herein, "sequentially administering" means that each dose of the PCSK9 inhibitor is administered to the subject at different time points, e.g., on different days separated by predetermined intervals (e.g., hours, days, weeks, or months). The methods comprise sequentially administering to the patient a single initial dose of a PCSK9 inhibitor, followed by one or more second doses of a PCSK9 inhibitor, and optionally followed by one or more third doses of a PCSK9 inhibitor.

The terms "initial dose", "second dose", and "third dose" refer to the temporal order of administration of the individual doses of the pharmaceutical composition comprising a PCSK9 inhibitor. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"); "second dose" is the dose administered after the initial dose; the "second dose" is the dose administered after the second dose. The initial, second and third doses may all contain the same amount of PCSK9 inhibitor, but may typically differ from each other in terms of frequency of administration. However, in certain embodiments, the amounts of PCSK9 inhibitor contained in the initial, second, and/or third doses are different from each other (e.g., adjusted up or down as appropriate) during treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of a treatment regimen as a "loading dose" followed by subsequent doses administered on a less frequent basis (e.g., a "maintenance dose").

According to an exemplary embodiment, each second and/or third dose is administered 1 to 26 (e.g., 1, 11/2, 2, 21/2, 3, 31/2, 4, 41/2, 5, 51/2, 6, 61/2, 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2, 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2, 22, 221/2, 23, 231/2, 24, 241/2, 25, 251/2, 26, 261/2, or more) weeks after the immediately preceding dose. As used herein, the phrase "immediately preceding dose" means a dose of antigen binding molecule that is administered to a patient in a sequence of multiple administrations, without intervening doses, prior to administration of the immediately next dose in the sequence.

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

In embodiments involving multiple third doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 2, 4, 6, 8, or more weeks after the immediately preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 1 to 2, 4, 6, 8, or more weeks after the immediately preceding dose. Alternatively, the frequency of the third and/or third dose administered to the patient may vary over the course of the treatment regimen. The frequency of administration can also be adjusted by the physician during the course of treatment according to the needs of the individual patient after clinical examination.

The methods of the invention include administration regimens that include an up-titration (up-titrate) option (also referred to herein as "dose alteration"). As used herein, "up-titration option" means that after receiving a particular number of doses of a PCSK9 inhibitor, if the patient has not achieved a specified decrease in one or more established treatment parameters, the dose of the PCSK9 inhibitor is increased thereafter. For example, in the case of a treatment regimen that includes administering a 75mg dose of an anti-PCSK 9 antibody to a patient at a frequency of once every two weeks, if after 8 weeks (i.e., 5 doses administered at

weeks

0, 2, 4, 6, and 8), the patient has not yet reached a serum LDL-C concentration of less than 70mg/dL, then the dose of anti-PCSK 9 antibody is increased thereafter to, for example, 150mg administered once every two weeks (e.g., from week 10 or week 12 or thereafter).

In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds PCSK9 is administered to the patient at a dose of about 75mg at a frequency of once every two weeks. In certain embodiments, if the patient's LDL-C measured after one or more, two or more, three or more, four or more, or five or more doses is <70mg/dL, then the about 75mg dose is maintained. In certain embodiments, if the patient's LDL-C, measured after one or more, two or more, three or more, four or more, or five or more doses, is still ≧ 70mg/dL, the about 75mg dose is discontinued, and the antibody or antigen-binding fragment thereof that specifically binds PCSK9 is subsequently administered to the patient at a dose of about 150mg at a frequency of once every two weeks.

In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds PCSK9 is administered to the patient at a dose of about 300mg at a frequency of once every four weeks. In certain embodiments, if the patient's LDL-C measured after one or more, two or more, three or more, four or more, or five or more doses is <70mg/dL, then an approximately 300mg dose is maintained. In certain embodiments, if the patient's LDL-C, measured after one or more, two or more, three or more, four or more, or five or more doses, is still ≧ 70mg/dL, the about 300mg dose is discontinued, and the patient is subsequently administered the antibody or antigen-binding fragment thereof that specifically binds PCSK9 at a dose of about 150mg at a frequency of once every two weeks.

In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds PCSK9 is administered to the patient at a dose of about 150mg at a frequency of once every two weeks.

In certain embodiments, when the antibody or antigen-binding fragment thereof that specifically binds PCSK9 is administered to the patient at a dose of about 150mg at a frequency of once every two weeks, the dose of about 150mg is discontinued if the patient's LDL-C measured after at least one dose or at least two, three, four, or five consecutive doses is <10, 15, 20, or 25mg/dL, followed by administration of the antibody or antigen-binding fragment thereof that specifically binds PCSK9 to the patient at a dose of about 75mg at a frequency of once every two weeks. While not wishing to be bound by theory, it is hypothesized that very low LDL-C levels (e.g., <10, 15, 20, or 25mg/dL) may exacerbate diabetes. In certain embodiments, a dose of about 150mg is administered to the patient at a constant dose. In certain embodiments, a dose of about 150mg is administered to the patient after dose adjustment as disclosed herein (e.g., from about 75mg every two weeks, or from about 300mg every four weeks).

Combination therapy

As described elsewhere herein, the methods may comprise administering a PCSK9 inhibitor to the patient in combination with ("in addition to" the PCSK9 inhibitor to the patient's previously prescribed Lipid Modification Therapy (LMT) "). LMTs include, but are not limited to, statins, fibrates, nicotinic acids (e.g., nicotinic acid and its derivatives), bile acid sequestrants, ezetimibe (ezetimibe), lomitapide (lomitamide), phytosterols, orlistat (orlistat), and the like. For example, a PCSK9 inhibitor may be administered to a patient in combination with a stable daily treatment statin regimen. Exemplary daily treatment statin regimens that may be administered in combination with a PCSK9 inhibitor in the context of the methods of the present invention include, for example, atorvastatin (10, 20, 40 or 80mg daily), (atorvastatin/ezetimibe 10/10 or 40/10mg daily), rosuvastatin (5, 10 or 20mg daily), cerivastatin (0.4 or 0.8mg daily), pitavastatin (1, 2 or 4mg daily), fluvastatin (20, 40 or 80mg daily), simvastatin (5, 10, 20, 40 or 80mg daily), simvastatin/ezetimibe (10/10, 20/10, 10/10 or 80/10mg daily), lovastatin (10, 20, 40 or 80mg daily), pravastatin (10, 20, 40 or 80mg daily), and combinations thereof. In certain embodiments, the statin treatment is the most tolerable statin treatment for the patient. Other LMTs that may be administered in combination with a PCSK9 inhibitor in the context of the methods of the present invention include, for example, (1) agents that inhibit cholesterol uptake and/or bile acid reabsorption (e.g., ezetimibe); (2) agents that increase lipoprotein catabolism (such as niacin); and/or (3) activators of LXR transcription factors that function in the elimination of cholesterol, such as 22-hydroxycholesterol.

According to certain embodiments, methods are provided that include administering a PCSK9 inhibitor (e.g., an anti-PCSK 9 antibody, such as aliskirumab, efuzumab, bococizumab, roducizumab, ralpancizumab, or LY3015014) in combination with an inhibitor of angiopoietin-like protein 3 (e.g., an anti-ANGPTL 3 antibody, such as REGN1500), an inhibitor of angiopoietin-like protein 4 (e.g., an anti-ANGPTL 4 antibody, such as an anti-ANGPTL 4 antibody referred to as "H1H 268P" or "H4H 284P" in U.S. patent No. 9,120,851), or an inhibitor of angiopoietin-like protein 8 (e.g., an anti-ANGPTL 8 antibody) to a patient.

According to certain embodiments, methods are provided that include administering a PCSK9 inhibitor (e.g., an anti-PCSK 9 antibody, such as aliskirumab, efuzumab, bococizumab, roducizumab, ralpancizumab, or LY3015014) to a patient in combination with an additional anti-diabetic therapy in addition to the insulin therapy. Exemplary additional anti-diabetic treatments include, but are not limited to:

(a) all drugs mentioned in Rote list 2016, (e.g., all antidiabetics mentioned in Rote list 2014 chapter 12), all antiobesity or appetite suppressants mentioned in Rote list 2016 chapter 06, all lipid lowering agents mentioned in Rote list 2016 chapter 58, all antihypertensives mentioned in Rote list 2016 chapter 17, all nephrotonics mentioned in Rote list, or all diuretics mentioned in Rote list 2016 chapter 36;

(b) pancreatic heightGlycemic like peptide 1(GLP-1) treatments, including GLP-1, GLP-1 analogs, and GLP-1 receptor agonists, such as: GLP-1(7-37), GLP-1(7-36) amides, lixisenatide, e.g.

) Exenatide (exenatide, e.g. Exendin-4, exendin-4, exendi,

Exenatide NexP), exenatide-LAR, liraglutide (e.g. liraglutide)

) Semaglutide, taloglutide, albiglutide, dulaglutide, albumin-glucagon-like peptide-1 (albugon), oxyntomodulin, geniposide (geniposide), ACP-003, CJC-1131, CJC-1134-PC, GSK-2374697, PB-1023, TTP-054, langenintide (HM-11260C), CM-3, GLP-1Eligen, 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-XT + glucagon-XTEN (VRS-859+ AMX-808), and polymer-bound analogs of GLP-1 and GLP-1;

(c) dual GLP-1/GIP agonists (e.g., RG-7697(MAR-701), MAR-709, BHM081, BHM089, BHM 098); dual GLP-1/glucagon receptor agonists (e.g., BHM-034, OAP-189(PF-05212389, TKS-1225), TT-401/402, ZP2929, LAPS-HMOXM25, MOD-6030);

(d) a dual GLP-1/gastrin agonist (e.g., ZP-3022);

(e) gastrointestinal peptides such as peptide YY3-36 (PYY3-36) or an analog thereof and Pancreatic Polypeptide (PP) or an analog thereof;

(f) glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, growth hormone releasing hormone antagonists or inverse agonists, xenin and analogs thereof;

(g) dipeptidyl peptidase-IV (DPP-4) inhibitors, such as: alogliptin (e.g. alogliptin)

) Linagliptin (linagliptin, e.g. linagliptin

) Saxagliptin (saxagliptin, e.g. SAxagliptin)

) Sitagliptin (sitagliptin, for example)

) Anegliptin (anagliptin), tigecycline (teneligliptin, e.g. teneligliptin

) Trelagliptin (trelagliptin), vildagliptin (vildagliptin, e.g. trelagliptin

) Gemigliptin, alogliptin (omarigliptin), Avoglliptin (evogliptin), Dougliptin (dutogliptin), DA-1229, MK-3102, KM-223, KRP-104, PBL-1427, Pinoxacin hydrochloride (Pinoxacin hydrochloride), and Ari-2243;

(h) sodium-dependent glucose transporter 2(SGLT-2) inhibitors, such as: canagliflozin (dapagliflozin), rhagliflozin (remogliflozin), remogliflozin etanate (remogliflozin), sergliflozin (sergliflozin), empagliflozin (empagliflozin), ipragliflozin (ipragliflozin), tolgliflozin (tofoglazin), rusagliflozin (lesagliflozin), elliflozin (ugliflazin), EGT-0001442, LIK-32066, SBM-TFC-039 and KGA-35 (DSP-3235);

(i) dual inhibitors of SGLT-2 and SGLT-1 (e.g., LX-4211, LIK 066);

(j) SGLT-1 inhibitors (e.g., LX-2761, KGA-3235) or SGLT-1 inhibitors in combination with anti-obesity drugs such as Ileal Bile Acid Transfer (IBAT) inhibitors (e.g., GSK-1614235+ GSK-2330672);

(k) biguanides (e.g., metformin, buformin, phenformin);

(l) Thiazolidinediones (e.g., pioglitazone (pioglitazone), rosiglitazone (rosiglitazone)), glitazone analogs (e.g., lobeglitazone);

(m) peroxisome proliferator-activated receptor (PPAR-) (α, gamma or α/gamma) agonists or modulators (e.g., saroglitazar)

) GFT-505) or a partial PPAR γ agonist (e.g., Int-131);

(n) sulfonylureas (e.g., tolbutamide, glibenclamide, glimepiride),

glipizide (glipizide) and meglitinides (meglitinides, such as nateglinide (nateglinide), repaglinide (repaglinide), mitiglinide (mitiglinide));

(o) α -glucosidase inhibitors (e.g., acarbose, miglitol, voglibose);

(q) amylin and amylin analogs (e.g. pramlintide,

)；

(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-Chem 1);

(q) GPR40 agonists (e.g., 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 absorbing TGR5(GPBAR1 ═ G protein-coupled bile acid receptor 1) agonists (e.g. INT-777, XL-475, SB 756050);

(t) diabetes immunotherapy, e.g., oral administration of C-C chemokine receptor type 2 (CCR-2) antagonists (e.g., CCX-140, JNJ-41443532), interleukin 1 β (IL-1 β) antagonists (e.g., AC-201), or oral administration of monoclonal antibodies (MoA) (e.g., methionine (methazamide), VVP808, PAZ-320, P-1736, PF-05175157, PF-04937319);

(v) anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, such as: nuclear factor kappa B inhibitors (e.g.

)；

(w) adenosine monophosphate-activated protein kinase (AMPK) stimulators, such as: imeglimin (PXL-008), Debio-0930(MT-63-78), R-118;

(x)11- β -inhibitors of hydroxysteroid dehydrogenase 1(11- β -HSD-1) (e.g., LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329, HSD-016, BI-135585);

(y) glucokinase activators (e.g., PF-04991532, TTP-399(GK1-399), GKM-001(ADV-1002401), ARRY-403(AMG-151), TAK-329, TMG-123, ZYGK 1);

(z) inhibitors of diacylglycerol O-acyltransferase (DGAT) (e.g., pradigastat (LCQ-908)), inhibitors of protein tyrosine phosphatase 1 (e.g., rodussquesmine), inhibitors of glucose-6-phosphatase, inhibitors of fructose-1, 6-bisphosphatase, inhibitors of glycogen phosphorylase, inhibitors of phosphoenolpyruvate carboxykinase, inhibitors of glycogen synthase kinase, inhibitors of pyruvate dehydrogenase kinase;

(aa) modulators of glucose transporter-4, somatostatin receptor 3 agonists (e.g., MK-4256);

(bb) One or more lipid lowering agents are also suitable as a combination partner, for example: 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase (HMG-CoA-reductase) inhibitors such as simvastatin (e.g.

) Atorvastatin (e.g.)

) Rosuvastatin (e.g. rosuvastatin)

) Pravastatin (e.g.

) Fluvastatin (e.g. fluvastatin

) Pitavastatin (e.g. pitavastatin

) Lovastatin (e.g.

) Mevastatin (mevastatin, e.g.)

) Rivastatin (rivastatin), cerivastatin

Fibrates such as bezafibrate (e.g. benzafibrate

Delayed), ciprofibrate (ciprofibrate, e.g.

) Fenofibrate (e.g. fenofibrate

) Gemfibrozil (e.g. gemfibrozil)

) Etofibrate (etofibrate), bisfibrate (simfibrate), chlorofibrate (ronifibrate), clinofibrate (clinofibrate), clofibrate (clofibrate), nicotinic acid and derivatives thereof (e.g. nicotinic acid, including extended release formulations of nicotinic acid), nicotinic acid receptor 1 agonists (e.g. GSK-256073), PPAR-delta agonists, acetyl coenzyme a-acetyltransferase (ACAT) inhibitors (e.g. avasimibe), cholesterol absorption inhibitors (e.g. ezetimibe, avasimibe), cholesterol absorption inhibitors (e.g. clofibrate, niacin receptor 1 agonists,

S-556971), a bile acid binding substance (e.g. cholestyramine (cholestyramine), colesevelam (colesevelam)), an Ileal Bile Acid Transport (IBAT) inhibitor (e.g. GSK-2330672, LUM-002), a microsomal triglyceride transfer protein (MTP) inhibitor (e.g. lomitapide (AEMITapede (AEGR-733)), SLx-4090, granotapide), a modulator of the proprotein convertase subtilisin/kexin type 9 (PCSK9) (e.g. alikumab (REGN/SAR 236553), AMG-145, LGT-209, PF-04950615, MPSK3169, LY3015014, ALD-306, ALN-PCS, BMS-962476, SPC5001, ISIS-4814, LG1B 20, LGT-210, 1D05, PCSK-PCSSK 469-2, PCSX-539 7, an ATP-962476, a selective modulator of the uptake of lipid receptor (e.g. a) receptor (e.g. a-567), a selective modulator of the liver receptor (e.g. a-567), a receptor (e.g. a-3646), a-729, ATP-75, ATP-borteosine.g. a-8, ATP-borteosine.g. a-7, ATP-a receptor (e.g. a) modulator) receptor (e.g. a selective modulator) receptor (e.g. a) 3, aAgents (e.g., BMS-823778, TAP-301, DRL-21994, DRL-21995), phospholipase A2(PLA2) inhibitors (e.g., Dapradil (Darapidib)),

Varespladib (varespladib), rilaplaib (rilaplaib)), ApoA-I enhancers (e.g., RVX-208, CER-001, MDCO-216, CSL-112), cholesterol synthesis inhibitors (e.g., ETC-1002), lipid metabolism modulators (e.g., BMS-823778, TAP-301, DRL-21994, DRL-21995) and omega-3 fatty acids and their derivatives (e.g., eicosapentaenoic ethyl (AMR101),

AKR-063、NKPL-66、PRC-4016、CAT-2003)；

(cc) bromocriptine (e.g. bromocriptine)

) Phentermine and phentermine formulations or combinations (e.g. Adipex-P, Ionamin, flupirtine, and flupirtine, respectively),

) Benzphetamine (benzphetamine, e.g. benzphetamine)

) Bupropion (diethyl propion, e.g. bupropion)

) Phendimetrazine (phendimitrazin, e.g. phendimetrazine

) Bupropion (bupropion) and combinations (e.g.

) Sibutramine (sibutramine, e.g. Sibutramine)

) Topiramate (topiramat, e.g. Topiramat

) Zonisamide (zonisamid, e.g.

) Tesofensine (tesofensine), opioid antagonists such as naltrexone (e.g. naltrexone)

Naltrexone + bupropion), cannabinoid receptor 1(CB1) antagonists (e.g., 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., Verneprill, S-234462), NPY4 agonists (e.g., PP-1420), β -3-adrenergic receptor agonists, leptin or leptin mimetics, agonists of the 5-hydroxytryptamine 2c (5HT2c) receptor (e.g., lorcaserin (lorcaserin),

) Pramlintide/metreleptin, lipase inhibitors such as cetistat (cetilistat, e.g. cetolistat)

) Orlistat (e.g.

) Angiogenesis inhibitors (e.g., ALS-L1023), betahistine and histamine H3 antagonists (e.g., HPP-404), AgRP (agouti-related protein) inhibitors (e.g., TTP-435), serotonin reuptake inhibitors such as fluoxetine (e.g., fluoxetine, e.g., fluoxetine)

) Duloxetine (duloxetine, e.g. for

) Inhibition of dual or triple monoamine uptakeFormulations (dopamine, norepinephrine and serotonin reuptake) such as sertraline (sertraline, e.g.

) Tesofensine (tesofensine), methionine aminopeptidase 2(MetAP2) inhibitors (e.g., Beloranib) and antisense oligonucleotides generated against fibroblast growth factor receptor 4(FGFR4) (e.g., ISIS-FGFR4Rx) or antiproliferative protein targeting peptides-1 (e.g., ISIS-FGFR4Rx)

) (ii) a And

(dd) nitric oxide donors, AT1 antagonists or angiotensin II (AT2) receptor antagonists, such as telmisartan (telmisartan, e.g. TELMISartan)

) Candesartan (e.g. candesartan)

) Valsartan (valsartan, e.g. valsartan)

) Losartan (losartan, e.g.

) Eprosartan (eprosartan, e.g. eprosartan)

) Irbesartan (irbesartan, e.g. irbesartan)

) Olmesartan (olmesartan, e.g.

) Tasosartan, azilsartan, e.g. azilsartan

) Dual angiotensin receptor blockers (dual ARBs), Angiotensin Converting Enzyme (ACE) inhibitors, ACE-2 activators, renin inhibitors, pro-renin inhibitors, Endothelin Converting Enzyme (ECE) inhibitors, endothelin receptor (ET1/ETA) blockers, endothelin antagonists, diuretics, aldosterone antagonists, aldosterone synthase inhibitors, α -receptor blockers, antagonists of α -2 adrenergic receptors, β -receptor blockers, mixed α -receptor/β -receptor blockers, calcium antagonists, Calcium Channel Blockers (CCB), nasal formulations of the calcium channel blocker diltiazem (e.g., CP-404), dual mineralocorticoids/CCB, centrally acting antihypertensive agents, inhibitors of neutral endopeptidase, aminopeptidase-a inhibitors, vasopeptide inhibitors, dual vasopeptide inhibitors such as neprilysin-ACE inhibitors or neprilysin-ECE inhibitors, dual receptor-ecen inhibitors, dual angiotensin converting enzyme (AT) inhibitors, dual Angiotensin Converting Enzyme (ACE) inhibitors, renin antagonists, renin inhibitors, angiotensin converting enzyme (as), angiotensin converting enzyme (AT) inhibitors, angiotensin converting enzyme (as), angiotensin converting enzyme inhibitors, angiotensin converting enzyme (aat) inhibitors, angiotensin converting enzyme (e) inhibitors, angiotensin converting enzyme (e) inhibitors

(ee) suitable combinations thereof.

In certain embodiments, the additional anti-diabetic treatment is a GLP-1 treatment (e.g., lixisenatide). In certain embodiments, GLP-1 therapy is formulated with methionine (e.g., L-methionine or D-methionine). In certain embodiments, polysorbates (e.g., polysorbate 20, polysorbate 80), poloxamers (e.g., poloxamer 188), benzalkonium chloride, histidine, lysine, and/or EDTA are absent or substantially absent from the GLP-1 therapeutic formulation. In certain embodiments, the GLP-1 therapeutic formulation is free or substantially free of surfactants such as polyols (e.g., polypropylene glycol, polyethylene glycol, poloxamers, Pluronics, Tetronics), portions of polyols, and fatty acid esters and ethers such as those of glycerol and sorbitol (e.g., span.rtm., tween.rtm., myrj.rtm., brij.rtm., cremophor.rtm.). Formulations for GLP-1 treatmentComprises a suitable preservative (e.g., phenol, m-cresol, benzyl alcohol and/or paraben) and a suitable tonicity modifier (e.g., glycerol, dextrose, lactose, sorbitol, mannitol, glucose, NaCl, calcium or magnesium compounds such as CaCl)₂). The concentration of glycerol, dextrose, lactose, sorbitol, mannitol and glucose is typically in the range of 100-250mM, with NaCl concentrations up to 150 mM.

In certain embodiments, the insulin treatment received by the patient is combined with an additional anti-diabetic treatment (e.g., any of the aforementioned anti-diabetic treatments that are not insulin treatments). For example, in certain embodiments, the anti-diabetic treatment comprises a combination of insulin treatment (e.g., insulin glargine) and GLP-1 treatment (e.g., lixisenatide). These treatments may be provided alone or in a single pharmaceutical composition. For example, insulin glargine and lixisenatide may be formulated in a single pharmaceutical composition (e.g.,

100/33) for daily injections.

In the context of this method, one or more additional therapeutically active components, e.g., any of the agents listed above or derivatives thereof, may be administered prior to, concurrently with, or shortly after the administration of the PCSK9 inhibitor; (for the purposes of this disclosure, such administration regimens are considered to be "in combination with" the additional therapeutically active component "administering the PCSK9 inhibitor). The methods of the present invention include pharmaceutical compositions and methods of use thereof, wherein the PCSK9 inhibitor is co-formulated with one or more additional therapeutically active components as described elsewhere herein.

Administering a PCSK9 inhibitor as an add-on therapy

The treatment methods of the invention include treating a patient with hypercholesterolemia and diabetes with a PCSK9 inhibitor (such as an antibody or antigen-binding fragment thereof that specifically binds PCKS 9), wherein the PCSK9 inhibitor can be administered as an add-on therapy to the patient's pre-existing insulin therapy and/or LMT (if applicable), such as an add-on therapy to the patient's pre-existing daily therapeutic insulin and/or statin regimen.

For example, the methods comprise an additive treatment regimen wherein the PCSK9 inhibitor is administered as an additive treatment to the same stable multiple daily insulin treatment regimen and/or daily treatment statin regimen (i.e., the same dosage of statins) that the patient received prior to receiving the PCSK9 inhibitor. In other embodiments, the PCSK9 inhibitor is administered as an add-on therapy to a therapeutic insulin and/or statin regimen that comprises a greater or lesser amount of insulin and/or statin than the dose of insulin and/or statin that the patient received prior to receiving the PCSK9 inhibitor. For example, after initiation of a treatment regimen comprising a PCSK9 inhibitor administered at a particular dosing frequency and dosage, the daily dose of insulin and/or statin administered or prescribed to the patient may be (a) maintained, (b) increased, or (c) decreased (e.g., up-titrate or down-titrate) as compared to the daily statin dose taken by the patient prior to initiation of a PCSK9 inhibitor treatment regimen, depending on the therapeutic needs of the patient.

Therapeutic efficacy

The method results in a reduction in serum levels of one or more lipid components selected from the group consisting of: LDL-C, ApoB100, non-HDL-C, total cholesterol, VLDL-C, triglycerides, lp (a), HDL-C, LDL particle count, LDL particle size, ApoC3, ApoA-1, triglyceride-rich lipoprotein cholesterol (TRL-C), and residual cholesterol. According to certain embodiments, administration of a pharmaceutical composition comprising a PCSK9 inhibitor to a patient results in a reduction in the average percentage from baseline of serum low density lipoprotein cholesterol (LDL-C) of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or higher; a reduction in average percentage from baseline of ApoB of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more; a reduction in average percentage from baseline of ApoB100 of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or higher; at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or higher from the average percentage of baseline non-HDL-C; at least about 10%, 15%, 20%, 25%, 30%, 35%, 40% or higher from the mean percentage of baseline total cholesterol; a reduction in mean percentage from baseline of VLDL-C of at least about 5%, 10%, 15%, 20%, 25%, 30% or more; a reduction in average percentage from baseline of triglycerides of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or higher; an average percentage reduction from baseline of LDL particle number of at least about 20%, 25%, 30%, 35%, 40%, 45%, 50% or higher; an average percent reduction from baseline of LDL particle size of at least about 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% or more; a reduction of at least about 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 9.0%, 10% or more from the average percentage of baseline of apolipoprotein C3(ApoC 3); an average percentage increase from baseline of HDL-C of at least about 1%, 2%, 3%, 4%, 5% or higher; an average percentage increase from baseline for ApoA-1 of at least about 1%, 2%, 3%, 4%, 5% or more; a reduction in mean percentage from baseline of TRL-C of at least about 5%, 10%, 15%, 20%, 25%, 30% or higher; and/or a reduction of at least about 5%, 10%, 15%, 20%, 25% or more from the average percentage of the baseline of lp (a).

The methods of the invention comprise treating a patient with hypercholesterolemia and T1DM receiving insulin treatment comprising administering to the patient multiple doses of an anti-PCSK 9 antibody or antigen-binding fragment thereof in an amount of about 75 to 150mg per dose, and at a dosing frequency of about once every two weeks or once every four weeks, or according to a dosing regimen of an up-titration dosing regimen disclosed herein. After about 8, 10, 12, 14, 16, 18, 20, 22, 24 or more weeks of treatment with an anti-PCSK 9 antibody, the patient may exhibit at least a 35%, 50% or 60% reduction in LDL-C levels from baseline. In certain embodiments, after one or more weeks of treatment with the anti-PCSK 9 antibody, the patient exhibits a decrease in LDL-C levels from baseline of about 35%, 50%, or 60% or more.

The methods of the invention further comprise treating a patient with hypercholesterolemia and T2DM receiving insulin treatment comprising administering to the patient multiple doses of an anti-PCSK 9 antibody or antigen-binding fragment thereof in an amount of about 75 to 150mg per dose, and at a dosing frequency of about once every two weeks or once every four weeks, or according to a dosing regimen of an up-titration dosing regimen disclosed herein. After about 8, 10, 12, 14, 16, 18, 20, 22, 24 or more weeks of treatment with an anti-PCSK 9 antibody, the patient may exhibit a reduction in LDL-C levels from baseline of at least 40%, 48%, or 54%. In certain embodiments, the patient exhibits a decrease in LDL-C levels from baseline of about 40%, 48%, or 54% or more after one or more weeks of treatment with the anti-PCSK 9 antibody.

As disclosed herein, the methods of the invention do not alter the diabetes parameters of the patient. For example, in certain embodiments, the method does not affect (e.g., does not change by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) the patient's hemoglobin A1c (HbA1c) level. In certain embodiments, the method does not affect (e.g., does not change by more than 2%, 4%, 6%, 8%, 10%, 12%, 15%, 18%, or 20%) the fasting blood glucose (FPG) level of the patient.

In further embodiments, the invention relates to the use of an antibody or antigen-binding fragment thereof that specifically binds human proprotein convertase subtilisin/kexin type 9 (PCSK9) for the treatment of hypercholesterolemia in a patient suffering from type 1 diabetes (T1 DM).

In yet further embodiments, the present invention relates to methods of treating hypercholesterolemia in a patient suffering from type 1 diabetes (T1 DM).

In one embodiment, the use and/or method comprises the steps of:

(a) selecting a high cardiovascular risk patient for insulin treatment having

(i) T1DM, and

(ii) treatment of poorly controlled hypercholesterolemia with maximally tolerated statins; and

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

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

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

In one embodiment of the use and/or method, the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs shown in

SEQ ID NOs

2, 3 and 4, and three light chain CDRs shown in

SEQ ID NOs

7, 8 and 10.

In one embodiment of the use and/or method, the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having the amino acid sequence of SEQ ID NO:1 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID NO: 6.

In one embodiment of the use and/or method, the antibody or antigen binding fragment thereof is selected from the group consisting of: alikumab, efuzumab, bococizumab, ludwizumab, ralpancizumab and LY 3015014.

In one embodiment of the use and/or method, the antibody or antigen-binding fragment thereof is aleucizumab.

In one embodiment, the use and/or method further comprises the steps of:

(c) administering one or more of the following doses of the antibody or antigen-binding fragment thereof to the patient about every two weeks if the level of LDL-C in the patient is below a threshold level, or about every two weeks if the level of LDL-C in the patient is greater than or equal to the threshold level, 150mg of the antibody or antigen-binding fragment thereof about every two weeks.

In one embodiment, the use and/or method further comprises the steps of:

(c) administering to the patient one or more of the following doses of 300mg of the antibody or antigen-binding fragment thereof about every four weeks if the level of LDL-C in the patient is below a threshold level, or about every two weeks of one or more of the following doses of 150mg of the antibody or antigen-binding fragment thereof if the level of LDL-C in the patient is greater than or equal to the threshold level.

In one embodiment of said use and/or method, said threshold level is 70 mg/dL.

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

In one embodiment of the use and/or method, the patient further receives concomitant Lipid Modification Therapy (LMT).

In one embodiment of the use and/or method, the LMT is selected from the group consisting of: statins, cholesterol absorption inhibitors, fibrates, nicotinic acids, omega-3 fatty acids, and bile acid sequestrants.

In one embodiment of the use and/or method, the LMT is a statin therapy.

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

In one embodiment of the use and/or method, the statin treatment is the most tolerated statin treatment.

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

In one embodiment of the use and/or method, the patient is intolerant to statins.

In one embodiment of the use and/or method, the insulin treatment is selected from the group consisting of: human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degluvium, insulin aspart and basal insulin.

In one embodiment of the use and/or method, the patient receives concomitant anti-diabetic therapy in addition to insulin therapy.

In one embodiment of the use and/or method, the additional concomitant antidiabetic treatment is selected from the group consisting of glucagon-like peptide 1(GLP-1) treatment, gastrointestinal peptides, glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, ghrelin antagonists or inverse agonists, xenin analogs, biguanides, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, α -glucosidase inhibitors, sodium-dependent glucose transporter 2(SGLT-2) inhibitors, SGLT-1 inhibitors, peroxisome proliferator-activated receptor (PPAR-) (α, γ or α/γ) agonists or modulators, amylin analogs, G protein coupled receptor 119 (GPR) agonists, GPR40 agonists, GPR120 agonists, GPR142 agonists, systemic or low absorption TGR5 agonists, agonists for the immunotherapy of diabetes, anti-inflammatory therapies, agonists for the metabolism disorder, modulators of the G protein coupled receptor 119 (GPR), GPR40 agonists, GPR120 agonists, GPR142 agonists, GPR 82 agonists, inhibitors of glucose-acyl kinase, glucose-acyl-acyltransferase agonists, inhibitors of glucose-mediated kinase, combinations thereof, and inhibitors of glucose-acyl-Acyltransferase (AMPK) inhibitors.

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

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

In one embodiment of the use and/or method, the antibody or antigen-binding fragment thereof reduces the apolipoprotein C3(ApoC3) level of the patient.

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

In one embodiment of the use and/or method, the antibody or antigen-binding fragment thereof:

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

(b) Does not affect fasting blood glucose (FPG) levels in the patient.

In further embodiments, the present invention relates to the use and/or method for treating hypercholesterolemia in a patient suffering from type 1 diabetes (T1DM), the method comprising the steps of:

(a) selecting a high cardiovascular risk patient for insulin treatment having

(i) T1DM, and

(ii) treatment of poorly controlled hypercholesterolemia with maximally tolerated statins;

In a further embodiment, the present invention relates to a method for treating hypercholesterolemia in a patient suffering from type 2 diabetes (T2DM), the method comprising the steps of:

(a) selecting a high cardiovascular risk patient for insulin treatment having

(i) T1DM, and

SEQ ID NOs

2, 3 and 4, and three light chain CDRs shown in

SEQ ID NOs

7, 8 and 10.

In one embodiment of the use and/or method, further comprising the steps of:

In one embodiment of said use and/or method, said threshold level is 70 mg/dL.

In one embodiment of the use and/or method, the LMT is a statin therapy.

In one embodiment of the use and/or method, the additional antidiabetic treatment is selected from the group consisting of glucagon-like peptide 1(GLP-1) treatment, gastrointestinal peptides, glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, ghrelin antagonists or inverse agonists, xenin analogs, biguanides, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, α -glucosidase inhibitors, sodium-dependent glucose transporter 2(SGLT-2) inhibitors, SGLT-1 inhibitors, peroxisome proliferator-activated receptor (PPAR-) (α, γ or α/γ) agonists or modulators, amylin analogs, G protein-coupled receptor 119 (GPR) agonists, GPR40 agonists, GPR120 agonists, GPR142 agonists, systemic or low absorption TGR5 agonists, agonists for diabetes immunotherapy, anti-inflammatory and metabolic syndrome therapies, activators, GPR40 agonists, GPR120 agonists, GPR142 agonists, DG-mediated inhibitors of glucose-acyl kinase, glucose-acyl kinase inhibitors, glucose-acyl-kinase inhibitors, and combinations thereof.

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

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

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

(b) Does not affect fasting blood glucose (FPG) levels in the patient.

In further embodiments, the present invention relates to the use and/or method for treating hypercholesterolemia in a patient suffering from type 2 diabetes (T2DM), the method comprising the steps of:

(a) selecting a high cardiovascular risk patient for insulin treatment having

(i) T2DM, and

(c) administering to the patient one or more of the following doses of 75mg of the antibody or antigen-binding fragment thereof about every two weeks if the level of LDL-C in the patient is less than 70mg/dL, or 150mg of the antibody or antigen-binding fragment thereof about every two weeks if the level of LDL-C in the patient is greater than or equal to 70mg/dL, wherein the antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO:1 and an LCVR having the amino acid sequence of SEQ ID NO:6, and wherein the patient receives concomitant insulin therapy.

In a further embodiment, the invention relates to the use of an antibody or antigen-binding fragment thereof that specifically binds human proprotein convertase subtilisin/kexin type 9 (PCSK9) for the treatment of hypercholesterolemia in patients suffering from type 2 diabetes (T2DM) and atherosclerotic cardiovascular disease (ASCVD).

In yet further embodiments, the invention relates to methods for treating hypercholesterolemia in a patient suffering from T2DM and ASCVD.

In one embodiment, the use and/or method comprises the steps of:

(a) selecting a high cardiovascular risk patient for insulin treatment having

(i)T2DM、

(ii) ASCVD and

(iii) treatment of poorly controlled hypercholesterolemia with maximally tolerated statins; and

In one embodiment of the use and/or method, the ASCVD is defined as Coronary Heart Disease (CHD), ischemic stroke, or peripheral arterial disease.

In one embodiment of the use and/or method, the CHD includes acute myocardial infarction, asymptomatic myocardial infarction, and unstable angina.

SEQ ID NOs

2, 3 and 4 and three light chain CDRs shown in

SEQ ID NOs

7, 8 and 10.

In one embodiment, the use and/or method further comprises the steps of:

In one embodiment of said use and/or method, said threshold level is 70 mg/dL.

In one embodiment of the use and/or method, the LMT is a statin therapy.

In one embodiment of the use and/or method, the statin treatment is a maximally tolerated dose statin treatment.

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

(b) Does not affect fasting blood glucose (FPG) levels in the patient.

In further embodiments, the present invention relates to the use and/or method for treating hypercholesterolemia in a patient suffering from type 2 diabetes (T2DM), the method comprising:

(a) selecting a high cardiovascular risk patient for insulin treatment having

(i)T2DM、

(ii) ASCVD and

(iii) treatment of poorly controlled hypercholesterolemia with maximally tolerated statins;

Examples

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

Example 1: generation of human antibodies against human PCSK9

Human anti-PCSK 9 antibodies were generated as described in U.S. patent No. 8,062,640. An exemplary PCSK9 inhibitor for use in the following examples is a human anti-PCSK 9 antibody designated "mAb 316P," also referred to as "REGN 727" or "alikumab. mAb316P has the following amino acid sequence features: comprises the amino acid sequence of SEQ ID NO: 5 and a light chain comprising SEQ ID No. 9; a Heavy Chain Variable Region (HCVR) comprising SEQ ID NO 1 and a Light Chain Variable Region (LCVR) comprising SEQ ID NO 6; heavy chain complementarity determining region 1 comprising SEQ ID NO:2 (HCDR1), HCDR2 comprising SEQ ID NO:3, HCDR3 comprising SEQ ID NO:4, light chain complementarity determining region 1 comprising SEQ ID NO:7 (LCDR1), LCDR2 comprising SEQ ID NO:8, and LCDR3 comprising SEQ ID NO: 10.

Example 2: randomized, double-blind, placebo-controlled, parallel-group study to evaluate efficacy and safety of aleucizumab in insulin-treated patients with type 1 or type 2 diabetes and with hypercholesterolemia with high cardiovascular risk that is not adequately controlled at the time of maximally tolerated LDL-C lowering therapy

Introduction to

More than 3.8 million people worldwide suffer from diabetes, most of which die from cardiovascular disease (CVD). Those with diabetes are at higher risk of CVD, have associated clinical complications and are at an earlier age, and have a life expectancy that is shortened by about 6 to 7 years, compared to persons without diabetes. In addition to the high human cost of the disease, CVD contributes significantly to the overall medical expenditure for these patients.

The study, designated Odyssey DM-insulin, included adult patients with type 1 or type 2 diabetes in insulin therapy with hypercholesterolemia at high Cardiovascular (CV) risk that was not adequately controlled at maximal tolerated doses of statin therapy with or without other Lipid Modification Therapy (LMT).

Purpose of study

The primary objectives of this study were: (a) evaluating the efficacy of aleuromab to reduce calculated low density lipoprotein cholesterol (LDL-C) after 24 weeks of treatment in a high cardiovascular risk patient with insulin-treated diabetes and with hypercholesterolemia that is not well controlled at the time of maximally tolerated LDL-C reduction treatment compared to placebo; and (b) evaluating the safety and tolerability of aleutizumab in patients with insulin-treated diabetes.

The secondary objective of this study was to evaluate the efficacy of aliskirumab on other lipid parameters (e.g., measured LDL-C, non-high density lipoprotein cholesterol (non-HDL-C), apolipoprotein b (Apo b), Total Cholesterol (TC), lipoprotein a (lp (a)), high density lipoprotein cholesterol (HDL-C), Triglyceride (TG) levels, triglyceride rich lipoprotein (TGRL), apolipoprotein a-1(Apo a-1), apolipoprotein C3(ApoC3), and LDL particle number and size) compared to placebo at

weeks

12 and 24.

Design of research

This is a phase 3b randomized, double-blind, placebo-controlled multinational and multicenter study to evaluate the efficacy and safety of aleucizumab administered by Subcutaneous (SC) injection in patients at high CV risk and with type 1 or type 2 diabetes and insulin treatment with hypercholesterolemia that is not adequately controlled by maximally tolerated LDL-C lowering therapy. The study consisted of a screening period of up to 3 weeks, a 24-week double-blind treatment period, and a safety observation period lasting 8 weeks after the end of the double-blind treatment period.

Unless patients are intolerant to statins, they take a stable, maximally tolerated dose of statin therapy with or without other Lipid Modification Therapy (LMT). The statin doses and dosage regimens, as well as the dose and dosage regimen of one or more other lipid-modifying therapies, if applicable, were stable throughout the study period (including 4 weeks prior to the screening period, during the screening period, and from screening to randomized assignment). Throughout the study from screening to week 24 visit (visit), patients were on a glucose and lipid regulated stable diet. Patients are receiving diabetes treatment according to local/regional standards of care.

Patients are stratified by type of diabetes (i.e., type 1 diabetes versus type 2 diabetes). Recruitment of patients with type 2 diabetes was completed when approximately 400 patients were randomly assigned. At the end of the targeted recruitment period, recruitment of patients with type 1 diabetes was completed.

If LDL-C was ≧ 70mg/dL (1.81mmol/L) at visit week 8, alikumab was administered subcutaneously at a starting dose of 75mg Q2W for 12 weeks, with a blind titration up-titration to alikumab 150mg Q2W at week 12. Patients with LDL-C <70mg/dL (1.81mmol/L) at visit week 8 continued to use alirocumab 75mg Q2W until the end of the treatment period.

Data from lipid parameters of blood samples were masked (mask) after random assignment. At the investigator's discretion, no attempt was made by either the investigator or the patient to independently evaluate the patient's lipid values after randomized assignment until after week 24 visits, except for patient safety.

Patients visited the study site at weeks-3, 0,8, 12, 20 and 24 with laboratory work at each visit. In addition, telephone calls were also made on

weeks

4 and 32.

Adverse Events (AEs) occurred within 70 days of the last dose of the study pharmaceutical product (IMP) were recorded. Patients with Severe Adverse Events (SAE) or adverse events of particular interest (AESI) were followed (follow up) until resolved, stable or dead.

Patient selection

A total of 517 patients were enrolled in the study, including 76 patients with T1DM and 441 patients with T2 DM.

Inclusion criteria

Patients enrolled in the study met all of the following criteria:

(1) a patient with type 1 or type 2 diabetes treated with insulin and having a level ≧ 70mg/dLLDL-C (1.81mmol/L) insufficiently controlled with or without other LMTs by a stable, maximal dose/schedule of statins that were tolerated by the patient for at least 4 weeks prior to the screening visit (week-3). The statin of the maximum dose/regimen tolerated by the patient is the dose/regimen tolerated by the registered patient based on the judgment of the researcher or attention. Some examples of acceptable causes for patients taking lower statin doses include, but are not limited to, adverse effects on higher doses, advanced age, low Body Mass Index (BMI), regional practices, local prescription information, or concomitant medications. The patient may have taken an alternate daily dose of statin as long as the dose is taken consistently (e.g., once a week, wednesday, friday, etc.). Concomitant treatment with more than 1 statin is not allowed. Patients with documented intolerance and therefore no longer receiving statin therapy are also eligible for this study at the discretion of the investigator. One or more reasons for not performing the maximum dose/regimen for statins (including statin intolerance) are recorded in the form of a medical report.

(2) Patients with age greater than or equal to 18 years or adult legal age at the time of the screening visit are selected, whichever is greater.

(3) Patients with type 1 or type 2 diabetes were diagnosed at least one year prior to the screening visit (week-3). Patients diagnosed with type 1 diabetes need to meet all of the following criteria:

(a) diagnosis before the age of 30 years;

(b) treatment with multiple daily injection regimens/basal-dietary insulin regimens or insulin pump regimens within 6 months after diagnosis; and

(c) c peptide <0.2pmol/mL at the time of the screening visit.

(4) Glycosylated hemoglobin (HbA1c) < 10% at screening visit (week-3). Patients with elevated HbA1c (up to 10%) were eligible if they were not scheduled for lower HbA1c during the study at the discretion of the investigator.

(5) Patients with a recorded history of CVD (including CHD and/or CHD risk equivalents) and/or at least one additional CV risk factor.

The medical history of CHD includes at least one of:

(a) acute Myocardial Infarction (MI);

(b) asymptomatic MI;

(c) unstable angina pectoris;

(d) coronary revascularization procedures (e.g., Percutaneous Coronary Intervention (PCI) or Coronary Artery Bypass Graft (CABG)); and

(e) clinically significant CHD is diagnosed by invasive or non-invasive tests (e.g., coronary angiography, pressure testing with a treadmill, stress echocardiography, or nuclear imaging).

CHD risk equivalents include at least one of:

(a) the recorded peripheral artery disease meets at least one of the following criteria:

(i) current intermittent claudication (lower limb muscular discomfort, which is reproducible and produced by exercise and relieved by rest within 10 minutes) (its origin is presumed to be atherosclerosis) with ankle-brachial index (ankle-brachial index) of either leg ≦ 0.90 at rest;

(ii) history of intermittent claudication (lower limb muscular discomfort, reproducible and produced by exercise and relieved by rest within 10 minutes) and at the same time intravascular procedures or surgical interventions in one or both legs due to atherosclerotic disease; and

(iii) a history of critical limb ischemia and at the same time thrombolytic, endovascular procedures or surgical intervention in one or both legs due to atherosclerotic disease; and

(b) a prior ischemic stroke was recorded with focal ischemic neurological deficits lasting more than 24 hours, the origin of which was considered atherosclerosis. Computed tomography or magnetic radio imaging must be performed to exclude bleeding and non-ischemic neurological diseases.

Cardiovascular risk factors include at least one of:

(a) hypertension (established on antihypertensive drugs);

(b) a current smoker;

(c) males up to 45 years old and females up to 55 years old;

(d) history of microalbuminuria/macroalbuminuria;

(e) a history of diabetic retinopathy (pre-proliferative or proliferative);

(f) family history of early onset CHD (father or brother before age 55; mother or sister before age 65);

(g) low HDL-C (male <40mg/dL (1.0mmol/L), female <50mg/dL (1.3 mmol/L)); and

(h) recorded eGFR of 15 ≦ eGFR lasting 3 months or longer (including screening visits)<60mL/min/1.73m²Limited Chronic Kidney Disease (CKD).

(6) Signed written informed consent.

Exclusion criteria

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

(1) exclusion criteria related to the study method:

(a) plan to start a new LMT or modify the dose of the current LMT during the study;

(b) no stable dose of LMT (including statin or other LMT) was performed until the screening visit (week-3) lasted at least 4 weeks or from screening to the randomized assignment unless the statin was intolerant, in which case statin treatment was not performed 4 weeks prior to/during the screening visit;

(c) nutraceuticals or over-the-counter medications that may affect lipids were not used at stable doses for at least 4 weeks prior to the screening visit (week-3) or between the screening and random assignment visits;

(d) use of red yeast rice products within 4 weeks of the screening visit (week-3) or between the screening and random assignment visits;

(e) systemic corticosteroids are used unless a stable regimen is used as an alternative treatment for pituitary/adrenal disorders for at least 6 weeks prior to randomization. Topical, intra-articular, nasal, inhalation, and ophthalmic steroid treatments are not considered "systemic" and are allowed;

(f) continuous hormone replacement therapy was used unless the regimen was already stable in the 6 weeks prior to the screening visit (week-3) and no schedule change of regimen during the study was planned;

(g) recent MI (within 3 months prior to the screening visit (week-3) or between the screening and randomly assigned visits), unstable angina resulting in hospitalization, uncontrolled arrhythmia, CABG, PCI, carotid surgery or stenting, stroke, Transient Ischemic Attack (TIA), intravascular procedure or surgical intervention for peripheral vascular disease;

(h) scheduled PCI, CABG, carotid or peripheral revascularization procedures were planned during the study;

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

(j) systolic pressure >180mmHg or diastolic pressure >110mmHg at screening or random assignment visit;

(k) patients who had received plasmapheresis treatment or were scheduled to receive plasmapheresis treatment within 2 months prior to the screening visit (week-3), between screening and randomized assignment;

(l) A known history of hemorrhagic stroke;

(m) a known history of loss of function (i.e., genetic mutation or sequence variation) of PCSK9 or a known history of homozygous familial hypercholesterolemia;

(n) active progression of new cancers or carcinomas within the past 5 years, in addition to adequately treated basal cell skin carcinoma, squamous cell skin carcinoma or cervical carcinoma in situ;

(o) a known medical history of a positive HIV test;

(p) patients who have taken any effective investigational drug within 1 month or 5 half-lives (whichever is longer);

(q) patients who had not previously received a cholesterol lowering diet instruction prior to the screening visit (week-3);

(r) patients who withdrawn consent during screening (starting from signed ICF);

(s) variable unstable body weight defined as >5kg as judged by the investigator within 2 months prior to the screening visit;

(t) BMI >45kg/m2 or plan to perform bariatric surgery, bariatric plan or start bariatric medication during the course of the study;

(u) recent start of weight loss medication (i.e., within 3 months prior to screening visit or between screening and random assignment) or recent weight loss surgery (within the past 6 months) and within an effective weight loss phase as judged by the investigator;

(v) patients who did not receive insulin treatment for at least 6 months prior to the screening visit or who did not undergo a stable insulin regimen for at least 3 months prior to the screening visit (i.e. change in insulin type, general timing/frequency of injection, mode or mode of administration such as basal (type 2 diabetes only), basal-meal, etc.), or the possibility of needing to change insulin type/frequency or mode of injection during the study;

(w) no stable insulin dose (i.e., more than 30% change in total daily insulin dose as judged by the researcher) for at least 3 months prior to screening, or the possibility of needing to reinforce an insulin/antihyperglycemic regimen during the course of the study as judged by the researcher (e.g., addition of new medications, schedule of titrating insulin doses, etc.);

(x) Other antihyperglycemic drugs administered by patients are unstable for at least 3 months prior to the screening visit;

(y) recent history of diabetes decompensation within 2 months prior to the screening visit (i.e. diabetic ketoacidosis or Hypertonic Hyperglycemic State (HHS));

(z) receiving or planning to receive renal replacement therapy (e.g., hemodialysis, renal transplantation, etc.) during the study;

(aa) there are any clinically significant uncontrolled endocrine diseases known to affect serum lipids or lipoproteins. Patients undergoing thyroid replacement therapy can be included if the dose of thyroxine is stable for at least 3 months prior to screening and the patient's sensitive thyroid stimulating hormone (s-TSH) level at the time of screening visit is within ± 10% of the normal range for the laboratory;

(bb) laboratory findings during screening (excluding randomly assigned laboratories, except pregnancy tests):

(i) serum TG >400mg/dL (4.52mmol/L) (allow 1 replicate laboratory);

(ii) positive serum or urine pregnancy test in women with fertility potential;

(iii) positive test for hepatitis b surface antigen or hepatitis c antibody;

(iv) eGFR <15mL/min/1.73m2 according to the 4 variable renal disease dietary Modification (MDRD) equation;

(v) ALT or AST >3 × ULN (1 replicate laboratory allowed); or

(vi) Creatine Phosphokinase (CPK) >3 × ULN (allowed 1 replicate laboratory); or

(cc) conditions/circumstances such as:

(i) patients with a short life expectancy;

(ii) the need for concomitant therapies that may deviate from the original assessment;

(iii) failure to meet specific protocol requirements (e.g., need for hospitalization, ability to make study visits, etc.);

(iv) the patient is a researcher or any co-researcher, research assistant, pharmacist, research coordinator, other staff or their relatives who is directly involved in performing the protocol;

(v) uncooperative or any situation that may cause the patient to be out of compliance with the study procedure;

(vi) any technical/administrative reasons that prevent patients in the study from being randomly assigned; or

(vii) Any clinically significant abnormalities identified at the time of screening that would preclude safe completion of the study or limit endpoint assessment in the judgment of the investigator or secondary investigator, such as major systemic disease, short-lived patients.

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

(2) Exclusion criteria related to active comparator (active comparator) and/or mandatory background therapy: all contraindications for background treatment or use of warning/preventive measures (as appropriate) shown in the corresponding national product label.

(3) Exclusion criteria associated with current knowledge of aliskirumab:

(a) allergy to aliskirumab or to any component of aliskirumab;

(b) pregnant or breast-fed women;

(c) women with fertility potential (in case of specific local requirements, as defined in the ICF and/or local protocol annex) and/or women who are unwilling or unable to undergo pregnancy tests, not protected by the high-efficiency fertility control method. Women with fertility potential must have a negative pregnancy test confirmed at the time of screening and inclusion in the visit. They must use an effective contraceptive method throughout the study treatment and for at least 10 weeks after the last IMP injection. The contraceptive method employed must meet the criteria for an efficient fertility control method according to "International Conference on harmony of technical requirements for Registration of Pharmaceuticals for Human uses. M3(R2): guiding on non-clinical safety endpoints for the connection of Human clinical practices and marking administration for pharmaceutical uses. ICH.2009jun: 1-25". Postmenopausal women must block menses for at least 12 months.

Study treatment

Pharmaceutical product for research

Sterile aliskirumab drug product is provided in an auto-injector (also known as a prefilled pen) in an aqueous buffer (pH 6.0) containing sucrose, histidine and polysorbate 20 at concentrations of 75mg/mL and 150mg/mL, both in 1mL volumes. Sterile placebo of alilimumab was prepared in 1mL volumes in the same formulation as alilimumab without the addition of protein in prefilled pens, for patients for injection training, and for those patients in the placebo treated group (arm). During screening, the patient (or another designated person) must undergo placebo self-injection training using a prefilled pen prior to the first administration of IMP.

For patients randomly assigned to alirocumab, the initial dose was 75mg administered subcutaneously Q2W once. If the LDL-C value at week 8 is ≧ 70mg/dL (1.81mmol/L), the dose is increased blindly to 150mg Q2W at week 12 for patients randomized to alikumab. Patients randomized to placebo were administered their injections subcutaneously throughout the 24 week treatment period of Q2W.

Route of administration and method

A pre-filled pen training guide (auto-injector training guide) is provided to the site and instructions for use (using the auto-injector) are provided to the patient. Each administration of IMP consisted of a 1mL subcutaneous injection in the outer region of the abdomen, thigh or upper arm (i.e., deltoid zone). If additional concomitant medications are injected at the same site planned for IMP injection, the patient is advised to use an alternate site to administer IMP.

IMP may be administered by self-injection or by another designated person (e.g., spouse, relative, etc.). If a given person should inject the patient with aliskirumab during the study, it should be ensured that the person is adequately trained prior to administering the injection. Anyone who plans to administer IMP is trained by researchers.

Instructions are provided to the patient (or another designated person (e.g., spouse, relative, etc.) to whom the injection is to be administered) during training and as needed during the course of the study. Close supervision and feedback is given at the first visit and, as needed, at other visits.

The used prefilled pen is discarded in a sharps container provided to the patient. It is recommended to take subcutaneous IMP injections in turn in the anatomical region (e.g., right thigh, then left thigh or right abdomen, then left abdomen). During the study, the patient also had the option of injecting in different anatomical areas (e.g., thigh then abdomen, or outer area of upper arm, etc.).

The patient is asked to store IMP in a refrigerator. The IMP should be placed in an external, safe location for about 30 to 40 minutes at room temperature prior to administration. Thereafter, IMP should be administered as soon as possible.

Timing of administration

During screening, the patient or designated person must undergo placebo self-injection training using a pre-filled pen prior to the first IMP injection.

At the time of the random assignment visit, the patient or another designated person (such as a spouse, relative, etc.) makes the first IMP injection on site under direct on-site staff supervision. Patients were monitored at the study site for at least 30 minutes after the first injection in the study. If the assigned person is changed during the study, the newly assigned person will be trained using placebo.

IMP subcutaneous injections, Q2W, were then performed outside the clinic until the last injection. If the injection is scheduled to occur on the same day of a site visit, the IMP is performed after the blood sampling is complete. In exceptional cases, this is also allowed if the patient prefers injections at the study site and can make provisions to accommodate administering injections on site.

IMP should be administered Q2W subcutaneously, ideally at approximately the same time of day. However, a window period of ± 3 days is acceptable. The time of day is based on the preference of the patient.

If an injection is delayed more than 7 days from the missed date or missed completely, either by mistake or by other circumstances, the patient is asked to return to the original schedule of IMP administration without administering a delayed injection. If the injection is delayed less than or equal to 7 days from the missed date, either by mistake or by other circumstances, the patient is asked to administer a delayed injection and then the original schedule of IMP administration is restarted.

Non-investigational drug

The following classes of drugs were identified as non-IMP because the drugs are background treatments or potential rescue drugs:

(a) a statin;

(b) cholesterol absorption inhibitors (ezetimibe);

(c) bile acid binding chelators (e.g., cholestyramine, colestipol (colestipol), colesevelam (colesevelam));

(d) nicotinic acids;

(e) fibrates (such as fenofibrate);

(f) omega-3 fatty acids (greater than or equal to 1000mg per day); and

(g) insulin.

For background LMTs, including statins, national product labels are followed on-site for patient safety monitoring and management. During the study, patients received stable, patient-tolerated maximum dose/regimen of statin therapy with or without other LMTs. The lipid spectra values were obtained by blind methods from samples obtained after random assignment. However, for safety reasons, the field is made to notify TG alarms with the aim of making decisions on the patient's background LMT.

From the screening visit (week-3) until week 24 visit, the background LMT did not change. During this time, no dose adjustments, stops or starts other statins or other LMTs are made unless all concerns (including but not limited to TG alarms issued by the central laboratory) overwhelmed by the discretion of the investigator warrant an exception to such a change. For TG alarms that have been confirmed by repeat testing, the researcher conducts a survey, manages the patient, and modifies the background LMT according to his/her medical judgment.

If the patient has tolerated the drug and maintains a stable dose, all fibrate is admitted. If the patient needs to introduce a fibrate during the study (i.e., as a rescue treatment in response to a TG alarm), the addition of fenofibrate is only allowed. Background LMT and insulin are provided by sponsors. Patients obtain these drugs according to local regulations.

Procedure for blind method

The alirocumab and placebo of alirocumab are provided in the same matched pre-filled pen and packaged identically, including a label to protect blindness. Each treatment kit is labeled with a number, which is generated by the sponsor's computer program. Treatment kit numbers were obtained by investigators at patient random assignment and subsequent patient visit via IVRS/IWRS schedule, which is available 24 hours a day, 7 days a week.

According to the double-blind design, study patients, researchers, and study field personnel remain blind to study treatment and have no access to random assignments (treatment codes) except as described below.

Adverse events

The treatment code was blinded by the pharmaco-alert department for reporting to the Health Authority (the Health Authority) any Suspected Unexpected Severe Adverse Reactions (SUSAR), i.e. severe adverse events that were unexpected (per the specific department of CIB) and reasonably associated with the use of IMP according to the discretion of the researcher and/or sponsor.

Lipid parameters

The lipid parameter values of blood samples obtained after a randomly assigned visit by the central laboratory were not communicated to the field, so that they could not infer the treatment group of their patients based on the obtained LDL-C levels. The sponsor's operations team cannot obtain lipid parameters relevant to patient identification until eventually a database lock occurs. For safety purposes, TG alarms with TG values ≧ 500mg/dL were sent to the investigator at any time after random assignment.

At the end of the double-blind treatment period (visit week 24), the investigators continued to manage the patient's lipids according to standard practice. Any lipid values after random assignment are written in the source file and are not shared with the sponsor.

anti-Alikumab antibodies

Patient anti-alirocumab antibody results were not communicated to the field while the study was ongoing. Until after final database lock occurs, the sponsor's operating team is unable to obtain anti-aleucirumab antibody results associated with the patient identification number. Laboratory technicians involved in determining patient anti-alikumab antibody titers were excluded from the operating team and established a method to prevent any potential blindness.

Randomly assigned code corruption during research

In the case of an AE, the password is destroyed in the case that knowledge of the IMP is required to treat the patient. Contact with the monitoring team/researcher is initiated, if possible, before the code is destroyed. All calls are recorded by the monitoring team, as appropriate, to include the date and time of the call, the name of the person contacted within the monitoring team, the patient ID, the requested documentation, and the decision whether to blind.

Code destruction may be performed at any time by using an appropriate module of an Interactive Voice Response System (IVRS)/interactive network response system (IWRS), depending on which system is used on-site, and/or by dialing any other telephone number provided by the sponsor for this purpose. However, prior to blinding a case, it is preferable to discuss the case in relation to the researcher. If the blinding method is corrupted, the researcher is asked to record the date, time, and reason for the code corruption and report this information on the appropriate page of the e-CRF. When the cause of the blindness was noted, the investigator did not provide any details about the nature of the IMP. The researcher does not reveal IMP details to the sponsor's representative or any staff until the database is closed. Further, when the table (e.g., AE, SAE) is completed, the study treatment is not disclosed on the table.

Code destruction material is also kept at the entity responsible for the "24 hour alarm system"; but the system should only be used in very exceptional cases (i.e., IVR/IWR systems cannot use or contact researchers and/or field workers). However, the preferred option is to use IVRS blindness. The availability of local code destruction material was reported to researchers by a clinical monitoring team. Each patient participating in the study was provided with a patient card including an associated "24 hour alarm system" telephone number. Sponsors are also allowed to blindly uncover some SAEs to comply with regulatory reporting requirements (i.e. for some SAEs that are both relevant and unexpected).

If the code is destroyed, the patient permanently stops IMP administration.

Method for assigning patients to treatment groups

A randomly assigned list of treatment kit numbers is generated centrally by the sponsor. IMP (aliskirumab 75 or 150mg kit, or placebo kit) was packaged according to this list.

The trial supply operations manager provides a randomly assigned list of treatment kit numbers and the research biometist provides a random assignment scheme to the centralized treatment distribution system provider. The centralized therapy distribution system provider then generates a patient randomized distribution list from which to distribute therapy to the patients.

During double-blind treatment, patients were randomly assigned to receive either placebo or aliskirumab. The random distribution ratio aliskirab to placebo was 2: 1. For each randomly assigned patient, there are several corresponding treatment kit numbers (re-visits provided) that are assigned by the centralized treatment distribution system. The random assignments were stratified by diabetes type (i.e., type 1 and type 2).

Treatment kit numbers were assigned using the centralized treatment assignment system at random assignment visits (day 1, week 0), then at week 12 as a resupply visit, and if necessary at a previously unscheduled visit.

For patients in the alirocumab treatment group, the treatment kit dispensed at week 12 was based on the patient's week 8 LDL-C level following the ascending titration rule. Scheduled data transfers between the central laboratory and the centralized treatment distribution system provider are planned with the aim of being performed blindly to the study site and the sponsor.

Before randomly assigning patients, the researcher or assignee must contact the centralized treatment assignment system.

Randomly assigned patients are defined as patients enrolled and assigned using the therapy kit number from the centralized therapy distribution system as recorded in their log file. Patients cannot be randomly assigned more than once during the study. If the treatment is used without contacting the centralized treatment distribution system, the patient is considered to have not been randomly distributed and withdrawn from the study.

Two types of centralized therapy distribution systems, IVRS and IWRS, are used, depending on the choice of site.

Packaging and label

For the double-blind treatment period, each double-blind treatment kit (aliskirumab or placebo to aliskirumab) was prepared to contain 6 prefilled pens in a package that the child could not open. To protect the blindness, all cases of double-blind treatment kits for injections have the same look and feel and are therefore labeled with double-blind labels.

In addition to the double-blind treatment kit for injection, a training kit containing 1 placebo prefilled pen of alirocumab was prepared for the purpose of instructing patients on administration of injections, which was performed prior to random assignment at the screening visit (week-3, first visit). A second injection training with placebo of aliskirumab was performed, if deemed necessary, prior to random assignment using an additional training kit. Injection training with placebo was performed and recorded in CRF, which included the situation if the designated person administering IMP to the patient changed during the study.

The package conforms to a predetermined schedule for administration. The contents of the tag comply with local regulatory specifications and requirements.

Storage condition and shelf life

Researchers or other authorized personnel (e.g., pharmacists) are responsible for storing IMPs in an insured and safe place according to local regulations, label specifications, policies, and procedures. Control of IMP storage conditions, particularly temperature control (e.g., refrigerated storage) and information about stability in use and instructions for handling IMPs are managed according to rules provided by the sponsor.

IMP is stored on site in refrigerators between +2 ℃ and +8 ℃ (36 ° F to 46 ° F). The temperature of the on-site refrigerator was checked daily and recorded on a log sheet. The IMP stored at the research site is kept in a properly locked room, subject to storage conditions identified on the tag by the researcher or designated personnel or other authorized personnel.

After supplying the IMP kit to the patient at the study site visit, appropriate provisions are made to transfer the IMP kit from the study site to the patient's refrigerator.

Study endpoint

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

End of primary efficacy

The primary efficacy endpoint was the percent change in LDL-C from baseline to week 24 in the intent-to-treat (ITT) population, using all LDL-C values regardless of whether treatment was followed (ITT was assessed). The percent change was defined as 100x (calculated LDL-C value at week 24-calculated LDL-C value at baseline)/calculated LDL-C value at baseline.

The baseline calculated LDL-C value was the last LDL-C level obtained before the first double-blind IMP injection. The calculated LDL-C at week 24 is the LDL-C level obtained within the week 24 analysis window. According to the above definition, all calculated LDL-C values between week 8 to week 24 (planned or unplanned, fasted or not) were allowed to be used, if appropriate, to provide values for the primary efficacy endpoint.

Primary safety endpoint

Safety parameters (AE, laboratory parameters, vital signs) were evaluated throughout the study. The observation of security data is as follows:

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

(b) treatment Emergency Adverse Event (TEAE) period was defined as the time from the first dose of double-blind IMP injection to the last dose of IMP injection +70 days (10 weeks) since the residual effect of treatment was expected until 10 weeks after the double-blind IMP was stopped; and

(c) the post-treatment period is defined as the time from the day after the end of the TEAE period until all SAE and AESI are resolved/stabilized, whichever comes later.

An AE is any unfortunate medical event in a patient administered a drug product or in a patient of a clinical study, and it does not necessarily have to have a causal relationship to this treatment.

SAE is any unfortunate medical event with the following characteristics, at any dose:

(a) leading to death;

(b) threatening life. The term "life threatening" in the definition of "severe" refers to an event in which a patient is at risk of death at the time of the event; if it is more severe, it does not refer to an event that is assumed to be likely to cause death;

(c) requiring hospitalization of hospitalized patients or extending existing hospitalization times;

(d) resulting in persistent or significant disability/incapacity;

(e) is a congenital abnormality/birth defect; or

(f) Is a medically important event.

In deciding whether urgent reporting is appropriate in other situations, such as important medical events that may not immediately be life threatening or result in death or hospitalization but may harm the patient or may require medical or surgical intervention (i.e., specific measures or corrective therapy), medical and scientific judgments are carried out to prevent one of the other outcomes listed in the above definitions.

The following list of medically significant events is intended to be used as a guide for determining which conditions must be considered medically significant events. This list is not exhaustive:

(a) intensive therapy in the emergency room or at home for allergic bronchospasm, cachexia (i.e., agranulocytosis, aplastic anemia, myelodysplasia, pancytopenia, etc.), or convulsions (seizures), epilepsy (epilepsy), seizures (epilepic fit, absence, etc.));

(b) the occurrence of drug dependence or drug abuse;

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

(d) suicidal ideation or any event suggestive of suicide;

(e) syncope, loss of consciousness (unless recorded as a result of blood sampling);

(f) bullous skin rash;

(g) cancer diagnosed during the study or exacerbated during the study;

(h) chronic neurodegenerative disease (newly diagnosed) or exacerbated during the study; and

(i) suspected of transmitting the infectious agent, if any suspected transmission of the infectious agent via a pharmaceutical product (e.g., product contamination).

Adverse events of particular interest (AESI) are AEs (severe or not) that need to be monitored, recorded and managed in a pre-specified manner. For this study, AESI was:

(a) increase of ALT: ALT ≧ 3 × ULN (if baseline ALT < ULN) or ALT ≧ 2 times the baseline value (if baseline ALT ≧ ULN);

(b) allergic events: allergic drug reactions and/or local injection site reactions are considered by the investigator as allergic (or having an allergic component), which requires a consultation with another physician to further evaluate hypersensitivity/allergy should be reported as AESI according to the investigator's medical judgment;

(c) pregnancy: pregnancy occurred in either the female patient or the male patient's partner (if allowed by the female partner and local regulatory authorities) during the study or within 70 days after the last dose of study medication. In all cases, pregnancy was recorded as AESI. Pregnancy is considered to be an SAE only if it meets one or more SAE criteria. In the case of pregnancy in female patients included in the study, study products were discontinued. Follow-up of pregnancy is mandatory until the outcome has been determined;

(d) symptomatic overdosing with IMP. Overdose (accidental or intentional) is an event suspected by the investigator or spontaneously notified by the patient (not counted based on systemic injections) and defined as at least two expected doses within an expected treatment interval (i.e., 2 or more injections administered within <7 calendar days), reported using the term "symptomatic overdose (accidental or intentional)", indicating conditions in parentheses (e.g., "symptomatic overdose (accidental)" or "symptomatic overdose (intentional)"). The patient is monitored and appropriate symptomatic treatment is established. The overdose condition is specified explicitly in the verbatim and symptoms, if any, are entered in a separate AE/SAE table. Asymptomatic overdose was required as a standard AE report;

(e) neurological events: neurological events requiring additional examinations/procedures and/or referrals to specialists are required as AESI reports. If the event does not require additional examination/procedures and/or referral to an expert, it is required to be reported as a standard AE; and

(f) neurocognitive events: all neurocognitive events were considered AESI.

Secondary efficacy endpoints

The key secondary endpoints of this study are as follows:

(a) calculated percentage change in LDL-C from baseline to week 24, using all LDL-C values during efficacy treatment (on-treatment estimun);

(b) percent change in measured LDL-C from baseline to week 24 (ITT assessed (ITT estimate));

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

(d) percent change in measured LDL-C from baseline to week 12 (ITT assessed);

(e) percent change from baseline to week 24 of non-HDL-C (ITT assessed);

(f) percentage change in Apo B from baseline to week 24 (ITT assessed);

(g) percent change in total cholesterol from baseline to week 24 (ITT assessed);

(h) proportion of patients reaching LDL-C <70mg/dL at week 24 (assessed in treatment);

(i) proportion of patients reaching LDL-C <50mg/dL at week 24 (assessed in treatment);

(j) proportion of patients who reached non-HDL-C <100mg/dL at week 24 (assessed in treatment);

(k) proportion of patients reaching non-HDL-C <80mg/dL at week 24 (assessed in treatment);

(l) Percent change in lp (a) from baseline to week 24 (ITT assessed);

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

(n) percent change in TG from baseline to week 24 (ITT assessed);

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

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

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

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

(b) absolute change in FPG from baseline to week 12 and 24 (ITT assessed and in treatment assessed);

(c) absolute change in total daily insulin dose from baseline to week 12 and 24 (ITT assessed and treatment assessed); and

(d) absolute change in number of glucose lowering treatments from baseline to week 12 and 24 (ITT assessed and in treatment assessed).

Other efficacy endpoints of the study included:

(a) calculated percentage change in LDL-C from baseline to week 12 (assessed in treatment);

(b) percent change in measured LDL-C from baseline to week 12 and 24 (assessed in treatment);

(c) percent change from baseline to week 12 (ITT and assessed in treatment) and to week 24 (assessed in treatment) of non-HDL, Apo B, total cholesterol, lp (a), HDL-C and TG;

(d) proportion of patients who reached a calculated LDL-C <50 and also <70mg/dL at week 12 (ITT assessed and assessed in treatment) and week 24 (ITT assessed);

(e) the proportion of patients with a 50% or more decrease in LDL-C compared to baseline (ITT assessed) calculated at week 12 and week 24;

(f) proportion of patients who reached a non-HDL-C <80mg/dL and also <100mg/dL at week 12 (ITT assessed and assessed in treatment) and week 24 (ITT assessed);

(g) proportion of patients who reached Apo B <80mg/dL at weeks 12 and 24 (ITT assessed and in-treatment assessed);

(h) percent change in LDL-C particle number and size from baseline to week 12 (ITT assessed and in treatment assessed) and week 24 (in treatment assessed);

(i) percent change in TGRL, Apo A-1 and Apo C-III from baseline to week 12 and to week 24 (ITT assessed and treatment assessed);

(j) absolute change in the ratios Apo B/Apo A-1 and TC/HDL-C from baseline to week 12 and to week 24 (ITT assessed and in-treatment assessed);

(k) proportion of patients who reached calculated LDL-C <70 and <50mg/dL at weeks 12 and 24 (ITT assessed and in-treatment assessed) according to baseline A1C < 8% or ≧ 8%; and

(l) The proportion of patients reaching calculated LDL-C <70mg/dL and <50mg/dL at week 12 and week 24 (ITT assessed and in-treatment assessed) according to baseline A1C < median A1C or ≧ median A1C.

Study procedure

The window period at week 0 was +3 days. The window period at week 8, week 12 and week 24 was ± 3 days. The window period at week 4, week 20 and week 32 was ± 7 days. For all visits on day 1/after the incoming visit, if one visit date changes, the next visit is made according to the original schedule outlined in figure 1.

Blood sampling

All blood sampling, including blood sampling for determining lipid parameters (e.g., TC, LDL-C, HDL-C, TG, non-HDL-C, ApoA, Apo B, Apo C-III, Lp (a), LDL particle size and number) and blood sampling for determining blood glucose, was performed in the morning, under fasting conditions (i.e., overnight, fasting for at least 10 to 12 hours, and smoking abstinence) for all field visits of the entire study, and prior to IMP injection. Alcohol consumption within 48 hours and intense physical exercise within 24 hours before blood sampling were prevented. If the patient is not in a fasting state, no blood sample is taken and a new appointment is given to the patient the next day (or as close as possible to that day) with fasting (see conditions above) indicated.

Laboratory testing

Laboratory data were collected according to the study schedule outlined in figure 1 and the following guidelines:

(a) hematology: all visits except week 4 and week 20; week 0 if applicable, and may be based on the clinical judgment of the investigator;

(b) chemistry: all visits except 3 rd and 6 th visits; this can be done at week 0, if applicable, and can be done based on the clinical judgment of the investigator, except for blood glucose that should be done on all patients at week 0;

(c) HbA1 c: screening and week 0, week 12 and week 24;

(d) lipidome (lipid panel): screening and

weeks

0,8, 12, 20 and 24;

(e) measured LDL-C quantified via β

weeks

0, 12 and 24;

(f) other lipid assessments (Apo B, Apo A-1, Apo C-III, LDL particle size and number, Lp [ a ]):

weeks

0, 12 and 24;

(g) liver group (liver panel): all visits except the 3 rd and 6 th visits; this can be done at week 0, if applicable, and can be based on the clinical judgment of the investigator. In case the total bilirubin value is above the normal range, differentiation into bound bilirubin and unbound bilirubin will occur automatically;

(h) creatine Phosphokinase (CPK): all visits except the 3 rd and 6 th visits; week 0 if applicable, and may be based on the clinical judgment of the investigator;

(i) hepatitis b surface antigen: screening only;

(j) hepatitis c antibody: at screening and week 24; in the case of an increase in ALT during the study, hepatitis C antibodies should be identified. If the hepatitis C antibody is positive during the study, a reflexive test is performed;

(k) pregnancy test (in women with fertility potential only): serum pregnancy test at screening only, urine pregnancy test at

week

0 and 24;

(l) Thyroid stimulating hormone: screening only patients who are taking thyroid hormone substitutes;

(m) C peptide: screening only;

(n) PCSK9 levels: only at week 0; and

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

Urine sampling

Urinalysis was performed at screening and at visit week 24. Test strips (dipsticks) were performed to test for pH, specific gravity and to assess the presence of blood, protein, glucose, ketones, nitrate, leukocyte esterase, urobilinogen and bilirubin. If the test strip is abnormal, standard microscopic examination is carried out. Microscopic evaluation of the presence or absence of Red Blood Cells (RBCs), red blood cell clumps, White Blood Cells (WBCs), WBC clumps, epithelial cells (transitional epithelial cells, renal tubular epithelial cells, and squamous epithelial cells), casts (hyaline casts, epithelial casts, WBC casts, RBC casts, granular casts, fat casts, cell casts, broad casts, wax-like casts), crystals (triphosphate crystals, calcium oxalate crystals, calcium phosphate crystals, calcium carbonate crystals, uric acid crystals, amorphous crystals, ammonium re-urate crystals, bilirubin crystals, leucine crystals, tyrosine crystals, cystine crystals), bacteria, yeast sprouts, yeast hyphae, trichomonas, oval fat bodies, fat, mucus, and sperm.

A spot urine test was performed on albumin and creatinine to calculate the albumin to creatinine ratio at the time of screening and at week 24 visit. Any clinically relevant abnormal laboratory values are immediately rechecked before any decision is made on the relevant patient.

Physical examination

General physical examination was performed. If a new clinically significant abnormality or worsening from baseline is detected after inclusion, the AE is reported and the patient is considered for further clinical studies and/or expert consultation according to the investigator's medical judgment.

Blood pressure and heart rate

Blood Pressure (BP) was measured in a sitting position using the same equipment (after the patient had comfortably rested in the sitting position for at least 5 minutes) on the same arm at approximately the same time of day under standard conditions. Values are recorded in e-CRF; systolic and diastolic pressures were recorded. At the first screening visit, the two-arm BP is measured. The arm with the highest diastolic pressure was determined at this visit and the BP on this arm was measured throughout the study. The highest value is recorded in the e-CRF.

Heart rate was measured while BP was measured.

Weight and height

Body weight is obtained with the patient wearing underwear or very light clothing and without shoes and with the bladder empty. The same scale was used throughout the study.

The altitude is measured because a self-reported altitude is unacceptable.

iTAQ questionnaire

iTAQ is a Patient Reported Outcome (PRO) measurement to assess treatment acceptability over a 4 week period prior to completion of the questionnaire. Patients were asked to complete at week 8 and week 24 visits.

Insulin diary

The patient was instructed to complete the insulin recording with the aim of recording his/her daily insulin dose (basal insulin and prandial insulin, if applicable) for at least 7 days before each visit and to take this information to the next study visit. For more than 7 days prior to study visit, the patient may record daily insulin doses, but only enter information collected on the last 7 days prior to each visit into the CRF.

Results

76 patients with T1DM and 441 patients with T2DM were co-enrolled.

All 76 patients with a random assignment of T1DM were treated and were therefore included in the safety population. 2 randomly assigned patients with T1DM (all in the aleuromab group) were not included in the intent-to-treat (ITT) population.

Of 441 patients with T2DM, 3 were untreated (alirocumab group 1, placebo group 2), and were therefore not included in the safety population. 12 randomly assigned patients with T2DM (aliskirumab group 7, placebo group 5) were not included in the ITT population.

If no calculated LDL-C value is available at baseline or within one of the analysis windows up to week 24, the patient is not included in the ITT population.

Study patients

Six (7.9%) patients with T1DM prematurely discontinued study treatment (3 in the alikumab group [ 5.9% ] (2 patients due to AE termination) and 3 in the placebo group [ 12.0% ] (2 patients due to AE termination.) all 3 patients in the alikumab group also did not complete the study period, whereas in the placebo group, 2 patients also did not complete the study period and 1 patient remained in the study until the study period was complete.

Table 2: treatment of patients with T1DM according to IVRS

The percentage was calculated using the number of patients randomly assigned as denominator.

39 (8.8%) patients with T2DM prematurely discontinued study treatment (29 in the alikumab group (9.9%), 10 in the placebo group (6.8%). of the 29 patients in the alikumab group, 18 patients also failed to complete the study, 11 patients remain in the study until the study period is complete. in the placebo group, 7 patients also failed to complete the study period, and 3 patients remain in the study until the study period is complete.

Table 3: treatment of patients with T2DM according to IVRS

The percentage was calculated using the number of patients randomly grouped as denominator.

Demographic and baseline characteristics

Baseline characteristics were generally similar in the alirocumab and placebo groups. 60.5% of randomly assigned patients with T1DM were male, while 54.2% of randomly assigned patients with T2DM were male. Patients with T1DM were younger with a mean age of 56.1(SD ═ 9.5), whereas patients with T2DM had a mean age of 64.0(SD ═ 9.1). The mean BMI of patients with T1DM was 30.0kg/m²(SD ═ 5.9), whereas a mean BMI of 32.6kg/m was observed for patients with T2DM²(SD＝5.06)。

Table 4: baseline demographics and patient characteristics-patients with type 1 diabetes according to IVRS

BMI: body mass index.

Table 5: baseline demographics and patient characteristics-patients with type 2 diabetes according to IVRS

BMI: body mass index.

Baseline calculated LDL-C was higher in patients with T1DM (mean 121.0mg/dL, SD 51.2) than in patients with T2DM (mean 110.4mg/dL, SD 37.3). The baseline triglyceride median (Q1: Q3) was 102.0mg/dL (76.5:135.0) in patients with T1DM lower than the median (Q1: Q3) in patients with T2DM at 147.0mg/dL (105.0: 212.0).

Table 6: baseline lipid parameters-quantitative summary in conventional units-patients with type 1 diabetes according to IVRS

With respect to other lipid parameters, T1DM patients showed a percent reduction in LDL-C particle number (LS mean) from baseline, 40.7% at week 12, 44.4% at week 24, and a percent reduction in LDL-C particle size, 2.3% at week 12, 2.3% at week 24. ApoC3 in these patients decreased by 6.9% at week 12 and 7.5% at week 24.

Table 7: baseline lipid parameters-quantitative summary in conventional units-patients with type 2 diabetes according to IVRS

With respect to other lipid parameters, T2DM patients showed a percent reduction in LDL-C particle number (LS mean) from baseline, 37.6% at week 12, 38.3% at week 24, and a percent reduction in LDL-C particle size, 2.6% at week 12, 2.8% at week 24. ApoC3 in these patients decreased by 6.3% at week 12 and 5.8% at week 24.

In patients with T1DM, the mean duration of diabetes and the mean duration of insulin use were similar between 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, the mean duration of diabetes and the mean duration of insulin use were similar between treatment groups. In T2DM, 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).

The duration of hypercholesterolemia was generally similar between treatment groups and between patients with T1DM and T2 DM.

As reported by the investigators, the proportion of patients with statin intolerance was 31.6% in patients with T1DM and 23.8% in patients with T2 DM.

The proportion of patients receiving fibrate at the time of random distribution was 2.6% in patients with T1DM and 8.8% in patients with T2 DM.

The proportion of patients receiving cholesterol absorption inhibitors (including ezetimibe) at random assignment was higher in the alirocumab group (13.6%) than in the placebo group (7.6%), particularly in patients with T2 DM: 45 patients (15.3%) versus 10 patients (6.8%).

Cardiovascular history and risk factors are generally similar between treatment groups. The following differences were observed between patients with T1DM and patients with T2 DM:

(1) ASCVD is more frequent in patients with T2DM (40.1% versus 21.1%), coronary heart disease (34.7% versus 15.8%) and stroke (8.2% versus 2.6%) than in patients with T1DM, while PAD (4.3% versus 9.2%) is less frequent in T2DM versus T1DM patients.

(2) In patients without ASCVD, 56.7% of patients with T1DM had target organ damage (microalbuminuria, macroalbuminuria) and/or CKD and/or retinopathy relative to 39.4% of patients with T2 DM. Still in patients without ASCVD, the following additional cardiovascular risk factors were observed:

(a) patients with T1DM are more frequent than patients with T2 DM: current smokers (20.0% vs. 14.0%), pre-proliferative diabetic retinopathy (36.7% vs. 12.9%) and proliferative diabetic retinopathy (20.0% vs. 5.7%).

(b) Patients with T1DM were less frequent than patients with T2 DM: hypertension (55.0% vs. 84.8%), microalbuminuria (10.0% vs. 19.7%), low HDL-C (16.7% vs. 28.0%).

(3) In 45% of patients with T1DM and 55.7% of patients with T2DM, the presence of 3 or more CV risk factors was observed in patients without ASCVD.

Overall, patients T1DM and T2DM were treated with high and medium strength statins in two treatment groups, with a higher proportion of patients treated with medium strength statins (58.9%). Overall, 59.0% of patients with T1DM and T2DM received statin treatment only.

Table 9: background lipid modification therapy at random assignment

Note that:

^aonly for patients currently taking statins.

^bIn combination with or without statins.

^cThe high-strength statin corresponds to 40 to 80mg daily of atorvastatin or 20 to 40mg daily of rosuvastatin or 80mg daily of simvastatin.

The medium strength statins correspond to 10 to 20mg daily of atorvastatin or 5 to 10mg daily of rosuvastatin or 20 to 40mg daily of simvastatin or 40 to 80mg daily of pravastatin or 40mg daily of lovastatin or 80mg daily of fluvastatin or 2 to 4mg daily of pitavastatin.

The low-strength statin corresponds to 10mg daily simvastatin or 10 to 20mg daily pravastatin or 20mg daily lovastatin or 20 to 40mg daily fluvastatin or 1mg daily pitavastatin. Patients receiving statins at more than one intensity level are calculated at the highest intensity level.

The number of patients randomly assigned was calculated as the denominator, in addition to each strength of statin (where% was calculated using the number of patients taking the statin as the denominator) and each daily dose category (where% was calculated using the number of patients taking the particular statin as the denominator).

Exposure of the safety population to the study drug product is summarized in table 10.

Table 10: exposure to study drug product-injections

Note that:

^apatients who were up-titrated at week 12 according to IVRS were thereafter treated with at least one injection of 150mg of aliskirumab (transfer). The denominator corresponds to patients with IVRS treatment after week 12 of at least one injection.

Patients were considered to be in the treatment group they actually received.

The duration of IMP injection exposure in weeks was defined as (last IMP injection date +14 days-first IMP injection date)/7, regardless of intermittent discontinuation.

Efficacy of

End of primary efficacy

The percent change in calculated LDL-C from baseline to week 24 in the ITT population of patients with T1DM and patients with T2DM, aleurozumab was superior to placebo (as shown in tables 11 and 12 and figures 2 and 3). In patients with T1DM, the proportion of individuals reaching LDL-C <70mg/dL (<1.8mmol/L) was 70.2% in the alikumab group, 5.1% in the placebo group (P <0.0001), and the proportion of individuals reaching LDL-C <50mg/dL (1.3mmol/L) was 55.1% in the alikumab group and 0% in the placebo group (P values not calculable). In patients with T2DM, the proportion of individuals reaching LDL-C <70mg/dL (<1.8mmol/L) was 76.4% in the alikumab group, 7.4% (P <0.0001) in the placebo group, and the proportion of individuals reaching LDL-C <50mg/dL (1.3mmol/L) was 50.7% in the alikumab group and 2.4% (P <0.0001) in the placebo group. Sensitivity analysis of primary efficacy endpoints showed similar results in both populations (data not shown).

Table 11: calculated percentage change in LDL-C from baseline to week 24: MMRM-patients with type 1 diabetes according to IVRS

Note that:

least Squares (LS) mean, Standard Error (SE) and p-value were obtained from MMRM (mixed effects model of repeated measurements) analysis.

The model includes fixed categorical effects (fixed categorical effects) for treatment groups, randomization layer (strata) from IVRS, time points, treatment group-to-time point interactions, layer-to-time point interactions, treatment group-to-layer-to-time point interactions, and continuous fixed covariates of baseline calculated LDL-C values and baseline value-to-time point interactions.

The MMRM model was performed on all patients in the ITT population (i.e., type 1 and type 2 diabetic patients).

MMRM models and baseline descriptions were performed on patients using baseline values and post-baseline values (post-baseline value) in at least one analysis window used in the model.

According to the fixed hierarchical approach used to ensure that the overall type I error rate is strongly controlled at the 0.05 level, p values are followed by '×' if statistically significant.

Table 12: calculated percentage change in LDL-C from baseline to week 24: MMRM-patients with type 2 diabetes according to IVRS

Note that:

The model includes the fixed classification effect of treatment groups, randomized layers according to IVRS, time points, and treatment group interaction with time points, layer interaction with time points, treatment group interaction with layer, and treatment group interaction with layer and time points, and continuous fixed covariates of baseline calculated LDL-C values and baseline value interaction with time points.

The MMRM model and baseline description were performed on the patient using the baseline values and post-baseline values in at least one analysis window used in the model.

Secondary efficacy endpoints

Aleucirumab resulted in significant reductions in non-HDL-C, ApoB, total cholesterol, and lp (a) levels from baseline to week 24 (differences from placebo), as well as an increase in HDL-C in the ITT population of patients with T1DM and patients with T2DM (as shown in tables 13 and 14, respectively).

Table 13: percent change from baseline of key secondary efficacy endpoint-patients with type 1 diabetes

At the endpoint triglyceride for participants with T2D and the endpoint HDL-C for participants with T1D, the stratified test was terminated, and therefore all subsequent statistical comparisons were not considered for statistical significance.

The P value is used for descriptive purposes only.

Apo, apolipoprotein; ITT, intent-to-treat; LDL-C, low density lipoprotein cholesterol; lp (a), lipoprotein (a); LS, least squares; non-HDL-C, non-high density lipoprotein cholesterol; TRL-C, triglyceride-rich lipoprotein cholesterol; SE, standard error; SD, standard deviation.

Table 14: percent change from baseline in key secondary efficacy endpoints-patients with type 2 diabetes

The P value is used for descriptive purposes only.

Diabetes-related endpoints

Overall, FPG and HbA1c and glucose lowering therapy remained stable over time in both treatment groups of patients with T1DM and patients with T2 DM.

With respect to HbA1c, in the T1DM cohort, in the alikumab group, the mean HbA1 c% was 7.84% (SD ═ 0.94) and the mean absolute change was-0.03% (0.6) at baseline, while in the placebo group, the mean HbA1c was 7.68% (0.78) and the mean absolute change was-0.23% (0.36) at baseline. In the T2DM cohort, mean HbA1c was 7.52% (0.96) at baseline and mean absolute change was 0.18% (0.74) in the alikumab group, while mean HbA1c was 7.54% (1.02) and mean absolute change was 0.06% (0.66) at baseline in the placebo group.

With respect to FPG, in the T1DM cohort, the mean FPG was 173mg/dL at baseline (SD 70.6) with a mean absolute change of-0.03 mg/dL (0.6) in the alikumab group, whereas in the placebo group, the mean FPG was 166.5mg/dL at baseline (75.6) with a mean absolute change of 14.6mg/dL (75.9). In the T2DM cohort, the mean FPG was 154.1mg/dL (50.1) at baseline and the mean absolute change was 9.5mg/dL (61.8) in the alikumab group, whereas in the placebo group the mean FPG was 153.5mg/dL (52.5) at baseline and the mean absolute change was 10.0mg/dL (47.0).

Safety feature

A total of 344 patients (51T 1DM and 293T 2DM) were exposed to aliskirumab and 170 patients (25T 1DM and 145T 2DM) were exposed to placebo.

Overall, the ratio of patients with any Treatment Emergent Adverse Event (TEAE) was similar across the treatment groups in the safety population of patients with T1DM or T2DM (see table 15).

Table 15: summary of adverse events profile: treating an emergency adverse event

Note that:

TEAE treatment of emergency adverse events; SAE: serious adverse events.

n (%) number and percentage of patients with at least one TEAE.

More frequently (≧ 10%) TEAE is reported in the following Systemic Organ Classification (SOC):

(a) infectious disease and infection (21.8% in aliskirumab versus 21.8% in placebo);

(b) gastrointestinal disorder (13.1% in alirocumab versus 12.4% in placebo);

(c) musculoskeletal and connective tissue diseases (21.5% in alikumab versus 15.9% in placebo); and

(d) systemic disease and site of administration (11.0% in aliskirumab versus 8.8% in placebo).

At PT levels, the most frequently reported TEAEs (> 2%) in the alikumab group and with a difference in incidence of > 0.5% compared to the placebo group were (in order of decreasing frequency in the alikumab group): myalgia (4.4% versus 1.8%), arthralgia (2.9% versus 1.8%), bronchitis (2.6% versus 0.6%), dizziness (2.6% versus 1.2%), and peripheral edema (2.0% versus 0.6%). In contrast, the most frequently reported TEAEs (> 2%) in the placebo group and with an incidence difference of > 0.5% compared to the alikumab group were: influenza (2.3% versus 2.9%), limb pain (1.7% versus 2.9%), hypoglycemia (1.7% versus 2.4%), cough (1.5% versus 2.9%), musculoskeletal pain (1.2% versus 2.4%), upper respiratory tract infections (0.9% versus 2.4%), hyperglycemia (0.9% versus 2.4%), and pneumonia (0.6% versus 2.4%).

Overall, 25 patients (7.3%) in the alirocumab group and 14 patients (8.2%) in the placebo group reported treatment for emergency SAE. SAE (at PT level) reported in more than 1 patient in any treatment group were pneumonia (1 patient in the Alikumab group (0.3%) versus 2 patients in the placebo group (1.2%), foraminal stenosis (2 patients in the Alikumab group (0.6%) versus no patients in the placebo group), and urinary tract infection (2 patients (0.6%) versus no patients in the placebo group). one death due to myocardial infarction was reported in T2DM patients in the placebo group 1 month after the first IMP dose administration (3 rd visit). Overall, 17 patients in the Alikumab group (4.9%) and 4 patients in the placebo group (2.4%) experienced a TEAE that caused permanent treatment discontinuation.at PT level, patients with permanent treatment discontinuation in more than one patient in the treatment group had a proportion of TEAE in the TEkumab group: 0.6 patients in the Alikumab group (0.6% versus 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%).

For adverse events of particular interest (AESI), an increase in ALT that meets the AESI criteria is defined as either ALT ≧ 3XULN (if baseline ALT < ULN) or ALT ≧ 2 times the baseline value (if baseline ALT ≧ ULN). These events were reported in 2 patients (0.6%) of the alirocumab group versus 1 patient (0.6%) of the placebo group.

Allergic drug reactions meeting AESI criteria are defined as allergic events that require consultation with additional physicians for further evaluation. These events were reported in 5 patients (1.5%) of the aliskirab group versus 4 patients (2.4%) of the placebo group. These reactions were primarily skin and subcutaneous tissue disorders, reported in 3 patients (0.9%) in the alirocumab group (1 atopic dermatitis, 1 eczema and 1 light-sensitive reaction) and 2 patients (1.2%) in the placebo group (1 dermatitis, 1 drug rash). Two other AESI allergic drug reactions in the aleucizumab group were drug hypersensitivity and eosinophilia.

Neurological events meeting the AESI criteria are defined as neurological events requiring additional examination/procedures and/or referrals to specialists. Such events were reported in 1 patient (0.3%) of the alirocumab group (paresthesia) versus 1 patient (0.6%) of the placebo group (dysphagia). Two events were reported in patients with T2 DM.

All neurocognitive events are considered AESI. Neurocognitive events grouped according to sponsor or FDA were reported in 4 patients (1.2%) of the alikumab group versus no patients in the placebo group. All events were reported in patient T2 DM: cognitive impairment was reported in 2 patients (0.6%) and memory impairment and amnesia were reported in 1 patient each (0.3%). Notably, 2 cognitive disorders also lead to permanent treatment discontinuation.

Local injection site reactions that meet the AESI criteria are defined as local injection site reactions that are allergic and require consultation by another physician, or as non-allergic local injection site reactions that are clinically significant (e.g., reactions of swelling or erythema >2.5cm in diameter; reactions that interfere with activity). The LISR (placebo versus injection site response to aliskirumab) confirmed by each investigator in relation to IMP ('eCRF') was reported in 6 patients (1.7%) of the aliskirumab cohort versus 8 patients (4.7%) of the placebo cohort. Local Injection Site Response (LISR) meeting AESI criteria, defined as the response that requires consultation with another physician for further evaluation, was not reported.

There was no report of symptomatic overdose or pregnancy.

Analysis of liver function tests (ALT, AST, ALP, total bilirubin), CPK and kidney function tests (creatinine, eGFR, BUN) did not show differences in the time course of any study parameters between treatment groups. During the study period, PCSA analysis did not identify PCSA with increased ALT in any treatment group. In patients with normal CPK values at baseline, an increase of >3ULN (and. ltoreq.10 ULN) was reported in 7 patients (2.1%) in the alirocumab group relative to 1 patient (0.6%) in the placebo group. All patients had T2 DM. No CPK increase >10ULN was reported.

Regardless of the baseline state, minor differences in the numerical values of the proportion of patients with mild, moderate or severe reductions in Glomerular Filtration Rate (GFR) during treatment were observed: mild, moderate, and severe reductions in GFR were 49.7%, 28.1%, and 3.8% of patients in the alikumab group, and 50.6%, 24.4%, and 3.6% of patients in the placebo group, respectively. Similarly, an increase in blood creatinine (> 30% and < 100%) was measured in 13 patients (3.8%) of the alikumab group versus 5 patients (3.0%) in the placebo group. None of the patients had an increase in creatinine > 100%. There were no meaningful differences in renal function.

No meaningful differences in vital signs were observed between treatment groups.

Example 3: analysis of individuals with type 2 diabetes mellitus and ASCVD from the Odyssey DM-insulin clinical trial

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

In this assay, we evaluated the efficacy and safety of alirocumab in individuals with T2DM, high LDL-C or non HDL-C, and determined ASCVD receiving the most tolerated statins in the DM-insulin study. DM-insulin study participants with ASCVD and T1DM were not included in this analysis because of the low individual numbers in this group (aleucirumab: n-11; placebo: n-5). As used in this example, ASCVD is defined as coronary heart disease (CHD; acute and asymptomatic Myocardial Infarction (MI) and unstable angina), ischemic stroke, or peripheral arterial disease.

Baseline and efficacy data were analyzed from the study. Efficacy assays included percent reduction from baseline at week 24 of non-HDL-C, LDL-C, ApoB and LDL-PN, and percent of individuals reaching non-HDL-C <100mg/dL (<2.59mmol/L), LDL-C <70mg/dL (<1.81mmol/L), and ApoB <80mg/dL at week 24. The intent-to-treat (ITT) analysis included all randomly assigned individuals with baseline LDL-C values and at least one LDL-C value up to week 24.

The analysis included 177 DM-insulin individuals with established ASCVD and T2DM (Table 16).

Table 16: baseline characteristics (randomly assigned population)

Diagnosis is performed by invasive/non-invasive tests.

Including patients with established HTN in anti-HTN drug therapy.^§Defined as the estimated glomerular filtration rate of 15-60mL/min/1.73m²。^∥Defined as microalbuminuria, macroalbuminuria, retinopathy and/or CKD.

One individual in the placebo group did not receive insulin at the time of random assignment and remained untreated with insulin for the duration of the study.

BMI, body mass index; DPP4, dipeptidyl peptidase 4; GLP-1, glucagon-like peptide 1;

GLT, glucose lowering therapy; FPG, fasting plasma glucose, HbA1c, glycated hemoglobin;

HTN, hypertension; SGLT2, sodium/glucose cotransporter 2.

Regardless of treatment allocation, in addition to ASCVD, 89.3% of the individuals analyzed also had a history of hypertension and 28.2% also had Chronic Kidney Disease (CKD). In total, 20.3% confirmed the history of ischemic stroke, 10.7% had Peripheral Arterial Disease (PAD). Regardless of treatment assignment, at baseline, the mean (standard deviation [ SD ]) non-HDL-C level was 144.2(46.2) mg/dL [3.73(1.20) mmol/L ]; the mean LDL-C level was 108.7(39.1) mg/dL [2.82(1.01) mmol/L ].

Efficacy of

At week 24, aliskirumab reduced non-HDL-C, ApoB, LDL-PN and LDL-C from baseline relative to controls (fig. 4). At week 24, a significantly higher proportion of individuals achieved non-HDL-C <100mg/dL (<2.59mmol/L), LDL-C <70mg/dL (<1.81mmol/L), and ApoB <80mg/dL (all P < 0.0001; fig. 5) relative to controls.

Safety feature

Safety analyses were performed in pooled populations of individuals with T2DM, high LDL-C or non-HDL-C and established ASCVD who received the most resistant statins in the DM-insulin study and individuals with T1DM or T2DM, high LD-C or non-HDL-C and established ASCVD who received the most resistant statins in the DM-Dyslipidemia (dylipdemia) study (see Chan et al (2017) Ann trans med.5(23):477, which is incorporated herein by reference in its entirety). In total, 66.4% (alirocumab) and 67.0% (control) individuals reported treatment for emergency adverse events (TEAE; table 17). The adverse event patterns were similar for both groups. The mean (SD) HbA1c levels were similar at baseline (Alikumab: 7.3[0.9 ]%; control: 7.3[0.9 ]%) and at week 24 (Alikumab: 7.6[1.2 ]%; control: 7.5[1.2 ]%; safety analysis) for each treatment group. FPG levels at baseline (Alikumab: 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 (Alikumab: 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) were also similar regardless of treatment assignment.

Table 17: safety summary

SAE, severe adverse events.

Conclusion

In individuals with T2DM and ASCVD who have high LDL-C levels despite the use of most tolerated statins, alirocumab significantly reduced the level of atherogenic cholesterol and LDL-PN relative to controls.

Sequence listing

<110> Senoffy Biotech Co

<120> methods of treating hyperlipidemia in diabetic patients by administering PCSK9 inhibitors

<130>602480:SA9-208TW

<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

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<170>PatentIn version 3.5

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

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

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Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val

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Ser Thr Ile Ser Gly Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Lys His Thr Leu Tyr

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

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Ala Lys Asp Ser Asn Trp Gly Asn Phe Asp Leu Trp Gly Arg Gly Thr

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Leu Val Thr Val Ser Ser

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Gly Phe Thr Phe Asn Asn Tyr Ala

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<213> Artificial sequence

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Ile Ser Gly Ser Gly Gly Thr Thr

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<213> Artificial sequence

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Ala Lys Asp Ser Asn Trp Gly Asn Phe Asp Leu

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<213> Artificial sequence

<220>

<223> description of the Artificial sequence the synthetic REGN727 heavy chain polypeptide

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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 Asn Asn Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val

35 40 45

Ser Thr Ile Ser Gly Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Lys His Thr Leu Tyr

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

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Ala Lys Asp Ser Asn Trp Gly Asn Phe Asp Leu Trp Gly Arg Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala AlaLeu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

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Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

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Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

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Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

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Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

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

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

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Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

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Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

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Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

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<210>6

<211>113

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<213> Artificial sequence

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<223> description of Artificial sequences synthetic Polypeptides

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

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

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Ser Asn Asn Arg Asn Phe Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln

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

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

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

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Tyr Tyr Thr Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

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<213> Artificial sequence

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Gln Ser Val Leu Tyr Arg Ser Asn Asn Arg Asn Phe

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<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

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Trp Ala Ser

1

<210>9

<211>220

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<213> Artificial sequence

<220>

<223> description of the Artificial sequence the synthetic REGN727 light chain polypeptide

<400>9

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

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

20 25 30

Ser Asn Asn Arg Asn Phe Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Asn Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser GlyVal

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 Thr Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu 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

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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 Cys

210 215 220

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<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

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Gln Gln Tyr Tyr Thr Thr Pro Tyr Thr

1 5

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<211>127

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic Polypeptides

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

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

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

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Ala Asn Ile Asn Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

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

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

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

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Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210>12

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>12

Gly Phe Thr Phe Ser Ser His Trp

1 5

<210>13

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>13

Ile Asn Gln Asp Gly Ser Glu Lys

1 5

<210>14

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<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>14

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

1 5 10 15

Gly Met Asp Val

20

<210>15

<211>112

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic Polypeptides

<400>15

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 Gln Ser Leu Leu His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg 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 ValTyr Tyr Cys Met Gln Thr

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

100 105 110

<210>16

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>16

Gln Ser Leu Leu His Ser Asn Gly Asn Asn Tyr

1 5 10

<210>17

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>17

Leu Gly Ser

1

<210>18

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>18

Met Gln Thr Leu Gln Thr Pro Leu Thr

1 5

<210>19

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>19

Gly Phe Thr Phe Ser Ser His Trp

1 5

<210>20

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>20

Ile Asn Gln Asp Gly Ser Glu Lys

1 5

<210>21

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>21

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

1 5 10 15

Gly Met Asp Val

20

<210>22

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>22

Gln Ser Leu His His Ser Asn Gly Asn Asn Tyr

1 5 10

<210>23

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>23

Leu Gly Ser

1

<210>24

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>24

Met Gln Thr Leu Gln Thr Pro Leu Thr

1 5

<210>25

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>25

Gly Phe Thr Phe Ser Ser His Trp

1 5

<210>26

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>26

Ile Asn Gln Asp Gly Ser Glu Lys

1 5

<210>27

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>27

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

1 5 10 15

Gly Met Asp Val

20

<210>28

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>28

Gln Ser Leu Leu His Ser Asn Gly Asn Asn Tyr

1 5 10

<210>29

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>29

Leu Gly Ser

1

<210>30

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>30

Met Gln Thr Leu Gln Thr Pro Leu Thr

1 5

<210>31

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>31

Gly Phe Thr Phe Ser Ser His Trp

1 5

<210>32

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>32

Ile Asn Gln Asp Gly Ser Glu Lys

1 5

<210>33

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<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

20

<210>34

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>34

Gln Ser Leu His His Ser Asn Gly Asn Asn Tyr

1 5 10

<210>35

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>35

Leu Gly Ser

1

<210>36

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>36

Met Gln Thr Leu Gln Thr Pro Leu Thr

1 5

<210>37

<211>131

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic VH, m2CX1D05 polypeptide

<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 7580

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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences, synthetic VH CDR1, m2CX1D05 peptide

<400>38

Gly Gly Thr Phe Asn Ser His Ala Ile Ser

1 5 10

<210>39

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis of VH CDR2, m2CX1D05 peptide

<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

20

<210>40

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences, synthetic VH CDR3, m2CX1D05 peptide

<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

20

<210>41

<211>213

<212>PRT

<213> Artificial sequence

<220>

<223> description of the Artificial sequence synthetic LC, m2CX1D05 polypeptide

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic VL CDR1, m2CX1D05 peptide

<400>42

Arg Ala Ser Gln Gly Ile Arg Ser Ala Leu Asn

1 5 10

<210>43

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic VL CDR2, m2CX1D05 peptide

<400>43

Leu Leu Ile Tyr Asn Gly Ser Thr Leu Gln Ser

1 5 10

<210>44

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic VL CDR3, m2CX1D05 peptide

<400>44

Gln Gln Phe Asp Gly Asp Pro

1 5

<210>45

<211>119

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic VH, 1B20 Polypeptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic VH CDR1; 1B20 peptide

<400>46

Gly Tyr Ser Phe Thr Asn Tyr Trp Ile Ser

1 5 10

<210>47

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic VH CDR2; 1B20 peptide

<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

20

<210>48

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic VH CDR3; 1B20 peptide

<400>48

Asp Tyr Trp Tyr Lys Pro Leu Phe Asp Ile

1 5 10

<210>49

<211>220

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic LC 1B20 Polypeptides

<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 AspAsn 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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic VL CDR1; 1B20 peptide

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic VL CDR2; 1B20 peptide

<400>51

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser

1 5 10

<210>52

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic VL CDR3; 1B20 peptide

<400>52

Gln Gln Tyr Ser Ser Phe Pro Ile

1 5

<210>53

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic variable heavy antibody region polypeptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis of the AX132 heavy chain CDR1 antibody Domain peptide

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX132 heavy chain CDR2 antibody region peptides

<400>55

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

15 10 15

Lys Phe Lys Gly Lys Ala Thr

20

<210>56

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX132 heavy chain CDR3 antibody region peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic variable light antibody region polypeptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic CDR1 antibody region peptides of AX213 and AX132 light chains

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic CDR2 antibody region peptides of AX213 and AX132 light chains

<400>59

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

1 5 10

<210>60

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic CDR3 antibody region peptides of AX132 and AX213 light chain

<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>PRT

<213> Artificial sequence

<220>

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX213 heavy chain CDR1 antibody region peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX213 heavy chain CDR2 antibody region peptides

<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 LysAla Thr

20

<210>64

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX213 heavy chain CDR3 antibody region peptides

<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>PRT

<213> Artificial sequence

<220>

<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 GlySer 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>PRT

<213> Artificial sequence

<220>

<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>PRT

<213> Artificial sequence

<220>

<400>67

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

1 5 10

<210>68

<211>16

<212>PRT

<213> Artificial sequence

<220>

<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>PRT

<213> Artificial sequence

<220>

<223> description of the Artificial sequences synthetic AX1 VH antibody sequence Polypeptides

<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 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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX1 VH CDR1 antibody sequence peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX1 VH CDR2 antibody sequence peptides

<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

20

<210>72

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX1 VH CDR3 antibody sequence peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of the Artificial sequences synthetic AX1 VL antibody sequence Polypeptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX1 VL CDR1 antibody sequence peptides

<400>74

Arg Ala Ser Gln Asp Ile Ser Arg Tyr Leu Ala

1 5 10

<210>75

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX1 AX9 AX189 VL CDR2 antibody sequence peptides

<400>75

Ala Ala Ser Ser Leu Gln Ser

1 5

<210>76

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX1 VL CDR3 antibody sequence peptides

<400>76

Ala Ala Tyr Asp Tyr Ser Leu Gly Gly Tyr Val

1 5 10

<210>77

<211>121

<212>PRT

<213> Artificial sequence

<220>

<223> description of the Artificial sequences synthetic AX9 AX189 VH antibody sequence polypeptide

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX9 AX189 VH CDR1 antibody sequence peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX9 AX189 VH CDR2 antibody sequence peptides

<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

20

<210>80

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX9 AX189 VH CDR3 antibody sequence peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of the Artificial sequence synthetic AX189 VL antibody sequence polypeptide

<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 9095

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

100 105

<210>82

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX189 VL CDR1 antibody sequence peptide

<400>82

Arg Ala Ser Gln Asp Val Ser Arg Tyr Leu Thr

1 5 10

<210>83

<211>7

<212>PRT

<213> Artificial sequence

<220>

<400>83

Ala Ala Ser Ser Leu Gln Ser

1 5

<210>84

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic AX189 VL CDR3 antibody sequence peptide

<400>84

Gln Ala Tyr Asp Tyr Ser Leu Ser Gly Tyr Val

1 5 10

<210>85

<211>115

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>86

Ser Tyr Gly Ile Ser

1 5

<210>87

<211>17

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>88

Gly Tyr Gly Met Asp Val

1 5

<210>89

<211>109

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>90

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser

1 5 10

<210>91

<211>7

<212>PRT

<213> Intelligent people

<400>91

Glu Val Ser Asn Arg Pro Ser

1 5

<210>92

<211>9

<212>PRT

<213> Intelligent people

<400>92

Asn Ser Tyr Thr Ser Thr Ser Met Val

1 5

<210>93

<211>123

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>94

Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn

1 5 10

<210>95

<211>17

<212>PRT

<213> Intelligent people

<400>95

Ser Ile Ser Ser Ser Ser Ser Tyr Ile Ser Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>96

<211>14

<212>PRT

<213> Intelligent people

<400>96

Asp Tyr Asp Phe Trp Ser Ala Tyr Tyr Asp Ala Phe Asp Val

1 5 10

<210>97

<211>111

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>98

Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10

<210>99

<211>7

<212>PRT

<213> Intelligent people

<400>99

Gly Asn Ser Asn Arg Pro Ser

1 5

<210>100

<211>11

<212>PRT

<213> Intelligent people

<400>100

Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Val

1 5 10

<210>101

<211>114

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>102

Gly Phe Thr Phe Ser Ser Tyr Gly Met His

1 5 10

<210>103

<211>17

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>104

Asp Arg Gly Leu Asp

1 5

<210>105

<211>113

<212>PRT

<213> Intelligent people

<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 TyrSer

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>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>107

Trp Ala Ser Thr Arg Glu Ser

1 5

<210>108

<211>9

<212>PRT

<213> Intelligent people

<400>108

Gln Gln Tyr Tyr Ser Thr Pro Trp Thr

1 5

<210>109

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic anti-PCSK 9 monoclonal antibody pJG04 (cloned LGT-209 and LGT-210) Vh heavy chain variable region (FR1-FR4) polypeptide

<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 7580

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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic anti-PCSK 9 monoclonal antibody clones LGT-209, LGT-210 and LGT-211 heavy chain CDR1 peptides

<400>110

Thr Met Tyr Met Ser

1 5

<210>111

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic anti-PCSK 9 monoclonal antibody clones LGT-209, LGT-210 and LGT-211 heavy chain CDR2 peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic anti-PCSK 9 monoclonal antibody pJG04 (cloned LGT-209 and LGT-210) Vh heavy chain complementarity determining region 3(CDR3) peptide

<400>112

Ser Tyr Tyr Tyr Tyr Asn Met Asp Tyr

1 5

<210>113

<211>106

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic anti-PCSK 9 monoclonal antibody pJG10 (cloned LGT-209 and LGT-211) Vk light chain variable region (FR1-FR4) polypeptide

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic anti-PCSK 9 monoclonal antibody clones LGT-209, LGT-210 and LGT-211 light chain CDR1 peptides

<400>114

Arg Ala Ser Gln Ser Val Ser Tyr Met His

1 5 10

<210>115

<211>7

<212>PRT

<213> Artificial sequence

<220>

<400>115

Gly Val Phe Arg Arg Ala Thr

1 5

<210>116

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic mouse anti-PCSK 9 monoclonal antibody LFU720 and anti-PCSK 9 monoclonal antibody clone LGT-209, LGT-210 and LGT-211 light chain CDR3 peptides

<400>116

Leu Gln Trp Ser Ser Asp Pro Pro Thr

1 5

<210>117

<211>118

<212>PRT

<213> Intelligent people

<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 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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>118

Gly Tyr Thr Phe Thr Ser Tyr

1 5

<210>119

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>119

Ser Pro Phe Gly Gly Arg

1 5

<210>120

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic variable heavy chain CDR peptides

<400>120

Glu Arg Pro Leu Tyr Ala Ser Asp Leu

1 5

<210>121

<211>107

<212>PRT

<213> Intelligent people

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic variable light chain CDR peptides

<400>122

Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala

1 5 10

<210>123

<211>7

<212>PRT

<213> Artificial sequence

<220>

<400>123

Ser Ala Ser Tyr Arg Tyr Thr

1 5

<210>124

<211>9

<212>PRT

<213> Artificial sequence

<220>

<400>124

Gln Gln Arg Tyr Ser Leu Trp Arg Thr

1 5

<210>125

<211>118

<212>PRT

<213> Intelligent people

<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 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>PRT

<213> Intelligent people

<400>126

Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His

1 5 10

<210>127

<211>17

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>128

Glu Arg Pro Leu Tyr Ala Met Asp Tyr

1 5

<210>129

<211>107

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>130

Lys Ala Ser Gln Asp Val His Thr Ala Val Ala

1 5 10

<210>131

<211>7

<212>PRT

<213> Intelligent people

<400>131

His Ala Ser Tyr Arg Tyr Thr

1 5

<210>132

<211>9

<212>PRT

<213> Intelligent people

<400>132

Gln Gln Arg Tyr Ser Leu Trp Arg Thr

1 5

<210>133

<211>118

<212>PRT

<213> Intelligent people

<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 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>PRT

<213> Intelligent people

<400>134

Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His

1 5 10

<210>135

<211>17

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>136

Glu Arg Pro Leu Tyr Ala Ser Asp Leu

1 5

<210>137

<211>107

<212>PRT

<213> Intelligent people

<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>PRT

<213> Intelligent people

<400>138

Lys Ala Ser Gln Asp Val His Thr Ala Val Ala

1 5 10

<210>139

<211>7

<212>PRT

<213> Intelligent people

<400>139

His Ala Ser Tyr Arg Tyr Thr

1 5

<210>140

<211>9

<212>PRT

<213> Intelligent people

<400>140

Gln Gln Arg Tyr Ser Leu Trp Arg Thr

1 5

<210>141

<211>118

<212>PRT

<213> mouse

<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>PRT

<213> mouse

<400>142

Gly Tyr Thr Phe Thr Ser Tyr

1 5

<210>143

<211>6

<212>PRT

<213> mouse

<400>143

Asn Pro Ser Asn Gly Arg

1 5

<210>144

<211>9

<212>PRT

<213> mouse

<400>144

Glu Arg Pro Leu Tyr Ala Met Asp Tyr

1 5

<210>145

<211>108

<212>PRT

<213> mouse

<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>PRT

<213> mouse

<400>146

Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210>147

<211>7

<212>PRT

<213> Artificial sequence

<220>

<400>147

Ser Ala Ser Tyr Arg Tyr Thr

1 5

<210>148

<211>9

<212>PRT

<213> mouse

<400>148

Gln Gln Arg Tyr Ser Thr Pro Arg Thr

1 5

<210>149

<211>115

<212>PRT

<213> mouse

<400>149

Glu Val Gln Leu Gln Gln Ser Gly Pro GluLeu 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>PRT

<213> mouse

<400>150

Gly Tyr Thr Phe Thr Asp Tyr

1 5

<210>151

<211>6

<212>PRT

<213> mouse

<400>151

Asn Pro Asn Asn Gly Gly

1 5

<210>152

<211>6

<212>PRT

<213> mouse

<400>152

Trp Leu Leu Phe Ala Tyr

1 5

<210>153

<211>108

<212>PRT

<213> mouse

<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 PheCys 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>PRT

<213> mouse

<400>154

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala

1 5 10

<210>155

<211>7

<212>PRT

<213> mouse

<400>155

Ser Ala Ser Tyr Arg Tyr Ser

1 5

<210>156

<211>9

<212>PRT

<213> mouse

<400>156

Gln Gln Phe Tyr Ser Tyr Pro Tyr Thr

1 5

<210>157

<211>123

<212>PRT

<213> mouse

<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>PRT

<213> mouse

<400>158

Gly Tyr Thr Phe Thr Asp Tyr

1 5

<210>159

<211>6

<212>PRT

<213> mouse

<400>159

Asn Pro Asn Asn Gly Gly

1 5

<210>160

<211>14

<212>PRT

<213> mouse

<400>160

Gly Gly Ile Tyr Tyr Arg Tyr Asp Arg Asn Tyr Phe Asp Tyr

1 5 10

<210>161

<211>107

<212>PRT

<213> mouse

<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>PRT

<213> mouse

<400>162

Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn

1 5 10

<210>163

<211>7

<212>PRT

<213> mouse

<400>163

Tyr Thr Ser Ser Leu His Ser

1 5

<210>164

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>164

Gln Gln Tyr Ser Lys Leu Pro Phe Thr

1 5

<210>165

<211>117

<212>PRT

<213> mouse

<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>PRT

<213> mouse

<400>166

Gly Phe Thr Phe Ser Asp Tyr

1 5

<210>167

<211>6

<212>PRT

<213> mouse

<400>167

Asn Tyr Asp Gly Ser Asn

1 5

<210>168

<211>8

<212>PRT

<213> mouse

<400>168

Glu Lys Phe Ala Ala Met Asp Tyr

1 5

<210>169

<211>108

<212>PRT

<213> mouse

<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>PRT

<213> mouse

<400>170

Lys Ala Ser Gln Asp Val Ser Asn Ala Leu Ala

1 5 10

<210>171

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis of variable light chain CDR peptides

<400>171

Ser Ala Ser Tyr Arg Tyr Thr

1 5

<210>172

<211>9

<212>PRT

<213> mouse

<400>172

Gln Gln His Tyr Ser Thr Pro Trp Thr

1 5

<210>173

<211>121

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic Polypeptides

<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 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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>174

Gly Phe Thr Phe Thr Arg His Thr Ile His

1 5 10

<210>175

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>176

Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr

1 5 10

<210>177

<211>108

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic Polypeptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>178

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210>179

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>179

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210>180

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>180

Gln Gln Ser Tyr Arg Ile Gln Pro Thr

1 5

<210>181

<211>121

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic Polypeptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>182

Gly Phe Thr Phe Ser Ser Thr Ala Ile His

1 5 10

<210>183

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>184

Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr

1 5 10

<210>185

<211>108

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic Polypeptides

<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 ProLys 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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>186

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210>187

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>187

Ser Ala Ser Phe Leu Tyr Ser

15

<210>188

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>188

Gln Gln Ser Tyr Pro Ala Leu His Thr

1 5

<210>189

<211>125

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic Polypeptides

<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 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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>190

Gly Phe Pro Phe Ser Lys Leu Gly Met Val

1 5 10

<210>191

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic Polypeptides

<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 TyrGln 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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<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>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>195

Gln Leu Ser Asn Leu Ala Ser

1 5

<210>196

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400>196

Tyr Gln Asn Leu Glu Leu Pro Leu Thr

1 5

<210>197

<211>2076

<212>DNA

<213> Intelligent people

<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 gcccagagca tcccgtggaa cctggagcgg 480

attacccctc cacggtaccg ggcggatgaa taccagcccc ccgacggagg 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 ccagcatggg gacccgtgtc cactgccacc aacagggcca cgtcctcaca 1680

ggctgcagct cccactggga ggtggaggac cttggcaccc acaagccgcc tgtgctgagg 1740

ccacgaggtc agcccaacca gtgcgtgggc 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>PRT

<213> Intelligent people

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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

115120 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

275280 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

435440 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 600605

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

1. A method for treating hypercholesterolemia in a patient having type 1 diabetes (T1DM), the method comprising:

(a) selecting a high cardiovascular risk patient receiving insulin treatment, said patient having (i) T1DM, and (ii) hypercholesterolemia that is not adequately controlled by maximally tolerated statin treatment; and

2. The method of claim 1, wherein 75mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

3. The method of claim 1, wherein 150mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

4. The method of claim 1, wherein 300mg of the antibody or antigen-binding fragment is administered to the patient every four weeks.

5. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs shown in SEQ ID NOs 2, 3, and 4, and three light chain CDRs shown in SEQ ID NOs 7, 8, and 10.

6. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having the amino acid sequence of SEQ ID No. 1, and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID No. 6.

7. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: alikumab, efuzumab, bococizumab, ludwizumab, ralpancizumab and LY 3015014.

8. The method of claim 7, wherein the antibody or antigen-binding fragment thereof is aleucizumab.

9. The method of any of the preceding claims, further comprising:

10. The method of any of the preceding claims, further comprising:

11. The method of claim 9 or 10, wherein the threshold level is 70 mg/dL.

12. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously.

13. The method of any one of the preceding claims, wherein the patient further receives concomitant Lipid Modification Therapy (LMT).

14. The method of claim 13, wherein the LMT is selected from the group consisting of: statins, cholesterol absorption inhibitors, fibrates, nicotinic acids, omega-3 fatty acids, and bile acid sequestrants.

15. The method of claim 14, wherein the LMT is a statin therapy.

16. The method of claim 15, wherein the statin is selected from the group consisting of: atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin and cerivastatin.

17. The method of any one of claims 14-16, wherein the statin treatment is the most tolerated statin treatment.

18. The method of claim 14, wherein the cholesterol absorption inhibitor is ezetimibe.

19. The method of any one of claims 1-14 and 18, wherein the patient is intolerant to statins.

20. The method of any one of the preceding claims, wherein the insulin treatment is selected from the group consisting of: human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degluvium, insulin aspart and basal insulin.

21. The method of any one of the preceding claims, wherein the patient receives concomitant antidiabetic therapy in addition to insulin therapy.

22. The method of claim 21, wherein the additional concomitant antidiabetic therapy is selected from the group consisting of glucagon-like peptide 1(GLP-1) therapy, gastrointestinal peptides, glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, ghrelin antagonists or inverse agonists, xenin analogs, biguanides, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, α -glucosidase inhibitors, sodium-dependent glucose transporter 2(SGLT-2) inhibitors, SGLT-1 inhibitors, peroxisome proliferator-activated receptor (PPAR-) (α, γ or α/γ) agonists or modulators, amylin analogs, G protein coupled receptor 119(GPR119) agonists, GPR40 agonists, GPR120 monophosphorus agonists, GPR142 agonists, systemic or low absorption TGR5 agonists, diabetic immune therapy, anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, anti-inflammatory agonists or agonists for the treatment of the GPR119, GPR β agonists, inhibitors of the glucose-acyl transferase, glucose-receptor activator, inhibitors of the glucose-releasing hormone, and their combinations.

23. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof reduces the LDL-C level of the patient by at least 40%.

24. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof reduces non-HDL-C levels in the patient by at least 35%.

25. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof reduces apolipoprotein C3(ApoC3) levels in the patient.

26. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof reduces the number and/or size of lipoprotein particles in the patient.

27. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof:

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

(b) Does not affect fasting blood glucose (FPG) levels in the patient.

28. A method for treating hypercholesterolemia in a patient having type 1 diabetes (T1DM), the method comprising:

29. A method for treating hypercholesterolemia in a patient having type 2 diabetes (T2DM), the method comprising:

30. The method of claim 29, wherein 75mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

31. The method of claim 29, wherein 150mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

32. The method of claim 29, wherein 300mg of the antibody or antigen-binding fragment is administered to the patient every four weeks.

33. The method of any one of claims 29-32, wherein the antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs set forth in SEQ ID NOs 2, 3, and 4 and the three light chain CDRs set forth in SEQ ID NOs 7, 8, and 10.

34. The method of any one of claims 29-33, wherein the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having the amino acid sequence of SEQ ID No. 1 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID No. 6.

35. The method of any one of claims 29-32, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: alikumab, efuzumab, bococizumab, ludwizumab, ralpancizumab and LY 3015014.

36. The method of claim 35, wherein the antibody or antigen-binding fragment thereof is aleucizumab.

37. The method of any one of claims 29-36, further comprising:

38. The method of any one of claims 29-36, further comprising:

39. The method of claim 37 or 38, wherein the threshold level is 70 mg/dL.

40. The method of any one of claims 29-39, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously.

41. The method of any one of claims 29-40, wherein the patient further receives concomitant Lipid Modification Therapy (LMT).

42. The method of claim 41, wherein the LMT is selected from the group consisting of: statins, cholesterol absorption inhibitors, fibrates, nicotinic acids, omega-3 fatty acids, and bile acid sequestrants.

43. The method of claim 42, wherein the LMT is a statin therapy.

44. The method of claim 43, wherein the statin is selected from the group consisting of: atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin and cerivastatin.

45. The method of any one of claims 42-44, wherein the statin treatment is a maximum tolerated dose statin treatment.

46. The method of claim 42, wherein the cholesterol absorption inhibitor is ezetimibe.

47. The method of any one of claims 29-42 and 46, wherein the patient is intolerant to statins.

48. The method of any one of claims 29-47, wherein the insulin treatment is selected from the group consisting of: human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degluvium, insulin aspart and basal insulin.

49. The method of any one of claims 29-48, wherein the patient receives concomitant anti-diabetic therapy in addition to insulin therapy.

50. The method of claim 49, wherein the additional anti-diabetic treatment is selected from the group consisting of glucagon-like peptide 1(GLP-1) treatment, gastrointestinal peptides, glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, ghrelin antagonists or inverse agonists, xenin analogs, biguanides, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, α -glucosidase inhibitors, sodium-dependent glucose transporter 2(SGLT-2) inhibitors, SGLT-1 inhibitors, peroxisome proliferator-activated receptor (PPAR-) (α, γ, or α/γ) agonists or modulators, amylin analogs, G protein coupled receptor 119(GPR119) agonists, GPR40 agonists, GPR120 agonists, GPR142 agonists, systemic or low absorption TGR5 agonists, diabetic immunotherapy, anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, adenosine kinase activators (GPR119), GPR40 agonists, GPR120 agonists, GPR142 agonists, GPR β agonists, inhibitors of glucose-acyl kinase, inhibitors of glucose-acyl transferase inhibitors, combinations thereof, and inhibitors of glucose-acyl transferase inhibitors of glucose-activating enzyme (GAG 3, and GLYC 3.

51. The method of any one of claims 29-50, wherein the antibody or antigen-binding fragment thereof reduces LDL-C levels in the patient by at least 40%.

52. The method of any one of claims 29-51, wherein said antibody or antigen-binding fragment thereof reduces non-HDL-C levels in said subject by at least 35%.

53. The method of any one of claims 29-52, wherein said antibody or antigen-binding fragment thereof reduces ApoC3 levels in said patient.

54. The method of any one of claims 29-53, wherein the antibody or antigen-binding fragment thereof reduces the number and/or size of lipoprotein particles in the patient.

55. The method of any one of claims 29-54, wherein the antibody or antigen-binding fragment thereof:

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

(b) Does not affect fasting blood glucose (FPG) levels in the patient.

56. A method for treating hypercholesterolemia in a patient having type 2 diabetes (T2DM), the method comprising:

(a) selecting a high cardiovascular risk patient receiving insulin treatment with (i) T2DM and (ii) hypercholesterolemia not adequately controlled by maximally tolerated statin treatment; and

57. A method for treating hypercholesterolemia in a patient having T2DM and atherosclerotic cardiovascular disease (ASCVD), the method comprising:

58. The method of claim 56, wherein said ASCVD is defined as Coronary Heart Disease (CHD), ischemic stroke, or peripheral artery disease.

59. The method of claim 58, wherein the CHD comprises acute myocardial infarction, asymptomatic myocardial infarction, and unstable angina.

60. The method of any one of claims 57-59, wherein 75mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

61. The method of any one of claims 57-59, wherein 150mg of the antibody or antigen-binding fragment is administered to the patient every two weeks.

62. The method of any one of claims 57-59, wherein 300mg of the antibody or antigen-binding fragment is administered to the patient every four weeks.

63. The method of any one of claims 57-62, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs shown in SEQ ID NOs 2, 3, and 4, and three light chain CDRs shown in SEQ ID NOs 7, 8, and 10.

64. The method of any one of claims 57-63, wherein the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having the amino acid sequence of SEQ ID NO:1 and a Light Chain Variable Region (LCVR) having the amino acid sequence of SEQ ID NO: 6.

65. The method of any one of claims 57-62, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: alikumab, efuzumab, bococizumab, ludwizumab, ralpancizumab and LY 3015014.

66. The method of claim 65, wherein the antibody or antigen-binding fragment thereof is Alikumab.

67. The method of any one of claims 57-66, further comprising:

68. The method of any one of claims 57-66, further comprising:

69. The method of claim 67 or 68, wherein the threshold level is 70 mg/dL.

70. The method of any one of claims 57-69, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously.

71. The method of any one of claims 57-70, wherein the patient further receives concomitant Lipid Modification Therapy (LMT).

72. The method of claim 71, wherein the LMT is selected from the group consisting of: statins, cholesterol absorption inhibitors, fibrates, nicotinic acids, omega-3 fatty acids, and bile acid sequestrants.

73. The method of claim 72, wherein the LMT is a statin therapy.

74. The method of claim 73, wherein said statin is selected from the group consisting of: atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, pitavastatin and cerivastatin.

75. The method of any one of claims 72-74, wherein the statin treatment is a maximum tolerated dose statin treatment.

76. The method of claim 72, wherein the cholesterol absorption inhibitor is ezetimibe.

77. The method of any one of claims 57-72 and 76, wherein the patient is intolerant to statins.

78. The method of any one of claims 57-77, wherein the insulin treatment is selected from the group consisting of: human insulin, insulin glargine, insulin glulisine, insulin detemir, insulin lispro, insulin degluvium, insulin aspart and basal insulin.

79. The method of any one of claims 57-78, wherein the patient receives concomitant anti-diabetic therapy in addition to insulin therapy.

80. The method of claim 79, wherein the additional anti-diabetic treatment is selected from the group consisting of glucagon-like peptide 1(GLP-1) treatment, gastrointestinal peptides, glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, ghrelin antagonists or inverse agonists, xenin analogs, biguanides, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, α -glucosidase inhibitors, sodium-dependent glucose transporter 2(SGLT-2) inhibitors, SGLT-1 inhibitors, peroxisome proliferator-activated receptor (PPAR-) (α, γ, or α/γ) agonists or modulators, amylin analogs, G protein coupled receptor 119(GPR119) agonists, GPR40 agonists, GPR120 agonists, GPR142 agonists, systemic or low absorption TGR5 agonists, diabetic immunotherapy, anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, adenosine kinase activators (GPR119), GPR40 agonists, GPR120 agonists, GPR142 agonists, GPR β agonists, inhibitors of glucose-acyl kinase, inhibitors of glucose-acyl transferase inhibitors, combinations thereof, and inhibitors of glucose-acyl transferase inhibitors of glucose-activating enzyme (GAG 3, and GLYC 3.

81. The method of any one of claims 57-80, wherein the antibody or antigen-binding fragment thereof reduces LDL-C levels in the patient by at least 40%.

82. The method of any one of claims 57-81, wherein said antibody or antigen-binding fragment thereof reduces non-HDL-C levels in said subject by at least 35%.

83. The method of any one of claims 57 to 82, wherein the antibody or antigen-binding fragment thereof reduces ApoC3 levels in the patient.

84. The method of any one of claims 57-83, wherein the antibody or antigen-binding fragment thereof reduces the number and/or size of lipoprotein particles in the patient.

85. The method of any one of claims 57-84, wherein the antibody or antigen-binding fragment thereof:

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

(b) Does not affect fasting blood glucose (FPG) levels in the patient.

86. A method for treating hypercholesterolemia in a patient having T2DM and ASCVD, the method comprising: