WO2017063593A1 - Anti-pcsk9 antibodies and uses thereof - Google Patents

Anti-pcsk9 antibodies and uses thereof Download PDF

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Publication number
WO2017063593A1
WO2017063593A1 PCT/CN2016/102209 CN2016102209W WO2017063593A1 WO 2017063593 A1 WO2017063593 A1 WO 2017063593A1 CN 2016102209 W CN2016102209 W CN 2016102209W WO 2017063593 A1 WO2017063593 A1 WO 2017063593A1
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Prior art keywords
seq
pcsk9
antigen
polypeptide sequence
binding fragment
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PCT/CN2016/102209
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French (fr)
Inventor
Lile LIU
Jing Gao
Xinxiu YANG
Zhiqiang Xu
Shaoping HU
Lini HUANG
Hongzhuan GU
Yu Zhang
Yangyang ZHAI
Tatchi Teddy YANG
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Harbour Biomed Limited
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Priority to CN201680060612.7A priority Critical patent/CN108350085B/en
Priority to JP2018519721A priority patent/JP6700387B2/en
Priority to US15/768,032 priority patent/US10858446B2/en
Priority to EP16854969.9A priority patent/EP3362482A4/en
Publication of WO2017063593A1 publication Critical patent/WO2017063593A1/en
Priority to HK18114467.5A priority patent/HK1255331A1/en

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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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name "Sequence Listing File", creation date of September 19, 2016 and having a size of about 76.7 kB.
  • the sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.
  • the invention relates to monoclonal anti-PCSK9 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies.
  • Methods of making the antibodies, and methods of using the antibodies to treat diseases including lipid disorders, metabolic diseases, hypercholesterolemia, inflammatory diseases and infectious diseases are also provided.
  • PCSK9 proprotein convertase subtilisin/kexin type 9a
  • PCSK9 Loss-of-function mutations in PCSK9 have been associated with a reduction of LDL-C levels and cardiovascular disease, while humans with high levels of PCSK9 have elevated levels of LDL-C and significantly enhanced risk of cardiovascular disease.
  • Gain-of-function mutants of PCSK9 are causatively associated with familial hypercholesterolaemia (Gencer et al., 2015, Swiss Med Wkly. 145:wl4094).In vitro experiments have shown that PCSK9 can reduce the expression level of LDLR on HepG2 cells. Similarly, an increase in PCSK9 levels in mice has reduced LDLR expression in the liver, while PCSK9 knockout mice have elevated LDLR expression.
  • PCSK9 is a promising therapeutic target in the management of lipid disorders.
  • Antibodies against PCSK9 are considered to be the greatest advance in cardiovascular disease prevention since statins, and it is thought that they can be used as therapeutics for treating, e.g., high cholesterol, hyperlipidemia, coronary heart disease, metabolic syndrome and acute coronary artery syndrome.
  • the invention satisfies this need by providing monoclonal antibodies that specifically bind PCSK9 with high affinity, induce LDL uptake by hepatocytes, and prevent LDLR degradation in vivo.
  • the fully human anti-PCSK9 antibodies of the invention have a higher affinity to PCSK9 than Alirocumab (Regeneron).
  • the invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that bind PCSK9.
  • the invention relates to an isolated monoclonal antibody or antigen- binding fragment thereof comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of:
  • the antibody or antigen-binding fragment thereof binds PCSK9specifically, preferably binds specifically to human PCSK9.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising:
  • the antibody or antigen-binding fragment thereof binds PCSK9, preferably binds specifically to human PCSK9.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region having a polypeptide sequence at least 85%, preferably at least 90%, more preferably at least 95% identical to one of SEQ ID NOs: 1, 9, 17, 25, 33 or 91- 108, or 41, or a light chain variable region having a polypeptide sequence at least 85%, preferably at least 90%, more preferably at least 95% identical to one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45.
  • the isolated monoclonal antibody or antigen-binding fragment thereof of the invention is human/rat chimeric.
  • the isolated monoclonal antibody or antigen-binding fragment thereof of the invention is human.
  • the isolated monoclonal antibody or antigen-binding fragment thereof of the invention further comprises a constant region, preferably a human heavy chain IgGl constant region, and a human antibody light chain kappa constant region.
  • the invention relates to an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the invention in another general aspect, relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the invention in another general aspect, relates to a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the invention in another general aspect, relates to a pharmaceutical composition, comprising an isolated monoclonal antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.
  • the invention relates to a method of blocking the binding of PCSK9 to LDLR, or a method of augmenting uptake of LDL, in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.
  • the invention in another general aspect, relates to a method of treating a disease, disorder or condition, preferably a lipid disorder, a metabolic disease, an inflammatory disease or an infectious disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.
  • the invention in another general aspect, relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment under conditions to produce the monoclonal antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture.
  • the invention in another general aspect, relates to a method of producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • Figure 1 shows effect of different batches of Immunogen A (hPCSK9-His) on LDL uptake inhibition in HepG2 cells;
  • Figure 2 shows the binding activity of chimeric anti-PCSK9 antibodies according to embodiments of the invention to Immunogen A (hPCSK9-His), as measured by ELISA;
  • Figure 3 shows the binding activity of chimeric anti-PCSK9 antibodies according to embodiments of the invention to recombinant human PCSK9 D374Y protein, as measured by ELISA;
  • Figure 4 shows the binding activity of chimeric anti-PCSK9 antibodies according to embodiments of the invention to recombinant cyno monkey PCSK9 protein, as measured by ELISA;
  • Figure 5 shows the inhibition of binding of recombinant PCSK9 protein to LDLR ECD protein by chimeric anti-PCSK9 antibodies according to embodiments of the invention, as measured by ELISA;
  • Figure 6 shows the inhibition of binding of recombinant PCSK9 D374Y protein to LDLR ECD protein by chimeric anti-PCSK9 antibodies according to embodiments of the invention, as measured by ELISA;
  • Figure 7 shows the inhibition of PCSK9 D374Y -induced down regulation of LDLR by chimeric anti-
  • PCSK9 antibodies according to embodiments of the invention, as determined by FACS;
  • Figure 8 shows the inhibition ofPCSK9 D374Y -induced LDL uptake in HepG2 cells by chimeric anti- PCSK9 antibodies according to embodiments of the invention
  • Figure 9 shows the binding activity of fully human anti-PCSK9 antibodies according to embodiments of the invention to Immunogen A (hPCSK9-His), as measured by ELISA;
  • Figure 10 shows the binding activity of fully human anti-PCSK9 antibodies according to embodiments of the invention to recombinant PCSK9 D374Y protein, as measured by ELISA;
  • Figure 11 shows the binding activity of fully human anti-PCSK9 antibodies according to embodiments of the invention to cyno monkey PCSK9, as measured by ELISA;
  • Figure 12 shows the inhibition of binding of recombinant PCSK9 protein to LDLR ECD protein by fully human anti-PCSK9 antibodies according to embodiments of the invention, as measured by ELISA;
  • Figure 13 shows the inhibition of binding of recombinant PCSK9 D374Y protein to LDLR ECD protein by fully human anti-PCSK9 antibodies according to embodiments of the invention, as measured by ELISA;
  • Figure 14 shows the inhibition of PCSK9 D374Y -induced LDL uptake in HepG2 cells by a fully human anti-PCSK9 antibody according to embodiments of the invention
  • Figure 15 shows the inhibition of PCSK9 D374Y -induced LDL uptake in HepG2 cells by a fully human anti-PCSK9 antibody according to embodiments of the invention
  • Figure 16 shows the inhibition of PCSK9 D374Y -induced LDL uptake in HepG2 cells by a fully human anti-PCSK9 antibody according to embodiments of the invention
  • Figure 17 shows the binding activity of a fully human anti-PCSK9 antibody according to embodiments of the invention to Immunogen A, as measured by ELISA;
  • Figure 18 shows the binding activity of a fully human anti-PCSK9 antibody according to embodiments of the invention to recombinant PCSK9 D374Y protein, as measured by ELISA;
  • Figure 19 shows the binding activity of a fully human anti-PCSK9 antibody according to embodiments of the invention to cyno monkey PCSK9, as measured by ELISA;
  • Figure 20 shows the inhibition of binding of recombinant PCSK9 protein to LDLR ECD protein by a fully human anti-PCSK9 antibody according to embodiments of the invention, as measured by ELISA;
  • Figure 21 shows the inhibition of binding of recombinant PCSK9 D374Y to LDLR ECD protein by a fully human anti-PCSK9 antibody according to embodiments of the invention, as measured by ELISA;
  • Figure 22 shows the inhibition of PCSK9 D374Y -induced LDL uptake in HepG2 cells by a fully human anti-PCSK9 antibody according to embodiments of the invention, as measured by ELISA;
  • FIGS 23 A &B show the inhibition of recombinant hPCSK9 protein-mediated LDLR degradation in mice by anti-PCSK9 antibodies according to embodiments of the invention: liver lysates from mice administered with recombinant human PCSK9 protein (hPCSK9) and anti-PCSK9 antibodies, control IgG or vehicle were subject to Western blot analysis using an antibody against mouse LDLR (mLDLR) and an antibody against mouse ⁇ -actin ( ⁇ -actin), the ratio of the density of the mLDLRband v.s. the density of the ⁇ -actinband on the Western Blot was plotted:
  • mLDLR mouse LDLR
  • ⁇ -actin mouse ⁇ -actin
  • Fig. 23 A the ratio of mLDLR/m -actin in mice administered with 30mg/kg, 3mg/kg, or 0.3mg/kg of 139G1C8, control IgG or vehicle;
  • Fig. 23 B the ratio of mLDLR/m -actin in mice administered with 30mg/kg, 3mg/kg, or 0.3mg/kg of 96F8C6, control IgG or vehicle;
  • FIG 24 shows the thermostability of fully human anti-PCSK9 antibodies according to embodiments of the invention, as measured by Differential Scanning Calorimetry (DSC);
  • Figure 25 shows the freeze/thaw stability of fully human anti-PCSK9 antibodies according to embodiments of the invention.
  • Figure 26 shows the solubility of fully human anti-PCSK9 antibodies according to embodiments of the invention.
  • Figure 27 A&B show the inhibition of PCSK9 D374Y -induced LDL uptake in HepG2 cells by various human anti-PCSK9 antibodies according to embodiments of the invention having improved affinity according to embodiments of the invention:
  • Figure 27 A inhibition of PCSK9 D374Y -induced LDL uptake in HepG2 cells by antibody clone AF-mab023_06, AF-mab023_12, or 96F8C6;
  • Figure 27 B inhibition of PCSK9 D374Y -induced LDL uptake in HepG2 cells by antibody clone AF-mab023_01, AF-mab023_l l, AF-mab023_14, or AF-mab023_18.
  • any numerical value such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term "about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • the invention generally relates to isolated anti-PCSK9 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies.
  • Methods of making the antibodies, and methods of using the antibodies to treat diseases including lipid disorders, metabolic diseases, hypercholesterolemia, inflammatory diseases and infectious diseases are also provided.
  • the antibodies of the invention possess one or more desirable functional properties, including but not limited to high- affinity binding to PCSK9, high specificity to PCSK9, the ability to block the binding of PCSK9 to LDLR, and the ability to stimulate uptake of LDL and prevent the degradation of LDLR induced by PCSK9.
  • the invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that bind PCSK9.
  • antibody is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.
  • the antibodies of the invention can be of any of the five major classes or corresponding sub-classes.
  • the antibodies of the invention are IgGl, IgG2, IgG3 or IgG4.
  • Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains.
  • the antibodies of the invention can contain a kappa or lambda light chain constant domain.
  • the antibodies of the invention include heavy and/or light chain constant regions from rat or human antibodies.
  • antibodies contain an antigen-binding region that is made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., CDR1, CDR2, and CDR3).
  • the light chain variable region domains are alternatively referred to as LCDR1, LCDR2 and LCRD3
  • the heavy chain variable region domains are alternatively referred to as HCDR1, HCRD2 and HCDR3.
  • an "isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to PCSK9 is substantially free of antibodies that do not bind to PCSK9). In addition, an isolated antibody is substantially free of other cellular material and/or chemicals.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the monoclonal antibodies of the invention can be made by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods.
  • the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.
  • the term "antigen-binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv 1 ), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab) an scFv dimer (bivalent diabody), a multi specific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure.
  • an antibody fragment such as, for example, a diabody, a Fab
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds.
  • the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the constant region of the heavy chain.
  • the antigen-binding fragment comprises Fab and F(ab').
  • single-chain antibody refers to a conventional single-chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids.
  • single domain antibody refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.
  • human antibody refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide.
  • humanized antibody refers to a non-human antibody that is modified to increase the sequence homology to that of a human antibody, such that the antigen-binding properties of the antibody are retained, but its antigenicity in the human body is reduced.
  • chimeric antibody refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable region of both the light and heavy chains often corresponds to the variable region of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.
  • PCSK9 refers to the proprotein convertase subtilisin/kexin type 9 protein, a secretory protein that is a soluble member of the mammalian proprotein convertase family of secretory serine endoproteases. Secreted PCSK9 binds to LDLR on the surface of cells. Upon internalization of the bound proteins, PCSK9 prevents LDLR from endocytic recycling and results in lysosomal degradation of both proteins (Rallidis and Lekakis, 2016, Hellenic J Cardiol. 57(2):86-91).
  • human PCSK9 refers to a PCSK9 originated from a human. An exemplary amino acid sequence of a human PCSK9 is represented in GenBank Accession No.
  • an antibody that "specifically binds to PCSK9” refers to an antibody that binds to a PCSK9, preferably a human PCSK9, with a KD of 1 ⁇ 10 ⁇ 7 M or less, preferably 1 ⁇ 10 ⁇ 8 M or less, more preferably 5 10 "9 M or less, 1 10 "9 M or less, 5 ⁇ 10 "10 M or less, or 1 ⁇ 10 "10 M or less.
  • KD refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods in the art in view of the present disclosure.
  • the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as a Octet RED96 system.
  • a biosensor system e.g., a Biacore® system
  • bio-layer interferometry technology such as a Octet RED96 system.
  • the invention relates to an isolated monoclonal antibody or antigen- binding fragment thereof comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of:
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising:
  • the antibody or antigen-binding fragment thereof binds PCSK9, preferably binds specifically to human PCSK9.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, comprising a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to one of SEQ ID NOs: 1, 9, 17, 25, 33 or 91- 108,or 41 , or a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45.
  • the isolated monoclonal antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region having the polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to SEQ ID NO: 1, 9, 17, 25, 33 or 41, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to SEQ ID NO: 5, 13, 21, 29, 37 or 45, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region having the polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to one of SEQ ID NOs: 33 or 91-108, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to SEQ ID NO: 37.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, comprising:
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 6, 7, 8, 2, 3, and 4, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 1, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 5.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 5.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDRl, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 14, 15, 16, 10, 11, and 12, respectively, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 9, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 13.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 13.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDRl, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 22, 23, 24, 18, 19, and 20, respectively, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 17, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 21.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 21.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDRl, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 30, 31, 32, 26, 27, and 28, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 25, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 29.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 25; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 29.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDRl, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 38, 39, 40, 34, 35, and 36, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 33, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 37.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 33; and a light chain variable region having the polypeptide sequence of SEQ ID NO:37.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising an LCDRl having the polypeptide sequence of SEQ ID NO: 38, an LCDR2 having the polypeptide sequence of SEQ ID NO: 39, an LCDR3 having the polypeptide sequence of SEQ ID NO: 40, an HCDR1 having the polypeptide sequence of SEQ ID NO: 34, an HCDR2 having the polypeptide sequence of SEQ ID NO: 35, and an HCDR3 having the polypeptide sequence of one selected from the group consisting of SEQ ID NOs: 36 and 73-90.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to one of SEQ ID NOs: 91-108, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 37.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of one of SEQ ID NOs: 91-108; and a light chain variable region having the polypeptide sequence of SEQ ID NO:37.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 46, 47, 48, 42, 43, and 44, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to SEQ ID NO: 41, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to SEQ ID NO: 45.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 41; and a light chain variable region having the polypeptide sequence of SEQ ID NO:45.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, wherein the antibody or antigen-binding fragment thereof is chimeric.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, wherein the antibody or antigen-binding fragment thereof is human.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, comprising a constant region, preferably a human heavy chain IgGl constant region (SEQ ID NO: 67), and a human antibody light chain, preferably a human light chain kappa constant region (SEQ ID NO: 69).
  • a constant region preferably a human heavy chain IgGl constant region (SEQ ID NO: 67)
  • a human antibody light chain preferably a human light chain kappa constant region (SEQ ID NO: 69).
  • the invention in another general aspect, relates to an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the coding sequence of a protein can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding monoclonal antibodies or antigen-binding fragments thereof of the invention can be altered without changing the amino acid sequences of the proteins.
  • the invention in another general aspect, relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector.
  • the vector is a recombinant expression vector such as a plasmid.
  • the vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication.
  • the promoter can be a constitutive, inducible or repressible promoter.
  • the invention relates to a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention. Any host cell known to those skilled in the art in view of the present disclosure can be used for recombinant expression of antibodies or antigen- binding fragments thereof of the invention. In some embodiments, the host cells are E.
  • the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.
  • the invention in another general aspect, relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions to produce a monoclonal antibody or antigen-binding fragment thereof of the invention, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant).
  • Expressed antibodies or antigen-binding fragments thereof can be harvested from the cells and purified according to conventional techniques known in the art and as described herein.
  • the invention in another general aspect, relates to a pharmaceutical composition, comprising an isolated monoclonal antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.
  • carrier refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term
  • pharmaceutically acceptable carrier refers to a non-toxic material that does not interfere with the effectiveness of a composition according to the invention or the biological activity of a composition according to the invention. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in an antibody pharmaceutical composition can be used in the invention.
  • the invention relates to a method of blocking the binding of PCSK9 to LDLR, or of augmenting LDL uptake in liver in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.
  • the functional activity of antibodies and antigen-binding fragments thereof that bind PCSK9 can be characterized by methods known in the art and as described herein.
  • Methods for characterizing antibodies and antigen-binding fragments thereof that bind PCSK9 include, but are not limited to, affinity and specificity assays including Biacore, ELISA, and OctetRed analysis; receptor ligand binding assays to detect blocking of the binding of PCSK9 to LDLR; assays to detect inhibition of LDLR degradation induced by PCSK9 in HepG2 cells; assays to detect increases in LDL uptake by HepG2 cells; experiments to detect the inhibition of LDLR degradation induced by PCSK9 in liver; etc.
  • the methods for characterizing antibodies and antigen-binding fragments thereof that bind PCSK9 include those described in Examples 2-15 below.
  • the invention in another general aspect, relates to a method of treating lipid disorders, metabolic diseases, hypercholesterolemia, inflammatory diseases and infectious diseases in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.
  • the term "subject" refers to an animal, and preferably a mammal.
  • the subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig or mouse) or a primate (e.g., a monkey, chimpanzee, or human).
  • the subject is a human.
  • the pharmaceutical composition comprises a therapeutically effective amount of the anti-PCSK9 antibody or antigen-binding fragment thereof.
  • therapeutically effective amount refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject.
  • a therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.
  • a therapeutically effective amount means an amount of the anti-PCSK9 antibody or antigen-binding fragment thereof that stimulates LDL uptake in a subject in need thereof. Also as used herein with reference to anti-PCSK9 antibodies or antigen-binding fragments thereof, a therapeutically effective amount means an amount of the anti- PCSK9 antibody or antigen-binding fragment thereof that results in treatment of a disease, disorder, or condition; prevents or slows the progression of the disease, disorder, or condition; or reduces or completely alleviates symptoms associated with the metabolic disease, disorder, or condition.
  • the disease, disorder or condition to be treated is a lipid disorder, hyperlipidemia, dyslipidemia, or a metabolic disease.
  • the disease, disorder or condition to be treated is a lipid disorder, including but not limited to, primary hyperlipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia.
  • the disease, disorder or condition to be treated is an inflammatory disease, such as sepsis, or an infectious diseases.
  • a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the
  • the therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • compositions described herein are formulated to be suitable for the intended route of administration to a subject.
  • the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.
  • the terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as lipid disorder, hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia, a metabolic disease, an inflammatory disease such as sepsis, or an infectious disease.
  • “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.
  • composition used in the treatment of a lipid disease, disorder or condition or a metabolic disease, disorder or condition can be used in combination with another treatment including, but not limited to, statins or other lipid lowering drugs.
  • the term "in combination,” in the context of the administration of two or more therapies to a subject, refers to the use of more than one therapy.
  • the use of the term “in combination” does not restrict the order in which therapies are administered to a subject.
  • a first therapy e.g., a composition described herein
  • a first therapy can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject.
  • the invention in another general aspect, relates to a method of producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising combining a monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • Embodiment 1 is an isolated monoclonal antibody or antigen-binding fragment thereof comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of:
  • the antibody or antigen-binding fragment thereof binds PCSK9, preferably binds specifically to human PCSK9.
  • Embodiment 2 is an isolated monoclonal antibody or antigen-binding fragment thereof comprising:
  • an HCDR3 having the polypeptide sequence of one of SEQ ID NOs: 36 and 73-90; wherein the antibody or antigen-binding fragment thereof binds PCSK9, preferably binds specifically to human PCSK9.
  • Embodiment 3 is the isolated monoclonal antibody or antigen-binding fragment of Embodiment 1 or 2, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to one of SEQ ID NOs: 1, 9, 17, 25, 33 or 91-108,or 41, or a light chain variable region having a polypeptide sequence at least 95% identical to one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45.
  • Embodiment 4 is the isolated monoclonal antibody or antigen-binding fragment of Embodiment 3, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 1, 9, 17, 25, 33 or 41, and a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 5, 13, 21, 29, 37 or 45 respectively.
  • Embodiment 5 is the isolated monoclonal antibody or antigen-binding fragment of Embodiment 3, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to one of SEQ ID NOs: 91-108, and a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 37.
  • Embodiment 6 is the isolated monoclonal antibody or antigen-binding fragment of Embodiment 4 or 5, comprising:
  • Embodiment 7 is the isolated monoclonal antibody or antigen-binding fragment of any of
  • Embodiment 8 is isolated monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 7, wherein the antibody or antigen-binding fragment thereof is human.
  • Embodiment 9 is the isolated antibody or antigen-binding fragment of Embodiment 8, comprising a human heavy chain IgGl constant region and a human antibody light chain kappa constant region.
  • Embodiment 10 is the isolated antibody or antigen-binding fragment of any of Embodiments 1 to 9, wherein the antibody or antigen-binding fragment binds to a human PCSK9 with a K D of 5 ⁇ 10 ⁇ 9 M or less, preferably a K D of 1 ⁇ 10 ⁇ 9 M or less, wherein the K D is measured by surface plasmon resonance analysis, such as by using a Biacore system, or by a bio-layer interferometry technology, such as by using a Octet RED96 system.
  • Embodiment 11 is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 10.
  • Embodiment 12 is a vector comprising the isolated nucleic acid of Embodiment 11.
  • Embodiment 13 is a host cell comprising the nucleic acid of Embodiment 12.
  • Embodiment 14 is a pharmaceutical composition, comprising the isolated monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 10 and a pharmaceutically acceptable carrier.
  • Embodiment 15 is a method of blocking binding of PCSK9 to LDLR, or augmenting uptake of LDL in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of Embodiment 14.
  • Embodiment 16 is a method of treating a lipid disease, disorder or condition, a metabolic disease, disorder or condition, an inflammatory disease, disorder or condition, or an infectious disease, disorder or condition in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of Embodiment 14.
  • Embodiment 17 is the method of Embodiment 16, further comprising administering to the subject an additional agent for treating the lipid disorder, metabolic disease, inflammatory disease, or infectious disease in the subject in need thereof.
  • Embodiment 18 is a method of treating a lipid disorder, metabolic disease, inflammatory disease, or infectious disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of Embodiment 14.
  • Embodiment 19 is the method of Embodiment 18, wherein the lipid disorder or metabolic disease is hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, or homozygous familial
  • hypercholesterolemia or wherein the inflammatory disease is sepsis.
  • Embodiment 20 is the method of any of Embodiments 18-19, further comprising administering to the subject an additional agent for treating the lipid disorder, metabolic disease, inflammatory disease, or infectious disease in the subject in need thereof.
  • Embodiment 21 is a method of producing the monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 10, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment under conditions to produce the monoclonal antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment from the cell or cell culture.
  • Embodiment 22 is a method of producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 10, comprising combining the monoclonal antibody or antigen-binding fragment with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • Embodiment 23 is an isolated monoclonal antibody or antigen-binding fragment of any of
  • Embodiments 1 to 10 for use in treating a lipid disorder, metabolic disease, inflammatory disease, or infectious disease, such as hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia or sepsis, in a subject in need thereof.
  • Embodiment 24 is a pharmaceutical composition of Embodiment 14 for use in treating a lipid disorder, metabolic disease, inflammatory disease, or infectious disease, such as hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia, or sepsis in a subject in need thereof.
  • Embodiment 25 is an use of an isolated monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 10 for the manufacture of a medicament for treating a lipid disorder, metabolic disease, inflammatory disease, or infectious disease, such as hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia, or sepsis, in a subject in need thereof.
  • Embodiment 26 is an use of a pharmaceutical composition of Embodiment 14 for the manufacture of a medicament for treating a lipid disorder, metabolic disease, inflammatory disease, or infectious disease, such as hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial
  • hypercholesterolemia or sepsis, in a subject in need thereof.
  • Human PCSK9 protein was used as an immunogen to generate anti-PCSK9 antibodies.
  • the use of human immunoglobulin transgenic mouse technology for the development and preparation of fully human antibodies was first described by Abgenix (xeno mouse and Medarex (HuMab "mouse”); Lonberg et al., 1994, Nature 368: 856-859; Lonberg and Huszar, 1995, Internal Rev. Immunol. 13 :65-93; Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci. 764:536-546).
  • Antibodies with high affinity (K D ⁇ 1 * 10 "9 M) to PCSK9 were obtained by carrying out pilot antibody production, purification and validation.
  • the antibodies, which are specific for PCSK9, are able to block the binding of PCSK9 to LDLR.
  • the amino acid sequences of the heavy and light chain variable regions of the generated anti- PCSK9 antibodies were determined using standard molecular biology methods and are summarized in Table 1.
  • Fully human versions of the anti-PCSK9 antibodies were generated.
  • the fully human anti-PCSK9 antibodies bound to the human PCSK9 with high affinity (K D ⁇ 1 * 10 "9 M), and blocked the binding of PCSK9 to LDLR.
  • the biological activities of the anti-PCSK9 antibodies were evaluated by receptor ligand blocking assay and LDL uptake assays, in which they blocked PCSK9 binding to LDLR, enhanced LDL uptake by HepG2 cells, and prevented PCSK9-induced LDLR degradation in vivo.
  • the anti-PCSK9 antibodies can be used for the treatment of lipid disorders, metabolic diseases, hypercholesterolemia, inflammatory diseases or infectious diseases.
  • HEK293 cells (Invitrogen) were transiently transfected using polyethylenimine (PEI, Polysciences) with the plasmid and expanded in FreeStyle 293 expression medium (Invitrogen) at 37 ° C. After 4 days of expansion, the culture medium was collected and centrifuged to remove cell components. The culture supernatant contained the recombinant His- tagged PCSK9. Imidazole was added to a final concentration of lOmM to the culture supernatant, which contained the recombinant His-tagged PCSK9. The sample was passed through a 0.22 micron sterile filter and subjected to Ni-NTA affinity chromatography.
  • PEI polyethylenimine
  • FreeStyle 293 expression medium Invitrogen
  • PCSK9-His His-tagged PCSK9 protein
  • Buffer A 1 x PBS, pH 7.2 containing 20 niM Imidazole
  • Buffer B Buffer A, 0.1% (v/v) Trition X100, 0.1% (v/v) Triton XI 14
  • His-tagged PCSK9 protein was eluted from the Ni-NTA affinity chromatography column with Buffer C [1 x PBS (pH7.2), 250mM imidizole].
  • the eluate was further purified using a Superdex 200 column (GE, Healthcare) to obtain highly pure His-tagged recombinant human PCSK9 protein.
  • the sample was dialyzed with 1 x PBS (pH7.2) at 4 °C overnight. After dialysis, the purified PCSK9-His was passed through a 0.22 micron sterile filter, aliquoted and stored at -80 " C.
  • Human Ig Fc does not interact with the mouse Fc receptor, so immune responses triggered in mice by hFc-containing immunogens are weak, resulting in low efficiency generation of monoclonal antibodies.
  • Harbour H2L2 transgenic mice were generated by introducing the gene encoding the human immunoglobulin (Ig) variable region and the gene encoding the rat Ig constant region into the mouse genome, such that the mice contained a chimeric Ig comprising hV-rC, while expression of the mouse Ig was disabled (WO 2010/070263 Al).
  • Harbour H2L2 transgenic mice are able to produce comparable immune responses and antibody titers to wild type mice (e.g., Balb/c).
  • the antibody titer and specificity in serum were determined by ELISA, and the results are shown in Table 4.
  • the blank control was 1% (w/w) BSA.
  • the OD450nm values shown in Table 4 are the serum titer values from 7 days after the third boost, as determined by ELISA.
  • the serum titer after the third boost usually reached at least 1 : 10,000.
  • the antibody titer and specificity in seram were determined by ELISA and FACS analysis, and the results are shown in Table 6.
  • Table 6 shows that seram from mice immunized with PCSK9 exhibited different levels of binding to Immunogen A. The highest seram dilution was about one million. The blank control was 1% (w/w) BSA.
  • the OD450nm values shown in Table 6 are the seram titer values from 7 days after the third boost, as determined by ELISA.
  • mice with specific immune response against hPCSK9 were selected for fusion and were given a final boost by intraperitoneal injection of lOOug hPCSK9-His. Three days later, the mice were sacrificed, and their splenocytes were collected. NH 4 OH was added to the splenocyte samples to a final concentration of 1% (w/w) to lyse red blood cells in the sample. The samples were centrifuged at 1000 rpm and washed three times with DMEM culture media.
  • the viability of the splenocytes was determined, and viable splenocytes were then fused with mouse myeloma cells SP2/0 (ATCC) at a ratio of 5: 1 by the high efficiency electric fusion method (see Methods in Enzymology, Vol. 220).
  • Fused cells were re-suspended in DMEM media containing 20% FBS and lx hypoxanthine- aminopterin-thymidine (HAT) medium (w/w), and the concentration was adjusted to 10 5 cells/200uL. 200uL of fused cells were added to each well of 96-well plate, which was incubated at 37 ° C, 5% C0 2 . 14 days after cell fusion, hybridoma supernatants were collected and screened by ELISA.
  • HAT lx hypoxanthine- aminopterin-thymidine
  • Clones with an OD450nm greater than 0.5 by ELISA were expanded in a 24-well plate containing DMEM with 10% (w/w) heat-inactivated FBS at 37 ° C, 5% (v /v) C0 2 . Supernatants were collected after 3 days of culturing. The antibody isotypes were determined, and their ability to bind to recombinant PCSK9 D374Y protein (gain-of-function mutant; prepared using the method of Example 1) was determined by ELISA (see Example 3).
  • a receptor ligand binding assay was performed to determine the blocking activity of the hybridoma supernatant (see Example 4), an assay to measure degradation of LDLR was performed (see Example 5), and a blocking assay to measure PCSK9-mediated cellular LDL uptake was carried out (see Example 6).
  • the supernatants were collected and preliminarily screened by assessing their ability to bind to recombinant human PCSK9, PCSK9 D374Y , cyno monkey PCSK9 (SEQ ID NO: 63, prepared using the method of Example 1) and mouse PCSK9 (UniProtKB ID Q80W65; SEQ ID NO: 65, prepared using the method of Example 1), as determined by ELISA, or their ability to block binding of PCSK9 or PCSK9 D374Y to LDLR.
  • the antibody concentrations from the hybridoma cells were low, about 1-lOug/mL, and there was a large variation in antibody concentrations.
  • the FBS and the components of the culture medium can interfere with the analysis. Therefore, it was necessary to perform small scale antibody production and purification (1-5 mg).
  • Hybridoma cells from Example 2 were cultured in T-75 culture flasks using Hybridoma serum free medium (Invitrogen), and passaged for 3 generations. Cells were transferred to 2L culture flasks when the hybridoma cells were in good condition. 500mL of production media was added to each culture flask, and the cell density was adjusted to 10 5 cells/mL. The culture flasks were placed onto a rotary incubator at 37 ° C with a rotating speed of 3 cycles per minute. Hybridoma cells were cultured for 14 days, after which the supernatant was collected and the cells were removed. The supernatants were then filtered through 0.45 micron filtration. The culture supernatants were then ready for purification or storage at -30 ° C.
  • Monoclonal antibodies were purified by passing the hybridoma culture supernatants through 2mL Protein G columns (GE Healthcare). Protein G columns were first equilibrated with PBS buffer (pH7.2), and the hybridoma culture supernatants were then applied to the equilibrated Protein G columns with a constant flow rate of 3mL/minute. The columns were then washed with 4 volumes of PBS buffer. The anti-PCSK9 antibodies were then eluted with elution buffer (0.1M acetate buffer, pH2.5), and the UV absorbance of the eluates were monitored using a UV detector (A280 UV absorption peak). 10% of 1.0M Tris-HCL buffer was added to the eluates to neutralize the pH, and the samples were sterile-filtered by passing them through 0.22 micron filters. Sterile-filtered purified anti- PCSK9 antibodies were obtained.
  • the concentrations of purified anti-PCSK9 antibodies were determined by UV absorbance (A280/1.4), and the purity and endotoxin level (Lonza kit) were measured. The results of the analyses are shown in Table 7.
  • the purified anti-PCSK9 antibodies had endotoxin concentrations less than 1.0 EU/mg.
  • Streptavidin (Cat No. 85878, Sigma) was diluted with PBS to a final concentration of lug/mL, lOOuL were added to each well of a 96-well ELISA plate that was then sealed with a plastic film and incubated overnight at 4 ° C. The next day, the plates were washed twice with wash buffer [0.01% (v/v) Tween 20], then blocked with blocking buffer [0.01% (v/v) 0.1% BSA (w/w) Tween 20] at room temperature for 2 hours.
  • the blocking buffer was aspirated away, and lOOuL of 0.5ug/mL biotinylated immunogen A (hPCSK9-His), biotinylated PCSK9 D374Y or biotinylated cyno PCSK9 proteins (Biotin labeling kit, Invitrogen) were added to each well and incubated at 37 ° C for 1 hour. Unbound PCSK9 proteins were removed by washing the plate three times with wash buffer [0.01% (v/v) Tween 20]. 200uL of purified PCSK9 antibodies from Example 2 were added to each well and incubated for 1 hour at 37 ° C.
  • the plates were washed twice with wash buffer [0.01% (v/v) Tween 20], and 200uL HRP-conjugated secondary antibody (Sigma) was added to each well and incubated for 2 hours at 37 ° C. After the plates were washed three times with wash buffer, lOOul TMB substrate were added to each well and incubated for 30 minutes at room temperature. lOOul stop solution (0. IN HC1) were added to each well to stop the reaction. The absorbance at 450nm was measured using an ELISA plate reader (384plus SpectraMax, Molecular Device). The results are shown in Figures 2-4 and in Tables 8-10.
  • the IgG control was rat IgG.
  • Table 10 Binding activities of chimeric anti-PCSK9 mAbs to Cyno Monkey PCSK9-His, as measured by ELISA
  • Both wild type PCSK9 and its mutant PCSK9 D374Y bind to LDLR, and the mutant form of PCSK9 D374Y has higher binding affinity to LDLR than the wild type version.
  • a receptor ligand binding assay was performed to determine the ability of the anti-PCSK9 antibodies to block the binding of PCSK9 and PCSK9 D374Y to LDLR.
  • LDLR ECD -His The extracellular domain of LDLR (SEQ ID NO: 64; corresponding to Ala22-Arg788 of full-length LDLR) was cloned along with a His tag (LDLR ECD -His; prepared using the method of Example 1). Purified LDLR ECD -His was diluted with PBS to a final concentration of l.Oug/ml, and lOOuL of diluted LDLR ECD -His were added to each well of 96-well plate that was then sealed with plastic film and incubated at 4 ° C overnight.
  • the plate was washed twice with wash buffer [PBS + 0.01% (v/v) Tween 20] and incubated with blocking buffer [PBS + 0.01% (v/v) BSA +1% Tween20 (w/w)] at room temperature for 2 hours.
  • the blocking buffer was aspirated, and 50uL purified anti-PCSK9 antibodies from Example 2 were added to each well of 96-well plate. 50ul of 0.5ug/mL biotinylated recombinant hPCSK9-His or biotinylated PCSK9 D374Y protein (biotinylated using the Biotin labeling kit, Invitrogen) were added to each well, mixed, and incubated at 37 ° C for 2 hours.
  • Example 5 Assay to measure PCSK9-mediated LDLR degradation
  • Log phase HepG2 cells were digested with trypsin, centrifuged and resuspended in MEM medium (Invitrogen Cat. No. 11095-080) supplemented with 10% (w/w) fetal bovine serum. The cell density was adjusted to 3xl0 5 cells/mL, and cells were added to each well of 24-well plate. After the places were incubated for 6 hours to allow the HepG2 cells to adhere, the culture medium was aspirated and replenished with MEM medium supplemented with 10% (w/w) LPDS (Kalen Biomedical LLC, 880100-2) and incubated overnight.
  • MEM medium Invitrogen Cat. No. 11095-080
  • PCSK9 D374Y and anti-PCSK9 antibodies from Example 2 were mixed and added to the 24-well plate, which was incubated for 4 hours at 37 ° C. MEM medium supplemented with 10% LPDS was removed, and the plates were washed twice with PBS. The HepG2 cells were dissociated from the plate using Versene solution (Invitrogen) and collected. The cells were washed twice with PBS, the cell counts were determined, and the cell density was adjusted to 2 10 6 cells/mL with PBS.
  • Blocking buffer [1% (w/w) FBS PBS] was added to the cell suspension, and lOOuL of the cell suspension were added to each well of 96-well FACS plate (FACS Calibur, BD) that was incubated on ice for 15 minutes, then centrifuged. The blocking buffer was aspirated away, and lOOuL of lug/uL LDLR antibody (Progen, 61009) was added to each well and incubated on ice for 15 minutes. The plates were washed twice with FACS buffer [1% (w/w) BSA HBSS], and lOOuL Alexa-488-conjugated anti-rabbit secondary antibody (Molecular Probes, A- 11034) were added to each well and incubated on ice for 15 minutes.
  • the plates were washed three times with FACS buffer and resuspended in lOOuL FACS buffer after the final wash.
  • the Mean Fluorescence Intensity (MFI) was determined using FACS Calibur (BD), and the results are shown in Figure 7 and in Table 13.
  • the IgG control was rat IgG, and the values in the table are the mean fluorescence intensities of the cell populations.
  • the results showed that the anti-PCSK9 antibodies inhibited PCSK9 D374Y -induced LDLR degradation in HepG2 cells in a dose dependent manner.
  • Example 6 Assay to measure inhibition of PCSK9-mediated LDL uptake in HepG2 cells
  • Log phase HepG2 cells were digested with trypsin, centrifuged and resuspended in MEM medium (Invitrogen Cat. No. 11095-080) supplemented with 10% (w/w) fetal bovine serum. The cell density was adjusted to 5xl0 5 cells/mL, and cells were added to a PDL-coated (Millipore, A-003-E) 96-well plate. After the places were incubated for 6 hours to allow the HepG2 cells to adhere, the culture medium was aspirated and replenished with MEM medium supplemented with 10% (w/w) LPDS (Kalen Biomedical LLC, 880100-2) and incubated overnight.
  • MEM medium Invitrogen Cat. No. 11095-080
  • Example 7 Determination of the amino acid sequences in the variable regions of the anti- PCSK9 antibodies
  • RNA isolation After the supernatants from the hybridoma subclones obtained from Example 2 were characterized (i.e., validation and determination of bioactivity, Examples 3-6), 5xl0 7 hybridoma cells were collected by centrifugation. lmL Trizol was added to the cell pellets, mixed and transferred to 1.5mL centrifuge tubes, and incubated at room temperature for 5 minutes. 0.2mL chloroform was added to the samples and vortexed for 15 seconds. After standing for 2 minutes, the mixtures were centrifuged at 12000g at 4°C for 5 minutes.
  • Reverse transcription and PCR lug of RNA and reverse transcriptase were added to a reaction mixture of a final volume of 20uL, and the mixture was incubated at 42°C for 60 minutes and then at 70°C for 10 minutes to terminate the reaction.
  • a 50uL PCR reaction mixture was prepared, containing luL cDNA, 25 pmol of each primer, luL DNA polymerase, 250umol dNTPs, and the buffer system.
  • the PCR program settings were as follows: denaturation at 95°C for 3 minutes, 35 cycles of denaturation (95°C for 30 seconds), annealing (55°C for 30 seconds) and elongation (72°C for 35 seconds), followed by a final extension at 72°C for 5 minutes to obtain the PCR product.
  • the commercially available reverse transcription kit used was PrimeScript RT Master Mix (Takara, RR036), and the commercially available Q5 ultra-fidelity polymerase PCR kit was from NEB (M0492).
  • Cloning and sequencing 5uL PCR products were examined by agarose gel electrophoresis, and the samples were recovered from the agarose gel using NucleoSpin Gel & PCR Clean-up kit (MACHEREY-NAGEL, 740609).
  • Ligation reaction To a 50ng sample, 50ng T vector, 0.5uL ligase, and luL buffer were added and brought to a final volume of lOuL with water. The reaction mixture was incubated at 16°C for 30 minutes using the T4 DNA Ligase (NEB, M0402).
  • 5uL ligation product was added to lOOuL competent cells (Ecos lOlcompetent cells, Yeastern, FYE607), which were incubated on ice for 5 minutes, heat shocked at 42°C for 1 minute and incubated on ice again for 1 minute.
  • Cells were recovered by adding 650uL SOC medium without antibiotics and incubating at 37°C in a shaking incubator for 30 minutes at 200 rpm. 200uL of each bacterial culture was spread onto an LB agar plate containing antibiotics at 37°C overnight. The next day, PCR reactions were set up using the T vector primers M13F and M13R.
  • Example 8 Conversion, expression and purification of fully human anti-PCSK9 antibody [00192]
  • Step 1 Plasmid construction and preparation: Sequences of the anti-PCSK9 antibody heavy and light chain variable regions were obtained according to Example 7. The anti-PCSK9 antibodies' heavy chain variable region sequences were subcloned into expression vectors containing a signal peptide and a human heavy chain IgGl constant region. The anti-PCSK9 antibodies' light chain variable region sequences were subcloned into expression vectors containing a signal peptide and a human antibody light chain kappa constant region. The recombinant plasmids were verified and confirmed by sequencing (the sequencing method was the same as in Example 7).
  • Alkaline lysis was performed using a reagent kit (MACHEREY-NAGEL) to improve the purity and quality of the recombinant plasmids, and the plasmids were filtered through 0.22uM filters (Millpore). The purified plasmids were used for transfection.
  • a reagent kit (MACHEREY-NAGEL) to improve the purity and quality of the recombinant plasmids, and the plasmids were filtered through 0.22uM filters (Millpore). The purified plasmids were used for transfection.
  • Step 2 HEK293E cells (Invitrogen) were cultured in FreeStyle 293 medium (Invitrogen) at 37 ° C, 130RPM, 8% C0 2 (v/v). HEK293E cells were adjusted to l-1.5xl0 6 /mL cell density for transfection. 10% (v/ v) F68 (Invitrogen) was added to the FreeStyle 293 medium to a final concentration of 0.1% (v/ v), as Medium A. 5mL of Medium A and 200ug/mL PEI (Sigma) were mixed to generate Medium B. 5mL of Medium A and lOOug/mL recombinant plasmid from step 1 were mixed to generate Medium C.
  • HEK293E cells were incubated overnight while shaking. The next day, peptone was added to a final concentration of 0.5% (w/v). On about day 5-7, the antibodies' titers were determined. On about day 6-7, the HEK293E cultures were centrifuged (30 minutes, 3500RPM), and the supernatants were collected and filtered through 0.22uM filters for purification.
  • Step 3 Antibody purification: Protein A columns (GE) were washed with 0.1M NaOH for 30 minutes or with 5 bed volumes of 0.5M NaOH to get rid of endotoxin. Columns that had not been used in a long time were soaked in 1M NaOH for at least 1 hour, washed with endotoxin-free water to a neutral pH, and washed with 10 bed volumes of 1% Triton X100. The columns were then equilibrated with 5 bed volumes of PBS (PBS phosphate buffer, pH7.2). The filtered supernatants from Step 2 were loaded onto the columns, and the flow through was collected, if necessary.
  • PBS PBS phosphate buffer, pH7.2
  • the columns were washed with 5 bed volume of PBS and then eluted with 5 bed volumes of 0.1M Glycine-HCl pH3.0.
  • the eluate containing anti-PCSK9 antibody was neutralized with 0.5 bed volume of 1M Tris-HCl (NaCl 1.5M) pH8.5.
  • the human anti-PCSK9 antibodies were dialyzed in IX PBS for 4 hours, to avoid endotoxin contamination. After dialysis, anti-PCSK9 antibodies' concentrations were determined by
  • Receptor ligand binding assays confirmed that fully human anti-PCSK9 antibodies, generated by conversion of the chimeric anti-PCSK9 antibodies, are able to block the binding of PCSK9 to LDLR.
  • the IgG control was human IgG.
  • Fully human anti-PCSK9 antibodies generated by conversion of the chimeric anti-PCSK9 antibodies, are able to inhibit PCSK9 D374Y -mediated reduction of LDL uptake.
  • Dissociation constants were determined by Octed red 96 (Fortiebio). The detailed operation and methods were followed according to the specifications of the instrument provided by the manufacturer. Briefly, an AHC sensor (Anti-human Fc sensor, Fortiebio) was used for the affinity determination. The anti-PCSK9 antibody was diluted to lOug/mL in PBS buffer (pH7.4) containing 0.1% (w/w) BSA and 0.02% (v/v) Tween 20 and incubated with the AHC sensor.
  • PBS buffer pH7.4
  • Anti-human Fc IgG was immobilized on flow cells 1 and 2: HBS-EP+ (lOmM HEPES, 150mM NaCl, 3mM EDTA, 0.05% P20, pH 7.4) was used as running buffer, and the immobilization of anti-human Fc IgG was carried out using the immobilization wizard template.
  • Flow cells 1 and 2 of a Series S CM5 sensor chip were activated with freshly -mixed 50mmol/L NHS and 200mmol L EDC. 20ug/mL of anti-human Fc IgG diluted in lOmM NaAC (pH 4.5) was injected into the activated flow cells 1 and 2. The remaining active coupling sites were blocked with 1M ethanolamine.
  • Recombinant His-tagged hPCSK9 protein was diluted to 50nM, followed by four 2-fold serial dilutions with HBS-EP+ buffer.
  • the His-tagged hPCSK9 protein concentrations were OnM, 3.125nM, 6.25nM, 12.5nM, 25nM and 50nM.
  • K D measurements were carried out with HBS-EP+ as the running buffer.
  • Each antibody was injected over the CM5 sensor flow cell 2 with a flow rate of lOuL/min to reach response 230 RU.
  • Prepared His-tagged hPCSK9 protein was then injected over flow cells 1 and 2, at a flow rate of 30uL/min for 180sec. Buffer flow was maintained for 400 seconds for dissociation measurements (30uL/min).
  • mice 9-10 week old C57BL/6 mice were housed and maintained under SPF conditions. Mice were randomized into 5 groups, with 4 mice in each group. At Day -1, mice were intraperitoneally administered 30mg/kg, 3mg/kg, or 0.3mg/kg test anti-PCSK9 antibody (e.g., 139G1C8 or 96F8C6) control IgG or vehicle. The next day, all mice were intravenuously administered recombinant human PCSK9 protein (30 ug/mouse). One hour after injection with PCSK9, the mice were sacrificed and their livers were harvested.
  • test anti-PCSK9 antibody e.g., 139G1C8 or 96F8C6
  • Example 12 Antibody thermostability, measured by differential scanning calorimetry (DSC)
  • Example 13 Antibody freeze/thaw stability
  • the freeze/thaw stabilities of the fully human anti-PCSK9 antibodies were characterized as follows. A lOOuL aliquot from the frozen stocks of each anti-PCSK9 antibody was thawed at room temperature. Once fully thawed, the samples were then rapidly frozen in the -80°C freezer and kept at -80°C for at least two hours before being thawed again at room temperature. The samples went through three identical freeze/thaw cycles. Visual inspection was used to check for precipitation. 20uL aliquots were removed from the samples for size-exclusion chromatography (SEC) analysis after three freeze/thaw cycles.
  • SEC size-exclusion chromatography
  • the solubility of the fully human anti-PCSK9 antibodies was characterized by concentrating lOmg IgG using centrifugal filters (Amicon Ultra-0.5mL 30K) at 14000g at 4°C down to >100mg/mL. 2mL or more of IgG was added into the centrifugal filters and concentrated at 14000g at 4°C.
  • the setting time of centrifuging was 2 min, 3 min, 5 min, 8 min, 15 min, and 20 min, and each time 20uL were aliquoted to a collection tube to measure the concentration with a nanodrop at A280. The centrifugation was finished when the concentration reached lOOmg/mL.
  • Step 1 CDR mutagenesis library construction and validation [00226] 1. Construction of phagemid expression vector and verification of scFv display: Sequences of the 98F8C6 anti-PCSK9 antibody heavy and light chain variable regions were obtained according to the methods of Example 6. Nucleic acids encoding the heavy chain variable region, linker and light chain variable region were assembled using overlapping PCR to generate an scFv. The PCR product was ligated into a phagemid vector, which was then transformed into TGI cells. Positive clones from ampicillin selection plates were selected, and their phagemid expression vectors were recovered and sequenced.
  • CDR positions were defined by Kabat numbering.
  • Random mutagenesis libraries were constructed using overlapping PCR. PCR products were digested using the restriction enzyme Sfil for 2 hours at 50 ° C and were then ligated into an Sfil- digested phagemid using T4 ligase at 16 ° C overnight. Each library was electro-transformed into TGI E.coli.
  • Step 2 Affinity -driven library panning
  • the CDR mutagenesis ScFv phage libraries were panned using biotinylated soluble hPCSK9 as antigen in solution phase, under equilibrium conditions.
  • the centrifuge tubes and streptavidin-magnetic to be used were first blocked with 2% MPBS (2% milk in PBS) for 2 hours at room temperature.
  • the libraries were pre- depleted with the beads in 2% MPBS and then mixed with biotin-hPCSK9 in the blocked tubes and incubated on a rotator at room temperature for 2 hours.
  • the concentration of antigen used was lOnM in the first round of panning, and was reduced 10-fold at a time in the subsequent rounds.
  • the ScFv-antigen-biotin complexes were mixed with streptavidin-beads and incubated for 15 minutes.
  • the beads were collected with a magnet, transferred to 1.5 microcentrifuge tubes, and washed 5 times each with MPBST (2% milk, 0.5% Tween20 in PBS), PBST (0.5% Tween 20 in PBS), MPBS (2% milk in PBS) and PBS.
  • Bound phage was eluted from the beads with ImL trypsin (lOug/mL in PBS) at 37°C for 30 minutes.
  • Beads were drawn to one side of the tube using a magnet, and the solution containing eluted phage was transferred to a tube containing 4mL TGI (A600-0.6) for titration of the panned library.
  • the output clones were sequenced to determine the enrichment of mutations. After 3 or 4 rounds of panning, the resulting clones were selected for screening.
  • VH and VL sequences from enriched clones were cloned into IgG expression vectors, inserted between leader sequence and constant region accordingly.
  • the resulting vectors were transfected into HEK293 cells by transient transfection. After 5-7 days, the IgGs in the culture supernatant were purified by affinity purification using Protein A column, as described in Example 8.
  • the VH sequences of the affinity -improved variants are listed in Table 30.
  • the light chain sequences of these variants were the same as those of the light chain of antibody 98F8C6, e.g., the LCDR 1-3 of SEQ ID NOs:38-40, respectively, or alight chain variable region of SEQ ID NO:37, or its coding sequence SEQ ID NO:58.

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Abstract

Anti-PCSK9 antibodies and antigen-binding fragments thereof are described. Also described are nucleic acids encoding the antibodies, compositions comprising the antibodies, and methods of producing the antibodies and using the antibodies for treating or preventing diseases such as lipid disorders, metabolic diseases, hypercholesterolemia, inflammatory diseases and infectious diseases.

Description

ANTI-PCSK9 ANTIBODIES AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is entitled to priority pursuant to Chinese Patent Application No. 201510673316.1, filed October 16, 2015, the disclosure of which is incorporated by reference herein in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name "Sequence Listing File", creation date of September 19, 2016 and having a size of about 76.7 kB. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates to monoclonal anti-PCSK9 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies. Methods of making the antibodies, and methods of using the antibodies to treat diseases including lipid disorders, metabolic diseases, hypercholesterolemia, inflammatory diseases and infectious diseases are also provided.
BACKGROUND OF THE INVENTION
[0004] In 2008, approximately 17 million people died from cardiovascular diseases, accounting for about one third of death worldwide. That number is expected to increase to 23.6 million by the year 2030 (Mozaffarian et al., 2015, Circulation. 13 l(4):e29-322). Various studies have shown a strong correlation between low density lipoprotein cholesterol (LDL-C) levels and the development of cardiovascular disease, due mainly to LDL-C playing a key in atherosclerosis (Rallidis and Lekakis, 2016, Hellenic J Cardiol. 57(2):86-91). It is therefore thought that reducing plasma LDL-C levels could be an effective way to prevent cardiovascular diseases. Statins have served as the primary treatment for hypercholesterolemia for many years and have been largely effective. However, statin treatment fails to result in target LDL-C levels in a significant number of high risk patients, and new therapeutics for the lowering of LDL-C levels are therefore needed.
[0005] The majority of LDL-C is cleared by the LDL receptor (LDLR) on hepatocytes. In 2003, proprotein convertase subtilisin/kexin type 9a (PCSK9) was discovered as a key factor in the LDL-C clearance pathway. PCSK9 is produced mainly in hepatocytes and is a secreted serine protease that binds to LDLR and results in internalization and lysosomal degradation of LDLR. The resulting decrease in concentration of LDLR proteins at the surface of hepatocytes causes a reduction in LDL-C clearance from the plasma. Thus, the presence of PCSK9 can inhibit the hepatic metabolism of plasma LDL-C. In addition to LDLR and ApoB, PCSK9 is the third gene to be associated with autosomal dominant Familial Hyperlipidemia (Rallidis and Lekakis, 2016, Hellenic J Cardiol. 57(2):86-91).
[0006] Loss-of-function mutations in PCSK9 have been associated with a reduction of LDL-C levels and cardiovascular disease, while humans with high levels of PCSK9 have elevated levels of LDL-C and significantly enhanced risk of cardiovascular disease. Gain-of-function mutants of PCSK9 are causatively associated with familial hypercholesterolaemia (Gencer et al., 2015, Swiss Med Wkly. 145:wl4094).In vitro experiments have shown that PCSK9 can reduce the expression level of LDLR on HepG2 cells. Similarly, an increase in PCSK9 levels in mice has reduced LDLR expression in the liver, while PCSK9 knockout mice have elevated LDLR expression. Taken together, the data suggests that PCSK9 is a promising therapeutic target in the management of lipid disorders. [0007] Antibodies against PCSK9 are considered to be the greatest advance in cardiovascular disease prevention since statins, and it is thought that they can be used as therapeutics for treating, e.g., high cholesterol, hyperlipidemia, coronary heart disease, metabolic syndrome and acute coronary artery syndrome.
[0008] Phase I and II clinical trials of fully human PCSK9 monoclonal antibody (mAb) from, e.g., Amgen and
Sanofi Regeneron, have been completed, and gave ranges of promising results. For example, studies of Alirocumab
(Sanofi/Regeneron) saw a reduction in LDL-C levels ranging from 39.2% to 67.9% (Gencer et al., 2015, Swiss Med
Wkly. 145:wl4094). Phase III clinical trials, with the objective of evaluating the effect of PCSK9 inhibition on the occurrence of cardiovascular events in patients with acute coronary syndrome are ongoing.
[0009] Despite the progress, there is a need in the art for more effective therapeutics comprising anti-PCSK9 antibodies that effectively inhibit the binding of PCSK9 to LDLR while causing minimal adverse side effects in humans.
BRIEF SUMMARY OF THE INVENTION
[0010] The invention satisfies this need by providing monoclonal antibodies that specifically bind PCSK9 with high affinity, induce LDL uptake by hepatocytes, and prevent LDLR degradation in vivo. In particular, the fully human anti-PCSK9 antibodies of the invention have a higher affinity to PCSK9 than Alirocumab (Regeneron).
[0011] In one general aspect, the invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that bind PCSK9.
[0012] According to a particular aspect, the invention relates to an isolated monoclonal antibody or antigen- binding fragment thereof comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of:
(1) SEQ ID NOs: 38, 39, 40, 34, 35, and 36, respectively
(2) SEQ ID NOs: 6, 7, 8, 2, 3, and 4, respectively;
(3) SEQ ID NOs: 14, 15, 16, 10, 11, and 12, respectively;
(4) SEQ ID NOs: 22, 23, 24, 18, 19, and 20, respectively;
(5) SEQ ID NOs: 30, 31, 32, 26, 27, and 28, respectively; or
(6) SEQ ID NOs: 46, 47, 48, 42, 43, and 44, respectively;
wherein the antibody or antigen-binding fragment thereof binds PCSK9specifically, preferably binds specifically to human PCSK9.
[0013] According to another particular aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising:
(1) an LCDR1 having the polypeptide sequence of SEQ ID NO: 38;
(2) an LCDR2 having the polypeptide sequence of SEQ ID NO: 39;
(3) an LCDR3 having the polypeptide sequence of SEQ ID NO: 40;
(4) an HCDR1 having the polypeptide sequence of SEQ ID NO: 34;
(5) an HCDR2 having the polypeptide sequence of SEQ ID NO: 35; and
(6) an HCDR3 having the polypeptide sequence of one of SEQ ID NOs: 36 and 73-90;
wherein the antibody or antigen-binding fragment thereof binds PCSK9, preferably binds specifically to human PCSK9.
[0014] According to another particular aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region having a polypeptide sequence at least 85%, preferably at least 90%, more preferably at least 95% identical to one of SEQ ID NOs: 1, 9, 17, 25, 33 or 91- 108, or 41, or a light chain variable region having a polypeptide sequence at least 85%, preferably at least 90%, more preferably at least 95% identical to one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45. [0015] According to one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the invention is human/rat chimeric.
[0016] According to another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the invention is human.
[0017] According to yet another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the invention further comprises a constant region, preferably a human heavy chain IgGl constant region, and a human antibody light chain kappa constant region.
[0018] In another general aspect, the invention relates to an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
[0019] In another general aspect, the invention relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
[0020] In another general aspect, the invention relates to a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
[0021] In another general aspect, the invention relates to a pharmaceutical composition, comprising an isolated monoclonal antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.
[0022] In another general aspect, the invention relates to a method of blocking the binding of PCSK9 to LDLR, or a method of augmenting uptake of LDL, in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.
[0023] In another general aspect, the invention relates to a method of treating a disease, disorder or condition, preferably a lipid disorder, a metabolic disease, an inflammatory disease or an infectious disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.
[0024] In another general aspect, the invention relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment under conditions to produce the monoclonal antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture.
[0025] In another general aspect, the invention relates to a method of producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
[0026] Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.
[0028] In the drawings:
[0029] Figure 1 shows effect of different batches of Immunogen A (hPCSK9-His) on LDL uptake inhibition in HepG2 cells;
[0030] Figure 2 shows the binding activity of chimeric anti-PCSK9 antibodies according to embodiments of the invention to Immunogen A (hPCSK9-His), as measured by ELISA;. [0031] Figure 3 shows the binding activity of chimeric anti-PCSK9 antibodies according to embodiments of the invention to recombinant human PCSK9D374Y protein, as measured by ELISA;
[0032] Figure 4 shows the binding activity of chimeric anti-PCSK9 antibodies according to embodiments of the invention to recombinant cyno monkey PCSK9 protein, as measured by ELISA;
[0033] Figure 5 shows the inhibition of binding of recombinant PCSK9 protein to LDLR ECD protein by chimeric anti-PCSK9 antibodies according to embodiments of the invention, as measured by ELISA;
[0034] Figure 6 shows the inhibition of binding of recombinant PCSK9D374Y protein to LDLR ECD protein by chimeric anti-PCSK9 antibodies according to embodiments of the invention, as measured by ELISA;
[0035] Figure 7 shows the inhibition of PCSK9D374Y-induced down regulation of LDLR by chimeric anti-
PCSK9 antibodies according to embodiments of the invention, as determined by FACS;
[0036] Figure 8 shows the inhibition ofPCSK9D374Y-induced LDL uptake in HepG2 cells by chimeric anti- PCSK9 antibodies according to embodiments of the invention;
[0037] Figure 9 shows the binding activity of fully human anti-PCSK9 antibodies according to embodiments of the invention to Immunogen A (hPCSK9-His), as measured by ELISA;
[0038] Figure 10 shows the binding activity of fully human anti-PCSK9 antibodies according to embodiments of the invention to recombinant PCSK9D374Y protein, as measured by ELISA;
[0039] Figure 11 shows the binding activity of fully human anti-PCSK9 antibodies according to embodiments of the invention to cyno monkey PCSK9, as measured by ELISA;
[0040] Figure 12 shows the inhibition of binding of recombinant PCSK9 protein to LDLR ECD protein by fully human anti-PCSK9 antibodies according to embodiments of the invention, as measured by ELISA;
[0041] Figure 13 shows the inhibition of binding of recombinant PCSK9D374Y protein to LDLR ECD protein by fully human anti-PCSK9 antibodies according to embodiments of the invention, as measured by ELISA;
[0042] Figure 14 shows the inhibition of PCSK9D374Y-induced LDL uptake in HepG2 cells by a fully human anti-PCSK9 antibody according to embodiments of the invention;
[0043] Figure 15 shows the inhibition of PCSK9D374Y-induced LDL uptake in HepG2 cells by a fully human anti-PCSK9 antibody according to embodiments of the invention;
[0044] Figure 16 shows the inhibition of PCSK9D374Y-induced LDL uptake in HepG2 cells by a fully human anti-PCSK9 antibody according to embodiments of the invention;
[0045] Figure 17 shows the binding activity of a fully human anti-PCSK9 antibody according to embodiments of the invention to Immunogen A, as measured by ELISA;
[0046] Figure 18 shows the binding activity of a fully human anti-PCSK9 antibody according to embodiments of the invention to recombinant PCSK9D374Y protein, as measured by ELISA;
[0047] Figure 19 shows the binding activity of a fully human anti-PCSK9 antibody according to embodiments of the invention to cyno monkey PCSK9, as measured by ELISA;
[0048] Figure 20 shows the inhibition of binding of recombinant PCSK9 protein to LDLR ECD protein by a fully human anti-PCSK9 antibody according to embodiments of the invention, as measured by ELISA;
[0049] Figure 21 shows the inhibition of binding of recombinant PCSK9D374Y to LDLR ECD protein by a fully human anti-PCSK9 antibody according to embodiments of the invention, as measured by ELISA;
[0050] Figure 22 shows the inhibition of PCSK9D374Y-induced LDL uptake in HepG2 cells by a fully human anti-PCSK9 antibody according to embodiments of the invention, as measured by ELISA;
[0051] Figures 23 A &B show the inhibition of recombinant hPCSK9 protein-mediated LDLR degradation in mice by anti-PCSK9 antibodies according to embodiments of the invention: liver lysates from mice administered with recombinant human PCSK9 protein (hPCSK9) and anti-PCSK9 antibodies, control IgG or vehicle were subject to Western blot analysis using an antibody against mouse LDLR (mLDLR) and an antibody against mouse β-actin (πιβ-actin), the ratio of the density of the mLDLRband v.s. the density of the ηιβ-actinband on the Western Blot was plotted:
Fig. 23 A: the ratio of mLDLR/m -actin in mice administered with 30mg/kg, 3mg/kg, or 0.3mg/kg of 139G1C8, control IgG or vehicle; and
Fig. 23 B: the ratio of mLDLR/m -actin in mice administered with 30mg/kg, 3mg/kg, or 0.3mg/kg of 96F8C6, control IgG or vehicle;
[0052] Figure 24 shows the thermostability of fully human anti-PCSK9 antibodies according to embodiments of the invention, as measured by Differential Scanning Calorimetry (DSC);
[0053] Figure 25 shows the freeze/thaw stability of fully human anti-PCSK9 antibodies according to embodiments of the invention;
[0054] Figure 26 shows the solubility of fully human anti-PCSK9 antibodies according to embodiments of the invention; and
[0055] Figure 27 A&B show the inhibition of PCSK9D374Y-induced LDL uptake in HepG2 cells by various human anti-PCSK9 antibodies according to embodiments of the invention having improved affinity according to embodiments of the invention:
Figure 27 A: inhibition of PCSK9D374Y-induced LDL uptake in HepG2 cells by antibody clone AF-mab023_06, AF-mab023_12, or 96F8C6; and
Figure 27 B: inhibition of PCSK9D374Y-induced LDL uptake in HepG2 cells by antibody clone AF-mab023_01, AF-mab023_l l, AF-mab023_14, or AF-mab023_18.
DETAILED DESCRIPTION OF THE INVENTION
[0056] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.
[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.
[0058] Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term "about." Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
[0059] The invention generally relates to isolated anti-PCSK9 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies. Methods of making the antibodies, and methods of using the antibodies to treat diseases including lipid disorders, metabolic diseases, hypercholesterolemia, inflammatory diseases and infectious diseases are also provided. The antibodies of the invention possess one or more desirable functional properties, including but not limited to high- affinity binding to PCSK9, high specificity to PCSK9, the ability to block the binding of PCSK9 to LDLR, and the ability to stimulate uptake of LDL and prevent the degradation of LDLR induced by PCSK9.
[0060] In a general aspect, the invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that bind PCSK9.
[0061] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Accordingly, the antibodies of the invention can be of any of the five major classes or corresponding sub-classes. Preferably, the antibodies of the invention are IgGl, IgG2, IgG3 or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, the antibodies of the invention can contain a kappa or lambda light chain constant domain. According to particular embodiments, the antibodies of the invention include heavy and/or light chain constant regions from rat or human antibodies. In addition to the heavy and light constant domains, antibodies contain an antigen-binding region that is made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2 and LCRD3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCRD2 and HCDR3.
[0062] As used herein, the term an "isolated antibody" refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to PCSK9 is substantially free of antibodies that do not bind to PCSK9). In addition, an isolated antibody is substantially free of other cellular material and/or chemicals.
[0063] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the invention can be made by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods. For example, the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.
[0064] As used herein, the term "antigen-binding fragment" refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv1), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab) an scFv dimer (bivalent diabody), a multi specific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds. According to particular embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the constant region of the heavy chain. According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab').
[0065] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.
[0066] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide.
[0067] As used herein, the term "humanized antibody" refers to a non-human antibody that is modified to increase the sequence homology to that of a human antibody, such that the antigen-binding properties of the antibody are retained, but its antigenicity in the human body is reduced.
[0068] As used herein, the term "chimeric antibody" refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable region of both the light and heavy chains often corresponds to the variable region of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.
[0069] As used herein, the term "PCSK9" refers to the proprotein convertase subtilisin/kexin type 9 protein, a secretory protein that is a soluble member of the mammalian proprotein convertase family of secretory serine endoproteases. Secreted PCSK9 binds to LDLR on the surface of cells. Upon internalization of the bound proteins, PCSK9 prevents LDLR from endocytic recycling and results in lysosomal degradation of both proteins (Rallidis and Lekakis, 2016, Hellenic J Cardiol. 57(2):86-91). The term "human PCSK9" refers to a PCSK9 originated from a human. An exemplary amino acid sequence of a human PCSK9 is represented in GenBank Accession No.
NP_777596.2.
[0070] As used herein, an antibody that "specifically binds to PCSK9" refers to an antibody that binds to a PCSK9, preferably a human PCSK9, with a KD of 1 χ 10~7 M or less, preferably 1 χ 10~8 M or less, more preferably 5 10"9 M or less, 1 10"9 M or less, 5 χ 10"10 M or less, or 1 χ 10"10 M or less. The term "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods in the art in view of the present disclosure. For example, the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as a Octet RED96 system.
[0071] The smaller the value of the KD of an antibody, the higher affinity that the antibody binds to a target antigen.
[0072] According to a particular aspect, the invention relates to an isolated monoclonal antibody or antigen- binding fragment thereof comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of:
(1) SEQ ID NOs: 38, 39, 40, 34, 35, and 36, respectively
(2) SEQ ID NOs: 6, 7, 8, 2, 3, and 4, respectively;
(3) SEQ ID NOs: 14, 15, 16, 10, 11, and 12, respectively;
(4) SEQ ID NOs: 22, 23, 24, 18, 19, and 20, respectively;
(5) SEQ ID NOs: 30, 31, 32, 26, 27, and 28, respectively; or
(6) SEQ ID NOs: 46, 47, 48, 42, 43, and 44, respectively;
wherein the antibody or antigen-binding fragment thereof binds PCSK9, preferably specifically binds to human PCSK9. [0073] According to another particular aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising:
(1) an LCDR1 having the polypeptide sequence of SEQ ID NO: 38;
(2) an LCDR2 having the polypeptide sequence of SEQ ID NO: 39;
(3) an LCDR3 having the polypeptide sequence of SEQ ID NO: 40;
(4) an HCDR1 having the polypeptide sequence of SEQ ID NO: 34;
(5) an HCDR2 having the polypeptide sequence of SEQ ID NO: 35; and
(6) an HCDR3 having the polypeptide sequence of one of SEQ ID NOs: 36 and 73-90;
wherein the antibody or antigen-binding fragment thereof binds PCSK9, preferably binds specifically to human PCSK9.
[0074] According to another particular aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, comprising a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to one of SEQ ID NOs: 1, 9, 17, 25, 33 or 91- 108,or 41 , or a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45. According to one preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region having the polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to SEQ ID NO: 1, 9, 17, 25, 33 or 41, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to SEQ ID NO: 5, 13, 21, 29, 37 or 45, respectively. According to another preferred aspect, the isolated monoclonal antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region having the polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to one of SEQ ID NOs: 33 or 91-108, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to SEQ ID NO: 37.
[0075] According to another particular aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, comprising:
a. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 5; b. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 13;
c. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 21;
d. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 25, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 29;
e. a heavy chain variable region having the polypeptide sequence of one selected from the group consisting of SEQ ID NOs: 33 and 91-108, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 37; or
f. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 41, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 45.
[0076] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 6, 7, 8, 2, 3, and 4, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 1, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 5. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 5.
[0077] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDRl, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 14, 15, 16, 10, 11, and 12, respectively, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 9, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 13. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 13.
[0078] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDRl, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 22, 23, 24, 18, 19, and 20, respectively, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 17, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 21. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 21.
[0079] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDRl, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 30, 31, 32, 26, 27, and 28, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 25, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 29. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 25; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 29.
[0080] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDRl, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 38, 39, 40, 34, 35, and 36, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 33, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 37. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 33; and a light chain variable region having the polypeptide sequence of SEQ ID NO:37.
[0081] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising an LCDRl having the polypeptide sequence of SEQ ID NO: 38, an LCDR2 having the polypeptide sequence of SEQ ID NO: 39, an LCDR3 having the polypeptide sequence of SEQ ID NO: 40, an HCDR1 having the polypeptide sequence of SEQ ID NO: 34, an HCDR2 having the polypeptide sequence of SEQ ID NO: 35, and an HCDR3 having the polypeptide sequence of one selected from the group consisting of SEQ ID NOs: 36 and 73-90.
[0082] In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to one of SEQ ID NOs: 91-108, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 37. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of one of SEQ ID NOs: 91-108; and a light chain variable region having the polypeptide sequence of SEQ ID NO:37.
[0083] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of SEQ ID NOs: 46, 47, 48, 42, 43, and 44, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to SEQ ID NO: 41, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably95% identical to SEQ ID NO: 45. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 41; and a light chain variable region having the polypeptide sequence of SEQ ID NO:45.
[0084] According to another particular aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, wherein the antibody or antigen-binding fragment thereof is chimeric.
[0085] According to another particular aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, wherein the antibody or antigen-binding fragment thereof is human.
[0086] According to another particular aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, comprising a constant region, preferably a human heavy chain IgGl constant region (SEQ ID NO: 67), and a human antibody light chain, preferably a human light chain kappa constant region (SEQ ID NO: 69).
[0087] In another general aspect, the invention relates to an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention. It will be appreciated by those skilled in the art that the coding sequence of a protein can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding monoclonal antibodies or antigen-binding fragments thereof of the invention can be altered without changing the amino acid sequences of the proteins.
[0088] In another general aspect, the invention relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of an antibody or antigen-binding fragment thereof in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the invention. [0089] In another general aspect, the invention relates to a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention. Any host cell known to those skilled in the art in view of the present disclosure can be used for recombinant expression of antibodies or antigen- binding fragments thereof of the invention. In some embodiments, the host cells are E. coli TGI or BL21 cells (for expression of, e.g., an scFv or Fab antibody), CHO-DG44 or CHO-K1 cells (for expression of, e.g., a full-length IgG antibody). According to particular embodiments, the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.
[0090] In another general aspect, the invention relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions to produce a monoclonal antibody or antigen-binding fragment thereof of the invention, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). Expressed antibodies or antigen-binding fragments thereof can be harvested from the cells and purified according to conventional techniques known in the art and as described herein.
[0091] In another general aspect, the invention relates to a pharmaceutical composition, comprising an isolated monoclonal antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.
[0092] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term
"pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effectiveness of a composition according to the invention or the biological activity of a composition according to the invention. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in an antibody pharmaceutical composition can be used in the invention.
[0093] In another general aspect, the invention relates to a method of blocking the binding of PCSK9 to LDLR, or of augmenting LDL uptake in liver in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.
[0094] The functional activity of antibodies and antigen-binding fragments thereof that bind PCSK9 can be characterized by methods known in the art and as described herein. Methods for characterizing antibodies and antigen-binding fragments thereof that bind PCSK9 include, but are not limited to, affinity and specificity assays including Biacore, ELISA, and OctetRed analysis; receptor ligand binding assays to detect blocking of the binding of PCSK9 to LDLR; assays to detect inhibition of LDLR degradation induced by PCSK9 in HepG2 cells; assays to detect increases in LDL uptake by HepG2 cells; experiments to detect the inhibition of LDLR degradation induced by PCSK9 in liver; etc. According to particular embodiments, the methods for characterizing antibodies and antigen-binding fragments thereof that bind PCSK9 include those described in Examples 2-15 below.
[0095] In another general aspect, the invention relates to a method of treating lipid disorders, metabolic diseases, hypercholesterolemia, inflammatory diseases and infectious diseases in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.
[0096] As used herein, the term "subject" refers to an animal, and preferably a mammal. According to particular embodiments, the subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig or mouse) or a primate (e.g., a monkey, chimpanzee, or human). In particular embodiments, the subject is a human. [0097] According to embodiments of the invention, the pharmaceutical composition comprises a therapeutically effective amount of the anti-PCSK9 antibody or antigen-binding fragment thereof. As used herein, the term "therapeutically effective amount" refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject. A therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.
[0098] As used herein with reference to anti-PCSK9 antibodies or antigen-binding fragments thereof, a therapeutically effective amount means an amount of the anti-PCSK9 antibody or antigen-binding fragment thereof that stimulates LDL uptake in a subject in need thereof. Also as used herein with reference to anti-PCSK9 antibodies or antigen-binding fragments thereof, a therapeutically effective amount means an amount of the anti- PCSK9 antibody or antigen-binding fragment thereof that results in treatment of a disease, disorder, or condition; prevents or slows the progression of the disease, disorder, or condition; or reduces or completely alleviates symptoms associated with the metabolic disease, disorder, or condition.
[0099] According to particular embodiments, the disease, disorder or condition to be treated is a lipid disorder, hyperlipidemia, dyslipidemia, or a metabolic disease. According to more particular embodiments, the disease, disorder or condition to be treated is a lipid disorder, including but not limited to, primary hyperlipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia. According to other particular embodiments, the disease, disorder or condition to be treated is an inflammatory disease, such as sepsis, or an infectious diseases.
[00100] According to particular embodiments, a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (ix) increase the survival of a subject with the disease, disorder or condition to be treated, or a symptom associated therewith; (xi) inhibit or reduce the disease, disorder or condition to be treated, or a symptom associated therewith in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.
[00101] The therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.
[00102] According to particular embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.
[00103] As used herein, the terms "treat," "treating," and "treatment" are all intended to refer to an
amelioration or reversal of at least one measurable physical parameter related to a lipid disease, disorder or condition or a metabolic disease, disorder or condition, which is not necessarily discernible in the subject, but can be discernible in the subject. The terms "treat," "treating," and "treatment," can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition. In a particular embodiment, "treat," "treating," and "treatment" refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as lipid disorder, hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia, a metabolic disease, an inflammatory disease such as sepsis, or an infectious disease. In a particular embodiment, "treat," "treating," and "treatment" refer to prevention of the recurrence of the disease, disorder, or condition. In a particular embodiment, "treat," "treating," and "treatment" refer to an increase in the survival of a subject having the disease, disorder, or condition. In a particular embodiment, "treat," "treating," and "treatment" refer to elimination of the disease, disorder, or condition in the subject.
[00104] According to particular embodiments, a composition used in the treatment of a lipid disease, disorder or condition or a metabolic disease, disorder or condition can be used in combination with another treatment including, but not limited to, statins or other lipid lowering drugs.
[00105] As used herein, the term "in combination," in the context of the administration of two or more therapies to a subject, refers to the use of more than one therapy. The use of the term "in combination" does not restrict the order in which therapies are administered to a subject. For example, a first therapy (e.g., a composition described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject.
[00106] In another general aspect, the invention relates to a method of producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising combining a monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
EMBODIMENTS
[00107] The invention provides also the following non-limiting embodiments.
[00108] Embodiment 1 is an isolated monoclonal antibody or antigen-binding fragment thereof comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of:
(1) SEQ ID NOs: 6, 7, 8, 2, 3, and 4, respectively;
(2) SEQ ID NOs: 14, 15, 16, 10, 11, and 12, respectively;
(3) SEQ ID NOs: 22, 23, 24, 18, 19, and 20, respectively;
(4) SEQ ID NOs: 30, 31, 32, 26, 27, and 28, respectively;
(5) SEQ ID NOs: 38, 39, 40, 34, 35, and 36, respectively; or
(6) SEQ ID NOs: 46, 47, 48, 42, 43, and 44, respectively;
wherein the antibody or antigen-binding fragment thereof binds PCSK9, preferably binds specifically to human PCSK9.
[00109] Embodiment 2 is an isolated monoclonal antibody or antigen-binding fragment thereof comprising:
(1) an LCDR1 having the polypeptide sequence of SEQ ID NO: 38;
(2) an LCDR2 having the polypeptide sequence of SEQ ID NO: 39;
(3) an LCDR3 having the polypeptide sequence of SEQ ID NO: 40;
(4) an HCDR1 having the polypeptide sequence of SEQ ID NO: 34;
(5) an HCDR2 having the polypeptide sequence of SEQ ID NO: 35;
(6) an HCDR3 having the polypeptide sequence of one of SEQ ID NOs: 36 and 73-90; wherein the antibody or antigen-binding fragment thereof binds PCSK9, preferably binds specifically to human PCSK9.
[00110] Embodiment 3 is the isolated monoclonal antibody or antigen-binding fragment of Embodiment 1 or 2, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to one of SEQ ID NOs: 1, 9, 17, 25, 33 or 91-108,or 41, or a light chain variable region having a polypeptide sequence at least 95% identical to one of SEQ ID NOs: 5, 13, 21, 29, 37 or 45.
[00111] Embodiment 4 is the isolated monoclonal antibody or antigen-binding fragment of Embodiment 3, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 1, 9, 17, 25, 33 or 41, and a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 5, 13, 21, 29, 37 or 45 respectively.
[00112] Embodiment 5 is the isolated monoclonal antibody or antigen-binding fragment of Embodiment 3, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to one of SEQ ID NOs: 91-108, and a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 37.
[00113] Embodiment 6 is the isolated monoclonal antibody or antigen-binding fragment of Embodiment 4 or 5, comprising:
(a) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 5;
(b) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 13;
(c) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 21;
(d) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 25, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 29;
(e) a heavy chain variable region having the polypeptide sequence of one of SEQ ID NOs: 33 or 91- 108, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 37; or
(f) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 41, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 45.
[00114] Embodiment 7 is the isolated monoclonal antibody or antigen-binding fragment of any of
Embodiments 1 to 6, wherein the antibody or antigen-binding fragment thereof is chimeric.
[00115] Embodiment 8 is isolated monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 7, wherein the antibody or antigen-binding fragment thereof is human.
[00116] Embodiment 9 is the isolated antibody or antigen-binding fragment of Embodiment 8, comprising a human heavy chain IgGl constant region and a human antibody light chain kappa constant region.
[00117] Embodiment 10 is the isolated antibody or antigen-binding fragment of any of Embodiments 1 to 9, wherein the antibody or antigen-binding fragment binds to a human PCSK9 with a KD of 5 χ 10~9 M or less, preferably a KD of 1 χ 10~9 M or less, wherein the KD is measured by surface plasmon resonance analysis, such as by using a Biacore system, or by a bio-layer interferometry technology, such as by using a Octet RED96 system.
[00118] Embodiment 11 is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 10.
[00119] Embodiment 12 is a vector comprising the isolated nucleic acid of Embodiment 11.
[00120] Embodiment 13 is a host cell comprising the nucleic acid of Embodiment 12.
[00121] Embodiment 14 is a pharmaceutical composition, comprising the isolated monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 10 and a pharmaceutically acceptable carrier. [00122] Embodiment 15 is a method of blocking binding of PCSK9 to LDLR, or augmenting uptake of LDL in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of Embodiment 14.
[00123] Embodiment 16 is a method of treating a lipid disease, disorder or condition, a metabolic disease, disorder or condition, an inflammatory disease, disorder or condition, or an infectious disease, disorder or condition in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of Embodiment 14.
[00124] Embodiment 17 is the method of Embodiment 16, further comprising administering to the subject an additional agent for treating the lipid disorder, metabolic disease, inflammatory disease, or infectious disease in the subject in need thereof.
[00125] Embodiment 18 is a method of treating a lipid disorder, metabolic disease, inflammatory disease, or infectious disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of Embodiment 14.
[00126] Embodiment 19 is the method of Embodiment 18, wherein the lipid disorder or metabolic disease is hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, or homozygous familial
hypercholesterolemia, or wherein the inflammatory disease is sepsis.
[00127] Embodiment 20 is the method of any of Embodiments 18-19, further comprising administering to the subject an additional agent for treating the lipid disorder, metabolic disease, inflammatory disease, or infectious disease in the subject in need thereof.
[00128] Embodiment 21 is a method of producing the monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 10, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment under conditions to produce the monoclonal antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment from the cell or cell culture.
[00129] Embodiment 22 is a method of producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 10, comprising combining the monoclonal antibody or antigen-binding fragment with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
[00130] Embodiment 23 is an isolated monoclonal antibody or antigen-binding fragment of any of
Embodiments 1 to 10 for use in treating a lipid disorder, metabolic disease, inflammatory disease, or infectious disease, such as hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia or sepsis, in a subject in need thereof.
[00131] Embodiment 24 is a pharmaceutical composition of Embodiment 14 for use in treating a lipid disorder, metabolic disease, inflammatory disease, or infectious disease, such as hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia, or sepsis in a subject in need thereof.
[00132] Embodiment 25 is an use of an isolated monoclonal antibody or antigen-binding fragment of any of Embodiments 1 to 10 for the manufacture of a medicament for treating a lipid disorder, metabolic disease, inflammatory disease, or infectious disease, such as hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia, or sepsis, in a subject in need thereof.
[00133] Embodiment 26 is an use of a pharmaceutical composition of Embodiment 14 for the manufacture of a medicament for treating a lipid disorder, metabolic disease, inflammatory disease, or infectious disease, such as hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial
hypercholesterolemia, or sepsis, in a subject in need thereof.
EXAMPLES [00134] The following examples of the invention are to further illustrate the nature of the invention. It should be understood that the following examples do not limit the invention and that the scope of the invention is to be determined by the appended claims.
[00135] Example 1 - Generation of anti-PCSK9 antibodies
[00136] Human PCSK9 protein was used as an immunogen to generate anti-PCSK9 antibodies. The use of human immunoglobulin transgenic mouse technology for the development and preparation of fully human antibodies was first described by Abgenix (xeno mouse and Medarex (HuMab "mouse"); Lonberg et al., 1994, Nature 368: 856-859; Lonberg and Huszar, 1995, Internal Rev. Immunol. 13 :65-93; Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci. 764:536-546).
[00137] Antibodies with high affinity (KD< 1 * 10"9M) to PCSK9 were obtained by carrying out pilot antibody production, purification and validation. The antibodies, which are specific for PCSK9, are able to block the binding of PCSK9 to LDLR. The amino acid sequences of the heavy and light chain variable regions of the generated anti- PCSK9 antibodies were determined using standard molecular biology methods and are summarized in Table 1.
[00138] Table 1. SEQ ID NOs of the amino acid sequences of the heavy chain variable regions, HCDRs, light chain variable regions and LCDRs of chimeric anti-PCSK9 antibodies of the invention
Figure imgf000017_0001
[00139] The heavy chain and light chain variable regions of the anti-PCSK9 antibodies listed in Table 1 are encoded by the nucleic acid sequences summarized in Table 2.
[00140] Table 2. SEQ ID NOs of the nucleic acid sequences of the heavy chain and light chain variable regions of chimeric anti-PCSK9 antibodies of the invention
SEQ ID NOs (nucleotides of the sequence corresponding to CDRs)
Clone ID Heavy Chain variable region Light Chain variable region
74C10A8 49 50
(CDR1 : nt 76-105) (CDR1 : nt 70-102)
(CDR2: nt 148-195) (CDR2: nt 148-168)
(CDR3 : nt 289-351) (CDR3 : nt 263-291)
76A1B 11 51 52
(CDR1 : nt 76-105) (CDR1 : nt 70-102)
(CDR2: nt 148-198) (CDR2: nt 148-168)
(CDR3 : nt 295-342) (CDR3 : nt 265-291)
139G1C5 53 54
(CDR1 : nt 76-108) (CDR1 : nt 70-102)
(CDR2: nt 151-198) (CDR2: nt 148-168)
(CDR3 : nt 295-321) (CDR3 : nt 265-291) 152G2F7 55 56
(CDR1 : nt 76-105) (CDR1 : nt 70-102)
(CDR2: nt 148-195) (CDR2: nt 148-168)
(CDR3 : nt 295-354) (CDR3 : nt 265-291)
96F8C6 57 58
(CDR1 : nt 76-105) (CDR1 : nt 70-102)
(CDR2: nt 148-198) (CDR2: nt 148-168)
(CDR3 : nt 295-357) (CDR3 : nt 265-291)
103C11E8 59 60
(CDR1 : nt 76-108) (CDR1 : nt 70-102)
(CDR2: nt 151-198) (CDR2: nt 148-168)
(CDR3 : nt 295-330) (CDR3 : nt 265-291)
[00141] Fully human versions of the anti-PCSK9 antibodies were generated. The fully human anti-PCSK9 antibodies bound to the human PCSK9 with high affinity (KD< 1 * 10"9M), and blocked the binding of PCSK9 to LDLR. The biological activities of the anti-PCSK9 antibodies were evaluated by receptor ligand blocking assay and LDL uptake assays, in which they blocked PCSK9 binding to LDLR, enhanced LDL uptake by HepG2 cells, and prevented PCSK9-induced LDLR degradation in vivo. The anti-PCSK9 antibodies can be used for the treatment of lipid disorders, metabolic diseases, hypercholesterolemia, inflammatory diseases or infectious diseases.
[00142] Example 2 - Preparation of anti-PCSK9 antibodies
[00143] (Step 1) Preparation of Immunogen A, His-tagged PCSK9 (PCSK9-His)
[00144] The coding sequence of human PCSK9 (hPCSK9; SEQ ID NO: 61), corresponding to the full-length protein sequence of SEQ ID NO: 62 (UniProtKB ID Q8NBP7), was cloned along with a His tag into pCpC vector (Invitrogen, #V044-50) using standard molecular biology cloning techniques (Sambrook and Russell, 1989, Molecular cloning: a laboratory manual, New York : Cold Spring Harbor Laboratory Press, 2nd ed.). HEK293 cells (Invitrogen) were transiently transfected using polyethylenimine (PEI, Polysciences) with the plasmid and expanded in FreeStyle 293 expression medium (Invitrogen) at 37°C. After 4 days of expansion, the culture medium was collected and centrifuged to remove cell components. The culture supernatant contained the recombinant His- tagged PCSK9. Imidazole was added to a final concentration of lOmM to the culture supernatant, which contained the recombinant His-tagged PCSK9. The sample was passed through a 0.22 micron sterile filter and subjected to Ni-NTA affinity chromatography. After equilibrating the sample with Buffer A [1 x PBS, pH 7.2 containing 20 niM Imidazole) and Buffer B [(Buffer A, 0.1% (v/v) Trition X100, 0.1% (v/v) Triton XI 14], His-tagged PCSK9 protein (PCSK9-His) was eluted from the Ni-NTA affinity chromatography column with Buffer C [1 x PBS (pH7.2), 250mM imidizole]. The eluate was further purified using a Superdex 200 column (GE, Healthcare) to obtain highly pure His-tagged recombinant human PCSK9 protein. The sample was dialyzed with 1 x PBS (pH7.2) at 4 °C overnight. After dialysis, the purified PCSK9-His was passed through a 0.22 micron sterile filter, aliquoted and stored at -80 "C.
[00145] To characterize the PCSK9-His immunogen, the sample's protein concentration and purity were determined, and the immunogen' s molecular weight and biological activity were determined.
[00146] The biological activity of the His-tagged hPCSK9 immunogen was assessed in a series of in vitro characterizations, and it was found that the immunogen displayed the bioactivity of PCSK9, and it inhibited LDL uptake by HepG2 cells in a dose-dependent manner. The two batches of His-tagged hPCSK9 immunogen that were produced demonstrated comparable bioactivity in the inhibition of HepG2 LDL uptake (see Figure 1). These two batches of biologically active His-tagged hPCSK9 immunogen were used for mouse immunizations. [00147] Table 3.Recombinant human PCSK9 inhibits LDL u take b He G2 cells
Figure imgf000019_0002
[00148] (Step 2) Preparation of Immunogen B, hPCS9 expressing construct
[00149] The coding sequence of human PCSK9 (hPCSK9; SEQ ID NO: 61) was subcloned into pcDNA3.1 vector (Invitrogen), and the resulting plasmid was coated onto a l.Oum colloidal gold bullet (Bio-RAD) for subsequent immunization using a Helios gene gun (Bio-rad No. 165-2431), following the instructions of the Helios gene gun data sheet.
[00150] (Step 3) Hybridoma cell fusion and antibody screening
[00151] Human Ig Fc does not interact with the mouse Fc receptor, so immune responses triggered in mice by hFc-containing immunogens are weak, resulting in low efficiency generation of monoclonal antibodies. Harbour H2L2 transgenic mice were generated by introducing the gene encoding the human immunoglobulin (Ig) variable region and the gene encoding the rat Ig constant region into the mouse genome, such that the mice contained a chimeric Ig comprising hV-rC, while expression of the mouse Ig was disabled (WO 2010/070263 Al). Harbour H2L2 transgenic mice are able to produce comparable immune responses and antibody titers to wild type mice (e.g., Balb/c).
[00152] (part 3A) 6-8 week old Harbour H2L2 transgenic mice (Beijing Weitong Lihua) were immunized with Immunogen A, and the mice were kept under Specific Pathogen Free (SPF) conditions. In the first immunization, 50ug of Immunogen A was injected into the abdominal cavity of each mouse along with 0.25mL Complete Freund's Adjuvant (CFA). To enhance the immune response, 50ug of Immunogen A was injected into the abdominal cavity of each mouse along with 0.25 Incomplete Freund's Adjuvant (IF A) two weeks after the first immunization, and subsequent boosts were administered 3 weeks apart. Blood samples were collected one week after immunization. The antibody titer and specificity in serum were determined by ELISA, and the results are shown in Table 4. The blank control was 1% (w/w) BSA. The OD450nm values shown in Table 4 are the serum titer values from 7 days after the third boost, as determined by ELISA. The serum titer after the third boost usually reached at least 1 : 10,000.
[00153] Table 4.Serum titers of Harbour H2L2 transgenic mice immunized with hPCSK9-His protein, as
Figure imgf000019_0001
Figure imgf000019_0003
[00154] (part 3B) 6-8 week old Harbour H2L2 transgenic mice (Beijing Weitong Lihua) were immunized with Immunogen A, and the mice were kept under SPF conditions. In the first immunization, 50ug of Immunogen A was injected into bottom of the tail (BOT) of each mouse along with 0.20ml of Gerbu adjuvant (Invitrogen). The first the boost was administered two weeks after the first immunization, and subsequent boosts were administered three weeks apart. Blood samples were collected one week after immunization. The antibody titer and specificity in seram were determined by ELISA, and the results are shown in Table 5. The blank control was 1% (w/w) BSA. The OD450nm values shown in Table 5 are the seram titer values from 7 days after the third boost, as determined by ELISA. The seram titer after the third boost usually reached at least 1 : 10,000.
[00155] Table 5. Seram titers of Harbour H2L2 transgenic mice immunized with hPCSK9-His protein, as determined
Figure imgf000020_0001
[00156] (part 3C) 6-8 week old Harbour H2L2 transgenic mice (Beijing Weitong Lihua) were immunized with Immunogen A, and the mice were kept under SPF conditions. In the first immunization, 50ug of Immunogen A was injected into the abdominal cavity of each mouse along with 0.25mL Complete Freund's Adjuvant (CFA). To enhance the immune response, 50ug of Immunogen A was injected into the abdominal cavity of each mouse along with 0.25 Incomplete Freund's Adjuvant (IF A) two weeks after the first immunization, and subsequent boosts were administered 3 weeks apart. Blood samples were collected one week after immunization. The antibody titer and specificity in seram were determined by ELISA and FACS analysis, and the results are shown in Table 6. Table 6 shows that seram from mice immunized with PCSK9 exhibited different levels of binding to Immunogen A. The highest seram dilution was about one million. The blank control was 1% (w/w) BSA. The OD450nm values shown in Table 6 are the seram titer values from 7 days after the third boost, as determined by ELISA.
[00157] Table 6. Seram titers of Harbour H2L2 transgenic mice immunized with hPCSK9-His protein, as determined
Figure imgf000020_0002
[00158] Prior to the completion of steps 3 A-C above, mice with specific immune response against hPCSK9 were selected for fusion and were given a final boost by intraperitoneal injection of lOOug hPCSK9-His. Three days later, the mice were sacrificed, and their splenocytes were collected. NH4OH was added to the splenocyte samples to a final concentration of 1% (w/w) to lyse red blood cells in the sample. The samples were centrifuged at 1000 rpm and washed three times with DMEM culture media. The viability of the splenocytes was determined, and viable splenocytes were then fused with mouse myeloma cells SP2/0 (ATCC) at a ratio of 5: 1 by the high efficiency electric fusion method (see Methods in Enzymology, Vol. 220).
[00159] Fused cells were re-suspended in DMEM media containing 20% FBS and lx hypoxanthine- aminopterin-thymidine (HAT) medium (w/w), and the concentration was adjusted to 105 cells/200uL. 200uL of fused cells were added to each well of 96-well plate, which was incubated at 37°C, 5% C02. 14 days after cell fusion, hybridoma supernatants were collected and screened by ELISA. Clones with an OD450nm greater than 0.5 by ELISA were expanded in a 24-well plate containing DMEM with 10% (w/w) heat-inactivated FBS at 37°C, 5% (v /v) C02. Supernatants were collected after 3 days of culturing. The antibody isotypes were determined, and their ability to bind to recombinant PCSK9D374Y protein (gain-of-function mutant; prepared using the method of Example 1) was determined by ELISA (see Example 3). A receptor ligand binding assay was performed to determine the blocking activity of the hybridoma supernatant (see Example 4), an assay to measure degradation of LDLR was performed (see Example 5), and a blocking assay to measure PCSK9-mediated cellular LDL uptake was carried out (see Example 6).
[00160] Based on the 24-well plate screening results, clones that bound to PCSK9, blocked the receptor-ligand interaction between PCSK9 and LDLR, and inhibited PCSK9-mediated reduction in LDL uptake were selected and subcloned. Subcloning was carried out by limited dilution in a 96-well plate with DMEM media containing 10% (v/v) FBS at 37°C and 5% (v/v) C02. After 10 days of culturing, the supernatants were collected and preliminarily screened by assessing their ability to bind to recombinant human PCSK9, PCSK9D374Y, cyno monkey PCSK9 (SEQ ID NO: 63, prepared using the method of Example 1) and mouse PCSK9 (UniProtKB ID Q80W65; SEQ ID NO: 65, prepared using the method of Example 1), as determined by ELISA, or their ability to block binding of PCSK9 or PCSK9D374Y to LDLR.
[00161] Clones that met the selection criteria were expanded in DMEM media containing 10% (w/w) FBS at 37°C, 5% (v/v) C02 and frozen in liquid nitrogen so that the hybridoma cells could be used for subsequent antibody production and purification.
[00162] (Step 4) Production and purification of lead candidate antibodies
[00163] The antibody concentrations from the hybridoma cells were low, about 1-lOug/mL, and there was a large variation in antibody concentrations. In addition, the FBS and the components of the culture medium can interfere with the analysis. Therefore, it was necessary to perform small scale antibody production and purification (1-5 mg).
[00164] Hybridoma cells from Example 2 were cultured in T-75 culture flasks using Hybridoma serum free medium (Invitrogen), and passaged for 3 generations. Cells were transferred to 2L culture flasks when the hybridoma cells were in good condition. 500mL of production media was added to each culture flask, and the cell density was adjusted to 105 cells/mL. The culture flasks were placed onto a rotary incubator at 37°C with a rotating speed of 3 cycles per minute. Hybridoma cells were cultured for 14 days, after which the supernatant was collected and the cells were removed. The supernatants were then filtered through 0.45 micron filtration. The culture supernatants were then ready for purification or storage at -30°C.
[00165] Monoclonal antibodies were purified by passing the hybridoma culture supernatants through 2mL Protein G columns (GE Healthcare). Protein G columns were first equilibrated with PBS buffer (pH7.2), and the hybridoma culture supernatants were then applied to the equilibrated Protein G columns with a constant flow rate of 3mL/minute. The columns were then washed with 4 volumes of PBS buffer. The anti-PCSK9 antibodies were then eluted with elution buffer (0.1M acetate buffer, pH2.5), and the UV absorbance of the eluates were monitored using a UV detector (A280 UV absorption peak). 10% of 1.0M Tris-HCL buffer was added to the eluates to neutralize the pH, and the samples were sterile-filtered by passing them through 0.22 micron filters. Sterile-filtered purified anti- PCSK9 antibodies were obtained.
[00166] The concentrations of purified anti-PCSK9 antibodies were determined by UV absorbance (A280/1.4), and the purity and endotoxin level (Lonza kit) were measured. The results of the analyses are shown in Table 7. The purified anti-PCSK9 antibodies had endotoxin concentrations less than 1.0 EU/mg.
[00167] Table 7. Quality Control analysis of Purified Chimeric PCSK9 mAbs from Hybridoma
Figure imgf000022_0001
[00168] Example 3 - Characterization of lead candidate antibodies
[00169] (part A) Detection of the binding of anti-PCSK9 antibodies to recombinant PCSK9 and PCSK9D374Y protein by ELISA
[00170] The binding of purified anti-PCSK9 antibodies from Example 2 to recombinant human PCSK9 protein (Immunogen A), PCSK9D374Y and cyno PCSK9 protein was analyzed by ELISA.
[00171] Streptavidin (Cat No. 85878, Sigma) was diluted with PBS to a final concentration of lug/mL, lOOuL were added to each well of a 96-well ELISA plate that was then sealed with a plastic film and incubated overnight at 4°C. The next day, the plates were washed twice with wash buffer [0.01% (v/v) Tween 20], then blocked with blocking buffer [0.01% (v/v) 0.1% BSA (w/w) Tween 20] at room temperature for 2 hours. The blocking buffer was aspirated away, and lOOuL of 0.5ug/mL biotinylated immunogen A (hPCSK9-His), biotinylated PCSK9D374Yor biotinylated cyno PCSK9 proteins (Biotin labeling kit, Invitrogen) were added to each well and incubated at 37°C for 1 hour. Unbound PCSK9 proteins were removed by washing the plate three times with wash buffer [0.01% (v/v) Tween 20]. 200uL of purified PCSK9 antibodies from Example 2 were added to each well and incubated for 1 hour at 37°C. The plates were washed twice with wash buffer [0.01% (v/v) Tween 20], and 200uL HRP-conjugated secondary antibody (Sigma) was added to each well and incubated for 2 hours at 37°C. After the plates were washed three times with wash buffer, lOOul TMB substrate were added to each well and incubated for 30 minutes at room temperature. lOOul stop solution (0. IN HC1) were added to each well to stop the reaction. The absorbance at 450nm was measured using an ELISA plate reader (384plus SpectraMax, Molecular Device). The results are shown in Figures 2-4 and in Tables 8-10. The IgG control was rat IgG.
[00172] Table 8. Binding activities of chimeric anti-PCSK9 mAbs to human PCSK9-His, as measured by ELISA
Figure imgf000022_0002
Ar itibody concentra Ltion (nM)
Clone ID 66.67 6.67 0.67 0.067 0.0067 0.00067 0.000067 0
74C10A8 2.98 2.99 2.89 1.15 0.30 0.17 0.19 0.22
76A1B11 2.85 2.85 2.66 0.86 0.27 0.21 0.20 0.18
139G1C5 2.93 2.94 2.87 1.55 0.36 0.21 0.19 0.22
152G2F7 2.91 2.91 2.42 0.60 0.25 0.21 0.18 0.17
96F8C6 2.86 2.89 2.73 1.00 0.30 0.18 0.17 0.20
IgG control 0.19 0.20 0.22 0.12 0.18 0.18 0.24 0.20
103C11E8 2.72 2.71 2.41 0.68 0.21 0.15 0.11 0.14
IgG control 0.11 0.10 0.09 0.09 0.10 0.09 0.11 0.10 [00173] Table 9. Binding activities of chimeric anti-PCSK9 mAbs to human PCSK9 , as measured by
ELISA
Figure imgf000023_0001
[00174] Table 10. Binding activities of chimeric anti-PCSK9 mAbs to Cyno Monkey PCSK9-His, as measured by ELISA
Figure imgf000023_0002
[00175] Example 4 - Determination of the ability of the anti-PCSK9 antibodies to block the binding of PCSK9 and PCSK9D374Yto LDLR
[00176] Both wild type PCSK9 and its mutant PCSK9D374Y bind to LDLR, and the mutant form of PCSK9D374Y has higher binding affinity to LDLR than the wild type version. A receptor ligand binding assay was performed to determine the ability of the anti-PCSK9 antibodies to block the binding of PCSK9 and PCSK9D374Y to LDLR.
[00177] The extracellular domain of LDLR (SEQ ID NO: 64; corresponding to Ala22-Arg788 of full-length LDLR) was cloned along with a His tag (LDLRECD-His; prepared using the method of Example 1). Purified LDLRECD-His was diluted with PBS to a final concentration of l.Oug/ml, and lOOuL of diluted LDLRECD-His were added to each well of 96-well plate that was then sealed with plastic film and incubated at 4°C overnight. The plate was washed twice with wash buffer [PBS + 0.01% (v/v) Tween 20] and incubated with blocking buffer [PBS + 0.01% (v/v) BSA +1% Tween20 (w/w)] at room temperature for 2 hours. The blocking buffer was aspirated, and 50uL purified anti-PCSK9 antibodies from Example 2 were added to each well of 96-well plate. 50ul of 0.5ug/mL biotinylated recombinant hPCSK9-His or biotinylated PCSK9D374Y protein (biotinylated using the Biotin labeling kit, Invitrogen) were added to each well, mixed, and incubated at 37°C for 2 hours. The plate was washed three times with wash buffer [0.01% (v/v) Tween 20] . lOOuL HRP -conjugated streptavidin (Sigma) were then added to each well and incubated at 37°C for 1 hour. The plate was then washed three times with wash buffer, and lOOuL TMB substrate were added to each well. After 15 minutes of incubation at room temperature, the reaction was stopped by adding 50uL stop solution (0. IN HC1). The absorbance at OD450nm was measured with an ELISA plate reader (384plus SpectraMax, Molecular Devices). The results, shown in Figures 5-6 and in Tables 11-12, demonstrate that the anti-PCSK9 antibodies can block the binding of PCSK9 and PCSK9D374Y to LDLR.
[00178] Table ll.Inhibition of binding of human PCSK9 to LDLR by chimeric anti-PCSK9 mAbs, as measured by ELISA
Figure imgf000024_0001
[00179] Table 12. Inhibition of binding of human PCSK9 J /4Yto LDLR by chimeric anti-PCSK9 mAbs, as measured by ELISA
Figure imgf000024_0002
[00180] Example 5 - Assay to measure PCSK9-mediated LDLR degradation
[00181] Log phase HepG2 cells (ATCC) were digested with trypsin, centrifuged and resuspended in MEM medium (Invitrogen Cat. No. 11095-080) supplemented with 10% (w/w) fetal bovine serum. The cell density was adjusted to 3xl05 cells/mL, and cells were added to each well of 24-well plate. After the places were incubated for 6 hours to allow the HepG2 cells to adhere, the culture medium was aspirated and replenished with MEM medium supplemented with 10% (w/w) LPDS (Kalen Biomedical LLC, 880100-2) and incubated overnight. The next day, 0.4ug/mL PCSK9D374Y and anti-PCSK9 antibodies from Example 2 were mixed and added to the 24-well plate, which was incubated for 4 hours at 37°C. MEM medium supplemented with 10% LPDS was removed, and the plates were washed twice with PBS. The HepG2 cells were dissociated from the plate using Versene solution (Invitrogen) and collected. The cells were washed twice with PBS, the cell counts were determined, and the cell density was adjusted to 2 106 cells/mL with PBS. Blocking buffer [1% (w/w) FBS PBS] was added to the cell suspension, and lOOuL of the cell suspension were added to each well of 96-well FACS plate (FACS Calibur, BD) that was incubated on ice for 15 minutes, then centrifuged. The blocking buffer was aspirated away, and lOOuL of lug/uL LDLR antibody (Progen, 61009) was added to each well and incubated on ice for 15 minutes. The plates were washed twice with FACS buffer [1% (w/w) BSA HBSS], and lOOuL Alexa-488-conjugated anti-rabbit secondary antibody (Molecular Probes, A- 11034) were added to each well and incubated on ice for 15 minutes. The plates were washed three times with FACS buffer and resuspended in lOOuL FACS buffer after the final wash. The Mean Fluorescence Intensity (MFI) was determined using FACS Calibur (BD), and the results are shown in Figure 7 and in Table 13. The IgG control was rat IgG, and the values in the table are the mean fluorescence intensities of the cell populations. The results showed that the anti-PCSK9 antibodies inhibited PCSK9D374Y-induced LDLR degradation in HepG2 cells in a dose dependent manner.
[00182] Table 13. Effect of chimeric anti-PCSK9 mAbs on human PCSK9D374Y-mediated down regulation of LDLR
Figure imgf000025_0001
[00183] Example 6 - Assay to measure inhibition of PCSK9-mediated LDL uptake in HepG2 cells
[00184] Log phase HepG2 cells (ATCC) were digested with trypsin, centrifuged and resuspended in MEM medium (Invitrogen Cat. No. 11095-080) supplemented with 10% (w/w) fetal bovine serum. The cell density was adjusted to 5xl05 cells/mL, and cells were added to a PDL-coated (Millipore, A-003-E) 96-well plate. After the places were incubated for 6 hours to allow the HepG2 cells to adhere, the culture medium was aspirated and replenished with MEM medium supplemented with 10% (w/w) LPDS (Kalen Biomedical LLC, 880100-2) and incubated overnight. lOug/mL of BODIPY® FL LDL (Invitrogen, L3483), 0.5ug/mL of PCSK9D374Y , and anti- PCSK9 antibodies from Example 2 were mixed and added to the 96-well plate, which was incubated for 6 hours. The culture medium was aspirated, the plates were washed twice with PBS, and the fluorescence intensity at 520nm/485nm was measured using an M5 plate reader (Molecular Device). The results, shown in Figure 8 and in Tables 14 and 15, indicated that the anti-PCSK9 antibodies blockedPCSK9D374Y-mediated inhibition of LDL uptake in a dose-dependent manner.
[00185] Table 14.Effect of chimeric anti-PCSK9 mAbs on human PCSK9D374Y-mediated inhibition of LDL uptake
Figure imgf000025_0002
Table 15.Effect of chimeric anti-PCSK9 mAbs on human PCSK9 J /4Y -mediated inhibition of LDL
Figure imgf000026_0001
[00187] Example 7 - Determination of the amino acid sequences in the variable regions of the anti- PCSK9 antibodies
[00188] Total RNA isolation: After the supernatants from the hybridoma subclones obtained from Example 2 were characterized (i.e., validation and determination of bioactivity, Examples 3-6), 5xl07 hybridoma cells were collected by centrifugation. lmL Trizol was added to the cell pellets, mixed and transferred to 1.5mL centrifuge tubes, and incubated at room temperature for 5 minutes. 0.2mL chloroform was added to the samples and vortexed for 15 seconds. After standing for 2 minutes, the mixtures were centrifuged at 12000g at 4°C for 5 minutes. The supernatants were collected and transferred to new 1.5mL centrifuge tubes, 0.5mL isopropyl alcohol was added and mixed gently, and the samples were incubated at room temperature for 10 minutes. The samples were centrifuged at 12000g at 4°C for 15 minutes. The supernatants were aspirated, and the precipitates were washed with lmL 75% (v/v) ethanol. The mixtures were centrifuged at 12000g at 4°C for 5 minutes, the supernatants were decanted, and the precipitates were air-dried. Total RNA was obtained by adding DEPC-treated water to the precipitates (55°C water bath for 10 minutes).
[00189] Reverse transcription and PCR: lug of RNA and reverse transcriptase were added to a reaction mixture of a final volume of 20uL, and the mixture was incubated at 42°C for 60 minutes and then at 70°C for 10 minutes to terminate the reaction. A 50uL PCR reaction mixture was prepared, containing luL cDNA, 25 pmol of each primer, luL DNA polymerase, 250umol dNTPs, and the buffer system. The PCR program settings were as follows: denaturation at 95°C for 3 minutes, 35 cycles of denaturation (95°C for 30 seconds), annealing (55°C for 30 seconds) and elongation (72°C for 35 seconds), followed by a final extension at 72°C for 5 minutes to obtain the PCR product. The commercially available reverse transcription kit used was PrimeScript RT Master Mix (Takara, RR036), and the commercially available Q5 ultra-fidelity polymerase PCR kit was from NEB (M0492).
[00190] Cloning and sequencing: 5uL PCR products were examined by agarose gel electrophoresis, and the samples were recovered from the agarose gel using NucleoSpin Gel & PCR Clean-up kit (MACHEREY-NAGEL, 740609). Ligation reaction: To a 50ng sample, 50ng T vector, 0.5uL ligase, and luL buffer were added and brought to a final volume of lOuL with water. The reaction mixture was incubated at 16°C for 30 minutes using the T4 DNA Ligase (NEB, M0402). 5uL ligation product was added to lOOuL competent cells (Ecos lOlcompetent cells, Yeastern, FYE607), which were incubated on ice for 5 minutes, heat shocked at 42°C for 1 minute and incubated on ice again for 1 minute. Cells were recovered by adding 650uL SOC medium without antibiotics and incubating at 37°C in a shaking incubator for 30 minutes at 200 rpm. 200uL of each bacterial culture was spread onto an LB agar plate containing antibiotics at 37°C overnight. The next day, PCR reactions were set up using the T vector primers M13F and M13R. Pipette tips were used to pick bacterial colonies and were dipped into the PCR reaction mixture and pipetted up and down. Half of the reaction mixture was transferred to an LB agar plate containing lOOnM ampicillin. After the PCR reactions, 5uL of the PCR products were removed and examined by agarose gel electrophoresis, and positive samples were sent for sequencing and analysis (Kabat, 1991, "Sequences of proteins of objective interest," the NIH, Bethesda, MD). The sequencing results are shown in Tables 1-2.
[00191] Example 8 - Conversion, expression and purification of fully human anti-PCSK9 antibody [00192] (Step 1) Plasmid construction and preparation: Sequences of the anti-PCSK9 antibody heavy and light chain variable regions were obtained according to Example 7. The anti-PCSK9 antibodies' heavy chain variable region sequences were subcloned into expression vectors containing a signal peptide and a human heavy chain IgGl constant region. The anti-PCSK9 antibodies' light chain variable region sequences were subcloned into expression vectors containing a signal peptide and a human antibody light chain kappa constant region. The recombinant plasmids were verified and confirmed by sequencing (the sequencing method was the same as in Example 7).
Alkaline lysis was performed using a reagent kit (MACHEREY-NAGEL) to improve the purity and quality of the recombinant plasmids, and the plasmids were filtered through 0.22uM filters (Millpore). The purified plasmids were used for transfection.
[00193] (Step 2) Transfection: HEK293E cells (Invitrogen) were cultured in FreeStyle 293 medium (Invitrogen) at 37°C, 130RPM, 8% C02 (v/v). HEK293E cells were adjusted to l-1.5xl06/mL cell density for transfection. 10% (v/ v) F68 (Invitrogen) was added to the FreeStyle 293 medium to a final concentration of 0.1% (v/ v), as Medium A. 5mL of Medium A and 200ug/mL PEI (Sigma) were mixed to generate Medium B. 5mL of Medium A and lOOug/mL recombinant plasmid from step 1 were mixed to generate Medium C. After 5 minutes of incubation, Medium B and Medium C were mixed and incubated for 15 minutes to generate Mixture D. lOmL of Mixture D were slowly added to lOOmL HEK293E cells with continuous stirring to avoid local accumulation of PEI.
HEK293E cells were incubated overnight while shaking. The next day, peptone was added to a final concentration of 0.5% (w/v). On about day 5-7, the antibodies' titers were determined. On about day 6-7, the HEK293E cultures were centrifuged (30 minutes, 3500RPM), and the supernatants were collected and filtered through 0.22uM filters for purification.
[00194] (Step 3) Antibody purification: Protein A columns (GE) were washed with 0.1M NaOH for 30 minutes or with 5 bed volumes of 0.5M NaOH to get rid of endotoxin. Columns that had not been used in a long time were soaked in 1M NaOH for at least 1 hour, washed with endotoxin-free water to a neutral pH, and washed with 10 bed volumes of 1% Triton X100. The columns were then equilibrated with 5 bed volumes of PBS (PBS phosphate buffer, pH7.2). The filtered supernatants from Step 2 were loaded onto the columns, and the flow through was collected, if necessary. The columns were washed with 5 bed volume of PBS and then eluted with 5 bed volumes of 0.1M Glycine-HCl pH3.0. The eluate containing anti-PCSK9 antibody was neutralized with 0.5 bed volume of 1M Tris-HCl (NaCl 1.5M) pH8.5. The human anti-PCSK9 antibodies were dialyzed in IX PBS for 4 hours, to avoid endotoxin contamination. After dialysis, anti-PCSK9 antibodies' concentrations were determined by
spectrophotometry or a reagent kit, the purities of the antibodies were determined by HPLC-SEC, and the levels of endotoxin were determined by an endotoxin test kit (Lonza). The fully human anti-PCSK9 antibodies were characterized, and the results are shown in Figures 9-11 and 17-19, and in Tables 16-21.
[00195] Table 16. Binding activities of fully human anti-PCSK9 mAbs to human PCSK9, as measured by ELISA
Figure imgf000027_0001
[00196] Table 17. Binding activities of fully human anti-PCSK9 mAbs to human PCSK9, as measured by ELISA
Figure imgf000028_0001
[00197] Table 18. Binding activities of fully human anti-PCSK9 mAbs to human PCSK9 J , as measured by ELISA
Figure imgf000028_0002
Table 19. Binding activities of fully human anti-PCSK9 mAbs to human PCSK9 U3 , as measured
Figure imgf000028_0003
[00199] Table 20. Binding activities of fully human anti-PCSK9 mAbs to Cyno Monkey PCSK9, as measured by ELISA
Figure imgf000028_0004
[00200] Table 21. Binding activities of fully human anti-PCSK9 mAbs to Cyno Monkey PCSK9, as measured by ELISA Antiboc y concen tration (nN 4)
Clone ID 66.67 6.67 0.67 0.067 0.0067 0.00067 0.000067 0
103C11E8 2.97 2.96 2.81 1.15 0.29 0.21 0.11 0.07
IgG control 0.81 0.15 0.08 0.06 0.07 0.07 0.07 0.07
[00201] Receptor ligand binding assays confirmed that fully human anti-PCSK9 antibodies, generated by conversion of the chimeric anti-PCSK9 antibodies, are able to block the binding of PCSK9 to LDLR. The results, shown in Figures 12-13 and 20-21, and in Tables 22-25, indicated that the fully human anti-PCSK9 antibodies can block the binding of PCSK9 and PCSK9D374Y to LDLR. The IgG control was human IgG.
[00202] Table 22.Inhibition of binding of human PCSK9 to LDLR by fully human anti-PCSK9 mAbs, as measured by ELISA
Figure imgf000029_0001
[00203] Table 23.Inhibition of binding of human PCSK9 to LDLR by fully human anti-PCSK9 mAbs, measured by ELISA
Figure imgf000029_0002
[00204] Table 24.Inhibition of binding of human PCSK9U374 Y to LDLR by fully human anti-PCSK9 mAbs, as measured by ELISA
Figure imgf000029_0003
[00205] Table 25.Inhibition of binding of human PCSK9 J /4Y to LDLR by fully human anti-PCSK9 mAbs, as measured by ELISA
Figure imgf000030_0001
[00206] Fully human anti-PCSK9 antibodies, generated by conversion of the chimeric anti-PCSK9 antibodies, are able to inhibit PCSK9D374Y-mediated reduction of LDL uptake. The results, shown in Figures 14-16 and 22, and in Tables 26-27, indicated that fully human anti-PCSK9 antibodies blocked PCSK9D374Y-mediated inhibition in LDL uptake in a dose-dependent manner. The IgG control was human IgG.
[00207] Table 26.Effect of fully human anti-PCSK9 mAbs on human PCSK9D374Y-mediated inhibition of LDL
Figure imgf000030_0002
[00208] Table 27.Effect of fully human anti-PCSK9 mAbs on human PCSK9 J -mediated inhibition of LDL
Figure imgf000030_0003
[00209] Example 9 - Determination of binding and dissociation constants by Octed Red 96
[00210] Dissociation constants were determined by Octed red 96 (Fortiebio). The detailed operation and methods were followed according to the specifications of the instrument provided by the manufacturer. Briefly, an AHC sensor (Anti-human Fc sensor, Fortiebio) was used for the affinity determination. The anti-PCSK9 antibody was diluted to lOug/mL in PBS buffer (pH7.4) containing 0.1% (w/w) BSA and 0.02% (v/v) Tween 20 and incubated with the AHC sensor. Five different concentrations of recombinant hPCSK9-His (two-fold serially diluted from a starting concentration of lOOnM or 400nM) were incubated with the antibody-loaded AHC sensor at 30°C for 3 minutes. The reaction mixture was further incubated in PBS buffer (pH7.4) containing 0.1% (v/w) BSA and 0.02% (v/v) Tween 20 at 30°C for 5 minutes. The association and dissociation signals of anti-PCSK9 antibodies to Immunogen A were recorded in real time using Octet Red 96. The affinity, association and dissociation constants were determined using Octet User software, and the results are shown in Table 28. [00211] Table 28.Binding kinetics and affinities of fully human anti-PCSK9 mAbs to human PCSK9-His protein, as determined by Octet Red 96
Figure imgf000031_0001
[00212] Example 10 - Determination of binding and dissociation constants by Biacore
[00213] Anti-human Fc IgG was immobilized on flow cells 1 and 2: HBS-EP+ (lOmM HEPES, 150mM NaCl, 3mM EDTA, 0.05% P20, pH 7.4) was used as running buffer, and the immobilization of anti-human Fc IgG was carried out using the immobilization wizard template. Flow cells 1 and 2 of a Series S CM5 sensor chip were activated with freshly -mixed 50mmol/L NHS and 200mmol L EDC. 20ug/mL of anti-human Fc IgG diluted in lOmM NaAC (pH 4.5) was injected into the activated flow cells 1 and 2. The remaining active coupling sites were blocked with 1M ethanolamine.
[00214] Recombinant His-tagged hPCSK9 protein was diluted to 50nM, followed by four 2-fold serial dilutions with HBS-EP+ buffer. The His-tagged hPCSK9 protein concentrations were OnM, 3.125nM, 6.25nM, 12.5nM, 25nM and 50nM. KD measurements were carried out with HBS-EP+ as the running buffer. Each antibody was injected over the CM5 sensor flow cell 2 with a flow rate of lOuL/min to reach response 230 RU. Prepared His-tagged hPCSK9 protein was then injected over flow cells 1 and 2, at a flow rate of 30uL/min for 180sec. Buffer flow was maintained for 400 seconds for dissociation measurements (30uL/min). To remove the tested antibody from the surface, lOmM glycine-HCl pH 1.5 was injected for 20 seconds (30uL/min). Flow cell 1 was used as reference flow cell. The above steps were repeated for each concentration of serially-diluted His-tagged hPCSK9 protein. The KD value for each antibody was evaluated using Biacore T200 evaluation software 1.0, and the data was fit with a 1: 1 binding model. The results are shown in Table 29.
[00215] Table 29.Binding kinetics and affinities of fully human anti-PCSK9 mAbs to human PCSK9-His
Clone ID KD (M) ka (l Ms) Mi/s)
74C10A8 ND ND ND
76A1B11 ND ND ND
139G1C5 7.09E-11 1.71E+05 2.40E-05
152G2F7 ND ND ND
96F8C6 2.33E-09 9.13E+04 3.92E-05
103C11E8 1.42E-09 3.82E+04 5.41E-05 Alirocumab, Regeneron 3.94E-10 3.18E+04 8.06E-05
Evolocumab, Amgen <1.22E-10 <1.0E+05 8.22E-04
[00216] Example 11 - Effect of PCSK9 antibody on PCSK9-mediated LDLR degradation
[00217] 9-10 week old C57BL/6 mice were housed and maintained under SPF conditions. Mice were randomized into 5 groups, with 4 mice in each group. At Day -1, mice were intraperitoneally administered 30mg/kg, 3mg/kg, or 0.3mg/kg test anti-PCSK9 antibody (e.g., 139G1C8 or 96F8C6) control IgG or vehicle. The next day, all mice were intravenuously administered recombinant human PCSK9 protein (30 ug/mouse). One hour after injection with PCSK9, the mice were sacrificed and their livers were harvested. Western blot analysis of liver lysates was performed using an antibody against mouse LDLR (mLDLR) and an antibody against mouse β-actin (πιβ-actin). The protein bands were quantified using densitometry, and the ratio of mLDLR to πιβ-actin is plotted. The results, shown in Figures 23 A and B, indicated that the anti-PCSK9 antibodies inhibited PCSK9-mediated LDLR degradation in vivo.
[00218] Example 12 - Antibody thermostability, measured by differential scanning calorimetry (DSC)
[00219] Fully human anti-PCSK9 antibodies were adjusted to lmg/mL and a final volume of about 700uL with sample buffer. The parameters were set up as follows (VP -DSC): Starting Temperature 30°C; Final Temperature 100°C; Scan rate 50°C/hour; Number of Rescans 0; PreScan Thermostat 3 min; PostScan Thermostat 0 min; Post Cycle Thermostat 25°C; Filtering Period 25 seconds; Feedback Mode/Gain None; Cell Refill Parameters 35°C. The resulting protein-buffer thermograms were processed by subtracting a corresponding buffer-buffer scan and subsequently fitting a baseline to the trace. The 7ms were recorded at each peak maxima observed in the thermograms using Origin™ 7.0 software. The results are shown in Figure 24.
[00220] Example 13 - Antibody freeze/thaw stability
[00221] The freeze/thaw stabilities of the fully human anti-PCSK9 antibodies were characterized as follows. A lOOuL aliquot from the frozen stocks of each anti-PCSK9 antibody was thawed at room temperature. Once fully thawed, the samples were then rapidly frozen in the -80°C freezer and kept at -80°C for at least two hours before being thawed again at room temperature. The samples went through three identical freeze/thaw cycles. Visual inspection was used to check for precipitation. 20uL aliquots were removed from the samples for size-exclusion chromatography (SEC) analysis after three freeze/thaw cycles. The stability of the fully human anti-PCSK9 antibodies before and after the freeze/thaw cycles were analyzed by HPLC-SEC characterization. The results, shown in Figure 25, demonstrated that after three freeze/thaw cycles, monomer IgGs accounted for more than 95% of each of the anti-PCSK9 antibodies tested.
[00222] Example 14 - Antibody solubility
[00223] The solubility of the fully human anti-PCSK9 antibodies was characterized by concentrating lOmg IgG using centrifugal filters (Amicon Ultra-0.5mL 30K) at 14000g at 4°C down to >100mg/mL. 2mL or more of IgG was added into the centrifugal filters and concentrated at 14000g at 4°C. The setting time of centrifuging was 2 min, 3 min, 5 min, 8 min, 15 min, and 20 min, and each time 20uL were aliquoted to a collection tube to measure the concentration with a nanodrop at A280. The centrifugation was finished when the concentration reached lOOmg/mL. For HPLC-SEC characterization, 6uL of concentrated samples were injected into an HPLC-SEC column, and the percentages of monomers and aggregates were determined based on the peak area. The results, shown in Figure 26, demonstrated that all of the fully human anti-PCSK9 antibodies tested had a solubility over lOOmg/mL and that monomer IgGs were higher than 95% for all of the anti-PCSK9 antibodies tested.
[00224] Example 15 - Affinity maturation of anti-PCSK9 antibodies
[00225] (Step 1) CDR mutagenesis library construction and validation [00226] 1. Construction of phagemid expression vector and verification of scFv display: Sequences of the 98F8C6 anti-PCSK9 antibody heavy and light chain variable regions were obtained according to the methods of Example 6. Nucleic acids encoding the heavy chain variable region, linker and light chain variable region were assembled using overlapping PCR to generate an scFv. The PCR product was ligated into a phagemid vector, which was then transformed into TGI cells. Positive clones from ampicillin selection plates were selected, and their phagemid expression vectors were recovered and sequenced.
[00227] 2. Design of CDR mutagenesis libraries: CDR positions were defined by Kabat numbering. In a typical antibody structure, the HCDR3 and LCDR3 domains provide the core interface for antigen recognition and are surrounded by other CDRs. Therefore, in the absence of structural information regarding which of the CRDs of the anti-PCSK9 antibodies contribute to antigen binding, HCDR3 and LCDR3 were initially targeted for library generation. Amino acid residues on the CDR3 loop were randomized using NNK primers (N = any nucleotide, K =
G,T). The mutation rate of each position was kept at 50% to control the number of mutations in each variant and therefore maximally retain the original binding epitope. Long CDRs (>10 amino acids) were separated into two or three overlapping segments. Due to the size of the HCDR3 of clone 96F8C6, it was separated into two overlapping segments, CDR-H3-1 and CDR-H3-2, and each segment was mutated in a separate random library. The primers for
CDR mutagenesis were as follows:
[00228] SEQ ID NO: 70 (CDRH3-l_mut-F)
gtgtattactgtgcgaga(NNKNNKNNKNNKNNKNNKNNKNN^
[00229] SEQ ID NO: 71 (CDRH3-2_mut-F)
attactatggttcggggagt(NNKNNKNN^
[00230] SEQ ID NO: 72 (CDRL3_mut-F)
attttgcaactMtactgc(NNKNNKNNKNNKNNKNNKNNKNNK
[00231] 3. Library construction: Random mutagenesis libraries were constructed using overlapping PCR. PCR products were digested using the restriction enzyme Sfil for 2 hours at 50°C and were then ligated into an Sfil- digested phagemid using T4 ligase at 16°C overnight. Each library was electro-transformed into TGI E.coli.
[00232] 4. Library validation: The size of each library was between 108 and 109 clones. 50 clones were sequenced for each library, and sequence alignments with the wild type sequence were used to evaluate the quality of the mutation libraries, such as assessment of the mutation positions and the mutation rates.
[00233] 5. Library packaging and titration: After the amplification of the bacteria libraries, helper phage was added to package phage particles for the following panning steps. The titers of the purified phage libraries were measured by counting newly -infected TGI clones.
[00234] (Step 2) Affinity -driven library panning
[00235] The CDR mutagenesis ScFv phage libraries were panned using biotinylated soluble hPCSK9 as antigen in solution phase, under equilibrium conditions. The centrifuge tubes and streptavidin-magnetic to be used were first blocked with 2% MPBS (2% milk in PBS) for 2 hours at room temperature. The libraries were pre- depleted with the beads in 2% MPBS and then mixed with biotin-hPCSK9 in the blocked tubes and incubated on a rotator at room temperature for 2 hours. The concentration of antigen used was lOnM in the first round of panning, and was reduced 10-fold at a time in the subsequent rounds. The ScFv-antigen-biotin complexes were mixed with streptavidin-beads and incubated for 15 minutes. The beads were collected with a magnet, transferred to 1.5 microcentrifuge tubes, and washed 5 times each with MPBST (2% milk, 0.5% Tween20 in PBS), PBST (0.5% Tween 20 in PBS), MPBS (2% milk in PBS) and PBS. Bound phage was eluted from the beads with ImL trypsin (lOug/mL in PBS) at 37°C for 30 minutes. Beads were drawn to one side of the tube using a magnet, and the solution containing eluted phage was transferred to a tube containing 4mL TGI (A600-0.6) for titration of the panned library. [00236] After each round of panning, the output clones were sequenced to determine the enrichment of mutations. After 3 or 4 rounds of panning, the resulting clones were selected for screening.
[00237] (Step 3) ELISA screening of affinity -enhanced binding variants
[00238] More than 500 single clones were selected after 3 or 4 rounds of panning. IPTG induction was used to induce expression of the ScFv variants, and clones with strong ELISA signal were sent for sequencing.
[00239] (Step 4) Eukaryotic production of affinity improved variants and affinity characterization
[00240] VH and VL sequences from enriched clones were cloned into IgG expression vectors, inserted between leader sequence and constant region accordingly. The resulting vectors were transfected into HEK293 cells by transient transfection. After 5-7 days, the IgGs in the culture supernatant were purified by affinity purification using Protein A column, as described in Example 8. The VH sequences of the affinity -improved variants are listed in Table 30. The light chain sequences of these variants were the same as those of the light chain of antibody 98F8C6, e.g., the LCDR 1-3 of SEQ ID NOs:38-40, respectively, or alight chain variable region of SEQ ID NO:37, or its coding sequence SEQ ID NO:58.
Figure imgf000034_0001
SEQ ID NO: 96
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 78
AF-mab023_06 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYFQSGSYNY
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYFQ YYYGMDV S GS YNYYYYGMD VWGQGTTVT VS S
SEQ ID NO: 97
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 79
AF-mab023_07 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYYQQGSYN
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYYQ YYYYGMDV QGSYNYYYYGMDVWGQGTTVTVSS
SEQ ID NO: 98
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 80
AF-mab023_08 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYIADGSYNY
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYIA YYYGMDV D GS YNYYYYGMD VWGQGTTVT VS S
SEQ ID NO: 99
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 81
AF-mab023_10 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYYDSGRYN
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYYD YYYYGMDV S GRYNYYYYGMD VWGQGTT VTVS S
SEQ ID NO: 100
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 82
AF-mab023_ll VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYYQEGSYN
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYYQ YYYYGMDV EGSYNYYYYGMD VWGQGTT VTVSS
SEQ ID NO: 101
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 83
AF-mab023_12 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYYTEGGYN
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYYT YYYYGMDV EGGYNYYYYGMD VWGQGTTVT VS S
SEQ ID NO: 102
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 84
AF-mab023_13 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYYTEGSYNY
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYYT YYYGMDV EGSYNYYYYGMD VWGQGTTVTVSS
SEQ ID NO: 103
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 85
AF-mab023_14 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYYADGGYN
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYYA YYYYGMDV DGGYNYYYYGMD VWGQGTTVTVSS SEQ ID NO: 104
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 86
AF-mab023_15 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYYQD GNYN
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYYQ YYYYGMDV DGNYNYYYYGMDVWGQGTTVTVSS
SEQ ID NO: 105
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 87
AF-mab023_16 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYYQD GKYN
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYYQ YYYYGMDV D GKYNYYYYGMD VWGQGTT VT VS S
SEQ ID NO: 106
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 88
AF-mab023_17 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYTGMGEYN
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYTG YYYYGMDV MGEYNYYYYGMD VWGQGTTVT VS S
SEQ ID NO: 107
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 89
AF-mab023_18 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYYADGDYN
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYYA YYYYGMDV D GD YNYYYYGMD VWGQGTT VTVS S
SEQ ID NO: 108
EVQLVESGGGLVQPGGSLRLSCAASGITFSSYWMSW SEQ ID NO: 90
AF-mab023_19 VRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTIS DRGTYYYASGKYN
RDIAKNSLYLQMNSLRAEDTAVYYCARDRGTYYYA YYYYGMDV S GKYNYYYYGMD VWGQGTT VTVS S
[00242] The final dissociation constant (KD) of the affinity -enhanced antibodies against human PCSK9 were measured by Biacore as described in Example 10, and the data are shown in Table 31. The effect of the affinity- enhanced antibodies on recombinant PCSK9D374Y-induced LDL uptake was determined, and the results are shown in Figures 27 A and B, and in Table 32.
[00243] Table 31. Binding kinetics and affinities of affinity-enhanced human anti-PCSK9 mAbs to human
Figure imgf000036_0001

Claims

AF-mab023 11 5.53E+05 1.44E-04 2.6E-10 AF-mab023 12 4.80E+05 8.47E-05 1.76E-10 AF-mab023 13 4.55E+05 1.78E-04 3.92E-10 AF-mab023 14 5.34E+05 1.64E-04 3.06E-10 AF-mab023 15 4.77E+05 1.82E-04 3.81E-10 AF-mab023 16 3.19E+05 1.43E-04 4.47E-10 AF-mab023 17 3.51E+05 1.09E-04 3.1E-10 AF-mab023 18 5.51E+05 1.61E-04 2.92E-10 AF-mab023 19 2.26E+05 3.51E-04 1.55E-09 [00244] Table 32.Effect of affinit -enhanced human anti-PCSK9 mAbson human PCSK9U374Y-mediated inhibition of LDL uptake [00245] While the invention has been described in detail, and with reference to specific embodiments thereof, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention. REFERENCES Mozaffarian et al., 2015, Circulation.13 l(4):e29-322 Gencer et al., 2015, Swiss Med Wkly. 145:wl4094 Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci. 764:536-546 Kabat, 1991, "Sequences of proteins of objective interest," the NIH, Bethesda, MD Lonberg and Huszar, 1995, Internal Rev. Immunol. 13 :65-93 Lonberg et al., 1994, Nature 368: 856-859 Rallidis and Lekakis, 2016, Hellenic J Cardiol. 57(2):86-91 Sambrook and Russell, 1989, Molecular cloning: a laboratory manual, New York : Cold Spring Harbor Laboratory Press, 2nd ed CLAIMS We claim:
1. An isolated monoclonal antibody or antigen-binding fragment thereof comprising:
(1) an LCDR1 having the polypeptide sequence of SEQ ID NO: 38;
(2) an LCDR2 having the polypeptide sequence of SEQ ID NO: 39;
(3) an LCDR3 having the polypeptide sequence of SEQ ID NO: 40;
(4) an HCDR1 having the polypeptide sequence of SEQ ID NO: 34;
(5) an HCDR2 having the polypeptide sequence of SEQ ID NO: 35; and
(6) an HCDR3 having the polypeptide sequence of one selected from the group consisting of SEQ ID NOs: 36 and 73-90;
wherein the antibody or antigen-binding fragment thereof binds PCSK9.
2. An isolated monoclonal antibody or antigen-binding fragment thereof comprising
LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of:
(1) SEQ ID NOs: 6, 7, 8, 2, 3 and 4, respectively
(2) SEQ ID NOs: 14, 15, 16, 10, 11, and 12, respectively;
(3) SEQ ID NOs: 22, 23, 24, 18, 19, and 20, respectively;
(4) SEQ ID NOs: 30, 31, 32, 26, 27, and 28, respectively; or
(5) SEQ ID NOs: 46, 47, 48, 42, 43, and 44, respectively; wherein the antibody or antigen-binding fragment thereof binds PCSK9.
3. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region having the polypeptide sequence of at least 85% identical to SEQ ID NO: 33, and a light chain variable region having the polypeptide sequence of at least 85% identical to SEQ ID NO: 37.
4. The isolated monoclonal antibody or antigen-binding fragment of any of claims 1 to 3, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to one selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33,
4 land 91-108, and a light chain variable region having a polypeptide sequence at least 95% identical to one selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37 and45, respectively.
5. The isolated monoclonal antibody or antigen-binding fragment of any one of claims 1 to 4, comprising:
a. a heavy chain variable region having the polypeptide sequence of one selected from the group consisting of SEQ ID NOs: 33 and 91-108, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 37
b. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 5; c. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 13;
d. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 21;
e. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 25, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 29; or
f. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 41, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 45.
6. The isolated monoclonal antibody or antigen-binding fragment of any of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is chimeric.
7. The isolated monoclonal antibody or antigen-binding fragment of any of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is human.
8. The isolated monoclonal antibody or antigen-binding fragment of claim 7, comprising a human heavy chain IgGl constant region, and a human antibody light chain kappa constant region constant region.
9. An isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment of any of claims 1 to 8.
10. A vector comprising the isolated nucleic acid of claim 9.
11. A host cell comprising the nucleic acid of claim 9.
12. A pharmaceutical composition, comprising the isolated monoclonal antibody or antigen- binding fragment of any of claims 1 to 8 and a pharmaceutically acceptable carrier.
13. A method of blocking binding of PCSK9 to LDLR, or augmenting uptake of LDL in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 12.
14. A method of treating a lipid disease, disorder or condition, a metabolic disease, disorder or condition, an inflammatory disease, disorder or condition, or an infectious disease, disorder or condition in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 12.
15. A method of treating a lipid disorder, metabolic disease or inflammatory disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 12, wherein the lipid disorder, metabolic disease or inflammatory disease is selected from the group consisting of hyperlipidemia, primary hyperlipidemia, dyslipidemia, mixed dyslipidemia, homozygous familial hypercholesterolemia, and sepsis.
16. A method of producing the monoclonal antibody or antigen-binding fragment of any of claims 1 to 8, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment under conditions to produce the monoclonal antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment from the cell or cell culture.
17. A method of producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment of any of claims 1 to 8, comprising combining the monoclonal antibody or antigen-binding fragment with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
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