WO2015092394A1 - Anticorps destinés à être utilisés pour traiter des états associés à des variants spécifiques de pcsk9 chez des populations de patients spécifiques - Google Patents

Anticorps destinés à être utilisés pour traiter des états associés à des variants spécifiques de pcsk9 chez des populations de patients spécifiques Download PDF

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Publication number
WO2015092394A1
WO2015092394A1 PCT/GB2014/053730 GB2014053730W WO2015092394A1 WO 2015092394 A1 WO2015092394 A1 WO 2015092394A1 GB 2014053730 W GB2014053730 W GB 2014053730W WO 2015092394 A1 WO2015092394 A1 WO 2015092394A1
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Prior art keywords
human
antibody
seq
pcsk9
ligand
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PCT/GB2014/053730
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English (en)
Inventor
Jasper Clube
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Kymab Limited
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Priority claimed from GB1322250.0A external-priority patent/GB2521355A/en
Priority claimed from GB1322253.4A external-priority patent/GB2521356B/en
Priority claimed from US14/138,446 external-priority patent/US8883157B1/en
Priority claimed from EP14172579.6A external-priority patent/EP2886558A1/fr
Priority claimed from US14/331,665 external-priority patent/US9023359B1/en
Priority claimed from US14/331,730 external-priority patent/US9914769B2/en
Priority claimed from US14/331,609 external-priority patent/US9051378B1/en
Priority claimed from US14/457,566 external-priority patent/US8945560B1/en
Priority claimed from US14/457,536 external-priority patent/US9017678B1/en
Priority claimed from US14/472,828 external-priority patent/US8986691B1/en
Priority claimed from US14/472,685 external-priority patent/US8992927B1/en
Priority claimed from US14/472,698 external-priority patent/US8986694B1/en
Priority claimed from US14/472,818 external-priority patent/US8980273B1/en
Priority claimed from EP14185297.0A external-priority patent/EP2975058A1/fr
Priority claimed from US14/500,233 external-priority patent/US9045548B1/en
Priority claimed from US14/507,368 external-priority patent/US9034332B1/en
Priority claimed from US14/536,049 external-priority patent/US9045545B1/en
Priority claimed from US14/537,403 external-priority patent/US9067998B1/en
Priority to DE112014005747.9T priority Critical patent/DE112014005747T5/de
Application filed by Kymab Limited filed Critical Kymab Limited
Priority to EP23207654.7A priority patent/EP4328245A2/fr
Priority to EP14199053.1A priority patent/EP2975059A1/fr
Priority to EP17199373.6A priority patent/EP3332790A1/fr
Priority to IES20140321A priority patent/IES86623B2/en
Publication of WO2015092394A1 publication Critical patent/WO2015092394A1/fr
Priority to DE202015008988.7U priority patent/DE202015008988U1/de
Priority to PCT/EP2015/066099 priority patent/WO2016008899A1/fr
Priority to DE202015008974.7U priority patent/DE202015008974U1/de

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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
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • the technology described herein relates to ligands, e.g., antibodies for the treatment of disease.
  • PCSK9 Proprotein convertase subtilisin kexin type 9
  • LDLR low density lipoprotein receptor
  • PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8) family of serine proteases (Seidah et al., 2003). Humans have nine prohormone-proprotein convertases that can be divided between the S8A and S8B subfamilies (Rawlings et al., 2006). Furin, PC1/PC3, PC2, PACE4, PC4, PC5/PC6 and PC7/PC8/LPC/SPC7 are classified in subfamily S8B.
  • Prohormone-proprotein convertases are expressed as zymogens and they mature through a multi step process.
  • the function of the pro-domain in this process is two-fold.
  • the pro- domain first acts as a chaperone and is required for proper folding of the catalytic domain (Ikemura et al., 1987).
  • Once the catalytic domain is folded autocatalysis occurs between the pro-domain and catalytic domain.
  • the pro-domain remains bound to the catalytic domain where it then acts as an inhibitor of catalytic activity (Fu et al., 2000).
  • maturation proceeds with a second autocatalytic event at a site within the pro-domain (Anderson et al., 1997). After this second cleavage event occurs the pro-domain and catalytic domain dissociate, giving rise to an active protease.
  • PCSK9 VFAQISIP (SEQ ID NO: 67)
  • VFAQISIP SEQ ID NO: 67
  • PCSK9 Purified PCSK9 is made up of two species that can be separated by non-reducing SDS-PAGE; the pro-domain at 17 Kd, and the catalytic plus C-terminal domains at 65 Kd. PCSK9 has not been isolated without its inhibitory pro-domain, and measurements of PCSK9's catalytic activity have been variable (Naureckiene et al., 2003; Seidah et al., 2003).
  • a PCSK9 polypeptide includes terminal residues, such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues and/or fusion protein residues.
  • PCSK9 has also been referred to as FH3, NARC1, HCHOLA3, proprotein convertase subtilisin/kexin type 9, and neural apoptosis regulated convertase 1.
  • the PCSK9 gene encodes a proprotein convertase protein that belongs to the proteinase K subfamily of the secretory subtilase family.
  • PCSK9 denotes both the proprotein and the product generated following autocatalysis of the proprotein.
  • the protein can be referred to as the "mature,” “cleaved”, “processed” or “active” PCSK9.
  • the protein can be referred to as the "inactive”, “pro-form”, or "unprocessed” form of PCSK9.
  • PCSK9 also encompasses PCSK9 molecules incorporating post-translational modifications of the PCSK9 amino acid sequence, such as PCSK9 sequences that have been glycosylated, PCSK9 sequences from which its signal sequence has been cleaved, PCSK9 sequence from which its pro domain has been cleaved from the catalytic domain but not separated from the catalytic domain (see, e.g., FIGS. 1A and IB of US20120093818A1).
  • the present invention provides for improved human patient diagnosis and therapy based on human PCSK9 variation. Importantly, the invention enables tailored medicines that address individual human patient genotypes or phenotypes.
  • PCSK9 sequences reveals that there is significant variation across diverse human populations and provides for the ability for correlation between individual human patients and tailored medical and diagnostic approaches addressing the target.
  • the technical applications of these findings, as per the present invention thus contribute to better treatment, prophylaxis and diagnosis in humans and provides for patient benefit by enabling personalized medicines and therapies. This provides advantages of better prescribing, less wastage of medications and improved chances of drug efficacy and better diagnosis in patients.
  • the patient receives drugs and ligands that are tailored to their needs - as determined by the patient's genetic or phenotypic makeup.
  • the invention provides for the genotyping and/or phenotyping of patients in connection with such treatment, thereby allowing a proper match of drug to patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment using drugs or ligands that are not matched to the patient (eg, poor efficacy and/or side-effects) and avoids pharmaceutical mis- prescription and waste.
  • the invention provides:-
  • a ligand that binds a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27 for use in a method comprising the step of using the ligand to target said PCSK9 in a human to treat and/or prevent a disease or condition mediated by PCSK9, the method comprising administering the ligand to the human.
  • a pharmaceutical composition or kit for treating and/or preventing a PCSK9- mediated condition or disease is provided.
  • a method of producing an anti-human PCSK9 antibody binding site comprising obtaining a plurality of anti-PCSK9 antibody binding sites, screening the antibody binding sites for binding to a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating an antibody binding site that binds in the screening step, and optionally producing a form f, c, r, p, m, e, h, aj or q PCSK9- binding fragment or derivative of the isolated antibody.
  • a method of producing an anti-human PCSK9 antibody comprising immunising a non-human vertebrate (eg, a mouse or a rat) with a human PCSK9 comprising an amino acid sequence selected from the group consisting of the amino acid sequences of forms f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating an antibody that binds a human PCSK9 comprising selected from the group consisting of forms f, c, r, p, m, e, h, ajarid q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3, and optionally producing a form f, c, r, p, m,
  • kits for PCSK9 genotyping a human comprising a nucleic acid (i) comprising a sequence of contiguous nucleotides that specifically hybridises to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof, or specifically hybridises to an antisense sequence or an RNA transcript of said sequence, wherein said sequence of contiguous nucleotides hybridises to at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28 or hybridises to an antisense sequence or an RNA transcript thereof; and/or (ii) comprising a sequence of at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or comprising an antisense sequence or RNA version of said contiguous nucleotides, wherein said sequence of contiguous nucle
  • an anti-PCSK9 ligand that binds a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q in the manufacture of a medicament for targeting said PCSK9 in a human to treat and/or prevent a disease or condition mediated by PCSK9.
  • a method of targeting a PCSK9 for treating and/or preventing a PCSK9-mediated disease or condition in a human comprising administering an anti-PCSK9 ligand to a human comprising a nucleotide sequence selected from the group consisting SEQ ID NOs: 29-37, whereby a PCSK9 encoded by said nucleotide sequence is targeted.
  • a method of treating and/or preventing a disease or condition mediated by PCSK9 in a human comprising targeting a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, a and ⁇ 7 by administering to the human a ligand that binds said PCSK9 thereby treating and/or preventing said disease or condition in the human.
  • a method of PCSK9 genotyping a nucleic acid sample of a human comprising identifying in the sample the presence of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof.
  • a method of PCSK9 typing a protein sample of a human comprising identifying in the sample the presence of a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q.
  • a method of treating and/or preventing in a human patient a cardiovascular disease or condition, or a disease or condition that is associated with elevated LDL cholesterol (eg, hypercholesterolemia), wherein the patient is receiving or has previously received statin treatment for said disease or condition comprising typing the patient using a method of the invention and administering a ligand according to the invention whereby the human is treated or said disease or condition is prevented; optionally also reducing or stopping statin treatment.
  • LDL cholesterol eg, hypercholesterolemia
  • an antibody or antibody fragment for use in a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, wherein the antibody or fragment comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation I474V or E670G in SEQ ID NO: l, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma-1 constant regions comprising such an Asp and Leu.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation 1474 or E670G in SEQ ID NO: 1, wherein the antibody or fragment comprises a human gamma-1 heavy chain constant region that comprises an Asp corresponding to position 204 of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein said human comprises (i) an IGHG1*01 human heavy chain constant region gene segment, or the human expresses antibodies comprising human gamma- 1 heavy chain constant regions comprising such an Asp and Leu and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation I474V or
  • an antibody or antibody fragment for use in a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, wherein the antibody comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation I474V or E670G in SEQ ID NO: l, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGHG2*01 human heavy chain constant region gene segment, or the human expresses
  • a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation 1474 or E670G in SEQ ID NO: 1, wherein the antibody or fragment comprises a human gamma-2 heavy chain constant region that comprises an amino acid selected from the group consisting of a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44 and wherein said human comprises (i) an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation
  • IGHG2*01 human heavy chain constant region gene segment or the human expresses antibodies comprising human gamma-2 heavy chain constant regions comprising such a Pro, Asn, Phe, Val and Ala and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation I474V or E670G in SEQ ID NO: 1.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • an antibody or antibody fragment for use in a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, wherein the antibody or fragment comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 50 or a Cys corresponding to position 87 of SEQ ID NO: 50, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation I474V or E670G in SEQ ID NO: l, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGKC*01 human light chain constant region gene segment, or the human expresses antibodies comprising human kappa light chain constant regions comprising such an Val and Cys.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation 1474 or E670G in SEQ ID NO: 1, wherein the antibody or fragment comprises a human kappa light chain constant region that comprises a Val corresponding to position 84 of SEQ ID NO: 50 or a Cys corresponding to position 87 of SEQ ID NO: 50 and wherein said human comprises (i) an IGKC*01 human light chain constant region gene segment, or the human expresses antibodies comprising human kappa light chain constant regions comprising such an Val and Cys and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation I474V or E670G in
  • an antibody or antibody fragment for use in a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, wherein the antibody or fragment comprises a human IGLC2*01 lambda light chain constant region, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation I474V or E670G in SEQ ID NO:l, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises a human IGLC2*01 lambda light chain constant region gene segment, or the human expresses antibodies comprising human IGLC2*01 lambda light chain constant regions.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation 1474 or E670G in SEQ ID NO: 1, wherein the antibody or fragment comprises a human IGLC2*01 lambda light chain constant region and wherein said human comprises (i) an IGLC2*01 human light chain constant region gene segment, or the human expresses antibodies comprising a human IGLC2*01 lambda light chain constant regions and (ii) a nucleotide sequence encoding said proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation I474V or E670G in SEQ ID NO: 1.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • an antibody or antibody fragment for use in a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof comprising a human variable domain that is derived from the recombination of a human VH gene segment, a human D gene segment and a human JH gene segment, wherein the VH gene segment is derived from the recombination of a human VH gene segment, a human D gene segment and a human JH gene segment, wherein the VH gene segment is selected from the group consisting of (i) IGHV1-18*01 and the genome of the human comprises a human IGHV1-18*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGHV1-18*01; or (ii) IGVH1-46*01 and the genome of the human comprises a human IGHV1-46*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGHV1-18
  • a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation 1474 or E670G in SEQ ID NO: 1, wherein (i) the antibody or fragment comprises a human variable domain that is derived from the recombination of a human VH gene segment, a human D gene segment and a human JH gene segment, wherein the VH gene segment is selected from the group consisting of (a) IGHV1-18*01 and the genome of the human comprises a human IGHV1-18*01 nucleotide sequence or the human expresses antibodies comprising variable domains derived from the recombination of human IGHV1- 18*01; and (b) IGVH1-46*01 and the genome of the human comprises a human IGHV1-46*01 nucleot
  • PCSK9 pro
  • a method for treating a PCSK9-mediated disease or condition in a human by targeting a PCSK9 that comprises a C-terminal domain amino acid polymorphism comprising administering to the human a ligand (eg, an antibody or fragment) that has been determined to specifically bind to a PCSK9 comprising a C-terminal domain comprising I474V or 670G (numbering according to SEQ ID NO: l); wherein the human expresses said PCSK9 or the genome of the human comprises a nucleotide sequence encoding said PCSK9; wherein said human is treated for said disease or condition.
  • a ligand eg, an antibody or fragment
  • a method of reducing cholesterol level or maintaining a previously reduced cholesterol level in a human in need thereof comprising: - a. Carrying out an initial treatment of said human for an initial treatment period by
  • an anti-human PCSK9 ligand eg, an antibody or fragment
  • an anti-human PCSK9 ligand eg, an antibody or fragment
  • the ligand has been determined to specifically bind to a PCSK9 comprising a C-terminal domain comprising I474V or 670G (numbering according to SEQ ID NO: l);
  • the human expresses said PCSK9 or the genome of the human comprises a nucleotide sequence encoding said PCSK9 and (iii) the human has received or is receiving statin treatment to lower or maintain cholesterol level; wherein the initial treatment comprises the administration of a single or multiple doses of the ligand to the human;
  • an antibody or antibody fragment for use in a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, wherein the antibody or fragment comprises a human (eg, gamma) heavy chain constant region that comprises a first amino acid that is encoded by a human (eg, gamma) heavy chain constant region gene segment SNP, and the antibody or fragment specifically binds a proprotein convertase
  • subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation I474V or E670G in SEQ ID NO: l, wherein the human comprises a nucleotide sequence encoding said PCSK9 amino acid sequence and comprises a human (eg, gamma) heavy chain constant region gene segment comprising said SNP, or the human expresses antibodies comprising human (eg, gamma) constant regions comprising said first amino acid.
  • PCSK9 amino acid sequence that comprises a C-terminal domain comprising a mutation I474V or E670G in SEQ ID NO: l
  • the human comprises a nucleotide sequence encoding said PCSK9 amino acid sequence and comprises a human (eg, gamma) heavy chain constant region gene segment comprising said SNP, or the human expresses antibodies comprising human (eg, gamma) constant regions comprising said first amino acid.
  • an antibody or antibody fragment for use in a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, wherein the antibody or fragment comprises a human (eg, kappa or lambda) light chain constant region that comprises a first amino acid that is encoded by a human (eg.
  • PCSK9 proprotein convertase subtilisin/kexin type 9 amino acid sequence that comprises a C- terminal domain comprising a mutation I474V or E670G in SEQ ID NO: l
  • the human comprises a nucleotide sequence encoding said PCSK9 amino acid sequence and comprises a human (eg, kappa or lambda respectively) light chain constant region gene segment comprising said SNP, or the human expresses antibodies comprising human (eg, kappa or lambda respectively) constant regions comprising said first amino acid.
  • an antibody or antibody fragment for use in a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, wherein the antibody or fragment comprises a human variable (eg, VH, VK or VA) domain that comprises a first amino acid that is encoded by a V (eg, VH, VK or VA respectively) gene segment SNP, and the antibody or fragment specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that comprises a C-terminal domain comprising a mutation I474V or E670G in SEQ ID NO:l, wherein the human comprises a nucleotide sequence encoding said PCSK9 amino acid sequence and comprises a human V (eg, VH, VK or VA respectively) gene segment comprising said SNP, or the human expresses antibodies comprising V (eg, VH, VK or VA respectively) domains comprising said first amino acid.
  • a human variable (eg, VH, VK or VA) domain that comprises
  • Figure 1 shows in silico modeling of PCSK9 surface variant residues.
  • Figure 2 depicts the cumulative allele frequency distribution across the 1000
  • Figure 3 depicts frameworks and CDRs encoded by VH3-23*04 as obtained from the IMGT database (available on the World Wide Web at www.IMGT.org).
  • Figure 3 discloses the nucleotide sequences as SEQ ID NOS 69, 69, 69, 71, 71, 72, 74, 76, 39 and 77, respectively, in order of appearance.
  • Figure 3 discloses the coded amino acid sequences as SEQ ID NOS 70, 70, 70, 70, 70, 70, 73, 75, 75, 38 and 78, respectively, in order of appearance.
  • Figure 4 depicts sequences of VH3-23*04.
  • the portion of VH3-23*04 comprising the FW1 residue change of rs56069819 (SEQ ID NO: 38).
  • the portion of the nucleic acid sequence encoding rs56069819 is depicted (SEQ ID NO: 39).
  • the FW1 encoded by VH3-23*04 is depicted (SEQ ID NO: 40).
  • the skilled person will know that SNPs or other changes that translate into amino acid variation can cause variability in conformation or activity of human targets to be addressed. This has spawned great interest in personalized medicine where genotyping and knowledge of protein and nucleotide variability is used to tailor medicines and diagnosis of patients more effectively.
  • the present invention provides for tailored pharmaceuticals and testing that specifically addresses rarer variant forms of a human target of interest (TOI), that target being human PCSK9.
  • TOI human target of interest
  • the present invention harnesses the power of human genetic variation analysis and rationally-designed sequence selection.
  • the technical applications of these approaches, as per the present invention contribute to better treatment, prophylaxis and diagnosis in humans and provides for patient benefit by providing choice and enabling personalized medicines and therapies. This provides advantages of better prescribing, less wastage of medications and improved chances of drug efficacy and better diagnosis in patients.
  • HapMap The International HapMap Consortium. 2003;
  • the HapMap Project is an international project that aims to compare the genetic sequences of different individuals to identify chromosomal regions containing shared genetic variants.
  • the HapMap www site provides tools to identify chromosomal regions and the variant therein, with options to drill down to population level frequency data.
  • the present invention involves the identification and cataloguing of naturally- occurring human genomic target sequence variants, including those found to be relatively low- frequency or rare variants that segregate with specific human ethnic populations and in many individual humans.
  • An aspect of the invention is based on rational design of sequence selection addressing the desirability to tailor medicaments and diagnostics to rarer, but yet still significant groups of human individuals that suffer from, or have the potential to suffer from (ie, who are at risk of), a disease or condition mediated or associated with the target of interest.
  • the inventor included considerations of the spread of prevalence of naturally-occurring target variant sequences across multiple, diverse human ethnic populations, as well as the importance of addressing such populations where many individuals are likely to display a genotype and/or phenotype of one or more of the variants being analysed.
  • the inventor saw the importance of adopting the art- recognised classifications of human ethnic populations, and in this respect the inventor based the analysis and design on the recognised human ethnic populations adopted by the 1000 Genomes Project, since this is a resource that is, and will continue to be, widely adopted by the scientific and medical community.
  • Figure 2 shows the cumulative allele frequency distribution across the 1000
  • Genomes Project databse of human VH3-23 alleles comprising SNP rs56069819 (such alleles denonted M C" and the most frequent allele (which does not comprise this SNP) denoted "A").
  • Indicated in the figure are those human PCSK9 variant forms (marked "Variants") that are found in the various sub-populations with above-average occurrence of human VH3-23 alleles comprising SNP rs56069819.
  • the inventor designed the following variant sequence selection criteria, these being criteria that the inventor realised would provide for useful medical drugs and diagnostics to tailored need in the human population.
  • PCSK9 variant sequences found in many individuals distributed across such many different ethnic populations.
  • the inventor's selection included, as a consideration, selection for nucleotide variation that produced amino acid variation in corresponding PCSK9 forms (ie, non-synonymous variations), as opposed to silent variations that do not alter amino acid residues in the target protein.
  • variants whose variant amino acid residues (versus the most common form of human PCSK9) are surface-exposed on the target are desirable for selection, since the inventor saw these as contributing to determining the topography of the target and potentially contributing to how and where ligand binding on the target occurs.
  • the cumulative human allele frequency is 30, 25, 20, 15, 10 or
  • the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to 15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about 5%.
  • the naturally-occurring human target variant sequences are found in at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different human ethnic populations (using the standard categorisation of the 1000 Genomes Project).
  • the naturally-occurring human target variant sequences are found in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 individuals distributed across such many different ethnic populations.
  • frequencies may be determined using bioinformatics.
  • frequencies may be determined by reference to a database comprising at least 1000 or 2000 human sequences.
  • heterozygous human genotype frequency means the cumulative frequency of all genotypes in the sample or database or in humans having one occurrence of the rare variant allele and one occurrence of another allele (heterozygous state), eg, genotype in 1000 Genomes database.
  • homozygous human genotype frequency means the cumulative frequency of two occurrences of the variant allele (homozygous state), eg, genotype in 1000 Genomes Project database.
  • total human genotype frequency means the total of heterozygous plus homozygous human genotype frequencies.
  • “cumulative human allele frequency” refers to the total of all occurrences of the variant allele in the sample or database or in humans, eg, in the 1000 Genomes Project database.
  • the criteria are applied with reference to one or more human genomic sequence databases as described herein.
  • the criteria are those as applied to the 1000 Genomes database.
  • the 1000 Genomes database release 13.
  • the 1000 Genomes database in its most recent version as at 1 October 2013.
  • the ethnic populations are selected from those identified in the 1000
  • N A Rosenberg etal (Science 20 December 2002: vol. 298 no. 5602 2342-2343) studied the genetic structure of human populations of differing geographical ancestry. In total, 52 populations were sampled, these being populations with:
  • the International HapMap Project Nature, 2003 Dec 18;426(6968):789-96, discloses that goal of the HapMap Project: to determine the common patterns of DNA sequence variation in the human genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them in DNA samples from populations with ancestry from parts of Africa, Asia and Europe.
  • the relevant human populations of differing geographical ancestry include Yoruba, Japanese, Chinese, Northern European and Western European populations. More specifically:-
  • a suitable sample of human populations used in the present invention is as follows: - (a) European ancestry
  • each human population is selected from a population marked "(a)" above.
  • each human population is selected from a population marked "(b)" above.
  • each human population is selected from a population marked "(c)" above.
  • the ethnic populations are selected from the group consisting of an ethnic population with European ancestry, an ethnic population with East Asian, an ethnic population with West African ancestry, an ethnic population with Americas ancestry and an ethnic population with South Asian ancestry.
  • the ethnic populations are selected from the group consisting of an ethnic population with Northern European ancestry; or an ethnic population with Western European ancestry; or an ethnic population with Toscani ancestry; or an ethnic population with British ancestry; or an ethnic population with Icelandic ancestry; or an ethnic population with Finnish ancestry; or an ethnic population with Iberian ancestry; or an ethnic population with Japanese ancestry; or an ethnic population with Chinese ancestry; or an ethnic population Vietnamese ancestry; or an ethnic population with Yoruba ancestry; or an ethnic population with Luhya ancestry; or an ethnic population with Gambian ancestry; or an ethnic population with Malawian ancestry; or an ethnic population with Native American ancestry; or an ethnic population with Afro- Caribbean ancestry; or an ethnic population with Mexican ancestry; or an ethnic population with Puerto Rican ancestry; or an ethnic population with Columbian ancestry; or an ethnic population with Peruvian
  • the invention provides useful anti-target ligands for addressing humans suffering from or likely to suffer from a disease or condition mediated or associated with PCSK9.
  • the ligand specifically binds to a PCSK9 variant as per the invention.
  • the ligand may inhibit or antagonise the activity of the PCSK9 target, eg, the ligand neutralises the target.
  • the skilled person will be familiar with neutralising ligands in general, such as antibodies or antibody fragments, and can readily test suitable ligands for specific binding and/or neutralisation of a target in vitro or in an in vivo assay.
  • the ligand is (or has been determined as) a neutraliser of the
  • determination is carried out in a human (eg, in a clinical trial). In an example, determination is carried out in a non-human, eg, in a mouse, rat, rabbit, pig, dog, sheep or non-human primate (eg, Cynomolgous monkey, rhesus monkey or baboon).
  • a human eg, in a clinical trial
  • determination is carried out in a non-human, eg, in a mouse, rat, rabbit, pig, dog, sheep or non-human primate (eg, Cynomolgous monkey, rhesus monkey or baboon).
  • An antibody "fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody.
  • antibody fragments include dAb, Fab, Fab' , F(ab' )2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
  • the ligand of the invention is or comprises an antibody or antibody fragment, for example an antibody or fragment comprising human variable regions (and optionally also human constant regions).
  • Anti-PCSK9 or PCSK9-binding or targeting antibodies and fragments can be prepared according to any known method, eg, using transgenic mice (eg, the KymouseTM or VelocimouseTM, or OmnimouseTM , XenomouseTM, HuMab MouseTM or MeMo MouseTM), rats (eg, the OmniratTM), camelids, sharks, rabbits, chickens or other non-human animals immunised with the PCSK9 followed optionally by humanisation of the constant regions and/or variable regions to produce human or humanised antibodies.
  • transgenic mice eg, the KymouseTM or VelocimouseTM, or OmnimouseTM , XenomouseTM, HuMab MouseTM or MeMo MouseTM
  • rats eg, the OmniratTM
  • display technologies can be used, such as yeast, phage or ribosome display, as will be apparent to the skilled person.
  • Standard affinity maturation eg, using a display technology, can be performed in a further step after isolation of an antibody lead from a transgenic animal, phage display library or other library.
  • suitable technologies are described in US20120093818 (Amgen, Inc), which is incorporated by reference herein in its entirety, eg, the methods set out in paragraphs [0309] to [0346].
  • a VELOCIMMUNETM or other mouse or rat can be challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies.
  • lymphatic cells such as B-cells
  • the lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest.
  • DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain.
  • Such an antibody protein may be produced in a cell, such as a CHO cell.
  • DNA encoding the antigen-specific chimaeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.
  • high affinity chimaeric antibodies are isolated having a human variable region and a mouse constant region.
  • the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc.
  • the mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgGl or IgG4 (for example, SEQ ID NO: 751, 752,753 in US2011/0065902 (which is incorporated by reference herein in its entirety), which sequences are incorporated herein by reference for use in the ligands of the present invention).
  • the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
  • the ligand of the invention is or comprises a nucleic acid, eg, RNA, eg, siRNA that hybridises under stringent condition to the PCSK9 variant sequence, eg, hybridises a nucleotide sequence comprising one or more nucleotides that are variant (versus the most common PCSK9 sequence, eg, with reference to the 1000 Genomes Project database).
  • RNA eg, siRNA that hybridises under stringent condition to the PCSK9 variant sequence
  • hybridises a nucleotide sequence comprising one or more nucleotides that are variant (versus the most common PCSK9 sequence, eg, with reference to the 1000 Genomes Project database).
  • the nucleic acid hybridises to a region immediately flanking a nucleotide that is variant compared to the corresponding nucleotide of the PCSK9 nucleotide sequence having the highest cumulative human allele frequency and/or the highest total human genotype frequency.
  • the nucleic acid hybridises to at two or more such variant nucleotides.
  • Specific hybridisation is under stringent conditions, as will be apparent to the skilled person, eg, conditions of 5xSSC, SxDenhardt's reagent, and 0.5% SDS at 65° C.
  • Target binding ability, specificity and affinity can be determined by any routine method in the art, eg, by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • Kd is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.
  • the surface plasmon resonance (SPR) is carried out at 25°C.
  • the SPR is carried out at 37°C.
  • the SPR is carried out at physiological pH, such as about pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (also referred to as HBS-EP)).
  • physiological pH such as about pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (also referred to as HBS-EP)).
  • the SPR is carried out at a physiological salt level, eg,
  • the SPR is carried out at a detergent level of no greater than
  • the SPR is carried out at 25°C or 37°C in a buffer at pH7.6,
  • the buffer can contain lOmM Hepes.
  • the SPR is carried out at 25°C or 37°C in HBS-EP.
  • HBS-EP is available from Teknova Inc (California; catalogue number H8022).
  • the affinity of the ligand is determined using SPR by
  • SPR surface plasmon resonance
  • Regeneration of the capture surface can be carried out with lOmM glycine at pH1.7. This removes the captured antibody and allows the surface to be used for another interaction.
  • the binding data can be fitted to 1: 1 model inherent using standard techniques, eg, using a model inherent to the ProteOn XPR36TM analysis software.
  • the ligand of the invention is contained in a medical container, eg, a vial, syringe, IV container or an injection device (eg, an intraocular or intravitreal injection device).
  • the ligand is in vitro, eg, in a sterile container.
  • the invention provides a kit comprising the ligand of the invention, packaging and instructions for use in treating or preventing or diagnosing in a human a disease or condition mediated by the PCSK9.
  • the instructions indicate that the human should be genotyped for a PCSK9 variant sequence of the invention before administering the ligand to the human.
  • the instructions indicate that the human should be phenotyped for a PCSK9 variant of the invention before administering the ligand to the human.
  • the human is of Chinese (eg, Han or CHS) ethnicity and the instructions are in Chinese (eg, Mandarin).
  • the instructions comprise directions to administer alirocumab or evolocumab to said human.
  • the invention addresses the need to treat humans having naturally-occurring rarer natural PCSK9 alleles, genotypes and phenotypes (rarer protein forms).
  • the invention provides the following aspects.
  • An anti-human PCSK9 ligand for use in a method of treating and/or preventing a PCSK9-mediated disease or condition in a human whose genome comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, wherein the method comprises administering the ligand to the human.
  • nucleotide sequence is selected from the group consisting of
  • SEQ ID Nos: 29-35 and 37 or selected from the group consisting of SEQ ID Nos: 29-32 and 34-37; or selected from the group consisting of SEQ ID Nos: 29-32, 34, 35 and 37.
  • These are naturally- occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above.
  • haplotype allele sequences that do not encode 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et / 2006).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31 and 37 that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et a/ 2005).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31, 32, 34, 35, 36 and 37 are selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37.
  • nucleotide sequence is SEQ ID NO 29.
  • the nucleotide sequence is SEQ ID NO 30.
  • the nucleotide sequence is SEQ ID NO 31.
  • the nucleotide sequence is SEQ ID NO 32.
  • the nucleotide sequence is SEQ ID NO 33.
  • the nucleotide sequence is SEQ ID NO 34.
  • the nucleotide sequence is SEQ ID NO 35.
  • the nucleotide sequence is SEQ ID NO 36.
  • the nucleotide sequence is SEQ ID NO 37.
  • the PCSK9 variant is not the most frequent.
  • the ligand, antibody, fragment or binding site of the invention is recombinant.
  • ligand of aspect 1 wherein the ligand has been or is determined as capable of binding a human PCSK9 selected from the group consisting forms f, c, r r p, m, e, h, aj and q.
  • the ligand binds (or has been determined to bind) two, three, four or more human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q.
  • the ligand comprises a protein domain that specifically binds to PCSK9, eg, a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q.
  • PCSK9 eg, a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q.
  • the term "specifically binds,” or the like, means that a ligand, eg, an antibody or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about lxlO "6 M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. An isolated antibody that specifically binds a human PCSK9 may, however, exhibit cross-reactivity to other antigens such as a PCSK9 molecule from another species. Moreover, multi-specific antibodies (e.g., bispecifics) that bind to human PCSK9 and one or more additional antigens are nonetheless considered antibodies that "specifically bind" PCSK9, as used herein.
  • the ligand comprises or consists of a protein that mimics the EGFA domain of the LDL receptor and specifically binds to PCSK9, eg, a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q.
  • PCSK9 eg, a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q.
  • the ligand antagonises PCSK9 eg, a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, aj and q.
  • the method comprises (before administering the ligand) the step of determining that the ligand is capable of binding a human PCSK9 selected from the group consisting forms f, c, r, p, m, e, h, ajanti q.
  • binding is determined by SPR. In an example of any aspect, binding is determined by ELISA.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • the terms "is determined”, “is genotyped” or “is phenotyped” and the like herein mean that the method comprises a step of such determining, genotyping or phenotyping.
  • a ligand that binds a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27 for use in a method comprising the step of using the ligand to target said PCSK9 in a human to treat and/or prevent a disease or condition mediated by PCSK9, the method comprising administering the ligand to the human.
  • the disease or condition is mediated by a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27.
  • amino acid sequence selected from the group consisting of
  • SEQ ID Nos: 4-23, 26 and 27 or selected from the group consisting of SEQ ID Nos: 4-14 and 18- 27; or selected from the group consisting of SEQ ID Nos: 4-14, 18-23, 26 and 27.
  • These are naturally-occurring sequences that do not comprise 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • amino acid sequence is SEQ ID NO: 18, 19 or 20, that comprises a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).
  • amino acid sequence selected from the group consisting of
  • amino acid sequence selected from the group consisting of
  • SEQ ID NOs: 10-14 and 18-27 are sequences that have a naturally-occurring combination of differences from SEQ ID NOs: 1-3 (form a) and which meet the criteria set out above.
  • amino acid sequence is SEQ ID NO: 4.
  • amino acid sequence is SEQ ID NO: 5.
  • amino acid sequence is SEQ ID NO: 6.
  • amino acid sequence is SEQ ID NO: 7.
  • amino acid sequence is SEQ ID NO: 8.
  • amino acid sequence is SEQ ID NO: 9.
  • amino acid sequence is SEQ ID NO: 10.
  • amino acid sequence is SEQ ID NO: 11.
  • amino acid sequence is SEQ ID NO: 12.
  • amino acid sequence is SEQ ID NO: 13.
  • amino acid sequence is SEQ ID NO: 14.
  • amino acid sequence is SEQ ID NO: 15.
  • amino acid sequence is SEQ ID NO: 16.
  • amino acid sequence is SEQ ID NO: 17.
  • amino acid sequence is SEQ ID NO: 18.
  • amino acid sequence is SEQ ID NO: 19.
  • amino acid sequence is SEQ ID NO: 20.
  • amino acid sequence is SEQ ID NO: 21.
  • amino acid sequence is SEQ ID NO: 22.
  • amino acid sequence is SEQ ID NO: 23.
  • amino acid sequence is SEQ ID NO: 24.
  • amino acid sequence is SEQ ID NO: 25.
  • amino acid sequence is SEQ ID NO: 26.
  • amino acid sequence is SEQ ID NO: 27.
  • nucleotide sequence is selected from the group consisting of
  • SEQ ID Nos: 29-35 and 37 or selected from the group consisting of SEQ ID Nos: 29-32 and 34-37; or selected from the group consisting of SEQ ID Nos: 29-32, 34, 35 and 37.
  • These are naturally- occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above.
  • haplotype allele sequences that do not encode 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et 2006).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31 and 37 that encode 670G which is a marker for severity of coronary atherosclerosis (Chen ef a/2005).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31, 32, 34, 35, 36 and 37 are selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37.
  • nucleotide sequence is SEQ ID NO 29.
  • the nucleotide sequence is SEQ ID NO 30.
  • the nucleotide sequence is SEQ ID NO 31.
  • the nucleotide sequence is SEQ ID NO 32.
  • the nucleotide sequence is SEQ ID NO 33.
  • the nucleotide sequence is SEQ ID NO 34.
  • the nucleotide sequence is SEQ ID NO 35.
  • the nucleotide sequence is SEQ ID NO 36.
  • the nucleotide sequence is SEQ ID NO 37.
  • ligand of any preceding aspect wherein the human has been or is genotyped as positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof.
  • nucleotide sequence is selected from the group consisting of
  • SEQ ID Nos: 29-35 and 37 or selected from the group consisting of SEQ ID Nos: 29-32 and 34-37; or selected from the group consisting of SEQ ID Nos: 29-32, 34, 35 and 37. [00207] These are naturally-occurring allele (ha lotype) sequences that do not encode
  • the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et a 2006).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31 and 37 that encode 670G which is a marker for severity of coronary atherosclerosis (Chen ef a/2005).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31, 32, 34, 35, 36 and 37 are selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37.
  • nucleotide sequence is SEQ ID NO: 29.
  • nucleotide sequence is SEQ ID NO: 30.
  • nucleotide sequence is SEQ ID NO: 31.
  • nucleotide sequence is SEQ ID NO: 32.
  • nucleotide sequence is SEQ ID NO: 33.
  • nucleotide sequence is SEQ ID NO: 34.
  • nucleotide sequence is SEQ ID NO: 35.
  • nucleotide sequence is SEQ ID NO: 36.
  • nucleotide sequence is SEQ ID NO: 37.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • the method comprises genotyping the human as positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof.
  • nucleotide sequence is selected from the group consisting of
  • SEQ ID Nos: 29-35 and 37 or selected from the group consisting of SEQ ID Nos: 29-32 and 34-37; or selected from the group consisting of SEQ ID Nos: 29-32, 34, 35 and 37.
  • These are naturally- occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above.
  • haplotype allele sequences that do not encode 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et / 2006).
  • the nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31 and 37 that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et a/ 005).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31, 32, 34, 35, 36 and 37 are selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37.
  • nucleotide sequence is SEQ ID NO: 29.
  • nucleotide sequence is SEQ ID NO: 30.
  • nucleotide sequence is SEQ ID NO: 31.
  • nucleotide sequence is SEQ ID NO: 32.
  • the nucleotide sequence is SEQ ID NO: 33.
  • nucleotide sequence is SEQ ID NO: 34.
  • nucleotide sequence is SEQ ID NO: 35.
  • nucleotide sequence is SEQ ID NO: 36.
  • nucleotide sequence is SEQ ID NO: 37.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • ligand of any preceding aspect wherein the human has been or is genotyped as heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof; optionally wherein the human has been or is genotyped as comprising the nucleotide sequence of SEQ ID NO: 28 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29- 37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof.
  • Heterozygous here means that in the human's genotype one allele comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof and other allele can be any PCSK9 (eg, form a, a'or an allele comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof).
  • PCSK9 eg, form a, a'or an allele comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof.
  • the method comprises (before administering the ligand) genotyping the human as heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof; optionally also genotyping the human as comprising the nucleotide sequence of SEQ ID NO: 28 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof.
  • nucleotide sequence is selected from the group consisting of
  • SEQ ID Nos: 29-35 and 37 or selected from the group consisting of SEQ ID Nos: 29-32 and 34-37; or selected from the group consisting of SEQ ID Nos: 29-32, 34, 35 and 37.
  • These are naturally- occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above.
  • haplotype allele sequences that do not encode 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et 2006).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31 and 37 that encode 670G which is a marker for severity of coronary atherosclerosis (Chen ef a/2005).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31, 32, 34, 35, 36 and 37 are selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37.
  • nucleotide sequence is SEQ ID NO 29.
  • nucleotide sequence is SEQ ID NO 30.
  • nucleotide sequence is SEQ ID NO 31.
  • the nucleotide sequence is SEQ ID NO 32.
  • the nucleotide sequence is SEQ ID NO 33.
  • nucleotide sequence is SEQ ID NO 34.
  • nucleotide sequence is SEQ ID NO 35.
  • nucleotide sequence is SEQ ID NO 36.
  • nucleotide sequence is SEQ ID NO 37.
  • Homozygous here means that in the human's genotype each allele comprises the same nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof.
  • the method comprises genotyping the human as homozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof.
  • nucleotide sequence is selected from the group consisting of
  • SEQ ID Nos: 29-35 and 37 or selected from the group consisting of SEQ ID Nos: 29-32 and 34-37; or selected from the group consisting of SEQ ID Nos: 29-32, 34, 35 and 37.
  • These are naturally- occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above.
  • haplotype allele sequences that do not encode 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et a 2006).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31 and 37 that encode 670G which is a marker for severity of coronary atherosclerosis (Chen ef a/2005).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31, 32, 34, 35, 36 and 37 are selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37.
  • nucleotide sequence is SEQ ID NO: 29.
  • nucleotide sequence is SEQ ID NO: 30.
  • nucleotide sequence is SEQ ID NO: 31.
  • nucleotide sequence is SEQ ID NO: 32.
  • nucleotide sequence is SEQ ID NO: 33.
  • nucleotide sequence is SEQ ID NO: 34.
  • nucleotide sequence is SEQ ID NO: 35.
  • nucleotide sequence is SEQ ID NO: 36.
  • nucleotide sequence is SEQ ID NO: 37.
  • ligand of any preceding aspect wherein the ligand comprises an antibody binding site that binds a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27 and optionally has been or is determined as capable of such binding.
  • the method comprises (before administering the ligand) the step of determining that the ligand is capable of binding to said human PCSK9.
  • the binding is specific binding.
  • the ligand binds
  • the PCSK9 (or has been determined as binding) to the PCSK9 with an affinity (Kd) of ImM, ⁇ , ⁇ or InM or less.
  • Kd affinity
  • the affinity is no less than 10, 100 or 1000 fM.
  • binding or affinity is determined by SPR or ELISA.
  • the disease or condition is mediated by a human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27.
  • amino acid sequence selected from the group consisting of
  • These are naturally-occurring sequences that do not comprise 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • the amino acid sequence is SEQ ID NO: 18, 19 or 20, that comprises a 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).
  • amino acid sequence selected from the group consisting of
  • amino acid sequence selected from the group consisting of
  • SEQ ID Nos: 10-14 and 18-27 or selected from the group consisting of SEQ ID Nos: 10-14, 18-23,
  • amino acid sequence is SEQ ID NO: 4.
  • amino acid sequence is SEQ ID NO: 5.
  • amino acid sequence is SEQ ID NO: 6.
  • amino acid sequence is SEQ ID NO: 7.
  • amino acid sequence is SEQ ID NO: 8.
  • amino acid sequence is SEQ ID NO: 9.
  • amino acid sequence is SEQ ID NO: 10.
  • amino acid sequence is SEQ ID NO: 11.
  • amino acid sequence is SEQ ID NO: 12.
  • amino acid sequence is SEQ ID NO: 13.
  • amino acid sequence is SEQ ID NO: 14.
  • amino acid sequence is SEQ ID NO: 15.
  • amino acid sequence is SEQ ID NO: 16.
  • amino acid sequence is SEQ ID NO: 17.
  • amino acid sequence is SEQ ID NO: 18.
  • amino acid sequence is SEQ ID NO: 19.
  • amino acid sequence is SEQ ID NO: 20.
  • amino acid sequence is SEQ ID NO: 21.
  • amino acid sequence is SEQ ID NO: 22.
  • amino acid sequence is SEQ ID NO: 23.
  • amino acid sequence is SEQ ID NO: 24.
  • amino acid sequence is SEQ ID NO: 25.
  • amino acid sequence is SEQ ID NO: 26.
  • amino acid sequence is SEQ ID NO: 27.
  • the ligand of aspect 11 wherein the ligand is an antibody or antibody fragment.
  • the antibody or antibody fragment is a PCSK9 antagonist, eg, neutralises PCSK9.
  • PCSK9 antagonist eg, neutralises PCSK9.
  • Examples of such antibodies are disclosed, for instance, in WO 2008/057457, WO2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/133647, WO 2009/100297, WO 2009/100318, WO 201 1/037791, WO 201 1/053759, WO 201 1/053783, WO 2008/125623, WO 2011/072263, WO 2009/055783, WO 2010/029513, WO 2011/11 1007, WO 2010/077854, the disclosures and sequences of such antibodies being incorporated herein in their entireties by refere for use in the invention.
  • One specific example is AMG 145 (Amgen), LY3015014 (Eli Li
  • the ligand is or comprises evolocumab.
  • the ligand is SAR236553/REGN727 (Sanofi Aventis/Regeneron) or a PCSK9-binding derivative thereof.
  • the ligand comprises or consists of a neutralizing antibody that binds to the PCSK9, wherein the antibody binds to PCSK9 and reduces the likelihood that PCSK9 binds to LDLR.
  • the ligand comprises or consists a ligand selected from evolocumab, lD05-IgG2 (Merck & Co.), ALN-PCS02 (Alnylam), RN316 (Pfizer- Rinat), LY3015014 (Eli Lilly) and alirocumab, or a PCSK9-binding derivative thereof.
  • the ligand is SAR236553/REGN727 (Sanofi Aventis/Regeneron) or a PCSK9-binding derivative thereof.
  • the ligand comprises a sequence of contiguous nucleotides that specifically hybridises to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof, or specifically hybridises to an antisense sequence or an RNA transcript of said sequence, wherein said sequence of contiguous nucleotides hybridises to at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28 or hybridises to an antisense sequence or an RNA transcript thereof respectively; and/or (ii) the ligand comprises a sequence of at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or is an antisense sequence or RNA version of said contiguous nucleotides, wherein said
  • nucleotide sequence is selected from the group consisting of
  • SEQ ID Nos: 29-35 and 37 or selected from the group consisting of SEQ ID Nos: 29-32 and 34-37; or selected from the group consisting of SEQ ID Nos: 29-32, 34, 35 and 37.
  • These are naturally- occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above.
  • haplotype allele sequences that do not encode 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta eta/ 2006).
  • SEQ ID NOs: 31 and 37 that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et a/ 005).
  • nucleotide sequence selected from the group consisting of
  • nucleotide sequence is SEQ ID NO: 29.
  • nucleotide sequence is SEQ ID NO: 30.
  • nucleotide sequence is SEQ ID NO: 31.
  • nucleotide sequence is SEQ ID NO: 32.
  • nucleotide sequence is SEQ ID NO: 33.
  • nucleotide sequence is SEQ ID NO: 34.
  • nucleotide sequence is SEQ ID NO: 35.
  • nucleotide sequence is SEQ ID NO: 36.
  • nucleotide sequence is SEQ ID NO: 37.
  • the ligand comprises at least 10, 11, 12, 13, 14, 15, 20, 25,
  • nucleotide sequence 30, 35, 40, 45, 50 or 100 contiguous nucleotides of said nucleotide sequence.
  • the ligand of any preceding aspect, wherein the disease or condition is hypercholesterolemia, hyperlipidemia, hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.
  • said disease or condition is hypercholesterolaemia.
  • hypocholesterolaemia refers to a condition in which cholesterol levels are elevated above a desired level. In some embodiments, this denotes that serum cholesterol levels are elevated. In some embodiments, the desired level takes into account various "risk factors" that are known to one of skill in the art (and are described or referenced in US20120093818).
  • the ligand of any preceding aspect wherein the human is identified as heterozygous for Familial Hypercholesterolemia, statin intolerant, statin uncontrolled, or at risk for developing hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.
  • Cholesterol levels are measured in milligrams (mg) of cholesterol per deciliter (dL) of blood in the United States and some other countries. Canada and most European countries measure cholesterol in millimoles (mmol) per liter (L) of blood. Below are general guideline ideal ranges and elevated ranges.
  • Elevated LDL cholesterol is, therefore, 160 mg/dL or above (4.1 mmol/L or above).
  • the method comprises (before administering the ligand) determining that the ligand is capable of such inhibition.
  • Inhibition determination is eg, inhibition in a blood or serum sample, at rtp, at pH7, at 37 degrees centigrade and/or under the physiological conditions of a human body.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • a pharmaceutical composition or kit for treating and/or preventing a PCSK9-mediated condition or disease (eg, as recited in aspect 14 or 15), the composition or kit comprising a ligand of any preceding aspect and optionally a statin (eg, cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin or pravastatin); and optionally in combination with a label or instructions for use to treat and/or prevent said disease or condition in a human (eg, covering treatment of a human as recited in aspect 18 or 19); optionally wherein the label or instructions comprise a marketing authorisation number (eg, an FDA or EMA authorisation number); optionally wherein the label or instructions comprise directions to administer alirocumab or evolocumab to said human; optionally wherein the kit comprises an IV or injection device that comprises the ligand (and, e
  • a method of producing an anti-human PCSK9 antibody binding site comprising obtaining a plurality of anti-PCSK9 antibody binding sites, screening the antibody binding sites for binding to a human PCSK9 selected from the group consisting of forms f, c, r f p, m, e, h, aj and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating an antibody binding site that binds in the screening step, and optionally producing a form f, c, r, p, m, e, h, aj or q PCSK9-binding fragment or derivative of the isolated antibody.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • the plurality of binding sites comprises or consists of a plurality of 4-chain antibodies or fragments thereof, eg, dAbs, Fabs or scFvs.
  • Suitable methods for producing pluralities of binding sites for screening include phage display (producing a phage display library of antibody binding sites), ribosome display (producing a ribosome display library of antibody binding sites), yeast display (producing a yeast display library of antibody binding sites), or immunisation of a non-human vertebrate (eg, a rodent, eg, a mouse or rat, eg, a VelocimouseTM, KymouseTM, XenomouseTM, Aliva MouseTM, HuMab MouseTM, OmnimouseTM, OmniratTM or MeMo MouseTM) with a PCSK9 epitope and isolation of a repertoire of antibody- producing cells (eg, a B-cell, plasma cell or plasmablast repertoire) and/or a repertoire of isolated antibodies.
  • a non-human vertebrate eg, a rodent, eg, a mouse or rat, eg, a VelocimouseTM, KymouseTM, Xenom
  • the method comprises selecting one or more antibody binding sites that each specifically binds to a human PCSK9 epitope comprising amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3.
  • the ligand specifically binds to an epitope comprising an amino acid that is variant compared to the corresponding amino acid of the PCSK9 encoded by SEQ ID NO: 1, 2 or 3.
  • the ligand specifically binds to an epitope comprising two or more such variant amino acids.
  • specific binding means binding with an affinity (Kd) of ImM, lOOnM, lOnM or InM or less, eg, as determined by SPR.
  • epitope is a region of an antigen that is bound by an antibody.
  • Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
  • a method of producing an anti-human PCSK9 antibody comprising immunising a non-human vertebrate (eg, a mouse or a rat) with a human PCSK9 comprising an amino acid sequence selected from the group consisting of the amino acid sequences of forms f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3 and isolating an antibody that binds a human PCSK9 comprising selected from the group consisting of forms f, c, r, p, m, e, h, aj and q or a catalytic or C-terminal domain or a peptide thereof that comprises amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3, and optionally producing a form f, c,
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • the method comprises isolating a cell (eg, B-cell, plasmablast, plasma cell or memory cell) comprising the nucleic acid, wherein the cell is obtained from a non- human vertebrate that has been immunised with the PCSK9 epitope.
  • a cell eg, B-cell, plasmablast, plasma cell or memory cell
  • the cell is obtained from a non- human vertebrate that has been immunised with the PCSK9 epitope.
  • kits for PCSK9 genotyping a human comprising a nucleic acid (i) comprising a sequence of 10 or more (eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) contiguous nucleotides that specifically hybridises to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C- terminal domain-encoding sequence thereof, or specifically hybridises to an antisense sequence or an RNA transcript of said sequence, wherein said sequence of contiguous nucleotides hybridises to at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28 or hybridises to an antisense sequence or an RNA transcript thereof; and/or (ii) comprising a sequence of at least 10 or more (eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) nucleo
  • nucleotide sequence is selected from the group consisting of
  • SEQ ID Nos: 29-35 and 37 or selected from the group consisting of SEQ ID Nos: 29-32 and 34-37; or selected from the group consisting of SEQ ID Nos: 29-32, 34, 35 and 37.
  • These are naturally- occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above.
  • haplotype allele sequences that do not encode 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et / 2006).
  • SEQ ID NOs: 31 and 37 that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et a/ 005).
  • nucleotide sequence selected from the group consisting of
  • nucleotide sequence is SEQ ID NO: 29.
  • the nucleotide sequence is SEQ ID NO: 30.
  • nucleotide sequence is SEQ ID NO: 31.
  • nucleotide sequence is SEQ ID NO: 32.
  • nucleotide sequence is SEQ ID NO: 33.
  • nucleotide sequence is SEQ ID NO: 34.
  • nucleotide sequence is SEQ ID NO: 35.
  • nucleotide sequence is SEQ ID NO: 36.
  • nucleotide sequence is SEQ ID NO: 37.
  • kits for PCSK9 genotyping or phenotyping a human wherein the kit comprises a ligand according to any one of aspects 1 to 19 or an antibody, fragment or derivative produced by the method of any one of aspects 21 to 23.
  • PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q in the manufacture of a medicament for treating and/or preventing a PCSK9-mediated disease or condition in a human whose genome comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, optionally for treating and/or preventing a PCSK9-mediated disease or condition in a human as recited in aspect 18 or 19.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q in the manufacture of a medicament for targeting said PCSK9 in a human to treat and/or prevent a disease or condition mediated by PCSK9, optionally for targeting PCSK9 in a human as recited in aspect 18 or 19.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • nucleotide sequence is selected from the group consisting of
  • SEQ ID Nos: 29-35 and 37 or selected from the group consisting of SEQ ID Nos: 29-32 and 34-37; or selected from the group consisting of SEQ ID Nos: 29-32, 34, 35 and 37.
  • These are naturally- occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above.
  • haplotype allele sequences that do not encode 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta eta/ 006).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31 and 37 that encode 670G which is a marker for severity of coronary atherosclerosis (Chen et a/ 005).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31, 32, 34, 35, 36 and 37 are selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37.
  • nucleotide sequence is SEQ ID NO: 29.
  • the nucleotide sequence is SEQ ID NO: 30.
  • nucleotide sequence is SEQ ID NO: 31.
  • nucleotide sequence is SEQ ID NO: 32.
  • nucleotide sequence is SEQ ID NO: 33.
  • nucleotide sequence is SEQ ID NO: 34.
  • nucleotide sequence is SEQ ID NO: 35.
  • nucleotide sequence is SEQ ID NO: 36.
  • nucleotide sequence is SEQ ID NO: 37.
  • the ligand can be any anti-PCSK9 ligand disclosed herein.
  • a method of targeting a PCSK9 for treating and/or preventing a PCSK9-mediated disease or condition in a human comprising administering an anti-PCSK9 ligand to a human comprising a nucleotide sequence selected from the group consisting SEQ ID NOs: 29-37, whereby a PCSK9 encoded by said nucleotide sequence is targeted.
  • the ligand can be any anti-PCSK9 ligand disclosed herein.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • a method of treating and/or preventing a disease or condition mediated by PCSK9 in a human comprising targeting a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, a and q by administering to the human a ligand that binds said PCSK9 thereby treating and/or preventing said disease or condition in the human.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • the ligand can be any anti-PCSK9 ligand disclosed herein.
  • nucleotide sequence is selected from the group consisting of
  • SEQ ID Nos: 29-35 and 37 or selected from the group consisting of SEQ ID Nos: 29-32 and 34-37; or selected from the group consisting of SEQ ID Nos: 29-32, 34, 35 and 37.
  • These are naturally- occurring allele (haplotype) sequences that do not encode 46L and which meet the criteria set out above.
  • haplotype allele sequences that do not encode 46L and which meet the criteria set out above.
  • These groups comprise variants that are associated with elevated LDL-C.
  • the nucleotide sequence is SEQ ID NO: 34, that encodes a 425S, which is associated with elevated LDL-C (Pisciotta et 2006).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31 and 37 that encode 670G which is a marker for severity of coronary atherosclerosis (Chen ef a/2005).
  • nucleotide sequence selected from the group consisting of
  • SEQ ID NOs: 31, 32, 34, 35, 36 and 37 are selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37.
  • the nucleotide sequence is SEQ ID NO: 29.
  • nucleotide sequence is SEQ ID NO: 30.
  • the nucleotide sequence is SEQ ID NO: 31.
  • the nucleotide sequence is SEQ ID NO: 32.
  • nucleotide sequence is SEQ ID NO: 33.
  • the nucleotide sequence is SEQ ID NO: 34.
  • nucleotide sequence is SEQ ID NO: 35.
  • nucleotide sequence is SEQ ID NO: 36.
  • nucleotide sequence is SEQ ID NO: 37.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • said forms are the pro-forms.
  • the method comprises genotyping the human as positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • any one of aspects 29 to 36 wherein the human has been or is genotyped as heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof; optionally wherein the human has been or is genotyped as comprising the nucleotide sequence of SEQ ID NO: 28 or the catalytic- or C-terminal domain-encoding sequence thereof and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof.
  • any one of aspects 29 to 38 comprises genotyping the human for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain-encoding sequence thereof before administering the ligand to the human, wherein the ligand is determined to be capable of binding to a PCSK9 encoded by said selected sequence.
  • a statin eg, cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin or pravastatin
  • a Forty-fourth Aspect The method of any one of aspects 29 to 43, wherein the ligand is administered by subcutaneous injection.
  • a method of PCSK9 genotyping a nucleic acid sample of a human the method comprising identifying in the sample the presence of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminal domain- encoding sequence thereof.
  • a method of PCSK9 typing a protein sample of a human comprising identifying in the sample the presence of a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q.
  • said forms are the mature forms.
  • said forms are the pro-forms.
  • the method comprises obtaining a PCSK9 protein sample from the human and then carrying out the identifying step.
  • LDL cholesterol eg, hypercholesterolemia
  • said reducing or stopping comprises reducing the dose and/or dosing frequency of statin.
  • a diagnostic, therapeutic or prophylactic kit comprising a ligand that is capable of binding to or has been or is determined as capable of binding to an amino acid sequence selected from SEQ ID NOs: 4-27 and instructions for carrying out the method of any one of aspects 46 to 49 and/or a label or instructions indicating or covering administration of the ligand to a human as defined in any one of aspects 1 to 19.
  • a diagnostic, therapeutic or prophylactic kit comprising a nucleic acid probe comprising a nucleotide sequence that specifically hybridises to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or an antisense sequence or NA transcript thereof and instructions for carrying out the method of aspect 45, 47 or 48.
  • the PCSK9 is human PCSK9, eg, a mature, cleaved, autocatalysed or active PCSK9.
  • the disease is a cardiovascular disease such as hyperlipidaemia.
  • the ligand specifically binds to human
  • PCSK9 eg, one or more of the rare PCSK9 variants disclosed herein (eg, one, two, three, more or all mature forms f, c, r, p, m, e, h, aj and ⁇ and optionally also the a and/or a'form.
  • the ligand specifically binds to mature form and/or cas well as form a.
  • Binding to each such form is, for example, respectively with a Kd of at least ImM, ⁇ , InM, ⁇ , ⁇ or lpM.
  • the ligand binds form a and a PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q, wherein the ligand binding to said selected form is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.
  • a Kd determined by SPR
  • both forms are mature forms.
  • both forms are pro-forms.
  • the ligand binds form and form f, wherein the ligand binding to form f ⁇ % with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.
  • a Kd determined by SPR
  • both forms are mature forms.
  • both forms are pro-forms.
  • the ligand binds form and form c, wherein the ligand binding to form c is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.
  • a Kd determined by SPR
  • both forms are mature forms.
  • both forms are pro-forms.
  • the ligand binds form a and form r, wherein the ligand binding to form ris with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.
  • a Kd determined by SPR
  • both forms are mature forms.
  • both forms are pro-forms.
  • the ligand binds form and form p, wherein the ligand binding to form p is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.
  • a Kd determined by SPR
  • both forms are mature forms.
  • both forms are pro-forms.
  • the ligand binds form and form m, wherein the ligand binding to form m is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.
  • a Kd determined by SPR
  • both forms are mature forms.
  • both forms are pro-forms.
  • the ligand binds form and form e, wherein the ligand binding to form e is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.
  • both forms are mature forms.
  • both forms are pro-forms.
  • the ligand binds form a and form h, wherein the ligand binding to form h is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.
  • both forms are mature forms.
  • both forms are pro-forms.
  • the ligand binds form a and form aj, wherein the ligand binding to form aj s with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.
  • a Kd determined by SPR
  • both forms are mature forms.
  • both forms are pro-forms.
  • the ligand binds form a and form q, wherein the ligand binding to form q is with a Kd (determined by SPR) that is at least 60, 70, 80, 90 or 95% of the Kd for binding to form a.
  • a Kd determined by SPR
  • both forms are mature forms.
  • both forms are pro-forms.
  • the ligand neutralises human PCSK9 eg, one or more of the rare PCSK9 variants disclosed herein (eg, one, two, three, more or all mature forms f, c, r, p, m, e, h, a and q) and optionally also the a and/or a'form.
  • the ligand neutralises mature form f and/ore as well as form a. Determination of neutralisation can be performed, for example, by any neutralisation assay method disclosed in US20120093818A1 (Amgen, Inc) or US20110065902A1 (Regeneron Pharmaceuticals, Inc).
  • Ligands of the invention that bind or target PCSK9 are useful, for example, for therapeutic and prophylactic applications disclosed in US20120093818A1 and US20110065902A1, these specific disclosures being
  • an antigen binding protein can inhibit, interfere with or modulate one or more biological activities of a PCSK9 (eg, one or more of the rare variants disclosed herein and optionally also the a and/or a'form).
  • a PCSK9 eg, one or more of the rare variants disclosed herein and optionally also the a and/or a'form.
  • ligand binds specifically to human PCSK9 (eg, one or more of the rare variants disclosed herein and optionally also the a and/or a'form) and/or substantially inhibits binding of human PCSK9 (eg, said one or more of the rare variants disclosed herein and optionally also the a and/or a'form) to LDLR by at least 20%, eg, 20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by measuring binding in an in vitro competitive binding assay).
  • the ligand is an antibody.
  • the ligand has a Kd of less (binding more tightly) than 10 "7 ,
  • Kd is determined using SPR.
  • the ligand has an IC50 for blocking the binding of LDLR to one or more of the rare PCSK9 variants disclosed herein (and optionally also the a and/or a'form) of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM.
  • the ligand has an IC50 for blocking the binding of LDLR to the a and/or a'form of PCSK9 that is no more than 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10-fold more (ie, more inhibitory) than the IC50 for blocking the binding of LDLR to one or more of the rare PCSK9 variants disclosed herein (eg, one or more PCSK9 proteins comprising a sequence selected from SEQ ID NOs: 4 to 27).
  • the rare PCSK9 variants disclosed herein eg, one or more PCSK9 proteins comprising a sequence selected from SEQ ID NOs: 4 to 27.
  • the ligand has an IC50 for blocking the binding of LDLR to (i) the a and/or a'form of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, eg, in the range of ImM to lpM (eg, ImM to ⁇ ; ⁇ to ⁇ ; InM to ⁇ ; or ⁇ to lpM) and (ii) one or more PCSK9 proteins comprising a sequence selected from SEQ ID NOs: 4 to 27 of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000
  • the ligand binds to the a and/or 'form of PCSK9 with a binding affinity (Kd) that is greater than up to 10%, greater than up to 20%, greater than up to 40%, greater than up to 50%, greater than up to 55%, greater than up to 60%, greater than up to 65%, greater than up to 70%, greater than up to 75%, greater than up to 80%, greater than up to 85%, greater than up to 90%, greater than up to 95% or greater than up to 100% (ie, is double) relative to binding to a PCSK9 comprising a sequence selected from SEQ ID NOs: 4 to 27.
  • Kd binding affinity
  • binding measurements can be made using a variety of binding assays known in the art, eg, using surface plasmon resonance (SPR), such as by BiacoreTM or using the ProteOn XPR36TM (Bio-Rad®), or using KinExA® (Sapidyne Instruments, Inc).
  • SPR surface plasmon resonance
  • BiacoreTM surface plasmon resonance
  • ProteOn XPR36TM Bio-Rad®
  • KinExA® KinExA®
  • the surface plasmon resonance (SPR) is carried out at 25°C.
  • the SPR is carried out at 37°C.
  • the SPR is carried out at physiological pH, such as about pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (also referred to as HBS-EP)).
  • physiological pH such as about pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (also referred to as HBS-EP)).
  • the SPR is carried out at a physiological salt level, eg,
  • the SPR is carried out at a detergent level of no greater than
  • the SPR is carried out at 25°C or 37°C in a buffer at pH7.6,
  • the SPR is carried out at 25°C or 37°C in HBS-EP.
  • HBS-EP is available from Teknova Inc (California; catalogue number H8022).
  • the affinity of the ligand which is an antibody is determined using
  • SPR surface plasmon resonance
  • Regeneration of the capture surface can be carried out with lOmM glycine at pH1.7. This removes the captured antibody and allows the surface to be used for another interaction.
  • the binding data can be fitted to 1: 1 model inherent using standard techniques, eg, using a model inherent to the ProteOn XPR36TM analysis software.
  • assaying or testing of a ligand of the invention is carried out at or substantially at pH7 (eg, for in tests and assays J and at or substantially at rtp.
  • IgG2 heavy chain constant domain of an anti-PCSK9 antibody of the present invention has the amino acid sequence as shown in SEQ ID NO: 154, FIG. 3KK of US20120093818A1, which sequence is incorporated herein by reference.
  • IgG4 heavy chain constant domain of an anti-PCSK9 antibody of the present invention has the amino acid sequence as shown in SEQ ID NO: 155, FIG. 3KK of US20120093818A1, which sequence and disclosure is incorporated herein by reference in its entirety.
  • a kappa light chain constant domain of an anti-PCSK9 antibody has the amino acid sequence as shown in SEQ ID NO: 157, FIG. 3KK of US20120093818A1, which sequence and disclosure is incorporated herein by reference in its entirety.
  • lambda light chain constant domain of an anti-PCSK9 antibody has the amino acid sequence as shown in SEQ ID NO: 156, FIG. 3KK of US20120093818A1, which sequence and disclosure is incorporated herein by reference in its entirety.
  • the ligand binds mature PCSK9, eg, a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a' form.
  • the ligand binds the catalytic domain of
  • PCSK9 eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or ⁇ ?'form.
  • the ligand binds the prodomain of PCSK9, eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a 'form.
  • the ligand binds to the V domain of PCSK9, eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a' form. In some embodiments, the ligand binds to the V domain of PCSK9 (eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or 'form) and prevents (or reduces, eg, by at least 10%) PCSK9 from binding to LDLR.
  • the ligand binds to the V domain of PCSK9 (eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a'form), and while it does not prevent (or reduce) the binding of PCSK9 to LDLR, the ligand prevents or reduces (eg, by at least 10%) the adverse activities mediated through PCSK9 on LDLR.
  • the V domain of PCSK9 eg, of a mature form of one or more of the rare variants disclosed herein and optionally also the a and/or a'form
  • the ligand is or comprises a fully human antibody.
  • the ligand comprises human variable regions or humanised variable regions.
  • the ligand of the invention specifically binds to an epitope of a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, and q, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the amino acid is selected from the group consisting of 46L, 53V, 425S, 443T, 474V, 619P and 670G (numbering as used in SEQ ID NO: l).
  • the amino acid is selected from the group consisting of 425S, 443T, 474V, 619P and 670G (numbering as used in SEQ ID NO: l).
  • the amino acid is selected from the group consisting of 425S and 443T (numbering as used in SEQ ID NO: l).
  • the amino acid is selected from the group consisting of 474V, 619P and 670G (numbering as used in SEQ ID NO: 1).
  • the PCSK9 form is the mature form.
  • the PCSK9 form is the pro-form.
  • the ligand also specifically binds to form a and/or a'.
  • the ligand specifically binds to an epitope of form TCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to an epitope of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form /-PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to an epitope of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form 3 PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to an epitope of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • ligand binds specifically to the pro-domain of a human PCSK9 selected from the group consisting of forms f, c, r r p, m, e, h, aj and q.
  • the ligand also specifically binds to the pro-domain of form a and/or a'.
  • the ligand specifically binds to the pro-domain of form PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the pro- domain of form c PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the pro-domain of form rPCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the pro-domain of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro-domain of form ay ' PCSIO, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the pro- domain of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • ligand binds specifically to the catalytic domain of a human
  • the ligand selected from the group consisting of forms f, c, r, p, m, e, h, aj and q.
  • the ligand also specifically binds to the catalytic domain of form a and/or a'.
  • the ligand specifically binds to the catalytic domain of form PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the catalytic domain of form C PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the catalytic domain of form rPCSK9 wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the catalytic domain of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form _? PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the catalytic domain of form ⁇ PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • ligand binds specifically to the C-terminal domain of a human
  • the ligand selected from the group consisting of forms f, c, r, p, m, e, h, aj and q.
  • the ligand also specifically binds to the C-terminal domain of form a and/or a'.
  • the ligand specifically binds to the C-terminal domain of form /TCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the C-terminal domain of form C PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the C-terminal domain of form /-PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the C-terminal domain of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form 5/ PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the C-terminal domain of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • ligand binds specifically to the substrate-binding groove of a human PCSK9 selected from the group consisting of forms f, c, r, p, m, e, h, aj and q (see
  • the ligand also specifically binds to the substrate-binding groove of form a and/or a'. In an embodiment, the ligand specifically binds to the Substrate- binding groove of form f PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the Substrate-binding groove of form C PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form r PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form p PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the Substrate-binding groove of form m PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form e PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form h PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand specifically binds to the Substrate- binding groove of form a PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a. In an embodiment, the ligand specifically binds to the Substrate-binding groove of form q PCSK9, wherein the epitope comprises at least one amino acid that is not found in form a.
  • the ligand is or comprises an antibody disclosed in Table 2 of
  • the PCSK9-binding ligand of the invention is selected from the antigen binding proteins disclosed in US20120093818A1 (Amgen, Inc), eg, in paragraphs [0009] to [0014] and [0058] to [0063] of US20120093818A1; all of these disclosures (including the sequences of such proteins) are incorporated herein by reference as though explicitly recited herein and for possible inclusion in one or more claims or for use in the present invention.
  • the ligand of the invention comprises an isolated antigen binding protein that binds PCSK9 comprising: A) one or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 from a CDRH1 in a sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (ii) a CDRH2 from a CDRH2 in a sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87,
  • the isolated antigen binding protein comprises at least one CDRH of A) and at least one CDRL of B). In some embodiments, the isolated antigen binding protein comprises at least two CDRH of A) and at least two CDRL of B). In some embodiments, the isolated antigen binding protein comprises said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.
  • the CDRH of A) is selected from at least one of the group consisting of: (i) a CDRH1 amino acid sequence selected from the CDRH1 in a sequence selected from the group consisting of SEQ ID NO: 67, 79, 89, and 49; (ii) a CDRH2 amino acid sequence selected from the CDRH2 in a sequence selected from the group consisting of SEQ ID NO: 67, 79, 89, and 49; (iii) a CDRH3 amino acid sequence selected from the CDRH3 in a sequence selected from the group consisting of SEQ ID NO: 67, 79, 89, and 49; and (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids.
  • the CDRL of B) is selected from at least one of the group consisting of: (i) a CDRL1 amino acid sequence selected from the CDRL1 in a sequence selected from the group consisting of SEQ ID NO: 12, 35, 32, and 23; (ii) a CDRL2 amino acid sequence selected from the CDRL2 in a sequence selected from the group consisting of SEQ ID NO: 12, 35, 32, and 23; (iii) a CDRL3 amino acid sequence selected from the CDRL3 in a sequence selected from the group consisting of SEQ ID NO: 12, 35, 32, and 23; and (iv) a CDRL of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids; or C) one or more heavy chain CDRHs of A) and one or more light chain CDRLs of B.
  • the CDRH of A) is selected from at least one of the group consisting of: (i) a CDRH1 amino acid sequence of the CDRH1 amino acid sequence in SEQ ID NO: 67; (ii) a CDRH2 amino acid sequence of the CDRH2 amino acid sequence in SEQ ID NO: 67; (iii) a CDRH3 amino acid sequence of the CDRH3 amino acid sequence in SEQ ID NO: 67; and (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids; said CDRL of B) is selected from at least one of the group consisting of: (i) a CDRL1 amino acid sequence of the CDRL1 amino acid sequence in SEQ ID NO: 12; (ii) a CDRL2 amino acid sequence of the CDRL2 amino acid sequence in SEQ ID NO: 12; (iii) a CDRL3 amino acid sequence of the
  • the antigen binding protein comprises A) a CDRH1 of the CDRH1 sequence in SEQ ID NO: 67, a CDRH2 of the CDRH2 sequence in SEQ ID NO: 67, and a CDRH3 of the CDRH3 sequence in SEQ ID NO: 67, and B) a CDRL1 of the CDRL1 sequence in SEQ ID NO: 12, a CDRL2 of the CDRL2 sequence in SEQ ID NO: 12, and a CDRL3 of the CDRL3 sequence in SEQ ID NO: 12.
  • the antigen binding protein comprises a heavy chain variable region (VH) having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or a light chain variable region (VL) having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46.
  • VH heavy chain variable region
  • the VH has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or the VL has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46.
  • the VH is selected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or the VL is selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46.
  • the PCSK9-targeting or binding ligand comprises or consists of AMG145 or 31H4, 16F12, 11F1, 8A3 or 21B12 disclosed in
  • the PCSK9-targeting or binding ligand comprises or consists of AMG145.
  • the AMG145 or other ligand of the invention is glycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos or Hek293 cell).
  • the ligand of the invention is produced in CHO.
  • Antibody ligands to PCSK9 are described in, for example, WO 2008/057457, WO 2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/125623, and US 2008/0008697.
  • the ligand is or comprises an antibody disclosed in the .Examples of US20110065902A1 (eg, 316P or 300N) or is a PCSK9-binding derivative thereof. All of these disclosures (including the sequences of such proteins and corresponding nucleotide sequences) are incorporated herein by reference as though explicitly recited herein and for possible inclusion in one or more claims or for use in the present invention.
  • the ligand is or comprises the variable domains of antibody 316P or 300N disclosed in US20110065902A1 or is (or comprises) such antibody or a PCSK9-binding derivative thereof. The foregoing reference is incorporated by reference herein in its entirety.
  • the ligand is or comprises the variable domains of antibody alirocumab or SAR236553/REGN727 (Sanofi Aventis/Regeneron) or is (or comprises) such antibody or a PCSK9-binding derivative thereof.
  • the antibody is glycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos or Hek293 cell).
  • the ligand is alirocumab or SAR236553/REGN727.
  • the ligand is or comprises the variable domains of antibody evolocumab or or is (or comprises) such antibody or a PCSK9-binding derivative thereof.
  • the antibody is glycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos or Hek293 cell).
  • the ligand is evolocumab.
  • the ligand is selected from evolocumab, lD05-IgG2 (Merck &
  • the ligand is selected from the following (sequences and definitions as per US2011/0065902, incorporated herein by reference in its entirety):-
  • an antibody or antigen-binding fragment thereof which specifically binds hPCSK9, wherein the antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pair having SEQ ID NOs: 218/226.
  • the antibody or antigen-binding fragment of concept 1 comprising heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.
  • the antibody or antigen-binding fragment of concept 2 comprising an HCVR having the amino acid sequence of SEQ ID NO: 218 and an LCVR having the amino acid sequence of SEQ ID NO: 226.
  • An antibody or antigen-binding fragment thereof which binds to the same epitope on hPCSK9 as an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.
  • An antibody or antigen-binding fragment thereof which competes for binding to hPCSK9 with an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.
  • the ligand is selected from the following (sequences and definitions as per US2012/0093818, incorporated herein by reference herein in its entirety):-
  • An isolated neutralizing antigen binding protein that binds to a PCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the neutralizing antigen binding protein decreases the LDLR lowering effect of PCSK9 on LDLR, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60.
  • An antigen binding protein that selectively binds to PCSK9, wherein said antigen binding protein binds to PCSK9 with a Kd that is less than 100 pM.
  • An antigen binding protein that binds to a PCSK 9 protein of SEQ ID NO: 303 in a first manner, wherein the antigen binding protein binds to a variant of PCSK9 in a second manner, wherein said PCSK9 variant has at least one point mutation at a position selected from the group consisting of: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 of SEQ ID NO: 303, wherein the first manner comprises a first EC50, a first Bmax, or a first EC50 and a first Bmax, wherein the second manner comprises a second EC50, a second Bmax, or a second EC50 and a second Bmax, and wherein a value for the first manner is different from a value for the second manner, and wherein the antigen binding protein comprises a light
  • the antigen binding protein of concept 6 wherein the first manner comprises a first Bmax, wherein the second manner comprises a second Bmax that is different from the first Bmax, and wherein said PCSK9 variant has at least one point mutation selected from the group consisting of: D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and Q554R.
  • a method of making an antigen binding protein that binds to a PCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the antigen binding protein decreases the LDLR lowering effect of PCSK9 on LDLR comprising:providing a host cell comprising a nucleic acid sequence that encodes the antigen binding protein; andmaintaining the host cell under conditions in which the antigen binding protein is expressed, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60.
  • a method for treating or preventing a condition associated with elevated serum cholesterol levels in a subject comprising administering to a subject in need thereof an effective amount of an isolated neutralizing antigen binding protein simultaneously or sequentially with an agent that elevates the availability of LDLR protein, wherein the isolated antigen binding protein binds to a PCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the neutralizing antigen binding protein decreases the LDLR lowering effect of PCSK9 on LDLR, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60.
  • An antigen binding protein that binds to PCSK9 wherein when the antigen binding protein is bound to PCSK9, the antibody is positioned 8 angstroms or less from at least one of the following residues of PCSK9: S153, S188, 1189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, 1154, T187, H193, E195, 1196, M201, V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372, C375, or C378, wherein the antigen binding protein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 46, and wherein the antigen binding protein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 60.
  • the ligand can be used for the treatment, therapy, prophylaxis and/or diagnosis of one or more diseases or conditions or susceptibility thereto, wherein such diseases or conditions comprise those disclosed in US20120093818A1 (Amgen, Inc) and US20110065902A1 (Regeneron Pharmaceuticals, Inc), eg, a disease or condition disclosed in paragraphs [0375] to [0383] of US20120093818A1, which disclosure is incorporated herein by reference in its entirety for inclusion in one more claims herein.
  • the ligand can be administered to a human characterised as described in
  • the ligand can be administered in a form or combination disclosed in
  • the ligand can be used in a method of diagnosis as set out in US20120093818A1
  • the ligand of the invention is a diagnostic tool.
  • the ligand can be used to assay the amount of PCSK9 present in a sample and/or subject.
  • such ligands need not be neutralizing ligands.
  • the diagnostic ligand is not a neutralizing ligand.
  • the diagnostic ligand binds to a different epitope than a neutralizing ligand binds to.
  • the two ligands do not compete with one another.
  • the ligands of the invention are used or provided in an assay kit and/or method for the detection of PCSK9 in mammalian tissues or cells in order to screen/diagnose for a disease or disorder associated with changes in levels of PCSK9.
  • the kit comprises a ligand that binds PCSK9 and means for indicating the binding of the ligand with PCSK9, if present, and optionally PCSK9 protein levels.
  • Various means for indicating the presence of a ligand can be used. For example, fluorophores, other molecular probes, or enzymes can be linked to the ligand and the presence of the ligand can be observed in a variety of ways.
  • the method for screening for such disorders can involve the use of the kit, or simply the use of one of the disclosed ligands and the determination of whether the ligand binds to PCSK9 in a sample.
  • high or elevated levels of PCSK9 will result in larger amounts of the ligand binding to PCSK9 in the sample.
  • degree of ligand binding can be used to determine how much PCSK9 is in a sample.
  • Subjects or samples with an amount of PCSK9 that is greater than a predetermined amount e.g., an amount or range that a person without a PCSK9 related disorder would have
  • the invention provides a method wherein the ligand is administered to a subject taking a statin, in order to determine if the statin has increased the amount of PCSK9 in the subject.
  • the ligand is a non-neutralizing ligand and is used to determine the amount of PCSK9 in a subject receiving an ABP and/or statin treatment.
  • the ligand of the invention can specifically bind human
  • PCSK9 (eg, one, two or more rare variant forms disclosed herein) and is characterized by at least one of: (i) capable of reducing serum total cholesterol at least about 25-35% and sustaining the reduction over at least a 24 day period relative to a predose level; (ii) capable of reducing serum LDL cholesterol at least about 65-80% and sustaining the reduction over at least a 24 day period relative to a predose level; (iii) capable of reducing serum LDL cholesterol at least about 40-70% and sustaining the reduction over at least a 60 or 90 day period relative to a predose level; (iv) capable of reducing serum triglyceride at least about 25-40% relative to predose level; (v) does not reduce serum HDL cholesterol or reduces serum HDL cholesterol no more than 5% relative to predose level.
  • an isolated nucleic acid molecule is provided and it encodes the ligand.
  • an expression vector is provided and comprises the nucleic acid molecule.
  • a pharmaceutical composition is provided and it can comprise the ligand and a pharmaceutically acceptable carrier.
  • a method is provided for treating a disease or condition which is ameliorated, improved, inhibited or prevented with a PCSK9 antagonist ligand of the invention. The method can comprise administering a therapeutic amount of the pharmaceutical composition or ligand to a subject in need thereof.
  • the subject is a human subject suffering from hypercholesterolemia, hyperlipidemia, indicated for LDL apheresis, identified as heterozygous for Familial Hypercholesterolemia, statin intolerant, statin uncontrolled, at risk for developing hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis and cardiovascular diseases.
  • a method of providing a treatment or therapy is provided to a subject.
  • the method comprises reducing serum cholesterol at least about 40-70% over at least 60 to 90 days.
  • a method of receiving treatment or therapy is provided, the method can comprise receiving a ligand thereof at a frequency of once every 60 to 90 days.
  • the invention provides a ligand of the invention which is or comprises an human antibody or antigen-binding fragment of a human antibody that specifically binds and inhibits human proprotein convertase subtilisin/kexin type 9 (hPCSK9, eg, one, two or more rare variant forms disclosed herein and optionally form a and/or form a), characterized by the ability to reduce serum LDL cholesterol in a human by 40-80% over a 24, 60 or 90 day period relative to predose levels, with little or no reduction in serum HDL cholesterol and/or with little or no measurable effect on liver function, as determined by ALT and AST measurements.
  • hPCSK9 human proprotein convertase subtilisin/kexin type 9
  • the ligand of the invention comprises an antibody or antigen- binding fragment of an antibody that specifically binds hPCSK9 and is characterized by at least one of:
  • the invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds hPCSK9 and is characterized by at least one of:
  • the antibody or antigen-binding fragment is characterized as exhibiting an enhanced binding affinity (KD) for hPCSK9 at pH 5.5 relative to the KD at pH 7.4, as measured by plasmon surface resonance.
  • KD enhanced binding affinity
  • the antibody or fragment thereof exhibits at least a 20-fold, at least a 40-fold or at least a 50-fold enhanced affinity for PCSK9 at an acidic pH relative to a neutral pH, as measured by surface plasmon resonance.
  • the antibody or antigen-binding fragment is characterized as not exhibiting an enhanced binding affinity for PCSK9 at an acidic pH relative to a neutral pH, as measured by surface plasmon resonance.
  • the antibody or fragment thereof exhibits a decreased binding affinity at an acidic pH.
  • the antibody or antigen-binding fragment binds human, human GOF mutation D374Y, cynomolgus monkey, rhesus monkey, mouse, rat and hamster PCSK9.
  • the antibody or antigen-binding fragment binds human and monkey PCSK9, but does not bind mouse, rat or hamster PCSK9.
  • the invention comprises an antibody or antigen-binding fragment of an antibody comprising one or more of a heavy chain variable region (HCVR), light chain variable region (LCVR), HCDRl, HCDR2, HCDR3 disclosed in any of paragraphs [023]-[037] of US2011/0065902, the disclosure of which is incorporated herein by reference in its entirety.
  • HCVR heavy chain variable region
  • LCVR light chain variable region
  • HCDRl light chain variable region
  • HCDR2 HCDR3 disclosed in any of paragraphs [023]-[037] of US2011/0065902, the disclosure of which is incorporated herein by reference in its entirety.
  • the invention comprises an antibody or antigen-binding fragment of an antibody which specifically binds hPCSK9, wherein the antibody or fragment comprises heavy and light chain CDR domains contained within heavy and light chain sequence pairs selected from the group consisting of SEQ ID NO (using the sequence numbering in
  • the CD sequences are contained within HCVR and LCVR selected from the amino acid sequence pairs of SEQ ID NO: 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 122/130, 138/140, 142/144, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 314/322, 330/332 and 334/336.
  • the CDR sequences are comprised within HCVR/LCVR sequences selected from SEQ ID NO: 90/92 or 218/226.
  • the invention features a pharmaceutical composition
  • a pharmaceutical composition comprising a ligand of the invention, wherein the ligand comprises or consists of a recombinant human antibody or fragment thereof which specifically binds hPCSK9 and a pharmaceutically acceptable carrier.
  • the invention features a composition which is a combination of a ligand of the invention (eg, an antibody or antigen-binding fragment of an antibody), and a second therapeutic agent.
  • the second therapeutic agent may be any agent that is advantageously combined with the ligand of the invention, for example, an agent capable of inducing a cellular depletion of cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase, such as, for example, cerovastatin, atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc; capable of inhibiting cholesterol uptake and or bile acid re-absorption; capable of increasing lipoprotein catabolism (such as niacin); and/or activators of the LXR transcription factor that plays a role in cholesterol elimination such as 22-hydroxycholesterol.
  • HMG 3-hydroxy-3-methylglutaryl
  • CoA coenzyme A
  • the invention provides a method for inhibiting hPCSK9 activity using the anti-PCSK9 ligand of the invention (eg, an antibody or antigen-binding portion of the antibody of the invention), wherein the therapeutic methods comprise administering a
  • the disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of PCSK9 activity.
  • Specific populations treatable by the therapeutic methods of the invention include subjects indicated for LDL apheresis, subjects with PCSK9-activating mutations (gain of function mutations, "GOF"), subjects with heterozygous Familial Hypercholesterolemia (heFH); subjects with primary hypercholesterolemia who are statin intolerant or statin uncontrolled; and subjects at risk for developing hypercholesterolemia who may be preventably treated.
  • dyslipidemia associated with secondary causes such as Type 2 diabetes mellitus, cholestatic liver diseases (primary biliary cirrhosis), nephrotic syndrome, hypothyroidism, obesity; and the prevention and treatment of atherosclerosis and cardiovascular diseases.
  • the ligand of the invention is useful to reduce elevated total cholesterol, non-HDL cholesterol, LDL cholesterol, and/or apolipoprotein B
  • the ligand (eg, antibody or antigen-binding fragment) of the invention may be used alone or in combination with a second agent, for example, an HMG-CoA reductase inhibitor and/or another lipid lowering drug.
  • isolated with reference to a ligand, antibody or protein, for example in any aspect, configuration, example or emodiment, means that a subject ligand, antibody, protein etc (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature.
  • an "isolated" ligand, antibody, protein etc constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample.
  • Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode such an isolated ligand, antibody protein etc.
  • the isolated ligand, antibody protein etc is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.
  • an "isolated" antibody is one that has been identified, separated and/or recovered from a component of its production environment (eg, naturally or recombinantly).
  • the isolated polypeptide is free of association with all other components from its production environment, eg, so that the antibody has been isolated to an FDA-approvable or approved standard.
  • Contaminant components of its production environment such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.
  • the invention encompasses the ligand (eg, antibody) conjugated to a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.
  • a therapeutic moiety such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.
  • Cytotoxin agents include any agent that is detrimental to cells. .
  • suitable cytotoxin agents and chemotherapeutic agents for forming immunoconjugates are known in the art, see for example, WO 05/103081, which is incorporated by reference herein in its entirety.
  • the antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific mAbs may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al. (1991) J. Immunol. 147:60-69.
  • the human anti-PCSK9 (eg, anti-PCSK9) mAbs can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein.
  • an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or a multispecific antibody with a second binding specificity.
  • An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference.
  • the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering).
  • the second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgGl antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N3845, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bi-specific antibody format described above are contemplate
  • the invention provides therapeutic methods for treating a human patient in need of a composition or ligand of the invention. While modifications in lifestyle and conventional drug treatment are often successful in reducing cholesterol levels, not all patients are able to achieve the recommended target cholesterol levels with such approaches.
  • Various conditions such as familial hypercholesterolemia (FH), appear to be resistant to lowering of LDL-C levels in spite of aggressive use of conventional therapy.
  • FH familial hypercholesterolemia
  • Homozygous and heterozygous familial hypercholesterolemia hoFH, heFH
  • hoFH homozygous familial hypercholesterolemia
  • hoFH heFH
  • patients diagnosed with hoFH are largely unresponsive to conventional drug therapy and have limited treatment options.
  • statins which reduce LDL-C by inhibiting cholesterol synthesis and upregulating the hepatic LDL receptor
  • a mean LDL-C reduction of only less than about 20% has been recently reported in patients with genotype-confirmed hoFH treated with the maximal dose of statins.
  • the addition of ezetimibe 10 mg/day to this regimen resulted in a total reduction of LDL-C levels of 27%, which is still far from optimal.
  • many patients are statin non-responsive, poorly controlled with statin therapy, or cannot tolerate statin therapy; in general, these patients are unable to achieve cholesterol control with alternative treatments. There is a large unmet medical need for new treatments that can address the short-comings of current treatment options.
  • Specific populations treatable by the therapeutic methods of the invention include patients indicated for LDL apheresis, subjects with PCSK9-activating (GOF) mutations, heterozygous Familial Hypercholesterolemia (heFH); subjects with primary hypercholesterolemia who are statin intolerant or statin uncontrolled; and subjects at risk for developing hypercholesterolemia who may be preventably treated.
  • GAF PCSK9-activating
  • heFH heterozygous Familial Hypercholesterolemia
  • subjects with primary hypercholesterolemia who are statin intolerant or statin uncontrolled
  • subjects at risk for developing hypercholesterolemia who may be preventably treated.
  • the invention provides therapeutic compositions comprising the anti-PCSK9 ligands, antibodies or antigen-binding fragments thereof of the present invention.
  • compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as
  • the dose may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like.
  • the ligand, eg, antibody, of the present invention is used for treating various conditions and diseases associated with PCSK9, including hypercholesterolemia, disorders associated with LDL and apolipoprotein B, and lipid metabolism disorders, and the like, in an adult patient, it is advantageous to intravenously administer the ligand or antibody of the present invention normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted.
  • composition invention provides the ligand by e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • the pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249: 1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
  • a liposome see Langer (1990) Science 249: 1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974).
  • a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g., ethanol
  • a polyalcohol e.g., propylene glycol, polyethylene glycol
  • a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • a pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition.
  • the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition.
  • the pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention.
  • .Examples include, but certainly are not limited to AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25TM pen, HUMALOGTM pen, HUMALIN 70/30TM pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPENTMI, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENTTM, OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIKTTM (sanofi-aventis, Frankfurt, Germany), to name only a few.
  • the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
  • dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • the amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.
  • the invention provides therapeutic methods in which the ligand, eg, antibody or antibody fragment, of the invention is useful to treat hypercholesterolemia associated with a variety of conditions involving hPCSK9.
  • the anti-PCSK9 ligands, eg, antibodies or antibody fragments, of the invention are particularly useful for the treatment of hypercholesterolemia and the like.
  • Combination therapies may include the anti-PCSK9 ligand of the invention with, for example, one or more of any agent that (1) induces a cellular depletion of cholesterol synthesis by inhibiting 3-hydroxy-3- methylglutaryl (HMG)-coenzyme A (CoA) reductase, such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin; (2) inhibits cholesterol uptake and or bile acid re-absorption; (3) increase lipoprotein catabolism (such as niacin); and activators of the LXR transcription factor that plays a role in cholesterol elimination such as 22-hydroxycholesterol or fixed combinations such as ezetimibe plus simvastatin; a statin with a bile resin (e.g.,
  • cholestyramine, colestipol, colesevelam a fixed combination of niacin plus a statin (e.g., niacin with lovastatin); or with other lipid lowering agents such as omega-3-fatty acid ethyl esters (for example, omacor).
  • statin e.g., niacin with lovastatin
  • other lipid lowering agents such as omega-3-fatty acid ethyl esters (for example, omacor).
  • Ligands of the invention are useful, for instance, in specific binding assays, for genotyping or phenotyping humans, affinity purification of the PCSK9 and in screening assays to identify other antagonists of PCSK9 activity. Some of the ligands of the invention are useful for inhibiting binding of PCSK9 to a congnate human receptor or protein, or inhibiting PCSK9-mediated activities.
  • the invention encompasses anti-PCSK9 (eg, PCSK9) antibody ligands having a modified glycosylation pattern.
  • modification to remove undesirable glycosylation sites may be useful, or e.g., removal of a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733).
  • ADCC antibody dependent cellular cytotoxicity
  • modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).
  • the invention features a pharmaceutical composition
  • a pharmaceutical composition comprising a ligand of the invention, wherein the ligand is or comprises a recombinant human antibody or fragment thereof which specifically binds the PCSK9 (eg, a rare variant as described herein) and a pharmaceutically acceptable carrier.
  • the invention features a composition which is a combination of an antibody ligand or antigen-binding fragment of an antibody of the invention, and a second therapeutic agent.
  • the second therapeutic agent may be any of an anti-inflammatory agent, an anti-angiogenesis agent, a painkiller, a diuretic, a chemotherapeutic agent, an antineoplastic agent, a vasodilator, a vasoconstrictor, a statin, a beta blocker, a nutrient, an adjuvant, an anti-obesity agent and an anti-diabetes agent.
  • “Pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the USA Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • a “pharmaceutically acceptable carrier, excipient, or adjuvant” refers to an carrier, excipient, or adjuvant that can be administered to a subject, together with an agent, e.g., any antibody or antibody chain described herein, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.
  • the invention features a method for inhibiting PCSK9 activity using the anti-PCSK9 ligand of the invention (eg, an antibody or antigen-binding portion of the antibody of the invention), wherein the therapeutic method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the ligand.
  • the disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of PCSK9 activity.
  • terapéuticaally effective amount is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).
  • SNPlex is a proprietary genotyping platform sold by Applied Biosystems.
  • Miniaturized assays such as microarrays with oligonucleotide reagents immobilized on small surfaces, are frequently proposed for large-scale mutation analysis and high- throughput genotyping (Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome (Wang DG, Fan JB, Siao G, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris MS, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson TJ, Lipshutz R, Chee M, Lander ES, Science.
  • the TaqManTM assay (Applied Biosystems), originally designed for quantitative realtime PCR, is a homogeneous, single-step assay also used in determination of mutation status of DNA (see, eg, A.A.
  • Cleavage separates a 5' - fluorophore from a 3' -quencher leading to detectable fluorescent signal.
  • the use of two allele- specific probes carrying different fluorophores permits SNP determination in the same tube without any post-PCR processing. Genotype is determined from the ratio of intensities of the two fluorescent probes at the end of amplification. Thus, rather than taking advantage of the full set of real-time PCR data as in quantitative studies, only end-point data are used.
  • TaqMan SNP genotyping in a high-throughput, automated manner is facilitated by the use of validated Pre-made TaqMan ® Genotyping assays, but Custom TaqMan ® Assays may also be used (High-throughput genotyping with single nucleotide polymorphisms, Ranade K, Chang MS, Ting CT, Pei D, Hsiao CF, Olivier M, Pesich R, Hebert J, Chen YD, Dzau VJ, Curb D, Olshen R, Risch N, Cox DR, Botstein D, Genome Res.
  • Single nucleotide polymorphisms can be determined using TaqManTM realtime PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification.
  • An algorithm for automatic genotype caling of SNPs using the full course of TaqMan real-time data is available for use (A. Callegaro eta/, Nucleic Acids Res. 2006; 34(7): e56, Published online 2006 April 14. doi: 10.1093/nar/gkll85, PMCID: PMC1440877).
  • the algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare.
  • “decrease”, “reduced”, or “reduction” are all used herein to mean a decrease by a statistically significant amount.
  • “reduce,” “reduction” or “decrease” typically means a decrease by at least 10% as compared to a reference level (e.g.
  • “reduction” does not encompass a complete reduction as compared to a reference level.
  • a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder. However, for example, for the purposes of lowering or reducing cholesterol level, for example, a reduction by about 5-10 points can be considered a "decrease" or "reduction.”
  • Inhibition refers and refers to decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more including 100% inhibition as compared to a reference level.
  • “Complete inhibition” refers to a 100% inhibition as compared to a reference level.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • an "increase” is a statistically significant increase in such level.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • “substantially” can refer to at least a 90% extent or degree of a characteristic or property of interest, e.g. at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or greater.
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, "individual,” “patient” and “subject” are used interchangeably herein.
  • the subject can be a non-human vertebrate, e.g. a primate, a rodent, a mouse, a rat, a pig, a sheep, a zebrafish, a frog, etc.
  • a non-human vertebrate e.g. a primate, a rodent, a mouse, a rat, a pig, a sheep, a zebrafish, a frog, etc.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples.
  • Mammals other than humans can be advantageously used as subjects that represent animal models of a disease or condition, e.g., a cardiovascular condition.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.
  • a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition.
  • a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
  • a "subject in need” or “human in need” of treatment for a particular condition can be a subject having that condition, such as increased cholesterol levels, diagnosed as having that condition, or at risk of developing that condition.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids with natural amino acids.
  • modified polypeptides one refers to polypeptides that include modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • polypeptide are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • protein and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins with the specified sequence.
  • One can also use peptide homologs, peptide orthologs, peptide paralogs, peptide fragments and other equivalents, variants, fragments, and analogs of the peptides as these terms are understood by one of ordinary skill in the art.
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid.
  • the nucleic acid can be either single-stranded or double-stranded.
  • a single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA. In some aspects one can also use analogs of nucleic acids.
  • nucleic acid probe refers to an isolated oligonucleotide molecule having a nucleic acid sequence which can hybridize to a target nucleic acid sequence, e.g. specifically hybridize to the target sequence.
  • a nucleic acid probe can further comprise a detectable label.
  • a nucleic acid probe can be attached to a solid surface.
  • a nucleic acid from is from about 5 nt to about 100 nt in length.
  • siRNA refers to a nucleic acid that forms an RNA molecule comprising two individual strands of RNA which are substantially complementary to each other.
  • the siRNA is at least about 15-40 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-40 nucleotides in length, and the double stranded siRNA is about 15-40 base pairs in length, preferably about 19-25 base nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).
  • a siRNA can be blunt- ended.
  • a siRNA can comprise a 3' and/or 5' overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides.
  • the length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand.
  • the siRNA molecules can also comprise a 3' hydroxyl group.
  • the siRNA can comprise a 5' phosphate group.
  • a siRNA has the ability to reduce or inhibit expression of a gene or target RNA when the siRNA is present or expressed in the same cell as the target gene, e.g. the target RNA.
  • siRNA-dependent post-transcriptional silencing of gene expression involves cutting the target RNA molecule at a site guided by the siRNA.
  • PCSK9 or "proprotein convertase subtilisin/kexin type 9” refers to a serine protease involved in regulating the levels of the low density lipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah and Prat, 2007). PCSK9 has been shown to directly interact with the LDLR protein, be endocytosed along with the LDLR, and co-immunofluoresce with the LDLR throughout the endosomal pathway (Lagace et al., 2006).
  • LDLR low density lipoprotein receptor
  • PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8) family of serine proteases (Seidah et al., 2003).
  • S8 subtilisin family of serine proteases
  • the sequence of PCSK9 for a variety of species is known, e.g., human PCSK9 (NCBI Gene ID No: 255738).
  • Nucleotide and polypeptide sequences for a number of PCSK9 isoforms are provided herein, e.g., SEQ ID NOs: 1-37.
  • PCSK9 exists as both a pro-form and a mature form.
  • Autocatalysis of the PCSK9 proform occurs between Glnl52 and Serl53 (VFAQISIP (SEQ ID NO: 67)) (Naureckiene et al., 2003), and has been shown to be required for its secretion from cells (Seidah et al., 2003).
  • the inactive form prior to this cleavage can be referred to herein as the "inactive”, “pro-form”, or "unprocessed” form of PCSK9.
  • the C-terminal fragment generated by the autocatalysis event can be referred to herein as the "mature,” “cleaved”, “processed” or “active” PCSK9.
  • Examples of pro-form and mature PCSK9 isoforms are provided herein, see, e.g. SEQ ID NOs: 1-27.
  • the "catalytic domain” of PCSK9 refers to the portion of a PCSK9 polypeptide corresponding to positions 153 to 449 of PCSK9, e.g. of SEQ ID NO: 1.
  • the "C-terminal domain” of PCSK9 refers to the portion of a PCSK9 polypeptide corresponding to positions 450-692 of PCSK9, e.g., of SEQ ID NO: 1.
  • a disease or condition "mediated by PCSK9” refers to a disease or condition which is caused by or characterized by a change in PCSK9, e.g. a change in expression level, a change in activity, and/or the presence of a variant or mutation of PCSK9.
  • diseases or conditions can include, for example, a lipid disorder,
  • the disease or condition is an inflammatory or autoimmune disease or condition.
  • a subject at risk of having or developing a disease or condition mediated by PCSK9 can be a subject exhibiting one or more signs or symptoms of such a disease or condition or having one or more risk factors for such a disease or condition, e.g. being overweight, having elevated cholesterol level, comprising one or more genetic polymorphisms known to predispose to the disease or condition, e.g., elevated cholesterol level, such as having a mutation in the LDLR (encoding low-density lipoprotein receptor) or APOB (encoding apolipoprotein B) or in the PCSK9 gene and/or having a family history of such a disease or condition.
  • LDLR encoding low-density lipoprotein receptor
  • APOB encoding apolipoprotein B
  • ligand refers to a molecule which can bind, e.g., specifically bind, to a second molecule or receptor.
  • a ligand can be, e.g., an antibody, antibody fragment, antibody portion, and/or affibody.
  • variant refers to a peptide or nucleic acid that differs from the polypeptide or nucleic acid (eg, the most common one in humans, eg, most frequent in a database as disclosed herein, such as the 1000 Genomes Project database) by one or more amino acid or nucleic acid deletions, additions, yet retains one or more specific functions or biological activities of the naturally occurring molecule.
  • Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size.
  • substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non- conventional amino acid.
  • amino acid substitutions are conservative.
  • polynucleotide or polypeptide refers to a polynucleotide or polypeptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild- type polynucleotide or polypeptide).
  • Variants of PCSK9 are provided elsewhere herein. Variants of PCSK9 can include the forms described herein as a, f, c, r, p, m, e h, aj, and q. Sequences of these variants are provided herein, see, e.g, SEQ ID NOs: l-27 and in Tables 1, 2 and 5.
  • “Modified variants” can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Some aspects use include insertion variants, deletion variants or substituted variants with substitutions of amino acids, including insertions and substitutions of amino acids and other molecules) that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins.
  • conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties.
  • Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • a “conservative substitution” of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non- polar, etc.) such that the substitution of even critical amino acids does not reduce the activity of the peptide, (i.e.
  • BBB blood brain barrier
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H.
  • substitutions suitable for amino acids on the exterior of a protein or peptide for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.
  • non-conservative amino acid substitutions are also encompassed within the term of variants.
  • an “antibody” refers to IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab') 2 , Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide- linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria. Antibodies can be humanized using routine technology.
  • an "antigen” is a molecule that is bound by a binding site on an antibody agent.
  • antigens are bound by antibody ligands and are capable of raising an antibody response in vivo.
  • An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof.
  • antigenic determinant refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.
  • an antibody fragment refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen.
  • An antibody fragment can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody.
  • an antibody fragment can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody.
  • an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL).
  • an antibody in another example, includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • antibody fragment encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies.
  • An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof).
  • Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.
  • antibody variable domain refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of Complementarity Determining Regions (CDRs; ie., CDR1, CDR2, and CDR3), and Framework Regions (FRs).
  • CDRs Complementarity Determining Regions
  • FRs Framework Regions
  • VH refers to the variable domain of the heavy chain.
  • VL refers to the variable domain of the light chain.
  • the amino acid positions assigned to CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or according to IMGT nomenclature.
  • D domain or region refers to the diversity domain or region of an antibody chain.
  • J domain or region refers to the joining domain or region of an antibody chain.
  • An antibody “gene segment”, e.g. a VH gene segment, D gene segment, or JH gene segment refers to oligonucleotide having a nucleic acid sequence that encodes that portion of an antibody, e.g. a VH gene segment is an oligonucleotide comprising a nucleic acid sequence that encodes a polypeptide VH domain.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed “framework regions" ("FR").
  • Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • antigen-binding fragment or "antigen-binding domain”, which are used interchangeably herein are used to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest.
  • binding fragments encompassed within the term "antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546; which is incorporated by reference herein in its entirety), which consists of a
  • the term "antibody binding site” refers to a polypeptide or domain that comprises one or more CDRs of an antibody and is capable of binding an antigen.
  • the polypeptide comprises a CDR3 (eg, HCDR3).
  • the polypeptide comprises CDRs 1 and 2 (eg, HCDR1 and 2) or CDRs 1-3 of a variable domain of an antibody (eg, HCDRsl-3).
  • the antibody binding site is provided by a single variable domain (eg, a VH or VL domain).
  • the binding site comprises a VH/VL pair or two or more of such pairs.
  • the term "specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
  • specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity.
  • specific binding can be "specific hybridization.”
  • a recombinant human(ized) antibody can be further optimized to decrease potential immunogenicity, while maintaining functional activity, for therapy in humans.
  • functional activity means a polypeptide capable of displaying one or more known functional activities associated with a recombinant antibody or antibody reagent thereof as described herein. Such functional activities include, e.g. the ability to bind to a target molecule.
  • immunizing refers to the step or steps of administering one or more antigens to an animal so that antibodies can be raised in the animal Generally, immunizing comprises injecting the antigen or antigens into the animal. Immunization can involve one or more administrations of the antigen or antigens. Suitable methods are prime-boost and RIMMS procedures as known to the skilled person in the art.
  • an "affibody” refers to a relatively small synthetic protein molecule that has high binding affinity for a target protein (e.g. for PCSK9 or a variant therefo).
  • Affibodies are composed of a three-helix bundle domain derived from the IgG-binding domain of staphylococcal protein A.
  • the protein domain consists of a 58 amino acid sequence, with 13 randomized amino acids affording a range of affibody variants.
  • an affibody weighs about 6 kDa while an antibody commonly weighs about 150 kDa)
  • an affibody molecule works like an antibody since its binding site is approximately equivalent in surface area to the binding site of an antibody.
  • K VH3-23*04 refers to a human VH domain variant comprising the polypeptide sequence of SEQ ID NO: 38.
  • VH3-23*04 has a valine residue instead of a leucine residue (see Figures 3 and 4; L24V, numbering including signal sequence; valine at position 5 shown in Figure 4) as a result of the presence of the rs56069819 SNP in the nucleic acid sequence encoding the VH domain.
  • rs56069819 refers to a mutation or variant in a VH gene segment from adenosine to cytosine (or thymine to guanine, depending upon the strand of DNA which is being read), resulting in the VH domain encoding VH3- 23*04.
  • Rs56069819 is depicted in Figure 4 and SEQ ID NO: 39, which demonstrate the T->G mutation (it is noted that the dbSNP entry for RS5606819 depicts the other strand, which comprises the A->C mutation). Further description of VH3-23*04 can be found, e.g., in US Patent Publication 2013/0071405; which is incorporated by reference herein in its entirety.
  • determine refers to ascertaining, e.g., by a quantitative or qualitative analysis.
  • has been determined can refer to ascertaining on the basis of previously obtained information or simultaneously obtained information.
  • selecting can include automation such as a computer implemented software program that upon input of the relevant data such as ethnicity or a panel of SNP data can make the determination based on the instructions set forth herein.
  • assaying refers to assessing, evaluating, quantifying, measuring, or characterizing an analyte, e.g., measuring the level of an analyte in a sample, identifying an analyte, or detecting the presence or absence of an analyte in a sample. In some embodiments, assaying refers to detecting a presence or absence of the analyte of interest. In some embodiments, assaying refers to quantifying an amount of an analyte, e.g., providing a measure of concentration or degree of analyte abundance. In some embodiments, assaying refers to enumerating the number of molecules of analyte present in a sample and/or specimen, e.g., to determine an analyte copy number.
  • multiplex refers to the carrying out of a method or process simultaneously and in the same reaction vessel on two or more, typically three or more, different target sequences, e.g. on two or more isoforms of PCSK9, or PCSK9 and an additional target.
  • a multiplex analysis typically includes analysis of 10-50; 10-100; 10-1000, 10-5000, 10-10000 reactions in a multiplex format, such as a multiwa!l, an array, or a multichannel reaction.
  • the analysis or multiplex analysis is also automated using robotics and typically software executed by a computer and may include a robotic handling of samples, automatic or robotic selection of positive or negative results, assaying for presence of absence of a target, such as a nucleic acid polymorphism or a protein variant.
  • a target such as a nucleic acid polymorphism or a protein variant.
  • biological sample or "test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a sample from a subject.
  • Exemplary biological samples include, but are not limited to, a biofluid sample; serum; plasma; urine; saliva; hair, epithelial cells, skin, a tumor biopsy and/or tissue sample etc.
  • the term also includes a mixture of the above-mentioned samples.
  • test sample or “biological sample” also includes untreated or pretreated (or pre-processed) biological samples.
  • the biological sample should typically comprise at least one cell comprising nucleic acids.
  • the test sample can be obtained by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells (e.g. isolated at a prior time point and isolated by the same or another person).
  • the test sample can be freshly collected or a previously collected, refrigerated, frozen or otherwise preserved sample.
  • the test sample can be an untreated test sample.
  • untreated test sample refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution.
  • Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof.
  • the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein.
  • a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein.
  • the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample.
  • a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof.
  • the test sample can be treated with a chemical and/or biological reagent.
  • Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing.
  • biomolecules e.g., nucleic acid and protein
  • One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing.
  • protease inhibitor which is generally used to protect or maintain the stability of protein during processing.
  • Genotyping refers to a process of determining the specific allelic composition of a cell and/or subject at one or more position within the genome, e.g. by determining the nucleic acid sequence at that position.
  • Genotyping refers to a nucleic acid analysis and/or analysis at the nucleic acid level.
  • phenotyping refers a process of determining the identity and/or composition of an expression product of a cell and/or subject, e.g. by determining the polypeptide sequence of an expression product.
  • Phenotyping refers to a protein analysis and/or analysis at the protein level.
  • nucleic acid amplification refers to the production of additional copies of a nucleic acid sequence and is typically carried out using polymerase chain reaction (PCR) or ligase chain reaction (LCR) technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N. Y.). Other methods for amplification are also contemplated in aspects of the invention.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • allele-specific amplification refers to a reaction (e.g., PCR reaction) in which at least one of the primers (e.g., allele-specific primer) is chosen from a polymorphic area of gene (e.g., single nucleotide polymorphism), with the polymorphism located at or near the primer's 3'-end.
  • a mismatched primer will not initiate amplification, whereas a matched primer will initiate amplification.
  • the appearance of an amplification product is indicative of the presence of the polymorphism.
  • sequencing refers to the determination of the exact order of nucleotide bases in a strand of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) or the exact order of amino acids residues or peptides in a protein. Nucleic acid sequencing can be done using Sanger sequencing or next-generation high-throughput sequencing.
  • next-generation sequencing refers to oligonucleotide sequencing technologies that have the capacity to sequence oligonucleotides at speeds above those possible with conventional sequencing methods (e.g. Sanger sequencing), due to performing and reading out thousands to millions of sequencing reactions in parallel.
  • Non-limiting examples of next-generation sequencing methods/platforms include Massively Parallel Signature Sequencing (Lynx Therapeutics); 454 pyro-sequencing (454 Life Sciences/ Roche Diagnostics); solid-phase, reversible dye-terminator sequencing (Solexa/Illumina): SOLiD technology (Applied Biosystems); Ion semiconductor sequencing (ION Torrent); DNA nanoball sequencing (Complete Genomics); and technologies available from Pacific Biosciences, Intelligen Bio-systems, Oxford Nanopore Technologies, and Helicos Biosciences.
  • Next-generation sequencing technologies and the constraints and design parameters of associated sequencing primers are well known in the art (see, e.g. Shendure, et al., "Next-generation DNA sequencing," Nature, 2008, vol. 26, No.
  • nucleic acid hybridization refers to the pairing of complementary RNA and DNA strands as well as the pairing of complementary DNA single strands.
  • nucleic acid hybridization can refer to a method of determining a nucleic acid sequence and/or identity by hybridizing a nucleic acid sample with a probe, e.g. Northern or Southern blot analysis or microarray analysis.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized ⁇ i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). For treatment to be effective a complete cure is not contemplated. The method can in certain aspects include cure as well.
  • the term "pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • compositions can be administered separately or simultaneously.
  • Separate administration refers to the two compositions being administered at different times, e.g. at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart.
  • One can also administer compositions at 24 hours apart, or even longer apart.
  • two or more compositions can be administered simultaneously, e.g. less than 10 or less than 5 minutes apart.
  • Compositions administered simultaneously can, in some aspects, be administered as a mixture, with or without similar or different time release mechanism for each of the components.
  • contacting refers to any suitable means for delivering, or exposing, an agent to at least one complex, enzyme, or cell.
  • Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.
  • Obtaining a biological sample from a subject can comprise physical removing a sample from a subject (e.g. drawing blood or taking a hair or saliva sample) without or without active participation from the subject; receiving a sample from a subject (e.g. the subject collects a saliva or hair sample themselves and provides it, e.g. in a container provided for the purpose); or procuring a sample from a storage facility, medical facility, or medical provider.
  • Obtain from the human or subject refers to an active step of, e.g., drawing blood or taking a tissue or cell sample.
  • cholesterol level refers to a level of one or more of total cholesterol, LDL cholesterol, HDL cholesterol, and/or triglycerides. Cholesterol levels can be the level of cholesterol in the blood of a subject.
  • maintaining a particular level refers to a process that results in the cholesterol level not increasing by more than 10% over time. Maintaining may also refer to maintaining a previously achieved level. For example, if a human has received statin treatment, one can maintain the cholesterol level achieved using the statin treatment.
  • the subject treated according to the methods described herein has previously had their cholesterol level reduced.
  • "previously reduced” indicates that at a prior point in time, the subject experienced a decrease in cholesterol levels.
  • the decrease can be due to administration of a pharmaceutical composition (e.g. administration of a composition as described herein or another composition, e.g. a statin) or due to another cause, e.g. a change in diet and/or exercise.
  • An existing treatment for high cholesterol levels is the administration of a statin.
  • a "statin” also known as HMG-CoA reductase inhibitors
  • HMG-CoA reductase inhibitors are inhibitors of the enzyme HMG-coA reductase, which mediates cholesterol production in the liver.
  • Statins by competitively binding HMG-CoA reductase, prevent the binding of HMG-CoA to the enzyme and thereby inhibit the activity of the reductase (e.g. the production of mevalonate).
  • statins can include atorvastatin (LIPITORTM), fluvastatin (LESCOLTM), lovastatin (MEVACORTM, ALTOCORTM), pravastatin (LIVALOTM), pravastatin (PRAVACHOLTM), rosuvastatin (CRESTORTM), and simvastatin (ZOCORTM).
  • Statins can be administered in combination with other agents, e.g. the combination of ezetimibe and simvastatin.
  • resistant to statin treatment or “reduced responsiveness to statin treatment” refers to a subject exhibiting a statistically significantly lower response to the administration of a statin as compared to a reference level.
  • the reference level can be, e.g., the average response for a population of subjects or the level of the individual subject at an earlier date.
  • a response to statin treatment is readily measured by one of skill in the art, e.g., measurement of cholesterol levels, changes in cholesterol levels, and/or HMG-CoA reductase activity.
  • detectable label refers to a molecule or moiety that can be detected, e.g. measured and/or determined to be present or absent.
  • Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g.
  • detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means.
  • the detectable labels used in the methods described herein can be
  • the detectable label can be linked by covalent or non-covalent means to the reagent.
  • a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules.
  • Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
  • the detectable label can be a fluorescent compound.
  • a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o- phthaldehyde, fluorescamine, Cy3TM, Cy5TM, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5TM, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyesTM, 6- carboxyfhiorescein (commonly known by the abbreviations FAM and F
  • a detectable label can be a radiolabel including, but not limited to 3 H, 12 1, 35 S, H C, 32 P, and 33 P.
  • a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase.
  • An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal.
  • Enzymes contemplated for use as a detectable label can include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene,
  • reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • a detectable tag such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • Other detection systems can also be used, for example, a biotin-streptavidin system.
  • the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate.
  • streptavidin peroxidase detection kits are commercially available, e. g.
  • a reagent can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • regulatory agency refers to one of the agencies responsible for evaluating, e.g, the safety and efficacy of a medical product and/or composition and controlling the sales/marketing of such products and/or compositions in a given area.
  • the Food and Drug Administration (FDA) in the US and the European Medicines Agency (EPA) in Europe are but two examples of such regulatory agencies.
  • Other non-limiting examples can include SDA, MPA, MHPRA, IMA, AN MAT, Hong Kong Department of Health-Drug Office, CDSCO, Medsafe, and KFDA.
  • an injection device refers to a device that is designed for carrying out injections, an injection including the steps of temporarily fluidically coupling the injection device to a person's tissue, typically the subcutaneous tissue. An injection further includes administering an amount of liquid drug into the tissue and decoupling or removing the injection device from the tissue.
  • an injection device can be an intravenous device or IV device, which is a type of injection device used when the target tissue is the blood within the circulatory system, e.g., the blood in a vein.
  • a common, but non-limiting example of an injection device is a needle and syringe.
  • a "buffer” refers to a chemical agent that is able to absorb a certain quantity of acid or base without undergoing a strong variation in pH.
  • Packaging refers to how the components are organized and/or restrained into a unit fit for distribution and/or use.
  • Packaging can include, e.g., boxes, bags, syringes, ampoules, vials, tubes, clamshell packaging, barriers and/or containers to maintain sterility, labeling, etc.
  • instructions refers to a display of written, printed or graphic matter on the immediate container of an article, for example the written material displayed on a vial containing a pharmaceutically active agent, or details on the composition and use of a product of interest included in a kit containing a composition of interest. Instructions set forth the method of the treatment as contemplated to be administered or performed.
  • a "solid surface” refers to an object suitable for the attachment of biomolecules.
  • a solid surface can include a particle (including, but not limited to an agarose or latex bead or particle or a magnetic particle), a bead, a nanoparticle, a polymer, a substrate, a slide, a coverslip, a plate, a dish, a well, a membrane, and/or a grating.
  • the solid surface can include many different materials including, but not limited to, polymers, plastics, resins, polysaccharides, silicon or silica based materials, carbon, metals, inorganic glasses, and membranes.
  • classification of a subject refers to determining if the subject has biological ancestors who originated in a particular geographical area, and are therefore likely to have particular genetic variants found in the populations which have historically occupied that area.
  • Classification can comprise, e.g. obtaining information on the subject's family, interviewing the subject or a family member regarding their biological family's ancestry, and/or genetic testing. Classification can be on the basis used for the 1000 Genomes Project, as will be familiar to the skilled person in the art.
  • the subject can be classified as being of a particular ancestry if at least the subject's genome comprises a substantial number of different alleles in common with other humans of that ancestry (eg, determined by reference to the 1000 Genomes Project database), for example, at least 10, 20, 30, 40, 50 or 100 or more alleles in common.
  • Abbreviations for particular ancestral groups are provided in Table 4.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
  • the invention addresses the need to treat humans having naturally-occurring rarer natural PCSK9 alleles, genotypes and phenotypes (rarer protein forms).
  • the invention provides the following aspects.
  • a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof comprising
  • a human comprising (i) a proprotein convertase subtilisin/kexin type 9 (PCSK9) nucleotide sequence selected from the group consisting of SEQ ID NOs: 29- 37; and/or (ii) a nucleotide sequence thereof encoding the catalytic domain or C- terminal domain of a PCSK9 protein; and
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • step (a) comprises selecting a human comprising a PCSK9 protein encoded by the nucleotide sequence of (i) or (ii).
  • the antibody or antibody fragment specifically binds said PCSK9 amino acid sequence.
  • the antibody or antibody fragment binds a second PCSK9 protein comprising an amino acid sequence encoded by (i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37; and/or (ii) a nucleotide sequence thereof encoding the catalytic domain or C-terminal domain of a PCSK9 protein.
  • the antibody comprises a VH domain derived from the recombination of a human VH gene segment, human D gene segments and a human JH segment, the VH gene segment comprising a nucleotide sequence that comprises a single nucleotide polymorphism with nucleotide C as indicated in rs56069819
  • the VH gene segment is VH3-23*04 (SEQ ID NO: 38) encoded by a sequence comprising SEQ ID NO: 39.
  • the antibody comprises a VH domain, wherein the VH domain comprises the framework 1 sequence of SEQ ID No. 40.
  • the human has been determined to comprise the nucleotide sequence of (i) and/or (ii).
  • the human has been determined to comprise a proprotein convertase subtilisin/kexin type 9 (PCSK9) variant protein encoded by the nucleotide sequence of (i) and/or (ii).
  • the method further comprises the step of determining that the human comprises the nucleotide sequence of (i) and/or (ii).
  • the determining step is performed before administration of the antibody to the human.
  • the method further comprises the step of determining that the human comprises a proprotein convertase subtilisin/kexin type 9 (PCSK9) variant protein encoded by the nucleotide sequence of (i) and/or (ii).
  • the determining step is performed before administration of the antibody to the human.
  • the step of determining comprises assaying a biological sample from the human for (i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37; and/or (ii) a nucleotide sequence encoding the catalytic domain or C-terminal domain of the PCSK9 variant protein.
  • the assaying comprises contacting the biological sample with
  • At least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides that can specifically hybridize to and identify in the biological sample a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain-encoding sequence thereof, or that specifically hybridizes to an antisense of said sequence, wherein said nucleic acid hybridizes to at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28 or hybridizes to an antisense sequence thereby forming a complex when at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminal domain- encoding sequence thereof is present; and/or
  • At least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or comprising an antisense sequence of said contiguous nucleotides, wherein said sequence of contiguous nucleotides comprises at least one nucleotide present in said selected sequence which is not present in SEQ ID NO: 28 thereby forming a complex when a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 is present; and
  • detecting the presence or absence of the complex determines that the human comprises the PCSK9 variant protein.
  • the assaying comprises nucleic acid amplification and optionally one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification.
  • the assaying is performed in a multiplex format.
  • the method further comprises obtaining the biological sample from the human.
  • said biological sample comprises serum, blood, feces, tissue, a cell, urine and/or saliva of said human.
  • said human is or has been further determined to be substantially resistant to statin treatment.
  • the human is receiving or has received statin treatment or has reduced responsiveness to statin treatment.
  • the human is further administered a statin.
  • said antibody or antibody fragment and said statin are administered separately or simultaneously.
  • said human is indicated as heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 and/or the nucleotide sequence thereof encoding the catalytic domain- or C-terminal domain-encoding sequence of a PCSK9 protein.
  • said human is further indicated as comprising the nucleotide sequence of SEQ ID NO: 28 and/or the catalytic domain- or C-terminal domain-encoding sequence thereof.
  • said human is indicated as homozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 and/or the catalytic domain- or C-terminal domain-encoding sequence thereof.
  • said human has been diagnosed with at least one condition selected from a lipid disorder, hyperlipoproteinemia, hyperlipidemia; dyslipidemia;
  • hypercholesterolemia a heart attack, a stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, claudication, type II diabetes, high blood pressure, and a cardiovascular disease or condition.
  • said method treats or prevents in said human at least one condition selected from a lipid disorder, hyperlipoproteinemia, hyperlipidemia; dyslipidemia;
  • hypercholesterolemia a heart attack, a stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, claudication, type II diabetes, high blood pressure, and a cardiovascular disease or condition.
  • a method of treating and/or preventing a disease or condition mediated by proprotein convertase subtilisin/kexin type 9 (PCSK9) in a human who has been determined to comprise and/or selected as comprising a PCSK9 variant protein comprising administering to the human a ligand that binds the PCSK9 variant protein to treat and/or prevent said disease or condition.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • paragraph 1 provides:
  • a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation as defined herein (eg, I474V or E670G) in SEQ ID NO: I, wherein the antibody comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment encoding a valine at the amino acid corresponding to position 5 of SEQ ID NO: 40 and wherein said human comprises (i) a VH gene segment encoding the framework 1 of SEQ ID NO: 40 and (ii) a nucleotide sequence encoding a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation in SEQ ID NO: 1.
  • paragraph 1 provides: - A method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof, the method comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation as defined herein (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody comprises a VL domain derived from the recombination of a human VL segment and a human JL segment, the human VL segment is a VA or VK disclosed herein and wherein said human comprises (i) said VL gene segment and (ii) a nucleotide sequence encoding a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation in SEQ ID NO: 1.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • paragraph 1 provides: -
  • a method of reducing cholesterol level or maintaining previously reduced cholesterol level in a human in need thereof comprising administering to said human an antibody or antibody fragment that specifically binds a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising a mutation as defined herein (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody comprises a C domain encoded by a human CH, CA or CK gene segment disclosed herein and wherein said human comprises (i) said C gene segment and (ii) a nucleotide sequence encoding a proprotein convertase subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain comprising said mutation in SEQ ID NO: 1.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • PCSK9 variant protein is selected from the group consisting of PCSK9 variant protein forms f, c, r, p, m, e, h, aj and q.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • the method of paragraph 5 further comprising assaying a biological sample from the human for (i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37; and/or (ii) a nucleotide sequence thereof encoding the catalytic domain or C- terminal domain of a PCSK9 protein.
  • the assaying comprises nucleic acid amplification and/or one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification.
  • the ligand is selected from an antibody, an antibody portion, an antibody fragment or an affibody.
  • the ligand is an antibody or antibody fragment that specifically binds to a human PCSK9 selected from forms f, c, m, e, h, p, q and aj, wherein the antibody or fragment comprises a VH domain derived from the
  • VH gene segment comprising a nucleotide sequence that comprises SNP rs56069819 (SEQ ID NO: 41).
  • said biological sample comprises serum, blood, feces, hair, tissue, cells, urine and/or saliva of said human.
  • SEQ ID NO: 29 and is classified as of ASW, YRI, GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry, then administering to said human a ligand that specifically binds said PCSK9 variant protein comprising a variant encoded by SEQ ID NO: 29; or
  • SEQ ID NO: 30 is classified as of ASW, YRI, GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry then administering to said human a ligand that specifically binds said PCSK9 variant protein comprising a variant encoded by SEQ ID NO: 30; or
  • SEQ ID NO: 32 and is classified as of ASW, GBR, TSI, CLM, JPT, PUR, IBS, FIN or CEU ancestry then administering to said human a ligand that specifically binds said PCSK9 variant protein comprising a variant encoded by SEQ ID NO: 32; or d.
  • SEQ ID NO: 33 and is classified as of LWK, ASW, YRI or CLM ancestry then
  • SEQ ID NO: 34 e. SEQ ID NO: 34, and is classified as of LWK, ASW or YRI ancestry then administering to said human a ligand that specifically binds said PCSK9 variant protein comprising a variant encoded by SEQ ID NO: 34; or
  • SEQ ID NO: 36 is classified as of LWK, ASW or YRI ancestry then administering to said human a ligand that specifically binds said PCSK9 variant protein comprising a variant encoded by SEQ ID NO: 36; or
  • SEQ ID NO: 37 is classified as of CHS, ASW, JPT, PUR or CHB ancestry then administering to said human a ligand that specifically binds said PCSK9 variant protein comprising a variant encoded by SEQ ID NO: 37.

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Abstract

Cette invention concerne des cibles d'intérêt humaines (TOI), des ligands anti-TOI, des kits et des compositions, ainsi qu'un procédé.
PCT/GB2014/053730 2013-12-17 2014-12-17 Anticorps destinés à être utilisés pour traiter des états associés à des variants spécifiques de pcsk9 chez des populations de patients spécifiques WO2015092394A1 (fr)

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DE112014005747.9T DE112014005747T5 (de) 2013-12-17 2014-12-17 Antikörper zur Verwendung bei der Behandlung von Zuständen, die mit spezifischen PCSK9 Varianten in spezifischen Patientenpopulationen in Beziehung stehen
EP23207654.7A EP4328245A2 (fr) 2014-07-15 2014-12-18 Anticorps utilisés dans le traitement de pathologies associées à des variants pcsk9 spécifiques dans des populations de patients spécifiques
EP14199053.1A EP2975059A1 (fr) 2014-07-15 2014-12-18 Anticorps pour utilisation dans le traitement de maladies liées à certains variants spécifiques de pcsk9 dans certaines populations spécifiques de patients
EP17199373.6A EP3332790A1 (fr) 2014-07-15 2014-12-18 Anticorps utilisés dans le traitement de pathologies associées à des variants pcsk9 spécifiques dans des populations de patients spécifiques
IES20140321A IES86623B2 (en) 2014-07-15 2014-12-22 An injectable antibody preparation for use in reducing or maintaining previously reduced cholesterol level
DE202015008988.7U DE202015008988U1 (de) 2014-07-15 2015-07-15 Targeting von humaner PCSK9 zur Cholesterinbehandlung
PCT/EP2015/066099 WO2016008899A1 (fr) 2014-07-15 2015-07-15 Ciblage de la pcsk9 humaine pour le traitement du cholestérol
DE202015008974.7U DE202015008974U1 (de) 2014-07-15 2015-07-15 Targeting von humaner PCSK9 zur Cholesterinbehandlung

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US201361916866P 2013-12-17 2013-12-17
US201361916862P 2013-12-17 2013-12-17
US61/916,866 2013-12-17
GB1322253.4 2013-12-17
GB1322250.0 2013-12-17
GB1322250.0A GB2521355A (en) 2013-12-17 2013-12-17 Human targets I
US61/916,862 2013-12-17
GB1322253.4A GB2521356B (en) 2013-12-17 2013-12-17 Antibodies for use in treating conditions related to specific PCKS9 variants and associated diagnostic methods
US14/138,446 2013-12-23
US14/138,446 US8883157B1 (en) 2013-12-17 2013-12-23 Targeting rare human PCSK9 variants for cholesterol treatment
US14/228,760 US8951523B1 (en) 2013-12-17 2014-03-28 Targeting rare human PCSK9 variants for cholesterol treatment
US14/228,760 2014-03-28
EP14172579.6A EP2886558A1 (fr) 2013-12-17 2014-06-16 Anticorps pour une utilisation dans le traitement de maladies liées à des variants spécifiques de PCSK9 dans des populations de patients spécifiques
EP14172578.8A EP2886557A1 (fr) 2013-12-17 2014-06-16 Anticorps pour utilisation dans le traitement de maladies liées à certains variants spécifiques de PCSK9 dans certaines populations spécifiques de patients
EP14172578.8 2014-06-16
EP14172579.6 2014-06-16
US14/331,609 US9051378B1 (en) 2014-07-15 2014-07-15 Targeting rare human PCSK9 variants for cholesterol treatment
US14/331,730 US9914769B2 (en) 2014-07-15 2014-07-15 Precision medicine for cholesterol treatment
US14/331,609 2014-07-15
US14/331,730 2014-07-15
US14/331,665 US9023359B1 (en) 2014-07-15 2014-07-15 Targeting rare human PCSK9 variants for cholesterol treatment
US14/331,665 2014-07-15
US14/457,536 US9017678B1 (en) 2014-07-15 2014-08-12 Method of treating rheumatoid arthritis using antibody to IL6R
US14/457,566 2014-08-12
US14/457,566 US8945560B1 (en) 2014-07-15 2014-08-12 Method of treating rheumatoid arthritis using antibody to IL6R
US14/457,536 2014-08-12
US14/472,685 US8992927B1 (en) 2014-07-15 2014-08-29 Targeting human NAV1.7 variants for treatment of pain
US14/472,818 US8980273B1 (en) 2014-07-15 2014-08-29 Method of treating atopic dermatitis or asthma using antibody to IL4RA
US14/472,698 2014-08-29
US14/472,828 US8986691B1 (en) 2014-07-15 2014-08-29 Method of treating atopic dermatitis or asthma using antibody to IL4RA
US14/472,698 US8986694B1 (en) 2014-07-15 2014-08-29 Targeting human nav1.7 variants for treatment of pain
US14/472,818 2014-08-29
US14/472,685 2014-08-29
US14/472,828 2014-08-29
US14/490,112 2014-09-18
US14/490,160 2014-09-18
EP14185297.0A EP2975058A1 (fr) 2014-07-15 2014-09-18 Anticorps pour leur utilisation dans le traitement de maladies liées à certains variants spécifiques de PCSK9 dans certaines populations spécifiques de patients
US14/490,160 US8999341B1 (en) 2014-07-15 2014-09-18 Targeting rare human PCSK9 variants for cholesterol treatment
US14/490,112 US9034331B1 (en) 2014-07-15 2014-09-18 Targeting rare human PCSK9 variants for cholesterol treatment
US14/490,091 US9068012B1 (en) 2014-07-15 2014-09-18 Targeting rare human PCSK9 variants for cholesterol treatment
US14/490,175 2014-09-18
EP14185297.0 2014-09-18
US14/490,091 2014-09-18
US14/490,175 US9040052B1 (en) 2013-12-17 2014-09-18 Precision Medicine by targeting rare human PCSK9 variants for cholesterol treatment
US14/500,397 2014-09-29
US14/500,233 US9045548B1 (en) 2014-07-15 2014-09-29 Precision Medicine by targeting rare human PCSK9 variants for cholesterol treatment
US14/500,233 2014-09-29
US14/500,397 US10618971B2 (en) 2013-12-17 2014-09-29 Targeting rare human PCSK9 variants for cholesterol treatment
US14/507,368 US9034332B1 (en) 2014-07-15 2014-10-06 Precision medicine by targeting rare human PCSK9 variants for cholesterol treatment
US14/507,368 2014-10-06
EP14190945.7 2014-10-29
EP14190945 2014-10-29
US14/536,049 2014-11-07
US14/536,049 US9045545B1 (en) 2014-07-15 2014-11-07 Precision medicine by targeting PD-L1 variants for treatment of cancer
US14/536,129 2014-11-07
US14/536,129 US9062105B1 (en) 2014-07-15 2014-11-07 Precision Medicine by targeting VEGF-A variants for treatment of retinopathy
US14/537,403 2014-11-10
US14/537,403 US9067998B1 (en) 2014-07-15 2014-11-10 Targeting PD-1 variants for treatment of cancer
US14/552,816 US10611849B2 (en) 2013-12-17 2014-11-25 Precision medicine by targeting rare human PCSK9 variants for cholesterol treatment
US14/552,816 2014-11-25
EP14196638 2014-12-05
EP14196641 2014-12-05
EP14196645.7 2014-12-05
EP14196641.6 2014-12-05
EP14196638.2 2014-12-05
EP14196645 2014-12-05

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