WO2013148284A1 - Anticorps qui se lient à un site de clivage de pcsk9 et leurs procédés d'utilisation - Google Patents

Anticorps qui se lient à un site de clivage de pcsk9 et leurs procédés d'utilisation Download PDF

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Publication number
WO2013148284A1
WO2013148284A1 PCT/US2013/031722 US2013031722W WO2013148284A1 WO 2013148284 A1 WO2013148284 A1 WO 2013148284A1 US 2013031722 W US2013031722 W US 2013031722W WO 2013148284 A1 WO2013148284 A1 WO 2013148284A1
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antibody
pcsk9
amino acid
seq
acid sequence
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PCT/US2013/031722
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English (en)
Inventor
Tao Sai
Joyce LAI
Daniel Kirchhofer
Andrew Peterson
Michael Terry LIPARI
Wei Li
Anita IZRAEL-TOMASEVIC
David Arnott
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Genentech, Inc.
F. Hoffmann-La Roche Ag
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Publication of WO2013148284A1 publication Critical patent/WO2013148284A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • PCSK9 prote convertase subtilisin/kexin type 9
  • LDLR hepatic LDL receptors
  • 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.
  • the invention provides anti-PCSK9 antibodies and methods of using the same.
  • 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.
  • 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.
  • an anti-PCSK9 antibody described herein is a monoclonal antibody.
  • an anti-PCSK9 antibody described herein is a human, humanized, or chimeric antibody.
  • an anti-PCSK9 antibody described herein is an antibody fragment.
  • an anti-PCSK9 antibody described herein comprises (a) HVR-
  • H3 comprising the amino acid sequence of SEQ ID NO:3
  • HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8
  • HVR-H2 comprising the amino acid sequence of SEQ ID NO:2.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the application provides an isolated nucleic acid encoding an anti- PCSK9 antibody described herein.
  • the application provides a host cell comprising a nucleic acid encoding an anti-PC SK9 antibody described herein.
  • 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).
  • 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.
  • 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.
  • the application provides a host cell comprising a nucleic acid encoding an anti-PC SK9 antibody described herein.
  • 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.
  • the application provides an immunoconjugate comprising an anti- PCSK9 antibody described herein and a cytotoxic agent.
  • the application provides a pharmaceutical formulation comprising an anti-PCSK9 antibody described herein and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • the second medicament elevates the level of LDLR.
  • the second medicament reduces the level of LDL-cholesterol.
  • 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.
  • the second medicament elevates the level of HDL-cholesterol.
  • 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.
  • the application provides a method for purifying PCSK9 cleaved at Arg218-Gln219, said method comprising
  • the application provides a method for purifying PCSK9 cleaved at Arg218-Gln219, said method comprising
  • the protease is hepsin or furin.
  • the application provides a purified composition of PCSK9 cleaved at Arg218-Gln219.
  • the cleaved PCSK9 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the total PCSK9 protein in the composition.
  • the cleaved PCSK9 is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the total protein in the composition.
  • the invention provides a method for determining the amount of cleaved PCSK9 present in a sample, comprising
  • the sample is a biological sample, such as, for example, human blood sample.
  • the amount of total PCSK9 in the sample determined using an antibody that binds to both intact and cleaved PCSK9.
  • FIG. 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.
  • B 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
  • FIG. 1 PCSK9 cleavage by a panel of serine proteases.
  • Ctrl untreated PCSK9; APC, activated protein C; HGFA, hepatocyte growth factor activator.
  • FIG. 3 Inhibition of protease-mediated PCSK9 cleavage by mutagenesis of '218- loop' residues and by antibody 3D5.
  • WT PCSK9 wildtype
  • R218A and R215A:R218A 2.6 ⁇ each
  • WT PCSK9 wildtype
  • R218A and R215A:R218A 2.6 ⁇ each
  • PCSK9 (1.9 ⁇ ) 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.
  • FIG. 4 Ab-3D5 epitope mapping.
  • 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,
  • LDLR by biolayer interferometry.
  • 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.
  • 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 (HiLoadTM 16/60 SuperdexTM 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.
  • FIG. 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 ⁇ -Cat CT; lanes 9-12).
  • FIG. 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.
  • M r relative molecular masses
  • FIG. 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.
  • FIG. 12 Cleaved PCSK9 forms degrade LDLR in mouse liver.
  • PCSK9 or with furin-cleaved PCSK9 (PCSK9c_fu).
  • PCSK9c_fu furin-cleaved PCSK9
  • 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 ⁇ g PCSK9 for 1 hour.
  • 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.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • Bind 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).
  • 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.
  • HVRs hypervariable regions
  • an antibody that binds to PCSK9 refers 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.
  • the extent of binding of an anti-PCSK9 antibody to an unrelated, non-PCSK9 protein is less than (RIA).
  • an antibody that binds to PCSK9 has a dissociation constant (Kd) of ⁇ ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 "8 M
  • an anti-PCSK9 antibody binds to an epitope of PCSK9 that is conserved among PCSK9 from different species.
  • 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.
  • 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.
  • 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.
  • 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.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • cleaved PCSK9 or "cPCSK9” refers to a species of PCSK9 that has been cleaved at Arg218-Gln219.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not
  • 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.
  • radioactive isotopes e.g., At , 1 , 1 , Y , Re , Re , Sm , Bi , P , adriamicin, vinca alkaloids (vincristine, vinblastine,
  • Antibody 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 refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • 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.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • 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.
  • FR 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.
  • 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.
  • host cell refers 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.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH
  • 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.
  • 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.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops").
  • 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.
  • CDRs complementarity determining regions
  • 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).
  • 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.
  • HVR residues and other residues in the variable domain 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.
  • 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).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • 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.
  • 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).
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • 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.
  • monoclonal antibody 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.
  • polyclonal antibody preparations 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.
  • 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.
  • 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.
  • 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.
  • 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 variable heavy domain
  • CHI heavy chain variable domain
  • CL constant light domain
  • ⁇ variable light domain
  • ⁇ lambda
  • 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.
  • % 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.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • 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.
  • pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • PCSK9 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.
  • PCSK9 activity or "biological activity” of PCSK9, as used herein, includes any biological effect of PCSK9.
  • PCSK9 activity includes the ability of PCSK9 to interact or bind to a substrate or receptor.
  • the biological activity of PCSK9 is the ability of PCSK9 to bind to a LDL-receptor (LDLR).
  • LDLR LDL-receptor
  • PCSK9 binds to and catalyzes a reaction involving LDLR.
  • PCSK9 activity includes the ability of PCSK9 to decrease or reduce the availability of LDLR.
  • the biological activity of PCSK9 includes the ability of PCSK9 to increase the amount of LDL in a subject.
  • 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.
  • 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.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • variable region 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).
  • FRs conserved framework regions
  • HVRs hypervariable regions
  • 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.
  • vector 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.”
  • 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.
  • 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.
  • 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.
  • 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.
  • HVR-H1 comprising the amino acid NO:2
  • HVR-H3 comprising the amino acid sequence of SEQ ID NO:3
  • HVR-Ll comprising the amino acid sequence of SEQ ID NO: 8
  • HVR-L2 comprising the amino acid sequence of SEQ ID NO:9
  • HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.
  • 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.
  • the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:3.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HVR-H2 comprising the amino acid sequence of SEQ ID NO:2
  • HVR-H3 comprising the amino acid sequence of SEQ ID NO:3
  • HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8
  • HVR-L2 comprising the amino acid sequence of SEQ ID NO:9
  • HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 10.
  • an anti-PCSK9 antibody is humanized.
  • 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
  • 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.
  • VH heavy chain variable domain
  • 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.
  • the anti-PCSK9 antibody comprises the VH sequence in SEQ ID NO:4, including post-translational modifications of that sequence.
  • 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.
  • an anti-PCSK9 antibody comprising 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.
  • VL light chain variable domain
  • 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.
  • the substitutions, insertions, or antibody comprises the VL sequence in SEQ ID NO: l 1, including post-translational modifications of that sequence.
  • 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.
  • an anti-PCSK9 antibody comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • 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.
  • the invention provides an antibody that binds to the same epitope as an anti-PC SK9 antibody provided herein.
  • 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.
  • 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.
  • an anti-PC SK9 antibody is a monoclonal antibody, including a chimeric, humanized or human antibody.
  • an anti-PCSK9 antibody is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab') 2 fragment.
  • the antibody is a full length antibody, e.g., an intact IgGl antibody or other antibody class or isotype as defined herein.
  • an anti-PC SK9 antibody may incorporate any of the features, singly or in combination, as described in Sections 1-7 below:
  • an antibody provided herein has a dissociation constant (Kd) of ⁇ ⁇ , ⁇ ⁇ ⁇ , ⁇ ⁇ ⁇ , ⁇ 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).
  • Kd dissociation constant
  • 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.
  • RIA radiolabeled antigen binding 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)).
  • MICROTITER multi-well plates (Thermo Scientific) are coated overnight with 5 ⁇ g/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).
  • a capturing anti-Fab antibody Cappel Labs
  • bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C).
  • 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 ⁇ /well of scintillant
  • 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).
  • 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.
  • EDC N-ethyl- N'- (3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/ml (-0.2 ⁇ ) before injection at a flow rate of 5 ⁇ /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.
  • association rates (k on ) and dissociation rates (k 0 ff) 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 k 0 ff/k on See, e.g., Chen et al, J. Mol. Biol. 293:865-881 (1999).
  • 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-AMINCOTM spectrophotometer (ThermoSpectronic) with a stirred cuvette.
  • a fluorescent quenching technique that measures the nm, 16 nm band-pass
  • Fab form 20 iiM anti-antigen antibody
  • a spectrometer such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCOTM spectrophotometer (ThermoSpectronic) with a stirred cuvette.
  • 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.
  • Fab fragment antigen binding protein
  • Fab' fragment antigen binding protein
  • Fab'-SH fragment antigen binding protein
  • Fv fragment antigen binding protein
  • scFv fragments fragment antigen binding fragment antigen binding protein fragments
  • 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.
  • 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.
  • recombinant host cells e.g. E. coli or phage
  • 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)).
  • 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.
  • a chimeric antibody is a "class switched" antibody in which the class or subclass has been fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • 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.
  • FJVRs e.g., CDRs, (or portions thereof) are derived from a non-human antibody
  • FRs or portions thereof
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • 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.
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • 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,
  • 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.
  • endogenous immunoglobulin loci have generally been inactivated.
  • 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).
  • 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
  • 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.
  • scFv single-chain Fv
  • Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
  • 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).
  • 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.
  • 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.
  • bispecific antibodies may bind to two different epitopes of PCSK9.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody
  • 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 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.
  • 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)
  • SDRs a-CDRs
  • 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.
  • 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.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR "hotspots" or SDRs.
  • 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.
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • 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.
  • 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.
  • ADEPT enzyme
  • 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.
  • 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.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • 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
  • 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.
  • antibody variants examples include 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
  • 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.
  • 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.
  • 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.
  • 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.
  • non-radioactive assays methods may be employed (see, for example, ACTITM 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.
  • 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.
  • 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).
  • 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).
  • 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
  • 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.
  • CDC Complement Dependent Cytotoxicity
  • Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286,
  • cysteine engineered antibodies e.g., "thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • 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.
  • 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.
  • 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.
  • 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),
  • PEG poly
  • 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.
  • conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided.
  • 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.
  • Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567.
  • 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).
  • one or more vectors e.g., expression vectors
  • a host cell comprising such nucleic acid is provided.
  • 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.
  • the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell).
  • a method of making an anti-PCSK9 antibody 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).
  • nucleic acid encoding an antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • 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.
  • 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.
  • 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 PLANTIBODIESTM technology for producing antibodies in transgenic plants).
  • Vertebrate cells may also be used as hosts.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the test antigen binding protein is present in excess.
  • Antigen binding proteins identified by competition assay 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.
  • a competing antigen binding protein when 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.
  • competition assays may be used to identify an antibody that competes with anti-PCSK9 antibody 3D5 for binding to PCSK9.
  • 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).
  • 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.
  • 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.
  • the application provides a method for determining differential binding of an anti-PCSK9 antibody to different forms of PCSK9, e.g., intact vs. cleaved.
  • 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.
  • 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.
  • 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.
  • a protease such as, for example, furin or hepsin
  • 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.
  • the methods may involve the use of control samples, such as, for example, molecular weight standards or characterized samples of PCSK9.
  • 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.
  • an anti-PCSK9 antibody binds human PCSK9 and prevents interaction with the LDLR.
  • 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).
  • 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.
  • 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.
  • 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.
  • ADC antibody-drug conjugate
  • 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.
  • an enzymatically active toxin or fragment thereof including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain
  • an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.
  • a radioactive atom to form a radioconjugate.
  • isotopes are available for the production of radioconjugates. Examples include At , 1 , 1 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • 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.
  • NMR nuclear magnetic resonance
  • 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
  • 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.
  • an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res.
  • 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).
  • cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB
  • 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.
  • a biological sample is blood, serum or other liquid samples of biological origin.
  • a biological sample comprises a cell or tissue.
  • an anti-PCSK9 antibody for use in a method of diagnosis or detection.
  • a method of detecting the presence of PCSK9 in a biological sample comprises detecting the presence of PCSK9 protein in a biological sample.
  • PCSK9 is human PCSK9.
  • 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.
  • 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
  • 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.
  • CVD cardiovascular disease
  • 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.
  • labeled anti-PCSK9 antibodies 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
  • Exemplary labels include, but are not limited to, the radioisotopes P, 14 C, 125 1, 3 H, and 131 I, 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.
  • luciferin 2,3-dihydrophthalazinediones
  • horseradish peroxidase HRP
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -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
  • HRP horseradish peroxidase
  • lactoperoxidase or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
  • the application provides a method for determing the amount of cleaved vs. intact PCSK9 in a sample.
  • 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.
  • 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.
  • the sample is biological sample from a subject, such as a mammalian subject.
  • 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.
  • 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.
  • 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
  • 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.
  • the sample is a biological sample from a subject, such as a mammalian subject.
  • the sample is a blood, serum or other sample from a human subject.
  • the furin cleavage sequence of PCSK9 215 RFHR-Q 219 (SEQ ID NO:38) harbors three naturally occurring gain-of-function mutations (see e.g., S. Benjannet, et al. .
  • 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.
  • compositions 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).
  • 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
  • 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.).
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • 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.
  • 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.
  • a statin such as, for example, atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and any combination thereof.
  • 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,
  • 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.
  • an anti-PCSK9 antibody for use as a medicament is provided.
  • an anti-PCSK9 antibody for use in treating conditions associated with a cholesterol related disorder is provided.
  • an anti-PC SK9 antibody for use in treating conditions associated with an elevated level of LDL-cholesterol is provided.
  • an anti-PCSK9 antibody for use in a method of treatment is provided.
  • 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.
  • 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.
  • the invention provides an anti-PCSK9 antibody for use in reducing LDL- cholesterol level in a subject.
  • the invention provides an anti-PCSK9 antibody for use in lowering serum LDL-cholesterol level in a subject.
  • 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-
  • 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.
  • 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.
  • 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.
  • the invention provides for the use of an anti-PC SK9 antibody in the manufacture or preparation of a medicament.
  • the medicament is for treatment of a cholesterol related disorder.
  • the cholesterol related disorder is hypercholesterolemia.
  • the medicament is for use in a method of treating hypercholesterolemia comprising administering to an individual having hypercholesterolemia an effective amount of the medicament.
  • the disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of PCSK9 activity.
  • diseases or disorders that are generally addressable (either treatable or preventable) through the use of statins can also be treated.
  • 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.
  • 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
  • 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.
  • 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.
  • the additional therapeutic agent is for preventing and/or treating atherosclerosis and/or cardiovascular diseases.
  • the additional therapeutic agent is for use in a method of reducing the risk of recurrent cardiovascular events.
  • the additional therapeutic agent is for elevating the level of HDL- cholesterol in a subject.
  • 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.
  • 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.
  • an antibody of the invention may be co-administered with at least one additional therapeutic agent.
  • additional therapeutic agent elevates the level of LDLR.
  • 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 ® .
  • an additional therapeutic agent is a HDL-cholesterol raising drugs.
  • 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 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.
  • 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.
  • about 1 ⁇ g/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 ⁇ g/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.
  • 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.
  • a flat-fixed dosing regimen is used to administer anti-PCSK9 antibody to an individual.
  • 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.
  • 150 mg, 300 mg, or 600 mg of anti-PCSK9 antibody is administered to an individual.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • 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.
  • 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.
  • 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.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as phosphat
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the purified composition of cleaved PCSK9 was obtained from a composition of PCSK9 that was initially expressed as a full-length PCSK9 protein.
  • the purified composition of cleaved PCSK9 is obtained from a composition of cleaved intact (e.g., full-length) PCSK9.
  • 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.
  • 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).
  • 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.
  • GYTFTDYYMN (SEQ IDNO: 1) 3D5 Heavy Chain CDR2
  • ATNWVFAY (SEQ ID NO: 3)
  • Example 1 The serine protease hepsin cleaves PCSK9 at the 'furin cleavage site'
  • 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)).
  • 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 215 RFHR-Q 219 (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.
  • 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).
  • the heavy chain and light chain variable region sequences for antibody 3D5 are shown in Fig. 6.
  • 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 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.
  • PCSK9c_fu 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.
  • 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 ( ⁇ -Cat CT) had a mass of -52,800 Da and started at Gln219 (Fig. 9).
  • the small fragment was the N-fragment (Serl53-Gln219).
  • 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 LDLR degradation by cleaved PCSK9 was measured in two systems, in a cellular HepG2 assay and in a mouse model of liver LDLR degradation.
  • PCSK9- wt reduced surface LDLR levels in a concentration-dependent fashion with a half-maximal reduction at 3.7-11 ⁇ (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 ⁇ g/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.
  • LDLR degradation model Liver LDLR levels were determined by quantitative immunoblotting after mice were injected with increasing doses of intact or cleaved PCSK9.
  • PCSK9c_hep and PCSK9c_fu a similar degree of inhibition was achieved at the 15-45 ⁇ g doses and 4545 ⁇ g dose, respectively, indicating about 2-3-fold reduced potencies of the cleaved forms compared to PCSK9-wt (Fig. 12).
  • 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).
  • 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.
  • 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.).
  • cDNAs 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
  • 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
  • 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% C0 2 incubator.
  • PCSK9 (50 mM Tris pH 8.0 and 150 mM NaCl) supplemented with 4 mM CaCl 2 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 CaCl 2 , or with hepsin, hepatocyte growth factor activator, matriptase in PCSK9 sample without CaCl 2 , for 6 h at 20°C. Neutralizing of hepsin could be
  • Inhibition assays with Ab-3D5 or Ab-7G7 was performed by incubating PCSK9 (1.9 ⁇ ) with 3 ⁇ 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 CaCl 2 for 6 h.
  • PCSK9 in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 mM CaCl 2 was treated with 80 nM furin for 20 h or with 40 nM hepsin for 6 h at room temperature.
  • Ab-3D5 (IgG) and anti-furin antibody (IgG) H-220, Santa Cruz Biotechnology
  • Ab-3D5 (IgG) and the hepsin antibody Ab25 (IgG) were added to the hepsin-treated samples to remove intact PCSK9 and hepsin, respectively.
  • PCSK9c_hep and PCSK9c_fu were pooled and stored at -80°C for subsequent functional studies, analysis by N-terminal sequencing and by mass spectrometry.
  • Antibodies 50 ⁇ g/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 CaCl 2 , 1 mg/ml BSA, 0.1%
  • 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.
  • 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;
  • HepG2 cells ATCC; Manassas, VA) were seeded into 48-well plates (Corning;
  • DMEM high glucose medium
  • Gibco Gibco; Grand Island, NY
  • LPDS penicillin/streptomycin
  • LPDS 10% FBS lipoprotein deficient serum
  • 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 ⁇ / ⁇ 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 ⁇ g/ml PCSK9-wt without or with 0.5 ⁇ Ab-3D5 were pre-incubated for 30 min and added to cells for 4 h at 37°C; or (iii) 15 ⁇ / ⁇ 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.
  • Biotin-LDLR biotinylated LDLR ectodomain
  • Biotin-LDLR 15 ⁇ g/ml was immobilized on the streptavidin biosensor and immersed into mixtures of 250 nM human PCSK9 pre-incubated for 30 min with 1 ⁇ Ab-3D5 or Ab-7G7 in Tris buffer. Steady-state binding values were determined and the results were expressed as percentage of the
  • the binding activity of furin-cleaved or hepsin-cleaved PCSK9 protein to LDLR was measured by a competitive ELISA. Briefly, 1 ⁇ g/mL of recombinant human LDLR
  • mice 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 ⁇ g of PCSK9 in PBS. In another experiment, mice were injected i.v.
  • 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.
  • TBS-T 10 mM Tris, pH 8.0, 150 mM NaCl, 0.

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Abstract

L'invention concerne des anticorps anti-PCSK9 qui se lient à un site de clivage de PCSK9 et leurs procédés d'utilisation.
PCT/US2013/031722 2012-03-29 2013-03-14 Anticorps qui se lient à un site de clivage de pcsk9 et leurs procédés d'utilisation WO2013148284A1 (fr)

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