Stable formulations containing anti-PCSK9 antibodies

Abstract

The present invention relates to methods of treating or preventing cholesterol-related diseases (such as hypercholesterolemia, hyperlipidemia or dyslipidemia) using antibodies against proprotein convertase subtilisin / kexin type 9 (PCSK9). Also described are formulations and methods of producing the formulations.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 61 / 642,363, filed May 3, 2012, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference Sequence Listing

[0004] This application incorporates by reference all subject matter contained in the attached sequence listing, which is in txt format, identified by the file name "A-1635-US-NP2_Seq_List.txt", created on May 1, 2013, and has a file size of 306 KB. Field of the Invention

[0005] The present invention relates to methods for treating or preventing cholesterol-related diseases (such as hypercholesterolemia, hyperlipidemia, or dyslipidemia) using antigen-binding proteins (including antibodies) directed against proprotein convertase subtilisin / kexin type 9 (PCSK9). Pharmaceutical formulations and methods for producing the formulations are also described. background

[0006] "Cholesterol-related diseases" (which include "serum cholesterol-related diseases") include any one or more of the following: hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and (in general) dyslipidemia, which can manifest as, for example, elevated total serum cholesterol, elevated LDL, elevated triglycerides, elevated VLDL, and / or low HDL. In fact, hypercholesterolemia is an established risk factor for coronary heart disease (CHD) in humans. Lowering low-density lipoprotein cholesterol (LDL-C) reduces cardiovascular risk and is the main goal of drug therapy for CHD. Statins (hydroxymethylglutaryl coenzyme A [HMGCoA] reductase inhibitors) are currently the first choice for the treatment of hypercholesterolemia. However, emerging data indicate that more aggressive hypercholesterolemia treatment is associated with a lower risk of CHD events. In addition, a proportion of patients are intolerant to or do not respond adequately to statin therapy. Therefore, new therapies that can be used alone or in combination with existing agents to more effectively lower LDL-C may be useful.

[0007] It is well established that the recycling of low-density lipoprotein receptors (LDLRs) on the surface of hepatocytes plays a key role in maintaining cellular and systemic cholesterol balance by regulating plasma LDL-C levels. More recently, it has been shown that proprotein convertase subtilisin / kexin type 9 (PCSK9) plays an important role in the recycling and regulation of LDLRs. PCSK9 is a member of the subtilisin family of serine proteases and is primarily expressed in the liver. After secretion, it causes post-translational downregulation of the LDLRs on the surface of hepatocytes by a mechanism involving direct binding to the LDLRs. Downregulation of the liver LDLRs, in turn, results in increased levels of circulating LDL-C. Therefore, PCSK9 may represent a target for inhibition by novel therapeutic agents in the context of hypercholesterolemia. A strong theoretical basis for the described approach can be obtained from studies in preclinical models and from the following findings: people with PCSK9 loss-of-function mutations have lower-than-normal cholesterol levels and a reduced incidence of CHD.

[0008] Overview of various implementation plans

[0009] In some aspects, the present invention includes a method for lowering a patient's serum LDL cholesterol level. The method comprises administering at least one anti-PCSK9 antibody as described herein to a patient in need thereof at a dose of about 10 mg to about 1400 mg. In some embodiments, the dose is about 10 mg to about 70 mg of at least one anti-PCSK9 antibody, administered once a week (QW). In some embodiments, the dose is about 14 mg to about 45 mg of at least one anti-PCSK9 antibody, administered once a week. In some embodiments, the dose is about 14 mg to about 35 mg of at least one anti-PCSK9 antibody, administered once a week. In some embodiments, the dose is about 70 mg to about 420 mg of at least one anti-PCSK9 antibody, administered once every 2 weeks (Q2W). In some embodiments, the dose is about 70 mg to about 350 mg of at least one anti-PCSK9 antibody, administered once every 2 weeks (Q2W). In some embodiments, the dose is about 105 mg to about 350 mg of at least one anti-PCSK9 antibody, administered once every 2 weeks (Q2W). In some embodiments, the dose is about 140 mg to about 280 mg of at least one anti-PCSK9 antibody administered once every 2 weeks (Q2W). In some embodiments, the dose is about 250 mg to about 480 mg of at least one anti-PCSK9 antibody administered once every 4 weeks (Q4W). In some embodiments, the dose is about 280 mg to about 420 mg of at least one anti-PCSK9 antibody administered once every 2 weeks (Q2W). In some embodiments, the dose is about 350 mg to about 420 mg of at least one anti-PCSK9 antibody administered once every 2 weeks (Q4W). In some embodiments, serum LDL cholesterol levels are reduced by at least about 30%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 35%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 40%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 45%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 50%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 55%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 60%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 75%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 70%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 75%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 80%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 85%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 90%.

[0010] In some aspects, the present invention includes a method of reducing serum LDL cholesterol levels in a patient, the method comprising administering to a patient in need thereof a dose of at least one anti-PCSK9 antibody, wherein the dose of the anti-PCSK9 antibody is administered on a schedule selected from the group consisting of: (1) at least about 14 mg per week (QW); (2) at least about 35 mg per week (QW); (3) at least about 70 mg per every other week (Q2W); (4) at least about 105 mg per every two weeks (Q2W); (5) at least about 140 mg per every other week (Q2W); (6) at least about 280 mg per every two weeks (Q2W); and (7) at least about 280 mg per every four weeks (Q4W); (8) at least about 350 mg per every four weeks (Q4W); and (9) at least about 420 mg per every four weeks (Q4W). In some embodiments, serum LDL cholesterol levels are reduced by at least about 30%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 35%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 40%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 45%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 50%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 55%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 60%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 65%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 70%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 75%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 80%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 85%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 90%.

[0011] In some aspects, the present invention includes a method of reducing a PCSK9 level in a patient, the method comprising administering to a patient in need thereof a dose of at least one anti-PCSK9 antibody, wherein the dose of the anti-PCSK9 antibody is administered according to a schedule selected from the group consisting of: (1) at least about 14 mg per week (QW); (2) at least about 35 mg per week (QW); (3) at least about 70 mg per every other week (Q2W); (4) at least about 105 mg per every two weeks (Q2W); (5) at least about 140 mg per every other week (Q2W); (6) at least about 280 mg per every other week (Q2W); and (7) at least about 280 mg per every four weeks (Q4W); (8) at least about 350 mg per every four weeks (Q4W); and (9) at least about 420 mg per every four weeks (Q4W). In some embodiments, the serum PCSK9 level is reduced by at least about 60%. In some embodiments, the serum PCSK9 value is reduced by at least about 65%. In some embodiments, the serum PCSK9 value is reduced by at least about 70%. In some embodiments, the serum PCSK9 value is reduced by at least about 75%. In some embodiments, the serum PCSK9 value is reduced by at least about 80%. In some embodiments, the serum PCSK9 value is reduced by at least about 85%. In some embodiments, the serum PCSK9 value is reduced by at least about 90%.

[0012] In some aspects, the present invention includes a method of reducing total cholesterol levels in a patient, the method comprising administering to a patient in need thereof a dose of at least one anti-PCSK9 antibody, wherein the dose of the anti-PCSK9 antibody is administered according to a schedule selected from the group consisting of: (1) at least about 14 mg per week (QW); (2) at least about 35 mg per week (QW); (3) at least about 70 mg per every other week (QW); (4) at least about 105 mg per every other week (QW); (5) at least about 140 mg per every other week (Q2W); (6) at least about 280 mg per every other week (Q2W); and (7) at least about 280 mg per every four weeks (Q4W); (8) at least about 350 mg per every four weeks (Q4W); and (9) at least about 420 mg per every four weeks (Q4W). In some embodiments, the total cholesterol level is reduced by at least about 20%. In some embodiments, the total cholesterol level is reduced by at least about 25%. In some embodiments, the total cholesterol level has been reduced by at least about 30%. In some embodiments, the total cholesterol level has been reduced by at least about 35%. In some embodiments, the total cholesterol level has been reduced by at least about 40%. In some embodiments, the total cholesterol level has been reduced by at least about 45%. In some embodiments, the total cholesterol level has been reduced by at least about 50%. In some embodiments, the total cholesterol level has been reduced by at least about 55%. In some embodiments, the total cholesterol level has been reduced by at least about 60%.

[0013] In some aspects, the present invention includes a method of reducing non-HDL cholesterol levels in a patient, the method comprising administering to a patient in need thereof a dose of at least one anti-PCSK9 antibody, wherein the dose of the anti-PCSK9 antibody is administered according to a schedule selected from the group consisting of: (1) at least about 14 mg per week (QW); (2) at least about 35 mg per week (QW); (3) at least about 70 mg per every other week (QW); (4) at least about 105 mg per every other week (QW); (5) at least about 140 mg per every other week (Q2W); (6) at least about 280 mg per every other week (Q2W); and (7) at least about 280 mg per every four weeks (Q4W); (8) at least about 350 mg per every four weeks (Q4W); and (9) at least about 420 mg per every four weeks (Q4W). In some embodiments, the non-HDL cholesterol level is reduced by at least about 30%. In some embodiments, the non-HDL cholesterol level is reduced by at least about 35%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 40%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 45%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 50%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 55%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 60%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 65%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 70%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 75%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 80%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 85%.

[0014] In some aspects, the present invention includes a method of reducing ApoB levels in a patient, the method comprising administering to a patient in need thereof a dose of at least one anti-PCSK9 antibody, wherein the dose of the anti-PCSK9 antibody is administered according to a schedule selected from the group consisting of: (1) at least about 14 mg weekly (QW); (2) at least about 35 mg weekly (QW); (3) at least about 70 mg every other week (QW); (4) at least about 105 mg every other week (QW); (5) at least about 140 mg every other week (Q2W); (6) at least about 280 mg every other week (Q2W); and (7) at least about 280 mg every four weeks (Q4W); (8) at least about 350 mg every four weeks (Q4W); and (9) at least about 420 mg every four weeks (Q4W). In some embodiments, the ApoB level is reduced by at least about 20%. In some embodiments, the ApoB level is reduced by at least about 25%. In some embodiments, ApoB levels are reduced by at least about 30%. In some embodiments, ApoB levels are reduced by at least about 35%. In some embodiments, ApoB levels are reduced by at least about 40%. In some embodiments, ApoB levels are reduced by at least about 45%. In some embodiments, ApoB levels are reduced by at least about 50%. In some embodiments, ApoB levels are reduced by at least about 55%. In some embodiments, ApoB levels are reduced by at least about 60%. In some embodiments, ApoB levels are reduced by at least about 65%. In some embodiments, ApoB levels are reduced by at least about 70%. In some embodiments, ApoB levels are reduced by at least about 75%.

[0015] In some aspects, the present invention includes a method of reducing lipoprotein A ("Lp(a)") levels in a patient, the method comprising administering to a patient in need thereof a dose of at least one anti-PCSK9 antibody, wherein the dose of the anti-PCSK9 antibody is administered according to a schedule selected from the group consisting of: (1) at least about 14 mg weekly (QW); (2) at least about 35 mg weekly (QW); (3) at least about 70 mg every other week (QW); (4) at least about 105 mg every other week (QW); (5) at least about 140 mg every other week (Q2W); (6) at least about 280 mg every other week (Q2W); and (7) at least about 280 mg every four weeks (Q4W); (8) at least about 350 mg every four weeks (Q4W); and (9) at least about 420 mg every four weeks (Q4W). In some embodiments, the Lp(a) level is reduced by at least about 10%. In some embodiments, Lp(a) levels are reduced by at least about 15%. In some embodiments, Lp(a) levels are reduced by at least about 20%. In some embodiments, Lp(a) levels are reduced by at least about 25%. In some embodiments, Lp(a) levels are reduced by at least about 30%. In some embodiments, Lp(a) levels are reduced by at least about 35%. In some embodiments, Lp(a) levels are reduced by at least about 40%. In some embodiments, Lp(a) levels are reduced by at least about 45%. In some embodiments, Lp(a) levels are reduced by at least about 50%. In some embodiments, Lp(a) levels are reduced by at least about 55%. In some embodiments, Lp(a) levels are reduced by at least about 60%. In some embodiments, Lp(a) levels are reduced by at least about 65%.

[0016] In some aspects, the present invention includes a method for treating or preventing a cholesterol-related disease in a patient, comprising administering to a patient in need thereof a dose of about 10 mg to about 480 mg of at least one anti-PCSK9 antibody described herein. In some embodiments, the dose is about 10 mg to about 70 mg of at least one anti-PCSK9 antibody administered once weekly (QW). In some embodiments, the dose is about 14 mg to about 35 mg of at least one anti-PCSK9 antibody administered once weekly. In some embodiments, the dose is about 70 mg to about 420 mg of at least one anti-PCSK9 antibody administered once every 2 weeks (Q2W). In some embodiments, the dose is about 70 mg to about 350 mg of at least one anti-PCSK9 antibody administered once every 2 weeks (Q2W). In some embodiments, the dose is about 105 mg to about 350 mg of at least one anti-PCSK9 antibody administered once every 2 weeks (Q2W). In some embodiments, the dose is about 140 mg to about 280 mg of at least one anti-PCSK9 antibody administered once every 2 weeks (Q2W). In some embodiments, the dose is about 250 mg to about 480 mg of at least one anti-PCSK9 antibody administered once every four weeks (Q4W). In some embodiments, the dose is about 280 mg to about 420 mg of at least one anti-PCSK9 antibody administered once every two weeks (Q2W). In some embodiments, the dose is about 350 mg to about 420 mg of at least one anti-PCSK9 antibody administered once every two weeks (Q4W). In some embodiments, serum LDL cholesterol levels are reduced by at least about 30%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 35%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 40%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 45%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 50%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 55%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 60%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 65%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 70%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 75%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 80%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 85%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 90%. In some embodiments, the cholesterol-related disease is hypercholesterolemia, hyperlipidemia, or dyslipidemia.

[0017] In some aspects, the present invention includes a method of treating or preventing a cholesterol-related disease in a patient, the method comprising administering to a patient in need thereof a dose of at least one anti-PCSK9 antibody, wherein the dose of the anti-PCSK9 antibody is administered according to a schedule selected from the group consisting of: (1) at least about 14 mg per week (QW); (2) at least about 35 mg per week (QW); (3) at least about 70 mg per every other week (QW); (4) at least about 105 mg per every other week (QW); (5) at least about 140 mg per every other week (Q2W); (6) at least about 280 mg per every other week (Q2W); and (7) at least about 280 mg per every four weeks (Q4W); (8) at least about 350 mg per every four weeks (Q4W); and (9) at least about 420 mg per every four weeks (Q4W). In some embodiments, serum LDL cholesterol levels are reduced by at least about 30%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 35%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 40%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 45%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 50%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 55%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 60%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 65%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 70%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 75%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 80%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 85%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 90%.

[0018] In some embodiments, the anti-PCSK9 antibody is 21B12, 26H5, 31H4, 8A3, 11F1, and / or 8A1.

[0019] In some embodiments, the cholesterol-related disease is hypercholesterolemia, hyperlipidemia, or dyslipidemia.

[0020] In some aspects, the invention includes pharmaceutical formulations comprising at least one anti-PCSK9 antibody selected from the group consisting of 21B12, 26H5, 31H4, 8A3, 11F1, and 8A1.

[0021] Other embodiments of the present invention will be readily apparent from the disclosure provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A The amino acid sequence of the mature form of PCSK9 is depicted with the prodomain underlined.

[0023] Figures 1B1 to 1B4 The amino acid and nucleic acid sequences of PCSK9 are depicted with the prodomain underlined and the signal sequence in bold.

[0024] Figures 2A-2D The following is a sequence comparison table of various light chains of different antigen-binding proteins. Figure 2C Continue Figure 2A The sequence that starts in . Figure 2D Continue Figure 2B The sequence that starts above.

[0025] Figures 3A-3D The following is a sequence comparison table of various heavy chains of different antigen binding proteins. Figure 3C Continue Figure 3A The sequence that starts in . Figure 3D Continue Figure 3B The sequence that starts above.

[0026] Figures 3E-3JJ The amino acid and nucleic acid sequences of the variable domains of some embodiments of the antigen binding proteins are described.

[0027] Figure 3KK The amino acid sequences of the various constant domains are depicted.

[0028] Figure 3LL-3BBB The amino acid and nucleic acid sequences of the variable domains of some embodiments of the antigen binding proteins are described.

[0029] Figure 3CCC-3JJJ A sequence comparison table of various heavy and light chains of some embodiments of antigen binding proteins.

[0030] Figure 4A This is the binding curve of the antigen binding protein to human PCSK9.

[0031] Figure 4B This is the binding curve of the antigen binding protein to human PCSK9.

[0032] Figure 4C This is the binding curve of the antigen binding protein and cynomolgus macaque PCSK9.

[0033] Figure 4D This is the binding curve of the antigen binding protein and cynomolgus macaque PCSK9.

[0034] Figure 4E This is the binding curve of the antigen binding protein to mouse PCSK9.

[0035] Figure 4F This is the binding curve of the antigen binding protein to mouse PCSK9.

[0036] Figure 5A Describes the results of SDS PAGE experiments involving PCSK9 and various antigen binding proteins showing the relative purity and concentration of the proteins.

[0037] Figure 5B and 5C Depicts a graph from a Biacore solution equilibrium assay for 21B12.

[0038] Figure 5D Graph depicting kinetics from a Biacore capture assay.

[0039] Figure 5E Depicts a bar graph depicting the binning results of the three ABPs.

[0040] Figure 6A Inhibition curve of antigen binding protein 31H4 IgG2 against PCSK9 in an in vitro PCSK9:LDLR binding assay.

[0041] Figure 6B Inhibition curve of antigen binding protein 31H4 IgG4 against PCSK9 in an in vitro PCSK9:LDLR binding assay.

[0042] Figure 6C Inhibition curve of antigen binding protein 21B12 IgG2 against PCSK9 in an in vitro PCSK9:LDLR binding assay.

[0043] Figure 6D Inhibition curve of antigen binding protein 21B12 IgG4 against PCSK9 in an in vitro PCSK9:LDLR binding assay.

[0044] Figure 7A Figure 3 is the inhibition curve of antigen binding protein 31H4 IgG2 in a cellular LDL uptake assay, which shows the effect of ABP in reducing the LDL uptake blocking effect of PCSK9.

[0045] Figure 7B Figure 3 is the inhibition curve of antigen binding protein 31H4 IgG4 in a cellular LDL uptake assay, which shows the effect of ABP in reducing the LDL uptake blocking effect of PCSK9.

[0046] Figure 7C Figure 2 is the inhibition curve of antigen binding protein 21B12 IgG2 in a cellular LDL uptake assay, which shows the effect of ABP in reducing the LDL uptake blocking effect of PCSK9.

[0047] Figure 7D Figure 2 is the inhibition curve of antigen binding protein 21B12 IgG4 in a cellular LDL uptake assay, which shows the effect of ABP in reducing the LDL uptake blocking effect of PCSK9.

[0048] Figure 8A Graph depicting the ability of ABP 31H4 to lower serum cholesterol in mice, where changes are relative to IgG control-treated mice (*p<0.01).

[0049] Figure 8B Graph depicting the ability of ABP 31H4 to lower serum cholesterol in mice, where changes are relative to time = 0 hours (#p, 0.05).

[0050] Figure 8C Graph depicting the effect of ABP 31H4 on HDL cholesterol levels in C57B1 / 6 mice (*p<0.01).

[0051] Figure 8D Graph depicting the effect of ABP 31H4 on HDL cholesterol levels in C57B1 / 6 mice (#p<0.05).

[0052] Figure 9 Western blot analysis depicting the ability of ABP 31H4 to increase the amount of liver LDLR protein present after various time points.

[0053] Figure 10A Graph depicting the ability (relative value) of antigen binding protein 31H4 to lower total serum cholesterol in wild-type mice.

[0054] Figure 10B Graph depicting the ability of antigen binding protein 31H4 to lower HDL in wild-type mice.

[0055] Figure 10C Graph depicting the ability of different antigen binding proteins 31H4 and 16F12 to lower serum cholesterol.

[0056] Figure 11A Describes the injection protocol used to test the duration and ability of antigen binding proteins to lower serum cholesterol.

[0057] Figure 11B To describe Figure 11A Figure 2 shows the results of the scheme in .

[0058] Figure 12A Depicted are LDLR levels in HepG2 cells in response to a combination of statins and ABP 21B12.

[0059] Figure 12BDepicted are LDLR levels in HepG2 cells in response to a combination of statins and ABP 31H4.

[0060] Figure 12C Depicted are LDLR levels in HepG2 cells in response to a combination of statins and ABP 25A7.1, a non-neutralizing antibody (as distinct from the neutralizing antibody "25A7").

[0061] Figure 12D Describes LDLR levels in HepG2 cells overexpressing PCSK9 in response to a combination of statins and ABP 21B12.

[0062] Figure 12E Describes LDLR levels in HepG2 cells overexpressing PCSK9 in response to a combination of statins and ABP 31H4.

[0063] Figure 12F Depicted are LDLR levels in HepG2 cells that overexpress PCSK9 in response to a combination of statins and ABP 25A7.1, a non-neutralizing antibody (as distinct from the neutralizing antibody "25A7").

[0064] Figure 13A Depicts the amino acid sequences of various light chains of different ABPs against PCSK9. A dot (.) indicates the absence of an amino acid.

[0065] Figure 13B Light chain cladogram depicting different ABPs targeting PCSK9.

[0066] Figure 13C Depicts the amino acid sequences of various heavy chains of different ABPs against PCSK9. A dot (.) indicates the absence of an amino acid.

[0067] Figure 13D Dendrogram depicting the heavy chain of different ABPs targeting PCSK9.

[0068] Figure 13E A comparison of light and heavy chain CDRs and the assignment of consensus sequence generating groups is described.

[0069] Figure 13F The consensus sequences for groups 1 and 2 are described.

[0070] Figure 13G The consensus sequences for groups 3 and 4 are described.

[0071] Figure 13H Depicts the consensus sequence for Groups 1 and 2. Dots (.) indicate identical residues.

[0072] Figure 13IDescribes the consensus sequence of Group 2. Dots (.) indicate consensus residues.

[0073] Figure 13J Depicts the consensus sequence for groups 3 and 4. Dots (.) indicate identical residues.

[0074] Figure 14 Graph showing reduction in LDL-c levels in patients receiving multiple doses of an anti-PCSK9 antibody (21B12).

[0075] Figure 15 Graph showing reduction in LDL-c levels in patients taking low- to moderate- and high-dose statins who received multiple doses of an anti-PCSK9 antibody (21B12).

[0076] Figure 16 Graph showing reduction in ApoB levels in patients receiving multiple doses of an anti-PCSK9 antibody (21B12).

[0077] Figure 17 Bar graph showing reductions in lipoprotein a ("Lp(a)") levels in patients taking low- to moderate- and high-dose statins who received multiple doses of an anti-PCSK9 antibody (21B12).

[0078] Figure 18 Graph showing reduction in LDL-c levels in patients with heterozygous familial hypercholesterolemia ("HeFH") who received multiple doses of an anti-PCSK9 antibody (21B12).

[0079] Figure 19 Graph showing reduction in PCSK9 levels in patients with heterozygous familial hypercholesterolemia ("HeFH") who received multiple doses of an anti-PCSK9 antibody (21B12).

[0080] Figure 20 Graph showing reduction in total cholesterol levels in patients with heterozygous familial hypercholesterolemia ("HeFH") who received multiple doses of an anti-PCSK9 antibody (21B12).

[0081] Figure 21 Graph showing reduction in non-HDL cholesterol levels in patients with heterozygous familial hypercholesterolemia ("HeFH") who received multiple doses of an anti-PCSK9 antibody (21B12).

[0082] Figure 22 Graph showing reduction in ApoB levels in patients with heterozygous familial hypercholesterolemia ("HeFH") who received multiple doses of an anti-PCSK9 antibody (21B12).

[0083] Figure 23Bar graph showing reduction in lipoprotein a ("Lp(a)") in patients with heterozygous familial hypercholesterolemia ("HeFH") who received multiple doses of an anti-PCSK9 antibody (21B12).

[0084] Figure 24A Graph showing pooled data associated with LDL-C lowering for patients from the four studies described in Examples 22-25 who received varying doses of an anti-PCSK9 antibody (21B12) every other week (Q2W) for 12 weeks.

[0085] Figure 24B Graph showing pooled data associated with LDL-C lowering for patients from the four studies described in Examples 22-25 who received varying doses of an anti-PCSK9 antibody (21B12) every four weeks (Q4W) for 12 weeks.

[0086] Figure 25A Bar graph showing pooled data associated with Lp(a) reduction in patients from the four studies described in Examples 22-25 who received different doses of an anti-PCSK9 antibody (21B12) every other week (Q2W) or every 4 weeks (Q4W) over 12 weeks.

[0087] Figure 25B Bar graph showing pooled data associated with HDL-C lowering for patients from the four studies described in Examples 22-25 who received different doses of anti-PCSK9 antibody (21B12) every other week (Q2W) or every 4 weeks (Q4W) over 12 weeks.

[0088] Figure 25C Bar graph showing pooled data associated with triglyceride lowering in patients from the four studies described in Examples 22-25 who received different doses of anti-PCSK9 antibody (21B12) every other week (Q2W) or every 4 weeks (Q4W) over 12 weeks.

[0089] Figure 25D Bar graph showing pooled data associated with VLDL-C lowering for patients from the four studies described in Examples 22-25 who received different doses of anti-PCSK9 antibody (21B12) every other week (Q2W) or every 4 weeks (Q4W) over 12 weeks.

[0090] Figure 26 Bar graph showing the viscosity of anti-PCSK9 antibody (21B12) formulations containing different stabilizers / excipients.

[0091] Figure 27Graph demonstrating that the stabilizer / excipient proline can reduce the viscosity of an anti-PCSK9 antibody (21B12) formulation with high protein concentration.

[0092] Figure 28A Graph showing the viscosity of the anti-PCSK9 antibody 21B12 at different concentrations in a formulation comprising 10 mM sodium acetate and 9% sucrose (pH 5.2) at 25°C and 40°C.

[0093] Figure 28B Graph showing the viscosity of different concentrations of the anti-PCSK9 antibody 21B12 in a formulation comprising 10 mM sodium acetate and 9% sucrose, pH 5.2, at 25°C and 40°C, compared to a formulation comprising 10 mM sodium acetate, 125 mM arginine, and 3% sucrose, pH 5.0, at 25°C and 40°C.

[0094] Figure 28C Graph showing the viscosity of different concentrations of the anti-PCSK9 antibody 21B12 in a formulation comprising 10 mM sodium acetate and 9% sucrose, pH 5.2, at 25°C and 40°C, compared to a formulation comprising 10 mM sodium acetate, 100 mM methionine, and 4% sucrose, pH 5.0, at 25°C and 40°C.

[0095] Figure 28D Graph showing the viscosity of different concentrations of anti-PCSK9 antibody 21B12 in a formulation comprising 10 mM sodium acetate and 9% sucrose (pH 5.2) at 25°C and 40°C, compared to a formulation comprising 10 mM sodium acetate and 250 mM proline (pH 5.0) at 25°C and 40°C.

[0096] Figure 29A FIG2 is a bar graph showing the number of 10 μm particles in different formulations of anti-PCSK9 antibody (ie, 21B12) over 6 months.

[0097] Figure 29B FIG2 is a bar graph showing the number of 25 μm particles in different formulations of anti-PCSK9 antibody (ie, 21B12) over 6 months.

[0098] Figure 30A FIG2 is a bar graph showing the number of 10 μm particles in different formulations of anti-PCSK9 antibody (ie, 11F1) over 4 months.

[0099] Figure 30B FIG2 is a bar graph showing the number of 25 μm particles in different formulations of anti-PCSK9 antibody (ie, 11F1) over 4 months.

[0100] Figure 31Graph illustrating the binding specificity of 11F1 to PCSKP, PCSK2, PCSK1, PCSK7, and furin in competition assays, where OD 450 Plotted on the vertical axis and the concentration of PCSK9 (ug / ml) is plotted on the horizontal axis.

[0101] Figure 32 Graph showing the dose-response curve of 11F1 inhibition of LDLR:D374Y PCSK9 binding in a competition assay, where OD 450 Plotted on the vertical axis and Log[11F1](pM) on the horizontal axis.

[0102] Figure 33 Graph depicting the dose-response curve for 11F1 inhibition of LDLR:WT PCSK9 binding in a competition assay, where OD 450 Plotted on the vertical axis and Log[11F1](pM) on the horizontal axis.

[0103] Figure 34 Graph depicting the dose-response curve for the ability of 11F1 to block the reduction in LDL uptake mediated by human D374Y PCSK9 in HepG2 cells, where relative fluorescence units (×10 4 ) is plotted on the vertical axis and Log[11F1](nM) is plotted on the horizontal axis.

[0104] Figure 35 Graph depicting the dose-response curve for the ability of 11F1 to block the reduction in LDL uptake mediated by human WT PCSK9 in HepG2 cells, with relative fluorescence units plotted (×10 4 ) is plotted on the vertical axis and Log[11F1](nM) is plotted on the horizontal axis.

[0105] Figure 36 Bar graph depicting the effects of 11F1 and 8A3 on serum non-HDL cholesterol in mice expressing human PCSK9 by AAV, with non-HDL-C serum concentration (mg / ml) plotted on the vertical axis and time after injection (days) plotted on the horizontal axis.

[0106] Figure 37 Bar graph depicting the effects of 11F1 and 8A3 on serum total cholesterol in mice expressing human PCSK9 by AAV, with serum total cholesterol (mg / ml) plotted on the vertical axis and time after injection (days) plotted on the horizontal axis.

[0107] Figure 38Bar graph depicting the effects of 11F1 and 8A3 on serum HDL cholesterol (HDL-C) in mice expressing human PCSK9 by AAV, with HDL-C (mg / ml) plotted on the vertical axis and time post-injection (days) plotted on the horizontal axis.

[0108] Figure 39 Graph depicting the IgG2, 8A3, and 11F1 antibody concentration profiles in mice expressing human PCSK9 by AAV, with serum antibody concentration (ng / mL) plotted on the vertical axis and time post-injection in days plotted on the horizontal axis.

[0109] Figure 40 Table summarizing the PK parameters of IgG2, 11F1, and 8A3 in mice expressing human PCSK9 from AAV.

[0110] Figure 41 Graph depicting the effect of a single subcutaneous administration of anti-KLH antibody (control), 21B12, 8A3, and 11F1 on serum LDL concentration (LDL-C) in cynomolgus monkeys, with LDL-C (mg / dl) plotted on the vertical axis and time post-administration in days on the horizontal axis.

[0111] Figure 42 Graph depicting the effect of a single subcutaneous administration of anti-KLH antibody (control), 21B12, 8A3, and 11F1 on serum total cholesterol in cynomolgus monkeys, with total cholesterol concentration (mg / dl) plotted on the vertical axis and time post-administration in days on the horizontal axis.

[0112] Figure 43 Graph depicting the effect of a single subcutaneous administration of anti-KLH antibody (control), 21B12, 8A3, and 11F1 on serum HDL cholesterol in cynomolgus monkeys, with HDL-C (mg / dl) plotted on the vertical axis and time post-administration in days on the horizontal axis.

[0113] Figure 44 Graph depicting the effects of a single subcutaneous administration of anti-KLH antibody (control), 21B12, 8A3, and 11F1 on serum triglycerides in cynomolgus monkeys, with serum triglyceride concentration (mg / dl) plotted on the vertical axis and time post-administration in days on the horizontal axis.

[0114] Figure 45 Graph depicting the effect of a single subcutaneous administration of anti-KLH antibody (control), 21B12, 8A3, and 11F1 on apolipoprotein B (ApoB) in cynomolgus monkeys, with APOB concentration (mg / dl) plotted on the vertical axis and time post-administration in days on the horizontal axis.

[0115] Figure 46 Graph depicting the mean pharmacokinetic profiles of anti-KLH antibody (control), 21B12, 8A3, and 11F1 in cynomolgus monkeys, with antibody concentration (ng / ml) plotted on the vertical axis and time post-administration in days on the horizontal axis.

[0116] Figure 47 Table summarizing the PK parameters of anti-KLH antibody (control), 21B12, 8A3, and 11F1 in cynomolgus monkeys.

[0117] DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

[0118] Disclosed herein are antigen binding proteins (e.g., antibodies and functional binding fragments thereof) that bind to PCSK9. In some embodiments, the antigen binding protein binds to PCSK9 in a different manner and prevents PCSK9 from functioning. In some embodiments, the antigen binding protein blocks or reduces the ability of PCSK9 to interact with other substances. For example, in some embodiments, the antigen binding protein binds to PCSK9 in a manner that prevents or reduces the likelihood that PCSK9 will bind to LDLR. In other embodiments, the antigen binding protein binds to PCSK9 but does not block the ability of PCSK9 to interact with LDLR. In some embodiments, the antigen binding protein is a human monoclonal antibody.

[0119] As will be appreciated by those skilled in the art, according to the present disclosure, altering the interaction between PCSK9 and LDLR can increase the amount of LDLR available for binding to LDL, which in turn can reduce the amount of serum LDL in a subject, thereby reducing the subject's serum cholesterol level. Thus, antigen binding proteins directed against PCSK9 can be used in various methods and formulations for treating subjects with elevated serum cholesterol levels, at risk of elevated serum cholesterol levels, or who may benefit from a reduction in their serum cholesterol levels. Thus, various methods and techniques for lowering serum cholesterol, maintaining serum cholesterol, or preventing an increase in serum cholesterol are also described herein. In some embodiments, the antigen binding protein allows binding between PCSK9 and LDLR, but the antigen binding protein prevents or reduces the adverse activity of PCSK9 on LDLR. In some embodiments, the antigen binding protein prevents or reduces the binding of PCSK9 to LDLR.

[0120] For convenience, the following sections generally summarize the various meanings of the terms used herein. According to this discussion, the overall aspects of antigen binding proteins are discussed, followed by specific examples showing the properties of various embodiments of antigen binding proteins and how they can be used.

[0121] Definition and implementation plan

[0122] It should be understood that both the above general description and the detailed description below are exemplary and illustrative only and do not limit the claimed invention. In this application, unless otherwise expressly stated, the use of the singular includes the plural. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms such as "includes" and "included" is not restrictive. In addition, unless otherwise expressly stated, terms such as "element" or "component" cover the elements and components that constitute a unit and the elements and components that constitute more than one subunit. In addition, the use of the term "part" may include a part or the entire part of a part.

[0123] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0124] The term "proprotein convertase subtilisin / kexin type 9" or "PCSK9" refers to the polypeptide set forth in SEQ ID NO: 1 and / or 3, or fragments thereof, as well as related polypeptides, including but not limited to allelic variants, splice variants, derivative variants, substitution variants, deletion variants, and / or insertion variants, including additions of an N-terminal methionine, fusion polypeptides, and interspecies homologs. In certain embodiments, the PCSK9 polypeptide includes terminal residues, such as but not limited to leader sequence residues, targeting residues, an amino-terminal methionine residue, a lysine residue, a tag residue, and / or a fusion protein residue. "PCSK9" is also known as FH3, NARC1, HCHOLA3, proprotein convertase subtilisin / kexin type 9, and neuronal apoptosis-regulated convertase 1. The PCSK9 gene encodes a proprotein convertase protein belonging to the proteinase K subfamily of the secreted subtilase family. The term "PCSK9" refers to both the proprotein and the products generated following autocatalysis of the proprotein. When referring only to the product of autocatalysis (e.g., for antigen binding proteins that selectively bind to cleaved PCSK9), the protein may be referred to as "mature," "cleaved," "processed," or "active" PCSK9. When referring only to the inactive form, the protein may be referred to as the "inactive," "proform," or "unprocessed" form of PCSK9. The term PCSK9 as used herein also includes naturally occurring alleles, such as the mutations D374Y, S127R, and F216L. The term PCSK9 also encompasses PCSK9 molecules that incorporate post-translational modifications of the PCSK9 amino acid sequence, such as PCSK9 sequences that have been glycosylated, pegylated, PCSK9 sequences in which the signal sequence has been cleaved, PCSK9 sequences in which the prodomain has been cleaved from the catalytic domain but not separated from the catalytic domain (e.g., Figure 1A and Figures 1B1 to 1B4 ).

[0125] The term "PCSK9 activity" includes any biological effect of PCSK9. In certain embodiments, PCSK9 activity includes the ability of PCSK9 to interact or bind to a substrate or receptor. In some embodiments, PCSK9 activity is represented by the ability of PCSK9 to bind to the LDL receptor (LDLR). In some embodiments, PCSK9 binds to LDLR and catalyzes reactions involving LDLR. In some embodiments, PCSK9 activity includes the ability of PCSK9 to alter (e.g., reduce) the availability of LDLR. In some embodiments, PCSK9 activity includes the ability of PCSK9 to increase the amount of LDL in a subject. In some embodiments, PCSK9 activity includes the ability of PCSK9 to reduce the amount of LDLR available for binding to LDL. In some embodiments, "PCSK9 activity" includes any biological activity caused by PCSK9 signaling.Exemplary activities include, but are not limited to, binding of PCSK9 to LDLR, PCSK9 enzymatic activity that cleaves LDLR or other proteins, binding of PCSK9 to proteins other than LDLR that promote the action of PCSK9, alteration of APOB secretion by PCSK9 (Sun X-M et al., "Evidence for effect of mutant PCSK9 on apoliprotein B secretion as the cause of unusually severe dominant hypercholesterolemia", Human Molecular Genetics 14:1161-1169, 2005 and Ouguerram K et al., "Apolipoprotein B100 metabolism in autosomal-dominant hypercholesterolemia related to mutations in PCSK9", Arterioscler thromb Vasc Biol. 24:1448-1453, 2004), and the role of PCSK9 in liver regeneration and neuronal cell differentiation (Seidah NG et al., "The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation", PNAS 100:928-933,2003), and the role of PCSK9 in hepatic glucose metabolism (Costet et al., “Hepatic PCSK9 expression is regulated by nutritional status via insulin and sterol regulatory element-binding protein 1c”. J.Biol.Chem .281(10):6211-18,2006).

[0126] As used herein, the term "hypercholesterolemia" refers to a condition in which cholesterol levels are elevated above a desired level. In some embodiments, this refers to an elevated serum cholesterol level. In some embodiments, the desired level takes into account various "risk factors" known to those of skill in the art (and described or referenced herein).

[0127] The terms "polynucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives; ribose modifications such as 2',3'-dideoxyribose; and internucleotide linkage modifications such as phosphorothioates, phosphorodithioates, selenophosphates, diselenophosphates, anilide phosphorothioates, anilide phosphates, and phosphoamino esters.

[0128] The term "oligonucleotide" means a polynucleotide comprising 200 or less nucleotides. In some embodiments, the oligonucleotide is 10 to 60 bases in length. In other embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 nucleotides in length. The oligonucleotide can be, for example, a single-stranded or double-stranded oligonucleotide for constructing a mutant gene. The oligonucleotide can be a sense or antisense oligonucleotide. The oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection and determination. The oligonucleotide can be, for example, used as a PCR primer, a cloning primer or a hybridization probe.

[0129] By "isolated nucleic acid molecule" is meant a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, which is not associated with all or a portion of a polynucleotide from which the isolated polynucleotide is found in nature, or which is linked to a polynucleotide to which it is not linked in nature. For the purposes of this disclosure, it is understood that a "nucleic acid molecule comprising a specified nucleotide sequence" does not include an entire chromosome. In addition to the specified sequence, an isolated nucleic acid molecule "comprising" a specified nucleic acid sequence may also include coding sequences for up to 10 or even up to 20 other proteins or portions thereof, or may include operably linked regulatory sequences that control the expression of the coding region of the nucleic acid sequence, and / or may include vector sequences.

[0130] Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of a nascent RNA transcript is referred to as the transcription direction; the sequence region from 5' to the 5' end of the RNA transcript on a DNA strand having the same sequence as the RNA transcript is referred to as the "upstream sequence"; the sequence region from 3' to the 3' end of the RNA transcript on a DNA strand having the same sequence as the RNA transcript is referred to as the "downstream sequence."

[0131] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of the coding sequence to which it is linked. The nature of the control sequence may depend on the host organism. In specific embodiments, the control sequence for prokaryotes may include a promoter, a ribosome binding site, and a transcription termination sequence. For example, the control sequence for eukaryotes may include a promoter containing one or more recognition sites for transcription factors, a transcription enhancer sequence, and a transcription termination sequence. A "control sequence" may include a leader sequence and / or a fusion partner sequence.

[0132] The term "vector" is intended to refer to any molecule or entity (eg, nucleic acid, plasmid, phage, or virus) used to transfer protein coding information into a host cell.

[0133] The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and / or controls (along with the host cell) the expression of one or more heterologous coding regions operably linked thereto. An expression construct may include, but is not limited to, sequences that affect or control the transcription, translation, and, if introns are present, RNA splicing of the coding region operably linked thereto.

[0134] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions under appropriate conditions. For example, a control sequence "operably linked" to a protein coding sequence in a vector is linked thereto so as to achieve expression of the protein coding sequence under conditions compatible with the transcriptional activity of the control sequence.

[0135] The term "host cell" means a cell that has been or is capable of being transformed with a nucleic acid sequence and that expresses a gene of interest. The term includes progeny of a parent cell, regardless of whether the progeny is identical to the original parent cell in morphology or genetic makeup, as long as the gene of interest is present.

[0136] The term "transfection" means the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52 :456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13 : 197. The described techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

[0137] The term "transformation" refers to a change in the genetic characteristics of a cell, and when a cell has been modified to contain new DNA or RNA, the cell has been transformed. For example, when a cell is genetically modified from its native state by introducing new genetic material via transfection, transduction or other techniques, the cell is transformed. After transfection or transduction, the transformed DNA can be physically integrated into the chromosome of the cell to recombinant with the DNA of the cell, or can be maintained for a short time in the form of an episomal element without replication, or can be independently replicated in the form of a plasmid. When the transformed DNA replicates with cell division, the cell is considered to have been "stably transformed."

[0138] The terms "polypeptide" or "protein" refer to a macromolecule having the amino acid sequence of a native protein (i.e., a protein produced by naturally occurring, non-recombinant cells); or a molecule produced by genetically engineered or recombinant cells and comprising a molecule having the amino acid sequence of a native protein, or a molecule in which one or more amino acids are missing from, one or more amino acids are added to, and / or one or more amino acids are substituted for the native sequence. The terms also include amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. The terms "polypeptide" and "protein" specifically encompass sequences of PCSK9 antigen-binding proteins, antibodies, or sequences in which one or more amino acids are missing from, one or more amino acids are added to, and / or one or more amino acids are substituted for the antigen-binding protein. The term "polypeptide fragment" refers to a polypeptide having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions compared to the full-length native protein. The fragments may also contain modified amino acids compared to the native protein. In certain embodiments, the fragments are approximately 5 to 500 amino acids in length. For example, the fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Suitable polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of PCSK9 binding antibodies, suitable fragments include, but are not limited to, CDR regions, variable domains of heavy and / or light chains, a portion of an antibody chain or its variable region including only two CDRs, and the like.

[0139] Reference to the term "isolated protein" means that the subject protein (1) is free from at least some of the other proteins with which it would normally be found, (2) is substantially free from other proteins from the same source, e.g., from the same species, (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other substances with which it is associated in nature, (5) is operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not associated in nature, or (6) does not occur in nature. Typically, an "isolated protein" comprises at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof, can encode the isolated protein. Preferably, the isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use.

[0140] The term "amino acid" includes its usual meaning in the art.

[0141] A "variant" of a polypeptide (eg, an antigen binding protein or antibody) comprises an amino acid sequence wherein one or more amino acid residues are inserted, deleted, and / or substituted relative to another polypeptide sequence. Variants include fusion proteins.

[0142] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by alignment and comparison of sequences. "Percent identity" means the percentage of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule in the compared molecules. For these calculations, gaps in the alignment, if any, are preferably processed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48 :1073.

[0143] When calculating percent identity, the sequences being compared are typically aligned in a manner that produces the largest match between the sequences. An example of a computer program that can be used to determine percent identity is the GCG program package (Devereux et al., 1984, Nucl. Acid Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to compare two polypeptides or polynucleotides to be determined for sequence identity percentage. The sequences are aligned to achieve the best match of their respective amino acids or nucleotides (as determined by the algorithm's "match span"). The algorithm uses a gap open penalty (calculated based on 3 × average diagonals, where "average diagonal" is the average value of the diagonals of the judgment matrix used; "diagonal" is the score or value assigned to each complete amino acid match by a specific judgment matrix); and a gap extension penalty (typically 1 / 10 × gap open penalty) and a judgment matrix (such as PAM250 or BLOSum 62). In certain embodiments, the algorithm also uses a standard judgment matrix (for the PAM 250 judgment matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSum62 judgment matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0144] Examples of parameters that can be used to determine percent identity of polypeptide or nucleotide sequences using the GAP program are the following:

[0145] Algorithm: Needleman et al., 1970, J. Mol. Biol. 48: 443-453

[0146] Judgment matrix: BLOSumm 62, from Henikoff et al., 1992, supra

[0147] Gap Penalty: 12 (but no end gap penalty)

[0148] Gap length penalty: 4

[0149] Similarity threshold: 0

[0150] Some alignment schemes used to align two amino acid sequences can only match a relatively short region of the two sequences, and this small aligned region can have a very high sequence identity, even if there is no significant relationship between the two full-length sequences. Therefore, if it is necessary to generate an alignment that spans at least 50 or other numbers of consecutive amino acids of the target polypeptide, the selected alignment method (GAP program) can be adjusted.

[0151] As used herein, the 20 conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), non-natural amino acids (e.g., α,α-disubstituted amino acids), N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components of the polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In polypeptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.

[0152] Similarly, unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of a nascent RNA transcript is referred to as the transcription direction; the sequence region from 5' to the 5' end of the RNA transcript on the DNA strand having the same sequence as the RNA is referred to as the "upstream sequence"; the sequence region from 3' to the 3' end of the RNA transcript on the DNA strand having the same sequence as the RNA is referred to as the "downstream sequence."

[0153] Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reverse or inverted forms of amino acid moieties.

[0154] Naturally occurring residues can be classified based on common side chain properties:

[0155] 1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0156] 2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0157] 3) Acidic: Asp, Glu;

[0158] 4) Basic: His, Lys, Arg;

[0159] 5) Residues that affect chain orientation: Gly, Pro; and

[0160] 6) Aromatic: Trp, Tyr, Phe.

[0161] For example, non-conservative substitutions may involve exchanging a member of one of these classes for a member of another class. Such substituted residues may, for example, be introduced into an area of ​​human antibody homologous to the non-human antibody or into a non-homologous area of ​​the molecule.

[0162] When making changes to the antigen binding protein or PCSK9 protein according to certain embodiments, the hydropathic index of the amino acids can be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0163] The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is understood in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids can be substituted for other amino acids with similar hydropathic indices or scores and still retain similar biological activity. In certain embodiments, when making changes based on the hydropathic index, substitutions of amino acids with a hydropathic index within ±2 are included. In certain embodiments, substitutions of amino acids with a hydropathic index within ±1 are included, and in certain embodiments, substitutions of amino acids with a hydropathic index within ±0.5 are included.

[0164] It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity, especially when the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as in the case of the present invention. In certain embodiments, the maximum local average hydrophilicity of a protein, as governed by the hydrophilicity of its neighboring amino acids, correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

[0165] The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in certain embodiments, substitutions of amino acids with hydrophilicity values ​​within ±2 are included, in certain embodiments, substitutions of amino acids with hydrophilicity values ​​within ±1 are included, and in certain embodiments, substitutions of amino acids with hydrophilicity values ​​within ±0.5 are included. Epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions."

[0166] Exemplary amino acid substitutions are listed in Table 1.

[0167] Table 1

[0168] Amino acid substitution

[0169]

[0170] The term "derivative" refers to a molecule that includes chemical modifications other than insertions, deletions, or substitutions of amino acids (or nucleic acids). In certain embodiments, a derivative comprises a covalent modification, including but not limited to chemical bonding to a polymer, lipid, or other organic or inorganic moiety. In certain embodiments, the chemically modified antigen-binding protein may have a longer circulating half-life than an unmodified antigen-binding protein. In certain embodiments, the targeting ability of the chemically modified antigen-binding protein to a desired cell, tissue, and / or organ may be improved. In some embodiments, the derivatized antigen-binding protein is covalently modified to include one or more water-soluble polymer linkers, including but not limited to polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, for example, the following U.S. Patent Nos.: 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In certain embodiments, the derivatized antigen binding protein comprises one or more polymers including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), and polyvinyl alcohol, as well as mixtures of such polymers.

[0171] In certain embodiments, the derivative is covalently modified with polyethylene glycol (PEG) subunits. In certain embodiments, one or more water-soluble polymers are bonded at one or more specific positions of the derivative, such as at the amino terminus. In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivative. In certain embodiments, PEG is used to improve the therapeutic ability of antigen-binding proteins. In certain embodiments, PEG is used to improve the therapeutic ability of humanized antibodies. Some such methods are, for example, discussed in U.S. Patent No. 6,133,426, which is hereby incorporated by reference herein for any purpose.

[0172] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptide compounds are referred to as "peptide mimetics" or "peptidomimetic compounds." Fauchere, J., Adv. Drug Res., 15:29 (1986); Veber and Freidinger, TINS, p. 392 (1985); and Evans et al., J. Med. Chem., 30:1229 (1987), which are incorporated herein by reference for any purpose. Such compounds are often developed using computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce similar therapeutic or prophylactic effects. In general, peptide mimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide having biochemical properties or pharmacological activity), such as a human antibody, but one or more peptide bonds are optionally replaced by a bond selected from the group consisting of: --CH2NH--, --CH2S--, --CH2-CH2--, --CH=CH- (cis and trans), --COCH2--, --CH(OH)CH2--, and --CH2SO-- by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine for L-lysine) can be used in certain embodiments to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or substantially identical consensus sequence variations can be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem., 61:387 (1992), incorporated herein by reference for any purpose); for example, by adding internal cysteine ​​residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

[0173] As used throughout this specification with respect to biological materials (eg, polypeptides, nucleic acids, host cells, and the like), the term "naturally occurring" means that the material is found in nature or is in a form of the material that is found in nature.

[0174] As used herein, an "antigen binding protein" ("ABP") refers to any protein that binds to a designated target antigen. In this application, the designated target antigen is a PCSK9 protein or a fragment thereof. "Antigen binding protein" includes, but is not limited to, antibodies and binding portions thereof, such as immunologically functional fragments. Peptibodies are another example of an antigen binding protein. As used herein, an "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) antigen binding protein is an antigen binding protein that comprises a portion of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specific antigen binding (regardless of how that portion is obtained or synthesized). The fragment is biologically active because it binds to the target antigen and can compete with other antigen binding proteins (including intact antibodies) for binding to a given epitope. In some embodiments, the fragment is a neutralizing fragment. In some embodiments, the fragment can block or reduce the likelihood of interaction between LDLR and PCSK9. In one aspect, the fragment will retain at least one CDR present in a full-length light or heavy chain, and in some embodiments, will comprise a single heavy and / or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of antigen-binding proteins (including intact antibodies). Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, diabodies (heavy chain variable domains and light chain variable domains on the same polypeptide linked by a short peptide linker that is too short to allow pairing between the two domains on the same chain), Fab', F(ab')2, Fv, domain antibodies, and single-chain antibodies, and can be derived from any mammalian origin, including, but not limited to, human, mouse, rat, camelid, or rabbit. It is further contemplated that a functional portion of the antigen-binding proteins disclosed herein (e.g., one or more CDRs) can be covalently bound to a second protein or small molecule to generate therapeutic agents directed to a specific target in vivo, possessing bifunctional therapeutic properties, or having an extended serum half-life. As will be appreciated by those skilled in the art, antigen-binding proteins may include non-protein components. In some sections of this disclosure, examples of ABPs are described herein using "numbers / letters / numbers" (e.g., 25A7). In these cases, the exact name indicates the specific antibody. That is, an ABP named 25A7 is not necessarily the same as an antibody named 25A7.1 (unless they are explicitly taught as the same in the specification, e.g., 25A7 and 25A7.3). As will be appreciated by those skilled in the art, in some embodiments, LDLR is not an antigen binding protein. In some embodiments, a binding subsection of LDLR is not an antigen binding protein, e.g., EGFa. In some embodiments, other molecules through which PCSK9 signals in vivo are not antigen binding proteins. Such embodiments will be explicitly identified as such.

[0175] Certain antigen-binding proteins described herein are antibodies or are derived from antibodies. In certain embodiments, the polypeptide structure of the antigen-binding protein is based on an antibody, including but not limited to monoclonal antibodies, bispecific antibodies, miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and their respective fragments. In some embodiments, the ABP comprises or consists of an avimer (tight binding peptide). These various antigen-binding proteins are further described herein.

[0176] The "Fc" region comprises two heavy chain fragments, which comprise the C H 1 and C H 2 domains. The two heavy chain fragments are connected by two or more disulfide bonds and by C H The three domains are held together by hydrophobic interactions.

[0177] The "Fab fragment" contains a light chain and a heavy chain C H 1 and variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0178] A "Fab' fragment" contains a light chain and a heavy chain containing the VH domain and C H 1 domain and C H 1 domain and C H The Fab′ domains form part of the region between the two heavy chains, thereby allowing for the formation of an interchain disulfide bond between the two heavy chains of the two Fab′ fragments to form an F(ab′) 2 molecule.

[0179] The "F(ab')2 fragment" contains two light chains and a C H 1 domain and C H The two heavy chains bind to a portion of the constant region between the two heavy chains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.

[0180] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0181] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference.

[0182] "Domain antibodies" are immunologically functional immunoglobulin fragments that contain only the variable region of a heavy chain or the variable region of a light chain. In some cases, two or more V H The two V domains of the bivalent domain antibody are covalently linked by a peptide linker to produce a bivalent domain antibody. H The regions may target the same or different antigens.

[0183] A "bivalent antigen-binding protein" or "bivalent antibody" comprises two antigen-binding sites. In some cases, the two binding sites have the same antigen-specificity. Bivalent antigen-binding proteins and bivalent antibodies can have bispecificity, see below. In certain embodiments, bivalent antibodies other than "multispecific" or "multifunctional" antibodies are generally understood to have each of their binding sites be identical.

[0184] A "multispecific antigen-binding protein" or "multispecific antibody" is an antigen-binding protein or antibody that targets more than one antigen or epitope.

[0185] "Bispecific," "dual-specific," or "bifunctional" antigen-binding proteins or antibodies are hybrid antigen-binding proteins or antibodies, respectively, that have two distinct antigen-binding sites. Bispecific antigen-binding proteins and antibodies are multispecific antigen-binding proteins and antibodies and can be produced by a variety of methods, including, but not limited to, hybridoma fusion or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79 :315-321; Kostelny et al., 1992, J. Immunol. 148 :1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind to two different epitopes that may reside on the same or different protein targets.

[0186] When the dissociation constant (K d )≤10 -7 When K is greater than 1, the antigen binding protein is said to “specifically bind” to its target antigen. d ≤5×10 -9 When K is 0, ABP binds to antigen specifically with high affinity, and when K is d ≤5×10 -10 When the K of the ABP is 0.05, the ABP specifically binds the antigen with "very high affinity". d ≤10 -9 In one embodiment, the dissociation rate is <1×10 -5 In other embodiments, the ABP binds to the K of human PCSK9. d will be between approximately 10-9 M and 10 -13 M, and in another embodiment, the ABP binds with a K d ≤5×10 -10 M. As will be appreciated by those skilled in the art, in some embodiments, any or all of the antigen-binding fragments can specifically bind to PCSK9.

[0187] An antigen binding protein is "selective" when it binds one target more tightly than it binds a second target.

[0188] "Antigen binding region" means a protein or a portion of a protein that specifically binds to a specified antigen (e.g., a paratope). For example, the portion of an antigen binding protein that contains amino acid residues that interact with the antigen and impart specificity and affinity to the antigen is referred to as an "antigen binding region." The antigen binding region typically includes one or more "complementary binding regions" ("CDRs"). Some antigen binding regions also include one or more "framework" regions. "CDRs" are amino acid sequences that contribute to antigen binding specificity and affinity. The "framework" region can help maintain the appropriate configuration of the CDRs to promote binding between the antigen binding region and the antigen. Structurally, the framework region can be located between the CDRs in an antibody. Examples of framework and CDR regions are shown in Figures 2A-3D, 3CCC-3JJJ. In some embodiments, the sequences of the CDRs of the light chain of antibody 3B6 are as follows: CDR1 TLSSGYSSYEVD (SEQ ID NO:279); CDR2 VDTGGIVGSKGE (SEQ ID NO:280); CDR3 GADHGSGTNFVVV (SEQ ID NO:281), and the FRs are as follows: FR1 QPVL TQPLFASASLGASVTLTC (SEQ ID NO:282); FR2 WYQQ RPGKGPRFVMR (SEQ ID NO:283); FR3 GIPDRFSVLGSGLNRYLTIKNIQEEDESDYHC (SEQ ID NO:284); and FR4 FGGGTKLTVL (SEQ ID NO:285).

[0189] In certain aspects, recombinant antigen-binding proteins that bind to PCSK9 (e.g., human PCSK9) are provided. In this context, a "recombinant antigen-binding protein" is a protein produced using recombinant technology, i.e., by expressing a recombinant nucleic acid as described herein. Methods and techniques for producing recombinant proteins are well known in the art.

[0190] The term "antibody" refers to a complete immunoglobulin of any isotype or a fragment thereof that can compete with a complete antibody for specific binding to a target antigen, and includes, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. An "antibody" is an antigen-binding protein. A complete antibody will typically comprise at least two full-length heavy chains and two full-length light chains, but in some cases may comprise fewer chains, such as antibodies naturally occurring in Camelidae that can comprise only heavy chains. An antibody may be derived from only a single source, or may be a "chimeric" antibody, i.e., different parts of an antibody may be derived from two different antibodies, as further described below. Antigen-binding proteins, antibodies, or binding fragments can be produced by recombinant DNA technology in a hybridoma, or by enzymatic or chemical cleavage of a complete antibody. Unless otherwise indicated, the term "antibody" includes derivatives, variants, fragments, and mutant proteins thereof in addition to antibodies comprising two full-length heavy chains and two full-length light chains, examples of which are described below. Furthermore, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof. In some embodiments, the term also encompasses peptibodies.

[0191] Naturally occurring antibody structural units typically comprise tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (in certain embodiments, approximately 25 kDa) and one full-length "heavy" chain (in certain embodiments, approximately 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of approximately 100 to 110 or more amino acids, which is generally responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant region, which may be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is similarly subdivided into subclasses, including but not limited to IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant regions are typically connected by a "J" region of about 12 or more amino acids, while the heavy chain also includes a "D" region of an additional about 10 amino acids. See, for example, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (incorporated herein by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair typically form an antigen binding site.

[0192] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions (also called complementarity determining regions or CDRs). The CDRs from the two chains of each pair are typically aligned by the framework regions, which enables binding to a specific epitope. From N-terminus to C-terminus, both the light chain variable region and the heavy chain variable region typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Amino acids are typically assigned to each domain according to the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)); or Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989).

[0193] In certain embodiments, the antibody heavy chain binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody light chain binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, a single variable domain specifically binds to the antigen in the absence of other variable domains.

[0194] In certain embodiments, a definitional description of the CDRs and identification of the residues that comprise the binding site of the antibody is achieved by analyzing the structure of the antibody and / or analyzing the structure of the antibody-ligand complex. In certain embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various analytical methods can be used to identify or roughly estimate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[0195] The Kabat definition is a standard for numbering residues in antibodies and is commonly used to identify CDR regions. See, for example, Johnson and Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the position of certain structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses an integrated suite of computer programs for modeling antibody structures produced by the Oxford Molecular Group. See, for example, Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); "AbM TM , A Computer Program for Modeling Variable Regions of Antibodies", Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of antibodies from the primary sequence using a combination of a knowledge database and ab initio methods as described by Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach", PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). Contact definitions are based on analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0196] By convention, the CDR regions in the heavy chain are usually referred to as H1, H2, and H3 and are numbered sequentially from the amino terminus to the carboxyl terminus. The CDR regions in the light chain are usually referred to as L1, L2, and L3 and are numbered sequentially from the amino terminus to the carboxyl terminus.

[0197] The term "light chain" includes full-length light chains and fragments thereof having variable region sequences sufficient to confer binding specificity. A full-length light chain includes variable region domains V L and constant region domain C L The variable region domain of the light chain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0198] The term "heavy chain" includes full-length heavy chains and fragments thereof having variable region sequences sufficient to confer binding specificity. A full-length heavy chain includes variable region domains V H and three constant region domains C H 1. C H 2 and C H 3. V H The domain is at the amino terminus of the polypeptide, and the C H The domain is at the carboxyl terminus, where C H 3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0199] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies with two different heavy chain / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including, but not limited to, hybridoma fusion or linking of Fab' fragments. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0200] Some mammalian species also produce antibodies with only a single heavy chain.

[0201] Each single immunoglobulin chain is typically made up of several "immunoglobulin domains," each consisting of approximately 90 to 110 amino acids and having a characteristic folding pattern. These domains are the basic units that make up antibody polypeptides. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically contains one or more domains that may be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. For example, an IgG heavy chain contains three C region domains, called C H 1. C H 2 and C H 3. The antibodies provided may have any of these isotypes and subtypes. In certain embodiments of the invention, the anti-PCSK9 antibodies are of the IgG2 or IgG4 subtype.

[0202] The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chains of an antibody, generally including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and approximately 100 to 110 amino-terminal amino acids in the light chain. In certain embodiments, the amino acid sequences of the variable regions of different antibodies vary widely, even between antibodies of the same species. The variable region of an antibody generally determines the specificity of a particular antibody for its target.

[0203] The terms "neutralizing antigen-binding protein" or "neutralizing antibody" refer to an antigen-binding protein or antibody, respectively, that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be accomplished, for example, by directly blocking the binding site on the ligand or by binding to the ligand and altering the ligand's binding ability through indirect means, such as structural or energetic changes in the ligand. In some embodiments, the terms may also refer to an antigen-binding protein that prevents the protein to which it binds from performing a biological function. When evaluating the binding and / or specificity of an antigen binding protein, such as an antibody or immunologically functional fragment thereof, the antibody or fragment can substantially inhibit the binding of a ligand to its binding partner when the excess antibody reduces the amount of binding partner that binds to the ligand by at least about 1-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-98%, 98-99%, or more than 99% (as measured in an in vitro competitive binding assay). In some embodiments, in the case of a PCSK9 antigen binding protein, the neutralizing molecule can attenuate the ability of PCSK9 to bind to LDLR. In some embodiments, neutralization ability is characterized and / or described via competition assays. In some embodiments, IC 50 or EC 50 The value describes the neutralizing ability. In some embodiments, ABPs 27B2, 13H1, 13B5, and 3C4 are non-neutralizing ABPs, 3B6, 9C9, and 31A4 are weak neutralizers, and the remaining ABPs in Table 2 are strong neutralizers. In some embodiments, the antibody or antigen binding protein neutralizes by binding to PCSK9 and preventing PCSK9 from binding to LDLR (or reducing the ability of PCSK9 to bind to LDLR). In some embodiments, the antibody or ABP neutralizes by binding to PCSK9 and still allowing PCSK9 to bind to LDLR, thereby preventing or reducing PCSK9-mediated LDLR degradation. Therefore, in some embodiments, a neutralizing ABP or antibody may still allow PCSK9 / LDLR binding, but will prevent (or reduce) subsequent LDLR degradation involving PCSK9.

[0204] The term "target" refers to a molecule or portion of a molecule that can be bound by an antigen binding protein. In certain embodiments, a target can have one or more epitopes. In certain embodiments, a target is an antigen. The use of "antigen" in the phrase "antigen binding protein" simply means that the protein sequence that constitutes the antigen can be bound by an antibody. In this context, it is not necessary that the protein be foreign or capable of inducing an immune response.

[0205] The term "competition" when used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., PCSK9 or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another antigen binding protein, for example: solid phase direct or indirect radioimmunoassays (RIAs); solid phase direct or indirect enzyme immunoassays (EIAs); sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology); 9 :242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137 : 3614-3619); solid phase direct labeling assay; solid phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling RIA using I-125 labeling (see, e.g., Morel et al., 1988, Molec. Immunol. 25 :7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176 :546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, the assay involves the use of purified antigens bound to a solid surface or cells carrying any of these solid surfaces, unlabeled test antigen-binding proteins, and labeled reference antigen-binding proteins. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antigen-binding proteins identified by competition assays (competing antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein and antigen-binding proteins that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antigen-binding protein to produce steric hindrance. Additional details about methods for determining competitive binding are provided in the Examples herein. Typically, when the competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antigen-binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0206] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antigen binding protein (including, for example, an antibody or an immunologically functional fragment thereof). In some embodiments, an antigen can be used to produce antibodies in an animal that can bind to that antigen. An antigen can have one or more epitopes that can interact with different antigen binding proteins (e.g., antibodies).

[0207] The term "epitope" includes any determinant that can be bound by an antigen binding protein (such as an antibody) or is bound to a T cell receptor. An epitope is a region of an antigen that is bound by an antigen binding protein that targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antigen binding protein. Most often, epitopes reside on proteins, but in some cases may reside on other types of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groups of molecules (such as amino acids, sugar side chains, phosphoryl or sulfonyl groups) and may have specific three-dimensional structural features and / or specific charge characteristics. In general, antibodies that are specific for a particular target antigen will preferentially identify epitopes on target antigens in a complex mixture of proteins and / or macromolecules.

[0208] As used herein, "substantially pure" means that the molecular species is the predominant species present, i.e., it is present in a greater molar amount than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition wherein the species of interest comprises at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, the species of interest is purified to substantial homogeneity, wherein contaminating species cannot be detected in the composition by conventional detection methods and thus the composition consists of a single detectable macromolecular species.

[0209] The term "agent" is used herein to refer to a compound, a mixture of compounds, a biomacromolecule, or an extract prepared from biological material.

[0210] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, such as by incorporation of a radiolabeled amino acid or attachment to a polypeptide having a biotin moiety detectable by labeled avidin (e.g., avidin streptavidin containing a fluorescent marker or enzymatically active avidin detectable by optical or colorimetric methods). In certain embodiments, the label or marker may also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags). In certain embodiments, the labels are linked by spacer arms of various lengths to reduce potential steric hindrance.

[0211] As used herein, the term "biological sample" includes, but is not limited to, any amount of material from a living thing or a formerly living thing. Such living things include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. Such materials include, but are not limited to, blood, serum, urine, cells, organs, tissues, bone, bone marrow, lymph nodes, and skin.

[0212] As used herein, the term "pharmaceutical composition" (or agent or drug) refers to a compound, composition, agent or drug that is capable of inducing a desired therapeutic effect when properly administered to a patient. It does not necessarily require more than one type of ingredient.

[0213] The term "therapeutically effective amount" refers to an amount of a PCSK9 antigen binding protein that is determined to produce a therapeutic response in a mammal. The therapeutically effective amount can be easily determined by a person skilled in the art.

[0214] As used herein, the term "modulator" is a compound that changes or alters the activity or function of a molecule. For example, a modulator can cause the magnitude of a certain activity or function of a molecule to increase or decrease compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor that reduces the magnitude of at least one activity or function of a molecule. Some exemplary activities and functions of molecules include, but are not limited to, binding affinity, enzymatic activity, and signal transduction. Some exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates, or small organic molecules. Peptibodies are described, for example, in U.S. Patent No. 6,660,843 (corresponding to PCT Application No. WO 01 / 83525).

[0215] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as those with a formally diagnosed disease, those with an unformally diagnosed disease, those receiving medical attention, those at risk of developing a disease, and the like.

[0216] The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and applications to reduce the risk that a subject will develop a disease or other risk factors. Treatment does not require a complete cure of the disease and encompasses embodiments that alleviate symptoms or reduce underlying risk factors.

[0217] The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it means that the likelihood of an event occurring has been reduced in the presence of a compound or method.

[0218] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and conversion (such as electroporation, liposome transfection). Enzymatic reaction and purification techniques can be performed according to manufacturer specifications or as generally completed in the art or as described herein. The aforementioned techniques and procedures can be performed generally according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclature used for analytical chemistry, synthetic organic chemistry and medicine and pharmaceutical chemistry as described herein and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry and medicine and pharmaceutical chemistry as described herein are well known in the art and commonly used. Standard techniques can be used for chemical synthesis, chemical analysis, drug preparation, preparation and delivery, and treatment patients.

[0219] Antigen binding proteins targeting PCSK9

[0220] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a serine protease involved in regulating the levels of low-density lipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah and Prat, 2007). PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8) family of serine proteases (Seidah et al., 2003). An exemplary human PCSK9 amino acid sequence is presented as Figure 1A SEQ ID NO: 1 (underlined to indicate the "pro" domain of the protein) and Figures 1B1 to 1B4 SEQ ID NO: 3 (signal sequence in bold and prodomain underlined). An exemplary human PCSK9 coding sequence is presented as SEQ ID NO: 2 ( Figures 1B1 to 1B4 As described herein, PCSK9 protein may also include fragments of the full-length PCSK9 protein. The structure of the PCSK9 protein was determined by two groups (Cunningham et al., Nature Structural & Molecular Biology, 2007, and Piper et al., Structure, 15:1-8, 2007), both of which are incorporated herein by reference in their entirety. PCSK9 comprises a signal sequence, an N-terminal prodomain, a subtilisin-like catalytic domain, and a C-terminal domain.

[0221] Provided herein are antigen binding proteins (ABPs) that bind to PCSK9 (including human PCSK9). In some embodiments, the provided antigen binding proteins are polypeptides comprising one or more complementarity determining regions (CDRs) as described herein. In some antigen binding proteins, the CDRs are embedded in "framework" regions that orient the CDRs so as to achieve their proper antigen binding properties. In some embodiments, the antigen binding proteins provided herein can interfere with, block, reduce, or modulate the interaction between PCSK9 and LDLR. Such antigen binding proteins are denoted as "neutralizing" antigen binding proteins. In some embodiments, binding between PCSK9 and LDLR still occurs even though the antigen binding protein is neutralizing and binds to PCSK9. For example, in some embodiments, the ABP prevents or reduces the adverse effects of PCSK9 on LDLR without blocking the LDLR binding site on PCSK9. Thus, in some embodiments, the ABP modulates or alters the ability of PCSK9 to cause LDLR degradation without necessarily preventing the binding interaction between PCSK9 and LDLR. Such ABPs can be particularly described as "non-competitive neutralizing" ABPs. In some embodiments, the neutralizing ABP binds to PCSK9 in a location and / or manner that prevents PCSK9 from binding to LDLR. Such ABPs can be particularly described as "competitive neutralizing" ABPs. Both of the above neutralizing agents can result in a greater amount of free LDLR present in the subject, thereby causing more LDLR to bind to LDL (and thereby reducing the amount of LDL in the subject). In turn, this reduces the amount of serum cholesterol present in the subject.

[0222] In some embodiments, the antigen binding proteins provided herein are capable of inhibiting PCSK9-mediated activity (including binding). In some embodiments, antigen binding proteins that bind to these epitopes particularly inhibit the interaction between PCSK9 and LDLR and other physiological effects mediated by PCSK9. In some embodiments, the antigen binding protein is a human antibody against PCSK9, such as a fully human antibody.

[0223] In some embodiments, the ABP binds to the catalytic domain of PCSK9. In some embodiments, the ABP binds to the mature form of PCSK9. In some embodiments, the ABP binds to the prodomain of PCSK9. In some embodiments, the ABP selectively binds to the mature form of PCSK9. In some embodiments, the ABP binds to the catalytic domain in a manner that prevents PCSK9 from binding or from efficiently binding to LDLR. In some embodiments, the antigen binding protein does not bind to the C-terminus of the catalytic domain. In some embodiments, the antigen binding protein does not bind to the N-terminus of the catalytic domain. In some embodiments, the ABP does not bind to the N-terminus or C-terminus of the PCSK9 protein. In some embodiments, the ABP binds to any of the epitopes bound by the antibodies discussed herein. In some embodiments, this can be determined by competition assays between the antibodies disclosed herein and other antibodies. In some embodiments, the ABP binds to an epitope bound by one of the antibodies described in Table 2. In some embodiments, the antigen binding protein binds to PCSK9 in a specific conformational state so as to prevent PCSK9 from interacting with LDLR. In some embodiments, the ABP binds to the V domain of PCSK9. In some embodiments, the ABP binds to the V domain of PCSK9 and prevents (or reduces) PCSK9 from binding to LDLR. In some embodiments, the ABP binds to the V domain of PCSK9, but it does not prevent (or reduce) PCSK9 from binding to LDLR, and the ABP prevents or reduces the adverse activity on LDLR mediated by PCSK9.

[0224] The antigen binding proteins disclosed herein have various utilities. For example, some antigen binding proteins are useful in specific binding assays, affinity purification of PCSK9 (particularly human PCSK9) or its ligands, and screening assays for identifying other antagonists of PCSK9 activity. Some antigen binding proteins are useful for inhibiting the binding of PCSK9 to LDLR, or inhibiting PCSK9-mediated activity.

[0225] Antigen binding proteins can be used in a variety of therapeutic applications as described herein. For example, in some embodiments, PCSK9 antigen binding proteins are useful in treating conditions associated with PCSK9, such as cholesterol-related diseases (or "serum cholesterol-related diseases"), such as hypercholesterolemia, as further described herein. Other uses of antigen binding proteins include, for example, diagnosing PCSK9-related diseases or conditions and screening assays to determine the presence or absence of PCSK9. Some antigen binding proteins described herein are useful in treating consequences, symptoms, and / or pathologies associated with PCSK9 activity.

[0226] In some embodiments, provided antigen-binding proteins comprise one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs). In some embodiments, the antigen-binding protein comprises (a) a polypeptide structure and (b) one or more CDRs inserted into and / or linked to the polypeptide structure. The polypeptide structure can take a variety of different forms. For example, it can be or comprise the framework of a naturally occurring antibody or a fragment or variant thereof, or it can be completely synthetic in nature. Examples of various polypeptide structures are further described below.

[0227] In certain embodiments, the polypeptide structure of the antigen-binding protein is an antibody or is derived from an antibody, including but not limited to monoclonal antibodies, bispecific antibodies, miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and portions or fragments of each of the above. In some cases, the antigen-binding protein is an immunological fragment of an antibody (e.g., Fab, Fab', F(ab')2, or scFv). Various structures are further described and defined herein.

[0228] Certain antigen binding proteins provided herein specifically and / or selectively bind to human PCSK9. In some embodiments, the antigen binding protein specifically and / or selectively binds to a human PCSK9 protein having residues 153-692 of SEQ ID NO: 3 and / or consisting of residues 153-692 of SEQ ID NO: 3. In some embodiments, the ABP specifically and / or selectively binds to a human PCSK9 protein having residues 31-152 of SEQ ID NO: 3 and / or consisting of residues 31-152 of SEQ ID NO: 3. In some embodiments, the ABP selectively binds to Figure 1A In some embodiments, the antigen binding protein specifically binds to at least a fragment of the PCSK9 protein and / or the full-length PCSK9 protein with or without a signal sequence.

[0229] In embodiments where the antigen binding protein is used for therapeutic applications, the antigen binding protein can inhibit, interfere with, or modulate one or more biological activities of PCSK9. In one embodiment, the antigen binding protein specifically binds to human PCSK9 and / or substantially inhibits the binding of human PCSK9 to LDLR by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or 85% or more (e.g., as measured by binding in an in vitro competitive binding assay). Some antigen binding proteins provided herein are antibodies. In some embodiments, the ABP has a K d Smaller (tighter binding) than 10-7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 In some embodiments, the IC of the ABP that blocks LDLR binding to PCSK9 (D374Y, high affinity variant) is 50 Less than 1 μM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM.

[0230] An example of the IgG2 heavy chain constant domain of the anti-PCSK9 antibody of the present invention has the amino acid sequence shown in SEQ ID NO: 154 in FIG3KK.

[0231] An example of the IgG4 heavy chain constant domain of the anti-PCSK9 antibody of the present invention has the amino acid sequence shown in SEQ ID NO: 155 in FIG3KK.

[0232] An example of an anti-PCSK9 antibody kappa light chain constant domain has Figure 3KK The amino acid sequence shown in SEQ ID NO: 157.

[0233] An example of an anti-PCSK9 antibody lambda light chain constant domain has Figure 3KK The amino acid sequence shown in SEQ ID NO: 156.

[0234] The variable regions of immunoglobulin chains typically exhibit the same overall structure, comprising relatively conserved framework regions (FRs) connected by three hypervariable regions (more commonly referred to as "complementarity determining regions" or CDRs). The CDRs from the two chains of each heavy chain / light chain pair mentioned above are typically aligned by the framework regions to form a structure that specifically binds to a specific epitope on a target protein (e.g., PCSK9). From N-terminus to C-terminus, naturally occurring light chain and heavy chain variable regions typically follow the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia and Lesk, 1987, J. Mol. Biol. 196 :901-917; Chothia et al., 1989, Nature 342 :878-883.

[0235] Various heavy and light chain variable regions are provided herein and described in Figures 2A-3JJ and 3LL-3BBB. In some embodiments, each of these variable regions can be connected to the above heavy chain and light chain constant regions to form a complete antibody heavy chain and light chain, respectively. In addition, the heavy chain and light chain sequences thus generated can each be combined to form a complete antibody structure.

[0236] Specific examples of some variable regions of the light and heavy chains of the provided antibodies and their corresponding amino acid sequences are summarized in Table 2.

[0237] Table 2: Exemplary heavy and light chain variable regions

[0238]

[0239]

[0240] In addition, each of the exemplary variable heavy chains listed in Table 2 can be combined with any of the exemplary variable light chains shown in Table 2 to form an antibody. Table 2 shows exemplary light and heavy chain pairs found in several antibodies disclosed herein. In some cases, the antibody comprises at least one variable heavy chain and one variable light chain from those listed in Table 2. In other cases, the antibody contains two identical light chains and two identical heavy chains. For example, an antibody or antigen binding protein may comprise one heavy chain and one light chain, two heavy chains, or two light chains. In some embodiments, the antigen binding protein comprises (and / or consists of) one, two, and / or three heavy chain CDRs and / or light chain CDRs from at least one sequence listed in Table 2 (the CDRs of the sequences are summarized in Figures 2A-3D ,and Figure 3CCC-3JJJ In some embodiments, all six CDRs (CDR1-3 (CDRL1, CDRL2, CDRL3) from the light chain and CDR1-3 (CDRH1, CDRH2, and CDRH3) from the heavy chain) are part of the ABP. In some embodiments, one, two, three, four, five, or more CDRs are included in the ABP. In some embodiments, one heavy chain CDR and one light chain CDR from the CDRs in the sequences in Table 2 are included in the ABP (the CDRs for the sequences in Table 2 are summarized in Figures 2A-3D In some embodiments, other segments (e.g., Figures 2A-2D 、 Figures 3A-3D as well as Figure 3CCC-3JJJ Examples of CDRs and FRs for the heavy and light chains indicated in Table 2 are summarized in Figures 2A-3D (and Figure 3CCC-3JJJ42, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, and 485. Optional heavy chain variable sequences (including CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4) can be selected from the following sequences: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, 60, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479 and 483. Figures 2A-3D In some entries, variations or alternative boundaries of the CDR and FR sequences are identified. These alternatives are identified with "v1" after the ABP name. Because most of these alternatives are minor in nature, only the segments with differences are shown in the table. It should be understood that the remaining segments of the light or heavy chain are the same as shown for the base ABP in other panels. Thus, for example, Figure 2C 19H9v1 and Figure 2A 19H9 in the same FR1, CDR1 and FR2, the only difference is indicated in Figure 2C For three nucleic acid sequences (ABP 26E10, 30B9, and 31B12), alternative nucleic acid sequences are provided in the figure. As will be appreciated by those skilled in the art, in practice, at most one of these sequences is required to generate an antibody or ABP. In fact, in some embodiments, only one or both of the specific heavy or light chain nucleic acids need to be present.

[0241] In some embodiments, the ABP is encoded by a nucleic acid sequence that encodes any of the protein sequences in Table 2.

[0242] In some embodiments, the ABP selectively binds to the LDLR-binding form of PCSK9 (e.g., the autocatalytic form of the molecule). In some embodiments, the antigen binding protein does not bind to the c-terminus of the catalytic domain (e.g., the c-terminal 5.5-10, 10-15, 15-20, 20-25, 25-30, 30-40 amino acids). In some embodiments, the antigen binding protein does not bind to the n-terminus of the catalytic domain (e.g., the n-terminal 5.5-10, 10-15, 15-20, 20-25, 25-30, 30-40 amino acids). In some embodiments, the ABP binds to amino acids within amino acids 1-100 of the mature form of PCSK9. In some embodiments, the ABP binds to amino acids within (and / or an amino acid sequence consisting of) the following amino acids: amino acids 31-100, 100-200, 31-152, 153-692, 200-300, 300-400, 452-683, 400-500, 500-600, 31-692, 31-449, and / or 600-692. In some embodiments, the ABP binds to the catalytic domain. In some embodiments, neutralizing and / or non-neutralizing ABPs bind to the prodomain. In some embodiments, the ABP binds to both the catalytic domain and the prodomain. In some embodiments, the ABP binds to the catalytic domain so as to block the region of the catalytic domain that interacts with the prodomain. In some embodiments, the ABP binds to a location or surface in the catalytic domain that interacts with the prodomain, as outlined in Piper et al. (Structure 15:1-8 (2007), which is hereby incorporated by reference in its entirety, including the structural representations therein). In some embodiments, the ABP binds to the catalytic domain and restricts the mobility of the prodomain. In some embodiments, the ABP binds to the catalytic domain without binding to the prodomain. In some embodiments, the ABP binds to the catalytic domain without binding to the prodomain, while preventing the prodomain from reorienting to allow PCSK9 to bind to LDLR. In some embodiments, the ABP binds to the same epitope as the epitope surrounding residues 149-152 of the prodomain in Piper et al. In some embodiments, the ABP binds to a groove on the V domain (as outlined in Piper et al.). In some embodiments, the ABP binds to a histidine-rich patch proximal to the groove on the V domain. In some embodiments, the antibody (that binds to the V domain) is not neutralizing. In some embodiments, the antibody that binds to the V domain is neutralizing. In some embodiments, the neutralizing ABP prevents PCSK9 from binding to LDLR. In some embodiments, the neutralizing ABP prevents PCSK9 from degrading LDLR but does not prevent PCSK9 from binding to LDLR (e.g., ABP 31A4).In some embodiments, the ABP binds to or blocks at least one histidine as depicted in Figure 4 of Piper et al. In some embodiments, the ABP blocks the catalytic triad in PCSK9.

[0243] In some embodiments, the antibodies selectively bind to a variant PCSK9 protein (e.g., D374Y) compared to wild-type PCSK9. In some embodiments, these antibodies bind to the variant at least twice as strongly as they bind to the wild-type, and preferably bind to the mutant at 2-5 times, 5-10 times, 10-100 times, 100-1000 times, 1000-10,000 times, or more than 10,000 times as strongly as they bind to the wild-type (e.g., via K d In some embodiments, the antibodies selectively inhibit the interaction of the variant D374Y PCSK9 with LDLR compared to the ability of wild-type PCSK9 to interact with LDLR. In some embodiments, the blocking strength of these antibodies on the ability of the variant to bind to LDLR is greater than the blocking strength on the ability of the wild-type, for example, the strength is at least twice that of the wild-type, and preferably the blocking strength on the mutant is 2-5 times, 5-10 times, 10-100 times, 100-1000 times or more than 1000 times that of the wild-type (e.g., via IC 50 In some embodiments, the antibody binds to and neutralizes both wild-type PCSK9 and variant forms of PCSK9 (such as D374Y) to a similar extent. In some embodiments, the antibody binds to PCSK9 to prevent the variant of LDLR from binding to PCSK9. In some embodiments, the variant of LDLR is at least 50% identical to human LDLR. It should be noted that variants of LDLR are known to those skilled in the art (e.g., Brown MS et al., "Calcium cages, acid baths and recycling receptors" Nature 388:629-630, 1997). In some embodiments, the ABP can increase the level of effective LDLR in heterozygous familial hypercholesterolemia (in which a loss-of-function variant of LDLR is present).

[0244] In some embodiments, the ABP binds to (but does not block) Figure 1A and / or Figure 1B1 to Figure 1B4 In some embodiments, the ABP binds to (but does not block) a PCSK9 variant that is at least 50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or 99% or more identical to a PCSK9 form described in the preceding text. Figure 1A and / or Figure 1B1 1B4 to a PCSK9 variant having at least 50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or more than 99% identity to the mature form of PCSK9 depicted in FIG. 1B4. In some embodiments, the ABP binds to and prevents Figure 1A and / or Figure 1B1 to Figure 1B4 PCSK9 variants having at least 50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99% or more identity to the PCSK9 forms described in

[0015] interact with LDLR. In some embodiments, the ABP binds to and prevents the interaction with LDLR. Figures 1B1 to 1B4 PCSK9 variants that have at least 50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or more than 99% identity to the mature form of PCSK9 described in

[0015] interact with LDLR. In some embodiments, the PCSK9 variants are human variants, such as variants at positions 474, E620G, and / or E670G. In some embodiments, the amino acid at position 474 is valine (as in other humans) or threonine (as in macaques and mice). Given the cross-reactivity data provided herein, it is believed that the antibodies of the present invention will readily bind to the above variants.

[0245] In some embodiments, the ABP binds to an epitope bound by one of the antibodies described in Table 2. In some embodiments, the antigen binding protein binds to PCSK9 in a specific conformational state so as to prevent PCSK9 from interacting with LDLR.

[0246] Humanized antigen binding proteins (e.g., antibodies)

[0247] As described herein, antigen binding proteins directed against PCSK9 may comprise humanized antibodies and / or portions thereof.An important practical application of the described strategy is the "humanization" of the mouse humoral immune system.

[0248] In certain embodiments, a humanized antibody is substantially non-immunogenic in humans. In certain embodiments, a humanized antibody has substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived. See, e.g., U.S. Patent 5,530,101; U.S. Patent 5,693,761; U.S. Patent 5,693,762; U.S. Patent 5,585,089.

[0249] In certain embodiments, amino acids of the antibody variable domain that can be modified without diminishing the natural affinity of the antigen binding domain while reducing its immunogenicity are identified. See, e.g., U.S. Patent Nos. 5,766,886 and 5,869,619.

[0250] In certain embodiments, modifications to antibodies by methods known in the art are generally designed to increase binding affinity for the target and / or reduce the immunogenicity of the antibody in the recipient. In certain embodiments, humanized antibodies are modified to eliminate glycosylation sites in order to increase the affinity of the antibody for its cognate antigen. See, for example, Co et al., Mol. Immunol., 30:1361-1367 (1993). In certain embodiments, humanized antibodies are produced using techniques such as "reconstruction", "superchimerism" or "veneering / surface resurfacing". See, for example, Vaswami et al., Annals of Allergy, Asthma, & Immunol. 81:105 (1998); Roguska et al., Prot. Engineer., 9:895-904 (1996); and U.S. Patent No. 6,072,035. In certain such embodiments, such techniques generally reduce antibody immunogenicity by reducing the number of foreign residues, but do not prevent anti-idiotypic and anti-allotypic responses following repeated administration of the antibody. Certain other methods for reducing immunogenicity are described, for example, in Gilliland et al., J. Immunol., 62(6):3663-71 (1999).

[0251] In some cases, humanizing an antibody can result in a loss of antigen binding ability. In certain embodiments, the humanized antibody is "backmutated." In certain such embodiments, the humanized antibody is mutated to include one or more amino acid residues found in the donor antibody. See, for example, Saldanha et al., Mol Immunol 36:709-19 (1999).

[0252] In certain embodiments, the complementarity determining regions (CDRs) of the light and heavy chain variable regions of antibodies against PCSK9 may be transplanted into framework regions (FRs) from the same or another species. In certain embodiments, the CDRs of the light and heavy chain variable regions of antibodies against PCSK9 may be transplanted into common human FRs. To generate common human FRs, in certain embodiments, FRs from several human heavy or light chain amino acid sequences are aligned to identify a common amino acid sequence. In certain embodiments, the FRs of the heavy or light chain of antibodies against PCSK9 are replaced with FRs from different heavy or light chains. In certain embodiments, rare amino acids in the FRs of the heavy and light chains of antibodies against PCSK9 are not replaced, while the remaining FR amino acids are replaced. Rare amino acids are specific amino acids in positions that are not normally found in FRs. In certain embodiments, the transplanted variable regions from antibodies against PCSK9 can be used with constant regions that are different from the constant regions of antibodies against PCSK9. In certain embodiments, the transplanted variable regions are part of a single-chain Fv antibody. CDR grafting is described, e.g., in U.S. Pat. Nos. 6,180,370, 6,054,297, 5,693,762, 5,859,205, 5,693,761, 5,565,332, 5,585,089, and 5,530,101 and in Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988); Winter, FEBS Letts., 430: 92-94 (1998), which are hereby incorporated by reference herein for any purpose.

[0253] Human antigen-binding proteins (e.g., antibodies)

[0254] As described herein, antigen binding proteins that bind to PCSK9 may comprise human (i.e., fully human) antibodies and / or portions thereof. In certain embodiments, nucleotide sequences encoding heavy and light chain immunoglobulin molecules and amino acid sequences constituting heavy and light chain immunoglobulin molecules are provided, particularly sequences corresponding to the variable regions. In certain embodiments, sequences corresponding to the complementary determining regions (CDRs) are provided, particularly sequences from CDR1 to CDR3. According to certain embodiments, hybridoma cell lines expressing the immunoglobulin molecules are provided. According to certain embodiments, hybridoma cell lines expressing the monoclonal antibodies are provided. In certain embodiments, the hybridoma cell line is selected from at least one cell line described in Table 2, e.g., 21B12, 16F12, and 31H4. In certain embodiments, purified human monoclonal antibodies against human PCSK9 are provided.

[0255] Large fragments of available human Ig loci can be used to transform mouse antibody production defective mouse strains and expect that the mice will produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can retain large variable gene diversity and appropriate regulation of antibody production and expression. By utilizing mice to achieve antibody diversification and selection and a mechanism for lacking immune tolerance to human proteins, human antibody repertoires regenerated in these mouse strains can produce high-affinity fully human antibodies against any relevant antigen (including human antigens). Using hybridoma technology, antigen-specific human MAbs with desired specificity can be produced and selected. Certain exemplary methods are described in WO 98 / 24893, U.S. Patent No. 5,545,807, EP 546073 and EP 546073.

[0256] In certain embodiments, constant regions from species other than human may be used as well as human variable regions.

[0257] The ability to clone and reconstruct megabase-sized human loci in yeast artificial chromosomes (YACs) and introduce them into the mouse germline provides a method for elucidating the functional components of very large or roughly mapped loci and for generating applicable human disease models. Furthermore, utilizing such technology to replace mouse loci with their human equivalents can provide an understanding of the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

[0258] Human antibodies avoid some of the problems associated with antibodies having murine or rat variable and / or constant regions. The presence of such murine or rat-derived proteins can lead to rapid clearance of the antibody or can cause the patient to develop an immune response against the antibody. To avoid the use of murine or rat-derived antibodies, fully human antibodies can be produced by introducing functional human antibody loci into rodents, other mammals, or animals so that the rodents, other mammals, or animals produce fully human antibodies.

[0259] Humanized antibodies are antibodies that, although initially containing non-human antibody amino acid sequences, have at least some of these non-human antibody amino acid sequences replaced by human antibody sequences. This is different from human antibodies, in which the antibodies are encoded (or can be encoded) by genes possessed by humans.

[0260] Antigen binding protein variants

[0261] Other antibodies provided are variants of the ABPs listed above formed by combinations or subportions of the variable heavy and variable light chains shown in Table 2 and comprising a variable light chain and / or a variable heavy chain each having at least 50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99% or more identity to an amino acid sequence (whole sequence or subportion of a sequence, e.g., one or more CDRs) of a sequence in Table 2. In some cases, the antibodies include at least one heavy chain and one light chain, while in other cases, the variant contains two identical light chains and two identical heavy chains (or subportions thereof). In some embodiments, Figures 2A-3D (and Figures 13A-13J Sequence comparisons (e.g., other embodiments in Figures 13A, 13C, 13F, 13G, 13H, 13I, and / or 13J) can be used to identify antibody segments that can be modified by observing those changes that affect binding and those that do not appear to affect binding. For example, by comparing similar sequences, it is possible to identify those segments that can be modified (e.g., specific amino acids) and how they can be modified while still retaining (or improving) the functionality of the ABP. In some embodiments, variants of the ABP include those consensus groups and sequences described in Figures 13A, 13C, 13F, 13G, 13H, 13I, and / or 13J, and positions identified as variable in the figures are allowed to vary. Figure 13A 、 13C The CDRs shown in 13F, 13G, and 13C were defined based on a hybrid combination of the Chothia method (based on the position of structural loop regions, see, e.g., "Standard conformations for the canonical structures of immunoglobulins", Bissan Al-Lazikani, Arthur M. Lesk and Cyrus Chothia, Journal of Molecular Biology, 273(4):927-948, November 7 (1997)) and the Kabat method (based on sequence variability, see, e.g., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 91-3242, Kabat et al., (1991)). Each residue determined by either method was included in the final CDR residue list (and is presented in Figure 13A 、 13C , 13F and 13G). Figure 13H 、 13IThe CDRs in Figures 13H and 13J were obtained by the Kabat method alone. Unless otherwise specified, the defined consensus sequences, CDRs, and FRs in Figures 13H-13J define and control the indicated CDRs and FRs of the ABP referenced in Figure 13.

[0262] In certain embodiments, the antigen binding protein comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from at least one of SEQ ID NOs: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In certain embodiments, the antigen binding protein comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from at least one of SEQ ID NOs: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In certain embodiments, the antigen binding protein comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 99% identical to an amino acid sequence selected from at least one of the following sequences: SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81 and 60.

[0263] In some embodiments, the antigen binding protein comprises a sequence that is at least 90%, 90-95%, and / or 95-99% identical to one or more CDRs from at least one of the following sequences: SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In some embodiments, there are 1, 2, 3, 4, 5, or 6 CDRs (each at least 90%, 90-95%, and / or 95-99% identical to the above sequences).

[0264] In some embodiments, the antigen binding protein comprises a sequence that is at least 90%, 90-95%, and / or 95-99% identical to one or more FRs from at least one of the following sequences: SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In some embodiments, there are 1, 2, 3, or 4 FRs (each at least 90%, 90-95%, and / or 95-99% identical to the above sequences).

[0265] In certain embodiments, the antigen binding protein comprises a light chain comprising a variable region comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from at least one of SEQ ID NOs: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In certain embodiments, the antigen binding protein comprises a light chain comprising a variable region comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from at least one of SEQ ID NOs: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In certain embodiments, the antigen binding protein comprises a light chain comprising a variable region comprising an amino acid sequence at least 99% identical to an amino acid sequence selected from at least one of SEQ ID NOs: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46.

[0266] In some embodiments, the antigen binding protein comprises a sequence that is at least 90%, 90-95%, and / or 95-99% identical to one or more CDRs from at least one of the following sequences: SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, there are 1, 2, 3, 4, 5, or 6 CDRs (each at least 90%, 90-95%, and / or 95-99% identical to the above sequences).

[0267] In some embodiments, the antigen binding protein comprises a sequence that is at least 90%, 90-95%, and / or 95-99% identical to one or more FRs from at least one of the following sequences: SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, there are 1, 2, 3, or 4 FRs (each at least 90%, 90-95%, and / or 95-99% identical to the above sequences).

[0268] In light of this disclosure, a skilled artisan will be able to use well-known techniques to determine suitable variants of the ABP as described herein. In certain embodiments, one skilled in the art can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions believed to be unimportant for activity. In certain embodiments, residues and portions of the molecule that are conserved between similar polypeptides can be identified. In certain embodiments, even regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without disrupting biological activity or adversely affecting polypeptide structure.

[0269] In addition, one skilled in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. Based on such comparisons, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues in similar proteins that are important for activity or structure. One skilled in the art can select chemically similar amino acids to replace the amino acid residues predicted to be important.

[0270] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence relative to structures in similar ABPs. Given this information, those skilled in the art can predict the alignment of the antibody's amino acid residues relative to the antibody's three-dimensional structure. In certain embodiments, those skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the protein's surface, as such residues may be involved in important interactions with other molecules. In addition, those skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if a change in a particular amino acid residue is found to result in impaired activity, an undesirable reduction in activity, or unsuitability, variants with such changes can be avoided. In other words, based on the information gathered from such routine experiments, those skilled in the art can readily determine which amino acids should be avoided for further substitution, either alone or in combination with other mutations.

[0271] Many scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996); Chou et al., Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384 (1979). In addition, computer programs are currently available to assist in the prediction of secondary structure. One method for predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins with a sequence identity greater than 30% or a similarity greater than 40% often have similar structural topologies. Recent developments in the Protein Structural Database (PDB) have provided enhanced predictability of secondary structure, including the number of potential folds within a polypeptide or protein structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) that the number of folds in a given polypeptide or protein is finite and that structure prediction will become significantly more accurate once a critical number of structures have been solved.

[0272] Other methods for predicting secondary structure include "threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-19 (1996)), "profiling" (Bowie et al., Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci. USA, 84(13):4355-4358 (1987)), and "evolutionary linkage" (see Holm, supra (1999) and Brenner, supra (1997)).

[0273] In certain embodiments, antigen-binding protein variants include glycosylation variants, wherein the number and / or type of glycosylation sites have been changed compared to the amino acid sequence of the parent polypeptide. In certain embodiments, protein variants include a greater or smaller number of N-linked glycosylation sites compared to the native protein. The N-linked glycosylation sites are characterized by Asn-X-Ser or Asn-X-Thr sequences, where the amino acid residue designated as X can be any amino acid residue except proline. Substitution of amino acid residues to generate this sequence provides potential new sites for adding N-linked carbohydrate chains. Alternatively, substitutions that eliminate this sequence will remove existing N-linked carbohydrate chains. Rearrangements of N-linked carbohydrate chains are also provided, wherein one or more N-linked glycosylation sites (typically those sites that occur naturally) are eliminated and one or more new N-linked sites are generated. Other preferred antibody variants include cysteine ​​variants, wherein one or more cysteine ​​residues are deleted or substituted with another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants may be useful when the antibody must be refolded into a biologically active conformation, such as after isolation from insoluble inclusion bodies. Cysteine ​​variants typically contain fewer cysteine ​​residues than the native protein and typically have an even number of cysteine ​​residues to minimize interactions due to unpaired cysteines.

[0274] According to certain embodiments, the amino acid substitution is a substitution that: (1) reduces susceptibility to proteolysis, (2) reduces susceptibility to oxidation, (3) alters binding affinity for forming a protein complex, (4) alters binding affinity, and / or (4) confers or alters other physicochemical or functional properties of the polypeptide. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence (in certain embodiments, in portions of the polypeptide outside of the domains forming intermolecular contacts). In certain embodiments, conservative amino acid substitutions generally do not substantially alter the structural characteristics of the parent sequence (e.g., the replacing amino acid should not tend to interrupt helices present in the parent sequence or disrupt other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structure are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature, 354:105 (1991), each of which is incorporated herein by reference.

[0275] In some embodiments, the variant is a variant of the nucleic acid sequence of the ABP disclosed herein. Those skilled in the art will appreciate that the above discussion can be used to identify, evaluate, and / or generate ABP protein variants and nucleic acid sequences encoding those protein variants. Thus, nucleic acid sequences encoding those protein variants (as well as nucleic acid sequences encoding the ABPs in Table 2 but different from those explicitly disclosed herein) are contemplated. For example, the ABP variant can be compared to SEQ ID NO: 152, 153, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 or at least one nucleic acid sequence represented by SEQ ID NO: 152, 153, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, At least one to six (and various combinations thereof) CDRs encoded by the nucleic acid sequences of 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 and 151 have at least 80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99% or more identity.

[0276] In some embodiments, if the nucleic acid sequence encoding a particular ABP (or the nucleic acid sequence itself) can be compared under stringent conditions with any nucleic acid sequence encoding a protein in Table 2 (such as, but not limited to, SEQ ID NO: 152, 153, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, and 151), then the antibody (or nucleic acid sequence encoding the same) is a variant. In one embodiment, suitable moderate stringency conditions include pre-washing in 5×SSC; 0.5% SDS; 1.0 mM EDTA (pH 8:0); hybridization overnight with 5×SSC at 50°C-65°C or, if cross-species homology exists, hybridization with 0.5×SSC at 45°C; followed by two washes each with 2×SSC, 0.5×SSC, and 0.2×SSC containing 0.1% SDS at 65°C for 20 minutes. The hybridizing DNA sequences are also within the scope of the present invention, as are the nucleotide sequences encoding the antibody polypeptides encoded by the hybridizing DNA sequences and the amino acid sequences encoded by these nucleic acid sequences due to code degeneracy. In some embodiments, variants of the CDRs include one or more CDRs within the sequences indicated above (a single CDR can be readily modified according to Figures 2A-3D and Figure 3CCC-3JJJThe term "selective hybridization" as used herein refers to nucleic acid sequences that hybridize to and the amino acid sequences encoded by those sequences (determined in other embodiments herein). The term "selective hybridization" as used herein means detectable and selective binding. The polynucleotides, oligonucleotides, and fragments thereof of the present invention selectively hybridize to nucleic acid chains under hybridization and wash conditions that minimize detectable binding to non-specific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the present invention and the relevant nucleic acid sequence will be at least 80%, and more typically will have preferably increasing homology, i.e., at least 85%, 90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is partial or complete identity between them. For example, 85% homology means that when the two sequences are aligned for maximum matching, 85% of the amino acids are identical. Gaps are allowed in the maximum match (in either of the two sequences being matched); gap lengths are preferably 5 or less, with 2 or less being more preferred. Alternatively, and preferably, two protein sequences (or polypeptide sequences derived therefrom of at least 30 amino acids in length) are homologous, as the term is used herein, if they have an alignment score (in standard deviation units) of 5 or more using the program ALIGN with a mutation data matrix and a gap penalty of 6 or more. See Dayhoff, MO, Atlas of Protein Sequence and Structure, pp. 101-110 (Vol. 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10. More preferably, two sequences, or portions thereof, are homologous if their amino acid identity is greater than or equal to 50% when optimally aligned using the ALIGN program. The term "corresponding to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contrast, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

[0277] Preparation of antigen-binding proteins (e.g., antibodies)

[0278] In certain embodiments, antigen binding proteins (e.g., antibodies) are generated by immunization with an antigen (e.g., PCSK9). In certain embodiments, antibodies are generated by immunization with full-length PCSK9, a soluble form of PCSK9, the catalytic domain alone, Figure 1A The antibodies of the present invention are produced by immunizing a mature form of PCSK9, a splice variant form of PCSK9, or a fragment thereof as shown in . In certain embodiments, the antibodies of the present invention may be polyclonal or monoclonal antibodies, and / or may be recombinant antibodies. In certain embodiments, the antibodies of the present invention are human antibodies prepared, for example, by immunizing a transgenic animal capable of producing human antibodies (see, for example, PCT Published Application No. WO93 / 12227).

[0279] In certain embodiments, certain strategies can be used to manipulate the intrinsic properties of an antibody, such as the affinity of the antibody for its target. Such strategies include, but are not limited to, using site-specific mutagenesis or random mutagenesis of polynucleotide molecules encoding the antibody to generate antibody variants. In certain embodiments, such generation is followed by screening for antibody variants that exhibit the desired change, such as increased or decreased affinity.

[0280] In certain embodiments, the amino acid residues targeted in the mutagenesis strategy are those in the CDRs. In certain embodiments, amino acids in the framework regions of the variable domains are targeted. In certain embodiments, the framework regions have been shown to contribute to the target binding properties of certain antibodies. See, for example, Hudson, Curr. Opin. Biotech., 9:395-402 (1999) and references therein.

[0281] In certain embodiments, smaller and more efficiently screened antibody variant libraries are generated by restricting random or site-directed mutagenesis to hypermutation sites in the CDRs, which are sites corresponding to regions that tend to mutate during the somatic affinity maturation process. See, for example, Chowdhury and Pastan, Nature Biotech., 17:568-572 (1999) and references therein. In certain embodiments, certain types of DNA elements can be used to identify hypermutation sites, including but not limited to certain direct and inverted repeats, certain consensus sequences, certain secondary structures, and certain palindromic sequences. For example, the DNA elements that can be used to identify hypermutation sites include, but are not limited to, four base sequences comprising, in sequence, purine (A or G), guanine (G), pyrimidine (C or T), adenosine or thymidine (A or T) (i.e., A / GGC / TA / T). Another example of a DNA element that can be used to identify hypermutation sites is the serine codon AGC / T.

[0282] Preparation of fully human ABPs (e.g., antibodies)

[0283] In certain embodiments, phage display technology is used to generate monoclonal antibodies. In certain embodiments, the technology produces fully human monoclonal antibodies. In certain embodiments, polynucleotides encoding single Fab or Fv antibody fragments are expressed on the surface of phage particles. See, for example, Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al., J Mol Biol 222: 581 (1991); U.S. Patent No. 5,885,793. In certain embodiments, phages are "screened" to identify those antibody fragments with affinity for the target. Therefore, some of the processes simulate immune selection by displaying antibody fragment repertoires on the surface of filamentous phages, and then selecting phages based on the binding of phages to the target. In some of the procedures, high-affinity functional neutralizing antibody fragments are isolated. In some of the embodiments (discussed in more detail below), a complete gene repertoire of human antibodies is generated by cloning naturally rearranged human V genes from peripheral blood lymphocytes. See, eg, Mullinax et al., Proc Natl Acad Sci (USA), 87:8095-8099 (1990).

[0284] According to certain embodiments, antibodies of the present invention are prepared using transgenic mice that have been embedded with a considerable portion of the genome that produces human antibodies but are defective in the production of endogenous murine antibodies. The mice are thus able to produce human immunoglobulin molecules and antibodies and are defective in the production of murine immunoglobulin molecules and antibodies. The technology for achieving this result is disclosed in the patents, applications, and references disclosed in this specification. In certain embodiments, methods such as those disclosed in PCT Publication No. WO 98 / 24893 or Mendez et al., Nature Genetics, 15: 146-156 (1997) can be used, and the applications and documents are hereby incorporated by reference herein for any purpose.

[0285] Generally speaking, fully human monoclonal ABPs (e.g., antibodies) specific for PCSK9 can be produced as follows. Transgenic mice containing human immunoglobulin genes are immunized with a relevant antigen (e.g., PCSK9), and antibody-expressing lymphocytes (e.g., B cells) are obtained from the mice. The recovered cells are fused with a myeloid cell line to prepare immortalized hybridoma cell lines, and the hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the relevant antigen. In certain embodiments, methods for preparing hybridoma cell lines that produce antibodies specific for PCSK9 are provided.

[0286] In certain embodiments, fully human antibodies are produced by exposing human splenocytes (B cells or T cells) to antigens in vitro, followed by reconstitution of the exposed cells in immunocompromised mice (e.g., SCID or nod / SCID). See, for example, Brams et al., J. Immunol. 160: 2051-2058 (1998); Carballido et al., Nat. Med., 6: 103-106 (2000). In certain of these methods, transplantation of human fetal tissue into SCID mice (SCID-hu) results in long-term hematopoiesis and development of human T cells. See, for example, McCune et al., Science, 241: 1532-1639 (1988); Ifversen et al., Sem. Immunol., 8: 243-248 (1996). In some cases, the humoral immune response in the chimeric mouse depends on the co-development of human T cells in the animal. See, e.g., Martensson et al., Immunol., 83:1271-179 (1994). In certain methods, human peripheral blood lymphocytes are transplanted into SCID mice. See, e.g., Mosier et al., Nature, 335:256-259 (1988). In certain such embodiments, when the transplanted cells are treated with a priming agent (e.g., Staphylococcal enterotoxin A (SEA)) or an anti-human CD40 monoclonal antibody, a higher B cell yield is detected. See, e.g., Martensson et al., Immunol., 84:224-230 (1995); Murphy et al., Blood, 86:1946-1953 (1995).

[0287] Thus, in certain embodiments, fully human antibodies can be produced by expressing recombinant DNA in a host cell or by expressing in a hybridoma cell. In other embodiments, antibodies can be produced using phage display technology as described herein.

[0288] By utilizing as described herein The antibodies described herein are prepared using a technique. The mice are thus capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies. The techniques for achieving the above-mentioned objectives are disclosed in the patents, applications, and references disclosed in the background section herein. However, in particular, a preferred embodiment of transgenic mice and antibodies produced therefrom is disclosed in the following documents: U.S. Patent Application No. 08 / 759,620 filed December 3, 1996, International Patent Application No. WO98 / 24893 published June 11, 1998, and WO 00 / 76310 published December 21, 2000, the disclosures of which are hereby incorporated herein by reference. See also Mendez et al., Nature Genetics, 15: 146-156 (1997), the disclosures of which are hereby incorporated herein by reference.

[0289] By using this technology, fully human monoclonal antibodies have been generated against a variety of antigens. The strain is immunized with a relevant antigen (e.g., PCSK9), lymphocytes (e.g., B cells) are recovered from the hyperimmunized mice, and the recovered lymphocytes are fused with a myeloid cell line to prepare an immortalized hybridoma cell line. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the relevant antigen. Provided herein are methods for generating multiple hybridoma cell lines that produce antibodies specific for PCSK9. In addition, provided herein are characterizations of the antibodies produced by the cell lines, including nucleotide and amino acid sequence analysis of the heavy and light chains of the antibodies.

[0290] Mouse The generation of strains is further discussed and described in the following documents: U.S. Patent Application No. 07 / 466,008 filed January 12, 1990, U.S. Patent Application No. 07 / 610,515 filed November 8, 1990, U.S. Patent Application No. 07 / 919,297 filed July 24, 1992, U.S. Patent Application No. 07 / 922,649 filed July 30, 1992, U.S. Patent Application No. 08 / 031,801 filed March 15, 1993, U.S. Patent Application No. 08 / 112,848 filed August 27, 1993, U.S. Patent Application No. 08 / 112,848 filed April 28, 1994, U.S. Patent Application No. 08 / 234,145 filed on June 5, 1995, U.S. Patent Application No. 08 / 376,279 filed on January 20, 1995, U.S. Patent Application No. 08 / 430,938 filed on April 27, 1995, U.S. Patent Application No. 08 / 464,584 filed on June 5, 1995, U.S. Patent Application No. 08 / 464,582 filed on June 5, 1995, U.S. Patent Application No. 08 / 463,191 filed on June 5, 1995, U.S. Patent Application No. 08 / 462,837 filed on June 5, 1995, U.S. Patent Application No. 08 / 486,853 filed on June 5, 1995, U.S. Patent Application No. 08 / 486,857 filed on June 5, 1995 No. 08 / 486,859, filed on June 5, 1995, No. 08 / 462,513, filed on June 5, 1995, No. 08 / 724,752, filed on October 2, 1996, No. 08 / 759,620, filed on December 3, 1996, U.S. Publication No. 2003 / 0093820, filed on November 30, 2001, and U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598, and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See also European Patent No. EP 0 463 151 B1 published on June 12, 1996, International Patent Application No. WO 94 / 02602 published on February 3, 1994, International Patent Application No. WO 96 / 34096 published on October 31, 1996, WO 98 / 24893 published on June 11, 1998, and WO 00 / 76310 published on December 21, 2000. The disclosures of each of the above-cited patents, applications, and references are hereby incorporated by reference herein in their entirety.

[0291] In an alternative approach, others, including GenPharm International, Inc., utilize a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked by incorporating fragments (single genes) from the Ig locus. Thus, one or more V H Gene, one or more D H Gene, one or more J HThe gene, μ constant region, and usually a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described in the following references: U.S. Patent No. 5,545,807 to Surani et al.; and U.S. Patent Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458, all to Lonberg and Kay; U.S. Patent Nos. 5,591,669 and 6,023,010 to Krimpenfort and Berns; U.S. Patent Nos. 5,612,205, 5,613,697, 5,614,680, 5,615,698, 5,616,617,619, 5,618,619,620, 5,619,619, 5,618,619,621, 5,619,619, 5,619,619, 5,619,619, 5,619,619, 5,619,619, 5,619,619, 5,619,619, 5 5,721,367 and 5,789,215; and U.S. Patent No. 5,643,763 to Choi and Dunn; and GenPharm International U.S. Patent Application Nos. 07 / 574,748 filed August 29, 1990, 07 / 575,962 filed August 31, 1990, 07 / 810,279 filed December 17, 1991, 07 / 853,408 filed March 18, 1992, 07 / 904,068 filed June 23, 1992, 07 / 990,860 filed December 16, 1992, 08 / 053,131 filed April 26, 1993, 08 / 096,762 filed July 22, 1993, 08 / 155,301 filed November 18, 1993, 08 / 161,739 filed December 3, 1993, 08 / 165,699 filed December 10, 1993, 08 / 209,741 filed March 9, 1994, the disclosures of which are hereby incorporated herein by reference. See also European Patent No. 0546073 Bl, International Patent Application Nos. WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852, and WO 98 / 24884, and U.S. Pat. No. 5,981,175, the disclosures of which are hereby incorporated by reference in their entireties. See further Taylor et al., 1992; Chen et al., 1993; Tuaillon et al., 1993; Choi et al., 1993; Lonberg et al., (1994); Taylor et al., (1994); and Tuaillon et al., (1995); Fishwild et al., (1996), the disclosures of which are hereby incorporated by reference in their entireties.

[0292] Kirin has also demonstrated that human antibodies can be produced from mice into which large fragments of chromosomes or entire chromosomes have been introduced by microcell fusion. See European Patent Application Nos. 773 288 and 843 961, the disclosures of which are hereby incorporated by reference. In addition, KM TM Kirin mice are the result of crossbreeding Kirin Tc mice with Medarex minilocus (Humab) mice. These mice have the human IgH transchromosome of Kirin mice and the kappa chain transgene of Genpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-102).

[0293] Human antibodies can also be obtained by in vitro methods. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (CAT), yeast display, and the like.

[0294] In some embodiments, the antibodies described herein have human IgG4 heavy chains as well as IgG2 heavy chains. Antibodies may also be of other human isotypes, including IgG1. Antibodies have high affinity, typically about 10 -6 to about 10 -13 M or 10 -13 Kd below M.

[0295] As will be appreciated, antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding specific antibodies can be used to transform suitable mammalian host cells. Transformation can be performed by any known method by which polynucleotides are introduced into host cells, including, for example, packaging the polynucleotides in a virus (or packaging them in a viral vector) and transducing the host cells with a virus (or vector) or by a transfection procedure known in the art, such as illustrated by U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (the patents are hereby incorporated by reference herein). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus.

[0296] Mammalian cell lines that can be used as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and many other cell lines. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with constitutive PCSK9 binding properties.

[0297] In certain embodiments, the antibodies and / or ABPs are produced by at least one of the following hybridomas: 21B12, 31H4, 16F12, any other hybridoma listed in Table 2 or disclosed in the Examples. In certain embodiments, the antigen binding protein binds to PCSK9 with a dissociation constant (K D ) is less than about 1 nM, such as 1000 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, and / or 1 pM to 0.1 pM or less.

[0298] In certain embodiments, the antigen binding protein comprises an immunoglobulin molecule of at least one of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotypes. In certain embodiments, the antigen binding protein comprises a human kappa light chain and / or a human heavy chain. In certain embodiments, the heavy chain is of the IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments, the antigen binding protein has been cloned for expression in mammalian cells. In certain embodiments, the antigen binding protein comprises a constant region in addition to any one of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotypes.

[0299] In certain embodiments, the antigen binding protein comprises a human lambda light chain and a human IgG2 heavy chain. In certain embodiments, the antigen binding protein comprises a human lambda light chain and a human IgG4 heavy chain. In certain embodiments, the antigen binding protein comprises a human lambda light chain and a human IgG1, IgG3, IgE, IgA, IgD, or IgM heavy chain. In other embodiments, the antigen binding protein comprises a human kappa light chain and a human IgG2 heavy chain. In certain embodiments, the antigen binding protein comprises a human kappa light chain and a human IgG4 heavy chain. In certain embodiments, the antigen binding protein comprises a human kappa light chain and a human IgG1, IgG3, IgE, IgA, IgD, or IgM heavy chain. In certain embodiments, the antigen binding protein comprises the variable region of an antibody linked to a constant region that is neither of the IgG2 nor IgG4 isotype. In certain embodiments, the antigen binding protein has been cloned for expression in mammalian cells.

[0300] In certain embodiments, conservative modifications to the heavy and light chains of an antibody from at least one of hybridoma lines 21B12, 31H4, and 16F12 (and corresponding modifications to the encoding nucleotides) will result in antibodies against PCSK9 with functional and chemical characteristics similar to those of the antibodies from hybridoma lines 21B12, 31H4, and 16F12. Conversely, in certain embodiments, substantial modification of the functional and / or chemical characteristics of an antibody against PCSK9 can be achieved by selecting substitutions in the amino acid sequences of the heavy and light chains that have a significantly different effect on maintaining (a) the structure of the molecular backbone in the area of ​​the substitution, e.g., a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the site of interest; or (c) the bulk of the side chain.

[0301] For example, a "conservative amino acid substitution" may involve replacing a native amino acid residue with a non-native residue so as to have little or no effect on the polarity or charge of the amino acid residue at that position. In addition, any native residue in the polypeptide may also be substituted with alanine, as previously described for "alanine scanning mutagenesis."

[0302] The desired amino acid substitutions (whether conservative or non-conservative) can be determined by one skilled in the art when such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify important residues for antibodies against PCSK9 or to increase or decrease the affinity of an antibody as described herein for PCSK9.

[0303] In certain embodiments, the antibodies of the present invention can be expressed in cell lines other than hybridoma cell lines. In certain embodiments, sequences encoding specific antibodies can be used to transform suitable mammalian host cells. According to certain embodiments, transformation can be performed by any known method for introducing polynucleotides into host cells, including, for example, packaging the polynucleotides in a virus (or packaging them in a viral vector) and transducing the host cells with the virus (or vector) or by transfection procedures known in the art, such as those illustrated in U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which are hereby incorporated by reference herein for any purpose). In certain embodiments, the transformation procedure used may depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus.

[0304] Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and many other cell lines. In certain embodiments, cell lines can be selected by determining which cell lines have high expression levels and produce antibodies with constitutive HGF binding properties. Expression vectors suitable for mammalian host cells are well known.

[0305] In certain embodiments, the antigen binding protein comprises one or more polypeptides. In certain embodiments, any of a variety of expression vector / host systems can be used to express polynucleotide molecules encoding polypeptides comprising one or more ABP components or the ABP itself. Such systems include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus (TMV)) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0306] In certain embodiments, polypeptides comprising one or more ABP components or the ABP itself are recombinantly expressed in yeast. Certain such embodiments utilize commercially available expression systems, such as the Pichia pastoris expression system (Invitrogen, San Diego, CA), following the manufacturer's instructions. In certain embodiments, such systems rely on a prepro-α sequence to direct secretion. In certain embodiments, transcription of the insert is driven by the alcohol oxidase (AOX1) promoter following induction with methanol.

[0307] In certain embodiments, secreted polypeptides comprising one or more ABP components or the ABP itself are purified from yeast growth medium. In certain embodiments, the methods used to purify polypeptides from yeast growth medium are the same as those used to purify polypeptides from bacterial and mammalian cell supernatants.

[0308] In certain embodiments, nucleic acids encoding polypeptides comprising one or more ABP components or the ABP itself are cloned into a baculovirus expression vector, such as pVL1393 (PharMingen, San Diego, CA). In certain embodiments, the vector can be used to infect Spodoptera frugiperda cells in sF9 protein-free medium and produce recombinant polypeptides according to the manufacturer's instructions (PharMingen). In certain embodiments, the polypeptide is purified and concentrated from the culture medium using a heparin-agarose column (Pharmacia).

[0309] In certain embodiments, polypeptides comprising one or more ABP components or the ABP itself are expressed in an insect system. Certain insect systems for polypeptide expression are well known to those skilled in the art. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes in fall armyworm cells or in Trichoplusia larvae. In certain embodiments, a nucleic acid molecule encoding a polypeptide can be inserted into a non-essential gene of the virus, such as the polyhedrin gene, and placed under the control of a promoter for that gene. In certain embodiments, successful insertion of the nucleic acid molecule results in inactivation of the non-essential gene. In certain embodiments, such inactivation produces a detectable characteristic. For example, inactivation of the polyhedrin gene results in the production of a virus lacking coat protein.

[0310] In certain embodiments, the recombinant virus can be used to infect Spodoptera frugiperda cells or Trichoplusia larvae. See, for example, Smith et al., J. Virol., 46:584 (1983); Engelhard et al., Proc. Nat. Acad. Sci. (USA), 91:3224-7 (1994).

[0311] In certain embodiments, polypeptides comprising one or more ABP components or the ABP itself prepared in bacterial cells are produced in the bacteria as insoluble inclusion bodies. In certain embodiments, host cells containing the inclusion bodies are collected by centrifugation; washed in 0.15 M NaCl, 10 mM Tris (pH 8), 1 mM EDTA; and treated with 0.1 mg / ml lysozyme (Sigma, St. Louis, MO) for 15 minutes at room temperature. In certain embodiments, the lysate is clarified by sonication, and cell debris is pelleted by centrifugation at 12,000 × g for 10 minutes. In certain embodiments, the pellet containing the polypeptide is resuspended in 50 mM Tris (pH 8) and 10 mM EDTA; layered with 50% glycerol; and centrifuged at 6000 × g for 30 minutes. In certain embodiments, the pellet can be resuspended in Mg-free water. ++ and Ca ++ In certain embodiments, the polypeptide is further purified by fractionating the resuspended pellet on a denaturing SDS polyacrylamide gel (see, e.g., Sambrook et al., supra). In certain embodiments, the gel can be soaked in 0.4 M KCl to visualize the protein, which can be excised and electroeluted in gel running buffer lacking SDS. According to certain embodiments, glutathione-S-transferase (GST) fusion proteins are produced in bacteria as soluble proteins. In certain embodiments, the GST fusion proteins are purified using a GST purification module (Pharmacia).

[0312] In certain embodiments, it is desirable to "refold" certain polypeptides, such as polypeptides comprising one or more ABP components or the ABP itself. In certain embodiments, the polypeptides are produced using certain recombinant systems discussed herein. In certain embodiments, the polypeptides are "refolded" and / or oxidized to form a desired tertiary structure and / or generate disulfide bonds. In certain embodiments, the structures and / or bonds are associated with a particular biological activity of the polypeptide. In certain embodiments, refolding is accomplished using any of a number of procedures known in the art. Exemplary methods include, but are not limited to, exposing a dissolved polypeptide reagent to a pH typically exceeding 7 in the presence of a chaotropic agent. An exemplary chaotropic agent is guanidine. In certain embodiments, the refolding / oxidation solution also contains a reducing agent and an oxidized form of that reducing agent. In certain embodiments, the reducing agent and its oxidized form are present in a ratio that produces a specific redox potential that allows disulfide shuffling. In certain embodiments, the shuffling allows for the formation of cysteine ​​bridges. Exemplary redox couples include, but are not limited to, cysteine / cystamine, glutathione / disulfide bis(GSH), copper chloride, dithiothreitol (DTT) / dithiane (DTT), and 2-mercaptoethanol (bME) / disulfide-bME. In certain embodiments, a cosolvent is used to improve refolding efficiency. Exemplary cosolvents include, but are not limited to, glycerol, polyethylene glycols of various molecular weights, and arginine.

[0313] In certain embodiments, a polypeptide comprising one or more ABP components or the ABP itself is substantially purified. Certain protein purification techniques are known to those skilled in the art. In certain embodiments, protein purification involves crude fractionation of the polypeptide portion from the non-polypeptide portion. In certain embodiments, the polypeptide is purified using chromatography and / or electrophoresis techniques. Exemplary purification methods include, but are not limited to, precipitation with ammonium sulfate; precipitation with PEG; immunoprecipitation; heat denaturation followed by centrifugation; chromatography, including, but not limited to, affinity chromatography (e.g., protein-A-Sepharose), ion exchange chromatography, size exclusion chromatography, and reverse phase chromatography; gel filtration; hydroxyapatite chromatography; isoelectric focusing; polyacrylamide gel electrophoresis; and combinations of these and other techniques. In certain embodiments, the polypeptide is purified by fast protein liquid chromatography or by high pressure liquid chromatography (HPLC). In certain embodiments, the purification steps may be varied or certain steps may be omitted, and still result in a method suitable for preparing a substantially purified polypeptide.

[0314] In certain embodiments, the degree of purification of a polypeptide preparation is quantified. Certain methods for quantifying the degree of purification are known to those skilled in the art. Certain exemplary methods include, but are not limited to, measuring the specific binding activity of a preparation by SDS / PAGE analysis and assessing the amount of polypeptide within the preparation. Certain exemplary methods for assessing the amount of purification of a polypeptide preparation include calculating the binding activity of the preparation and comparing it to the binding activity of the starting extract. In certain embodiments, the result of such calculation is expressed as a "fold purification." The unit used to express the amount of binding activity depends on the specific assay performed.

[0315] In certain embodiments, a polypeptide comprising one or more ABP components or the ABP itself is partially purified. In certain embodiments, partial purification can be accomplished by using fewer purification steps or by utilizing different forms of the same general purification scheme. For example, in certain embodiments, cation exchange column chromatography performed using an HPLC apparatus will generally produce a "purification fold" greater than the same technique using a low-pressure chromatography system. In certain embodiments, methods that produce a lower degree of purification may have advantages in terms of overall recovery of the polypeptide or maintenance of the binding activity of the polypeptide.

[0316] In some cases, the electrophoretic migration of a polypeptide under different SDS / PAGE conditions can sometimes be significantly different. See, for example, Capaldi et al., Biochem. Biophys. Res. Comm., 76:425 (1977). It should be understood that the apparent molecular weight of a purified or partially purified polypeptide may be different under different electrophoretic conditions.

[0317] Exemplary epitopes

[0318] Epitopes bound by anti-PCSK9 antibodies are provided. In some embodiments, epitopes bound by the presently disclosed antibodies are particularly suitable. In some embodiments, antigen binding proteins that bind to any of the epitopes bound by the antibodies described herein are suitable. In some embodiments, epitopes bound by any of the antibodies listed in Table 2 and Figures 2 and 3 are particularly suitable. In some embodiments, the epitope is on the PCSK9 catalytic domain.

[0319] In certain embodiments, a PCSK9 epitope can be used to prevent (e.g., reduce) binding of an anti-PCSK9 antibody or antigen binding protein to PCSK9. In certain embodiments, a PCSK9 epitope can be used to reduce binding of an anti-PCSK9 antibody or antigen binding protein to PCSK9. In certain embodiments, a PCSK9 epitope can be used to substantially inhibit binding of an anti-PCSK9 antibody or antigen binding protein to PCSK9.

[0320] In certain embodiments, a PCSK9 epitope can be used to isolate an antibody or antigen-binding protein that binds to PCSK9. In certain embodiments, a PCSK9 epitope can be used to generate an antibody or antigen-binding protein that binds to PCSK9. In certain embodiments, a PCSK9 epitope or a sequence comprising a PCSK9 epitope can be used as an immunogen to generate an antibody or antigen-binding protein that binds to PCSK9. In certain embodiments, a PCSK9 epitope can be administered to an animal, from which antibodies that bind to PCSK9 can subsequently be obtained. In certain embodiments, a PCSK9 epitope or a sequence comprising a PCSK9 epitope can be used to interfere with normal PCSK9-mediated activities, such as the association of PCSK9 with LDLR.

[0321] In some embodiments, the antigen binding proteins disclosed herein specifically bind to the N-terminal prodomain, the subtilisin-like catalytic domain, and / or the C-terminal domain. In some embodiments, the antigen binding proteins bind to the substrate binding groove of PCSK-9 (described in Cunningham et al., which is incorporated herein by reference in its entirety).

[0322] In some embodiments, domains / regions containing residues that are in contact with or buried by the antibody can be identified by mutating specific residues in PCSK9 (e.g., wild-type antigen) and determining whether the antigen binding protein can bind to the mutated or variant PCSK9 protein. By making many single mutations, residues can be identified that play a direct role in binding or are close enough to the antibody that the mutation can affect the binding between the antigen binding protein and the antigen. Based on the knowledge of these amino acids, domains or regions of the antigen that contain residues that are in contact with the antigen binding protein or covered by the antibody can be elucidated. Such domains may include the binding epitope of the antigen binding protein. One specific example of this general method utilizes an arginine / glutamate scanning protocol (see, e.g., Nanevicz, T. et al., 1995, J. Biol. Chem., 270 :37,21619-21625 and Zupnick, A. et al., 2006, J.Biol.Chem., 281 :29, 20464-20473). Generally, arginine and glutamic acid are substituted (usually individually) for amino acids in the wild-type polypeptide because these amino acids are charged and bulky and therefore have the potential to disrupt binding between the antigen-binding protein and the antigen in the region of the antigen where the mutation is introduced. Arginine present in the wild-type antigen is replaced with glutamic acid. A variety of such single mutants are obtained and the collected binding results are analyzed to determine which residues affect binding.

[0323] An alteration (e.g., a decrease or increase) in binding between an antigen binding protein and a variant PCSK9 as used herein refers to binding affinity (e.g., as measured by known methods such as a Biacore assay or a bead-based assay as described below in the Examples), EC 50 There is a change and / or there is a change (e.g., a decrease) in the overall binding capacity of the antigen binding protein (e.g., as evidenced by a decrease in Bmax in a plot of antigen binding protein concentration versus antigen concentration). A significant change in binding indicates that the mutated residue is directly involved in binding to the antigen binding protein or is in close proximity to the binding protein when the binding protein is bound to the antigen.

[0324] In some embodiments, a significant reduction in binding means that the binding affinity, EC50, and / or capacity of the antigen binding protein to the mutant PCSK9 antigen is reduced by greater than 10%, greater than 20%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% relative to the binding between the antigen binding protein and wild-type PCSK9 (e.g., as set forth in SEQ ID NO: 1 and / or SEQ ID NO: 303). In certain embodiments, binding is reduced below the limit of detectability. In some embodiments, a significant reduction in binding is indicated when the binding of the antigen binding protein to the variant PCSK9 protein is less than 50% (e.g., less than 40%, 35%, 30%, 25%, 20%, 15%, or 10%) of the binding observed between the antigen binding protein and the wild-type PCSK9 protein (e.g., the protein having SEQ ID NO: 1 and / or SEQ ID NO: 303). Such binding measurements can be performed using a variety of binding assays known in the art.

[0325] In some embodiments, antigen binding proteins are provided that exhibit significantly lower binding to variant PCSK9 proteins in which residues in a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303) are substituted with arginine or glutamic acid. In some embodiments, the antigen binding protein has significantly reduced or increased binding to a variant PCSK9 protein having any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 244) of the following mutations: R207E, D208R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R, A413R, E582R, D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and Q554R, compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303). In the shorthand notation used herein, the format is: wild-type residue:position in polypeptide:mutated residue, where the residues are numbered as in SEQ ID NO:1 or SEQ ID NO:303.

[0326] In some embodiments, the antigen binding protein has significantly reduced or increased binding to a mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, or more) mutations at the following positions as set forth in SEQ ID NO: 1: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554, as compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303). In some embodiments, the antigen binding protein has reduced or increased binding to a mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, or more) mutations at the following positions as set forth in SEQ ID NO: 1: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554, as compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303). In some embodiments, the antigen binding protein has substantially reduced or increased binding to a mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, or more) mutations at the following positions within SEQ ID NO: 1: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554, as compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303).

[0327] In some embodiments, the ABP has significantly reduced or increased binding to a mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, etc.) of the following mutations in SEQ ID NO: 1 or SEQ ID NO: 303: R207E, D208R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R, A413R, E582R, D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and Q554R, compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303).

[0328] In some embodiments, the binding of the ABP to a mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, etc.) of the following mutations in SEQ ID NO: 1 or SEQ ID NO: 303 is significantly reduced or increased compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303): R207E, D208R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R, A413R, and E582R. In some embodiments, binding is reduced. In some embodiments, the reduction in binding is observed as a change in EC50. In some embodiments, the change in EC50 is an increase in the numerical value of EC50 (and therefore, a decrease in binding).

[0329] In some embodiments, the binding of the ABP to a mutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, etc.) of the following mutations in SEQ ID NO: 1 is significantly reduced or increased compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303): D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and Q554R. In some embodiments, binding is reduced. In some embodiments, the reduction in binding is observed as a change in Bmax. In some embodiments, the shift in Bmax is a decrease in the maximum signal generated by the ABP. In some embodiments, for an amino acid that is to be part of an epitope, a decrease in Bmax of at least 10%, for example, a decrease of at least any of the following amounts can indicate, in some embodiments, that the residue is part of an epitope: 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%.

[0330] Although the variant forms just listed are mentioned with respect to the wild-type sequence shown in SEQ ID NO: 1 or SEQ ID NO: 303, it should be understood that in allelic variants of PCSK9, the amino acid at the indicated position may be different. Antigen binding proteins that exhibit significantly lower binding to such allelic forms of PCSK9 are also encompassed. Thus, in some embodiments, any of the above embodiments may be used with allelic sequences, rather than just with Figure 1A The wild-type sequence shown in was compared.

[0331] In some embodiments, the antigen binding protein has significantly reduced binding to a variant PCSK9 protein in which the residue at the selected position in the wild-type PCSK9 protein is mutated to any other residue. In some embodiments, the arginine / glutamic acid substitutions described herein are used at the identified positions. In some embodiments, alanine is used at the identified positions.

[0332] As indicated above, residues directly involved in binding or covered by the antigen binding protein can be identified based on the scanning results. Thus, these residues can indicate a domain or region of SEQ ID NO: 1 (or SEQ ID NO: 303 or SEQ ID NO: 3) containing the binding region to which the antigen binding protein binds. As can be seen from the results summarized in Example 39, in some embodiments, the antigen binding protein binds to a domain containing at least one of the following amino acids of SEQ ID NO: 1 or SEQ ID NO: 303: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554. In some embodiments, the antigen binding protein binds to a region containing at least one of the following amino acids: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 of SEQ ID NO: 1 or SEQ ID NO: 303.

[0333] In some embodiments, the antigen binding protein binds to a region containing at least one of the following amino acids: 162, 164, 167, 207, and / or 208 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, more than one (e.g., 2, 3, 4, or 5) of the identified residues are part of the region bound by the ABP. In some embodiments, the ABP competes with ABP 21B12.

[0334] In some embodiments, the antigen binding protein binds to a region containing at least amino acid 185 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, the ABP competes with ABP 31H4.

[0335] In some embodiments, the antigen binding protein binds to a region containing at least one of the following amino acids: 439, 513, 538, and / or 539 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, more than one (e.g., 2, 3, or 4) of the identified residues are part of the region bound by the ABP. In some embodiments, the ABP competes with ABP 31A4.

[0336] In some embodiments, the antigen binding protein binds to a region containing at least one of the following amino acids: 123, 129, 311, 313, 337, 132, 351, 390, and / or 413 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, more than one (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of the identified residues are part of the region bound by the ABP. In some embodiments, the ABP competes with ABP 12H11.

[0337] In some embodiments, the antigen binding protein binds to a region containing at least one of the following amino acids: 582, 519, 521, and / or 554 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, more than one (e.g., 2, 3, or 4) of the identified residues are part of the region bound by the ABP. In some embodiments, the ABP competes with ABP 3C4.

[0338] In some embodiments, the antigen binding protein binds to the aforementioned regions within a fragment or full-length sequence of SEQ ID NO: 1 or SEQ ID NO: 303. In other embodiments, the antigen binding protein binds to a polypeptide consisting of these regions. Reference to "SEQ ID NO: 1 or SEQ ID NO: 303" indicates that one or both of these sequences can be used or are relevant. The phrase does not imply that only one should be used.

[0339] As indicated above, the above description refers to specific amino acid positions with reference to SEQ ID NO:1. However, throughout the specification, reference is generally made to the Pro / Cat domain beginning at position 31 provided in SEQ ID NO:3. As indicated below, SEQ ID NO:1 and SEQ ID NO:303 lack the signal sequence of PCSK9. Thus, any comparison between these different disclosures should take this numbering difference into account. Specifically, any amino acid position in SEQ ID NO:1 will correspond to an amino acid position that adds 30 amino acids to the protein in SEQ ID NO:3. For example, position 207 of SEQ ID NO:1 corresponds to position 237 of SEQ ID NO:3 (the full-length sequence, and the numbering system generally used in this specification). Table 39.6 summarizes how the above-indicated positions with reference to SEQ ID NO:1 (and / or SEQ ID NO:303) correspond to SEQ ID NO:3 (which includes the signal sequence). Therefore, any of the above-indicated embodiments described with reference to SEQ ID NO:1 (and / or SEQ ID NO:303) are described with reference to SEQ ID NO:3 by indicating the corresponding position.

[0340] In some embodiments, ABP 21B12 binds to an epitope comprising residues 162-167 (e.g., residues D162-E167 of SEQ ID NO: 1). In some embodiments, ABP 12H11 binds to an epitope comprising residues 123-132 (e.g., S123-T132 of SEQ ID NO: 1). In some embodiments, ABP 12H11 binds to an epitope comprising residues 311-313 (e.g., A311-D313 of SEQ ID NO: 1). In some embodiments, the ABP may bind to an epitope comprising any of these sequence chains.

[0341] Competitive antigen binding protein

[0342] In another aspect, an antigen binding protein is provided that competes for specific binding to PCSK9 with an exemplified antibody or functional fragment that binds to an epitope described herein. The antigen binding protein may also bind to the same epitope or overlapping epitopes as an exemplified antigen binding protein. It is expected that antigen binding proteins and fragments that compete with or bind to the same epitope as the exemplified antigen binding proteins will exhibit similar functional properties. The exemplified antigen binding proteins and fragments include those described above, including those having the heavy and light chains, variable region domains, and CDRs included in Table 2 and / or Figures 2-3. Thus, as a specific example, the provided antigen binding proteins include those that compete with antibodies or antigen binding proteins having:

[0343] (a) All six CDRs listed for the antibodies listed in Figures 2-3;

[0344] (b) the VH and VL listed for the antibodies listed in Table 2; or

[0345] (c) Two light chains and two heavy chains as specified for the antibodies listed in Table 2.

[0346] Therapeutic drug preparations and administration

[0347] The present invention provides pharmaceutical formulations containing antigen-binding proteins to PCSK9. As used herein, a "pharmaceutical formulation" is a sterile composition of a pharmaceutically active drug (i.e., at least one antigen-binding protein to PCSK9) suitable for parenteral administration (including but not limited to intravenous, intramuscular, subcutaneous, nebulized, intrapulmonary, intranasal, or intrathecal administration) to a patient in need thereof and includes only pharmaceutically acceptable excipients, diluents, and other additives deemed safe by the Federal Drug Administration (FDAA) or other foreign regulatory agencies. Pharmaceutical formulations include liquid (e.g., aqueous) solutions that can be administered directly, as well as lyophilized powders that can be reconstituted into a solution by adding a diluent prior to administration. The term "pharmaceutical formulation" specifically excludes compositions for topical administration to a patient, compositions for oral ingestion, and compositions for parenteral nutrition.

[0348] In certain embodiments, the pharmaceutical formulation is a stable pharmaceutical formulation. As used herein, the phrase "stable pharmaceutical formulation," "stable formulation," or "pharmaceutical formulation is stable" refers to a pharmaceutical formulation of a biologically active protein that exhibits no more than 5% increased aggregation and / or reduced loss of biological activity when stored at 2-8°C for at least 1 month, or 2 months, or 3 months, or 6 months, or 1 year, or 2 years, compared to a control formulation sample. Formulation stability can be readily determined by one skilled in the art using a number of standard assays, including, but not limited to, size exclusion HPLC ("SEC-HPLC"), cation exchange HPLC (CEX-HPLC), Microscopic particle detection by light obscuration ("HIAC") and / or visual inspection.

[0349] In certain embodiments, the pharmaceutical formulation comprises any of the antigen binding proteins to PCSK9 described in Table 2 and Figures 2 and / or 3. In some embodiments, the pharmaceutical formulation comprises any of 21B12, 26H5, 31H4, 8A3, 11F1, or 8A1.

[0350] In some embodiments, the pharmaceutical formulation comprises more than one different antigen binding protein to PCSK9. In certain embodiments, the pharmaceutical formulation comprises more than one antigen binding protein to PCSK9, wherein the antigen binding protein to PCSK9 binds to more than one epitope. In some embodiments, the different antigen binding proteins do not compete with each other for binding to PCSK9. In some embodiments, any of the antigen binding proteins described in Table 2 and Figures 2 and / or 3 can be combined together in a pharmaceutical formulation.

[0351] In certain embodiments, antigen binding proteins and / or therapeutic molecules directed against PCSK9 are linked to half-life extending vehicles known in the art. Such vehicles include, but are not limited to, polyethylene glycol, glycogen (e.g., glycosylation of ABP), and dextran. Such vehicles are described, for example, in U.S. Application No. 09 / 428,082 (now U.S. Patent No. 6,660,843) and published PCT Application No. WO 99 / 25044, which are hereby incorporated by reference herein for any purpose.

[0352] In certain embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations employed. In some embodiments, the formulation materials are for subcutaneous (sc) and / or intravenous (IV) administration. In certain embodiments, pharmaceutical formulations comprise formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition.

[0353] In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., proline, arginine, lysine, methionine, taurine, glycine, glutamine, or asparagine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, sodium phosphate ("NaOAC"), Tris-HCl, Tris buffer, citrate, phosphate buffer, phosphate buffered saline (i.e., PBS buffer), or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, sucrose, fructose, lactose, mannose, trehalose, or dextrin); proteins (e.g., serum albumin, gelatin or immunoglobulin); coloring agents, flavoring agents and diluents; emulsifiers; hydrophilic polymers (such as polyvinyl pyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates (such as polysorbate 20, polysorbate 80), triton, tromethamine,

[00145] The invention also includes the following ingredients: lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th edition, edited by A.R. Gennaro, Mack Publishing Company (1995)).

[0354] In certain embodiments, the optimal pharmaceutical formulation will be determined by one skilled in the art based on, for example, the intended route of administration, delivery form, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In certain embodiments, the formulation may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antibodies of the invention.

[0355] In one aspect, pharmaceutical formulations comprise high concentrations of antigen binding proteins directed against PCSK9. In certain embodiments, the ABP concentration is within the range of about 70 mg / ml to about 250 mg / ml, such as about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 100 mg / ml, about 120 mg / ml, about 130 mg / ml, about 140 mg / ml, about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, about 200 mg / ml, about 210 mg / ml, about 220 mg / ml, about 230 mg / ml, about 240 mg / ml, or about 250 mg / ml, and including all values ​​therebetween. In some embodiments, the concentration of 21B12, 26H5, or 31H4 is in the range of about 100 mg / ml to about 150 mg / ml, for example, 100 mg / ml, about 100 mg / ml, about 120 mg / ml, about 130 mg / ml, about 140 mg / ml, or about 150 mg / ml. In some embodiments, the concentration of 8A3, 11F1, or 8A1 is in the range of about 140 mg / ml to about 220 mg / ml, for example, 140 mg / ml, about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, about 200 mg / ml, about 210 mg / ml, about 220 mg / ml, or about 250 mg / ml.

[0356] On the other hand, the pharmaceutical formulation comprises at least one buffer, such as sodium acetate, sodium chloride, phosphate, phosphate buffered saline ("PBS") and / or a Tris buffer having a pH of about 7.0-8.5. The buffer is used to maintain a physiologically suitable pH. In addition, the buffer can be used to enhance the isotonicity and chemical stability of the pharmaceutical formulation. In certain embodiments, the buffer is in the range of about 0.05 mM to about 40 mM, for example, about 0.05 mM, about 0.1 mM, about 0.5 mM, about 1.0 mM, about 5.0 mM, about 10 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM or about 100 nM buffer, including all values ​​therebetween. In certain embodiments, the buffer is NaOAC. Exemplary pHs for the pharmaceutical formulation include about 4 to about 6, or about 4.8 to about 5.8, or about 5.0 to about 5.2, or about 5, or about 5.2.

[0357] In certain embodiments, the pharmaceutical formulation is isotonic, wherein the osmolality is in the range of about 250 to about 350 milliosmol / kg, for example, about 250 mOsm / kg, about 260 mOsm / kg, about 270 mOsm / kg, about 280 mOsm / kg, about 290 mOsm / kg, about 300 mOsm / kg, about 310 mOsm / kg, about 320 mOsm / kg, about 330 mOsm / kg, about 340 mOsm / kg, or about 350 mOsm / kg, and including all values ​​therebetween. As used herein, "osmolality" is a measure of the ratio of solute to volume fluid. In other words, it is the number of molecules and ions (or molecules) per kilogram of solution. Osmolality can be measured on an analytical instrument called an osmometer, such as the Advanced Instruments 2020 Multi-Sample Osmometer (Norwood, MA). The Advanced Instruments 2020 Multi-Sample Osmometer measures osmolality using the freezing point depression method. As the osmotic solute in a solution increases, the temperature at which the solution solidifies decreases. Osmolality can also be measured using any other method known in the art and in any other device known in the art, such as using linear extrapolation.

[0358] In yet another aspect, the pharmaceutical formulation comprises at least one surfactant, including but not limited to polysorbate-80, polysorbate-60, polysorbate-40, and polysorbate-20. In certain embodiments, the pharmaceutical formulation comprises a surfactant at a concentration ranging from about 0.004% to about 10% weight / volume ("w / v") of the formulation, for example, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 5%, or about 10% w / v surfactant of the formulation. In certain embodiments, the pharmaceutical formulation comprises polysorbate 80 at a concentration ranging from about 0.004% to about 0.1% w / v of the formulation. In certain embodiments, the pharmaceutical formulation comprises polysorbate 20 at a concentration ranging from about 0.004% to about 0.1% w / v of the formulation.

[0359] In certain embodiments, the pharmaceutical formulation comprises at least one stabilizer, such as a polyhydroxy hydrocarbon (including but not limited to sorbitol, mannitol, glycerol, and galactitol) and / or a disaccharide (including but not limited to sucrose, lactose, maltose, and trehalose) and / or an amino acid (including but not limited to proline, arginine, lysine, methionine, and taurine) and / or benzyl alcohol, in an amount of about 0.5% to about 10% w / v of the formulation. In certain embodiments, the pharmaceutical formulation comprises a stabilizer at a concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% sucrose. In certain embodiments, the pharmaceutical formulation comprises a stabilizer at a concentration of about 5% sucrose. In certain embodiments, the pharmaceutical formulation comprises a stabilizer at a concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% sorbitol. In certain embodiments, the pharmaceutical formulation comprises a stabilizer at a concentration of about 9% sorbitol. In certain embodiments, the pharmaceutical formulation comprises a stabilizer at a concentration of about 1%, about 2%, about 3%, about 4%, or about 5% proline, arginine, lysine, methionine, and / or taurine. In certain embodiments, the pharmaceutical formulation comprises a stabilizer at a concentration of between about 2% and 3% proline. In certain embodiments, the pharmaceutical formulation comprises a stabilizer at a concentration of about 1%, about 2%, about 3%, about 4%, or about 5% benzyl alcohol. In certain embodiments, the pharmaceutical formulation comprises a stabilizer at a concentration of between about 1% and 2% benzyl alcohol.

[0360] On the one hand, the pharmaceutical preparation has a viscosity of less than about 30 centipoise (cP), as measured at room temperature (i.e. 25°C). As used herein, "viscosity" is the resistance to flow of a fluid and can be measured in units of centipoise (cP) or milliPascal-seconds (mPa-s), where at a given shear rate, 1 cP = 1 mPa-s. Viscosity can be measured using a viscometer, such as a Brookfield Engineering LVT type dial viscometer. Viscosity can also be measured using any other method known in the art and in any other device known in the art (e.g., absolute, kinematic or dynamic viscosity or absolute viscosity). In certain embodiments, the viscosity level of the pharmaceutical preparation is less than about 25 cP, about 20 cP, about 18 cP, about 15 cP, about 12 cP, about 10 cP, about 8 cP, about 6 cP, about 4 cP, about 2 cP, or about 1 cP.

[0361] On the one hand, the pharmaceutical formulation is stable as measured by at least one stability assay known to those skilled in the art, such as an assay that examines the biophysical or biochemical characteristics of the bioactive protein over time. As mentioned above, the stable pharmaceutical formulation of the present invention is a pharmaceutical formulation of a bioactive protein that exhibits an increase in aggregation and / or a decrease in bioactivity loss of no more than 5% when stored at 2-8°C for at least 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years compared to a control formulation sample. In certain embodiments, size exclusion HPLC ("SEC-HPLC") is used to measure pharmaceutical formulation stability. SEC-HPLC separates proteins based on the differences in the hydrodynamic volume of the protein. Molecules with larger hydrodynamic protein volumes elute earlier than molecules with smaller volumes. In the case of SEC-HPLC, a stable pharmaceutical formulation should exhibit an increase in high molecular weight species of no more than about 5% compared to a control sample. In certain other embodiments, a pharmaceutical formulation should exhibit an increase in high molecular weight species of no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, no more than about 0.5% compared to a control sample.

[0362] In certain embodiments, the stability of a pharmaceutical formulation is measured using cation exchange HPLC (CEX-HPLC). CEX-HPLC separates proteins based on differences in their surface charge. Charged isoforms of anti-PCSK9 ABP are separated on a cation exchange column at a set pH and eluted using a salt gradient. The eluate is monitored by UV absorbance. The distribution of charged isoforms is assessed by measuring the percentage of the peak area of ​​each isoform relative to the total peak area. In the case of CEX-HPLC, a stable pharmaceutical formulation should exhibit a decrease in the major isoform peak of no more than about 5% compared to a control sample. In certain other embodiments, a stable pharmaceutical formulation should exhibit a decrease in the major isoform peak of no more than about 3% to about 5% compared to a control sample. In certain embodiments, a pharmaceutical formulation should exhibit a decrease in the major isoform peak of no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, or no more than about 0.5% compared to a control sample.

[0363] In certain embodiments, using Detection of microscopic particles by light obscuration ("HIAC")Measuring the stability of pharmaceutical formulations. An electronic liquid-borne particle counting system (HIAC / Royco 9703 or equivalent) containing a light obscuration sensor (HIAC / Royco HRLD-150 or equivalent) and a liquid sampler can quantify the number of particles in a given test sample and their size range. When particles in the liquid pass between the light source and the detector, they weaken or "obscure" the light beam falling on the detector. When the concentration of particles is within the normal range of the sensor, these particles are detected one by one. Each particle passing through the detection area reduces the incident light on the light detector and the voltage output of the light detector decreases momentarily. The change in voltage is recorded as an electrical pulse, which is converted by the instrument into the number of particles present. The method is non-specific and measures particles regardless of their source. The particle sizes monitored are typically 10 μm and 25 μm. In the case of HIAC, a stable pharmaceutical formulation should exhibit no more than 6,000 10 μm particles per container (or unit) compared to a control sample. In certain embodiments, a stable pharmaceutical formulation should exhibit no more than 5000, no more than 4000, no more than 3000, no more than 2000, or no more than 1000 10 μm particles per container (or unit) compared to a control sample. In other embodiments, a stable pharmaceutical formulation should exhibit no more than 600 25 μm particles per container (or unit) compared to a control sample. In certain embodiments, a stable pharmaceutical formulation should exhibit no more than 500, no more than 400, no more than 300, no more than 200, no more than 100, or no more than 50 25 μm particles per container (or unit) compared to a control sample.

[0364] In certain embodiments, pharmaceutical formulation stability is measured using visual assessment. Visual assessment is a qualitative method for describing the visible physical characteristics of a sample. Depending on the characteristics being evaluated (e.g., color, clarity, the presence of particles or foreign matter), the sample is observed relative to the black and / or white background of the inspection room. The sample is also observed relative to a milky white reference standard and a color reference standard. In the case of a visual assessment, a stable pharmaceutical formulation should exhibit no significant change in color, clarity, particles, or the presence of foreign matter compared to a control sample.

[0365] One aspect of the present invention is a pharmaceutical formulation comprising: (i) about 70 mg / ml to about 250 mg / ml of an antigen binding protein to PCSK9; (ii) about 0.05 mM to about 40 mM buffer, such as sodium acetate ("NaOAC") serving as a buffer; (iii) about 1% to about 5% proline, arginine, lysine, methionine, or taurine (also known as 2-aminoethanesulfonic acid) and / or 0.5% to about 5% benzyl alcohol serving as a stabilizer; and (iv) about 0.004% to about 10% w / v of the formulation of a nonionic surfactant (including but not limited to, polysorbate-80, polysorbate-60, polysorbate-40, and polysorbate-20); wherein the pH of the formulation is in the range of about 4.0 to 6.0. In certain other embodiments, a pharmaceutical formulation of the invention comprises (i) at least about 70 mg / ml, about 100 mg / ml, about 120 mg / ml, about 140 mg / ml, about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, about 200 mg / ml of an anti-PCSK9 antibody; (ii) about 10 mM NAOAC; (iii) about 0.01% polysorbate 80; and (iv) about 2%-3% proline (or about 250 mM to about 270 mM proline), wherein the pH of the formulation is about 5. In certain other embodiments, the pharmaceutical formulations of the invention comprise (i) at least about 70 mg / ml, about 100 mg / ml, about 120 mg / ml, about 140 mg / ml of the anti-PCSK9 antibody 21B12, 26H5, and / or 31H4; (ii) about 10 mM NAOAC; (iii) about 0.01% polysorbate 80; and (iv) about 2%-3% proline (or about 250 mM to about 270 mM proline), wherein the pH of the formulation is about 5. In certain other embodiments, the pharmaceutical formulations of the invention comprise (i) at least about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, about 200 mg / ml of anti-PCSK9 antibody 8A3, 11F1, and / or 8A1; (ii) about 10 mM NAOAC; (iii) about 0.01% polysorbate 80; and (iv) about 2%-3% proline (or about 250 mM to about 270 mM proline), wherein the pH of the formulation is about 5.

[0366] One aspect of the invention is a pharmaceutical formulation comprising (i) at least about 70 mg / ml to about 250 mg / ml of an anti-PCSK9 antibody; (ii) about 5 mM to about 20 mM of a buffer, such as NAOAC; (iii) about 1% to about 10% w / v of the formulation of a polyhydroxy hydrocarbon (such as sorbitol) or a disaccharide (such as sucrose); and (iv) about 0.004% to about 10% w / v of the formulation of a surfactant, such as polysorbate 20 or polysorbate 80; wherein the pH of the formulation is in the range of about 4.8 to 5.8; and wherein the pharmaceutical formulation optionally comprises about 80 mM to about 300 mM proline, arginine, lysine, methionine, or taurine and / or 0.5% to about 5% benzyl alcohol to reduce viscosity. In certain other embodiments, the pharmaceutical formulations of the invention comprise (i) at least about 70 mg / ml to about 250 mg / ml of an anti-PCSK9 antibody; (ii) about 10 mM NAOAC; (iii) about 9% sucrose; and (iv) about 0.004% polysorbate 20, wherein the pH of the formulation is about 5.2. In certain other embodiments, the pharmaceutical formulations of the invention comprise (i) at least about 70 mg / ml, about 100 mg / ml, about 120 mg / ml, about 140 mg / ml, about 160 mg / ml, about 180 mg / ml, about 200 mg / ml of an anti-PCSK9 antibody; (ii) about 15 mM NAOAC; (iii) about 9% sucrose; and (iv) about 0.01% polysorbate 20, wherein the pH of the formulation is about 5.2. In certain other embodiments, a pharmaceutical formulation of the invention comprises (i) at least about 70 mg / ml, about 100 mg / ml, about 120 mg / ml, about 140 mg / ml, about 160 mg / ml, about 180 mg / ml, about 200 mg / ml of an anti-PCSK9 antibody; (ii) about 20 mM NAOAC; (iii) about 9% sucrose; and (iv) about 0.01% polysorbate 20, wherein the pH of the formulation is about 5.2. In certain other embodiments, the pharmaceutical formulations of the invention comprise (i) at least about 70 mg / ml, about 100 mg / ml, about 120 mg / ml, about 140 mg / ml, about 160 mg / ml, about 180 mg / ml, about 200 mg / ml of an anti-PCSK9 antibody; (ii) about 10 mM NAOAC; (iii) about 9% sucrose; (iv) about 0.01% polysorbate 80; and (v) about 250 mM proline, wherein the pH of the formulation is about 5.

[0367] The pharmaceutical preparations of the present invention can be administered in combination therapy (i.e., in combination with other agents). In certain embodiments, the combination therapy comprises an antigen binding protein capable of binding to PCSK9 in combination with at least one anti-cholesterol agent. Agents include, but are not limited to, chemical preparations, antibodies, antigen binding regions, and combinations and conjugates thereof synthesized in vitro. In certain embodiments, the agent may act as an agonist, antagonist, allosteric modulator, or toxin. In certain embodiments, the agent may inhibit or stimulate its target (e.g., receptor or enzyme activation or inhibition), thereby promoting the expression of LDLR to increase or reduce serum cholesterol levels.

[0368] In certain embodiments, antigen binding proteins to PCSK9 can be administered before, concurrently with, and after treatment with a cholesterol (serum and / or total cholesterol) lowering agent. In certain embodiments, antigen binding proteins to PCSK9 can be administered prophylactically to prevent or slow the onset of hypercholesterolemia, heart disease, diabetes, and / or any cholesterol-related disease. In certain embodiments, antigen binding proteins to PCSK9 can be administered to treat existing hypercholesterolemia conditions. In some embodiments, the ABP delays the onset of disease and / or disease-related symptoms. In some embodiments, the ABP is provided to a subject who does not have any symptoms of any cholesterol-related disease or its subset.

[0369] In certain embodiments, antigen binding proteins to PCSK9 are used together with specific therapeutic agents to treat various cholesterol-related diseases, such as hypercholesterolemia. In certain embodiments, two, three, or more agents may be administered, depending on the condition and the degree of treatment desired. In certain embodiments, the agents may be provided together by inclusion in the same formulation. In certain embodiments, the one or more agents and the antigen binding protein to PCSK9 may be provided together by inclusion in the same formulation. In certain embodiments, the agents may be formulated separately and provided together by inclusion in a treatment kit. In certain embodiments, the agents and the antigen binding protein to PCSK9 may be formulated separately and provided together by inclusion in a treatment kit. In certain embodiments, the agents may be provided separately.

[0370] In certain embodiments, formulations comprising an antigen binding protein to PCSK9 with or without at least one other therapeutic agent can be prepared for storage by mixing the selected agent having the desired purity with an optional formulating agent (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or aqueous solution. Additionally, in certain embodiments, formulations comprising an antigen binding protein to PCSK9 with or without at least one other therapeutic agent can be formulated as a lyophilizate using appropriate excipients.

[0371] In certain embodiments, when parenteral administration is contemplated, the therapeutic formulation may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired antigen binding protein for PCSK9 together with or without other therapeutic agents in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water in which the antigen binding protein for PCSK9 is formulated into a sterile isotonic solution that has been appropriately preserved together with or without at least one other therapeutic agent. In certain embodiments, the preparation may involve the desired molecule being formulated together with an agent that enables controlled or sustained release of the product so that the product can be delivered via reservoir injection, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. In certain embodiments, hyaluronic acid may also be used, and it may have the effect of promoting duration in the circulation. In certain embodiments, an implantable drug delivery device may be used to introduce the desired molecule.

[0372] In certain embodiments, pharmaceutical formulations can be formulated for inhalation. In certain embodiments, antigen binding proteins to PCSK9 can be formulated as dry powders for inhalation, with or without at least one other therapeutic agent. In certain embodiments, inhalation solutions comprising antigen binding proteins to PCSK9, with or without at least one other therapeutic agent, can be formulated with a propellant for aerosol delivery. In certain embodiments, the solution can be aerosolized for inhalation. Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0373] In certain embodiments, the pharmaceutical formulation may involve an effective amount of an antigen binding protein to PCSK9, with or without at least one other therapeutic agent, mixed with a non-toxic excipient suitable for the manufacture of tablets. In certain embodiments, a solution in unit dosage form can be prepared by dissolving the tablet in sterile water or another appropriate vehicle. In certain embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or acacia; or lubricants such as magnesium stearate, stearic acid or talc.

[0374] Other pharmaceutical formulations will be readily apparent to those skilled in the art, including formulations directed to antigen binding proteins for PCSK9 in the form of sustained or controlled delivery formulations, with or without at least one other therapeutic agent. In certain embodiments, techniques for preparing a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and reservoir injections are also known to those skilled in the art. See, for example, PCT Application No. PCT / US93 / 00829, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical formulations. In certain embodiments, sustained release formulations may include a semipermeable polymer matrix in the form of a shaped article, such as a film or microcapsule. Sustained-release matrices may include polyesters, hydrogels, polylactides (US 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma-ethyl L-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate (Langer et al., supra) or poly-D (-) -3-hydroxybutyric acid (EP 133,988). In certain embodiments, sustained-release formulations may also include liposomes, which can be prepared by any of several methods known in the art. See, eg, Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

[0375] Pharmaceutical preparations to be administered in vivo are generally sterile. In certain embodiments, this can be accomplished by filtration through a sterile filtration membrane. In certain embodiments, when the preparation is lyophilized, sterilization using this method can be performed before or after lyophilization and reconstitution. In certain embodiments, preparations for parenteral administration can be stored in lyophilized form or in solution form. In certain embodiments, parenteral preparations are typically placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle.

[0376] In certain embodiments, once the pharmaceutical formulation has been formulated, it can be stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, or in a dehydrated or lyophilized powder. In certain embodiments, the formulation can be stored in a ready-to-use form or in a form (e.g., lyophilized form) to be reconstituted prior to administration.

[0377] In certain embodiments, once the pharmaceutical formulation has been formulated, it can be stored in a pre-filled syringe as a solution or suspension in a ready-to-use form.

[0378] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit may contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits are included that contain single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyosyringes).

[0379] In certain embodiments, the effective amount of a pharmaceutical formulation comprising an antigen binding protein to PCSK9, with or without at least one other therapeutic agent, to be employed therapeutically will depend, for example, on the treatment context and the subject. Those skilled in the art will appreciate that appropriate therapeutic dosage levels according to certain embodiments will therefore vary depending, in part, on the delivered molecule, the indication for which the antigen binding protein to PCSK9 is being used, the use or absence of at least one other therapeutic agent, the route of administration, and the patient's size (weight, body surface or organ size) and / or condition (age and overall health). In certain embodiments, a clinician can titrate the dose and modify the route of administration to achieve the optimal therapeutic effect.

[0380] In certain embodiments, the formulation can be administered locally via implantation of a membrane, sponge, or another suitable material onto which the desired molecule has been adsorbed or encapsulated. In certain embodiments, when an implant device is used, the device can be implanted in any suitable tissue or organ, and delivery of the desired molecule can be achieved via diffusion, timed-release bolus, or continuous administration.

[0381] Dosage and administration schedule

[0382] Any antigen binding protein against PCSK9 described in Table 2 and Figures 2 and / or 3 can be administered to a patient according to the methods of the present invention. In some embodiments, the antigen binding protein against PCSK9 includes 21B12, 26H5, 31H4, 8A3, 11F1, or 8A1.

[0383] The amount of an antigen binding protein to PCSK9 (e.g., an anti-PCSK9 antibody) administered to a patient according to the methods of the present invention is generally a therapeutically effective amount. The amount of the ABP may be expressed in milligrams of antibody (i.e., mg) or in milligrams of antibody per kilogram of patient body weight (i.e., mg / kg). In certain embodiments, a typical dosage of the antigen binding protein to PCSK9 may range from about 0.1 μg / kg to about 100 mg / kg or more of the antigen binding protein to PCSK9. In certain embodiments, the dosage may range from 0.1 μg / kg to about 100 mg / kg; or 1 μg / kg to about 100 mg / kg; or 5 μg / kg to about 100 mg / kg of the antigen binding protein to PCSK9; or 1 mg / kg to about 50 mg / kg of the antigen binding protein to PCSK9; or 2 mg / kg to about 20 mg / kg of the antigen binding protein to PCSK9; or 2 mg / kg to about 10 mg / kg of the antigen binding protein to PCSK9.

[0384] In certain embodiments, the amount (or dose) of an antigen binding protein to PCSK9 can be in the range of at least about 10 mg to about 1400 mg; or about 14 mg to about 1200 mg; or about 14 mg to about 1000 mg; or about 14 mg to about 800 mg; or about 14 mg to about 700 mg; or about 14 mg to about 480 mg; or about 20 mg up to about 480 mg; or about 70 mg up to about 480 mg; or about 80 mg to about 480 mg; or about 90 mg to about 480 mg; or about 100 mg to about 480 mg; or about 105 mg to about 480 mg; or about 110 mg to about 480 mg; or about 115 mg to about 480 mg; or about 120 mg to about 480 mg; or about 125 mg to about 480 mg; or about 130 mg to about 480 mg; or about 135 mg to about 480 mg; or about 140 mg to about 480 mg; or about 145 mg to about 480 mg; or about 150 mg to about 480 mg; or about 160 mg to about 480 mg; or about 170 mg to about 480 mg; or about 180 mg to about 480 mg; or about 190 mg to about 480 mg; or about 200 mg to about 480 mg; or about 210 mg to about 480 mg; or about 220 mg or about 330 mg to about 480 mg; or about 340 mg to about 480 mg; or about 350 mg to about 480 mg; or about 360 mg to about 480 mg; or about 370 mg to about 480 mg; or about 380 mg to about 480 mg; or about 390 mg to about 480 mg; or about 400 mg to about 480 mg; or about 410 mg to about 480 mg; or about 420 mg or about 430 mg to about 480 mg; or about 440 mg to about 480 mg; or about 450 mg to about 480 mg; or about 460 mg to about 480 mg; or about 470 mg to about 480 mg of antigen binding protein to PCSK9.

[0385] In certain embodiments, the frequency of administration will take into account the pharmacokinetic parameters of the antigen binding protein for PCSK9 and / or any other therapeutic agent in the formulation used. In certain embodiments, the clinician will administer the formulation until the dose that achieves the desired effect is reached. In certain embodiments, therefore, a single dose of the formulation may be administered, or two, three, four, or more doses (which may or may not contain the same amount of the desired molecule) may be administered over time, or the formulation may be administered as a continuous infusion via an implantable device or catheter. The formulation may also be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices and automatic syringe delivery devices may be used to deliver the pharmaceutical formulations of the present invention. Further refinement of the appropriate dose is routinely performed by a person of ordinary skill in the art and is within the scope of the tasks routinely performed by them. In certain embodiments, the appropriate dose may be determined using appropriate dose-response data. In some embodiments, the amount and frequency of administration may take into account the desired cholesterol level (serum and / or overall) to be obtained and the subject's current cholesterol level, LDL level, and / or LDLR level, which can all be obtained by methods well known to those skilled in the art.

[0386] In certain embodiments, an antigen binding protein to PCSK9 is administered once a week (QW) to a patient in need thereof at a dose of at least about 10 mg; or up to about 14 mg; or up to about 20 mg; or up to about 35 mg; or up to about 40 mg; or up to about 45 mg; or up to about 50 mg; or up to about 70 mg.

[0387] In some other embodiments, the following doses of an antigen binding protein to PCSK9 are administered to a patient in need thereof once every other week (or every two weeks): at least about 70 mg; or up to about 100 mg; or up to about 105 mg; or up to about 110 mg; or up to about 115 mg; or up to about 120 mg; or up to about 140 mg; or up to about 160 mg; or up to about 200 mg; or up to about 250 mg; or up to 280 mg; or up to 300 mg; or up to 350 mg; or up to 400 mg; or up to 420 mg.

[0388] In certain other embodiments, the following doses of an antigen binding protein to PCSK9 are administered to a patient in need thereof once every four weeks (or once a month): at least about 250 mg; or up to about 280 mg; or up to about 300 mg; or up to about 350 mg; or up to about 400 mg; or up to about 420 mg; or up to about 450 mg; or up to 480 mg.

[0389] In some embodiments, serum LDL cholesterol levels are reduced by at least about 30%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 40%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 50%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 55%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 60%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 65%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 70%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 75%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 80%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 85%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 90%.

[0390] In some embodiments, the serum LDL cholesterol level is reduced by at least about 30%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days, relative to the pre-dose level.

[0391] In some embodiments, the serum LDL cholesterol level is reduced by at least about 35%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days relative to the pre-dose level.

[0392] In some embodiments, the serum LDL cholesterol level is reduced by at least about 40%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days relative to the pre-dose level.

[0393] In some embodiments, the serum LDL cholesterol level is reduced by at least about 45%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days, relative to the pre-dose level.

[0394] In some embodiments, the serum LDL cholesterol level is reduced by at least about 50%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days, relative to the pre-dose level.

[0395] In some embodiments, the serum LDL cholesterol level is reduced by at least about 55%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days, relative to the pre-dose level.

[0396] In some embodiments, the serum LDL cholesterol level is reduced by at least about 60%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days, relative to the pre-dose level.

[0397] In some embodiments, the serum LDL cholesterol level is reduced by at least about 65%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days relative to the pre-dose level.

[0398] In some embodiments, the serum LDL cholesterol level is reduced by at least about 70%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days, relative to the pre-dose level.

[0399] In some embodiments, the serum LDL cholesterol level is reduced by at least about 75%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28, or at least about 31 days relative to the pre-dose level.

[0400] In some embodiments, the serum LDL cholesterol level is reduced by at least about 80%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28, or at least about 31 days relative to the pre-dose level.

[0401] In some embodiments, the serum LDL cholesterol level is reduced by at least about 85%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28, or at least about 31 days relative to the pre-dose level.

[0402] In some embodiments, the serum LDL cholesterol level is reduced by at least about 90%, and the reduction persists for a period of at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 25 days, at least about 28 days, or at least about 31 days relative to the pre-dose level.

[0403] Certain therapeutic applications

[0404] As will be appreciated by those skilled in the art, diseases associated with, involving, or that may be affected by: altered cholesterol, LDL, LDLR, PCSK9, VLDL-C, apoprotein B ("ApoB"), lipoprotein A ("Lp(a)"), triglycerides, HDL-C, non-HDL-C, and total cholesterol levels can be addressed by the antigen binding proteins to PCSK9 described herein. In one aspect, antigen binding proteins to PCSK9 can be used in methods for treating and / or preventing and / or reducing the risk of diseases associated with, or having significant elevated serum cholesterol levels. In one aspect, antigen binding proteins to PCSK9 can be used in methods for treating and / or preventing and / or reducing the risk of diseases associated with, or having significant elevated PCSK9 values. In one aspect, antigen binding proteins to PCSK9 can be used in methods for treating and / or preventing and / or reducing the risk of diseases associated with, or having significant elevated total cholesterol levels. In one aspect, antigen binding proteins against PCSK9 can be used in methods for treating and / or preventing and / or reducing the risk of diseases associated with, or having significant significance for, elevated non-HDL cholesterol levels. In one aspect, antigen binding proteins against PCSK9 can be used in methods for treating and / or preventing and / or reducing the risk of diseases associated with, or having significant significance for elevated ApoB levels. In one aspect, antigen binding proteins against PCSK9 can be used in methods for treating and / or preventing and / or reducing the risk of diseases associated with, or having significant significance for elevated Lp(a) levels. In one aspect, antigen binding proteins against PCSK9 can be used in methods for treating and / or preventing and / or reducing the risk of diseases associated with, or having significant significance for elevated triglyceride levels. In one aspect, antigen binding proteins against PCSK9 can be used in methods for treating and / or preventing and / or reducing the risk of diseases associated with, or having significant significance for elevated VLDL-C levels.

[0405] In one aspect, antigen binding proteins directed against PCSK9 are used to regulate a patient's serum LDL cholesterol level. In some embodiments, antigen binding proteins directed against PCSK9 are used to reduce the amount of serum LDL cholesterol from abnormally high levels or even normal levels. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 30%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 35%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 40%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 45%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 50%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 55%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 60%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 65%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 70%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 75%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 80%. In certain embodiments, serum LDL cholesterol levels are reduced by at least about 85%. In some embodiments, serum LDL cholesterol levels are reduced by at least about 90%.

[0406] In one aspect, antigen binding proteins directed against PCSK9 are used to modulate a patient's serum PCSK9 levels. In certain embodiments, the antigen binding proteins directed against PCSK9 are neutralizing. In some embodiments, antigen binding proteins directed against PCSK9 are used to lower PCSK9 levels from abnormally high levels or even normal levels. In some embodiments, serum PCSK9 levels are reduced by at least about 60%. In some embodiments, serum PCSK9 levels are reduced by at least about 65%. In some embodiments, serum PCSK9 levels are reduced by at least about 70%. In some embodiments, serum PCSK9 levels are reduced by at least about 75%. In some embodiments, serum PCSK9 levels are reduced by at least about 80%. In some embodiments, serum PCSK9 levels are reduced by at least about 85%. In some embodiments, serum PCSK9 levels are reduced by at least about 90%.

[0407] On the one hand, antigen binding proteins to PCSK9 are used to regulate the total cholesterol level of a patient. In certain embodiments, antigen binding proteins to PCSK9 are neutralizing. In some embodiments, antigen binding proteins to PCSK9 are used to reduce the amount of total cholesterol from abnormally high levels or even normal levels. In some embodiments, total cholesterol levels are reduced by at least about 20%. In some embodiments, total cholesterol levels are reduced by at least about 25%. In some embodiments, total cholesterol levels are reduced by at least about 30%. In some embodiments, total cholesterol levels are reduced by at least about 35%. In some embodiments, total cholesterol levels are reduced by at least about 40%. In some embodiments, total cholesterol levels are reduced by at least about 45%. In some embodiments, total cholesterol levels are reduced by at least about 50%. In some embodiments, total cholesterol levels are reduced by at least about 55%. In some embodiments, total cholesterol levels are reduced by at least about 60%.

[0408] In one aspect, antigen binding proteins against PCSK9 are used to regulate a patient's non-HDL cholesterol levels. In certain embodiments, the antigen binding proteins against PCSK9 are neutralizing. In some embodiments, antigen binding proteins against PCSK9 are used to lower non-HDL cholesterol from abnormally high levels or even normal levels. In some embodiments, non-HDL cholesterol levels are reduced by at least about 30%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 35%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 40%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 50%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 55%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 60%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 65%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 70%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 75%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 80%. In some embodiments, non-HDL cholesterol levels are reduced by at least about 85%.

[0409] In one aspect, antigen binding proteins against PCSK9 are used to modulate a patient's ApoB levels. In certain embodiments, the antigen binding proteins against PCSK9 are neutralizing. In some embodiments, antigen binding proteins against PCSK9 are used to reduce the amount of ApoB from abnormally high levels or even normal levels. In some embodiments, ApoB levels are reduced by at least about 25%. In some embodiments, ApoB levels are reduced by at least about 30%. In some embodiments, ApoB levels are reduced by at least about 35%. In some embodiments, ApoB levels are reduced by at least about 40%. In some embodiments, ApoB levels are reduced by at least about 45%. In some embodiments, ApoB levels are reduced by at least about 50%. In some embodiments, ApoB levels are reduced by at least about 55%. In some embodiments, ApoB levels are reduced by at least about 60%. In some embodiments, ApoB levels are reduced by at least about 65%. In some embodiments, ApoB levels are reduced by at least about 70%. In some embodiments, ApoB levels are reduced by at least about 75%.

[0410] In one aspect, antigen binding proteins against PCSK9 are used to modulate a patient's Lp(a) levels. In certain embodiments, antigen binding proteins against PCSK9 are neutralizing. In some embodiments, antigen binding proteins against PCSK9 are used to reduce the amount of Lp(a) from abnormally high levels or even normal levels. In some embodiments, Lp(a) levels are reduced by at least about 5%. In some embodiments, Lp(a) levels are reduced by at least about 10%. In some embodiments, Lp(a) levels are reduced by at least about 15%. In some embodiments, Lp(a) levels are reduced by at least about 20%. In some embodiments, Lp(a) levels are reduced by at least about 25%. In some embodiments, Lp(a) levels are reduced by at least about 30%. In some embodiments, Lp(a) levels are reduced by at least about 35%. In some embodiments, Lp(a) levels are reduced by at least about 40%. In some embodiments, Lp(a) levels are reduced by at least about 45%. In some embodiments, Lp(a) levels are reduced by at least about 50%. In some embodiments, Lp(a) levels are reduced by at least about 55%. In some embodiments, Lp(a) levels are reduced by at least about 60%. In some embodiments, Lp(a) levels are reduced by at least about 65%.

[0411] As will be appreciated by those skilled in the art, the antigen binding proteins against PCSK9 of the present invention may be therapeutically useful for treating and / or preventing cholesterol-related diseases. In some embodiments, "cholesterol-related diseases" (including "serum cholesterol-related diseases") include any one or more of the following: familial hypercholesterolemia, non-familial hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and general dyslipidemia, which may manifest as, for example, elevated total serum cholesterol, elevated LDL, elevated triglycerides, elevated VLDL, and / or low HDL. Some non-limiting examples of primary and secondary dyslipidemias that can be treated using ABPs, alone or in combination with one or more other agents, include metabolic syndrome, diabetes, familial combined hyperlipidemia, familial hypertriglyceridemia, familial hypercholesterolemia (including heterozygous hypercholesterolemia, homozygous hypercholesterolemia), familial apolipoprotein B-100 deficiency, polygenic hypercholesterolemia, remnant removal disease, hepatic lipase deficiency, dyslipidemia secondary to any of the following: dietary indiscretion, hypothyroidism, medications (including estrogen and progesterone therapy, beta-blockers, and thiazide diuretics), nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage disease, liver cancer, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency, and alcohol-induced hypertriglyceridemia. ABP is also applicable to the prevention or treatment of atherosclerotic diseases, such as cardiovascular death, non-cardiovascular death or all-cause death, coronary heart disease, coronary artery disease, peripheral arterial disease, stroke (ischemic and hemorrhagic), angina or cerebrovascular disease and acute coronary syndrome, myocardial infarction and unstable angina. In some embodiments, ABP is applicable to reducing the risk of fatal and non-fatal heart attack, fatal and non-fatal stroke, certain types of cardiac surgery, hospitalization for heart failure, chest pain in patients with heart disease and / or cardiovascular events due to confirmed heart disease (such as previous heart attack, previous cardiac surgery and / or chest pain with signs of arterial blockage) and / or transplant-related vascular disease. In some embodiments, ABP is applicable to preventing or reducing the cardiovascular risk due to elevated CRP or hsCRP. In some embodiments, ABP and methods can be used to reduce the risk of recurrent cardiovascular events.

[0412] As will be appreciated by those skilled in the art, conditions or diseases that are typically treated (treatable or preventable) with statins may also benefit from the use of the antigen-binding proteins of the present invention. Furthermore, in some embodiments, diseases or conditions that may benefit from preventing cholesterol synthesis or increased LDLR expression may also be treated using various embodiments of the antigen-binding proteins. Furthermore, as will be appreciated by those skilled in the art, the use of anti-PCSK9 antibodies may be particularly useful for treating diabetes. Not only is diabetes a risk factor for coronary heart disease, but insulin also increases PCSK9 expression. That is, people with diabetes have elevated plasma lipid levels (which may be associated with high PCSK9 levels) and may benefit from lowering those levels. This is generally discussed in more detail in Costet et al. ("Hepatic PCSK9 Expression is Regulated by Nutritional Status via Insulin and Sterol Regulatory Element-binding Protein 1C," J. Biol. Chem., 281: 6211-6218, 2006), which is incorporated herein by reference in its entirety.

[0413] In some embodiments, antigen binding proteins are administered to subjects with diabetes, abdominal aortic aneurysm, atherosclerosis, and / or peripheral vascular disease to reduce their serum cholesterol levels to a safer range. In some embodiments, antigen binding proteins are administered to patients at risk of developing any of the diseases described herein. In some embodiments, ABPs are administered to subjects who smoke or have smoked in the past (i.e., former smokers), have high blood pressure, or a family history of early heart attacks.

[0414] In some embodiments, according to the 2004 NCEP treatment goals, if the subject is in moderate or higher risk, then ABP is administered to the subject. In some embodiments, if the subject's LDL cholesterol level is greater than 160 mg / dl, then ABP is administered to the subject. In some embodiments, if the subject's LDL cholesterol level is greater than 130 (and according to the 2004 NCEP treatment goals, it has a moderate or moderately high risk), then ABP is administered. In some embodiments, if the subject's LDL cholesterol level is greater than 100 (and according to the 2004 NCEP treatment goals, it has a high or very high risk), then ABP is administered. In some embodiments, if the subject's LDL cholesterol level is greater than 80 mg / dL, then ABP is administered. In some embodiments, if the subject's LDL cholesterol level is greater than 70 mg / dL, then ABP is administered.

[0415] The physician will be able to select the appropriate treatment indication and target lipid level based on the individual profile of the specific patient. A well-accepted standard for guiding the treatment of hyperlipidemia is the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of the High Blood Cholesterol in Adults (Adult Treatment Panel III) Final Report, National Institutes of Health, NIH Publication No. 02-5215 (2002), the printed content of which is hereby incorporated by reference in its entirety.

[0416] In some embodiments, antigen binding proteins directed against PCSK9 are used to treat or prevent hypercholesterolemia, hyperlipidemia, or dyslipidemia and / or to prepare medicaments for hypercholesterolemia, hyperlipidemia, or dyslipidemia and / or other cholesterol-related diseases (such as those indicated herein). In certain embodiments, antigen binding proteins directed against PCSK9 are used to treat or prevent conditions in which PCSK9 activity is normal, such as hypercholesterolemia. In such conditions, for example, reduction of PCSK9 activity to below normal activity can provide a therapeutic effect.

[0417] Combination therapy

[0418] In certain embodiments, methods of treating cholesterol-related diseases, such as hypercholesterolemia, hyperlipidemia, or dyslipidemia, are provided, comprising administering a therapeutically effective amount of one or more antigen binding proteins to PCSK9 and another therapeutic agent. In certain embodiments, the antigen binding protein to PCSK9 is administered prior to administering the at least one other therapeutic agent. In certain embodiments, the antigen binding protein to PCSK9 is administered concurrently with administering the at least one other therapeutic agent. In certain embodiments, the antigen binding protein to PCSK9 is administered after administering the at least one other therapeutic agent.

[0419] Therapeutic agents (in addition to the antigen binding protein) include, but are not limited to, at least one other cholesterol (serum and / or systemic cholesterol) lowering agent. In some embodiments, the agent increases the expression of LDLR, has been observed to increase serum HDL levels, lower LDL levels, or lower triglyceride levels. Exemplary agents include, but are not limited to, statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, Rosuvastatin, simvastatin), Nicotinic acid (Niacin) (NIACOR, NIASPAN (extended-release niacin), SLO-NIACIN (extended-release niacin), CORDAPTIVE (laropiprant), Fibric acids (LOPID (Gemfibrozil), TRICOR (fenofibrate), Bile acid sequestrants

[00135] These include, but are not limited to, QUESTRAN (cholestyramine), colesevelam (WELCHOL), COLESTID (colestipol), cholesterol absorption inhibitors (ZETIA (ezetimibe), niacin in combination with a statin (ADVICOR (LOVASTATIN and NISPANN), statin in combination with an absorption inhibitor (VYTORIN (ZOCOR and AZETIA), and / or lipid modulators. In some embodiments, the ABP is combined with a PPAR gamma agonist, a PPAR alpha / gamma agonist, a squalene synthase inhibitor, a CETP inhibitor, an antihypertensive agent, an antidiabetic agent (e.g., sulfonylureas, insulin, GLP-1 analogs, DDPIV inhibitors, e.g., metformin), an ApoB modulator (e.g., mipomersan), an MTP inhibitor, and / or arteriosclerosis obliterans treatment.In some embodiments, ABP is combined with an agent that increases a subject's LDLR protein level, such as statins, certain cytokines like oncostatin M, estrogens, and / or certain herbal ingredients (such as berberine). In some embodiments, ABP is combined with an agent that increases a subject's serum cholesterol level, such as certain antipsychotics, certain HIV protease inhibitors, dietary elements (such as high fructose, sucrose, cholesterol, or certain fatty acids), and certain nuclear receptor agonists and antagonists of RXR, RAR, LXR, FXR. In some embodiments, ABP is combined with an agent that increases a subject's PCSK9 level, such as statins and / or insulin. The combination of the two can mitigate the undesirable side effects of other agents with the ABP.

[0420] In certain embodiments, antigen binding proteins directed against PCSK9 can be used with at least one therapeutic agent for inflammation. In certain embodiments, antigen binding proteins directed against PCSK9 can be used with at least one therapeutic agent for immune diseases. Exemplary therapeutic agents for inflammatory and immune diseases include, but are not limited to, inhibitors of cyclooxygenase type 1 (COX-1) and cyclooxygenase type 2 (COX-2); small molecule modulators of 38 kDa mitogen-activated protein kinase (p38-MAPK); and small molecule modulators of intracellular molecules involved in inflammatory pathways, including, but not limited to, JNK, IKK, NF-κB, ZAP70, and ICK. Certain exemplary therapeutic agents for inflammation are described, for example, in CA Dinarello and LL Moldawer, Proinflammatory and Anti-Inflammatory Cytokines in Rheumatoid Arthritis: A Primer for Clinicians, 3rd Edition (2001), Amgen Inc., Thousand Oaks, CA.

[0421] Diagnostic applications

[0422] In some embodiments, the ABP is used as a diagnostic tool. The ABP can be used to analyze the amount of PCSK9 present in a sample and / or subject. As will be appreciated by those skilled in the art, the ABP need not be a neutralizing ABP. In some embodiments, the diagnostic ABP is not a neutralizing ABP. In some embodiments, the epitope bound by the diagnostic ABP is different from the epitope bound by the neutralizing ABP. In some embodiments, the two ABPs do not compete with each other.

[0423] In some embodiments, the ABPs disclosed herein are used or provided in assay kits and / or methods for detecting PCSK9 in mammalian tissues or cells to screen for / diagnose conditions or diseases associated with altered levels of PCSK9. The kit comprises an ABP that binds PCSK9 and a means for indicating the binding of the ABP to PCSK9 (if present) and, optionally, the level of PCSK9 protein. Various means for indicating the presence of the ABP can be used. For example, fluorophores, other molecular probes, or enzymes can be attached to the ABP, and the presence of the ABP can be observed in a variety of ways. Methods for screening for such diseases can involve using the kit, or simply using one of the disclosed ABPs and determining whether the ABP binds to PCSK9 in a sample. As will be appreciated by those skilled in the art, high or elevated levels of PCSK9 will result in greater amounts of the ABP binding to PCSK9 in a sample. Thus, the extent of ABP binding can be used to determine how much PCSK9 is present in a sample. A subject or sample having an amount of PCSK9 greater than a predetermined amount (e.g., an amount or range that a person without a PCSK9-related disease would have) can be characterized as having a PCSK9-mediated disease. In some embodiments, an ABP is administered to a subject taking a statin to determine whether the statin has increased the amount of PCSK9 in the subject.

[0424] In some embodiments, the ABP is a non-neutralizing ABP and is used to determine the amount of PCSK9 in a subject receiving ABP and / or statin therapy. Example

[0425] The following examples, including the experiments performed and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the present invention.

[0426] Example 1

[0427] Immunization and titration

[0428] Generation of anti-PCSK9 antibodies and hybridomas

[0429] For mice containing human immunoglobulin genes Mouse (Abgenix, Fremont, CA) was used to generate antibodies against the mature form of PCSK9 (described as Figure 1A The two groups Mice (Groups 1 and 2) were used to generate antibodies against PCSK9. Group 1 included the XMG2-KL strain. Mice that produce fully human IgG2 κand IgG2λ antibodies. Group 1 mice were immunized with human PCSK9. PCSK9 was prepared using standard recombinant techniques using the GenBank sequence as a reference (NM_174936). Group 2 included the XMG4-KL strain. Mice that produce fully human IgG4 κ Group 2 mice were also immunized with human PCSK9.

[0430] Both groups of mice were injected 11 times with antigen according to the schedule in Table 3. At the time of the initial immunization, each mouse was injected with a total of 10 μg of antigen, delivered intraperitoneally into the abdomen. This was followed by a booster dose of 5 μg and the injection method was staggered between intraperitoneal injection into the abdomen and subcutaneous injection at the base of the tail. For intraperitoneal injections, the antigen was injected with Gold (Sigma, catalog number T2684) was prepared as an emulsion, and for subcutaneous injection, the antigen was mixed with alum (aluminum phosphate) and CpG oligomers. At the 2nd to 8th and 10th injections, each mouse was injected with a total of 5 μg of antigen in alum gel adjuvant. The final injection of 5 μg of antigen per mouse was delivered in phosphate-buffered saline and delivered to two sites, 50% IP to the abdomen and 50% SQ at the base of the tail. The immunization schedule is summarized in Table 3 shown below.

[0431] Table 3

[0432]

[0433]

[0434] The protocol for titering XenoMouse animals was as follows: Costar 3368 medium-binding plates were coated with neutravadin at 8 μg / ml (50 μl / well) and incubated overnight at 4°C in 1× PBS / 0.05% azide. The plates were washed with RO water using a TiterTek 3-cycle wash. Each plate was blocked with 250 μl of 1× PBS / 1% milk and incubated at room temperature for at least 30 minutes. The blocker was washed with RO water using a TiterTek 3-cycle wash. 2 μg / ml of b-human PCSK9 was then captured in 50 μl of 1× PBS / 1% milk / 10 mM Ca₂+ (assay diluent) per well and incubated at room temperature for 1 hour. The plates were then washed with RO water using a TiterTek 3-cycle wash. For the primary antibody, sera were titrated in duplicate starting at 1:100 and then 1:3. This was done in 50 μl of assay diluent per well and incubated at room temperature for 1 hour. This was followed by a TiterTek 3-cycle wash with RO water. The secondary antibody was goat anti-human IgG Fc HRP at 400 ng / ml in 50 μl of assay diluent per well. This was incubated at room temperature for 1 hour. This was followed by a TiterTek 3-cycle wash with RO water and patted dry with a paper towel. For the substrate, a one-step TMB solution (Neogen, Lexington, Kentucky) was used (50 μl / well) and allowed to develop for 30 minutes at room temperature.

[0435] The protocol followed in the ELISA assay was as follows: For samples containing b-PCSK9 without a V5His tag, the following protocol was used: Costar 3368 medium binding plates (Corning Life Sciences) were used. Each plate was coated with 8 μg / ml of neutravidin (50 μl / well) in 1×PBS / 0.05% azide. Each plate was incubated overnight at 4°C. The plates were then washed using a Titertek M384 plate washer (Titertek, Huntsville, AL). Three wash cycles were performed. Each plate was blocked with 250 μl of 1×PBS / 1% milk and incubated at room temperature for approximately 30 minutes. The plates were then washed using an M384 plate washer. Three wash cycles were performed. The capture was b-hu PCSK9 without a V5 tag and incubated in 1×PBS / 1% milk / 10 mM CaCl2. 2+2 μg / ml was added to the plate (40 μl / well). The plates were then incubated for 1 hour at room temperature. Three wash cycles were performed. The serum was titrated in duplicate starting from 1:100 at a 1:3 ratio, and row H contained blank serum. The titration was performed in assay diluent at a volume of 50 μl per well. The plates were incubated for 1 hour at room temperature. Three wash cycles were then performed. The plate was added to 1× PBS / 1% milk / 10 mM CaCl2. 2+ 100ng / ml (1:4000) goat anti-human IgG Fc HRP (50 μl / well) in 5% paraformaldehyde was added to the plate and incubated at room temperature for 1 hour. Using a 3-cycle wash method, each plate was washed again. Each plate was then patted dry with a paper towel. Finally, 1 step of TMB (Neogen, Lexington, Kentucky) (50 μl / well) was added to the plate and quenched with 1N hydrochloric acid (50 μl / well) after 30 minutes at room temperature. The OD value was immediately read at 450nm using a Titertek plate reader.

[0436] Positive controls for detection of plate-bound PCSK9 included soluble LDL receptor (R&D Systems, catalog #2148LD / CF) and a polyclonal rabbit anti-PCSK9 antibody (Caymen Chemical #10007185) titrated 1:3 in duplicate starting at 3 μg / ml in assay diluent. LDLR was detected using goat anti-LDLR (R&D Systems, catalog #AF2148) and rabbit anti-goat IgGFc HRP at a concentration of 400 ng / ml; the rabbit polyclonal antibody was detected using goat anti-rabbit IgGFc at a concentration of 400 ng / ml in assay diluent. Negative controls included native XMG2-KL and XMG4-KL sera titrated 1:3 in duplicate starting at 1:100 in assay diluent.

[0437] For samples containing b-PCSK9 with a V5His tag, the following protocol was used: Costar 3368 medium binding plates (Corning Life Sciences) were used. Each plate was coated with 8 μg / ml of neutravidin (50 μl / well) in 1×PBS / 0.05% azide. Each plate was incubated overnight at 4°C. The plates were then washed using a Titertek M384 plate washer (Titertek, Huntsville, AL). Three wash cycles were performed. Each plate was blocked with 250 μl of 1×PBS / 1% milk and incubated at room temperature for approximately 30 minutes. The plates were then washed using an M384 plate washer. Three wash cycles were performed. The capture object was b-huPCSK9 with a V5 tag and incubated in 1×PBS / 1% milk / 10 mM CaCl2. 2+2 μg / ml was added (40 μl / well). The plates were then incubated for 1 hour at room temperature. Three wash cycles were performed. The serum was titrated in duplicate starting from 1:100 at 1:3, and row H was the blank serum. The titration was performed in the assay diluent at a volume of 50 μl per well. The plates were incubated for 1 hour at room temperature. The plates were then washed using an M384 plate washer operated using a three-cycle wash method. 50 μl / well of 1× PBS / 1% milk / 10 mM Ca was added. 2+ 400 ng / ml goat anti-human IgG Fc HRP in 5% HCl was added to the plate and incubated at room temperature for 1 hour. Three wash cycles were used to wash each plate again. Each plate was then patted dry with a paper towel. Finally, 1-step TMB (Neogen, Lexington, Kentucky) (50 μl / well) was added to the plate and the plate was quenched with 1N hydrochloric acid (50 μl / well) after 30 minutes at room temperature. OD values ​​were immediately read at 450 nm using a Titertek plate reader.

[0438] Positive controls were LDLR and rabbit anti-PCSK9 titrated 1:3 in duplicate starting at 3 μg / ml in assay diluent. LDLR was detected with goat anti-LDLR (R&D Systems, catalog number AF2148) and rabbit anti-goat IgG Fc HRP at a concentration of 400 ng / ml; the rabbit polyclonal antibody was detected with goat anti-rabbit IgG Fc at a concentration of 400 ng / ml in assay diluent. Human anti-His 1.2,3 and anti-V51.7.1 were titrated 1:3 in duplicate starting at 1 μg / ml in assay diluent; both were detected with goat anti-human IgG Fc HRP at a concentration of 400 ng / ml in assay diluent. Negative controls were native XMG2-KL and XMG4-KL sera titrated 1:3 in duplicate starting at 1:100 in assay diluent.

[0439] Mice immunized with soluble antigen as described were tested for antibody titers against human PCSK9 by ELISA assay. Table 4 summarizes the ELISA data and indicates that some mice appear to be specific for PCSK9. See, for example, Table 4. Therefore, at the end of the immunization program, 10 mice (indicated in bold in Table 4) were selected for harvest, and splenocytes and lymphocytes were isolated from the spleen and lymph nodes, respectively, as described herein.

[0440] Table 4

[0441] Overview of ELISA results

[0442]

[0443] Example 2

[0444] Lymphocyte recovery, B cell isolation, fusion and hybridoma generation

[0445] This example summarizes how to recover immune cells and how to generate hybridomas. Selected immunized mice were sacrificed by cervical dislocation and draining lymph nodes from each group were harvested and pooled. B cells were dissociated from lymphoid tissue by grinding in DMEM to release cells from the tissue and the cells were suspended in DMEM. The cells were counted and 0.9 ml of DMEM per 100 million lymphocytes was added to the cell pellet to gently but completely resuspend the cells.

[0446] Lymphocytes were mixed with non-secretory myeloma P3X63Ag8.653 cells (Kearney et al., (1979) J. Immunol. 123, 1548-1550) purchased from ATCC with a catalog number of CRL 1580 at a ratio of 1:4. The cell mixture was gently precipitated by centrifugation at 400 × g for 4 minutes. After decanting the supernatant, the cells were gently mixed using a 1 ml pipette. Preheated PEG / DMSO solution (catalog number P7306) from Sigma (1 ml per million B cells) was slowly added over 1 minute under gentle stirring, followed by mixing for 1 minute. Preheated IDMEM (2 ml per million B cells) (DMEM without glutamine, L-glutamine, penicillin / streptomycin, MEM non-essential amino acids (all from Invitrogen)) was then added over 2 minutes under gentle stirring. Preheated IDMEM (10 ml per million B cells) was finally added over 3 minutes. 6 B cells in 8 ml).

[0447] The fused cells were briefly centrifuged at 400 × g for 6 minutes and resuspended in 20 ml of selection medium (DMEM (Invitrogen), 15% FBS (Hyclone), supplemented with L-glutamine, penicillin / streptomycin, MEM non-essential amino acids, sodium pyruvate, 2-mercaptoethanol (all from Invitrogen), HA-aziserine hypoxanthine, OPI (oxaloacetate, pyruvate, bovine insulin) (all from Sigma), and IL-6 (Boehringer Mannheim)) per million B cells. The cells were incubated at 37°C for 20-30 minutes, then resuspended in 200 ml of selection medium and cultured in T175 flasks for 3-4 days before being plated in 96-well plates. Thus, hybridomas that produce antigen-binding proteins against PCSK9 were generated.

[0448] Example 3

[0449] Selecting PCSK9 Antibodies

[0450] This example summarizes how various PCSK9 antigen binding proteins were characterized and selected. Secreted antibodies (produced by the hybridomas generated in Examples 1 and 2) were evaluated for binding to PCSK9. Antibodies were selected based on binding data and inhibition of PCSK9 binding to LDLR and affinity. Binding to soluble PCSK9 was analyzed by ELISA as described below. (Surface Plasmon Resonance) quantification of binding affinity.

[0451] Primary screening

[0452] Primary screening was performed for antibodies that bind to wild-type PCSK9. Primary screening was performed on two harvests. Primary screening consisted of an ELISA assay and was performed using the following protocol:

[0453] Costar 3702 medium binding 384-well plates (Corning Life Sciences) were used. Each plate was coated with 4 μg / ml neutravidin in 1×PBS / 0.05% azide at a volume of 40 μl per well. Each plate was incubated overnight at 4°C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three wash cycles were performed. Each plate was blocked with 90 μl 1×PBS / 1% milk and incubated at room temperature for approximately 30 minutes. The plates were then washed. Three wash cycles were performed again. The capture sample was biotinylated PCSK9 without a V5 tag and incubated in 1×PBS / 1% milk / 10 mM Ca. 2+ 0.9 μg / ml was added to each well in a volume of 40 μl. The plates were then incubated at room temperature for 1 hour. The plates were then washed using a Titertek plate washer using a 3-cycle wash method. 10 μl of the supernatant was transferred to 40 μl of 1×PBS / 1% milk / 10 mM CaCl2. 2+ The plates were washed again using a Titertek plate washer with a 3-cycle wash cycle. 40 μl / well of 1×PBS / 1% milk / 10 mM CaCl2 was added. 2+ Goat anti-human IgG Fc POD at a concentration of 100 ng / ml (1:4000) was added to the plate and incubated at room temperature for 1 hour. Three wash cycles were used to wash each plate again. Finally, 40 μl / well of one-step TMB (Neogen, Lexington, Kentucky) was added to the plate and quenched with 40 μl / well of 1N hydrochloric acid after 30 minutes at room temperature. OD values ​​were immediately read at 450 nm using a Titertek plate reader.

[0454] The primary screen resulted in a total of 3104 antigen-specific hybridomas identified from both harvests. Based on the highest ELISA OD, 1500 hybridomas per harvest were further rounded to reach a total of 3000 positives.

[0455] Confirmatory screening

[0456] 3000 positives were then screened for binding to wild-type PCSK9 to confirm the generation of stable hybridomas. Screening was performed as follows: Costar 3702 medium binding 384-well plates (Corning Life Sciences) were used. Each plate was coated with 3 μg / ml of neutravidin in 1×PBS / 0.05% azide at a volume of 40 μl per well. Each plate was incubated overnight at 4°C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three wash cycles were performed. Each plate was blocked with 90 μl of 1×PBS / 1% milk and incubated at room temperature for approximately 30 minutes. The plates were then washed using an M384 plate washer. Three wash cycles were performed. The capture sample was b-PCSK9 without the V5 tag and incubated in 1×PBS / 1% milk / 10 mM CaCl2. 2+ 0.9 μg / ml was added to each well in a volume of 40 μl. The plates were then incubated at room temperature for 1 hour. The plates were then washed using a 3-cycle wash method. 10 μl of the supernatant was transferred to 40 μl of 1×PBS / 1% milk / 10 mM CaCl2. 2+ The plates were washed again using a Titertek plate washer with a 3-cycle wash cycle. 40 μl / well of 1×PBS / 1% milk / 10 mM CaCl2 was added. 2+ Goat anti-human IgG Fc POD at a concentration of 100 ng / ml (1:4000) was added to the plate and incubated at room temperature for 1 hour. Each plate was washed again using a Titertek plate washer operated using a 3-cycle wash method. Finally, 40 μl / well of one-step TMB (Neogen, Lexington, Kentucky) was added to the plate and quenched with 40 μl / well of 1N hydrochloric acid after 30 minutes at room temperature. The OD value was immediately read at 450 nm using a Titertek plate reader. In the second screening, a total of 2441 positives were found. These antibodies were then used in subsequent screenings.

[0457] Mouse cross-reactivity screening

[0458] The panel of hybridomas was then screened for cross-reactivity with mouse PCSK9 to determine if the antibodies could bind to both human and mouse PCSK9. The cross-reactivity screen was performed using the following protocol: Costar 3702 medium binding 384-well plates (Corning Life Sciences) were used. Each plate was coated with neutravidin at 3 μg / ml in 1×PBS / 0.05% azide at a volume of 40 μl per well. The plates were incubated overnight at 4°C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three wash cycles were performed. Each plate was blocked with 90 μl of 1×PBS / 1% milk and incubated at room temperature for approximately 30 minutes. The plates were then washed using a Titertek plate washer. Three wash cycles were performed. The capture sample was biotinylated mouse PCSK9 and incubated in 1×PBS / 1% milk / 10 mM CaCl2. 2+ 1 μg / ml was added to each plate at a volume of 40 μl per well. Each plate was then incubated at room temperature for 1 hour. The plates were then washed using a Titertek plate washer using a 3-cycle wash method. 50 μl of the supernatant was transferred to each plate and incubated at room temperature for 1 hour. The plates were washed again using a 3-cycle wash method. 40 μl / well of a 1× PBS / 1% milk / 10 mM CaCl2 solution was added. 2+ Goat anti-human IgG Fc POD at a concentration of 100 ng / ml (1:4000) was added to the plate and incubated at room temperature for 1 hour. Three wash cycles were used to wash each plate again. Finally, 40 μl / well of one-step TMB (Neogen, Lexington, Kentucky) was added to the plate and quenched with 40 μl / well of 1N hydrochloric acid after 30 minutes at room temperature. OD values ​​were immediately read at 450 nm using a Titertek plate reader. 579 antibodies were observed to cross-react with mouse PCSK9. These antibodies were then used in subsequent screening.

[0459] D374Y mutant binding screening

[0460] The D374Y mutation in PCSK9 has been documented in human populations (e.g., Timms KM et al., "Amutation in PCSK9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree," Hum. Genet. 114:349-353, 2004). To determine whether the antibodies are specific for wild-type or also bind to the D374Y form of PCSK9, samples were then screened for binding to mutant PCSK9 sequences containing the D374Y mutation. The screening protocol was as follows: Costar 3702 medium binding 384-well plates (Corning Life Sciences) were used for screening. Each plate was coated with 4 μg / ml neutravidin in 1× PBS / 0.05% azide at a volume of 40 μl per well. The plates were incubated overnight at 4°C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three wash cycles were performed. Each plate was blocked with 90 μl of 1×PBS / 1% milk and incubated at room temperature for approximately 30 minutes. The plates were then washed using a Titertek plate washer. Three wash cycles were performed. Each plate was washed with 1×PBS / 1% milk / 10 mM CaCl2. 2+ The plates were coated with biotinylated human PCSK9 D374Y at a concentration of 1 μg / ml in PBS and incubated at room temperature for 1 hour. The plates were then washed using a Titertek plate washer. Three wash cycles were performed. The supernatant of late-stage exhausted hybridoma cultures was diluted in PBS / milk / CaCl2. 2+ Dilute 1:5 (10 ml plus 40 ml) and incubate at room temperature for 1 hour. Then, add 40 μl / well of 1×PBS / 1% milk / 10 mM Ca 2+ Rabbit anti-human PCSK9 (Cayman Chemical) and human anti-His 1.2.3 (1:2) were titrated onto each plate and incubated at room temperature for 1 hour. The plates were then washed using a Titertek plate washer for 3 wash cycles. 40 μl / well of 1× PBS / 1% milk / 10 mM CaCl2 was added. 2+ Add 100 ng / ml (1:4000) goat anti-human IgG Fc HRP to the plate and incubate the plate for 1 hour at room temperature. Add 40 μl / well of 1×PBS / 1% milk / 10 mM Ca 2+Goat anti-rabbit IgG Fc HRP at a concentration of 100 ng / ml (1:4000) in PBS was added to the plate and incubated at room temperature for 1 hour. Each plate was then washed using a Titertek plate washer. Three wash cycles were performed. Finally, 40 μl / well of one-step TMB (Neogen, Lexington, Kentucky) was added to the plate and quenched with 40 μl / well of 1N hydrochloric acid after 30 minutes at room temperature. The OD value was immediately read at 450 nm using a Titertek plate reader. More than 96% of the positive hits for wild-type PCSK9 also bound to mutant PCSK9.

[0461] Large-scale receptor ligand blocking screen

[0462] To screen for antibodies that block PCSK9 binding to LDLR, an assay was developed using the D374Y PCSK9 mutant. The mutant was used in this assay because it has a higher binding affinity for LDLR, allowing the development of a more sensitive receptor ligand blocking assay. The receptor ligand blocking screen was performed using the following protocol: Screening was performed using Costar 3702 medium-binding 384-well plates (Corning Life Sciences). Each plate was coated with 2 μg / ml goat anti-LDLR (R&D catalog number AF2148) in 1×PBS / 0.05% azide at a volume of 40 μl per well. Each plate was incubated overnight at 4°C. Each plate was then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three wash cycles were performed. Each plate was blocked with 90 μl of 1×PBS / 1% milk and incubated at room temperature for approximately 30 minutes. Each plate was then washed using a Titertek plate washer. Three wash cycles were performed. The capture sample was LDLR (R&D, catalog number 2148LD / CF) and was in 1×PBS / 1% milk / 10 mM Ca 2+0.4 μg / ml was added to the plate at a volume of 40 μl per well. Each plate was then incubated at room temperature for 1 hour and 10 minutes. Simultaneously, 20 ng / ml of biotinylated human D374Y PCSK9 was incubated with 15 microliters of hybridoma depleted supernatant in Nunc polypropylene plates, with the depleted supernatant concentration diluted 1:5. Each plate was then pre-incubated at room temperature for approximately 1 hour and 30 minutes. Each plate was then washed using a Titertek plate washer operating with a 3-cycle wash method. 50 μl / well of the pre-incubation mixture was transferred to an LDLR-coated ELISA plate and incubated at room temperature for 1 hour. To detect LDLR-bound b-PCSK9, 40 μl / well of 500 ng / ml avidin-streptavidin-HRP in assay diluent was added to each plate. Each plate was incubated at room temperature for 1 hour. Each plate was washed again using the Titertek plate washer. Three wash cycles were performed. Finally, 40 μl / well of one-step TMB (Neogen, Lexington, Kentucky) was added to the plate and quenched with 40 μl / well of 1N hydrochloric acid after 30 minutes at room temperature. The OD value was immediately read at 450 nm using a Titertek plate reader. 384 antibodies were screened and identified to fully block the interaction between PCSK9 and LDLR, and 100 antibodies strongly blocked the interaction (OD < 0.3). These antibodies inhibited the binding interaction of PCSK9 and LDLR by greater than 90% (greater than 90% inhibition).

[0463] Receptor-ligand binding assays for a subset of blockers

[0464] The receptor ligand assay was then repeated using the mutant enzymes for the 384-member neutralizer subset identified in the first large-scale receptor ligand inhibition assay. The same protocol as that used in the large-scale receptor ligand blocking screen was employed in the 384-member blocker subset screening assay. This replicate screen validated the initial screening data.

[0465] This screen of a subset of 384 members identified 85 antibodies that blocked the interaction between the mutant PCSK9 enzyme and the LDLR by greater than 90%.

[0466] Receptor ligand binding assay with blockers that bind wild-type PCSK9 but not the D374Y mutant

[0467] In an initial panel of 3,000 suppositories (sup), 86 antibodies showed specific binding to wild-type PCSK9 but not to the huPCSK9(D374Y) mutant. These 86 suppositories were tested for their ability to block wild-type PCSK9 binding to the LDLR receptor. The following protocol was used: Screening was performed using Costar 3702 medium-binding 384-well plates (Corning Life Sciences). Each plate was coated with 10 μg / ml of anti-His 1.2.3 in 1×PBS / 0.05% azide at a volume of 40 μl per well. Each plate was incubated overnight at 4°C. The plates were then washed using a Titertek plate washer (Titertek, Huntsville, AL). Three wash cycles were performed. Each plate was blocked with 90 μl of 1×PBS / 1% milk and incubated at room temperature for approximately 30 minutes. The plates were then washed using a Titertek plate washer. Three wash cycles were performed. LDLR (R&D Systems #2148LD / CF or R&D Systems #2148LD) in 1×PBS / 1% milk / 10 mM Ca 2+ 5 μg / ml was added to the plate at a volume of 40 μl per well. Each plate was then incubated at room temperature for 1 hour. The plates were then washed using a Titertek plate washer using a 3-cycle wash cycle. Simultaneously, biotinylated human wild-type PCSK9 was preincubated with hybridoma depleted supernatant in Nunc polypropylene plates. 22 μl of hybridoma supernatant was transferred to 33 μl of depleted supernatant at a concentration of 583 ng / ml in 1× PBS / 1% milk / 10 mM Ca₂+, resulting in a final b-PCSK9 concentration of 350 ng / ml and a final depleted supernatant dilution of 1:2.5. Each plate was preincubated at room temperature for approximately 1 hour and 30 minutes. 50 μl / well of the preincubation mixture was transferred to the LDLR-captured ELISA plate and incubated at room temperature for 1 hour. The plates were then washed using a Titertek plate washer using a 3-cycle wash cycle. Add 40 μl / well of 500 ng / ml avidin streptavidin HRP in assay diluent to each plate. Incubate each plate at room temperature for 1 hour. Then wash each plate using a Titertek plate washer. Perform 3 wash cycles. Finally, add 40 μl / well of one-step TMB (Neogen, Lexington, Kentucky) to the plate and quench with 40 μl / well of 1N hydrochloric acid after 30 minutes at room temperature. Read the OD value immediately at 450 nm using a Titertek plate reader.

[0468] Filter results

[0469] Based on the results of the assay, several hybridoma lines were identified as producing antibodies that interact with PCSK9. A manageable number of clones were isolated from each line using limiting dilution. The clones are represented by the hybridoma line number (e.g., 21B12) and the clone number (e.g., 21B12.1). In general, no differences were detected between different clones of a particular line by the functional assays described herein. In a few cases, clones expressing differences in functional assays were identified from a particular line, for example, 25A7.1 was found not to block PCSK9 / LDLR but 25A7.3 (referred to herein as 25A7) was neutralizing. The isolated clones were each expanded in 50-100 ml of hybridoma culture medium and grown to exhaustion (i.e., cell viability was less than about 10%). The concentration and potency of antibodies against PCSK9 in the supernatants of those cultures were determined by ELISA and by in vitro functional assays as described herein. As a result of the screening described herein, the hybridoma with the highest titer of antibodies against PCSK9 was identified. The selected hybridomas are displayed in Figures 2A-3D And Table 2.

[0470] Example 4.1

[0471] Production of human 31H4 IgG4 antibody from hybridomas

[0472] This example generally describes how to produce an antigen-binding protein from a hybridoma line. The production protocol used was to generate 50 ml of spent supernatant followed by Protein A purification. Integra production was used for scale-up and was performed later. Hybridoma line 31H4 was grown in a T75 flask in 20 ml of culture medium (Integra medium, Table 5). When the hybridoma reached near confluence in the T75 flask, it was transferred to an Integra flask (Integra Biosciences, Integra CL1000, catalog number 90005).

[0473] Integra flasks are cell culture flasks that are divided into two chambers by a membrane, namely a small chamber and a large chamber. Hybridoma cells from the 31H4 hybridoma strain were cultured in a volume of 20-30 ml in Integra medium (for the composition of Integra medium, see Table 5) at a rate of 1×10 6 A minimum cell density of 100 cells is placed in the small chamber of an Integra flask. Separate Integra medium (1 L) is placed in the large chamber of an Integra flask. The membrane separating the two chambers is permeable to small molecular weight nutrients but impermeable to hybridoma cells and the antibodies produced by those cells. Therefore, the hybridoma cells and the antibodies produced by those hybridoma cells remain in the small chamber.

[0474] After one week, the culture medium was removed from the two chambers of the Integra flask and replaced with fresh Integra culture medium. The culture medium collected from the small chamber was retained separately. After the second week of growth, the culture medium was collected from the small chamber again. The hybridoma strain culture medium collected from the first week was merged with the hybridoma strain culture medium collected from the second week. The collected hybridoma strain culture medium samples were centrifuged to remove cells and debris (at 3000 rpm for 15 minutes) and filtered (0.22 μm) to obtain the supernatant. The clarified conditioned medium was loaded onto a protein A-agarose column. Optionally, the culture medium can be concentrated first and then loaded onto a protein A agarose column. Non-specific binding substances were removed by thorough PBS washing. The antibody protein bound to the protein A column was recovered by eluting the protein A column with standard acidic antibody (such as 50 mM citrate, pH 3.0). Aggregated antibody proteins in the Protein A Sepharose pool were removed by size exclusion chromatography or bound ion exchange chromatography on an anion exchange resin (e.g., Q-Sepharose resin). Specific IEX conditions for the 31H4 protein were Q-Sepharose HP, pH 7.8-8.0. The antibody was eluted using a 10 mM to 500 mM NaCl gradient over 25 column volumes.

[0475] Table 5 Culture medium composition

[0476] INTEGRA medium HSFM 10% ultra-low IgG serum 2mmol / L L-glutamine 1% NEAA 4g / L glucose

[0477] Example 4.2

[0478] Production of recombinant 31H4 human IgG2 antibody from transfected cells

[0479] This example summarizes how to produce the 31H4 IgG2 antibody from transfected cells. 293 cells for transient expression and CHO cells for stable expression were transfected with plasmids encoding the 31H4 heavy and light chains. Conditioned medium from the transfected cells was recovered by removing cells and cell debris. The clarified conditioned medium was loaded onto a Protein A-Sepharose column. Optionally, the medium can be concentrated and then loaded onto the Protein A-Sepharose column. Nonspecific binders were removed by thorough PBS washing. Antibody proteins bound to the Protein A column were recovered by standard acidic antibody elution (e.g., 50 mM citrate, pH 3.0) from the Protein A column. Aggregated antibody proteins in the Protein A-Sepharose pool were removed by size exclusion chromatography or bound ion exchange chromatography on an anion exchange resin (e.g., Q-Sepharose resin). Specific IEX conditions for the 31H4 protein are Q-Sepharose HP, pH 7.8-8.0. The antibody was eluted with a NaCl gradient from 10 mM to 500 mM in 25 column volumes.

[0480] Example 5

[0481] Production of human 21B12 IgG4 antibody from hybridomas

[0482] This example summarizes how to produce antibody 21B12 IgG4 from hybridoma. Hybridoma strain 21B12 was grown in culture medium (Integra culture medium, Table 5) in a T75 flask. When the hybridomas were close to confluence in the T75 flasks, they were transferred to Integra flasks (Integra Biosciences, Integra CL1000, catalog number 90005).

[0483] Integra flasks are cell culture flasks that are divided into two chambers by a membrane, namely a small chamber and a large chamber. Hybridoma cells from the 31H4 hybridoma strain were cultured in a volume of 20-30 ml in Integra medium (for the composition of Integra medium, see Table 5) at a rate of 1×10 cells per ml. 6 A minimum cell density of 10 cells is placed in the small chamber of an Integra flask. Separate Integra medium (1 L) is placed in the large chamber of an Integra flask. The membrane separating the two chambers is permeable to small molecular weight nutrients but impermeable to hybridoma cells and the antibodies produced by those cells. Therefore, the hybridoma cells and the antibodies produced by those hybridoma cells are retained in the small chamber.

[0484] After one week, the culture medium is removed from both chambers of the Integra flask and replaced with fresh Integra culture medium. The culture medium collected from the small chamber is retained separately. After the second week of growth, the culture medium is collected from the small chamber again. The hybridoma strain culture medium collected from week 1 is combined with the hybridoma strain culture medium collected from week 2. The collected hybridoma strain culture medium samples are centrifuged to remove cells and debris (at 3000 rpm for 15 minutes) and the resulting supernatant is filtered (0.22 μm). The clarified conditioned medium is loaded onto a protein A agarose column. Optionally, the culture medium is first concentrated and then loaded onto a protein A agarose column. Non-specific binding substances are removed by thorough PBS washing. The antibody protein bound to the protein A column is recovered by eluting the antibody from the protein A column with a standard acidic antibody (e.g., 50 mM citrate, pH 3.0). The aggregated antibody protein in the protein A agarose pool is removed by size exclusion chromatography or bound ion exchange chromatography performed on an anion exchange resin (e.g., Q agarose resin). Specific IEX conditions for 21B12 protein were Q-Sepharose HP, pH 7.8-8.0. The antibody was eluted using a 10 mM to 500 mM NaCl gradient over 25 column volumes.

[0485] Example 6

[0486] Production of human 21B12 IgG2 antibody from transfected cells

[0487] This example outlines how to produce the 21B12 IgG2 antibody from transfected cells. Cells (293 cells for transient expression and CHO cells for stable expression) were transfected with plasmids encoding the 21B12 heavy and light chains. Conditioned culture medium from the hybridoma cells was recovered by removing cells and cell debris. The clarified conditioned culture medium was loaded onto a Protein A-Sepharose column. Optionally, the culture medium can be concentrated and then loaded onto the Protein A-Sepharose column. Non-specific binding was removed by a thorough PBS wash. Antibody proteins bound to the Protein A column were recovered by standard acidic antibody elution (50 mM citrate, pH 3.0) from the Protein A column. Aggregated antibody proteins in the Protein A-Sepharose pool were removed by size exclusion chromatography or bound ion exchange chromatography on a cation exchange resin (e.g., SP-Sepharose resin). Specific IEX conditions for the 21B12 protein are SP-Sepharos...

Claims

1. A stable formulation comprising: at least one monoclonal antibody that specifically binds to PCSK9, wherein PCSK9 comprises the amino acids of SEQ ID NO 1, wherein the amount of the monoclonal antibody is 80 mg / ml to 140 mg / ml; and a pharmaceutically acceptable buffer in an amount of 0.05 mM to 40 mM; and a nonionic surfactant in an amount of 0.004% w / v to 10% w / v; and at least one pharmaceutically acceptable stabilizer, wherein the pH of the stable formulation is between 4.0 and 6.0, wherein the pharmaceutically acceptable stabilizer is selected from (i) proline, arginine, lysine and taurine in an amount of 1% to 5% w / v, and / or (ii) proline in an amount of 1% to 5% w / v and benzyl alcohol in an amount of 0.5% to 5% w / v, and wherein the monoclonal antibody comprises a light chain polypeptide and a heavy chain polypeptide, the light chain polypeptide comprising the following CDRs: a light chain CDR1 as CDR1 in SEQ ID NO: 158, a light chain CDR2 as CDR2 in SEQ ID NO: 162, and a light chain CDR3 as CDR3 in SEQ ID NO: 395; and the heavy chain polypeptide comprising the following CDRs: a heavy chain CDR1 as CDR1 in SEQ ID NO: 308 or SEQ ID NO: 368, a heavy chain CDR2 as CDR2 in SEQ ID NO: 175, and a heavy chain CDR3 as CDR3 in SEQ ID NO:

180.

2. The stable formulation of claim 1, wherein the pharmaceutically acceptable buffer is selected from the group consisting of glutamate, phosphate, phosphate-buffered saline, sodium acetate, sodium citrate, and Tris buffer.

3. The stable formulation of claim 1, wherein the pharmaceutically acceptable buffer is sodium acetate.

4. The stable formulation of claim 3, wherein the pharmaceutically acceptable buffer is present in an amount of 10-20 mM.

5. The stable formulation of any one of claims 1 to 4, wherein the nonionic surfactant is polysorbate 80 or polysorbate 20.

6. The stable formulation of claim 5, wherein the pharmaceutically acceptable surfactant is polysorbate 80.

7. The stable formulation of claim 5, wherein the pharmaceutically acceptable surfactant is polysorbate 20.

8. The stable formulation of any one of claims 1-4, wherein the nonionic surfactant is present in an amount of 0.004% w / v to 0.01% w / v.

9. The stable formulation of any one of claims 1-4, wherein the pharmaceutically acceptable stabilizer is proline and benzyl alcohol.

10. The stable formulation of any one of claims 1-4, further comprising sucrose.

11. The stable formulation of any one of claims 1-4, wherein the stabilizer is proline.

12. The stable formulation of claim 11, wherein the proline is present in an amount between 2% to 3% w / v.

13. The stable formulation of any one of claims 1-4, wherein the pH of the formulation is between 5.0 and 5.

5.

14. The stable formulation of any one of claims 1-4, wherein the monoclonal antibody comprises: (a) a light chain variable domain comprising the amino acid sequence of the light chain variable domain of SEQ ID NO: 297, and (b) a heavy chain variable domain comprising the amino acid sequence of the heavy chain variable domain of SEQ ID NO:

298.

15. The stable formulation of any one of claims 1-4, wherein the monoclonal antibody comprises: A light chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

49.

16. The stable formulation of any one of claims 1-4, wherein the stable formulation comprises a viscosity of 30 cP or less at 25°C.

17. The stable formulation of any one of claims 1-4, wherein the amount of monoclonal antibody is 120 mg / ml to 140 mg / ml and the stable formulation comprises a viscosity of 12 cP or less at 25°C.

18. The stable formulation of any one of claims 1-4, comprising an osmolality between 250 mOsmol / kg and 350 mOsmol / kg.

19. The stable formulation of any one of claims 1-4, wherein the stable formulation remains stable for at least 3, 6, 12, or 24 months.

20. The stable formulation of any one of claims 1-4, wherein the monoclonal antibody comprises: a light chain variable region comprising the light chain variable region of SEQ ID NO: 297 and a heavy chain variable region comprising the heavy chain variable region of SEQ ID NO: 298, and wherein the amount of the monoclonal antibody is 120 mg / ml.

21. The stable formulation of any one of claims 1-4, wherein the monoclonal antibody comprises: a light chain variable region comprising the light chain variable region of SEQ ID NO: 297 and a heavy chain variable region comprising the heavy chain variable region of SEQ ID NO: 298, and wherein the amount of the monoclonal antibody is 140 mg / ml.

22. A stable formulation comprising: (a) a monoclonal antibody in an amount of 80 mg / ml to 200 mg / ml, the monoclonal antibody comprising: a light chain variable region comprising SEQ ID NO:23 and a heavy chain variable region comprising SEQ ID NO:49; (b) 20 mM sodium acetate; (c) between 2.0% and 3.0% w / v proline; (d) 0.01% w / v polysorbate 20 or polysorbate 80; and (e) pH 5.

0.

23. A stable formulation comprising: (a) a monoclonal antibody in an amount of 120 mg / ml to 150 mg / ml, the monoclonal antibody comprising: i) a light chain variable region that is the light chain variable region of SEQ ID NO: 297, and a heavy chain variable region that is the heavy chain variable region of SEQ ID NO: 298, (b) 20 mM sodium acetate; (c) between 2.0% and 3.0% w / v proline; (d) 0.01% w / v polysorbate 20 or polysorbate 80; and (e) pH 5.

0.

24. A stable formulation comprising: a monoclonal antibody that specifically binds to PCSK9, wherein PCSK9 comprises the amino acid of SEQ ID NO 1, the monoclonal antibody being present in an amount of 100 mg / ml to 150 mg / ml; and a pharmaceutically acceptable buffering agent in an amount of 0.05 mM to 40 mM; and a pharmaceutically acceptable surfactant in an amount of 0.004% w / v to 20% w / v; and 0.5% w / v to 10% w / v of at least one pharmaceutically acceptable stabilizer, wherein the pH of the stable formulation is between 4.0 and 6.0, wherein the pharmaceutically acceptable stabilizer is selected from proline, arginine, lysine, and taurine, and wherein the monoclonal antibody comprises: a) a light chain variable region and a heavy chain variable region, the light chain variable region comprising the amino acid sequence of the light chain variable region being SEQ ID NO: 297, and the heavy chain variable region being the heavy chain variable region of SEQ ID NO: 298, or b) the light chain complementary determining region (CDR) of the CDRL1 sequence in SEQ ID NO:23, CDRL2 of the CDRL2 sequence in SEQ ID NO:23, and CDRL3 of the CDRL3 sequence in SEQ ID NO:23, and the heavy chain complementary determining region (CDR) of the CDRH1 sequence in SEQ ID NO:49, CDRH2 of the CDRH2 sequence in SEQ ID NO:49, and CDRH3 of the CDRH3 sequence in SEQ ID NO:

49.

25. The formulation of claim 24, wherein the monoclonal antibody comprises a light chain complementary determining region (CDR) of the CDRL1 sequence in SEQ ID NO:23, CDRL2 of the CDRL2 sequence in SEQ ID NO:23, and CDRL3 of the CDRL3 sequence in SEQ ID NO:23, and a heavy chain complementary determining region (CDR) of the CDRH1 sequence in SEQ ID NO:49, CDRH2 of the CDRH2 sequence in SEQ ID NO:49, and CDRH3 of the CDRH3 sequence in SEQ ID NO:

49.

26. The formulation of claim 25, wherein a) the heavy chain comprises: a CDR1 comprising SEQ ID NO: 308, a CDR2 comprising SEQ ID NO: 175, and a CDR3 comprising SEQ ID NO: 180; or b) the heavy chain comprises: a CDR1 comprising SEQ ID NO: 368, a CDR2 comprising SEQ ID NO: 175, and a CDR3 comprising SEQ ID NO: 180; and / or c) the light chain comprises: a CDR1 comprising SEQ ID NO:158, a CDR2 comprising SEQ ID NO:162, and a CDR3 comprising SEQ ID NO:

395.

27. The formulation of any one of claims 25-26, wherein each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition, the AbM definition, or a contact definition of the CDR.

28. The formulation of any one of claims 25-26, wherein each CDR is defined according to Kabat's CDR definition or Chothia's CDR definition.

29. The formulation of claim 24, wherein the monoclonal antibody comprises: A light chain variable region comprising the amino acid sequence of the light chain variable region of SEQ ID NO: 297, and a heavy chain variable region which is the heavy chain variable region of SEQ ID NO:

298.

30. The formulation of any one of claims 24-26 or 29, wherein the monoclonal antibody further comprises: (a) the light chain constant sequence of SEQ ID NO: 156; (b) the light chain constant sequence of SEQ ID NO: 157; (c) the heavy chain constant sequence of SEQ ID NO: 154; (d) the heavy chain constant sequence of SEQ ID NO: 155; (e) the light chain constant sequence of SEQ ID NO: 156 and the heavy chain constant sequence of SEQ ID NO: 154; (f) the light chain constant sequence of SEQ ID NO: 157 and the heavy chain constant sequence of SEQ ID NO: 154; (g) the light chain constant sequence of SEQ ID NO: 156 and the heavy chain constant sequence of SEQ ID NO: 155; or (h) the light chain constant sequence of SEQ ID NO:157 and the heavy chain constant sequence of SEQ ID NO:

155.

31. The formulation of any one of claims 24-26 or 29, wherein the pharmaceutically acceptable buffer is selected from the group consisting of glutamate, phosphate, phosphate-buffered saline, sodium acetate, sodium citrate, and Tris buffer.

32. The formulation of claim 31, wherein the pharmaceutically acceptable buffer is sodium acetate.

33. The formulation of any one of claims 24-26 or 29, wherein the pharmaceutically acceptable buffer is present in an amount of 10-20 mM.

34. The formulation of any one of claims 24-26 or 29, wherein the pharmaceutically acceptable surfactant is polysorbate 80 or polysorbate 20; optionally, the pharmaceutically acceptable surfactant is present in an amount of 0.004% w / v to 0.01% w / v.

35. The formulation of claims 24-26 or 29, wherein the stabilizer is proline, optionally present in an amount between 2% and 3% w / v.

36. The formulation of any one of claims 24-26 or 29, wherein the formulation a) pH between 5.0 and 5.5; b) comprising a viscosity of 30 cP or less at 25°C; c) comprising a monoclonal antibody in an amount of 120 mg / ml to 150 mg / ml and comprising a viscosity of 12 cP or less at 25°C; and / or d) comprises an osmolality between 250 mOsmol / kg and 350 mOsmol / kg.

37. The formulation of any one of claims 24-26 or 29, wherein the formulation remains stable for at least 3, 6, 12 or 24 months.

38. The formulation of claim 37, comprising: (a) the monoclonal antibody in an amount of 120 mg / ml to 150 mg / ml; (b) 10-20 mM sodium acetate; (c) between 2.0% and 3.0% w / v proline; (d) 0.01% w / v polysorbate 20 or polysorbate 80; and (e) a pH of 5.0 to 5.

5.

39. The formulation of claim 38, comprising: (b) 10 mM sodium acetate; (c) between 2.0% and 3.0% w / v proline; (d) 0.01% w / v polysorbate 20 or polysorbate 80; and (e) pH 5.

0.

40. The formulation of claim 39, comprising: (a) 120 mg / ml or (b) 140 mg / ml of the monoclonal antibody.

41. Use of a formulation as defined in any one of claims 24 to 40 for the preparation of a medicament for treating or preventing a cholesterol related disease in a patient.

42. The use of claim 41, wherein the cholesterol-related disease is selected from the group consisting of familial hypercholesterolemia, non-familial hypercholesterolemia, hyperlipidemia and dyslipidemia.

43. The use of claim 42, wherein the familial hypercholesterolemia comprises heterozygous familial hypercholesterolemia and homozygous familial hypercholesterolemia.

44. The use according to any one of claims 41 to 43, wherein The patient is administered the following doses: (a) up to 140 mg of the antibody once every two weeks (Q2W); (b) up to 420 mg of said antibody once every two weeks (Q2W); or (c) up to 420 mg of the antibody once every four weeks (Q4W).

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