WO2013148284A1 - Antibodies that bind to a pcsk9 cleavage site and methods of use - Google Patents

Abstract

The invention provides anti-PCSK9 antibodies that bind to a PCSK9 cleavage site and methods of using the same.

Description

ANTIBODIES THAT BIND TO A PCSK9 CLEAVAGE SITE

AND METHODS OF USE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial Nos.

61/617,615, filed March 29, 2012 and 61/642,972, filed May 4, 2012, which applications are hereby incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted in ASCII format via EFS-Web and hereby incorporated by reference in its entirety. Said ASCII copy, created on March 14, 2013, is named P4896WO_PCTSequenceListing.txt and is 29,480 bytes in size.

BACKGROUND

PCSK9 (proprotein convertase subtilisin/kexin type 9) is a member of the proprotein convertase family and a ligand of hepatic LDL receptors (LDLR) (N.G. Seidah, et al. Biol Chem 387, 871-877 (2006)). PCSK9 prevents LDLR recycling by directing the ligand:receptor complex for lysosomal degradation, resulting in reduced LDL-c clearance and increased plasma LDL-c levels. The importance of PCSK9 in lipid metabolism is strongly supported by human genetics and by physiologic studies in mice and monkeys (reviewed in J.D. Horton, Jet al. J Lipid Res 50 Suppl, S172-177 (2009); N.G. Seidah, Expert Opin Ther Targets 13, 19-28 (2009); P. Costet, et al. Trends Biochem Sci 33, 426-434 (2008); C.J. Duff and N.M. Hooper, Expert Opin Ther Targets 15, 157-168 (2011)). Moreover, the strong reduction in coronary heart disease due to loss-of-function mutations in the PCSK9 gene (J.C. Cohen, et al. N Engl J Med 354, 1264-1272 (2006)) provides a strong rationale for the development of PCSK9 inhibitors for dyslipidemia.

SUMMARY

The invention provides anti-PCSK9 antibodies and methods of using the same.

In one aspect, the application provides an isolated antibody that binds to PCSK9, wherein the antibody binds to intact PCSK9 with at least 100-fold greater affinity than it binds to PCSK9 cleaved at Arg218-Gln219.

In certain embodiments, the anti-PCSK9 antibodies described herein bind to an epitope of PCSK9 comprising, consisting essentially of, or consisting of amino acid residues Thr214 an epitope of PCSK9 comprising, consisting essentially of, or consisting of amino acid residues Glu211 through Ala220.

In certain embodiments, an anti-PCSK9 antibody described herein is a monoclonal antibody.

In certain embodiments, an anti-PCSK9 antibody described herein is a human, humanized, or chimeric antibody.

In certain embodiments, an anti-PCSK9 antibody described herein is an antibody fragment.

In certain embodiments, an anti-PCSK9 antibody described herein comprises (a) HVR-

H3 comprising the amino acid sequence of SEQ ID NO:3, (b) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2.

In certain embodiments, an anti-PCSK9 antibody described herein comprises (a) HVR- HI comprising the amino acid sequence of SEQ ID NO: 1 , (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3.

In certain embodiments, an anti-PCSK9 antibody described herein comprises (a) HVR- Ll comprising the amino acid sequence of SEQ ID NO: 8; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

In certain embodiments, an anti-PCSK9 antibody described herein comprises (a) HVR- Hl comprising the amino acid sequence of SEQ ID NO: l, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:8; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

In certain embodiments, an anti-PCSK9 antibody described herein comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11 ; or (c) a VH sequence as in (a) and a VL sequence as in (b).

In certain embodiments, an anti-PCSK9 antibody described herein comprises (a) a VH sequence of SEQ ID NO: 4, (b) a VL sequence of SEQ ID NO: 11, or (c) a VH sequence of SEQ ID NO:4 and a VL sequence of SEQ ID NO: 11. antibody.

In another aspect, the application provides an isolated nucleic acid encoding an anti- PCSK9 antibody described herein.

In another aspect, the application provides a host cell comprising a nucleic acid encoding an anti-PC SK9 antibody described herein. In certain embodiments, the nucleic acid encoding an anti-PC SK9 antibody comprises (a) a nucleotide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6 or 7; (b) a nucleotide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13 or 14; or (c) a sequence as in (a) and a sequence as in (b). In certain embodiments, the nucleic acid encoding an anti-PCSK9 antibody comprises (a) the nucleotide sequence of SEQID NO: 6; (b) the nucleotide sequence of SEQ ID NO: 13, or (c) the nucleotide sequence of SEQ ID NO: 6 and the nucleotide sequence of SEQ ID NO: 13. In certain embodiments, the nucleic acid encoding an anti-PCSK9 antibody comprises (a) the nucleotide sequence of SEQID NO: 7; (b) the nucleotide sequence of SEQ ID NO: 14, or (c) the nucleotide sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 14.

In another aspect, the application provides a host cell comprising a nucleic acid encoding an anti-PC SK9 antibody described herein.

In another aspect, the application provides a method of producing an anti-PC SK9 antibody described herein comprising culturing a host cell comprising a nucleic acid encoding an anti-PC SK9 antibody described herein so that the antibody is produced. In certain embodiments, the method further comprises recovering the antibody from the host cell.

In another aspect, the application provides an immunoconjugate comprising an anti- PCSK9 antibody described herein and a cytotoxic agent.

In another aspect, the application provides a pharmaceutical formulation comprising an anti-PCSK9 antibody described herein and a pharmaceutically acceptable carrier.

In another aspect, the application provides a method of reducing the LDL-cholesterol level in a subject, said method comprising administering to the subject an effective amount of an anti-PC SK9 antibody described herein.

In another aspect, the application provides a method of treating a cholesterol related disorder in a subject, said method comprising administering to the subject an effective amount of an anti-PC SK9 antibody described herein. a subject, said method comprising administering to the subject an effective amount of an anti- PCSK9 antibody described herein.

In certain embodiments, the methods described herein further comprise administering to the subject an effective amount of a second medicament, wherein the anti-PC SK9 antibody is the first medicament. In certain embodiments, the second medicament elevates the level of LDLR. In certain embodiments, the second medicament reduces the level of LDL-cholesterol. In certain embodiments, the second medicament comprises a statin, such as, for example, a statin selected from the group consisting of atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and any combination thereof. In certain embodiments, the second medicament elevates the level of HDL-cholesterol.

In another aspect, the application provides a method of inhibiting binding of PCSK9 to LDLR in a subject, said method comprising administering to the subject an effective amount of an anti-PC SK9 antibody described herein.

In another aspect, the application provides a method for purifying PCSK9 cleaved at Arg218-Gln219, said method comprising

(a) contacting a sample of PCSK9 with an anti-PCSK9 antibody described herein; and

(b) isolating the unbound PCSK9 from the bound PCSK9.

In certain embodiments, the application provides a method for purifying PCSK9 cleaved at Arg218-Gln219, said method comprising

(a) contacting the sample of PCSK9 with a protease;

(b) contacting a sample of PCSK9 with an anti-PCSK9 antibody described herein; and (b) isolating the unbound PCSK9 from the bound PCSK9.

In certain embodiments, the protease is hepsin or furin.

In another aspect, the application provides a purified composition of PCSK9 cleaved at Arg218-Gln219. In certain embodiments, the cleaved PCSK9 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the total PCSK9 protein in the composition. In certain embodiments, the cleaved PCSK9 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the total protein in the composition.

In another aspect, the invention provides a method for determining the amount of cleaved PCSK9 present in a sample, comprising

(a) determining the level of total PCSK9 in a sample;

(b) determing the level of intact PCSK9 in the sample using an anti-PCSK9 antibody described herein; and intact PCSK9 in the sample from the amount of total PCSK9 in the sample, In certain embodiments, the sample is a biological sample, such as, for example, human blood sample. In certain embodiments, the amount of total PCSK9 in the sample determined using an antibody that binds to both intact and cleaved PCSK9.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. PCSK9 domains and protease cleavage sites. A. Diagram showing the three main PCSK9 domains, the prodomain, catalytic (Cat) and C-terminal (CT) domains. The N- segment (Serl53-Arg218) comprises the N-terminal portion of the catalytic domain up to the furin cleavage site Arg218-Gln219. The molecular masses determined by mass spectrometry are indicated for the N-segment, the ANCat_CT (= Cat_CT lacking the N-segment) and for the Cat CT. B. The '218-loop' comprising the P4 - P4' amino acid sequence according to the Schechter and Berger nomenclature (Schechter et al. Biochem Biophys Res Commun 27, 157- 162 (1967)). The primary cleavage site for furin and hepsin (Arg218-Gln219) is indicated by solid arrow, the secondary cleavage site that is specific for hepsin (Arg215-Phe216) is indicated by a dashed arrow. Figure IB discloses SEQ ID NO:20.

Figure 2. PCSK9 cleavage by a panel of serine proteases. A. PCSK9 (1.9 μΜ) was incubated with 40 nM of various serine proteases for 6 h at room temperature and reaction products analyzed by SDS-PAGE (non-reducing conditions) followed by gel staining with SimplyBlue SafeStain. The individual bands are indicated according to the nomenclature in Fig. 1 A and the determined N-terminal sequences are in parenthesis. The last lane shows hepsin that was pretreated with the inhibitory anti-hepsin antibody Ab25 before incubation with PCSK9. Ctrl, untreated PCSK9; APC, activated protein C; HGFA, hepatocyte growth factor activator. B. Comparison of proteolytic activities of furin and hepsin towards PCSK9.

PCSK9 (1.9 μΜ) was incubated for 6 h with increasing protease concentrations (soluble hepsin and soluble furin) and analyzed by SDS-PAGE (right panel). Disappearance of the -60 kDa PCSK9 band (= Cat CT domain) was quantified and plotted against protease concentration (left panel). The calculated concentrations for 50% PCSK9 cleavage were 11.3 nM for hepsin and 79.6 nM for furin.

Figure 3. Inhibition of protease-mediated PCSK9 cleavage by mutagenesis of '218- loop' residues and by antibody 3D5. A. PCSK9 wildtype (WT) and PCSK9 mutants R218A and R215A:R218A (2.6 μΜ each) were incubated for 6 h with 40 nM of hepsin (upper panel) sites, Arg218-Gln219 and Arg215-Phe216, whereas furin only cleaves at Arg218-Gln219 (see also Figure IB). B. PCSK9 (1.9 μΜ) was preincubated with antibodies 3D5 or 7G7 for 20 min before treatment with 40 nM hepsin or with 80 nM furin for 6 h. Results indicate that antibody 3D5 completely inhibited PCSK9 cleavage by either protease, whereas antibody 7G7 did not.

Figure 4. Ab-3D5 epitope mapping. A. Tryptic digestion protection mass spectrometry. Tryptic digests of PCSK9 alone or of PCSK9:Ab-3D5 complexes were analyzed on a MALDI- TOF/TOF mass spectrometer. The sequences of the identified peptides are shown in pink (peptides present in digests of both PCSK9 alone and PCSK9:Ab-3D5 complex) and in blue (peptides not observed in PCSK9:Ab-3D5 complex). The latter peptides all end with Arg215 or with Arg218 (highlighted in red). The protection of trypsin cleavage by Ab-3D5 at these positions suggests that part of the Ab-3D5 epitope is encompassed by the '218-loop'. Figure 4A discloses SEQ ID NOs: 21-33, respectively, in order of appearance. B. Epitope mapping by overlapping peptide library screening. Overlapping peptides spanning the entire PCSK9 sequence were synthesized and binding to Ab-3D5 measured in an ELISA (GenScript,

Piscataway NJ). Ab-3D5 only bound to two peptides, peptide #60 and peptide #61. The consensus binding sequence was Glu211-Ala220 (highlighted), which encompasses the '218- loop'. The residues Arg215 and Arg218 are boxed. Figure 4B discloses SEQ ID NOs: 34-35, respectively, in order of appearance.

Figure 5. Inhibition of PCSK9 function by Ab-3D5. A. Inhibition of PCSK9 binding to

LDLR by biolayer interferometry. LDLR ectodomain was immobilized on the sensor of an Octet Red384 system (ForteBio; Menlo Park, CA) and binding to PCSK9 alone (Ctrl) or preincubated with Ab-3D5 or the non-blocking Ab-7G7 was measured. Results are the average of n=3 ± SD. The apparent increased binding of Ab-7G7 is due to the increased mass of the PCSK9:Ab-7G7 complex (vs Ctrl = PCSK9 alone) bound to the LDLR-loaded sensor tip. B. Ab-3D5 prevents LDLR degradation in HepG2 cells. HepG2 cells were treated for 4 h with buffer alone (Ctrl), with PCSK9 alone (-Ab) or with PCSK9 preincubated with increasing concentrations of the non-blocking Ab-7G7 or with Ab-3D5. Surface LDLR levels were quantified by FACS analysis and expressed as percent of control levels.

Figure 6. Alignment of 3D5 heavy chain variable region amino acid sequence (SEQ ID

NO: 4) with IGHV1 mouse germline heavy chain amino acid sequence (SEQ ID NO: 36) (top panel) and alignment of 3D5 light chain variable region amino acid sequence (SEQ ID NO: 11) with IGKV1 mouse germline light chain amino acid sequence (SEQ ID NO: 37) (bottom panel). PCSK9 was treated with furin (80 iiM) for 20 h. After addition of anti-furin IgG and Ab-3D5, proteins were separated on a S-200 size exclusion columns (HiLoad™ 16/60 Superdex™ prep grade, GE Healthcare). The high molecular weight complexes of intact PCSK9:Ab-3D5 were separated from the pure furin-cleaved PCSK9 that eluted in later fractions.

Figure 8. Purification and analysis of furin-cleaved PCSK9. A. PCSK9 was treated with furin for 20 h, after which an anti-furin antibody and Ab-3D5 were added and the protein mixture was applied to a quantitative S-200 size exclusion column. The first elution peak contained the complex formed of Ab-3D5 with residual intact PCSK9 (-60 kDa Cat CT; lanes 1-4) and was separated from the later eluting pure cleaved PCSK9 (~ 50 kDa ΔΝ-Cat CT; lanes 9-12). B. Pooled fractions (9-12) of pure furin-cleaved PCSK9 (= PCSK9c_fu) were compared to intact PCSK9 (= PCSK9) by analytical size exclusion chromatography. The elution volumes for PCSK9c_fu and PCSK9 were identical (12.72 and 12.78 ml), both having a deduced mass of 77 kDa.

Figure 9. Mass spectra of furin- and hepsin-cleaved PCSK9. Left panel shows the peaks of Cat_CT and AN Cat CT and right panel the peaks of the N-segment for intact (top), hepsin-cleaved (middle) and furin-cleaved PCSK9 (see Figure 1 A for domain nomenclature). Peaks are labeled with relative molecular masses (M_r) deconvoluted from electrospray time-of- fiight mass spectra, and their assigned amino acid residues in PCSK9. The N-segment accounts for the reduced M_r of AN Cat CT compared to Cat CT.

Figure 10. Cleaved PCSK9 forms reduce LDLR levels on HepG2 surface. A. HepG2 cells were treated for 4 h with intact increasing concentrations of intact PCSK9 (PCSK9) or furin-cleaved PCSK9 (PCSK9c_fu) and cell surface LDLR was quantified by FACS analysis using an anti-LDLR antibody. B. Same experimental protocol as in Fig. 6A for the comparison of intact PCSK9 with hepsin-cleaved PCSK9 (PCSK9c_hep). Results are the average ± SD of at least three independent experiments.

Figure 11. Shows the effects of antibody 3D5 on intact and hepsin-cleaved PCSK9 activity in a HepG2 assay.

Figure 12. Cleaved PCSK9 forms degrade LDLR in mouse liver. A. C57BL/6 mice in groups of n=3 were injected i.v. with PBS or with the indicated doses of intact PCSK9

(PCSK9) or with furin-cleaved PCSK9 (PCSK9c_fu). After 1 h livers were harvested and LDLR levels of pooled liver lysates (n=3/group) determined by quantitative immunob lotting. B. Same experimental protocol as in Fig. 7A for the comparison of intact PCSK9 vs hepsin- expression.

Figure 13. Shows that antibody 3D5 neutralizes PCSK9 activity in a mouse model. Mice received 20 mg/kg antibodies two hours prior to injection of 30 μg PCSK9 for 1 hour.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

I. DEFINITIONS

An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

"Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which refiects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

The terms "anti-PCSK9 antibody" and "an antibody that binds to PCSK9" refer to an antibody that is capable of binding PCSK9 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PCSK9. In one embodiment, the extent of binding of an anti-PCSK9 antibody to an unrelated, non-PCSK9 protein is less than (RIA). In certain embodiments, an antibody that binds to PCSK9 has a dissociation constant (Kd) of < ΙμΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10^"8 M

-8 -13 -9 -13

or less, e.g. from 10^" M to 10^" M, e.g., from 10^" M to 10^" M). In certain embodiments, an anti-PCSK9 antibody binds to an epitope of PCSK9 that is conserved among PCSK9 from different species.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')₂; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG₂, IgG₃, IgG₄, IgAi, and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "cleaved PCSK9" or "cPCSK9" refers to a species of PCSK9 that has been cleaved at Arg218-Gln219.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not

.21 1 _T131 _T125 _v90 _D 186 _D 188 _c 153 „.212 _n32 limited to, radioactive isotopes (e.g., At , 1 , 1 , Y , Re , Re , Sm , Bi , P , adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term "Fc region" herein is used to define a C-terminal region of an

immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH

Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al, supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al, supra.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term "hypervariable region" or "HVR," as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues," or "SDRs," which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-Ll, a- CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31- 34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

The term "intact PCSK9" refers to a species of PCSK9 that has not been cleaved at Arg218-Gln219, e.g., the full length PCSK9 corresponding to residues 1-692 of PCSK9.

An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al, J. Chromatogr. B 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-PCSK9 antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such molecule(s) present at one or more locations in a host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

"Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region

(VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, concerning the use of such therapeutic products.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNLX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are program.

The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject., A

pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term "PCSK9," as used herein, refers to any native PCSK9 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed PCSK9 as well as any form of PCSK9 that results from processing in the cell. The term also encompasses naturally occurring variants of PCSK9, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human PCSK9 is shown in SEQ ID NO: 15.

The term "PCSK9 activity" or "biological activity" of PCSK9, as used herein, 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 certain embodiments, the biological activity of PCSK9 is the ability of PCSK9 to bind to a LDL-receptor (LDLR). In certain embodiments, PCSK9 binds to and catalyzes a reaction involving LDLR. In certain embodiments, PCSK9 activity includes the ability of PCSK9 to decrease or reduce the availability of LDLR. In certain embodiments, the biological activity of PCSK9 includes the ability of PCSK9 to increase the amount of LDL in a subject. In certain embodiments, the biological activity of PCSK9 includes the ability of PCSK9 to decrease the amount of LDLR that is available to bind to LDL in a subject. In certain embodiments, the biological activity of PCSK9 includes the ability of PCSK9 to decrease the amount of LDLR that is available to bind to LDL. In certain embodiments, biological activity of PCSK9 includes any biological activity resulting from PCSK9 signaling.

As used herein, "treatment" (and grammatical variations thereof such as "treat" or

"treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6^th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J.

Immunol. 150:880-887 (1993); Clarkson et al, Nature 352:624-628 (1991).

The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

II. COMPOSITIONS AND METHODS

In one aspect, the invention is based, in part, on the discovery of an antibody that binds differentially to the cleaved vs. intact form of PCSK9. Antibodies of the invention are useful, e.g., for determining the level of intact vs. cleaved PCSK9 in a sample, for purifying the cleaved form of PCSK9, and for reducing LDL-cholesterol levels in a subject.

A. Exemplary Anti-PC SK9 Antibodies

In one aspect, the invention provides isolated antibodies that bind to the intact form of PCSK9 but not the form of PCSK9 that is cleaved at Arg218-Gln219. In certain embodiments, an anti-PCSK9 antibody binds to intact PCSK9 with an affinity at least 100-fold, 250-fold, 500-fold, 750-fold, or 1000-fold greater than the affinity of the antibody for PCSK9 cleaved at Arg218-Gln219.

In one aspect, the invention provides an anti-PCSK9 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid NO:2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; (d) HVR-Ll comprising the amino acid sequence of SEQ ID NO: 8; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-Hl comprising the amino acid sequence of SEQ ID NO: 1 ; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:3. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:2. In a further embodiment, the antibody comprises (a) HVR-Hl comprising the amino acid sequence of SEQ ID NO: l; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3.

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-Ll comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In one embodiment, the antibody comprises (a) HVR-Ll comprising the amino acid sequence of SEQ ID NO: 8; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-Hl comprising the amino acid sequence of SEQ ID NO: 1 , (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:3; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-Ll comprising the amino acid sequence of SEQ ID NO: 8, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10. comprising the amino acid sequence of SEQ ID NO: l; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 10.

In any of the above embodiments, an anti-PCSK9 antibody is humanized. In one embodiment, an anti-PCSK9 antibody comprises HVRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g. a human immunoglobulin

framework or a human consensus framework.

In another aspect, an anti-PCSK9 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-PC SK9 antibody comprising that sequence retains the ability to bind to differentially to the intact vs. cleaved forms of PCSK9. In certain

embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:4. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-PCSK9 antibody comprises the VH sequence in SEQ ID NO:4, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hl comprising the amino acid sequence of SEQ ID NO: l, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3.

In another aspect, an anti-PCSK9 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-PC SK9 antibody comprising that sequence retains the ability to bind to bind differentially to the intact vs. cleaved forms of PCSK9. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 11. In certain embodiments, the substitutions, insertions, or antibody comprises the VL sequence in SEQ ID NO: l 1, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-Ll comprising the amino acid sequence of SEQ ID NO: 8; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

In another aspect, an anti-PCSK9 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:4 and SEQ ID NO: l 1, respectively, including post-translational modifications of those sequences.

In a further aspect, the invention provides an antibody that binds to the same epitope as an anti-PC SK9 antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an anti-PCSK9 antibody comprising a VH sequence of SEQ ID NO:4 and a VL sequence of SEQ ID NO: 11. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of PCSK9 consisting of amino acids 214-219 or 211-220 of PCSK9.

In a further aspect of the invention, an anti-PC SK9 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, an anti-PCSK9 antibody is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab')₂ fragment. In another embodiment, the antibody is a full length antibody, e.g., an intact IgGl antibody or other antibody class or isotype as defined herein.

In a further aspect, an anti-PC SK9 antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of < ΙμΜ, < ΙΟΟ ηΜ, < ΙΟ ηΜ, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10^"8 M or less, e.g. from 10^"8 M to 10^"13 M, e.g., from 10^"9 M to 10^"13 M).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution binding affinity of Fabs for antigen is measured by equilibrating Fab unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al, J. Mol. Biol. 293:865-881(1999)). To establish conditions for the assay, MICROTITER multi-well plates (Thermo Scientific) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or

26 pM [ 125 I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al, Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20^®) in PBS. When the plates have dried, 150 μΐ/well of scintillant

(MICROSCINT-20™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmon resonance assays using a BIACORE^®-2000 or a BIACORE ^®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at -10 response units (RU). Briefly,

carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl- N'- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (-0.2 μΜ) before injection at a flow rate of 5 μΐ/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 μΐ/min.

Association rates (k_on) and dissociation rates (k₀ff) are calculated using a simple one-to-one Langmuir binding model (BIACORE Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k₀ff/k_on See, e.g., Chen et al, J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10^ M^~l s^~l by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the nm, 16 nm band-pass) at 25°C of a 20 iiM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibody fragment.

Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')₂, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g.,

Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half- life, see U.S.

Patent No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129- 134 (2003); and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al, Nat. Med. 9: 129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which FJVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in Riechmann et al, Nature 332:323-329 (1988); Queen et al, Proc. Nat 'lAcad. Sci. USA 86: 10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al, Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489- 498 (1991) (describing "resurfacing"); DaU'Acqua et al, Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68 (2005) and Klimka et al, Br. J. Cancer, 83 :252-260 (2000) (describing the "guided selection" approach to FR shuffling).

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol,

151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al, J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al, J. Biol. Chem. 271 :22611-22618 (1996)). 4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous

immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, e.g., U.S.

Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M

MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol, 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006)

(describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below. Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in

Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al, ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al, Nature 348:552- 554; Clackson et al, Nature 352: 624-628 (1991); Marks et al, J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004); Lee et al, J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.

Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol, 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities is for PCSK9 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of PCSK9. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al, EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross- linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al, Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al, J. Immunol., 148(5): 1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see,e.g. Gruber et al, J.

Immunol, 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies," are also included herein (see, e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a "Dual Acting FAb" or "DAF" comprising an antigen binding site that binds to PCSK9 as well as another, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "conservative substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE 1

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by

constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al, ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted. more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells ( 1989) Science, 244 : 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.

Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C -terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody. b) Glycosylation variants

In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%> or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US

2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO

2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).

Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al, Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function.

Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). c) Fc region variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non- limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat 7 Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al, Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al, J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96^® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat 'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al, Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al, Int'l. Immunol. 18(12): 1759-1769 (2006)).

Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al, J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or

334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J.

Immunol. 164: 4178-4184 (2000).

Antibodies with increased half lives and improved binding to the neonatal Fc receptor

(FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J.

Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)), are described in

US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286,

303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260;

U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants. In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.

Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No.

7,521,541. e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3- dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n- vinyl pyrrolidone)poly ethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed. B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acid encoding an anti-PCSK9 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell). In one embodiment, a method of making an anti-PCSK9 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of an anti-PCSK9 antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al, Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al, J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al, Annals N. Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR^" CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Anti-PC SK9 antibodies provided herein may be identified, screened for, or

characterized for their physical/chemical properties and/or biological activities by various assays known in the art. 1. Binding assays and other assays

In one aspect, an anti-PC SK9 antibody of the invention is tested for its PCSK9 binding activity, e.g., by known methods such as ELISA, Western blot, etc. Numerous types of competitive binding assays can be used to determine if an anti-PCSK9 antibody competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay {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 labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (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 labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabelled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. In certain embodiments, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

In one aspect of the invention, competition assays may be used to identify an antibody that competes with anti-PCSK9 antibody 3D5 for binding to PCSK9. In certain embodiments, epitope) that is bound by anti-PCSK9 antibody 3D5. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, immobilized PCSK9 is incubated in a solution comprising a first labeled antibody that binds to PCSK9 {e.g., anti-PCSK9 antibody 3D5) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to PCSK9. The second antibody may be present in a hybridoma supernatant. As a control, immobilized PCSK9 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to PCSK9, excess unbound antibody is removed, and the amount of label associated with immobilized PCSK9 is measured. If the amount of label associated with immobilized PCSK9 is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to PC SK9. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

In certain embodiments, the application provides a method for determining differential binding of an anti-PCSK9 antibody to different forms of PCSK9, e.g., intact vs. cleaved. For example, the method may involve separating different forms of PCSK9 on a gel, blotting the fragments, and then contacting the blot with an anti-PC SK9 antibody to determine which forms of PCSK9 are bound by the antibody. Alternatively, the method may involve, contacting a sample of PCSK9 with an antibody, separating the forms of PCSK9 bound to the antibody from the forms of PCSK9 not bound to the antibody, and then identifying which forms of PCSK9 were bound by the antibody as compared to which forms of PCSK9 were not bound to the antibody. In certain embodiments, the methods may further comprise contacting PCSK9 with a protease, such as, for example, furin or hepsin, to produce a sample of PCSK9 containing different cleavage forms. In certain embodiments, the invention provides a method for determining differential binding of an anti-PC SK9 antibody to different forms of PCSK9 by contacting the anti-PCSK9 antibody with different samples of purified forms of PCSK9 (e.g., a purified intact PCSK9 sample and a purified cleaved PCSK9 sample) and determing which forms of PCSK9 are bound by the antibody. In certain embodiments, the methods may involve the use of control samples, such as, for example, molecular weight standards or characterized samples of PCSK9. In one aspect, assays are provided for identifying anti-PCSK9 antibodies thereof having biological activity. Biological activity of the anti-PC SK9 antibodies may include, e.g., blocking, antagonizing, suppressing, interfering, modulating and/or reducing one or more biological activities of PCSK9. Antibodies having such biological activity in vivo and/or in vitro are provided.

In certain embodiments, an anti-PCSK9 antibody binds human PCSK9 and prevents interaction with the LDLR. In certain embodiments, an anti-PCSK9 antibody binds specifically to human PCSK9 and/or substantially inhibits binding of human PCSK9 to LDLR by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by measuring binding in an in vitro competitive binding assay). In certain embodiments, the invention provides isolated anti-PCSK9 antibodies which specifically bind to PCSK9 and which antagonize the PCSK9-mediated effect on LDLR levels when measured in vitro using the LDLR down regulation assay in HepG2 cells disclosed herein. D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-PC SK9 antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al, Cancer Res.

53:3336-3342 (1993); and Lode et al, Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al, Current Med. Chem. 13:477-523 (2006); Jeffrey et al, Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al, Bioconj. Chem. 16:717-721 (2005); Nagy et al, Proc. Natl. Acad. Sci. USA 97:829-834 (2000);

Dubowchik et al, Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al, J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; a CC1065.

In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive

211 131 125 isotopes are available for the production of radioconjugates. Examples include At , 1 , 1 , Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When the

radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine- 123 again, iodine- 131, indium-I l l, fluorine- 19, carbon- 13, nitrogen- 15, oxygen- 17, gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026. The linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020) may be used. such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo- SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-PCSK9 antibodies provided herein is useful for detecting the presence of PCSK9 in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample is blood, serum or other liquid samples of biological origin. In certain embodiments, a biological sample comprises a cell or tissue.

In one embodiment, an anti-PCSK9 antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of PCSK9 in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of PCSK9 protein in a biological sample. In certain embodiments, PCSK9 is human PCSK9. In certain embodiments, the method comprises contacting the biological sample with an anti-PC SK9 antibody as described herein under conditions permissive for binding of the anti-PCSK9 antibody to PCSK9, and detecting whether a complex is formed between the anti- PCSK9 antibody and PCSK9. Such method may be an in vitro or in vivo method. In one embodiment, an anti-PCSK9 antibody is used to select subjects eligible for therapy with an anti-PCSK9 antibody, e.g. where PCSK9 or LDL-cholesterol is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of the invention include cholesterol related disorders (which includes "serum cholesterol related disorders"), including any one or more of the following: hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular diseases, Alzheimers disease and generally dyslipidemias, which can be manifested, for example, by an elevated total serum cholesterol, elevated LDL, elevated triglycerides, elevated very low density lipoprotein

(VLDL), and/or low HDL. In one aspect, the invention provides a method for treating or preventing hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, cardiovascular disease (CVD) or coronary heart disease, in an individual comprising administering to the individual an effective amount of anti-PC SK9 antibody. In certain treating or preventing hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, CVD or coronary heart disease, in a subject. The invention further provides the use of an effective amount of an anti-PC SK9 antibody that antagonizes extracellular or circulating PCSK9 in the manufacture of a medicament for treating or preventing

hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, CVD or coronary heart disease, in an individual.

In certain embodiments, labeled anti-PCSK9 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or

32 molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes P, ¹⁴C, ¹²⁵1, ³H, and ¹³¹I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP,

lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

In certain embodiments, the application provides a method for determing the amount of cleaved vs. intact PCSK9 in a sample. For example, the method may involve determing the total amount of PCSK9 in a sample (e.g., the total amount of cleaved and intact PCSK9), and determing the amount of intact PCSK9 in the sample using an antibody as described herein, wherein the amount of cleaved PCSK9 in the sample may be determined by subtracting the amount of intact PCSK9 in the sample from the total amount of PCSK9 in the sample. In certain embodiments, the total amount of PCSK9 in the sample may be determined using an antibody that binds to both the cleaved and intact forms of PCSK9. An exemplary antibody that binds to both the cleaved and intact forms of PCSK9 is antibody YW508.20.33 described in PCT/US2011/066593. In certain embodiments, the sample is biological sample from a subject, such as a mammalian subject. In exemplary embodiments, the sample is a blood, serum or other sample from a human subject. sample contains a PCSK9 variant having a mutation in the region comprising amino acid residues 214-219 or 211-220 of PCSK9. For example, the method may involve contacting a sample with an antibody as described herein, and determining whether the antibody binds to the PCSK9 in the sample, wherein if the anti-PCSK9 antibody does not bind to PCSK9, then the sample contains a variant of PCSK9 containing a mutation in the region consisting of amino acid residues 214-219 or 211-220 or PCSK9. In certain embodiments, the method may involve contacting the sample with an antibody that binds to a region of PCSK9 distinct from amino acid residues 211-220 as a control (e.g., to confirm that PCSK9 is present in the sample). An exemplary antibody that binds to a different region of PCSK9 is antibody

YW508.20.33 described in PCT/US2011/066593. In other embodiments, the method may involve contacting an anti-PCSK9 antibody as described herein with wild-type PCSK9 as a control to confirm that the antibody is working and that assay conditions are suitable for binding. In certain embodiments, the sample is a biological sample from a subject, such as a mammalian subject. In exemplary embodiments, the sample is a blood, serum or other sample from a human subject. The furin cleavage sequence of PCSK9 ²¹⁵RFHR-Q²¹⁹ (SEQ ID NO:38) harbors three naturally occurring gain-of-function mutations (see e.g., S. Benjannet, et al. . J Biol Chem 281, 30561-30572 (2006); R. Essalmani, et al. J Biol Chem 286, 4257-4263 (2011); J. Cameron, et al. J Intern Med 263, 420-431 (2008); M. Abifadel, et al. Nat Genet 34, 154-156 (2003); and D. Allard, et al. Hum Mutat 26, 497 (2005)). Accordingly, the methods described herein are useful for identifying individuals carrying such gain-of-function mutations, e.g., individuals having or who may be at risk for developing hypercholesterolemia. In certain embodiments, the methods further comprise treating such individuals with an anti-PCSK9 antibody that binds to a region outside of the furin cleavage sequence, such as, for example, antibody YW508.20.33 described in PCT/US2011/066593.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-PC SK9 antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers {Remington 's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as

polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX^®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosammoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a statin, such as, for example, atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and any combination thereof. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres,

microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington 's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-PCSK9 antibodies provided herein may be used in therapeutic methods. In one aspect, an anti-PC SK9 antibody for use as a medicament is provided. In another aspect, an anti-PCSK9 antibody for use in treating conditions associated with a cholesterol related disorder is provided. In certain embodiments, an anti-PC SK9 antibody for use in treating conditions associated with an elevated level of LDL-cholesterol is provided. In certain embodiments, an anti-PCSK9 antibody for use in a method of treatment is provided. In certain embodiments, the invention provides an anti-PCSK9 antibody for use in a method of treating an individual having conditions associated with an elevated level of LDL-cholesterol comprising administering to the individual an effective amount of the anti-PC SK9 antibody. In certain embodiments, the methods and uses described herein further comprise administering to the individual an effective amount of at least one additional therapeutic agent, e.g., statin. In certain embodiments, the invention provides an anti-PCSK9 antibody for use in reducing LDL- cholesterol level in a subject. In further embodiments, the invention provides an anti-PCSK9 antibody for use in lowering serum LDL-cholesterol level in a subject. In certain

embodiments, the invention provides an anti-PCSK9 antibody for use in increasing availability of LDLR in a subject. In certain embodiments, the invention provides an anti-PCSK9 antibody for use in inhibiting binding of PCSK9 to LDLR in a subject. In certain embodiments, the invention provides an anti-PC SK9 antibody for use in a method of reducing LDL-cholesterol level in an individual comprising administering to the individual an effective of the anti-

PCSK9 antibody to reduce the LDL-cholesterol level. In certain embodiments, the invention provides an anti-PC SK9 antibody for use in a method of lowering serum LDL-cholesterol level in an individual comprising administering to the individual an effective amount of the anti- PCSK9 antibody to lower the serum LDL-cholesterol level. In certain embodiments, the invention provides an anti-PCSK9 antibody for use in a method of increasing availability of LDLR in an individual comprising administering to the individual an effective amount of the anti-PCSK9 antibody to increase availability of LDLR. In certain embodiments, the invention provides an anti-PCSK9 antibody for use in a method of inhibiting binding of PCSK9 to LDLR PCSK9 antibody to inhibit the binding of PCSK9 to LDLR. An "individual" according to any of the above embodiments is preferably a human.

In a further aspect, the invention provides for the use of an anti-PC SK9 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of a cholesterol related disorder. In certain embodiments, the cholesterol related disorder is hypercholesterolemia. In another embodiment, the medicament is for use in a method of treating hypercholesterolemia comprising administering to an individual having hypercholesterolemia an effective amount of the medicament.

In certain embodiments, the disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of PCSK9 activity. In certain embodiments, diseases or disorders that are generally addressable (either treatable or preventable) through the use of statins can also be treated. In certain embodiments, disorders or disease that can benefit from the prevention of cholesterol synthesis or increased LDLR expression can also be treated by anti-PC SK9 antibodies of the present invention. In certain embodiments, individuals treatable by the anti-PCSK9 antibodies and therapeutic methods of the invention include individuals indicated for LDL apheresis, individuals with PCSK9-activating mutations (gain of function mutations, "GOF"), individuals with

heterozygous Familial Hypercholesterolemia (heFH), individuals with primary

hypercholesterolemia who are statin intolerant or statin uncontrolled, and individuals at risk for developing hypercholesterolemia who may be preventably treated. Other indications include dyslipidemia associated with secondary causes such as Type 2 diabetes mellitus, cholestatic liver diseases (primary biliary cirrhosis), nephrotic syndrome, hypothyroidism, obesity, and the prevention and treatment of atherosclerosis and cardiovascular diseases.

In certain embodiments, the methods and uses described herein further comprise administering to the individual an effective amount of at least one additional therapeutic agent, e.g., statin. In certain embodiments, the additional therapeutic agent is for preventing and/or treating atherosclerosis and/or cardiovascular diseases. In certain embodiment, the additional therapeutic agent is for use in a method of reducing the risk of recurrent cardiovascular events. In certain embodiments, the additional therapeutic agent is for elevating the level of HDL- cholesterol in a subject.

In a further aspect, the invention provides pharmaceutical formulations comprising any of the anti-PC SK9 antibodies provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the anti-PCSK9 a pharmaceutical formulation comprises any of the anti-PCSK9 antibodies provided herein and at least one additional therapeutic agent, e.g., statin.

Antibodies of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody of the invention may be co-administered with at least one additional therapeutic agent. In certain embodiments, such additional therapeutic agent elevates the level of LDLR. In certain embodiments, an additional therapeutic agent is a LDL-cholesterol lowering drugs such as statin, e.g., atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, or any combination thereof, e.g., VYTORIN^®, ADVICOR^® or SIMCOR^®. In certain embodiments, an additional therapeutic agent is a HDL-cholesterol raising drugs.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the anti-PC SK9 antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.

An antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

Anti-PCSK9 antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg- lOmg/kg) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered.

In certain embodiments, a flat-fixed dosing regimen is used to administer anti-PCSK9 antibody to an individual. Depending on the type and severity of the disease an exemplary flat- fixed dosage might range from 10 to 1000 mg of anti-PCSK9 antibody. One exemplary dosage of the antibody would be in the range from about 10 mg to about 600 mg. Another exemplary dosage of the antibody would be in the range from about 100 mg to about 600 mg. In certain embodiments, 150 mg, 300 mg, or 600 mg of anti-PCSK9 antibody is administered to an individual. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeutic methods may be carried out using an immunoconjugate of the invention in place of or in addition to an anti- PCSK9 antibody. In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-PCSK9 antibody. I. Cleaved PCSK9 Compositions

In another aspect, the application provides a method for purifying PCSK9 cleaved at Arg218-Gln219. An exemplary method may involve contacting a sample of PCSK9 with an anti-PCSK9 antibody described herein, and removing the bound PCSK9 from the unbound PCSK9. The unbound PCSK9 corresponds to the cleaved form of PCSK9, whereas the bound form corresponds to the intact form of PCSK9. In certain embodiments, the PCSK9 may first be treated with a protease, such as, for example, hepsin or furin, to increase the portion of cleaved PCSK9 in the starting material. Arg218-Gln219. In certain embodiments, the cleaved PCSK9 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the total PCSK9 protein in the composition (e.g., the amount of intact or other forms of PCSK9 in the sample is less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the total PCSK9 protein in the composition). In certain embodiments, the cleaved PCSK9 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the total protein in the composition. In certain embodiments, the purified composition of cleaved PCSK9 is no more than 5-fold, 4-fold, 3-fold, or 2-fold less potent than intact PCSK9 in reducing LDLR surface levels on HepG2 cells. In certain embodiments, the purified composition of cleaved PCSK9 has at least 10%, 15%, 20%, 25%, 30%, 33%, 35%, or 40% of the activity of intact PCSK9 in reducing LDLR surface levels on HepG2 cells. In certain embodiments, the purified composition of cleaved PCSK9 has between 20-40%>, 25-35%, or 30-35% of the activity of intact PCSK9 in reducing LDLR surface levels on HepG2 cells. In certain embodiments, the purified composition of cleaved PCSK9 does not contain

recombinant PCSK9 protein having an N-terminus at Gln219. In certain embodiments, the purified composition of cleaved PCSK9 was not obtained by expression of a recombinant protein consisting of the same fragment of PCSK9 (e.g., same amino acid residues) as contained in the cleaved PCSK9 protein. In certain embodiments, the purified composition of cleaved PCSK9 was obtained from a composition of PCSK9 that was initially expressed as a full-length PCSK9 protein. In certain embodiments, the purified composition of cleaved PCSK9 is obtained from a composition of cleaved intact (e.g., full-length) PCSK9.

In another aspect, the application provides a purified composition of PCSK9 having an N-terminus at Glu219, wherein the PCSK9 is produced by purifying PCSK9 cleaved at Arg218-Gln219 from a composition comprising both intact (e.g., full length) PCSK9 and PCSK9 cleaved at Arg218-Glu219. In certain embodiments, the PCSK9 having an N-terminus at Glu219 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the total PCSK9 protein in the composition (e.g., the amount of intact or other forms of PCSK9 in the sample is less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the total PCSK9 protein in the composition). In certain embodiments, the PCSK9 having an N-terminus at Glu219 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the total protein in the composition. 3D5 Heavy Chain CDR1

GYTFTDYYMN (SEQ IDNO: 1) 3D5 Heavy Chain CDR2

GDINPNNGGATYNQKFND (SEQID NO: 2)

3D5 Heavy Chain CDR3

ATNWVFAY (SEQ ID NO: 3)

3D5 heavy chain variable region

EVOLOOSGPELVKPGASVKISCKASGYTFTDYYMNWV OSHG SLEWIGDINPNNG GATYNOKFNDKATLTVD SSSTAYMELRSLTSEDSAVYYCATNWVFAYWGOGTLV TVS A (SEQ ID NO: 4)

3D5 full length heavy chain amino acid sequence

MGWSCIILFLVATATGAYAEVOLOQSGPELVKPGASVKISCKASGYTFTDYYMNWV OSHG SLEWIGDINPNNGGATYNOKFNDKATLTVD SSSTAYMELRSLTSEDSAVYY CAT WVFAYWGQGTLVTVSAASTKGPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPV TLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDK IEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPDVQI SWFVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNNKDLPAPI ERTISKPKGSVRAPQVYVLPPPEEEMTK QVTLTCMVTDFMPEDIYVEWTNNGKTELN YKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGK

(SEQ ID NO: 5)

3D5 heavy chain variable region nucleotide sequence

GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGA AGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAATTGGGTG AAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATG GTGGTGCTACCTACAACCAGAAGTTCAATGACAAGGCCACATTGACTGTTGACAA GTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCA GTCTATTACTGTGCAACTAACTGGGTGTTTGCTTACTGGGGCCAAGGGACTCTGGT CACTGTCTCTGCA (SEQ ID NO: 6)

3D5 full length heavy chain nucleotide sequence

ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGCGTACGC TGAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTG AAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTACTACATGAATTGGGT GAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAAT GGTGGTGCTACCTACAACCAGAAGTTCAATGACAAGGCCACATTGACTGTTGACA AGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGC AGTCTATTACTGTGCAACTAACTGGGTGTTTGCTTACTGGGGCCAAGGGACTCTGG TCACTGTCTCTGCAGCCTCCACCAAGGGCCCATCGGTCTATCCACTGGCTCCTGTG CCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACA CCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTA ACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAA GCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGACCCACAATCAAGCCCT GTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATC TTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATG TGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTG AACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAAC AGTACTCTACGCGTGGTC AGTGCCCTCCCCATCC AGCACCAGGACTGGATGAGTGG CAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGA ACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCC ACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGAC TTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAA ACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGC AAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAG TGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCC GGGTAAATGA (SEQ ID NO: 7) 3D5 Light Chain CDR1

KSSQSLLHSDGKTYLN (SEQ ID NO: 8)

3D5 Light Chain CDR2

LVSKLDS (SEQ ID NO: 9)

3D5 Light Chain CDR3

WQGTHFPWT (SEQ ID NO: 10)

3D5 light chain variable region

DWMTOTPLTLSVTIGOPASISCKSSOSLLHSDGKTYLNWLLORPGOSPK LIYLVSK LDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWOGTHFPWTFGGGTKLEI (SEP ID NO: 11)

3D5 full length light chain amino acid sequence

MGWSCIILFLVATATGVHSDWMTOTPLTLSVTIGOPASISCKSSOSLLHSDGKTYLN WLLORPGOSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWOGT HFPWTFGGGTKLEIKRADAAPTVSIFPPSSEOLTSGGASWCFLNNFYPKDINV WKID GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRNEC (SEQ ID NO: 12)

3D5 light chain variable region nucleotide sequence

GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGC CTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTACATAGTGATGGAAAGACATATT TCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAG ATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTATTGC TGGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATAA AACGGGCT (SEQ ID NO : 13)

3D5 full length light chain nucleotide sequence

ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTC AGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAG CCTCC ATCTCTTGCAAGTC AAGTC AGAGCCTCTTACATAGTGATGGAAAGAC AT AT TTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGT GTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACA GATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTATTG CTGGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATA AAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTT AACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACA TCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAG TTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACG TTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACA AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA (SEQ ID NO: 14)

Human PCSK9 Amino Acid Sequence

MGTVSSRRSW WPLPLLLLLL LLLGPAGARA QEDEDGDYEE LVLALRSEED GLAEAPEHGT TA FHRCAKD PWRLPGTYW VLKEETHLSQ SERTARRLQA QAARRGYLTK ILHVFHGLLP GFLVKMSGDL LELALKLPHV DYIEEDSSVF AQSIPWNLER I PPRYRADE YQPPDGGSLV EVYLLDTSIQ SDHREIEGRV MVTDFENVPE EDGTRFHRQA SKCDSHGTHL AGWSGRDAG VAKGASMRSL RVLNCQGKGT VSGTLIGLEF IRKSQLVQPV GPLWLLPLA GGYSRVLNAA CQRLARAGVV LVTAAGNFRD DACLYSPASA PEVITVGATN AQDQPVTLGT LGTNFGRCVD LFAPGEDIIG ASSDCSTCFV SQSGTSQAAA HVAGIAAMML SAEPELTLAE LRQRLIHFSA KDVINEAWFP EDQRVLTPNL VAALPPSTHG AGWQLFCRTV WSAHSGPTRM A AVARCAPD EELLSCSSFS RSGKRRGERM EAQGGKLVCR AHNAFGGEGV YAIARCCLLP QANCSVHTAP PAEASMGTRV HCHQQGHVLT GCSSHWEVED LGTHKPPVLR PRGQPNQCVG HREAS IHASC CHAPGLECKV KEHGIPAPQE QVTVACEEGW TLTGCSALPG SHVLGAYAV DNTCWRSRD VSTTGSTSEG AVTAVAICCR SRHLAQASQE LQ (SEQ ID NO: 15)

III. EXAMPLES

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Example 1: The serine protease hepsin cleaves PCSK9 at the 'furin cleavage site'

Mature PCSK9 is composed of the prodomain that is non-covalently attached to the subtilisin-like catalytic domain (Cat), which is followed by the C-terminal domain (CT) (Fig. Enzymatic activity of PCSK9 is required for auto-catalytic processing of the single-chain PCSK9 precursor in the endoplasmic reticulum, but not for LDLR binding and LDLR degradation. Although the CRD is not directly involved in PCSK9 binding, it interacts with cell surface proteins, such as Annexin A2, to allow for proper internalization of the

ligand:receptor complex. In the endosomal low pH compartment the CRD may also engage in additional LDLR interactions to increase binding affinity. Posttranslational modifications of PCSK9 include phosphorylation, sulfation and glycosylation, none of which are essential for function. A posttranslational modification with a potential functional impact is the cleavage of the R218-Q219 peptide bond in the catalytic domain (Fig. IB) by the membrane-bound proprotein convertase furin (S. Benjannet, et al. (2006)). This cleavage site is located in surface loop, the '218-loop', which is not resolved in structures of unliganded PCSK9 and, therefore, appears to have inherent flexibility favoring scissile peptide exposure to proteases. In-vivo studies with hepatocyte-directed furin k/o mice corroborated the in vitro findings and suggested that furin is the main PCSK9 processing protease in-vivo. When analyzed by SDS-PAGE the cleaved PCSK9 shows a shift of the 60kDa (=Cat_CT) band to a lower 50kDa species that lacks the Serl53-Arg218 fragment (Fig. IB) and results from cellular assays suggested that cleaved PCSK9 is no longer able to degrade LDLR. This complete lack of activity was attributed to the loss of the N-segment, as well as the prodomain from PCSK9 (S. Benjannet, et al. (2006)).

The cleaved form circulates in human plasma and constitutes 15-40% of total circulating PCSK9. Cleaved PCSK9 was also found in mouse plasma contributing 30-50% to total PCSK9 levels. The furin cleavage sequence ²¹⁵RFHR-Q²¹⁹ (SEQ ID NO: 38) harbors the three naturally occurring gain-of-function mutations R215H, F216L and R218S. Patient plasma samples and in-vitro mutagenesis studies consistently show that these mutations lead to reduced levels of cleaved PCSK9. The impaired cleavage of mutant PCSK9 by furin is consistent with gain-of-function and increased LDL-c levels associated with these mutations.

We investigated whether furin cleavage of PCSK9 affects receptor binding or post- ligation events, such as endocytosis of the PCSK9c:LDLR complex or lysosomal degradation. The identification of a monoclonal antibody 3D5 that differentially recognized furin-cleaved and intact PCSK9 allowed us to obtain highly purified cleaved PCSK9 generated by treatment of PCSK9 with furin as well as with hepsin, a type II transmembrane protease having increased proteolytic activity towards PCSK9. rather inefficient in cleaving PCSK9 at the Arg218-Gln219 site. In order to identify a more efficient protease we tested a panel of trypsin-like serine proteases having preference for a basic PI residue (nomenclature according to Schechter et al. Biochem Biophys Res Commun 27, 157-162 (1967); see Fig. IB). The results showed that none of the examined proteases appreciably cleaved PCSK9 during a 6 h incubation, except for soluble furin (referred to as 'furin') and the soluble form of hepsin (referred to as 'hepsin'), a type II transmembrane serine protease (Fig. 2A). The cleavage by hepsin was specific, since pre-treatment with the neutralizing antibody Ab25 completely inhibited PCSK9 cleavage (Fig. 2A). Hepsin appeared more efficient than furin, since most of the intact 60 kDa PCSK9 band (Cat CT) was converted into the 50 kDa cleaved form (AN_Cat_CT), while less than 50% was converted by furin. N-terminal sequencing showed that for both furin and hepsin, the AN Cat CT domain (50 kDa) started with ²¹⁹QASK (SEQ ID NO: 16) and the ~ 12kDa fragment (N-fragment in

153

Fig. 2A) started with SIP. Therefore, hepsin cleaved at the same site as furin, i.e. at the Arg218-Gln219 peptide bond (Fig. IB). The relative cleavage efficiencies of hepsin and furin were quantified by measuring the disappearance of the intact 60 kDa band by densitometry. The results showed that during a 6 h reaction period, hepsin cleaved 50% of PCSK9 at a concentration of 11 nM compared to 80 nM for furin, indicating that hepsin was 7-fold more efficient (Fig. 2B).

To find out whether hepsin specifically cleaved at the Arg218-Gln219 site, we mutated the Arg218 residue to Ala, which should abolish cleavage. However, hepsin still cleaved PCSK9, albeit at a reduced rate, whereas furin was no longer able to cleave PCSK9 (Fig. 3A). An additional mutation of the proximal Arg215 residue was made to generate the double mutant R215A:R218A. This mutant was completely resistant to cleavage by hepsin (Fig. 3 A), suggesting that hepsin cleaved at two sites Arg215-Phe216 and Arg218-Gln219 separated by only 3 residues, whereas furin only cleaved at the latter site (Fig. IB).

Example 2: Identification of an antibody that differentially recognizes intact and furin- cleaved PCSK9

In order to generate highly purified cleaved PCSK9, we wished to identify an antibody that would only recognize intact but not cleaved PCSK9 for use in affinity purification of the cleaved form. Monoclonal antibodies derived from immunizing PCSK9 ^{~ ~} mice immunized with recombinant PCSK9 wildtype were analyzed for their ability to inhibit PCSK9 cleavage. One of these antibodies, Ab-3D5, completely prevented the cleavage by both furin and hepsin, This was examined by two different epitope mapping approaches. First, in an epitope excision experiment PCSK9 alone or in complex with Ab-3D5 was digested with trypsin for various time periods. The peptide fragments were identified by mass spectrometry and mapped onto the PCSK9 sequence (Fig. 4A). The results indicated that Ab-3D5 (in the PCSK9:Ab-3D5 complex) protected trypsin-mediated cleavage at the Arg215-Phe216 and Arg218-Gln219 sites, which were readily cleaved when PCSK9 was digested alone. Therefore, the Ab-3D5 epitope

214 219

encompassed the TRFHRQ (SEQ ID NO: 17) sequence. Second, a panel of overlapping synthetic peptides covering the entire PCSK9 amino acid sequence was prepared (Genscript, Piscataway NJ). ELISA experiments measuring binding of Ab-3D5 to the biotinylated peptides captured on streptavidin-coated plates showed that Ab-3D5 only bound to two

211 220 peptides spanning the '218-loop', with the consensus sequence EDGTRFHRQA (SEQ ID NO: 18) (Fig. 4B). The results from both approaches were in good agreement and clearly indicated that the Ab-3D5 epitope encompasses the PCSK9 '218-loop', which harbors the hepsin- and furin cleavage sites.

The recent crystal structure of the LDLR:PCSK9 complex revealed that the EGF(B) domain of LDLR is located close to the '218-loop', suggesting that Ab-3D5 may interfere with LDLR binding. Indeed, biolayer interferometry binding experiments demonstrated that Ab-3D5 completely inhibited LDLR binding to PCSK9 (Fig. 5A). In contrast, Ab-7G7, whose binding site did not overlap with that of Ab-3D5 according to competition binding experiments, did not inhibit LDLR binding to PCSK9 (Fig. 5 A). Additional studies with HepG2 cells further showed that Ab-3D5 inhibited PCSK9-mediated LDLR degradation in a concentration- dependent manner, whereas Ab-7G7 did not (Fig. 5B). Therefore, it is very probable that by binding to the '218-loop', Ab-3D5 sterically interfered with the LDLR binding.

The binding of Ab-3D5 to intact PCSK9 and to cleaved PCSK9, obtained by treatment of intact PCSK9 by hepsin (6 h) or furin (20 h), was determined by biolayer interferometry. Hepsin and furin were removed in a re-purification step, but the preparations still contained residual intact PCSK9. The affinity constants are summarized in Table 1 below showing that Ab-3D5 bound with high affinity to intact PCSK9, but did not bind to hepsin- or furin-cleaved PCSK9 at concentrations up to 285-fold and 170-fold, respectively, above the Κ_Ό value of the intact form. Control experiments with Ab-7G7 showed that this antibody bound to both forms with similar affinities (Table 1). These findings suggested that Ab-3D5 differentially recognized intact and cleaved PCSK9 and, thus, could be utilized for affinity purification of the cleaved form.

*The cleaved PCSK9 still contained residual intact PCSK9.

The heavy chain and light chain variable region sequences for antibody 3D5 are shown in Fig. 6.

Example 3: Purification and analysis of cleaved PCSK9

The residual intact PCSK9 in hepsin- and furin-treated preparations was removed by addition of Ab-3D5. The formed complexes of intact PCSK9 and Ab-3D5 (high molecular weight peak) could be easily separated from the cleaved (uncomplexed) form by size exclusion chromatography (Fig. 7). The elution fractions analyzed by SDS-PAGE showed the clear separation of intact PCSK9 co-eluting with Ab-3D5 IgG (the intact PCSK9:Ab-3D5 complex; fractions 1-4) and the cleaved form eluting in later fractions (Fig. 8A). The pooled fractions of the cleaved PCSK9 contained the 50 kDa AN Cat CT (starting with Gln219), the -15 kDa prodomain and the N-fragment (Serl53-Arg218). However, these fractions did not contain any detectable intact PCSK9. Similar results were obtained with hepsin-cleaved PCSK9.

The pooled fractions of the purified furin-cleaved PCSK9 (referred to as PCSK9c_fu) were re-applied to a S-200 analytical size exclusion column and compared to untreated PCSK9 (PCSK9-wt). Both proteins eluted at the same elution volume (12.72 and 12.78 ml) with a deduced mass of ~ 77 kDa (Fig. 8B). This suggested that furin cleavage did not result in the loss of a major PCSK9 fragment. This was further investigated by electrospray mass spectrometry of PCSK9c_fu and of hepsin-cleaved purified PCSK9 (referred to as

PCSK9c_hep). Intact PCSK9, which was used as reference material, gave a single peak of 60,516 Da corresponding to the Cat CT domain (Fig. 9) and several pro-domain peaks (13,756-14,000 Da) were observed, but no peaks in the mass range of the N-segment. For PCSK9c_hep and PCSK9c_fu, peaks that corresponded to the prodomain similar to intact

PCSK9 and to two complementary portions of the Cat CT domain were observed, a large and a small fragment. The large fragment (ΔΝ-Cat CT) had a mass of -52,800 Da and started at Gln219 (Fig. 9). The small fragment was the N-fragment (Serl53-Gln219). For PCSK9c_hep Serl53-Phe215 (Fig. 9). This result was in exellent agreement with the mutagenesis experiments (Fig. 3 A), indicating that furin cleaved only at the Arg218-Gln219 site, while hepsin additionally cleaved at the Arg215-Phe216 site. The combined masses of the small and large fragments amounted to 60,535 Da (+1 Da for PCSK9c_hep), which was essentially the same mass as the intact Cat CT domain (-60,516 Da). Therefore, the cleavage of PCSK9 by furin or hepsin did not result in the loss of any sizeable fragments, except for the partial excision and the probable loss of the three amino acid stretch Phe216-Arg218 in case of PCSK9c_hep. This is consistent with the identical masses of untreated and protease-treated PCSK9 observed by size exclusion chromatography. Therefore, hepsin and furin only produced internal cleavages and the Serl53-Gln218 segment (Serl53-Arg215 for hepsin) remained non- covalently attached to the catalytic domain. These segments, like the non-covalently attached prodomain, only dissociate from the catalytic domain under denaturing conditions by SDS- PAGE.

Example 4: Binding of cleaved PCSK9 to LDLR

Competition binding ELISA experiments showed that both cleaved PCSK9 forms retained much of the LDLR binding function, having only slightly reduced binding affinities compared to intact PCSK9 (Table 2 below). Compared to the respective intact PCSK9 controls (PCSK9-wt), the affinity losses (IC₅₀ wt/IC₅₀ cleaved) of PCSK9c_fu and PCSK9_hep were 1.4- and 1.1 -fold. Moreover, biolayer interferometry experiments showed that the intact PCSK9 controls (PCSK9-wt) bound to immobilized soluble LDLR with Κ_Ό values of 130 and 177 nM (Table 3 below). The determined Κ_Ό values for PCSK9c_hep and PCSK9c_fu were 268 nM and 357 nM, respectively. The affinity losses (Κ_Ό wt/K_O cleaved) were 2.1- and 2.0- fold, respectively. The results indicated that cleavage at the '218-loop' had only a moderate effect on LDLR binding.

Table 2. LDLR Competition Binding ELISA.

Analyte koff

(10⁴ x M^'V¹) (10 x s^"1) (10 ⁹ x M)

PCSK9-wt 4.7 ± 1.0 61.2 ± 13.1 129.7 ± 17.4

PCSK9c_hep 3.2 ± 0.6 85.8 ± 18.0 267.7 ± 10.9

PCSK9-wt 3.6 ± 0.5 62.4 ± 12.4 177.0 ± 38.9

PCSK9c_fu 3.9 ± 0.5 138.5 ± 2.7 356.5 ± 50.7

Example 5: LDLR degradation by PCSK9-C

The LDLR degradation by cleaved PCSK9 was measured in two systems, in a cellular HepG2 assay and in a mouse model of liver LDLR degradation. In the HepG2 assays, PCSK9- wt reduced surface LDLR levels in a concentration-dependent fashion with a half-maximal reduction at 3.7-11 μ^πιΐ (Fig. 10). Both PCSK9c_fu and PCSK9c_hep also reduced LDLR surface levels in a concentration-dependent fashion. Both cleaved forms were equally potent with half-maximal activity at 11-33 μg/ml (Fig. 10). Similar to the intact PCSK9 the maximal inhibition was 80-90%, but required about 3-fold higher concentrations for the cleaved forms.

In HepG2 cells Ab-3D5 was shown to potently and effectively inhibit LDLR degradation that was mediated by intact PCSK9 (Fig. 4B). In contrast, Ab-3D5 was unable to inhibit LDLR degradation mediated by PCSK9c_hep (Fig. 11), consistent with the antibody's loss of binding to the cleaved PCSK9.

The activities of the cleaved PCSK9 forms were further examined in a mouse liver

LDLR degradation model. Liver LDLR levels were determined by quantitative immunoblotting after mice were injected with increasing doses of intact or cleaved PCSK9. PCSK9-wt degraded LDLR in a dose-dependent fashion; more than 90%> reduction was achieved at doses above 15μg. For PCSK9c_hep and PCSK9c_fu a similar degree of inhibition was achieved at the 15-45μg doses and 4545μg dose, respectively, indicating about 2-3-fold reduced potencies of the cleaved forms compared to PCSK9-wt (Fig. 12). These findings suggested that PCSK9- c retained substantial LDLR binding and degradation activity.

Example 6: Neutralizing Activity of 3D5 Antibody in Mouse Model

The PCSK9-neutralizing activity of Ab-3D5 was examined in a mouse model of liver

LDLR degradation in which i.v. injection of human PCSK9 reduced liver LDLR levels to 23% (Rag control). Administration of 20 mg/kg of Ab-3D5 prevented LDLR degradation (77% of (Fig. 13).

The experiments described herein provide evidence that furin-cleaved PCSK9 retains substantial activity in-vitro and in-vivo. Therefore, liver LDLR levels may be under the control of two different species of circulating PCSK9, the intact and the cleaved forms. Based on the surmised functional incompetence of cleaved PCSK9, it was unexpected that both furin- and hepsin-cleaved PCSK9 was able to degrade LDLR on HepG2 cells and in mouse liver.

Biochemical and biophysical experiments established that furin-mediated cleavage at Arg218- Gln219 is an internal cleavage event without the loss of any fragment. The N-fragment spanning the N-terminal Serl53 to the Arg218 as well as the prodomain remained firmly attached to the catalytic domain. The same is true for hepsin cleavage, except that the

217 218 additional cleavage at Arg215-Phe216 likely resulted in the partial loss of the FHR trip ep tide. Material and Methods

Reagents

Soluble human furin and soluble LDLR ectodomain were from R & D Systems, factor Xa, activated protein C and thrombin from Haematologic Technologies and factor Xlla from Enzyme Research Laboratories. Soluble Hepsin, hepatocyte growth factor activator and matriptase were expressed and purified as described (R. Ganesan, et al. Protein Eng Des Sel 25, 127-133 (2012); D. Kirchhofer, et al. J Biol Chem 278, 36341-36349 (2003)). The neutralizing anti-hepsin antibody Ab25 was described recently (R. Ganesan, et al.).

Construction, expression and purification of wildtype and mutant PCSK9 proteins

Human PCSK9 complementary deoxyribonucleic acids (cDNAs) containing a histidine (His)₈ C-terminal tag (SEQ ID NO: 19) was cloned into a mammalian expression vector

(pRK5). Human PCSK9 R215A: R218A mutant was made by site-directed mutagenesis using QuikChange Lightning (Aligent Technologies; Santa Clara, CA). Human PCSK9 R218A mutant was constructed by ACTG Inc. (Wheeling, IL) using site-directed mutagensis. Mutants were confirmed by DNA sequencing. The recombinant human PCSK9 proteins (wild type and mutants) were transiently expressed in Chinese hamster ovary (CHO) cells and purified from conditioned media by affinity chromatography using a nickel nitrilotriacetic agarose column (Qiagen; Germantown, MD) followed by gel filtration on a Sephacryl S 200 column

(GE Healthcare; Piscataway, NJ). Eight to twelve week-old PCSK9 mice (UNQ 6075.ko.PCSK9.2965) were immunized with recombinant human PCSK9. The immunogen was resuspended in monophosphoryl lipid A/trehalose dicorynomycolate adjuvant and injected (2 μg protein/injection) via footpad at 3 to 4 day intervals for a total of 8 boosts. Three days after the final boost, lymphocytes from spleens and lymph nodes were harvested for fusion with SP2/0 myeloma cells (American Type Culture Collection) using the Cyto Pulse CEEF-50 apparatus (Cyto Pulse Sciences). Fused cells were cultured in ClonaCell-HY Medium C (StemCell Technologies) overnight, then centrifuged, resuspended in 10 ml ClonaCell-HY Medium C and gently mixed with 90 ml Methylcellulose-based ClonaCell-HY Medium D (StemCell Technologies) containing HAT components. The fused cells were plated into 100 mm Petri dishes (Becton Dickinson) and allowed to grow in 37°C in a 7% C0₂ incubator. After 7-10 days, single hybridoma clones were picked by ClonePix (Genetix, United Kingdom) and transferred into 96-well cell culture plates (Becton Dickinson) with 200 microliter/well ClonaCell-HY Medium E (StemCell Technologies). Hybridoma culture media were changed prior to ELISA screening. All ELISA positive clones were further screened on the functional assays. After at least two rounds of single cell subcloning by limiting dilution the final clones, including Ab-3D5 and Ab-7G7, were scaled up and supernatants collected for antibody purification. The hybridoma supernatants were purified by Protein A affinity chromatography, then sterile filtered (Nalge Nunc International) and stored at 4°C in PBS.

Cleavage of PCSK9 by recombinant furin and hepsin

Unless otherwise indicated, all cleavage reactions were performed in PCSK9 buffer (50 mM Tris pH 8.0 and 150 mM NaCl) supplemented with 4 mM CaCl₂ and reaction products were analyzed by SDS-PAGE under non-reducing conditions. PCSK9 (1.9 uM) was incubated with 40 nM of furin, factor Xa, factor Xlla, activated protein C, or thrombin in PCSK9 buffer supplemented with 4 mM CaCl₂, or with hepsin, hepatocyte growth factor activator, matriptase in PCSK9 sample without CaCl₂, for 6 h at 20°C. Neutralizing of hepsin could be

accomplished by preincubating 40 nM of hepsin with 3 μΜ of hepsin-specific antibody-25 (R. Ganesan, et al. Protein Eng Des Sel 25, 127-133 (2012)) for 20 min prior to the addition of PCSK9.

Experiments with PCSK9 mutants R218A and R215A:R218A (2.6 uM) were carried out in PCSK9 buffer supplemented with 4 mM CaCl₂ by incubation with either 40 nM hepsin for 6 h or with 80 nM furin for 20 h. Proteins were analyzed by SDS-PAGE followed by staining with SimplyBlue SafeStain (Invitrogen). incubation of PCSK9 (1.9 uM) with increasing concentrations of hepsin or furin for 6 h in PCSK9 buffer supplemented with 4 mM CaCl₂, followed by SDS-PAGE and densitometry of intact PCSK9 bands by the NIH ImageJ software. Results are the average ± SD of three experiments.

Inhibition assays with Ab-3D5 or Ab-7G7 was performed by incubating PCSK9 (1.9 μΜ) with 3 μΜ of either antibody for 20 min prior to the addition of 80 nM furin or 40 nM hepsin in PCSK9 buffer supplemented with 4 mM CaCl₂ for 6 h.

Purification of cleaved PCSK9

PCSK9 in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 mM CaCl₂ was treated with 80 nM furin for 20 h or with 40 nM hepsin for 6 h at room temperature. Then, Ab-3D5 (IgG) and anti-furin antibody (IgG) (H-220, Santa Cruz Biotechnology) were added to the furin-treated samples to remove intact PCSK9 and furin, respectively. Ab-3D5 (IgG) and the hepsin antibody Ab25 (IgG) were added to the hepsin-treated samples to remove intact PCSK9 and hepsin, respectively. After 30 min incubation the mixtures were applied to S-200 size exclusion columns (HiLoad™ 16/60 Superdex™ prep grade, GE Healthcare) and elution fractions were collected and analyzed by SDS-PAGE. Fractions containing pure cleaved PCSK9

(PCSK9c_hep and PCSK9c_fu, respectively) were pooled and stored at -80°C for subsequent functional studies, analysis by N-terminal sequencing and by mass spectrometry.

Initially, cleaved forms were produced without Ab-3D5 affinity purification and used for the binding experiments with Ab-3D5 and Ab-7G7 (Table 1). For these experiments we used the same protocol as described above, except that Ab-3D5 was not added to the cleaved material. Therefore, these cleaved PCSK9 preparations (designated PCSK9c_hep* and PCSK9c_fu*) still contained residual intact PCSK9.

Affinity measurements by biolayer interferometry

Binding affinities of Ab-3D5 and Ab-7G7 to cleaved PCSK9 forms were analyzed by biolayer interferometry using an Octet Red384 system (ForteBio; Menlo Park, CA). For these measurements we used hepsin- and furin-cleaved PCSK9 (PCSK9-c_hep* and PCSK9-c_fu*) that was not affinity-purified by Ab-3D5 and, thus, still contained residual intact PCSK9. Antibodies (50 μg/ml) were immobilized on anti-murine IgG Fc capture biosensors (ForteBio) in Tris-buffer (50 mM Tris, pH 7.5, 300 mM NaCl, 2 mM CaCl₂, 1 mg/ml BSA, 0.1%

Tween-20). Measurements of the association and dissociation constants were carried out in the presence of various concentrations of cleaved and intact PCSK9 (0-1 μΜ). Kinetic parameters kon, ϊϊ and K were calculated from a nonlinear fit of the data using the Octet software For experiments with cleaved PCSK9 forms, LDLR (ectodomain; R & D Systemts) was biotinylated according to manufacturer's instructions (Thermo Scientific) and immobilized on streptavidin biosensors. Measurements of the association and dissociation constants were carried out in the presence of increasing concentrations of PCSK9c_hep or PCSK9c_fu in Tris- buffer and Κ_Ό values determined as described. For each set of PCSK9c_hep and of PCSK9c_fu measurements parallel control experiments with PCSK9-wt were carried out. Therefore, there are two PCSK9-wt Κ_Ό values, one for PCSK9c_hep experiments (n=3 experiments) and one for PCSK9c_fu experiments (n=7-8 experiments).

Tryptic digestion protection experiments

PCSK9 with Ab-3D5 (1 : 1 molar ratio) in 50 mM Tris, pH 8.0, 150 mM NaCl were incubated overnight at 4° C. The PCSK9:Ab-3D5 complex was repurified on a S-200 size exclusion column and then digested with trypsin (Promega) in 50 mM Tris, pH 8.0, 50 mM NaCl, pH 8 at an enzyme : substrate ratio of 1 : 1000 (w/w). PCSK9 and Ab-3D5 were also digested under the same conditions. Aliquots of the resulting samples collected at different time points were treated with tm(2-carboxyethyl)phosphine (2 mM final concentration) to reduce disulfide bonds and were analyzed in parallel with non-reduced samples. Samples were mixed 1 : 1 with sinapinic acid matrix (100 mM in methanol :acetonitrile: water at a ratio of 56:36:8; Agilent technologies), applied to the sample stage of a MALDI-TOF/TOF mass spectrometer (4800, Applied Biosystems) and acquired in linear mode with delayed extraction. 1200 laser shots were averaged for each spectrum acquired. The cleavage sites on PCSK9 and PCSK9:Ab-3D5 complex at 5, 15, 30, 60, 120, 240 min digestions were determined by mapping observed peptide masses onto the sequence of PCSK9 with the program GPMAW (Lighthouse data, v8.0). The identities of selected peptides were confirmed by tandem mass spectrometry on the same instrument.

Mass spectrometry

PCSK9wt, PCSK9c_hep and PCSK9c_fu were analyzed by reverse phase liquid chromatography-electrospray quadrupole time-of-flight mass spectrometry (6520; Agilent Technologies). Raw spectra were deconvoluted using Mass Hunter software (v.B.04.00;

Agilent Technologies).

Cell surface LDLR assay with HepG2 cells

HepG2 cells (ATCC; Manassas, VA) were seeded into 48-well plates (Corning;

Corning, NY) at 1 x 10⁵ cells per well in high glucose medium (DMEM, Gibco; Grand Island, NY) containing 2 mM glutamine (Sigma), penicillin/streptomycin (Gibco) and 10% FBS lipoprotein deficient serum (LPDS, Intracel; Frederick, MD).

After 24 h, the cells were treated as follows: (i) the indicated concentrations of PCSK9- wt, PCSK9c_hep or PCSK9c_fu were added to the cells and incubated at 37°C for 4 h; (ii) 100 μ§/ι 1 PCSK9c_hep was pre-incubated with the indicated concentrations of Ab-3D5 antibody for 30 min and added to the cells for 4 h at 37°C; as additional controls, 15 μg/ml PCSK9-wt without or with 0.5 μΜ Ab-3D5 were pre-incubated for 30 min and added to cells for 4 h at 37°C; or (iii) 15 μ§/ι 1 PCSK9-wt was pre-incubated with the indicated concentrations of Ab7G7 or Ab-3D5 for 30 min and added to the cells for 4 h at 37°C.

Cells were rinsed with PBS and detached using cell dissociation buffer (Gibco). The cells were collected, centrifuged, and incubated with 1 :20 anti-LDLR antibody (Progen Biotechnik; Heidelberg, Germany) in FACS buffer (1% BSA in PBS) on ice for 10 min. The samples were then washed with PBS and incubated with 1 :200 goat anti-mouse IgG (H + L) Alexa Fluor 488 (Invitrogen; Carlsbad, CA) on ice for 5 min. After two PBS washes cells were re-suspended in PBS containing 10 μg/ml of propidium iodide and analyzed on a dual laser flow cytometer (FACScan, Becton Dickinson; Franklin Lakes, NJ). Relative fluorescence units (RFUs) were used to quantify LDLR expression levels on the HepG2 cell surface. Cell surface LDLR levels were expressed as percent of LDLR levels measured in the absence of PCSK9 (=control).

Competitive binding assays by biolayer interferometry

For antibody competition binding experiments we biotinylated LDLR ectodomain (biotin-LDLR) according to the manufacturer's instructions. Biotin-LDLR (15 μg/ml) was immobilized on the streptavidin biosensor and immersed into mixtures of 250 nM human PCSK9 pre-incubated for 30 min with 1 μΜ Ab-3D5 or Ab-7G7 in Tris buffer. Steady-state binding values were determined and the results were expressed as percentage of the

steady-state binding of human PCSK9 control. The results were the average ± SD of three independent experiments.

ELISA for measuring PCSK9 binding to LDLR

The binding activity of furin-cleaved or hepsin-cleaved PCSK9 protein to LDLR was measured by a competitive ELISA. Briefly, 1 μg/mL of recombinant human LDLR

(ectodomain; R&D Systems; Minneapolis, MN) in coating buffer (50 mM sodium carbonate, pH 9.6) was coated overnight on a 384-well MaxiSorp plate (Nalge Nunc International;

Rochester, NY) at 4°C. On the next day, 0.5 μg/ml of biotinylated PCSK9-wt in assay buffer mixed with an equal volume of serially diluted PCSK9c_hep or PCSK9c_fu and incubated for 30 min at room temperature. Then the pre-mixed solutions were added to LDLR-coated plates and incubated for 2 hours at room temperature. The binding of biotinylated PCSK9-wt to coated LDLR was detected by sequential additions of streptavidin-horseradish peroxidase (GE Healthcare; Buckinghamshire, UK) and substrate 3, 3', 5, 5'-tetramethyl benzidine

(TMBE-1000, Moss; Pasadena, MD). The mean absorbance values from duplicate wells were plotted as a function of antibody concentration and the data were fitted to a four-parameter equation for each antibody using KaleidaGraph (Synergy Software; Reading, PA).

Mouse model of liver LDLR degradation

Eight weeks old male C57BL/6 mice were purchased from Jackson Laboratory and housed for 2 weeks before starting the experiment. Mice were randomized into 3 groups (3-4 mice/group) based on body weight. In one experiment, mice were given either vehicle (V), or 20 mg/kg of a control mouse IgG (Rag), or 20mg/kg of Ab-3D5, or 20mg/kg of Ab-7G7 through the i.v. route. After 2 h, mice were dosed i.v. with 30 μg of PCSK9 in PBS. In another experiment, mice were injected i.v. with either PBS (vehicle control) or four doses (3 ug, 15 ug, 45 ug, or 90 ug) of PCSK9 or PCSK9_fu or PCSK9_hep. After lh livers were harvested and snap frozen. Approximately 200 mg of each liver was homogenized in Extraction Buffer 1 supplemented with Protease Inhibitor Cocktail (ProteoExtract Native Membrane Protein Extraction Kit, Calbiochem) using the TissueLyser (Qiagen) according to manufacturer's instructions. Lysates were centrifuged and the cell pellet was resuspended in Extraction Buffer II supplemented with Protease Inhibitor Cocktail (Calbiochem). After 30 min of gentle agitation at 4°C, the samples were centrifuged and the supernatants containing the membrane proteins were quantified using the Bradford assay. 4X SDS sample buffer was added. For each group (n = 3-4), liver proteins were pooled for a total of 100 μg of protein and boiled for 5 min. The samples were loaded onto a 4-12% Bis-Tris Midi gel and proteins separated by SDS- PAGE. After transfer to nitrocellulose membranes using the iBlot (Invitrogen), membranes were blocked with 5% nonfat milk for 1 h at room temperature. The blots were incubated with 1 :200 anti-LDLR (Abeam) in 5% nonfat milk overnight at 4 °C. Blots were washed three times with TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 15 min. Blots were then incubated with 1 :5000 anti-rabbit horseradish peroxidase (GE Healthcare) in 5% nonfat milk for 1 h. After washing with TBS-T, proteins were visualized using ECL-Plus (GE Healthcare) and exposure to XAR film (Kodak). The membranes were then washed with TBS- T and incubated with 1 :5000 anti-transferrin receptor (Invitrogen) for 2 hours at room horseradish peroxidase (GE Healthcare) for 1 h and washed again. Proteins were visualized using ECL Plus and exposure to XAR film. Quantification was performed using the ImageJ (NIH) program on scanned film, where LDLR was normalized to transferrin receptor.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims

1. An isolated antibody that binds to PCSK9, wherein the antibody binds to intact PCSK9 with at least 100-fold greater affinity than it binds to PCSK9 cleaved at Arg218- Gln219.

2. The antibody of claim 1, wherein the antibody binds to an epitope of PCSK9 comprising amino acid residues Thr214 through Gln219.

3. The antibody of claim 1 or 2, which is a monoclonal antibody.

4. The antibody of any one of claims 1-3, which is a human, humanized, or chimeric antibody.

5. The antibody of any one of claims 1-4, which is an antibody fragment.

6. The antibody of any one of claims 1-5, wherein the antibody comprises (a) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (b) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2.

7. The antibody of any one of claims 1-6, wherein the antibody comprises (a)

HVR-H1 comprising the amino acid sequence of SEQ ID NO: l, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3.

8. The antibody of any one of claims 1-6, further comprising (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

9. The antibody of claim 1, comprising (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:8; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

10. The antibody of any one of claims 1-9, comprising (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11 ; or (c) a VH sequence as in (a) and a VL sequence as in (b).

11. The antibody of any one of claims 1-10, comprising a VH sequence of SEQ ID

NO: 4.

12. The antibody of any one of claims 1-10, comprising a VL sequence of SEQ ID NO: 11.

14. The antibody of any one of claims 1-4 or 6-13, which is a full length IgGl antibody.

15. Isolated nucleic acid encoding the antibody of any one of claims 1-14.

16. A host cell comprising the nucleic acid of claim 15.

17. A method of producing an antibody comprising culturing the host cell of claim 16 so that the antibody is produced.

18. The method of claim 17, further comprising recovering the antibody from the host cell.

19. An immunoconjugate comprising the antibody of any one of claims 1-14 and a cytotoxic agent.

20. A pharmaceutical formulation comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.