1D05 PCSK9 antagonists

专利摘要:

公开号:AU2009212262A1
申请号:U2009212262
申请日:2009-02-06
公开日:2009-08-13
发明作者:Jeffrey T. Blue；Jon H. Condra；Rose M. Cubbon；Feng Dong；Sonia M. Gregory；Holly A. Hammond；Yan Li；Peter Luo；Henryk Mach；Denise K. Nawrocki；Laura Orsatti；Shilpa Pandit；Laurence B. Peterson；Joseph C. Santoro；Ayesha Sitlani；Kevin Wang；Dana D. Wood；Heidi Yoder；Pingyu Zhong
申请人:Istituto di Ricerche di Biologia Molecolare P Angeletti SpA；Merck Sharp and Dohme Corp；
IPC主号:C07K16-40

专利说明:
WO 2009/100297 PCT/US2009/033341 TITLE OF THE INVENTION iD05 PCSK9 ANTAGONISTS CROSS-REFERENCE TO RELATED APPLICATIONS 5 This application claims the benefit of U.S. Provisional Application No. 61/063,949, filed on February 7, 2008, and 61/066,577, filed February 21, 2008. STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not Applicable. 10 REFERENCE TO MICROFICHE APPENDIX Not Applicable. BACKGROUND OF THE INVENTION 15 Proprotein convertase subtilisin-kexin type 9 (hereinafter called "PCSK9"), also known as neural apoptosis-regulated convertase 1 ("NARC-1"), is a proteinase K-like subtilase identified as the 9 th member of the secretary subtilase family; see Seidah et al,, 2003 PNAS 100:928-933. The gene for PCSK9 localizes to human chromosome 1p33-p34.3; Seidah et al, supra. PCSK9 is expressed in cells capable of proliferation and differentiation including, for 20 example, hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons; Seidah et al, supra. Original synthesis of PCSK9 is in the form of an inactive enzyme precursor, or zymogen, of- 72-kDa which undergoes autocatalytic, intramolecular processing in the endoplasmic reticulum ("ER") to activate its functionality. This internal processing event has 25 been reported to occur at the SSVFAQ N SIPWNL 1 58 motif (SEQ ID NOs: 19 and 20, respectively); Benjannet et al., 2004 J Biol. Chem. 279:48865-48875, Such internal processing has been reported as a requirement of exit from the ER; Benjannet et al., supra; Seidah et al., supra. The cleaved and, thereby, activated protein is secreted in association with the cleaved peptide; supra. 30 The sequence for human PCSK9 (-22-kb long with 12 exons encoding a 692 amino acid protein) can be found in one instance at Deposit No. NP_777596.2. Human, mouse and rat PCSK9 nucleic acid sequences have been deposited; see, e.g., GenBank Accession Nos.: AX127530 (also AX207686), NP_705793 (also Q80W65), and P59996, respectively. PCSK9 possesses several domains found in other proprotein convertases, including an N-terminal signal 35 sequence, a pro domain, a catalytic domain and a cysteine-rich C terminal domain. The PCSK9 catalytic domain shares high sequence similarity with the proteinase K family of subtilases and, notably, a catalytic triad of D186, H226 and S386. - 1 - WO 2009/100297 PCT/US2009/033341 PCSK9 is disclosed and/or claimed in several patent publications including, but not limited to the following: PCT Publication Nos. WO 01/31007, WO 01/57081, WO 02/14358, WO 01/98468, WO 02/102993, WO 02/102994, WO 02/46383, WO 02/90526, WO 01/77137, and WO 01/34768; US Publication Nos. US 2004/0009553 and US 2003/0119038, and European 5 Publication Nos. EP 1 440 981, EP 1 067 182, and EP 1 471 152. PCSK9 has been ascribed a role in the differentiation of hepatic and neuronal cells (Seidah et at., supra.), is highly expressed in embryonic liver, and has been strongly implicated in cholesterol homeostasis, Studies have suggested a specific role for PCSK9 in cholesterol biosynthesis or uptake. In a study of cholesterol-fed rats, Maxwell et al. found that PCSK9 was 10 downregulated in a similar manner to three other genes involved in cholesterol biosynthesis, Maxwell et al, 2003 J Lipid Res. 44:2109-2119. The expression of PCSK9 has, in fact, been shown to be regulated by sterol regulatory element-binding proteins ("SREBP"), as seen with other genes involved in cholesterol metabolism; supra. Later support for these findings came about through a study of PCSK9 transcriptional regulation which demonstrated that such 15 regulation was quite typical of other genes implicated in lipoprotein metabolism; Dubuc et al., 2004 Arterioscler Thromb. Vasc. Biol. 24:1454-1459. Statins have been shown to upregulate PCSK9 expression in a manner attributed to the cholesterol-lowering effects of the drugs; supra. Moreover, it has been shown that PCSK9 promoters possess two conserved sites involved in cholesterol regulation, a sterol regulatory element and an SpI site; supra. 20 Several lines of evidence demonstrate that PCSK9, in particular, lowers the amount of hepatic LDLR protein and thus compromises the liver's ability to remove LDL cholesterol from the circulation. Adenovirus-mediated overexpression of PCSK9 in the livers of mice results in the accumulation of circulating LDL-C due to a dramatic loss of hepatic LDLR protein, with no effect on LDLR mRNA levels; Benjannet et al, 2004 Ji Biol. Chem. 279:48865 25 48875; Maxwell & Breslow, 2004 PNAS 101:7100-7105; Park et al., 2004 J Biol Chem. 279:50630-50638; and Lalanne et al., 2005 J Lipid Res. 46:1312-1319. The effect of PCSK9 overexpression on raising circulating LDL-C levels in mice is completely dependent on the expression of LDLR, again, indicating that the regulation of LDL-C by PCSK9 is mediated through downregulation of LDLR protein. In agreement with these findings, mice lacking 30 PCSK9 or in which PCSK9 mRNA has been lowered by antisense oligonucleotide inhibitors have higher levels of hepatic LDLR protein and a greater ability to clear circulating LDL-C; Rashid et al, 2005 PNAS 102:5374-5379; and Graham et al., 2007 J. Lipid Res. 48(4):763-767. In addition, lowering PCSK9 levels in cultured human hepatocytes by siRNA also results in higher LDLR protein levels and an increased ability to take up LDL-C; Benjannet et al., 2004 J 35 Biol. Chem. 279:48865-48875; and Lalanne et al., 2005 ] Lipid Res. 46:1312-1319. Together, these data indicate that PCSK9 action leads to increased LDL-C by lowering LDLR protein levels. -2- WO 2009/100297 PCT/US2009/033341 A number of mutations in the gene PCSK9 have also been conclusively associated with autosomal dominant hypercholesterolemia ("ADH"), an inherited metabolism disorder characterized by marked elevations of low density lipoprotein ("LDL") particles in the plasma which can lead to premature cardiovascular failure; see Abifadel et al., 2003 Nature Genetics 5 34:154-156; Timms et al, 2004 Hum. Genet. 114:349-353; Leren, 2004 Clin. Genet. 65:419-422. A later-published study on the Si 27R mutation of Abifadel et al, supra, reported that patients carrying such a mutation exhibited higher total cholesterol and apoB 100 in the plasma attributed to (1) an overproduction of apoB 100-containing lipoproteins, such as low density lipoprotein ("LDL"), very low density lipoprotein ("VLDL") and intermediate density lipoprotein ("IDL"), 10 and (2) an associated reduction in clearance or conversion of said lipoproteins; Ouguerram et al., 2004 Arterioscler. Thromb. Vase. Biol. 24:1448-1453. Accordingly, there can be no doubt that PCSK9 plays a role in the regulation of LDL. The expression or upregulation of PCSK9 is associated with increased plasma levels of LDL cholesterol, and the corresponding inhibition or lack of expression of PCSK9 is associated 15 with reduced LDL cholesterol plasma levels. Decreased levels of LDL cholesterol associated with sequence variations in PCSK9 have been found to confer protection against coronary heart disease; Cohen, 2006 N. Engl. J Med. 354:1264-1272. The identification of compounds and/or agents effective in the treatment of cardiovascular affliction is highly desirable. In clinical trials, reductions in LDL cholesterol 20 levels have been directly related to the rate of coronary events; Law et al, 2003 BMJ 326:1423 1427. More recently, the moderate lifelong reduction in plasma LDL cholesterol levels was found to correlate with a substantial reduction in the incidence of coronary events; Cohen et al., supra. This was the case even in populations with a high prevalence of non-lipid-related cardiovascular risk factors; supra. Accordingly, there is great benefit to be reaped from the 25 managed control of LDL cholesterol levels. The present invention advances these interests by providing antagonists of PCSK9 of use for inhibiting the activities of PCSK9 and the corresponding role PCSK9 plays in various therapeutic conditions. 30 SUMMARY OF THE INVENTION The present invention relates to antagonists of PCSK9 and, in particular embodiments, those antagonists that inhibit both human and murine PCSK9 and those exhibiting preferential targeting of processed PCSK9. Broadly, protein-specific antagonists of PCSK9 (or "PCSK9-specific antagonists" as referred to herein) are PCSK9 protein binding molecules or 35 molecules effective in the selective binding of PCSK9 and inhibition of PCSK9 function. These molecules are of import in the treatment of conditions associated with or impacted by PCSK9 function, including, but not limited to hypercholesterolemia, coronary heart disease, metabolic -3 - WO 2009/100297 PCT/US2009/033341 syndrome, acute coronary syndrome and related conditions. PCSK9-specific antagonists are characterized by selective recognition and binding to PCSK9. PCSK9-specific antagonists do not show significant binding to proteins other than PCSK9, other than in those specific instances where the antagonist is supplemented or designed to confer an additional, distinct specificity to 5 the PCSK9-specific binding component. PCSK9-specific antagonists forming particular embodiments hereof comprise (a) a heavy chain variable region comprising a CDR3 domain comprising SEQ ID NO: 17 or an equivalent of SEQ ID NO: 17, said equivalent characterized as having one or more conservative amino acid substitutions in the CDR3 domain; and/or (b) a light chain variable region comprising 10 a CDR3 domain comprising SEQ ID NO: 7 or an equivalent of SEQ ID NO: 7, said equivalent characterized as having one or more conservative amino acid substitutions in the CDR3 domain. In specific embodiments, PCSK9-specific antagonists bind to human and/or murine PCSK9 with a KD of 1.2 X 10-6 M or less. In more specific embodiments, PCSK9-specific antagonists bind to human and/or murine PCSK9 with a KD of 1 X 1 0-7 M or less. In additional embodiments, 15 PCSK9-specific antagonists bind to human and/or murine PCSK9 with a KD of 1 X 10-8 M or less. In further embodiments, PCSK9-specific antagonists bind to human and/or murine PCSK9 with a KD of 5 X 10-9 M or less, or of 1 X 10-9 M or less. In select embodiments, PCSK9 specific antagonists bind to human and/or murine PCSK9 with a KD of 1 X 10-10 M or less, a KD of I X 10-11 M or less, or a KD of 1 X 10-12 M or less. In specific embodiments, PCSK9 20 specific antagonists do not bind proteins other than PCSK9 at the above levels indicated for binding to PCSK9. Particular embodiments of the present invention include PCSK9-specific antagonists which exhibit binding to PCSK9 at one of the above prescribed levels and compete for binding to PCSK9 with ID05 antibody molecules. ID05 antibody molecules form important 25 PCSK9-specific antagonists hereof. 1D05 antibody molecules are characterized as comprising a (i) heavy chain variable region ("VH") comprising SEQ ID NO: 11; and (ii) a light chain variable region ("VL") comprising SEQ ID NO: 27. Said VH and VL regions comprise the full complement of disclosed CDRs 1, 2 and 3 for the VH (SEQ ID NOs: 13, 15 and 17) and VL regions (SEQ ID NOs: 3, 5 and 7), respectively. Examples of 1D05 antibody molecules include 30 without limitation: (i) a Fab which comprises a light chain comprising SEQ ID NO: 1 and an Fd chain comprising amino acids 1-233 of SEQ ID NO: 9 (or SEQ ID NO: 9); and (ii) a full length antibody molecule which comprises a light chain comprising SEQ ID NO: 26 and a heavy chain comprising SEQ ID NO: 25. PCSK9-specific antagonists are effective in counteracting PCSK9-dependent 35 inhibition of cellular LDL-uptake, and particularly human and/or murine PCSK9-dependent inhibition of cellular LDL uptake. Repeatedly, PCSK9-specific antagonist ID05 has demonstrated dose-dependent inhibition of the effects of PCSK9 on LDL uptake. Accordingly, -4- WO 2009/100297 PCT/US2009/033341 the disclosed PCSK9-specific antagonists are of import for lowering plasma LDL cholesterol levels. The disclosed antagonists also have utility for various diagnostic purposes, including the detection and quantification of PCSK9. Select 1D05 antagonists are, in particular, useful because of their cross-reactivity with both human and murine PCSK9. This quality enables 5 particular 1 D05 antagonists to be studied pharmacologically in murine models without having to ensure that the mice express human PCSK9. In such experiments, the murine model is sufficiently representative of the native activity of the targeted protein and the antagonists inhibition thereof. In specific embodiments, the present invention encompasses PCSK9-specific 10 antagonists. In particular embodiments, the present invention encompasses antibody molecules comprising disclosed heavy and/or light chain variable regions, equivalents of said regions having one or more conservative amino acid substitutions, and homologs thereof. Select embodiments comprise isolated PCSK9-specific antagonists that comprise the disclosed CDR domains or sets of the heavy and/or light chain CDR domains, and equivalents of such domains 15 characterized as having one or more conservative amino acid substitutions. As will be appreciated by those skilled in the art, fragments of PCSK9-specific antagonists that retain the ability to antagonize PCSK9 may be inserted into various frameworks; see, e.g., U.S. Patent No. 6,818,418 and references contained therein, the collective disclosures of which are incorporated herein by reference, which discuss various scaffolds which may be used to display antibody loops 20 previously selected on the basis of antigen binding. In the alternative, genes encoding for VL and VH may be joined, using recombinant methods, for example using a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules, otherwise known as single chain Fvs ("ScFVs"); see, e.g., Bird et al., 1988 Science 242: 423-426, and Huston et al., 1988 Proc. Natl. Acad. Sci. USA 85:5879-5883, 25 the disclosures of which are incorporated herein by reference. PCSK-9 specific antagonists and fragments may be in the form of various non antibody-based scaffolds, including but not limited to avimers (Avidia); DARPins (Molecular Partners); Adnectins (Adnexus), Anticalins (Pieris) and Affibodies (Affibody). The use of alternative scaffolds for protein binding is well appreciated in the scientific literature, see, e.g., 30 Binz & Plfckthun, 2005 Curr. Opin. Biotech. 16:1-11; the disclosure of which is incorporated herein by reference. Accordingly, non-antibody-based scaffolds or antagonist molecules comprising (i) the disclosed heavy and/or light chain variable region CDR3 sequences (SEQ ID NOs: 17 and 7, respectively), (ii) the disclosed heavy chain variable CDRI, CDR2 and CDR3 sequences or the disclosed light chain variable CDRI, CDR2 and CDR3 sequences: CDR1 (SEQ 35 ID NOs: 13 and 3, respectively), CDR2 (SEQ ID NOs: 15 and 5, respectively) and CDR3 (SEQ ID NOs; 17 and 7, respectively, (iii) the full complement (SEQ ID NOs; 13, 15, 17, 3, 5 and 7) of disclosed heavy and light chain CDRs within a variable region framework of a human heavy -5 - WO 2009/100297 PCT/US2009/033341 and/or light chain sequence, respectively, or (iv) the disclosed heavy and/or light chain variable regions SEQ ID NO: 11 and/or SEQ ID NO: 27 form important embodiments of the present invention, where such scaffolds or antagonist molecules exhibit selectivity for PCSK9 and counteract PCSK9-dependent inhibition of cellular LDL-uptake. 5 In another aspect, the present invention provides nucleic acid encoding the disclosed PCSK9-specific antagonists and, in particular embodiments, PCSK9-specific antagonists which comprise the disclosed heavy and light chains, the disclosed variable heavy and light regions and select components thereof (including CDRs 1, 2 and/or 3), particularly the disclosed respective CDR3 regions. In another aspect, the present invention provides vectors 10 comprising said nucleic acid. The present invention, additionally, provides isolated cell(s) comprising nucleic acid encoding disclosed PCSK9-specific antagonists. In another aspect, the present invention provides isolated cell(s) comprising a polypeptide or vector of the present invention. The present invention provides methods for making PCSK9-specific antagonists 15 disclosed herein including but not limited to antibodies, antigen binding fragments, derivatives, chimeric molecules, fusions of any of the foregoing with another polypeptide, or alternative structures/compositions capable of specifically binding PCSK9 which comprise the disclosed sequences. The methods comprise: (i) incubating a cell comprising nucleic acid encoding the PCSK9-specific antagonist(s), or which comprises individual nucleic acids encoding one or more 20 components thereof, said nucleic acids which, when expressed, collectively produce the antagonist(s), under conditions that allow for the expression and/or assembly of the PCSK9 specific antagonist(s), and (ii) isolating said antagonist(s) from the cell. One of skill in the art can obtain PCSK9-specific antagonists disclosed herein using standard recombinant DNA techniques as well. 25 The present invention provides a method for antagonizing the activity or function of PCSK9 or a noted effect of PCSK9 which comprises contacting a cell, population of cells, or tissue sample of interest expressing PCSK9 (or treated with or having therein human or murine PCSK9) with a PCSK9-specific antagonist disclosed herein under conditions that allow said antagonist to bind to PCSK9. Specific embodiments of the present invention include such 30 methods wherein the cell is a human or murine cell, Additional embodiments are wherein the cell expresses human or murine-derived PCSK9. In another aspect, the present invention provides a method for antagonizing the activity or function of PCSK9 or a noted effect of PCSK9 in a subject exhibiting a condition associated with PCSK9 activity, or a condition where the functioning of PCSK9 is 35 contraindicated for a particular subject, which comprises administering to the subject a therapeutically effective amount of a PCSK9-specific antagonist of the present invention in a pharmaceutical or other composition. -6- WO 2009/100297 PCT/US2009/033341 The present invention, thus, encompasses a method of treating a condition associated with PCSK9 activity, or a condition wherein the functioning of PCSK9 is contraindicated for a particular subject, which comprises administering to the subject a therapeutically effective amount of a PCSK9-specific antagonist of the present invention in a 5 pharmaceutical or other composition. In select embodiments, the condition is hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome or related conditions. In specific embodiments, the present invention encompasses a method of administering a disclosed PCSK9-specific antagonist to a subject which comprises delivering a 10 therapeutically effective amount of a pharmaceutical or other composition comprising a PCSK9 specific antagonist as disclosed herein. In another aspect, the present invention provides a pharmaceutical composition or other composition comprising a PCSK9-specific antagonist of the invention characterized as comprising a pharmaceutically acceptable carrier including but not limited to an excipient, 15 diluent, stabilizer, buffer, or alternative designed to facilitate administration of the antagonist in the desired amount to the treated individual. The following table offers a generalized outline of the sequences discussed in the present application. The Sequence Listing including all notations, sequences and features forms 20 as express part of the disclosure hereof: Table 1 SEQ ID NO: DESCRIPTION SEQ ID NO: 1 LIGHT CHAIN ("LC"); iD05 SEQ ID NO: 2 LIGHT CHAIN ("LC") NUCLEIC ACID; ID05 SEQ ID NO: 3 VL CDR1; ID05 SEQ ID NO: 4 VL CDR1 NUCLEIC ACID; ID05 SEQ ID NO: 5 VL CDR2; ID05 SEQ ID NO: 6 VL CDR2 NUCLEIC ACID; 1D05 SEQ ID NO: 7 VL CDR3; ID05 SEQ ID NO: 8 VL CDR3 NUCLEIC ACID; ID05 SEQ ID NO: 9 Fd CHAIN inclusive of linkers and tags; 1D05 SEQ ID NO: 10 Fd CHAIN NUCLEIC ACID; 1D05 SEQ ID NO: II VH; 1D05 -7- WO 2009/100297 PCT/US2009/033341 SEQ ID NO: DESCRIPTION SEQ ID NO: 12 VH NUCLEIC ACID; ID05 SEQ ID NO: 13 VH CDRI; ID05 SEQ ID NO: 14 VH CDR1 NUCLEIC ACID; ID05 SEQ ID NO: 15 VH CDR2; 1DO5 SEQ ID NO: 16 VH CDR2 NUCLEIC ACID; 1D05 SEQ ID NO: 17 VH CDR3; ID05 SEQ ID NO: 18 VH CDR3 NUCLEIC ACID; ID05 SEQ ID NO: 19 FRAGMENT OF PROCESSING SITE SEQ ID NO: 20 FRAGMENT OF PROCESSING SITE SEQ ID NO: 21 Constant domain of IgG1 SEQ ID NO: 22 Constant domain of IgG2 SEQ ID NO: 23 Constant domain of IgG4 SEQ ID NO: 24 Constant domain of IgG2m4 SEQ ID NO: 25 1D05 IgG2m4 Heavy Chain ("HC") SEQ ID NO: 26 1DO5 IgG Light (Kappa) Chain SEQ ID NO: 27 VL; 1D05 SEQ ID NO: 28 VL NUCLEIC ACID; 1D05 SEQ ID NO: 29 1D05 IgG2m4 HC NUCLEIC ACID SEQ ID NO: 30 1D05 IgG LC NUCLEIC ACID SEQ ID NO: 31 1D05 IgG2m4 HC PLASMID SEQ ID NO: 32 ID05 IgG LC PLASMID SEQ ID NO: 33 PRIMER SEQ ID NO: 34 PRIMER SEQ ID NO: 35 PRIMER SEQ ID NO: 36 PRIMER SEQ ID NO: 37 1D05 EPITOPE DOMAIN SEQ ID NO: 38 PORTION OF PCSK9 SEQUENCE IN FIGURE SEQ ID NO: 39 HUMAN EPITOPE AREA SEQ ID NO: 40 CONSENSUS SEQUENCE SEQ ID NO: 41 MURINE EPITOPE AREA SEQ ID NO: 42 SECONDARY FOOTPRINT EPITOPE SEQ ID NO;43 ID05 Variant VH CDRI Sequence SEQ ID NO:44 ID05 Variant VH CDR2 Sequence SEQ ID NO:45 ID05 Variant VH CDR3 Sequence -8- WO 2009/100297 PCT/US2009/033341 SEQ ID NO: DESCRIPTION SEQ ID NO:46 1 D05 Variant VL CDRl Sequence SEQ ID NO:47 1 D05 Variant VL CDR2 Sequence SEQ ID NO:48 I DOS Variant VL CDR3 Sequence SEQ ID NO:49 VL; ID05 Variant Sequence SEQ ID NO:50 VH; ID05 Variant Sequence SEQ ID NO:51 VH; ID05 Variant Sequence H32Y SEQ ID NO:52 VH; ID05 Variant Sequence M48A SEQ ID NO:53 VH; ID05 Variant Sequence M48L SEQ ID NO:54 VH; I D05 Variant SequenceH99Y SEQ ID NO:55 VH; ID05 Variant Sequence M48L/M109L/MI 15L SEQ ID NO:56 VH; ID05 Variant SequenceM48V SEQ ID NO:57 VI; ID05 Variant Sequence N50D SEQ ID NO:58 VI; ID05 Variant Sequence N50Q SEQ ID NO:59 VI; ID05 Variant SequenceN50T SEQ ID NO:60 VI; iD05 Variant SequenceN50Y -9-- WO 2009/100297 PCT/US2009/033341 BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 illustrates Fab expression vector pMORPH_x9_MH encoding the mPCSK9 2CX 1 DOS Fab heavy and light chains. FIGURE 2 illustrates the activity of 1D05 in a PCSK9-LDLR interaction TR 5 FRET assay. Both the Fab and IgG of 1 D05 are potent and inhibit the interaction fully. For the experiment, [AF647-PCSK9] = 1 OnM, [Eu-sLDLR] 1.5 nM (-20000 counts at FI620nm). FIGURES 3A-3D illustrate (i) 1D05 (Fab)s dose-dependent inhibition of murine PCSK9-dependent loss of cellular LDL-uptake (FIGURE 3A); (ii) ID05 (IgG)'s dose-dependent inhibition of murine PCSK9-dependent loss of cellular LDL-uptake (FIGURE 3B); (iii) 1D05 10 (Fab)'s dose-dependent inhibition of human PCSK9-dependent loss of cellular LDL-uptake (FIGURE 3C); and (iv) 1 D05 (IgG)'s dose-dependent inhibition of human PSCK9-dependent loss of cellular LDL-uptake (FIGURE 3D). 1D05 clearly cross-reacts with both human and mouse PCSK9. FIGURES 3A-3D have two controls: (i) a cell only control, showing the basal level of cellular LDL uptake, and (ii) a cell + PCSK9 (5 gg/ml) control which shows the level of 15 PCSK9-dependent loss of LDL-uptake. The titration experiments which contain I D05 and PCSK9 were done at a fixed concentration of PCSK9 (5 pig/ml) and increasing concentrations of ID05 shown in the graphs. As shown, ID05 can inhibit the effect of PCSK9 on cellular LDL uptake. IC50s for ID05 (Fab) are 97 and 144 nM for mouse and human PCSK9 protein, respectively. IC50s for ID05 (IgG) are 85 and 79 nM for mouse and human PCSK9 protein, 20 respectively. FIGURES 4A and 4B illustrate inhibition of PCSK9 internalization by 1D05 (Fab and IgG, respectively) and restoration of LDL uptake. HepG2 cells were plated and AlexaFluor labeled PCSK9 and LDL were then added to cells and incubated at 37 0 C for 4 hrs. Following incubation, the amount of PCSK9 or LDL internalized by cells was determined using cofocal 25 microscopy. Controls included the addition of cells alone (No treatment), and only AF-labeled PCSK9 in addition to 50X (250 pig/ml) unlabeled PCSK9 (50X Cold Wt). In addition to 5 jig/ml wild-type AF-labeled PCSK9 and 10ptg/ml AF-labeled LDL, increasing amounts of either the 1 D05 Fab (panel A) or IGG (panel B) were added, resulting in subsequent inhibition of PCSK9 internalization into cells and a recovery of LDL uptake. Together, these studies demonstrate that 30 both the Fab and IgG prevent PCSK9 internalization into cells. FIGURE 5 illustrates the LDL levels for each mouse represented by a set of connected symbols; the change in LDL (postbleed - prebleed) being shown as an average for each treatment group (A mg/dL). Treatment with PBS had no effect on LDL measurements (-4 mg/dL, 5% reduction). In contrast, serum LDL was reduced 20% with ID05 whole IgG (-19 35 mg/dL) and 34% with Fab fragments of 1D05 (-24 mg/dL). FIGURE 6 illustrates a sequence comparison of the constant domains of IgGI (SEQ ID NO: 21; Fe domain of which is represented by residues 110-130 of SEQ ID NO: 21), -10- WO 2009/100297 PCT/US2009/033341 IgG2 (SEQ ID NO: 22, Fe domain of which is represented by residues 107-326 of SEQ ID NO: 22), IgG4 (SEQ ID NO: 23; Fe domain of which is represented by residues 107-327 of SEQ ID NO: 23) and IgG2m4 (SEQ ID NO: 24; Fe domain of which is represented by residues 107-326 of SEQ ID NO: 24) isotypes. 5 FIGURES 7A and 7B illustrate peptide fragments originated by limited proteolysis of a) wt-PCSK9 and b) 1 D05/wt-PCSK9 complex with AspN for 5 minutes. The star in Figure 7A highlights the peptide fragment present in the wt-PCSK9 spectrum which is not detected in the 1 D05/wt-PCSK9 spectrum. The aspartic acid residue 169 is hence protected in the complex. 10 FIGURES 8A-8H illustrate peptide fragments originated by limited proteolysis of wt-PCSK9 (FIGURES 8A-8D) and 1D05/wt-PCSK9 complex (FIGURES 8E-8H)with GluC for 15 minutes. FIGURES 9A-9D illustrate peptide fragments originated by limited proteolysis with Trypsin of a) wt-PCSK9 and b) 1D05/wt-PCSK9 complex for 5 minutes. FIGURES 9C-D 15 illustrate the zoom views of the same spectra, respectively. The stars in Figures 9A and 9C highlight the peptide fragments present in the wt-PCSK9 spectrum which are not detected in the 1 D05/wt-PCSK9 spectrum. FIGURE 10 illustrates residues of the primary sequence of the wt-PCSK9 catalytic domain involved in binding with ID05 neutralizing Fab. The peptide fragments of wt 20 PCSK9 protected in limited proteolysis experiments by 1DO5 binding are boxed. FIGURE 11 illustrates peptides of the wt-PCSK9 catalytic domain involved in binding with ID05 neutralizing Fab. The peptides of wt-PCSK9 protected in limited proteolysis experiments by 1D05 binding are depicted in the segments between R194 and R199, and A168 and R165 in the zoom view of the structure of wt-PCSK9. 25 FIGURES 12A and 12B illustrate sequence alignment between the identified general epitope areas of human (SEQ ID NO: 39) and murine (SEQ ID NO: 41) PCSK9, A consensus sequence (SEQ ID NO: 40) is provided as the second sequence of FIGURE 12A. As evident in the Figures, the residues included in the protected peptide 194-199 are conserved among the two species while the residues in peptide 165-169 are not. 30 FIGURE 13 illustrates an analysis of I D05 and a distinct antibody I B20 in a PCSK9-1D05 interaction TR-FRET assay. Both Fabs are potent and inhibit the interaction fully. For this experiment, [AF647-a-V5]= 10 nM, [PCSK9]= 10 nM, and [Eu(8044)-1D05(IgG)] -1.5 nM (~18000 counts at F 1620 nm). FIGURES 14A-D illustrates that I B20 is a full inhibitor of PCSK9 function in the 35 Exopolar assay. - 11 - WO 2009/100297 PCT/US2009/033341 FIGURE 15 illustrates 1 D05 lowering LDL-C by - 50% in rhesus at 3 mpk. Plotted are %LDL changes in serum at the different time points tested, post a single IV dose of antibody treatment. FIGURE 16 illustrates the pharmacokinetic profile of ID05 at the dose levels 5 shown. Plotted are the serum drug (iD05) levels at time points tested following a single IV dose of antibody. The half-life of I D05 is 77 hr. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antagonists of PCSK9 and, in particular 10 embodiments, those antagonists that inhibit both human and murine PCSK9 and those that preferentially target processed PCSK9. Protein-specific antagonists of PCSK9 (or "PCSK9 specific antagonists") in accordance herewith are effective in the selective binding to and inhibition of PCSK9 function and, thus, are of import in the treatment of conditions associated with or impacted by PCSK9 function, including, but not limited to, hypercholesterolemia, 15 coronary heart disease, metabolic syndrome, acute coronary syndrome and related conditions. Use of the term "antagonist" refers to the fact that the subject molecule can antagonize the functioning of PCSK9. Use of the term "antagonizing" or derivatives thereof refers to the act of opposing, counteracting, inhibiting, neutralizing or curtailing one or more functions of PCSK9, Reference herein to PCSK9 function or PCSK9 activity refers to any function or activity that is 20 driven by, requires, or is exacerbated or enhanced by PCSK9. PCSK9-specific antagonists as described herein have proven to be effective for counteracting human and/or murine PCSK9 dependent inhibition of cellular LDL-uptake. One important embodiment hereof relates to ID05 antibody molecules. Such I DOS antibody molecules are characterized as comprising a (i) heavy chain variable region 25 ("VH") comprising SEQ ID NO: 11; and (ii) a light chain variable region ("VL") comprising SEQ ID NO: 27. Said VH and VL regions comprise the full complement of disclosed CDRs 1, 2 and 3 for the VH (SEQ ID NOs: 13, 15 and 17) and VL regions (SEQ ID NOs: 3, 5 and 7), respectively. Examples of 1 D05 antibody molecules include without limitation: (i) a Fab which comprises a light chain comprising SEQ ID NO: 1 and an Fd chain comprising amino acids I 30 233 of SEQ ID NO: 9 (or SEQ ID NO: 9); and (ii) a full length antibody molecule which comprises a light chain comprising SEQ ID NO: 26 and a heavy chain comprising SEQ ID NO: 25. The select group of ID05 antibodies demonstrate that PCSK9-specific antagonists as disclosed herein effectively inhibit both human and murine PCSK9 and may be studied pharmacologically in murine models absent the expression of human PCSK9. 35 The CDR definitions arrived at and disclosed herein were defined using the Morphosys software program Sequence Analysis Software ("SAS"). Applicants wish to note, however, that various other methods are available to delineate and define the start and end points - 12 - WO 2009/100297 PCT/US2009/033341 of the CDR sequences, including but not limited to Kabat, 1991 Sequences of Proteins of Immunological Interest, 5 th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Clothia et al., 1987 J Mol. Biol. 196:901-917; Clothia et al., 1989 Nature 342:877-883; Lefranc, 1997 Immunol. Today, 18:509; and'Chen et al., 1999 J Mol. Biol. 5 293:865-881. These and other methods have been reviewed and are well within the realm of skills possessed by those in the art; see, e.g., Honegger & Pluckthun, 2001 J. Mol. Biol. 309:657 670. While the current inventors have employed the SAS software to define the CDRs, the present invention fully encompasses the different definitions around the sequences and the varying CDR delineations arrived at through use of any different analysis software or methods. 10 Said use and resulting CDR definitions based on the presently disclosed sequences is fully within the scope of the present disclosure and anticipated herein. PCSK9-specific molecules also have utility for various diagnostic purposes in the detection and quantification of PCSK9. Disclosed PCSK9-specific antagonists are, furthermore, unique in that select 15 embodiments have demonstrated a preferential recognition of processed PCSK9, the active form of PCSK9. PCSK9-specific antagonists as disclosed herein are desirable molecules for lowering plasma LDL cholesterol levels and are of utility for any primate, mammal or vertebrate of commercial or domestic veterinary importance. PCSK9-specific antagonists are of utility as 20 well to inhibit the activity of PCSK9 in any population of cells or tissues possessing the LDL receptor. The utility of the disclosed antagonists is directly measurable by assays readily available to the skilled artisan. Means for measuring LDL uptake are described in the literature; see, e.g., Barak & Webb, 1981 J Cell Biol. 90:595-604, and Stephan & Yurachek, 1993 J Lipid Res. 34:325330. In addition, means for measuring LDL cholesterol in plasma is well described 25 in the literature; see, e.g., McNamara et al., 2006 Clinica Chimica Acta 369:158-167. The particular impact of the disclosed antagonists on cellular LDL uptake may also be measured through a method which comprises providing purified PCSK9 and labeled LDL particles to a cell sample; providing a PCSK9 antagonist to the cell sample; incubating said cell sample for a period of time sufficient to allow LDL particle uptake by the cells; quantifying the amount of 30 label incorporated into the cell; and identifying those antagonists that result in an increase in the amount of quantified label taken up by the cells as compared with that observed when PCSK9 is administered alone. An additional method for measuring the impact of the disclosed antagonists comprises providing purified PCSK9 and labeled LDL particles to a cell sample; providing a PCSK9 antagonist to the cell sample; incubating said cell sample for a period of time sufficient 35 to allow LDL particle uptake by the cells; isolating cells of the cell sample by removing the supernate; reducing non-specific association of labeled LDL particles (whether to the plate, the cells, or anything other than the LDL receptor); lysing the cells; quantifying the amount of label -13 - WO 2009/100297 PCT/US2009/033341 retained within the cell lysate; and identifying those antagonists that result in an increase in the amount of quantified label taken up by the cells as compared with that observed when PCSK9 is administered alone. Antagonists that result in an increase in the amount of quantified label are PCSK9 antagonists. 5 Any type of cell bearing the LDL receptor can be employed in the above methods including, but not limited to HEK cells, HepG2 cells, and CHO cells, LDL particles derived from any source are of use in the above-described assays. In particular assays, the LDL particles are fresh particles derived from blood. This can be accomplished by any method available to the skilled artisan including, but not limited to, the method of Havel et al., 1955 J Clin. Invest. 34: 10 1345-1353. The LDL particles may be labeled with fluorescence. The labeled LDL particles may have incorporated therein visible wavelength excited fluorophore 3,3' dioctadecylindocarbocyanine iodide (dil(3)) to form the highly fluorescent LDL derivative dil(3) LDL. Any label which enables the skilled artisan to detect LDL in the cellular lysate may be used. An LDL analog may be used that would only become detectable (e.g., become fluorescent 15 or fluoresce at a different wavelength, etc.) when metabolized intracellularly or, for instance, if it were to become associated with (or dissociated from) other molecules in the process of becoming internalized (e.g. a FRET assay, in which an LDL analog would become associated with a secondary fluor, or else be dissociated from a quencher). Any means available in the art for detecting internalization of labeled LDL particles can be employed. The incubation time for the 20 LDL particles and PCSK9 with the cells is an amount of time sufficient to allow LDL particle uptake by the cells. This time may be within the range of 5 minutes to 360 minutes. The concentration of PCSK9 added to the cells may be in the range of I nM to 5 gM and, in specific methods, be in the range of 0.1 nM to 3 ptM. One specific means by which the skilled artisan can determine a range of concentrations for a particular PCSK9 protein is to develop a dose response 25 curve in the LDL-uptake assay. A concentration of PCSK9 can be selected that promotes close to maximal loss of LDL-uptake and is still in the linear range of the dose response curve. Typically, this concentration is ~ 5 times the EC-50 of the protein extracted from the dose response curve. The concentrations can vary by protein. Broadly, PCSK9-specific antagonists as defined herein selectively recognize and 30 specifically bind to PCSK9, An antibody is typically said to specifically bind an antigen when the dissociation constant is <1 M, preferably 5100 nM and most preferably §10 nM. Use of the terms "selective" or "specific" herein, further, refers to the fact that the disclosed antagonists do not show significant binding to proteins other than PSCK9, except in those specific instances where the antagonist is supplemented or designed to confer an additional, distinct specificity to 35 the PCSK9-specific binding portion (as, for example, in bispecific or bifunctional molecules where the molecule is designed to bind two molecules or effect two functions, at least one of which is to specifically bind PCSK9). In specific embodiments, PCSK9-specific antagonists - 14 - WO 2009/100297 PCT/US2009/033341 bind to human and/or murine PCSK9 with a KD of 1.2 X Io-6 M or less. In more specific embodiments, PCSK9-specific antagonists bind to human and/or murine PCSK9 with a KD of 5 X 10-7 M or less, of 2 X Io-7 M or less, or of I X 10-7 M or less. In additional embodiments, PCSK9-specific antagonists bind to human and/or murine PCSK9 with a KD of 1 X 10-8 M or 5 less. In further embodiments, PCSK9-specific antagonists bind to human and/or murine PCSK9 with a KD of 5 X 10-9 M or less, or of I X 10-9 M or less. In select embodiments, PCSK9 specific antagonists bind to human and/or murine PCSK9 with a KD of 1 X 10-10 M or less, a KD of 1 X 10-11 M or less, or a KD of 1 X 10-12 M or less. In specific embodiments, PCSK9 specific antagonists do not bind proteins other than PCSK9 at the above KDs. KD refers to the 10 dissociation constant obtained from the ratio of Kd (the dissociation rate of a particular binding molecule-target protein interaction) to Ka (the association rate of the particular binding molecule-target protein interaction), or Kd/Ka which is expressed as a molar concentration (M). KD values can be determined using methods well established in the art. A preferred method for determining the KD of a binding molecule is by using surface plasmon resonance, for example 15 employing a biosensor system such as a BiacoreTM (GE Healthcare Life Sciences) system. PCSK9-specific antagonists disclosed herein have been shown to dose dependently inhibit human and/or murine PCSK9 dependent effects on LDL uptake. Accordingly, PCSK9-specific antagonists as disclosed herein are characterized by their ability to counteract PCSK9-dependent inhibition of LDL uptake into cells. This uptake of LDL into cells 20 by the LDL receptor is referred to herein as "cellular LDL uptake". In specific embodiments, PCSK9-specific antagonists counteract or antagonize human and/or murine PCSK9-dependent inhibition of LDL uptake into cells, exhibiting an IC50 of less than 1.0 X 10-6 M, or, in order of preference, less than 1 X 10-7 M, 1 X 10-8 M, I X 10-9 M, 1 X 10-10 M, 1 X 10-11 M and 1 X 10-12 M. The extent of inhibition by any PCSK9-specific antagonist may be measured 25 quantitatively in statistical comparison to a control, or via any alternative method available in the art for assessing a negative effect on, or inhibition of, PCSK9 function (i.e., any method capable of assessing antagonism of PCSK9 function). In specific embodiments, the inhibition is at least about 10% inhibition. In other embodiments, the inhibition is at least 20%, 30%, 40%, 50%, 60%, 70,%, 80%, 90%, or 95%. Accordingly, PCSK9-specific antagonists capable of effecting 30 these levels of inhibition of PCSK9 function form particular embodiments hereof. A PCSK9-specific antagonist in accordance herewith can be any binding molecule that specifically binds human and/or murine PCSK9 protein including, but not limited to, antibody molecules as defined below, any PCSK9-specific binding structure, any polypeptide or nucleic acid structure that specifically binds PCSK9, and any of the foregoing incorporated into 35 various protein scaffolds; including but not limited to, various non-antibody-based scaffolds, and various structures capable of affording or allowing for selective binding to PCSK9 including but not limited to small modular immunopharmaceuticals (or "SMIPs"; see, Haan & Maggos, 2004 - 15- WO 2009/100297 PCT/US2009/033341 Biocentury Jan 26); Immunity proteins (see, e.g., Chak et al., 1996 Proc. Natl. Acad. Sci. USA 93:6437-6442); cytochrome b562 (see Ku and Schultz, 1995 Proc. Natl. Acad Sci. USA 92:6552-6556); the peptide a2p8 (see Barthe et al., 2000 Protein Sci. 9:942-955); avimers (Avidia; see Silverman et al, 2005 Nat. Biotechnol. 23:1556-1561); DARPins (Molecular 5 Partners; see Binz et al., 2003 J. Mol. Biol. 332:489-503; and Forrer et al, 2003 FEBS Lett. 539:2-6); Tetranectins (see, Kastrup et al., 1998 Acta. Crystallogr. D. Riot. Crystallogr. 54:757 766); Adnectins (Adnexus; see, Xu et al., 2002 Chem. Biol. 9:933-942), Anticalins (Pieris; see Vogt & Skerra, 2004 Chemobiochem. 5:191-199; Beste et al, 1999 Proc. Natl. A cad Sci. USA 96:1898-1903; Lamla & Erdmann, 2003 J Mol. Biol. 329:381-388; and Lamla & Erdmann, 2004 10 Protein Expr. Purif 33:39-47); A-domain proteins (see North & Blacklow, 1999 Biochemistry 3 8:3926-3935), Lipocalins (see Schlehuber & Skerra, 2005 Drug Discov, Today 10:23-33); Repeat-motif proteins such as Ankyrin repeat proteins (see Sedgwick & Smerdon, 1999 Trends Biochem. Sci. 24:311-316; Mosavi et al, 2002 Proc. Nat. Acad. Sci. USA 99:16029-16034; and Binz et al, 2004 Nat. Biotechnol 22:575-5 82); Insect Defensin A (see Zhao et al., 2004 Peptides 15 25:629-635); Kunitz domains (see Roberts et al., 1992 Proc. Natl. Acad Sci. USA 89:2429-2433; Roberts et al, 1992 Gene 121:9-15; Dennis & Lazarus, 1994 J BioL Chem. 269:22129-22136; and Dennis & Lazarus, 1994£1 Biol. Chem. 269:22137-22144); PDZ-Domains (see Schneider et al., 1999 Nat. Biotechnol. 17:170-175); Scorpion toxins such as Charybdotoxin (see Vita et al, 1998 Biopolymers 47:93-100); 10 th fibronectin type III domain (or 1OFn3; see Koide et al, 1998 20 J Mol. Biol. 284:1141-1151, and Xu et al., 2002 Chem. Biol. 9:933-942); CTLA-4 (extracellular domain; see Nuttall et al., 1999 Proteins 36:217-227; and Irving et al., 2001 J Immunol. Methods 248:31-45); Knottins (see Souriau et aL, 2005 Biochemistry 44:7143-7155 and Lehtio et al., 2000 Proteins 41:316-322); Neocarzinostatin (see Heyd et al. 2003 Biochemistry 42:5674 5683); carbohydrate binding module 4-2 (CBM4-2; see Cicortas et al., 2004 Protein Eng. Des. 25 Sel. 17:213-221); Tendamistat (see McConnell & Hoess, 1995 J Mol. Biol. 250:460-470, and Li et al., 2003 Protein Eng. 16:65-72); T cell receptor (see Holler et al., 2000 Proc. Natl. Acad. Sc. USA 97:5387-5392; Shusta et al., 2000 Nat. Biotechnol. 18:754-759; and Li et al., 2005 Nat. Biotechnol 23:349-354); Affibodies (Affibody; see Nord et al., 1995 Protein Eng. 8:601-608; Nord et al, 1997 Nat. Biotechnol 15:772-777; Gunneriusson et al, 1999 Protein Eng. 12:873 30 878); and other selective binding proteins or scaffolds recognized in the literature; see, e.g., Binz & Pluckthun, 2005 Curr. Opin. Biotech. 16:1-11; Gill & Damle, 2006 Curr. Opin. BiotechnoL 17:1-6; Hosse et al, 2006 Protein Science 15:14-27; Binz et aL, 2005 Nat. Biotechnol 23:1257 1268; Hey et al., 2005 Trends in Biotechnol. 23:514-522; Binz & Pluckthun, 2005 Curr. Opin. Biotech. 16:459-469; Nygren & Skerra, 2004 J Immunolog. Methods 290:3-28; Nygren & 35 Uhlen, 1997 Curr. Opin. Struck. Biol. 7:463-469; the disclosures of which are incorporated herein by reference. Antibodies and the use of antigen-binding fragments is well defined and understood in the literature. The use of alternative scaffolds for protein binding is well -16- WO 2009/100297 PCT/US2009/033341 appreciated in the scientific literature as well, see, e.g., Binz & Pltickthun, 2005 Curr. Opin. Biotech. 16:1-11; Gill & Damle, 2006 Curr. Opin. Biotechnol 17:1-6; Hosse et al, 2006 Protein Science 15:14-27; Binz et al, 2005 Nat. BiotechnoL 23:1257-1268; Hey et al., 2005 Trends in Biotechnol. 23:514-522; Binz & Pluckthun, 2005 Curr. Opin. Biotech. 16:459-469; Nygren & 5 Skerra, 2004 J. Immunolog. Methods 290:3-28; Nygren & Uhlen, 1997 Curr. Opin. Struct. Biol. 7:463-469; the disclosures of which are incorporated herein by reference, Accordingly, non antibody-based scaffolds or antagonist molecules in accordance herewith exhibiting selectivity for PCSK9 that counteract PCSK9-dependent inhibition of cellular LDL-uptake form important embodiments of the present invention. Aptamers (nucleic acid or peptide molecules capable of 10 selectively binding a target molecule) are one specific example. They can be selected from random sequence pools or identified from natural sources such as riboswitches. Peptide aptamers, nucleic acid aptamers (e.g., structured nucleic acid, including both DNA and RNA based structures) and nucleic acid decoys can be effective for selectively binding and inhibiting proteins of interest; see, e.g., Hoppe-Seyler & Butz, 2000 J. Mol Med 78:426-430; Bock et al., 15 1992 Nature 355:564-566; Bunka & Stockley, 2006 Nat. Rev. MicrobioL 4:588-596; Martell et al., 2002 Molec. Ther. 6:30-34; Jayasena, 1999 Clin. Chem. 45:1628-1650; the disclosures of which are incorporated herein by reference. Importantly, the binding site (or epitope) for 1D05 on PCSK9 was identified through limited proteolysis and mass spectrometry ("LP-MS"). The limited proteolysis mass 20 spectrometry analysis involved incubating wild-type PCSK9 ("wt-PCSK9") and a complex of wt PCSK9 and 1 D05 with endoproteinase enzymes of different specificity in carefully controlled conditions. Under such conditions, the endoproteases cleaved only exposed primary cleavage sites. The experiment was designed so that the binding of 1 D05 to wt-hPCSK9 masked surface residues normally exposed on wt-hPCSK9 not bound to the antibody. Such masked residues 25 provided insight into the binding domain of ID05. Through such experiments, a novel neutralizing epitope conformational in nature and represented by peptides RYRAD (SEQ ID NO: 42) AND REIEGR (SEQ ID NO: 37) was identified. This epitope falls within PCSK9's catalytic domain and provides a novel target epitope for which to identify additional effective PCSK9 antagonists. Identification of additional PCSK9-specific antagonists binding this epitope is of 30 significant interest given 1 D05's PCSK9-neutralizing activity. One means of identifying antagonists and particularly antibodies that bind to the identified ID05 epitope or an overlapping epitope is through a competition or similar assay where the candidate antibody or binding molecule would have to out-compete 1DO5 for the epitope. Competitive antagonists encompassed herein are molecules that inhibit (i.e., prevent or 35 interfere with in comparison to a control) or reduce ID05 binding by at least 50%, 60%, 70%, and 80% in order of increasing preference (even more preferably, at least 90% and, most preferably, at least 95%) at 1 jiM or less with ID05 at or below its KD, and in particular those - 17 - WO 2009/100297 PCT/US2009/033341 molecules that antagonize (i) PCSK9 binding to the LDL receptor, (ii) PCSK9 internalization into cells, or (iii) both PCSK9 binding to the LDL receptor and PCSK9 internalization into cells. Competition between binding members may be readily assayed in vitro for example using ELISA and/or by monitoring the interaction of the antibodies with PCSK9 in solution. The exact means 5 for conducting the analysis is not critical. PCSK9 may be immobilized to a 96-well plate or may be placed in a homogenous solution. In specific embodiments, the ability of unlabeled candidate antibody(ies) to block the binding of labeled 1 DO5 can be measured using radioactive, enzyme or other labels. In the reverse assay, the ability of unlabeled antibodies to interfere with the interaction of labeled 1 D05 with PCSK9 wherein said 1 D05 and PCSK9 are already bound is 10 determined. In specific embodiments, (i) PCSK9 is contacted with labeled 1D05 (an antibody molecule which comprises a VL comprising SEQ ID NO: 27 and a VH comprising SEQ ID NO: 11); (ii) PCSK9 is contacted with the candidate antibody or pool of antibodies; and (iii) antibodies capable of interrupting or preventing complexes between PCSK9 and 1 D05 are identified. The readout in such an example is through measurement of bound label. ID05 and 15 the candidate antibody(ies) may be added in any order or at the same time. A specific assay that may be run is that of Example 13 where the activity of an antibody found to bind to the same epitope domain as ID05 is illustrated. Antibodies identified as 1D05 competitors in the above or other suitable assays may be tested for the ability to antagonize or neutralize (i) PCSK9 binding to the LDL receptor; 20 and/or (ii) PCSK9 internalization into cells. These parameters may be measured through the use of assays similar to that employed or described in the current specification. In specific embodiments, the inhibition demonstrated by the competing antibody is at least about 10% inhibition. In other embodiments, the inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. 25 The present invention specifically encompasses PCSK9-specific antagonists and particularly monoclonal antibody molecules (and their corresponding amino acid and nucleic acid sequences) that selectively bind to the epitope identified for 1 D05 or an overlapping epitope interfering with ID05's binding to PCSK9, Critical residues for ID05 binding that were identified on the epitope of PCSK9 are those residues corresponding to residues Arg194, Glul97 30 and Arg199 of human PCSK9. The narrow epitope comprising these amino acid residues is represented by SEQ ID NO: 37 and falls within the area of SEQ ID NO: 39 of human PCSK9 and SEQ ID NO: 41 of murine PCSK9. A secondary footprint of the antibody is represented by SEQ ID NO: 42. Monoclonal antibodies that specifically bind to the conformational epitope represented by SEQ ID NO: 37 and SEQ ID NO:42 or an overlapping epitope antagonize or 35 neutralize (i) PCSK9 binding to the LDL receptor; (ii) PCSK9 internalization into cells, or (iii) both. Accordingly, monoclonal antibodies that bind to an epitope on PCSK9 which comprises and/or consists of: SEQ ID NO: 37, SEQ ID NO: 39 or SEQ ID NO: 41 form important - 18- WO 2009/100297 PCT/US2009/033341 embodiments of the present invention. Specific embodiments of the present invention relate to monoclonal antibodies that recognize the following epitopes on PCSK9: SEQ ID NO: 37 and SEQ ID NO: 42, A monoclonal antibody molecule in accordance herewith may be an intact (complete or full length) antibody, a substantially intact antibody, or a portion or fragment of an 5 antibody comprising an antigen-binding portion, e.g., a Fab fragment, Fab' fragment or F(ab')2 fragment of a murine antibody or of a chimeric antibody or of a humanized antibody or of a human antibody, Monoclonal, as used herein, refers to a homogeneous or substantially homogeneous (or pure) antibody population (i.e,, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, more preferably at least about 97% or 98%, or most preferably at least 99% of the 10 antibodies in the population are identical and would compete in an ELISA assay for the same antigen or epitope. In specific embodiments of the present invention, the present invention provides monoclonal antibodies that (i) compete for binding to PCSK9 with a ID05 antibody molecule, reducing ID05 binding by at least 50% at 1 pM or less with ID05 at or below its KD, (ii) block PCSK9 binding to the LDL receptor, (iii) inhibit PCSK9 internalization into the cell, 15 and (iv) comprise a specific antigen-binding region, VH, VL, set of CDRs or heavy CDR3, heavy and/or light chain or any variant of these components described herein. Additional embodiments provide PCSK9-specific antagonists including but not limited to monoclonal antibodies that recognize/bind to SEQ ID NO: 37, SEQ ID NO: 39 or SEQ ID NO: 41, wherein the PCSK9 specific antagonists bind to human and/or murine PCSK9 with a KD of 1.2 X 10-6 M or less, and 20 wherein the PCSK9-specific antagonist competes with 1D05 for binding to PCSK9. In specific embodiments hereof, the PCSK9-specific antagonists are further defined by one or more of the following qualities: they (i) reduce ID05 binding by at least 50% at 1 pM or less with ID05 at or below its KD, (ii) block PCSK9 binding to the LDL receptor, (iii) inhibit PCSK9 internalization into the cell, and/or (iv) comprise a specific antigen-binding region, VH, VL, set of CDRs or 25 heavy CDR3, heavy and/or light chain or any variant of these components described herein. In specific embodiments, the PCSK9-specific antagonists in accordance with the above comprise (i) the disclosed heavy and/or light chain variable region CDR3 sequences (SEQ ID NOs: 17 and 7, respectively), (ii) the disclosed heavy and/or light chain variable regions CDR1 (SEQ ID NOs: 13 and 3, respectively), CDR2 (SEQ ID NOs: 15 and 5, respectively) and CDR3 (SEQ ID NOs; 17 30 and 7, respectively, (iii) the full complement (SEQ ID NOs; 13, 15, 17, 3, 5 and 7) of disclosed heavy and light chain CDRs within a variable region framework of a human heavy and/or light chain sequence; (iv) the disclosed VL and/or VH regions (SEQ ID NOs: 27 and 11, respectively); (v) the disclosed light and/or Fd chains (SEQ ID NO: 1 and amino acids 1-233 of SEQ ID NO: 9 (or SEQ ID NO: 9)), or (vi) the disclosed light and/or heavy chains (SEQ ID NOs: 26 and 25). In 35 specific embodiments, the PCSK9-specific antagonists bind to/recognize both SEQ ID NOs: 37 and SEQ ID NO: 42. -19- WO 2009/100297 PCT/US2009/033341 In any of the above assays for identifying antibodies binding the same or overlapping epitope region as ID05, binding of the known binder (i.e., ID05 antibody molecule known to bind residues Argl94, Glul97 and Arg199 of SEQ ID NO: 37) as compared to the binding of the candidate binder should be distinguishable. This can (but need not) be 5 accomplished through the use of labels on either or both molecules as will be readily appreciated by the skilled artisan. Labels, as used herein, refer to another molecule or agent incorporated into/affixed to the antibody molecule, In one embodiment, the label is a detectable marker, e.g., a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that 10 can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14C, 1 5 N, 35S, 90Y, 9 9 Tc, I 1 In, 1251, 1311), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, -galactosidase, luciferase, alkaline 15 phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, 20 daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. A 1D05 antibody used for the competition assays may be any antibody molecule 25 which is of the I D05 description provided herein (i.e. any antibody molecule selective for PCSK9 which comprises a VL comprising SEQ ID NO: 27 and a VH comprising SEQ ID NO: 11). Examples of such antibodies include without limitation (i) a Fab which comprises a light chain comprising SEQ ID NO: I and an Fd chain comprising amino acids 1-233 of SEQ ID NO: 9 (or SEQ ID NO: 9); (ii) a full length antibody molecule which comprises a light chain 30 comprising SEQ ID NO: 26 and a heavy chain comprising SEQ ID NO: 25. Peptides or peptidomimetics based on the regions corresponding to SEQ ID NO: 39 or SEQ ID NO: 41 (and in select embodiments the areas corresponding to SEQ ID NO: 37 and SEQ ID NO: 42) should have antagonistic properties by preventing the interaction of PCSK9 with LDLR. Importantly, peptides that comprise SEQ ID NO: 37 and SEQ ID NO: 42 should 35 generate neutralizing antibodies able to inhibit PCSK9 binding to LDLR and/or inhibit PCSK9 internalization into cells. -20- WO 2009/100297 PCT/US2009/033341 In specific embodiments, peptides encompassed herein comprise SEQ ID NO: 39 OR SEQ ID NO: 41. In select embodiments, the peptides comprise SEQ ID NO: 37 and are less than 50 amino acids. In certain embodiments, the peptides comprise both SEQ ID NO: 37 and SEQ ID NO: 42 and are 40 amino acids or less. In more specific embodiments, the peptides 5 comprise SEQ ID NO: 37 and are less than 40 amino acids, less than 30 amino acids, less than 20 amino acids, or less than 10 amino acids. Screening of peptides of the invention may be carried out utilizing competition assays as described above. If the peptide being tested competes with a 1 D05 antibody molecule (i.e. any antibody molecule selective for PCSK9 which comprises a VL comprising SEQ ID NO: 10 27 and a VH comprising SEQ ID NO: 11) as shown by a decrease in binding of such ID05 antibody molecule then it is likely that the peptide and ID05 bind to the same, or a closely related, epitope. Still another way to determine whether a peptide has the specificity of the 1 D05 antibody molecule is to pre-incubate the ID05 antibody molecule with PCSK9 with which it is normally reactive, and then add the peptide being tested with demonstrated specificity for 15 PCSK9 to determine whether the peptide is inhibited in its ability to bind PCSK9. If the peptide being tested is inhibited then, in all likelihood, it has the same, or a functionally equivalent, epitope and specificity as the 1 D05 antibody molecule. Using routine procedures as outlined throughout the instant specification and well known to those of ordinary skill in the art, one can then determine whether a peptide which binds 20 to PCSK9 is useful by determining whether the peptide is blocks PCSK9 from binding to the LDL receptor and/or prevents PCSK9 internalization into cells. Expression and selection of any of the PCSK9-specific antagonists described in the present application may be achieved using suitable technologies including, but not limited to phage display (see, e.g., International Application Number WO 92/01047, Kay et al,, 1996 Phage 25 Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press), yeast display, bacterial display, T7 display, and ribosome display (see, e.g,, Lowe & Jermutus, 2004 Curr. Pharm Biotech. 517-527). Particular PCSK9-specific antagonists forming part of the present invention are antibody molecules or antibodies. "Antibody molecule" or "Antibody" as described herein refers 30 to an immunoglobulin-derived structure with selective binding to human and/or murine PCSK9 including, but not limited to, a full length or whole antibody, an antigen binding fragment (a fragment derived, physically or conceptually, from an antibody structure), a derivative of any of the foregoing, a fusion of any of the foregoing with another polypeptide, or any alternative structure/composition which incorporates any of the foregoing for purposes of selectively 35 binding to/inhibiting the function of PCSK9. "Whole" antibodies or "full length" antibodies refer to proteins that comprise two heavy (H) and two light (L) chains inter-connected by disulfide bonds which comprise: (1) in -21- WO 2009/100297 PCT/US2009/033341 terms of the heavy chains, a variable region (abbreviated herein as "VH") and a heavy chain constant region which comprises three domains, CHI, CH2, and CH3; and (2) in terms of the light chains, a light chain variable region (abbreviated herein as "VL") and a light chain constant region which comprises one domain, CL. 5 Antibody fragments and, more specifically, antigen binding fragments are molecules possessing an antibody variable region or segment thereof (which comprises one or more of the disclosed CDR 3 domains, heavy and/or light within framework regions of heavy and/or light chains, as appropriate), which confers selective binding to PCSK9, and particularly human and/or murine PCSK9. Antibody fragments containing such an antibody variable region 10 include, but are not limited to the following antibody molecules: a Fab, a F(ab')2, a Fd, a Fv, a scFv, bispecific antibody molecules (antibody molecules comprising a PCSK9-specific antibody or antigen binding fragment as disclosed herein linked to a second functional moiety having a different binding specificity than the antibody, including, without limitation, another peptide or protein such as an antibody, or receptor ligand), a bispecific single chain Fv dimer, an isolated 15 CDR3, a minibody, a 'scAb', a dAb fragment, a diabody, a triabody, a tetrabody, a minibody, and artificial antibodies based upon protein scaffolds, including but not limited to fibronectin type III polypeptide antibodies (see, e.g., U.S. Patent No. 6,703,199 and International Application Numbers WO 02/32925 and WO 00/34784) or cytochrome B; see, e.g., Nygren et al., 1997 Curr. Opinion Struct. Biol. 7:463-469; the disclosures of which are incorporated herein by reference. 20 The antibody portions or binding fragments may be natural, or partly or wholly synthetically produced. Such antibody portions can be prepared by various means known by one of skill in the art, including, but not limited to, conventional techniques, such as papain or pepsin digestion. The term "isolated" as used herein in reference to antibody molecules, PCSK9 specific antagonists in general, encoding nucleic acid or other describes a property as it pertains 25 to the disclosed PCSK9-specific antagonists, nucleic acid or other that makes them different from that found in nature. The difference can be, for example, that they are of a different purity than that found in nature, or that they are of a different structure or form part of a different structure than that found in nature. A structure not found in nature, for example, includes recombinant human immunoglobulin structures including, but not limited to, recombinant human 30 immunoglobulin structures with optimized CDRs. Other examples of structures not found in nature are PCSK9-specific antagonists or nucleic acid substantially free of other cellular material. Isolated PCSK9-specific antagonists are generally free of other protein-specific antagonists having different protein specificities (i.e., possess an affinity for other than PCSK9). In one particular aspect, the present invention provides isolated PCSK9-specific 35 antagonists which antagonize PCSK9 function. In particular embodiments, said PCSK9-specific antagonists inhibit human and/or murine PCSK9's antagonism of cellular LDL uptake by interfering with PCSK9 binding to the LDL receptor and resultant PCSK9 cell internalization. - 22 - WO 2009/100297 PCT/US2009/033341 Disclosed PCSK9-specific antagonists, thus, form desirable molecules for lowering plasma LDL cholesterol levels; see, e.g,, Cohen et al., 2005 Nat. Genet. 37:161-165 (wherein significantly lower plasma LDL cholesterol levels were noted in individuals heterozygous for a nonsense mutation in allele PCSK9); Rashid et al., 2005 Proc. Natl. Acad. Sci. USA 102:5374-5379 5 (wherein PCSK9-knockout mice evidenced increased numbers of LDLRs in hepatocytes, accelerated plasma LDL clearance, and significantly lower plasma cholesterol levels); and Cohen et al., 2006 N. Engl. J Med. 354:1264-1272 (wherein humans heterozygous for mutated, loss of function, PCSK9 exhibited a significant reduction in the long-term risk of developing atherosclerotic heart disease). 10 Through repeat experiments, iD05 antibody molecules as disclosed herein dose dependently inhibited the effects of both human and/or murine PCSK9 on LDL uptake. In specific embodiments, the present invention, thus, encompasses PCSK9-specific antagonists and, in more specific embodiments, antibody molecules comprising the heavy and/or light chain variable regions (SEQ ID NO: 11 and 27, respectively) contained within these ID05 antibody 15 molecules or the heavy and/or light chains, e.g., amino acids 1-233 of SEQ ID NO: 9 (or SEQ ID NO: 9) and SEQ ID NO: 1, respectively, or SEQ ID NOs: 25 and 26, respectively, as well as equivalents (characterized as having one or more conservative amino acid substitutions that do not degrade the PCSK9-selective property of ID05) or homologs thereof. Particular embodiments comprise isolated PCSK9-specific antagonists that comprise the CDR domains 20 disclosed herein or sets of heavy and/or light chain CDR domains disclosed herein, or equivalents thereof, characterized as having one or more conservative amino acid substitutions. Use of the terms "domain" or "region" herein simply refers to the respective portion of the antibody molecule wherein the sequence or segment at issue will reside or, in the alternative, currently resides. 25 In specific embodiments, the present invention provides isolated PCSK9-specific antagonists and, in more specific embodiments, antibody molecules comprising a heavy chain variable region which comprises SEQ ID NO: 11; equivalents thereof characterized as having one or more conservative amino acid substitutions, and homologs thereof. The disclosed antagonists should counteract or inhibit human and/or murine PCSK9-dependent inhibition of 30 cellular LDL uptake. In specific embodiments, the present invention provides homologs of the disclosed antagonists characterized as being at least 90% identical over the heavy chain variable region to SEQ ID NO: 11; said antagonists which inhibit human and/or murine PCSK9 dependent inhibition of cellular LDL uptake by at least 10%. In specific embodiments, the present invention provides isolated PCSK9-specific 35 antagonists and, in more specific embodiments, antibody molecules comprising a light chain variable region which comprises SEQ ID NO: 27; equivalents thereof characterized as having one or more conservative amino acid substitutions, and homologs thereof. The disclosed - 23 - WO 2009/100297 PCT/US2009/033341 antagonists should counteract or inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake. In specific embodiments, the present invention provides homologs of the disclosed antagonists characterized as being at least 90% identical over the light chain variable region to SEQ ID NO: 27; said antagonists which inhibit human and/or murine PCSK9 5 dependent inhibition of cellular LDL uptake by at least 10%. In specific embodiments, the present invention provides isolated PCSK9-specific antibody molecules which comprise a heavy chain variable region comprising SEQ ID NO: 11 and a light chain variable region comprising SEQ ID NO: 27; or equivalents thereof characterized as having one or more conservative amino acid substitutions in the prescribed 10 sequences. Specific embodiments are said antagonists which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. In specific embodiments, the present invention provides homologs of the disclosed antagonists characterized as being at least 90% identical over the heavy and light chain variable regions to SEQ ID NOs: 11 and 27, respectively; said antagonists which inhibit human and/or murine PCSK9-dependent inhibition 15 of cellular LDL uptake by at least 10%. In particular embodiments, the present invention provides isolated PCSK9 specific antagonists and, in more specific embodiments, PCSK9 antibody molecules that comprise variable heavy CDR3 sequence SEQ ID NO: 17; and equivalents thereof characterized as having one or more conservative amino acid substitutions; specific embodiments of which 20 inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. Specific embodiments provide isolated antagonists which additionally comprise in the heavy chain variable region CDR1 and/or CDR2 sequences comprising SEQ ID NO: 13 and/or SEQ ID NO: 15, respectively; or equivalents thereof characterized as having one or more conservative amino acid substitutions in any one ore more of the CDR sequences. In specific 25 embodiments, the present invention provides homologs of the disclosed antagonists characterized as being at least 90% identical over the CDR3 sequences or within each of the CDRI, CDR2 and CDR3 sequences to SEQ ID NO: 17 or SEQ ID NOs: 13, 15 and 17, respectively, as appropriate; said antagonists which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. 30 In particular embodiments, the present invention provides isolated PCSK9 specific antagonists and, in more specific embodiments, antibody molecules which comprise variable light CDR3 sequence which comprises SEQ ID NO: 7; and equivalents thereof characterized as having one or more conservative amino acid substitutions; specific embodiments of which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at 35 least 10%. Specific embodiments provide isolated antagonists which additionally comprise in the light chain variable region CDR1 and/or CDR2 sequences comprising SEQ ID NO: 3 and/or SEQ ID NO: 5, respectively; or an equivalent thereof characterized as having one or more - 24 - WO 2009/100297 PCT/US2009/033341 conservative amino acid substitutions in any one or more of the CDR sequences. In specific embodiments, the present invention provides homologs of the disclosed antagonists characterized as being at least 90% identical over the CDR3 sequences or within each of the CDR 1, CDR2 and CDR3 sequences to SEQ ID NO: 7 or SEQ ID NOs: 3, 5 and 7, respectively, as appropriate; said 5 antagonists which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. In particular embodiments, the present invention provides isolated PCSK9 specific antagonists and, in more specific embodiments, antibody molecules which comprise heavy chain variable region CDR3 sequence and light chain variable region CDR3 sequence 10 comprising SEQ ID NOs: 17 and 7, respectively; or equivalents thereof characterized as having one or more conservative amino acid substitutions in any one or more of the CDR3 sequences; specific embodiments of which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. In specific embodiments, the present invention provides homologs of the disclosed antagonists characterized as being at least 90% identical over the 15 heavy and light chain variable region CDR3 sequences to SEQ ID NOs: 17 and 7, respectively; said antagonists which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. Specific embodiments provide isolated PCSK9-specific antagonists and, in more specific embodiments, antibody molecules which comprise heavy chain variable region CDR1, 20 CDR2, and CDR3 sequences and light chain variable region CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 13, 15, 17, 3, 5 and 7, respectively; and equivalents thereof characterized as having one or more conservative amino acid substitutions in any one or more of the CDR sequences; specific embodiments of which inhibit human and/or murine PCSK9 dependent inhibition of cellular LDL uptake by at least 10%. In specific embodiments, the 25 present invention provides homologs of the disclosed antagonists characterized as being at least 90% identical over the heavy and light chain variable region CDR1, CDR2 and CDR3 sequences to SEQ ID NOs: 13, 15, 17, 3, 5 and 7, respectively; said antagonists which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. One particular aspect of the present invention encompasses isolated PCSK9 30 specific antagonists and, in more specific embodiments, antibody molecules which are variants of that disclosed above which comprise a heavy chain variable region CDR3 sequence of SEQ ID NO: 45 wherein the CDR3 sequence is not SEQ ID NO: 17; specific embodiments of which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. Further embodiments hereof additionally comprise heavy chain variable region CDR1 35 sequence of SEQ ID NO: 43 wherein the variant sequence is not SEQ ID NO: 13 and/or heavy chain variable region CDR2 sequence of SEQ ID NO: 44 wherein the variant sequence is not SEQ ID NO: 15; specific embodiments of which inhibit human and/or murine PCSK9-dependent - 25 - WO 2009/100297 PCT/US2009/033341 inhibition of cellular LDL uptake by at least 10%. In other embodiments, the present invention encompasses heavy chain variable region sequence comprising CDRl, CDR2, and CDR3 sequence which, respectively, comprises SEQ ID NOs: 43, 44 and 45 in the respective regions, which are, respectively, not SEQ ID NOs: 13, 15 and 17; specific embodiments of which inhibit 5 human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. Another aspect of the present invention encompasses isolated PCSK9-specific antagonists and, in more specific embodiments, antibody molecules which are variants of that disclosed above which comprise a light chain variable region CDR3 sequence of SEQ ID NO: 48 wherein the CDR3 sequence is not SEQ ID NO: 7; specific embodiments of which inhibit human 10 and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. Further embodiments hereof additionally comprise light chain variable region CDR1 sequence of SEQ ID NO: 46 wherein the variant sequence is not SEQ ID NO: 3 and/or light chain variable region CDR2 sequence of SEQ ID NO: 47 wherein the variant sequence is not SEQ ID NO: 5; specific embodiments of which inhibit human and/or murine PCSK9-dependent inhibition of cellular 15 LDL uptake by at least 10%. In other embodiments, the present invention encompasses light chain variable region sequence comprising CDR1, CDR2 and CDR3 sequence which, respectively, comprises SEQ ID NOs: 46, 47 and 48 in the respective regions, which are, respectively, not SEQ ID NOs: 3, 5 and 7; specific embodiments of which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. 20 Additional distinct embodiments encompass isolated PCSK9-specific antagonists which comprise: (a) a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequence, wherein (i) the CDR1 sequence comprises SEQ ID NO: 13 or SEQ ID NO: 43; SEQ ID NO: 43 being different in sequence from SEQ ID NO: 13; (ii) the CDR2 sequence comprises SEQ ID NO: 15 or SEQ ID NO: 44; SEQ ID NO: 44 being different in sequence from SEQ ID 25 NO: 15; and (iii) the CDR3 sequence comprises SEQ ID NO: 17 or SEQ ID NO: 45; SEQ ID NO: 45 being different in sequence from SEQ ID NO: 17; and/or (b) a light chain variable region comprising CDR1, CDR2 and CDR3 sequence, wherein (i) the CDR1 sequence comprises SEQ ID NO: 3 or SEQ ID NO: 46; SEQ ID NO: 46 being different in sequence from SEQ ID NO: 3; (ii) the CDR2 sequence comprises SEQ ID NO: 5 or SEQ ID NO: 47; SEQ ID NO: 47 being 30 different in sequence from SEQ ID NO: 5; and (iii) the CDR3 sequence comprises SEQ ID NO: 7 or SEQ ID NO: 48; SEQ ID NO: 48 being different in sequence from SEQ ID NO: 7; specific embodiments of which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. Other aspects of the present invention encompass isolated PCSK9-specific 35 antagonists and, in more specific embodiments, antibody molecules which are variants of that disclosed above which comprise (i) a heavy chain variable region sequence comprising CDR1, CDR2, and CDR3 sequence which, respectively, comprises SEQ ID NOs: 43, 44 and 45 in the - 26 - WO 2009/100297 PCT/US2009/033341 respective regions, which are, respectively, not SEQ ID NOs:13, 15 and 17; and (ii) a light chain variable region sequence comprising CDR1, CDR2 and CDR3 sequence which, respectively, comprises SEQ ID NOs: 46, 47 and 48 in the respective regions, which are, respectively, not SEQ ID NOs: 3, 5 and 7; specific embodiments of which inhibit human and/or murine PCSK9 5 dependent inhibition of cellular LDL uptake by at least 10%. In specific embodiments herein the CDRs are in place of the corresponding regions of 1 D05 with out without conservative amino acid substitutions; specific embodiments of which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. In particular embodiments, the present invention encompasses isolated PCSK9 10 specific antagonists and, in more specific embodiments, antibody molecules comprising heavy and/or light chain variable regions comprising SEQ ID NOs: 50 and 49, respectively; said variants SEQ ID NOs which are not SEQ ID NOs: 11 and 27, respectively; specific embodiments of which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. 15 Specific embodiments include any isolated PCSK9-specific antagonist and, in more specific embodiments, antibody molecules which comprise heavy chain variable region sequence found in any of SEQ ID NOs: 51-56, optionally comprising a light chain variable region sequence disclosed herein (e.g,. SEQ ID NO: 27); specific embodiments of which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. 20 Other embodiments include any isolated PCSK9-specific antagonist and, in more specific embodiments, antibody molecules which comprise light chain variable region sequence found in any of SEQ ID NOs: 57-60, optionally comprising a heavy chain variable region sequence disclosed herein (e.g,. SEQ ID NO: 11); specific embodiments of which inhibit human and/or murine PCSK9-dependent inhibition of cellular LDL uptake by at least 10%. 25 Particular embodiments are isolated PCSK9-specific antagonists which comprise the above-described VH and VL regions in a full length antibody. Specific embodiments herein further comprise a series of amino acids selected from the group consisting of: SEQ ID NO: 21 (IgG1), SEQ ID NO: 22 (IgG2), SEQ ID NO: 23 (IgG4) and SEQ ID NO: 24 (IgG2m4). Conservative amino acid substitutions, as one of ordinary skill in the art will 30 appreciate, are substitutions that replace an amino acid residue with one imparting similar or better (for the intended purpose) functional and/or chemical characteristics. Antagonists bearing such conservative amino acid substitutions can be tested for retained or better activity using functional assays available in the art or described herein. PCSK9-specific antagonists possessing one or more conservative amino acid substitutions which retain the ability to selectively bind to 35 human PCSK9 and antagonize PCSK9 functioning at a level the same or better than 1 D05 antibody molecules as described herein are referred to herein as "functional equivalents" of the disclosed antagonists and form specific embodiments of the present invention. Conservative - 27 - WO 2009/100297 PCT/US2009/033341 amino acid substitutions are often ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), 5 uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such modifications are not designed to significantly reduce or alter the binding or functional inhibition characteristics of 10 the PCSK9-specific antagonist, albeit they may improve such properties. The purpose for making a substitution is not significant and can include, but is by no means limited to, replacing a residue with one better able to maintain or enhance the structure of the molecule, the charge or hydrophobicity of the molecule, or the size of the molecule. For instance, one may desire simply to substitute a less desired residue with one of the same polarity or charge. Such modifications 15 can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. One specific means by which those of skill in the art accomplish conservative amino acid substitutions is alanine scanning mutagenesis as discussed in, for example, MacLennan et al., 1998 Acta PhysioL Scand SuppL 643:55-67, and Sasaki et al., 1998 Adv. Biophys. 35:1-24. 20 In another aspect, the present invention provides isolated PCSK9-specific antagonists and, in more specific embodiments, antibody molecules which comprise heavy and/or light chain variable regions comprising amino acid sequences that are homologous to the corresponding amino acid sequences of the disclosed antibodies, wherein the antibody molecules inhibit PCSK9-dependent inhibition of cellular LDL uptake. Specific embodiments are 25 antagonists which comprise heavy and/or light chain variable regions which are at least 90% identical to disclosed heavy and/or light chain variable regions, respectively. Reference to "at least 90% identical" includes at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100% identical sequences along the full length of the molecule disclosed herein. PCSK9-specific antagonists with amino acid sequences homologous to the amino 30 acid sequences of antagonists described herein are typically produced to improve one or more of the properties of the antagonist without negatively impacting its specificity for PCSK9. One method of obtaining such sequences, which is not the only method available to the skilled artisan, is to mutate sequence encoding the PCSK9-specific antagonist or specificity-determining region(s) thereof, express an antagonist comprising the mutated sequence(s), and test the encoded 35 antagonist for retained function using available functional assays including those described herein. Mutation may be by site-directed or random mutagenesis. As one of skill in the art will appreciate, however, other methods of mutagenesis can readily bring about the same effect. For -28- WO 2009/100297 PCT/US2009/033341 example, in certain methods, the spectrum of mutants are constrained by non-randomly targeting conservative substitutions based on either amino acid chemical or structural characteristics, or else by protein structural considerations. In affinity maturation experiments, several such mutations may be found in a single selected molecule, whether they are randomly or non 5 randomly selected. There are also various structure-based approaches toward affinity maturation as demonstrated in, e.g., U.S. Patent No. 7,117,096, PCT Pub, Nos.: WO 02/084277 and WO 03/099999; the disclosures of which are incorporated herein by reference. As used herein, the percent homology between two amino acid or nucleic acid sequences is equivalent to the percent identity between the two sequences, and these two terms 10 will be used interchangeably throughout. As used herein, % identity of two nucleic acid or amino acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990 J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleic 15 acid sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to an amino acid sequence disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al., 1997 Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, 20 the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. Utilization of components of one or more disclosed PCSK9-specific molecules to produce other binding molecules with similar or better specificity is well within the realm of one skilled in the art. This can be accomplished, for example, using techniques of recombinant DNA technology. One specific example of this involves the introduction of DNA encoding the 25 immunoglobulin variable region, or one or more of the CDRs, of an antibody to the variable region, constant region, or constant region plus framework regions, as appropriate, of a different immunoglobulin. Such molecules form important aspects of the present invention. Specific immunoglobulins or the corresponding sequences, into which particular disclosed sequences may be inserted or, in the alternative, form the essential part of, include but are not limited to the 30 following antibody molecules which form particular embodiments of the present invention: a Fab (monovalent fragment with variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains), a F(ab')2 (bivalent fragment comprising two Fab fragments linked by a disulfide bridge or alternative at the hinge region), a Fd (VH and CH1 domains), a Fv (VL and VH domains), a scFv (a single chain Fv where VL and VH are joined by a linker, e.g., a 35 peptide linker, see, e.g., Bird et al., 1988 Science 242:423-426, Huston et al, 1988 PNAS USA 85:5879-5883), a bispecific antibody molecule (an antibody molecule comprising a PCSK9 specific antibody or antigen binding fragment as disclosed herein linked to a second functional -29- WO 2009/100297 PCT/US2009/033341 moiety having a different binding specificity than the antibody, including, without limitation, another peptide or protein such as an antibody, or receptor ligand), a bispecific single chain Fv dimer (see, e.g, PCT/US92/09965), an isolated CDR3, a minibody (single chain-CH3 fusion that self assembles into a bivalent dimer of about 80 kDa), a 'scAb' (an antibody fragment containing 5 VH and VL as well as either CL or CH), a dAb fragment (VH domain, see, e.g., Ward et al., 1989 Nature 341:544-546, and McCafferty et al., 1990 Nature 348:552-554; or VL domain; Holt et al., 2003 Trends in Biotechnology 21:484-489), a diabody (see, e.g., Holliger et al., 1993 PNAS USA 90:6444-6448 and International Application Number WO 94/13804), a triabody, a tetrabody, a minibody (a scFv joined to a CH3; see, e.g., Hu et al., 1996 Cancer Res. 56:3055 10 3061), IgG, IgGI, IgG2, IgG3, IgG4, IgM, IgD, IgA, IgE or any derivatives thereof, and artificial antibodies based upon protein scaffolds, including but not limited to fibronectin type III polypeptide antibodies (see, e.g., U.S. Patent No. 6,703,199 and International Application Number WO 02/32925) or cytochrome B; see, e.g., Koide et al., 1998 J Molec. Biol. 284:1141 1151, and Nygren et al., 1997 Current Opinion in Structural Biology 7:463-469; the disclosures 15 of which are incorporated herein by reference. Certain antibody molecules including, but not limited to, Fv, scFv, diabody molecules or domain antibodies (Domantis) may be stabilized by incorporating disulfide bridges to line the VH and VL domains, see, e.g., Reiter et al, 1996 Nature Biotech. 14:1239-1245; the disclosure of which is incorporated herein by reference. Bispecific antibodies may be produced using conventional technologies (see, e.g., Holliger & 20 Winter, 1993 Current Opinion BiotechnoL 4:446-449, specific methods of which include production chemically, or from hybrid hybridomas) and other technologies including, but not limited to, the BiTETM technology (molecules possessing antigen binding regions of different specificity with a peptide linker) and knobs-into-holes engineering (see, e.g., Ridgeway et al., 1996 Protein Eng. 9:616-621; the disclosure of which is incorporated herein by reference). 25 Bispecific diabodies may be produced in E. coli, and these molecules as other PCSK9-specific antagonists, as one of skill in the art will appreciate, may be selected using phage display in the appropriate libraries (see, e.g, International Application Number WO 94/13804; the disclosure of which is incorporated herein by reference). Variable domains, into which CDRs of interest are inserted, may be obtained from 30 any germ-line or rearranged human variable domain. Variable domains may also be synthetically produced. The CDR regions can be introduced into the respective variable domains using recombinant DNA technology. One means by which this can be achieved is described in Marks et al., 1992 Bio/Technology 10:779-783; the disclosure of which is incorporated herein by reference. A variable heavy domain may be paired with a variable light domain to provide an 35 antigen binding site. In addition, independent regions (e.g., a variable heavy domain alone) may be used to bind antigen. The artisan is well aware, as well, that two domains of an Fv fragment, VL and VH, while perhaps coded by separate genes, may be joined, using recombinant methods, -30- WO 2009/100297 PCT/US2009/033341 by'a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (scFvs). Specific embodiments provide the CDR(s) in germline framework regions. Framework regions, including but not limited to human framework regions, are known to those 5 of skill in the art (e.g., a human or non-human framework). The framework regions may be naturally occurring or consensus framework regions. In one aspect, the framework region of an antibody of the invention is human (see, e.g., Clothia et al., 1998 J. Mol. Biol. 278:457-479 for a listing of human framework regions; said disclosure of which is incorporated herein by reference in its entirety). Specific embodiments herein provide heavy chain variable CDR3 SEQ ID NO: 10 17 into VH 1 A3 in place of the relevant CDR. Specific embodiments herein provide heavy chain variable CDRl, CDR2 and/or CDR3 sequences ( SEQ ID NO:s 13, 15 and 17, respectively) into VHIA_3 in place of the relevant CDRs. Specific embodiments herein provide light chain variable CDR3 SEQ ID NO: 7 into VK1_4 in place of the relevant CDR, Specific embodiments herein provide light chain variable CDR1, CDR2 and/or CDR3 sequences ( SEQ 15 ID NO:s 3, 5 and 7, respectively) into VKl_4 in place of the relevant CDRs. Specific embodiments further provide heavy chain variable CDR3 SEQ ID NO: 17 and light chain variable CDR3 SEQ ID NO: 7 into VH1A_3 and VK1_4 germline sequences, respectively. Further embodiments, provide heavy chain variable CDRI, CDR2 and/or CDR3 sequences ( SEQ ID NO:s 13, 15 and 17, respectively) into VH1A_3 in place of the relevant CDRs; and light 20 chain variable CDRI, CDR2 and/or CDR3 sequences ( SEQ ID NO:s 3, 5 and 7, respectively) into VK1_4 in place of the relevant CDRs. The present invention encompasses antibody molecules that are human, humanized, deimmunized, chimeric and primatized. The invention also encompasses antibody molecules produced by the process of veneering; see, e.g., Mark et al., 1994 Handbook of 25 Experimental Pharmacology, vol. 113: The pharmacology of monoclonal Antibodies, Springer Verlag, pp. 105-134; the disclosure of which is incorporated herein by reference. "Human" in reference to the disclosed antibody molecules specifically refers to antibody molecules having variable and/or constant regions derived from human germline immunoglobulin sequences, wherein said sequences may, but need not, be modified/altered to have certain amino acid 30 substitutions or residues that are not encoded by human germline immunoglobulin sequence. Such mutations can be introduced by methods including, but not limited to, random or site specific mutagenesis in vitro, or by somatic mutation in vivo. Specific examples of mutation techniques discussed in the literature are that disclosed in Gram et al, 1992 PNAS USA 89:3 576 3580; Barbas et al., 1994 PNAS USA 91:3809-3813, and Schier et al., 1996 J. Mol. Biol. 35 263:551-567; the disclosures of which are incorporated herein by reference. These are only specific examples and do not represent the only available techniques. There are a plethora of mutation techniques in the scientific literature which are available to, and widely appreciated by, -31- WO 2009/100297 PCT/US2009/033341 the skilled artisan. "Humanized" in reference to the disclosed antibody molecules refers specifically to antibody molecules wherein CDR sequences derived from another mammalian species, such as a mouse, are grafted onto human framework sequences. "Primatized" in reference to the disclosed antibody molecules refers to antibody molecules wherein CDR 5 sequences of a non-primate are inserted into primate framework sequences, see, e.g., WO 93/02108 and WO 99/55369; the disclosures of which are incorporated herein by reference. Specific antibodies of the present invention are monoclonal antibodies and, in particular embodiments, are in one of the following antibody formats: IgD, IgA, IgE, IgM, IgG 1, IgG2, IgG3, IgG4 or any derivative of any of the foregoing. The language "derivatives thereof' 10 or "derivatives" in this respect includes, inter alia, (i) antibodies and antibody molecules with conservative modifications in one or both variable regions (i e., VH and/or VL), (ii) antibodies and antibody molecules with manipulations in the constant regions of the heavy and/or light chains, and/or (iii) antibodies and antibody molecules that contain additional chemical moieties which are not normally a part of the immunoglobulin molecule (e.g., pegylation). 15 Manipulations of the variable regions can be within one or more of the VH and/or VL CDR regions. Site-directed mutagenesis, random mutagenesis or other method for generating sequence or molecule diversity can be utilized to create mutants which can subsequently be tested for a particular functional property of interest in available in vitro or in vivo assays including those described herein. 20 Antibodies of the present invention also include those in which modifications have been made to the framework residues within VH and/or VL to improve one or more properties of the antibody of interest. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an 25 antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. Such "backmutated" antibodies are also intended to be encompassed by the invention. Another type of framework modification involves mutating one or more residues 30 within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al; the disclosure of which is incorporated herein by reference. In addition or alternative to modifications made within the framework or CDR 35 regions, antibodies of the invention may be engineered to include modifications within the Fc or constant regions, where present, typically to alter one or more functional properties of the - 32 - WO 2009/100297 PCT/US2009/033341 antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen dependent cellular cytotoxicity. The concept of generating "hybrids" or "combinatorial" IgG forms comprising various antibody isotypes to hone in on desired effector functionality has generally been 5 described; see, e.g., Tao et al., 1991 J. Exp. Med 173:1025-1028. A specific embodiment of the present invention encompasses antibody molecules that possess specific manipulations in the Fc region which have been found to result in reduced or altered binding to FcyR receptors, Clq or FcRn on the part of the antibody. The present invention, therefore, encompasses antibodies in accordance with the present description that do not provoke (or provoke to a lesser extent) 10 antibody-dependent cellular cytotoxicity ("ADCC"), complement-mediated cytotoxicity ("CMC"), or form immune complexes, while retaining normal pharmacokinetic ("PK") properties. Specific embodiments of the present invention provide an antibody molecule as defined in accordance with the present invention which comprises, as part of its immunoglobulin structure, SEQ ID NO: 24 and, in particular embodiments, residues 107-326 of SEQ ID NO: 24 15 as part of the immunoglobulin structure. The present invention encompasses antibody molecules which comprise: (i) a light chain comprising SEQ ID NO: 1, and (ii) a heavy chain comprising SEQ ID NO: 11 in sequence with (adjacent to) or followed by a series of amino acids selected from the group consisting of: SEQ ID NO: 21 (Ig 1), SEQ ID NO: 22 (IgG2), SEQ ID NO: 23 (IgG4) and SEQ ID NO: 24 (IgG2m4). FIGURE 6 illustrates a comparison of sequence 20 comprising SEQ ID NO: 24, particularly IgG2m4, with IgG1, IgG2, and IgG4. Amino acid sequences for mature, secreted anti-PCSK9 IgG2m4 heavy and light chains can be found as SEQ ID NOs: 25 and 26, respectively. Antibody molecules encoded at least in part by said sequence are encompassed herein. Specific PCSK9-specific antagonists may carry a detectable label, or may be 25 conjugated to a toxin (e.g., a cytotoxin), a radioactive isotope, a radionuclide, a liposome, a targeting moiety, a biosensor, a cationic tail, or an enzyme (e.g., via a peptidyl bond or linker). Such PCSK9-specific antagonist compositions form an additional aspect of the present invention. In another aspect, the present invention provides isolated nucleic acid encoding disclosed PCSK9-specific antagonists. "Isolated" as mentioned prior refers to the property of the 30 thing referred to that makes them different from that found in nature. The difference can be, for example, that they are of a different purity than that found in nature, or that they are of a different structure or form part of a different structure than that found in nature. An example of nucleic acid not found in nature is, for example, nucleic acid substantially free of other cellular material. The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or 35 substantially pure form. In specific instances, a nucleic acid may be isolated when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, for example, using standard techniques, including without limitation, alkaline/SDS - 33 - WO 2009/100297 PCT/US2009/033341 treatment, CsCI banding, column chromatography, agarose gel electrophoresis and other suitable methods known in the art. The nucleic acid may include DNA (inclusive of cDNA) and/or RNA. Nucleic acids of the present invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic 5 mice carrying human immunoglobulin genes), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library. The present invention encompasses isolated nucleic acid encoding disclosed 10 variable heavy and/or light chains and select components thereof, particularly the disclosed variable or respective CDR regions and, in particular CDR3. In specific embodiments hereof, the CDR(s) are provided within antibody framework regions and, in particular embodiments, human framework regions. Specific embodiments provide isolated nucleic acid encoding the CDR(s) into germline framework regions including, but not limited to, human germline framework 15 regions. Specific embodiments herein provide isolated nucleic acid encoding heavy chain CDR SEQ ID NO: 17 (in specific embodiments, said nucleic acid of which comprises SEQ ID NO: 18) into VH1A_3 in place of the nucleic acid encoding the relevant CDR. Specific embodiments herein provide nucleic acid encoding heavy chain variable CDRI, CDR2 and/or CDR3 sequences SEQ ID NOs: 13, 15 and 17, respectively (and, in particular embodiments, said nucleic acid of 20 which comprises SEQ ID NOs: 14, 16 and 18, respectively) into VH I A_3 in place of the relevant CDRs. Specific embodiments herein provide isolated nucleic encoding light chain CDR SEQ ID NO: 7 (in specific embodiments, said nucleic acid of which comprises SEQ ID NO: 8) into VKI_4 in place of the nucleic acid encoding the relevant CDR. Specific embodiments herein provide nucleic acid encoding light chain variable CDR1, CDR2 and/or CDR3 sequences SEQ 25 ID NOs: 3, 5 and 7, respectively (and, in particular embodiments, said nucleic acid of which comprises SEQ ID NOs: 4, 6 and 8, respectively) into VKJ_4 in place of the relevant CDRs. Specific embodiments further provide heavy chain variable CDR3 SEQ ID NO: 17 (and, in particular embodiments, said nucleic acid of which comprises SEQ ID NO: 18) and light chain variable CDR3 SEQ ID NO: 7 (and, in particular embodiments, said nucleic acid of which 30 comprises SEQ ID NO: 8) into VH1A_3 and VK1_4 germline sequences, respectively. Further embodiments provide heavy chain variable CDRI, CDR2 and/or CDR3 sequences SEQ ID NOs: 13, 15 and 17, respectively (and, in particular embodiments, said nucleic acid of which comprises SEQ ID NOs: 14, 16 and 18, respectively) into VHIA _3 in place of the relevant CDRs; and light chain variable CDR1, CDR2 and/or CDR3 sequences SEQ ID NOs: 3, 5 and 7, respectively (and, 35 in particular embodiments, said nucleic acid of which comprises SEQ ID NOs: 4, 6 and 8, respectively) into VKl_4 in place of the relevant CDRs. - 34- WO 2009/100297 PCT/US2009/033341 The isolated nucleic acid encoding the variable regions can be provided within any desired antibody molecule format including, but not limited to, the following: F(ab')2, a Fab, a Fv, a scFv, bispecific antibody molecules (antibody molecules comprising a PCSK9-specific antibody or antigen binding fragment as disclosed herein linked to a second functional moiety 5 having a different binding specificity than the antibody, including, without limitation, another peptide or protein such as an antibody, or receptor ligand), a bispecific single chain Fv dimer, a minibody, a dAb fragment, diabody, triabody or tetrabody, a minibody, IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgA, IgE or any derivatives thereof Specific embodiments provide isolated nucleic acid which encodes PCSK9 10 specific antagonists and, in more specific embodiments, antibody molecules comprising a heavy chain variable domain which comprises SEQ ID NO: 11; specific embodiments of which comprise nucleic acid sequence SEQ ID NO: 12. Specific embodiments of the present invention provide isolated nucleic acid encoding PCSK9-specific antagonists and, in more specific embodiments, antibody molecules, which additionally comprise: (i) nucleic acid encoding heavy 15 chain CDR1 amino acid sequence SEQ ID NO: 13 (specific embodiments of which comprise nucleic acid SEQ ID NO: 14) and/or (ii) nucleic acid encoding heavy chain CDR2 amino acid sequence SEQ ID NO: 15 (specific embodiments of which comprise nucleic acid SEQ ID NO: 16). Specific embodiments provide isolated nucleic acid encoding PCSK9-specific antagonists and, in more specific embodiments, antibody molecules comprising a light chain variable domain 20 which comprises SEQ ID NO: 27; specific embodiments of which comprise nucleic acid sequence SEQ ID NO: 28. Specific embodiments of the present invention provide isolated nucleic acid encoding PCSK9-specific antagonists and, in more specific embodiments, antibody molecules, which additionally comprise: (i) nucleic acid encoding light chain CDRI amino acid sequence SEQ ID NO: 3 (specific embodiments of which comprise nucleic acid SEQ ID NO: 4) 25 and/or (ii) nucleic acid encoding light chain CDR2 amino acid sequence SEQ ID NO: 5 (specific embodiments of which comprise nucleic acid SEQ ID NO: 6). Specific embodiments provide isolated nucleic acid encoding PCSK9-specific antagonists and, in more specific embodiments, antibody molecules which comprise a heavy chain variable domain which comprises SEQ ID NO: 11; specific embodiments of which comprise nucleic acid sequence SEQ ID NO: 12; and a 30 light chain variable domain which comprises SEQ ID NO: 27; specific embodiments of which comprise nucleic acid sequence SEQ ID NO: 28. Specific embodiments provide isolated nucleic acid encoding (i) heavy chain CDRI, CDR2 and/or CDR3 sequences (SEQ ID NOs: 13, 15 and 17, respectively; specific embodiments of which comprise nucleic acid SEQ ID NOs: 14, 16 and/or 18, respectively) preferably in a framework region (including but not limited to a human 35 framework region); and (ii) light chain CDR1, CDR2 and/or CDR3 sequences (SEQ ID NO: 3, 5 and 7, respectively; specific embodiments of which comprise nucleic acid SEQ ID NOs: 4, 6 and/or 8, respectively) preferably in a framework region (including but not limited to a human -35- WO 2009/100297 PCT/US2009/033341 framework region). The present invention further provides in specific embodiments, homologs of the antagonists disclosed above, characterized as being at least 90% identical over the heavy and/or light chain variable regions, or the CDR regions, as appropriate, whichever is present to the corresponding sequences of ID05. 5 Additional embodiments provide isolated nucleic acid encoding PCSK9-specific antagonists and, in more specific embodiments, antibody molecules which comprise a light chain comprising SEQ ID NO: 1 (specific embodiments of which comprise nucleic acid SEQ ID NO; 2) and a heavy chain or Fd chain comprising amino acids 1-233 of SEQ ID NO: 9, or SEQ ID NO: 9 (specific embodiments of which comprise nucleic acid 1-699 of SEQ ID NO: 10, or SEQ 10 ID NO: 10, respectively). Further embodiments provide isolated nucleic acid encoding PCSK9 specific antagonists and, in more specific embodiments, antibody molecules which comprise a light chain comprising SEQ ID NO: 26 (specific embodiments of which comprise SEQ ID NO: 30) and a heavy chain comprising SEQ ID NO: 25 (specific embodiments of which comprise SEQ ID NO: 29). The present invention further provides in specific embodiments, homologs of 15 the antagonists disclosed above, characterized as being at least 90% identical over the heavy and/or light chains to the corresponding sequences of 1DO5. Specific embodiments of the present invention encompass nucleic acid encoding antibody molecules that possess manipulations in the Fc region which result in reduced or altered binding to FEcyR receptors, Cl q, or FcRn on the part of the antibody. One specific embodiment 20 of the present invention is isolated nucleic acid which encodes for antibody molecules comprising as part of their immunoglobulin structure SEQ ID NO: 24 and, in particular embodiments, residues 107-326 of SEQ ID NO: 24. In specific embodiments, synthetic PCSK9 specific antagonists can be produced by expression from nucleic acid generated from oligonucleotides synthesized and assembled within suitable expression vectors; see, e.g., 25 Knappick et al, 2000 J. Mol. Biol. 296:57-86, and Krebs et al., 2001 J Immunol. Methods 254:67-84. The present invention encompasses nucleic acid encoding antibody molecules which comprise: (i) nucleic acid encoding a light chain comprising SEQ ID NO: 1 (specific embodiments of which comprise nucleic acid SEQ ID NO: 2), and (ii) nucleic acid encoding a 30 heavy chain comprising SEQ ID NO: 11 (specific embodiments of which comprise nucleic acid SEQ ID NO: 12) followed in sequence by (adjacent to) a set of nucleotides encoding for a set of amino acids selected from the group consisting of: SEQ ID NO: 21 (IgG I), SEQ ID NO: 22 (IgG2), SEQ ID NO: 23 (IgG4) and SEQ ID NO: 24 (IgG2m4). Nucleotide sequences for mature, secreted anti-PCSK9 IgG2m4 heavy and light chains can be found as SEQ ID NOs: 29 35 and 30, respectively. Plasmid sequences comprising heavy and light chain iD05 anti-PCSK9 IgG2m4 antibody molecules can be found as SEQ ID NOs: 31 and 32, respectively. Nucleic acid encoding such antibody molecules form important embodiments hereof - 36 - WO 2009/100297 PCT/US2009/033341 Also included within the present invention are isolated nucleic acids comprising nucleotide sequences which are at least about 90% identical and more preferably at least about 95% identical to the full length of the nucleotide sequences described herein, and which nucleotide sequences encode PCSK9-specific antagonists which inhibit PCSK9-dependent 5 inhibition of cellular LDL uptake by at least 10%. Reference to "at least about 90% identical" throughout the application includes at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical. The invention further provides isolated nucleic acid at least a portion of which hybridizes to the complement of nucleic acid consisting of SEQ ID NO: 12 and/or SEQ ID NO: 10 28 under stringent hybridization conditions, said nucleic acid of which confers upon antibody molecules the ability to specifically bind PCSK9 and antagonize PCSK9 function, and PCSK9 specific antagonists expressed employing said nucleic acid. Methods for hybridizing nucleic acids are well-known in the art; see, e.g., Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1989. Stringent hybridization conditions involve hybridizing at 15 68*C in 5x SSC/5x Denhardt's solution (or equivalent)/1.0% SDS, and washing in 0.2x SSC/0.1% SDS at room temperature. Moderately stringent conditions include washing in 3x SSC at 42*C. The parameters of salt concentration and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. The skilled artisan can manipulate various hybridization and/or washing conditions to specifically target nucleic acid in 20 the hybridizing portion that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 12 and/or SEQ ID NO: 28. Basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, 1989 and Ausubel et al. (eds), Current Protocols in Molecular Biology, John Wiley & 25 Sons, Inc., sections 2.10 and 6.3-6.4, 1995 (the disclosures of which are incorporated herein by reference), and can be readily determined by those having ordinary skill in the art. PCSK9 antagonists having one or more variable regions comprising nucleic acid which hybridizes to the complement of nucleic acid consisting of SEQ ID NO: 12 and/or SEQ ID NO: 28 under stringent hybridization conditions should be effective in antagonizing one or more functions of PCSK9. 30 Said antagonists and encoding nucleic acid, thus, form important embodiments of the present invention. In another aspect, the present invention provides vectors comprising the nucleic acid disclosed herein. Vectors in accordance with the present invention include, but are not limited to, plasmids and other expression constructs (eg., phage or phagemid, as appropriate) 35 suitable for the expression of the desired antibody molecule at the appropriate level for the intended purpose; see, e.g., Sambrook & Russell, Molecular Cloning: A Laboratory Manual: 3rd Edition, Cold Spring Harbor Laboratory Press; the disclosure of which is incorporated herein by -37- WO 2009/100297 PCT/US2009/033341 reference. For most cloning purposes, DNA vectors may be used. Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, bacterial artificial chromosomes, and other forms of episomal or integrated DNA. It is well within the purview of the skilled artisan to determine an appropriate vector for a particular gene transfer, 5 generation of a recombinant PCSK9-specific antagonist, or other use. In specific embodiments, in addition to a recombinant gene, the vector may also contain an origin of replication for autonomous replication in a host cell, appropriate regulatory sequences, such as a promoter, a termination sequence, a polyadenylation sequence, an enhancer sequence, a selectable marker, a limited number of useful restriction enzyme sites, and/or other sequences as appropriate and the 10 potential for high copy number. Examples of expression vectors for the production of protein specific antagonists are well known in the art; see, e.g., Persic et al., 1997 Gene 187:9-18; Boel et al., 2000 J Immunol. Methods 239:153-166, and Liang et al, 2001 J. Immunol. Methods 247:119-130; the disclosures of which are incorporated herein by reference. If desired, nucleic acid encoding the antagonist may be integrated into the host chromosome using techniques well 15 known in the art; see, e.g., Ausubel, Current Protocols in Molecular Biology John Wiley & Sons, 1999, and Marks et al., International Application Number WO 95/17516. Nucleic acid may also be expressed on plasmids maintained episomally or incorporated into an artificial chromosome; see, e.g., Csonka et al, 2000 J. Cell Science 113:3207-3216; Vanderbyl et al, 2002 Molecular Therapy 5:10. Specifically with regards to antibody molecules, the antibody 20 light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes may be inserted into the same expression vector. Nucleic acid encoding any PCSK9-specific antagonist or component thereof can be inserted into an expression vector using standard methods (e.g., ligation of complementary restriction sites on the nucleic acid fragment and vector, or blunt end ligation if no restriction sites are present). Another specific example of 25 how this may be carried out is through use of recombinational methods, e.g. the Clontech "InFusion" system, or Invitrogen "TOPO" system (both in vitro), or intracellularly (e.g. the Cre Lox system). Specifically with regards to antibody molecules, the light and heavy chain variable regions can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of 30 the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector comprising nucleic acid encoding a PCSK9-specific antagonist can encode a signal peptide that facilitates secretion of the antagonist from a host cell. The nucleic acid can be cloned into the vector such that the nucleic 35 acid encoding a signal peptide is linked in-frame adjacent to the PCSK9-specific antagonist encoding nucleic acid. The signal peptide may be an immunoglobulin or a non-immunoglobulin signal peptide. Any technique available to the skilled artisan may be employed to introduce the - 38 - WO 2009/100297 PCT/US2009/033341 nucleic acid into the host cell; see, e.g., Morrison, 1985 Science, 229:1202. Methods of subcloning nucleic acid molecules of interest into expression vectors, transforming or transfecting host cells containing the vectors, and methods of making substantially pure protein comprising the steps of introducing the respective expression vector into a host cell, and 5 cultivating the host cell under appropriate conditions are well known. The PCSK9-specific antagonist so produced may be harvested from the host cells in conventional ways. Techniques suitable for the introduction of nucleic acid into cells of interest will depend on the type of cell being used. General techniques include, but are not limited to, calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using viruses 10 appropriate to the cell line of interest (e.g., retrovirus, vaccinia, baculovirus, or bacteriophage). In another aspect, the present invention provides isolated cell(s) comprising nucleic acid encoding disclosed PCSK9-specific antagonists. A variety of different cell lines are contemplated herein and can be used for the recombinant production of PCSK9-specific antagonists, including but not limited to those from prokaryotic organisms (e.g., . coli, Bacillus, 15 and Streptomyces) and from eukaryotic (e.g., yeast, Baculovirus, and mammalian); see, e.g, Breitling et al., Recombinant antibodies, John Wiley & Sons, Inc. and Spektrum Akademischer Verlag, 1999; the disclosure of which is incorporated herein by reference. Plant cells, including transgenic plants, and animal cells, including transgenic animals (other than humans), comprising the nucleic acid or antagonists disclosed herein are also contemplated as part of the present 20 invention. Suitable mammalian cells or cell lines including, but not limited to, those derived from Chinese Hamster Ovary (CHO cells, including but not limited to DHFR-CHO cells (described in Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA 77:4216-4220) used, for example, with a DHFR selectable marker (e.g., as described in Kaufman and Sharp, 1982 Mol. Biol. 159:601-621), NSO myeloma cells (where a GS expression system as described in WO 25 87/04462, WO 89/01036, and EP 338,841 may be used), COS cells, SP2 cells, HeLa cells, baby hamster kidney cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells, and others comprising the nucleic acid or antagonists disclosed herein form additional embodiments of the present invention; the preceding cited disclosures of which are incorporated herein by reference. Specific embodiments of the present invention comprising 30 nucleic acid encoding disclosed PCSK9-specific antagonists include, but are not limited to, . coli; see, e.g., PlUckthun, 1991 Bio/Technology 9:545-55 1, or yeast, such as Pichia, and recombinant derivatives thereof (see, e.g., Li et al., 2006 Nat. Biotechnol. 24:210-215); the preceding disclosures of which are incorporated herein by reference. Specific embodiments of the present invention relate to eukaryotic cells comprising nucleic acid encoding the disclosed 35 PCSK9-specific antagonists, see, Chadd & Chamow, 2001 Current Opinion in Biotechnology 12:188-194, Andersen & Krummen, 2002 Current Opinion in Biotechnology 13:117, Larrick & Thomas, 2001 Current Opinion in Biotechnology 12:411-418; the disclosures of which are - 39 - WO 2009/100297 PCT/US2009/033341 incorporated herein by reference. Specific embodiments of the present invention relate to mammalian cells comprising nucleic acid encoding the disclosed PCSK9-specific antagonists which are able to produce PCSK9-specific antagonists with proper post translational modifications. Post translational modifications include, but are by no means limited to, disulfide 5 bond formation and glycosylation. Another type of post translational modification is signal peptide cleavage. Preferred embodiments herein have the appropriate glycosylation; see, e.., Yoo et al, 2002 J Immunol. Methods 261:1-20; the disclosure of which is incorporated herein by reference. Naturally occurring antibodies contain at least one N-linked carbohydrate attached to a heavy chain. Id. Different types of mammalian host cells can be used to provide for efficient 10 post-translational modifications. Examples of such host cells include Chinese Hamster Ovary (CHO), HeLa, C6, PCl2, and myeloma cells; see, Yoo et al., 2002 J. Immunol. Methods 261:1 20, and Persic et al, 1997 Gene 187:9-18; the disclosures of which are incorporated herein by reference. In another aspect, the present invention provides isolated cell(s) comprising a 15 polypeptide of the present invention. In another aspect, the present invention provides a method of making a PCSK9 specific antagonist of the present invention, which comprises incubating a cell comprising nucleic acid encoding the PCSK9-specific antagonist, or a heavy and/or light chain or a fragment thereof (e.g., VH and/or VL, or one or more of the disclosed heavy and/or light chain variable 20 region CDRs) of a desired PCSK9-specific antagonist (dictated by the desired antagonist) with specificity for human and/or murine PCSK9 under conditions that allow the expression of the PCSK9-specific antagonist, or the expression and assembly of said heavy and/or light chains or fragment into a PCSK9-specific antagonist, and isolating said PCSK9-specific antagonist from the cell. One example by which to generate particular desired heavy and/or light chain sequence 25 or fragment is to first amplify (and modify) the germline heavy and/or light chain variable sequences or fragment using PCR. Germline sequence for human heavy and/or light variable regions are readily available to the skilled artisan, see, e.g., the "Vbase" human germline sequence database, and Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; 30 Tomlinson, I.M. et al., 1992 "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops" J Mol. Biol. 227:776-798; and Cox, J.P.L. et al., 1994 "A Directory of Human Germ-line VK Segments Reveals a Strong Bias in their Usage" Eur. J Immunol. 24:827-836; the disclosures of which are incorporated herein by reference. Mutagenesis of germline sequences may be carried out using standard 35 methods, e.g., PCR-mediated mutagenesis where the mutations are incorporated into PCR primers, or site-directed mutagenesis. If full-length antibodies are desired, sequence is available for the human heavy chain constant region genes; see, e.g., Kabat. E.A. et al, 1991 Sequences of -40- WO 2009/100297 PCT/US2009/033341 Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. Fragments containing these regions may be obtained, for example, by standard PCR amplification. Alternatively, the skilled artisan can avail him/herself of vectors already encoding heavy and/or light chain constant regions. 5 Available techniques exist to recombinantly produce other antibody molecules which retain the specificity of an original antibody. A specific example of this is where DNA encoding the immunoglobulin variable region or the CDRs is introduced into the constant regions, or constant regions and framework regions, or simply the framework regions, of another antibody molecule; see, e.g., EP-184,187, GB 2188638, and EP-239400; the disclosures of which 10 are incorporated herein by reference. Cloning and expression of antibody molecules, including chimeric antibodies, are described in the literature; see, e.g., EP 0120694 and EP 0125023; the disclosures of which are incorporated herein by reference. Antibody molecules in accordance with the present invention may, in one instance, be raised and then screened for characteristics identified herein using known 15 techniques. Basic techniques for the preparation of monoclonal antibodies are described in the literature, see, e.g., Kohler and Milstein (1975, Nature 256:495-497); the disclosure of which is incorporated herein by reference. Fully human monoclonal antibodies can be produced by available methods. These methods include, but are by no means limited to, the use of genetically engineered mouse strains which possess an immune system whereby the mouse antibody genes 20 have been inactivated and in turn replaced with a repertoire of functional human antibody genes, while leaving other components of the mouse immune system unchanged. Such genetically engineered mice allow for the natural in vivo immune response and affinity maturation process which results in high affinity, full human monoclonal antibodies. This technology is well known in the art and is fully detailed in various publications, including but not limited to U.S. Patent 25 Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,249 (assigned to GenPharm International and available through Medarex, under the umbrella of the "UltraMab Human Antibody Development System"); as well as U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and related family members (assigned to Abgenix, disclosing their XenoMouse@ technology); the disclosures of which are incorporated 30 herein by reference. See also reviews from Kellerman and Green, 2002 Curr. Opinion in Biotechnology 13:593-597, and Kontermann & Stefan, 2001 Antibody Engineering, Springer Laboratory Manuals; the disclosures of which are incorporated herein by reference. Alternatively, a library of PCSK9-specific antagonists in accordance with the present invention may be brought into contact with PCSK9, and ones able to demonstrate 35 specific binding selected. Functional studies can then be carried out to ensure proper functionality, e.g., inhibition of PCSK9-dependent inhibition of cellular LDL uptake. There are various techniques available to the skilled artisan for the selection of protein-specific molecules -41- WO 2009/100297 PCT/US2009/033341 from libraries using enrichment technologies including, but not limited to, phage display (e.g, see technology from Cambridge Antibody Technology ("CAT") disclosed in U.S. Patent Nos. 5,565,332; 5,733,743; 5,871,907; 5,872,215; 5,885,793; 5,962,255; 6,140,471; 6,225,447; 6,291,650; 6,492,160; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081, as well as other 5 U.S. family members and/or applications which rely on priority filing GB 9206318, filed May 24, 1992; see also Vaughn et al., 1996, Nature Biotechnology 14:309-314), ribosome display (see, e.g., Hanes and PluckthOn, 1997 Proc. Natl. Acad. Sci. 94:4937-4942), bacterial display (see, e.g., Georgiou, et al., 1997 Nature Biotechnology 15:29-34) and/or yeast display (see, e.g., Kieke, et al., 1997 Protein Engineering 10:1303-1310); the preceding disclosures of which are 10 incorporated herein by reference. A library, for example, can be displayed on the surface of bacteriophage particles, with nucleic acid encoding the PCSK9-specific antagonist or fragment thereof expressed and displayed on its surface. Nucleic acid may then be isolated from bacteriophage particles exhibiting the desired level of activity and the nucleic acid used in the development of desired antagonist. Phage display has been thoroughly described in the 15 literature; see, e.g., Kontermann & Stefan, supra, and International Application Number WO 92/01047; the disclosures of which are incorporated herein by reference. Specifically with regard to antibody molecules, individual heavy or light chain clones in accordance with the present invention may also be used to screen for complementary heavy or light chains, respectively, capable of interaction therewith to form a molecule of the combined heavy and light chains; see, 20 e.g., International Application Number WO 92/01047. Any method of panning which is available to the skilled artisan may be used to identify PCSK9-specific antagonists. Another specific method for accomplishing this is to pan against the target antigen in solution, e.g. biotinylated, soluble PCSK9, and then capture the PCSK9-specific antagonist-phage complexes on streptavidin-coated magnetic beads, which are then washed to remove nonspecifically-bound 25 phage. The captured phage can then be recovered from the beads in the same way they would be recovered from the surface of a plate, (e.g. DTT) as described herein. PCSK9-specific antagonists may be purified by techniques available to one of skill in the art. Titers of the relevant antagonist preparation, ascites, hybridoma culture fluids, or relevant sample may be determined by various serological or immunological assays which 30 include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody ("ELISA") techniques and radioimmunoassay ("RIA") techniques. The present invention relates in part to methods employing PCSK9-specific antagonists described herein for antagonizing PCSK9 function; said methods of which are further described below. Use of the term "antagonizing" throughout the present application refers to the 35 act of opposing, inhibiting, counteracting, neutralizing or curtailing one or more functions of PCSK9. Inhibition or antagonism of one or more of PCSK9-associated functional properties can be readily determined according to methodologies known to the art (see, e.g,, Barak & Webb, -42 - WO 2009/100297 PCT/US2009/033341 1981 J. Cell Biol. 90:595-604; Stephan & Yurachek, 1993 J. Lipid Res. 34:325330; and McNamara et al., 2006 Clinica Chimica Acta 369:158-167) as well as those described herein. Inhibition or antagonism will effectuate a decrease in PCSK9 activity relative to that seen in the absence of the antagonist or, for example, that seen when a control antagonist of irrelevant 5 specificity is present. Preferably, a PCSK9-specific antagonist in accordance with the present invention antagonizes PCSK9 functioning to the point that there is a decrease of at least 10%, of the measured parameter including but not limited to the activities disclosed herein, and more preferably, a decrease of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95% of the measured parameter. Such inhibition/antagonism of PCSK9 functioning is particularly effective 10 in those instances where PCSK9 functioning is contributing at least in part to a particular phenotype, disease, disorder or condition which is negatively impacting the subject. In one aspect, the present invention provides a method for antagonizing the activity of PCSK9, which comprises contacting a cell, population of cells or tissue sample capable of being affected by PCSK9 (i.e., which expresses and/or comprises LDL receptors) with 15 a PCSK9-specific antagonist disclosed herein under conditions that allow said antagonist to bind to PCSK9 when present and inhibit PCSK9's inhibition of cellular LDL uptake. Specific embodiments of the present invention include such methods wherein the cell is a human cell. Additional embodiments of the present invention include such methods wherein the cell is a murine cell. 20 In another aspect, the present invention provides a method for antagonizing the activity of PCSK9 in a subject, which comprises administering to the subject a therapeutically effective amount of a PCSK9-specific antagonist of the present invention. In specific embodiments, the methods for antagonizing PCSK9 function are for the treatment of a PCSK9 associated disease, disorder or condition or, alternatively, a disease, disorder or condition that 25 could benefit from the effects of a PCSK9 antagonist. The medicament would be useful in a subject(s) exhibiting a condition associated with PCSK9 activity, or a condition where the functioning of PCSK9 is contraindicated for a particular subject. In select embodiments, the condition may be hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome or related conditions. 30 The present invention, thus, contemplates the use of PCSK9-specific antagonists described herein in various methods of treatment where antagonizing PCSK9 function is desirable. The method of treatment can be prophylactic or therapeutic in nature. In specific embodiments, the present invention relates to a method of treatment for a condition associated with/attributed to PCSK9 activity, or a condition where the functioning of PCSK9 is 35 contraindicated for a particular subject, which comprises administering to the subject a therapeutically effective amount of a PCSK9-specific antagonist of the present invention. In - 43 - WO 2009/100297 PCT/US2009/033341 select embodiments, the condition may be hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome or related conditions. Methods of treatment in accordance with the present invention comprise administering to an individual a therapeutically (or prophylactically) effective amount of a 5 PCSK9-specific antagonist of the present invention. Use of the terms "therapeutically effective" or "prophylactically effective" in reference to an amount refers to the amount necessary at the intended dosage to achieve the desired therapeutic/prophylactic effect for the period of time desired. The desired effect may be, for example, amelioration of at least one symptom associated with the treated condition. These amounts will vary, as the skilled artisan will appreciate, 10 according to various factors, including but not limited to the disease state, age, sex and weight of the individual, and the ability of the PCSK9-specific antagonist to elicit the desired effect in the individual. The response may be documented by in vitro assay, in vivo non-human animal studies, and/or further supported from clinical trials. The PCSK9-specific antagonist may be administered as a pharmaceutical 15 composition. The present invention, thus, provides a pharmaceutically acceptable composition comprising a PCSK9-specific antagonist of the invention and a pharmaceutically acceptable carrier including but not limited to an excipient, diluent, stabilizer, buffer, or alternative designed to facilitate administration of the antagonist in the desired format and amount to the treated individual. 20 The pharmaceutical composition may be formulated by any number of strategies known in the art, see, e.g., McGoff and Scher, 2000 Solution Formulation ofProteins/Peptides: In - McNally, E.J., ed. Protein Formulation and Delivery. New York, NY: Marcel Dekker; pp. 139-158; Akers & Defilippis, 2000, Peptides and Proteins as Parenteral Solutions. In Pharmaceutical Formulation Development of Peptides and Proteins. Philadelphia, PA: Taylor 25 and Francis; pp. 145-177; Akers et al., 2002, Pharm. Biotechnol. 14:47-127. A pharmaceutically acceptable composition suitable for patient administration will contain an effective amount of the PCSK9-specific antagonist in a formulation which both retains biological activity while also promoting maximal stability during storage within an acceptable temperature range. The antagonist-based pharmaceutically acceptable composition may, in particular 30 embodiments, be in liquid or solid form, or in the form of gas particles or aerosolized particles. Any technique for production of liquid or solid formulations may be utilized. Such techniques are well within the realm of the abilities of the skilled artisan. Solid formulations may be produced by any available method including, but not limited to, lyophilization, spray drying, or drying by supercritical fluid technology. Solid formulations for oral administration may be in 35 any form rendering the antagonist accessible to the patient in the prescribed amount and within the prescribed period of time. The oral formulation can take the form of a number of solid formulations including, but not limited to, a tablet, capsule, or powder. Solid formulations may - 44 - WO 2009/100297 PCT/US2009/033341 alternatively be lyophilized and brought into solution prior to administration for either single or multiple dosing according to methods well known to the skilled artisan. Antagonist compositions should generally be formulated within a biologically relevant pH range and may be buffered to maintain a proper pH range during storage. Both liquid and solid formulations 5 generally require storage at lower temperatures (e.g., 2-8"C) in order to retain stability for longer periods. Formulated antagonist compositions, especially liquid formulations, may contain a bacteriostat to prevent or minimize proteolysis during storage, including but not limited to effective concentrations (e.g., 1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat may be contraindicated for some patients. 10 Therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component. Additional components may be added to either a buffered liquid or solid antagonist formulation, including but not limited to sugars as a cryoprotectant (including but not limited to polyhydroxy hydrocarbons such as sorbitol, mannitol, glycerol, and dulcitol and/or disaccharides such as sucrose, lactose, maltose, or trehalose) and, in some instances, a 15 relevant salt (including but not limited to NaCl, KCl, or LiCl). Such antagonist formulations, especially liquid formulations slated for long term storage, will rely on a useful range of total osmolarity to both promote long term stability at temperatures of, for example, 2-8*C or higher, while also making the formulation useful for parenteral injection. As appropriate, preservatives, stabilizers, buffers, antioxidants and/or other additives may be included. The formulations may 20 contain a divalent cation (including but not limited to MgCl2, CaCI2, and MnCl2); and/or a non ionic surfactant (including but not limited to Polysorbate-80 (Tween 8OTM), Polysorbate-60 (Tween 60TM), Polysorbatc-40 (Tween 40TM), and Polysorbate-20 (Tween 20TM), polyoxyethylene alkyl ethers, including but not limited to Brij 58TM, Brij35TM, as well as others such as Triton X-100TM, Triton X- 1l4TM, NP40TM, Span 85 and the Pluronic series of non-ionic 25 surfactants (e.g., Pluronic 121)). Any combination of such components form specific embodiments of the present invention. Pharmaceutical compositions in liquid format may include a liquid carrier, e.g., water, petroleum, animal oil, vegetable oil, mineral oil, or synthetic oil. The liquid format may also include physiological saline solution, dextrose or other saccharide solution or glycols, such 30 as ethylene glycol, propylene glycol or polyethylene glycol. Preferably, the pharmaceutical composition may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free with suitable pH, tonicity, and stability. Pharmaceutical compositions may be formulated for administration after dilution in isotonic vehicles, for example, Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's 35 Injection. One aspect of the present invention is a pharmaceutical composition which comprises: (i) about 50 to about 200 mg/mL of a PCSK9-specific antagonist described herein; -45- WO 2009/100297 PCT/US2009/033341 (ii) a polyhydroxy hydrocarbon (including but not limited to sorbitol, mannitol, glycerol and dulcitol) and/or a disaccharide (including but not limited to sucrose, lactose, maltose and trehalose); the total of said polyhydroxy hydrocarbon and/or disaccharide being about 1% to about 6% weight per volume ("w/v")of the formulation; (iii) about 5 mM to about 200 mM of 5 histidine, imidazole, phosphate or acetic acid which serves as a buffering agent to prevent pH drift over the shelf life of the pharmaceutical composition and as a tonicity modifier; (iv) about 5 mM to about 200 mM of arginine, proline, phenylalanine, alanine, glycine, lysine, glutamic acid, aspartic acid or methionine to counteract aggregation; (v) about 0.0 1M to about 0.1 M of hydrochloric acid ("HCl")in an amount sufficient to achieve a pH in the range of about 5.5 to 10 about 7.5; and (vi) a liquid carrier including but not limited to sterile water, petroleum, animal oil, vegetable oil, mineral oil, synthetic oil, physiological saline solution, dextrose or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol; wherein said pharmaceutical composition has a pH in the range of about 5,5 to about 7.5; and wherein said pharmaceutical composition optionally comprises about 0.0 1% to about 1% w/v of 15 the formulation of a non-ionic surfactant (including but not limited to Polysorbate-80 (Tween 80TM), Polysorbate-60 (Tween 60TM), Polysorbate-40 (Tween 40TM), and Polysorbate-20 (Tween 20TM), polyoxyethylene alkyl ethers, including but not limited to Brij 58TM, Brij35TM, as well as others such as Triton X-100TM, Triton X-I 14TM, NP40TM, Span 85 and the Pluronic series of non ionic surfactants (e.g., Pluronic 121)). 20 HCl may be added as free acid, Histidine-HCI or Arginine-HCl. Where supplied as Histidine-HCl or Arginine-HCl, the total amounts of Histidine or Arginine in the HCI form should be that specified above. Accordingly, some or all of the HCl depending on the amounts of Histidine and/or Arginine may be supplied as Histidine-HCl and/or Arginine-HCI; as appropriate. Use of the term "about" with respect to amounts disclosed in the specification 25 means within 10% of the specified numbers provided. A range provided as, for example" in "about 50 to about 200" expressly includes as distinct embodiments each number within said range. As such in the above example, embodiments including but not limited to those having 50, 100, 125, 150 and 200 form specific embodiments herein. Pharmaceutical compositions as disclosed herein have general applicability despite the mode of administration. In specific 30 embodiments, the disclosed pharmaceutical compositions are useful for subcutaneous administration as a liquid or upon reconstitution of a lyophilized form. In specific embodiments, PCSK9-specific antagonists employed in the disclosed formulations may be pegylated or form part of fusion proteins. Specific aspects of the present invention relate to the above disclosed 35 pharmaceutical compositions which comprise: (i) about 50 to about 200 mg/mL of a PCSK9 specific antagonist described herein; (ii) about 1% to about 6% (in particular embodiments from about 2% to about 6%) w/v mannitol, trehalose or sucrose; (iii) about 10 mM to about 100 mM - 46 - WO 2009/100297 PCT/US2009/033341 of histidine; (iv) about 25 mM to about 100 mM of arginine or proline; (v) about 0.02 M to about 0.05M of hydrochloric acid ("HCI")in an amount sufficient to achieve a pH in the range of about 5.8 to about 7; and (vi) a liquid carrier including but not limited to sterile water, petroleum, animal oil, vegetable oil, mineral oil, synthetic oil, physiological saline solution, dextrose or 5 other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol; wherein said pharmaceutical composition has a pH in the range of about 5.8 to about 7; and wherein said pharmaceutical composition optionally comprising about 0.0 1% to about 1% w/v of the formulation of a non-ionic surfactant (including but not limited to Polysorbate-80 (Tween 8OTM), Polysorbate-60 (Tween 60TM), Polysorbate-40 (Tween 40TM), and Polysorbate-20 10 (Tween 20TM), polyoxyethylene alkyl ethers, including but not limited to Brij 58TM, Brij35TM, as well as others such as Triton X-100TM, Triton X- I14TM, NP4OTM, Span 85 and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121)). Specific embodiments provide pharmaceutical compositions which comprise: (i) 50 to 200 mg/mL of a PCSK9-specific antagonist described herein; (ii) about 1% to about 6% (in 15 particular embodiments from about 2% to about 6%) w/v mannitol, trehalose or sucrose; (iii) about 10 mM to about 150 mM of histidine; (iv) about 10 mM to about 150 mM of arginine or proline; (v) about 0.03 M to about 0.05 M of hydrochloric acid ("HCl")in an amount sufficient to achieve a pH in the range of about 5.8 to about 6.5; and (vi) a liquid carrier including but not limited to sterile water, petroleum, animal oil, vegetable oil, mineral oil, synthetic oil, 20 physiological saline solution, dextrose or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol; wherein said pharmaceutical composition has a pH in the range of about 5.8 to about 6.5; and wherein said pharmaceutical composition optionally comprising about 0.01% to about 1% w/v of Polysorbate-80 (Tween 8OTM) or Polysorbate-20 (Tween 20TM). 25 Specific embodiments herein provide pharmaceutical compositions which comprise: (i) 50 to 200 mg/mL of a PCSK9-specific antagonist described herein; (ii) about 1% to about 6% (in particular embodiments from about 2% to about 6%) w/v sucrose; (iii) about 25 mM to about 100 mM of histidine; (iv) about 25 mM to about 100 mM of arginine; (v) about 0.040 M to about 0.045 M of hydrochloric acid ("HCl")in an amount sufficient to achieve a pH 30 of about 6; and (vi) sterile water; wherein said pharmaceutical composition has a pH of about 6; and wherein said pharmaceutical composition optionally comprising about 0.01% to about 1% w/v of Polysorbate-80 (Tween 80TM) or Polysorbate-20 (Tween 20TM). In specific embodiments thereof, the levels of histidine and arginine are within 25 mM of each other and, in other embodiments are the same. 35 Specific embodiments herein provide pharmaceutical compositions which comprise (i) 50 to 200 mg/mL of a PCSK9-specific antagonist described herein; (ii) sucrose, histidine and arginine in one of the following amounts: (a) about 1% w/v sucrose, about 10 mM -47- WO 2009/100297 PCT/US2009/033341 histidine and about 25 mM arginine; (b) about 2% w/v sucrose, about 25 mM histidine and about 25 mM arginine; (c) about 3% w/v sucrose, about 50 mM histidine and about 50 mM arginine; or (d) about 6% w/v sucrose, about 100 mM histidine and about 100 mM arginine; (iii) about 0.04 mol or, alternatively, about 1.46 g of HCl; and (iv) sterile water; wherein said pharmaceutical 5 composition has a pH of about 6; and wherein said pharmaceutical composition optionally comprising about 0.01% to about 1% w/v of Polysorbate-80 (Tween 8OTM) or Polysorbate-20 (Tween 20TM). Specific embodiments herein are wherein the amounts of sucrose, histidine and arginine in (ii) above are that described in (c) or (d), Specific embodiments herein provide pharmaceutical compositions as described 10 which comprise 50 to 200 mg/ml of any one of the various PCSK9-specific antagonists described herein, For purposes of exemplification of one distinct embodiment thereof, and not to be construed as a limitation, is the following: a pharmaceutical formulation as described above which comprises: a PCSK9-specific antagonist which comprises: (a) a light chain comprising SEQ ID NO: 26; and (b) a heavy chain comprising SEQ ID NO: 25; wherein said PCSK9 15 specific antagonist is an antibody molecule that antagonizes PCSK9's inhibition of cellular LDL uptake. Particular embodiments herein are pharmaceutical compositions according to the above description which are lyophilized and reconstituted. In specific embodiments, said protein concentration in said lyophilized and reconstituted solution is up to 2-fold higher than in the pre 20 lyophilized composition. In specific embodiments, the protein or PCSK9-specific antagonist concentration in the lyophilized and/or reconstituted pharmaceutical composition is in the range of about 50 mg/mL to about 300 mg/mL. Diluents useful for reconstituting the lyophilized pharmaceutical compositions include but are not limited to sterile water, bacteriostatic water for injection ("BWFI"), phosphate-buffered saline, a sterile saline solution, physiological saline 25 solution, Ringer's solution or dextrose solution and may in specific embodiments contain 0.01 1% (w/v) of Polysorbate-80 (Tween 80TM) or Polysorbate-20 (Tween 20TM). In specific embodiments, lyophilized powder can be reconstituted with 1/60.2X original volume (or 0.167 mL) up to lX (lmL). Exemplary embodiments of the present invention are pharmaceutical 30 compositions as described herein which are stable. Other embodiments of the present invention are pharmaceutical compositions as described herein which are stable to lyophilization and reconstitution. Various methods are available to the skilled artisan to prepare lyophilized compositions; see, e.g., Martin & Mo, 2007 "Stability Considerations for Lyophilized Biologics" Amer. Pharm. Rev. "Stable" as used herein refers to the property of the protein or PCSK9 35 specific antagonist to retain its physical or chemical stability, conformational integrity, or its ability to exhibit less denaturation, protein clipping, aggregation, fragmentation, acidic variant formation or loss of biological activity compared with a control sample at a temperature in the -48- WO 2009/100297 PCT/US2009/033341 range of 4-374C for at least about 30 days. Other embodiments remain stable for up to 3 months, 6 months, 12 months, 2 years or longer periods at the above temperatures. In specific embodiments the formulation exhibits no significant changes at 2-8*C for at least 6 months, and preferably 12 months, 2 years or longer, in order of preference. Specific embodiments 5 experience less than 10% or, in particular embodiments, less than 5% of denaturation, protein clipping, aggregation, fragmentation, acidic variant formation or loss of biological activity compared with a control sample at a temperature in the range of 25-45*C (or alternatively 2-8*C) for at least about 30 days, 3 months, 6 months, 12 months, 2 years or longer, Stability of the formulations can be tested via several means known to the skilled artisan including, but not 10 limited to Size Exclusion Chromatography (SEC-HPLC) to measure aggregation and fragmentation, Dynamic Light Scattering (DLS) to measure particle size of concentrated samples, capillary SDS-PAGE to measure fragmentation and capillary iso-electric focusing (cIEF) or cation exchange chromatography ("CEX") to measure acidic variants formation. Techniques suitable for the analysis of protein stability are well understood by those of skill in the art: see 15 review in Peptide and Protein Drug Delivery, 247-30 1, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, 1993 Adv. Drug Delivery Rev. 10:29-90. Pharmaceutical compositions as described herein should be sterile. There are various techniques available to the skilled artisan to accomplish this including, but not limited to, filtration through sterile filtration membranes. In specific embodiments, employing lyophilized 20 and reconstituted compositions, this may be done prior to or following lyophilization and reconstitution. Dosing of antagonist therapeutics is well within the realm of the skilled artisan, see, e.g., Lederman et al., 1991 Int. J. Cancer 47:659-664; Bagshawe et al., 1991 Antibody, Inmunoconjugates and Radiopharmaceuticals 4:915-922, and will vary based on a number of 25 factors including but not limited to the particular PCSK9-specific antagonist utilized, the patient being treated, the condition of the patient, the area being treated, the route of administration, and the treatment desired. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective therapeutic amount of the antagonist. Dosage ranges may be from about 0.01 to 100 mg/kg, and more usually 0.05 to 25 mg/kg, of the host body weight. For example, 30 dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. For purposes of illustration, and not limitation, in specific embodiments, a dose of 5 mg to 2.0 g may be utilized to deliver the antagonist systemically. Optimal precision in achieving concentrations of antagonist within a range that yields efficacy without toxicity requires a regimen based on the 35 kinetics of the drug's availability to the target site(s). This involves a consideration of the distribution, equilibrium, and elimination of the PCSK9-specific antagonist. Antagonists described herein may be used alone at appropriate dosages. Alternatively, co-administration or - 49 - WO 2009/100297 PCT/US2009/033341 sequential administration of other agents may be desirable. It will be possible to present a therapeutic dosing regime for the PCSK9-specific antagonists of the present invention in conjunction with alternative treatment regimes. For example, PCSK9-specific antagonists may be used in combination or in conjunction with other drugs (therapeutic and/or prophylactic), 5 including but not limited to cholesterol-lowering drugs, for example, cholesterol absorption inhibitors (e.g., Zetia®) and cholesterol synthesis inhibitors (e.g., Zocor* and Vytorin®), The present invention contemplates such combinations and they form an important embodiment hereof. Accordingly, the present invention relates to methods of treatment as described above where the PCSK9-specific antagonist is administered/ delivered simultaneously with, following 10 or prior to another drug or drugs (therapeutic and/or prophylactic), including but not limited to cholesterol-lowering drugs, cholesterol absorportion inhibitors and cholesterol absorption inhibitors. Individuals (subjects) capable of treatment as described herein include primates, human and non-human, and include any non-human mammal or vertebrate of commercial or 15 domestic veterinary importance, The PCSK9-specific antagonist may be administered to an individual by any route of administration appreciated in the art, including but not limited to oral administration, administration by injection (specific embodiments of which include intravenous, subcutaneous, intraperitoneal or intramuscular injection), administration by inhalation, intranasal, or topical 20 administration, either alone or in combination with other agents designed to assist in the treatment of the individual. The PCSK9-specific antagonist may also be administered by injection devices, injector pens, needleless devices; and subcutaneous patch delivery systems. The route of administration should be determined based on a number of considerations appreciated by the skilled artisan including, but not limited to, the desired physiochemical 25 characteristics of the treatment. Treatment may be provided on a daily, weekly, biweekly, or monthly basis, or any other regimen that delivers the appropriate amount of PCSK9-specific antagonist to the individual at the prescribed times such that the desired treatment is effected and maintained. The formulations may be administered in a single dose or in more than one dose at separate times. 30 Also contemplated are methods of using the disclosed antagonists in the manufacture of a medicament for treatment of a PCSK9-associated disease, disorder or condition or, alternatively, a disease, disorder or condition that could benefit from the effects of a PCSK9 antagonist, The medicament would be useful in a subject(s) exhibiting a condition associated with PCSK9 activity, or a condition where the functioning of PCSK9 is contraindicated fr a 35 particular subject. In select embodiments, the condition may be hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome or related conditions. - 50 - WO 2009/100297 PCT/US2009/033341 PCSK9-specific antagonists disclosed herein may also be used as a method of diagnosis of PCSK9. In select embodiments, the present invention encompasses methods of identifying or quantifying the level of PCSK9 present in a sample (including but not limited to a biological sample, e.g., serum or blood) which comprises contacting the sample with a PCSK9 5 specific antagonist described herein and detecting or quantifying, respectively, binding to PCSK9. The PCSK9-specific antagonist may be used in various assay formats known to the skilled artisan and may form part of a kit (the general features of a kit of which are further described below). The present invention further provides for the administration of disclosed anti 10 PCSK9 antagonists for purposes of gene therapy. Through such methods, cells of a subject are transformed with nucleic acid encoding a PCSK9-specific antagonist of the invention. Subjects comprising the nucleic acids then produce the PCSK9-specific antagonists endogenously. Previously, Alvarez, et al, Clinical Cancer Research 6:3081-3087, 2000, introduced single-chain anti-ErbB2 antibodies to subjects using a gene therapy approach. The methods disclosed by 15 Alvarez, et al, supra, may be easily adapted for the introduction of nucleic acids encoding an anti-PCSK9 antibody of the invention to a subject. Nucleic acids encoding any PCSK9-specific antagonist may be introduced to a subject. The nucleic acids may be introduced to the cells of a subject by any means known 20 in the art. In preferred embodiments, the nucleic acids are introduced as part of a viral vector. Examples of preferred viruses from which the vectors may be derived include lentiviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, alphavirus, influenza virus, and other recombinant viruses with desirable cellular tropism. Various companies produce viral vectors commercially, including, but by no 25 means limited to, Avigen, Inc. (Alameda, Calif.; AAV vectors), Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford 30 BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors). Methods for constructing and using viral vectors are known in the art ( see, e.g., Miller, et al, Rio Techniques 7:980-990, 1992). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously, and thus are not infectious, in the 35 target cell. Preferably, the replication defective virus is a minimal virus, i.e., it retains only the sequences of its genome which are necessary for encapsidating the genome to produce viral particles. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Use -51- WO 2009/100297 PCT/US2009/033341 of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of vectors comprising attenuated or defective DNA virus sequences include, but are not limited to, a defective herpes virus vector (Kanno et al, Cancer Gen. Ther. 5 6:147-154, 1999; Kaplitt et al, J. Neurosci. Meth. 71:125-132, 1997 and Kaplitt et al, J. Neuro Onc. 19:137-147, 1994). Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Attenuated adenovirus vectors, such as the vector described by Strafford-Perricaudet et al, J Clin. Invest. 90:626-630, 1992 are 10 desirable in some instances. Various replication defective adenovirus and minimum adenovirus vectors have been described (PCT Publication Nos. W094/26914, W094/28938, W094/28152, W094/12649, W095/02697 and W096/22378). The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero et al, Gene 101:195, 1991; EP 185573; Graham, EMBO J 3:2917, 15 1984; Graham et al, J Gen. Virol. 36:59, 1977). The adeno-associated viruses (AAV) are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human 20 pathologies. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see Daly, et al, Gene Ther. 8:1343-1346, 2001, Larson et al, Adv. Exp. Med. Bio. 489:45-57, 2001; PCT Publication Nos. WO 91/18088 and WO 93/09239; US Patent Nos. 4,797,368 and 5,139,941 and EP 488528B1). In another embodiment, the gene can be introduced in a retroviral vector, e.g., as 25 described in US Patent Nos. 5,399,346, 4,650,764, 4,980,289, and 5,124,263; Mann et al, Cell 33:153, 1983; Markowitz et al, J. Virol., 62:1120, 1988; EP 453242 and EP178220. The retroviruses are integrating viruses which infect dividing cells. Lentiviral vectors can be used as agents for the direct delivery and sustained expression of nucleic acids encoding a PCSK9-specific antagonist of the invention in several 30 tissue types, including brain, retina, muscle, liver and blood. The vectors can efficiently transduce dividing and nondividing cells in these tissues, and maintain long-term expression of the PCSK9-specific antagonist. For a review, see Zufferey et al, J. Virol. 72:9873-80, 1998 and Kafri et al, Curr. Opin. Mol. Ther. 3:316-326, 2001. Lentiviral packaging cell lines are available and known generally in the art. They facilitate the production of high-titer lentivirus vectors for 35 gene therapy. An example is a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 106 IU/ml for at least 3 to 4 days; - 52 - WO 2009/100297 PCT/US2009/033341 see Kafri et al, J. Virol 73:576-584, 1999. The vector produced by the inducible cell line can be concentrated as needed for efficiently transducing nondividing cells in vitro and in vivo. Sindbis virus is a member of the alphavirus genus and has been studied extensively since its discovery in various parts of the world beginning in 1953. Gene 5 transduction based on alphavirus, particularly Sindbis virus, has been well-studied in vitro (see Straus et al, Microbiol. Rev., 58:491-562, 1994; Bredenbeek et al, i Virol., 67:6439-6446, 1993; Ij ima et al, Int. J Cancer 80:110-118, 1999 and Sawai et a[, Biochim. Biophyr. Res. Comm. 248:315-323, 1998. Many properties of alphavirus vectors make them a desirable alternative to other virus-derived vector systems being developed, including rapid engineering of expression 10 constructs, production of high-titered stocks of infectious particles, infection of nondividing cells, and high levels of expression (Strauss et al, 1994 supra). Use of Sindbis virus for gene therapy has been described. (Wahlfors et al, Gene. Ther. 7:472-480, 2000 and Lundstrom, J Recep. Sig. Transduct. Res. 19(1-4):673-686, 1999. In another embodiment, a vector can be introduced to cells by lipofection or with 15 other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for in vivo and in vitro transfection of a gene encoding a marker (Feigner et al, Proc. Natl. Acad, Sci. USA 84:7413-7417, 1987 and Wang et al, Proc. Nat. Acad Sci. USA 84:7851-7855, 1987). Useful lipid compounds and compositions for transfer of nucleic acids are described in PCT Publication Nos. WO 95/18863 and WO 96/17823, and in US Patent 20 No. 5,459,127. It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wilson, et al, J. 25 Biol Chem. 267:963-967, 1992; Williams et al, Proc. Nat. Acad Sci. USA 88:2726-2730, 1991). Other reagents commonly used for transfection of plasmids include, but are by no means limited to, FuGene, Lipofectin, and Lipofectamine. Receptor-mediated DNA delivery approaches can also be used (Wu et al, J Biol. Chem. 263:14621-14624, 1988). US Patent Nos, 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection 30 facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Vilquin et al, Gene Ther. 8:1097, 2001; Payen et al, Exp. Hematol. 29:295-300, 2001; Mir, Bioelectrochemistry 53:1-10, 2001; PCT Publication Nos. WO 99/01157, WO 99/01158 and WO 99/01175). Pharmaceutical compositions suitable for such gene therapy approaches and 35 comprising nucleic acids encoding an anti-PCSK9 antagonist of the present invention are included within the scope of the present invention. - 53 - WO 2009/100297 PCT/US2009/033341 In another aspect, the present invention provides a method for identifying, isolating, quantifying or antagonizing PCSK9 in a sample of interest using a PCSK9-specific antagonist of the present invention. The PCSK9-specific antagonists may be utilized as research tools in immunochemical assays, such as Western blots, ELISAs, radioimmunoassay, 5 immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art (see, e.g., Immunological Techniques Laboratory Manual, ed. Goers, J. 1993, Academic Press) or various purification protocols. The antagonists may have a label incorporated therein or affixed thereto to facilitate ready identification or measurement of the activities associated therewith. One skilled in the art is readily familiar with the various types of detectable labels 10 (e.g., enzymes, dyes, or other suitable molecules which are either readily detectable or cause some activity/result that is readily detectable) which are or may be useful in the above protocols. An additional aspect of the present invention are kits comprising PCSK9-specific antagonists or pharmaceutical compositions disclosed herein and instructions for use. Kits typically but need not include a label indicating the intended use of the contents of the kit. The 15 term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit. In specific embodiments wherein the pharmaceutical composition is provided lyophilized, the kit may include sterile water or saline for reconstitution of the formulation into liquid form. In specific embodiments, the amount of water or saline is from about 0.1 ml to 1.0 ml. 20 The following examples are provided to illustrate the present invention without limiting the same hereto: EXAMPLE 1 25 ISOLATION OF RECOMBINANT Fab DISPLAY PHAGE Recombinant Morphosys HuCAL Gold Fab phage display libraries (see, eg., Knappik et al., 2000 J. Mol. Biol. 296:57-86) were panned against immobilized recombinant murine PCSK9 through a process which is briefly described as follows: Phage Fab display libraries were first divided into 3 pools: one pool of VH2 + VH4 + VH5, another of VH1 + VH6, 30 and a third pool of VH3. The phage pools and immobilized PCSK9 protein were blocked with nonfat dry milk. For the first round of panning, each phage pool was bound independently to V5-, His-tagged PCSK9 protein immobilized in wells of Nunc Maxisorp plate. Immobilized phage PCSK9 complexes were washed sequentially with (1) PBS/0.5% Tween T M 20 (Three quick 35 washes); (2) PBS/0.5% Tween TM 20 (One 5 min. incubation with mild shaking); (3) PBS (Three quick washes); and (4) PBS (Two 5-min. incubations with mild shaking). Bound phages were eluted with 20 mM DTT and all three eluted phage suspensions were combined into one tube. E. -54- WO 2009/100297 PCT/US2009/033341 coli TG1 were infected with eluted phages. Pooled culture of phagemid-bearing cells (chloramphenicol-resistant) were grown up, and frozen stock of phagemid-bearing culture were made. Phage were rescued from culture by co-infection with helper phage, and phage stock for next round of panning were made, 5 For the second round of panning, phages from Round 1 were bound to immobilized, blocked V5-, His-tagged PCSK9 protein. Immobilized phage-PCSK9 complexes were washed sequentially with (1) PBS/0.05% Tween tM 20 (One quick wash); (2) PBS/0.05% Tween TM 20 (Four 5 min. incubations with mild shaking); (3) PBS (One quick wash); and (4) PBS (Four 5-min. incubations with mild shaking). Bound phages were eluted, E coli TGI cells 10 were infected, and phage were rescued as in Round I. For the third round of panning, phages from Round 2 were bound to immobilized, blocked V5-His-tagged PCSK9 protein. Immobilized phage-PCSK9 complexes were washed sequentially with (1) PBS/0.05% Tween T M 20 (Ten quick washes); (2) PBS/0.05% Tween T M 20 (Five 5 min. incubations with mild shaking); (3) PBS (Ten quick washes); and (4) PBS (Five 5 15 min. incubations with mild shaking). Bound phages were eluted and . coli TGI cells were infected as in Round 1. Phagemid-infected cells were grown overnight and phagemid DNA was prepared. XbaI-EcoRI inserts from Round 3 phagemid DNA were subeloned into Morphosys Fab expression vector pMORPHx9 MH to yield plasmid pMORPHx9 20 MH/mPCSK9 2 CX1 D05 (see, e.g., FIGURE 1), and a library of Fab expression clones was generated in E. coli TG1 F-. Transformants were spread on LB + chloramphenicol + glucose plates and grown overnight to generate bacterial colonies. Individual transformant colonies were picked and placed into wells of two 96-well plates for growth and screening for Fab expression. 25 EXAMPLE 2 ELISA SCREENING OF BACTERIALLY EXPRESSED FABS Cultures of individual transformants were IPTG-induced and grown overnight for Fab expression. Culture supernatants (candidate Fabs) were incubated with purified V5-, His tagged PCSK9 protein immobilized in wells of 96-well Nunc Maxisorp plates, washed with 30 0.1% TweenTM 20 in PBS using a plate washer, incubated with HRP-coupled anti-Fab antibody, and washed again with PBS/Tween T M 20. Bound HRP was detected by addition of TMP substrate, and A450 values of wells were read with a plate reader. Negative controls were included as follows: Controls for nonspecific Fab binding on each plate were incubated with parallel expressed 35 preparations of anti-EsB, an irrelevant Fab. Growth medium only. Positive controls for ELISA and Fab expression were included as follows: - 55 - WO 2009/100297 PCT/US2009/033341 EsB antigen was bound to three wells of the plate and subsequently incubated with anti-EsB Fab. To control for Fabs reacting with the V5 or His tags of the recombinant PCSK9 antigen, parallel ELISAs were performed using V5-, His-tagged secreted alkaline phosphatase protein (SEAP) expressed in the same cells as the original PCSK9 antigen and similarly purified. Putative 5 PCSK9-reactive Fabs were identified as yielding > 3X background values when incubated with PCSK9 antigen but negative when incubated with SEAP. Clones scoring as PCSK9-reactive in the first round of screening were consolidated onto a single plate, re-grown in triplicate, re induced with IPTG, and re-assayed in parallel ELISAs vs. PCSK9 and SEAP. Positive and negative controls were included as described above. Clones scoring positive in at least 2 of 3 10 replicates were carried forward into subsequent characterizations. In cases of known or suspected mixed preliminary clones, cultures were re-purified by streaking for single colonies on 2xYT plates with chloramphenicol, and liquid cultures from three or more separate colonies were assayed again by ELISAs in triplicate as described above. 15 EXAMPLE 3 DNA SEQUENCE DETERMINATION OF PCSK9 ELISA-POSITIVE FAB CLONES Bacterial culture for DNA preps was made by inoculating 1.2 ml 2xYT liquid media with chloramphenicol from master glycerol stocks of positive Fabs, and growing overnight. DNA was prepared from cell pellets centrifuged out of the overnight cultures using 20 the Qiagen Turbo Mini preps performed on a BioRobot 9600. ABI Dye Terminator cycle sequencing was performed on the DNA with Morphosys defined sequencing primers and run on an ABI 3100 Genetic Analyzer, to obtain the DNA sequence of the Fab clones. DNA sequences were compared to each other to determine unique clone sequences and to determine light and heavy chain subtypes of the Fab clones. 25 EXAMPLE 4 EXPRESSION AND PURIFICATION OF FABS FROM UNIQUE PCSK9 ELISA-POSITIVE CLONE Fabs from ELISA-positive clone m2CXlD05 and the EsB (negative control) Fab 30 were expressed by IPTG-induction in E. coli TGlF- cells. Cultures were lysed and the His tagged Fabs were purified by immobilized metal ion affinity chromatography (IMAC), and proteins were exchanged into 25mM HEPES pH 7.3/150 mM NaCl by centrifugal diafiltration. Proteins were analyzed by electrophoresis on Caliper Lab-Chip 90 and by conventional SDS PAGE, and quantified by Bradford protein assay. Purified Fab protein was re-assayed by ELISA 35 in serial dilutions to confirm activity of purified Fab. Positive and Negative controls were run as before. Purified Fab preparations were then analyzed as described below. -56- WO 2009/100297 PCT/US2009/033341 EXAMPLE 5 CONVERSION OF m2CXlD05 FAB TO FULL LENGTH IgG The DNA sequence encoding the m2CXID05 light chain variable region was amplified by polymerase chain reaction from plasmid template 5 pMORPHx9_MH/mPCSK9_2_CX1_DO5, using primers: ACAGATGCCAGATGCGATATCCAGATGACCCAGA (SEQ ID NO: 33) and TGCAGCCACCGTACGTTTAATTTCAACTTTCGTACC (SEQ ID NO: 34). The product of this amplification was cloned into plasmid pVIJNSA-GS-FB-LCK that had been previously digested with FspI and BmtI, using the InFusion cloning system (Clontech), The resulting 10 plasmid was verified by DNA sequencing across the variable region. Endotoxin-free plasmid preparations were made using the Qiagen Endo-Free plasmid maxiprep kit. The DNA sequence encoding the heavy chain variable region of pMORPHx9_MH/mPCSK9_2_CXI_DO5 was amplified by polymerase chain reaction using primers: ACAGGTGTCCACTCGCAGGTGCAATTGGTTCAGTCT (SEQ ID NO: 35) and 15 GCCCTTGGTGGATGCTGAGCTAACCGTCACCAGGGT (SEQ ID NO: 36), and the amplified product was cloned into plasmid pV I JNSA-BF-HCG2M4 that had been previously digested with FspI and BmtI. The resulting plasmid was verified by DNA sequencing across the variable region. Endotoxin-free plasmid preparations were made using the Qiagen Endo-Free plasmid maxiprep kit. 20 Full-length IgG was obtained by co-transfection of HEK293 cells with the ID05 light chain- and heavy-chain-encoding plasmids, following by Protein A purification of the expressed IgG. EXAMPLE 6 25 KINETIC EVALUATION OF FAB:PCSK9 INTERACTIONS WITH SURFACE PLASMON RESONANCE ("SPR") SPR measurements were performed using a BiacoreTM (Pharmacia Biosensor AB, Uppsala, Sweden) 2000 system. Sensor chip CM5 and Amine Coupling Kit for immobilization were from BiacoreT. 30 Anti-Fab IgG (Human specific) (Sigma, catalog #15260) was covalently coupled to surfaces 1 and 2 of a Sensor Chip CM5 via primary amine groups, using the immobilization wizard with the "Aim for immobilization" option using BiacoreTM Amine Coupling Kit (cat# BR 1000-50. A target immobilization of 5000 RU was specified. The wizard uses a 7 minute activation with a 1:1 mixture of 100 mM NHS (N-Hydroxysuccinimide) and 400 mM EDC (I 35 ethyl-3-(3-dimethylaminopropyl)-carbodiimide), injects the ligand in several pulses to achieve the desired level, then deactivates the remaining surface with a 7 minute pulse of ethanolamine. - 57 - WO 2009/100297 PCT/US2009/033341 Anti-PCSK9 Fabs were captured on capture surface 2, and surface 1 was used as a reference for kinetic studies of Fab:PCSK9 interactions. Each Fab was captured by flowing a 500 ng/ml solution at 5 or 10 pd/min for 1-1.5 minutes to reach a target RL for an R max of 100 150 RU for the reaction. 5-10 concentrations of hPCSK9v5His or mPCSK9v5His antigens were 5 flowed across the surface at 30 p/minute for 3-4 minutes. 15-60 minutes dissociation time was allowed before regeneration of the Anti-Fab surface with a 30 second pulse of 10 mM glycine pH 2.0. BiaEvaluation Software was used to evaluate the sensograms from the multiple concentration of PCSK9 antigen analyzed with each Fab, to estimate the kinetics constants of the 10 Fab:PCSK9 interactions. The kinetic constants were determined as follows: Table 2 Fab Antige k,, (1/Ms x 10J kl 10s x I x K (iM) Human 2.4 11. m2CXID05 Fab PCSK9 0.22 ± 0.01 7 ± 0.05 5 ± 0.75 mean (N=3) Murine 2.5 3.3 m2CXID05 Fab PCSK9 0.86 ± 0.02 7 ± 0.19 5 ±0,39 mean (N=3) - 58 - WO 2009/100297 PCT/US2009/033341 EXAMPLE 7 KINETIC EVALUATION OF IgG:PCSK9 INTERACTIONS WITH SURFACE PLASMON RESONANCE ("SPR") SPR measurements were performed using a BiacoreTM (Pharmacia Biosensor AB, 5 Uppsala, Sweden) 2000 system. Sensor chip CM5 and Amine Coupling Kit for immobilization were from BiacoreTM. A goat Anti-Human IgG (Caltag, catalog 1H10700) was covalently coupled to surfaces I and 2 of a Sensor Chip CM5 via primary amine groups, using the immobilization wizard with the "Aim for immobilization" option using BiacoreTM Amine Coupling Kit (cat# BR 10 1000-50. A target immobilization of 5000 RU was specified. The wizard uses a 7 minute activation with a 1:1 mixture of 100 mM NHS (N-Hydroxysuccinimide) and 400 mM EDC (1 ethyl-3-(3-dimethylaminopropyl)-carbodiimide), injects the ligand in several pulses to achieve the desired level, then deactivates the remaining surface with a 7 minute pulse of ethanolamine. Anti-PCSK9 IgGs were captured on capture surface 2, and surface 1 was used as a 15 reference for kinetic studies of IgG:PCSK9 interactions. IgG was captured by flowing a 10 nM solution at 10 p1/min for 1-1.5 minutes to reach a target RL for an R max of 100-150 RU for the reaction. 5-10 concentrations of hPCSK9v5His or mPCSK9v5His antigens were flowed across the surface at 30 or 60 pl/minute for 4 minutes. 15-60 minutes dissociation time was allowed before regeneration of the Anti-IgG surface with a 60 second pulse of 10 mM Glycine pH 1.7. 20 BiaEvaluation Software was used to evaluate the sensograms from the multiple concentration of PCSK9 antigen analyzed with each IgG, to estimate the kinetics constants of the IgG:PCSK9 interactions, The kinetic constants were determined as follows: Table 3 km (1/Ms x igG Antien k10 (1/s x 10 K (nM) 1D05IgG2m4 hPCSK9 0.88 ±0.01 3.16 ±0.27 3.6 0.33 mean (N=2) 1D05 IgG2m4 mPCSK9 0.67 ±006 2.15 ± 0.16 3.2 0.06 mean (N=2) 25 EXAMPLE 8 PCSK9-LDLR TR-FRET ASSAY FOR ID05 This assay is a variant of the one described in Fisher et al., 2007 J. Biol. Chem. 282:20502-20512. AlexaFluor647-labeled PCSK9 (final concentration 10 nM) was combined 30 with varying amounts of 1 D05 and to this was added Eu(8044)-labeled LDLR ectodomain to a final concentration of ~1.5 nM (sufficient to give -20,000 counts at F1620 nM on the Rubystar) in 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.1 mM CaCI2, 0.05% (w/v) BSA in a total volume of 50 p.L using 96 well black Dynatech U bottom plates. After at least 90 minutes of equilibration, samples were read in a Rubystar reader (BMG Corp.) using 20 flashes per well, a - 59 - WO 2009/100297 PCT/US2009/033341 50 usec integration delay, and a 200 usec total integration time. Data were expressed as the ratio of (F1665/F1620 x 10000) and an IC50 for I D05 was determined from the inflection point of a sigmoidal dose-response curve using a standard four parameter fit. FIGURE 2 illustrates the activity of 1D05 in the PCSK9-LDLR interaction TR 5 FRET assay. Both the Fab and IgG of 1DO5 are potent and inhibit the PCSK9-LDLR interaction fully. EXAMPLE 9 EXOPOLAR ASSAY: EFFECTS OF EXOGENOUS PCSK9 ON CELLULAR LDL UPTAKE 10 On day 1, 30,000 HEK cells/well were plated in a 96 well polyD-lysine coated plate. On day 2, the media was switched to no-serum containing DMEM media. On day 3, the media was removed and the cells were washed with OptiMEM. Purified PCSK9 was added in 100 pl of DMEM media containing LPDS and dI-LDL. The plates were incubated at 37 0 C for 6.5 hrs. The cells were washed quickly in TBS containing 2 mg/ml BSA; then washed in TBS 15 BSA for 2 minutes; and then washed twice (but quickly) with TBS. The cells were lysed in 100 dl RIPA buffer. Fluorescence was then measured in the plate using an Ex 520, Em 580 nm. The total cellular protein in each well was measured using a BCA Protein Assay and the fluorescence units were then normalized to total protein. The Exopolar Assay is effective for characterizing variant effects on LDL uptake; 20 see Table 4 below illustrating how the potencies of PCSK9 mutants correlate with plasma LDL cholesterol in the Exopolar Assay. Table 4 EC-50 (nMV) Mutation Gain/Loss LDL-C (mg/d ) Exopolar S127R Gain 277 14 D374Y Gain 388 1.3 Wild-type 140 51 R46L Loss 116 78 Results: m2CX 1D05, both Fab and IgG, dose-dependently inhibited the effects of 25 both human and murine PCSK9 on LDL uptake; an effect which was reproducibly observed. The amount of PCSK9 added to the cells was ~100-320 nM. m2CX1D05 (Fab) comprises a light chain of SEQ ID NO: 1 (comprising a VL of SEQ ID NO: 27) and a Fd chain of SEQ ID NO: 9 inclusive of linkers and tags (comprising a VH of SEQ ID NO: 11). 30 M2CX1D05 (IgG) comprises a light chain of SEQ ID NO: 26, and a heavy chain comprising SEQ ID NO: 25. - 60 - WO 2009/100297 PCT/US2009/033341 FIGURES 3A-3D illustrate (i) ID05 (Fab)s dose-dependent inhibition of murine PCSK9-dependent loss of cellular LDL-uptake (FIGURE 3A); (ii) iD05 (IgG)'s dose-dependent inhibition of murine PCSK9-dependent loss of cellular LDL-uptake (FIGURE 3B); (iii) ID05 (Fab)'s dose-dependent inhibition of human PCSK9-dependent loss of cellular LDL-uptake 5 (FIGURE 3C); and (iv) ID05 (IgG)'s dose-dependent inhibition of human PSCK9-dependent loss of cellular LDL-uptake (FIGURE 3D). iD05 clearly cross reacts with both human and mouse PCSK9. FIGURES 3A-3D have two controls: (i) a cell only control, showing the basal level of cellular LDL uptake, and (ii) a cell + PCSK9 (5 pg/ml) control which shows the level of PCSK9-dependent loss of LDL 10 uptake. The titration experiments which contain IDO5 and PCSK9 were done at a fixed concentration of PCSK9 (5 pg/ml) and increasing concentrations of 1 D05 shown in the graphs, ID05 can inhibit the effect of PCSK9 on cellular LDL uptake. IC50s for 1D05 (Fab) are 97 and 144 nM for mouse and human PCSK9 protein, respectively. IC50s for ID05 (IgG) are 85 and 79 nM for mouse and human PCSK9 protein, respectively. 15 EXAMPLE 10 PCSK9 CELLULAR UPTAKE The assay that follows was carried out according to the methods of Fisher et al., 2007 J. Biol. Chem. 282: 20502-12. 20 Cells treated with Alexa Fluor 647-labeled PCSK9 were imaged as follows. CHO cells were plated on poly-D-lysine-coated 96-well optical CVG sterile black plates (Nunc) at a density of 20,000 cells/well, Cells were plated in F-12K medium (nutrient mixture, Kaighn's modification (1x)) (Invitrogen) containing 100 units of penicillin and 100 tg/ml streptomycin sulfate and supplemented with 10% FBS. Plates were incubated overnight at 37 *C and 5% 25 C02. The following morning, the medium was removed and replaced with 100 pl of F- 12K medium containing 100 units of penicillin and 100 ptg/ml streptomycin sulfate. After 18 h, the medium was removed. Purified PCSK9 protein was labeled with Alexa Fluor 647 as described under "Experimental Procedures." Alexa Fluor 647-labeled PCSK9 (1, 5, or 20 pg/ml) was added in 50 p1 of F-12K medium containing 10% lipoprotein-deficient serum to the cells. The 30 plates were incubated at 37 *C for 4 h, and the cells were washed quickly with Tris-buffered saline before imaging. To label cellular nuclei, Hoechst 33342 at a final concentration of 0.1 pig/ml was added to each well. The plates were run on an Opera imager (Evotec Technologies GmbH, Hamburg, Germany) with a x40 water immersion objective. Images were captured using excitation wavelengths of 405 nm for fluorescent nuclei and 635 nm for Alexa Fluor 647-labeled 35 PCSK9. For each well, 11 individual fields containing >500 cells were captured for two emission wavelengths. The data were analyzed using a customized algorithm written using the Acapella language (Evotec Technologies GmbH). The algorithm identified and marked the -61- WO 2009/100297 PCT/US2009/033341 nuclear and cytoplasmic areas of individual cells, followed by measurement of the total cytoplasmic intensity of the cell. The intensity was expressed in arbitrary fluorescent units. For testing the 1 D05 Ab, the identical procedure was used, but with either HEK293 or HepG2 cells. For HepG2 cells, the plates would not have been poly-D-lysine coated. 5 5 pg/ml of AF647-labeled WT PCSK9 was added along with a titration of Fab ranging from 50 jtg/ml down. Using this procedure, we obtained IC50 values of roughly 80nM for the Fab in both cell types. Results: FIGURES 4A and 4B illustrate inhibition of PCSK9 internalization by the Fab 1 D05 and IgG I D05 and restoration of LDL uptake. 10 EXAMPLE 11 In Vivo ASSAY Both Fab fragments and whole IgG of human ID05 were tested in vivo in mice and changes in the level of LDL cholesterol were monitored. The mice used in these studies 15 were (136 x B6-Tg(CETP) Ldlrtml)FI mice which are hemizygous for the transgenic (Tg) expression of human CETP (which mice lack) as well as the disruption of the LDL receptor (tml). These mice are particularly useful because of their human-like lipid profiles and LDL-rich nature. Each mouse was bled twice, once at the beginning of the study to establish 20 individual baseline levels of LDL cholesterol ("pre") and a second time 3 hours later ("post") to assess what changes took place in LDL levels after treatment. Each mouse received two IV doses of Dulbecco's PBS as a vehicle control, ID05 IgG (0.5 mg), or ID05 Fab fragments (0,5 mg) over the course of 3 hours. The ID05 whole IgG was centrifuged at 230,000 x g to remove aggregates immediately prior to injection. 25 In FIGURE 5, the LDL levels for each mouse are represented by a set of connected symbols and the change in LDL (postbleed - prebleed) is shown as an average for each treatment group (A mg/dL). Treatment with PBS had no effect on LDL measurements (-4 mg/dL, 5% reduction). In contrast, serum LDL was reduced 20% with 1 DO5 whole IgG (-19 mg/dL) and 34% with Fab fragments of 1 D05 (-24 mg/dL). 30 EXAMPLE 12 LIMITED PROTEOLYSIS The limited proteolysis mass spectrometry strategy consists in the incubation of wt-hPCSK9 and I DO5/wt-hPCSK9 complex (substrates) with endoproteinase enzymes of 35 different specificity in carefully controlled conditions (i.e., low enzymes concentration and short digestion time). Under these conditions, the endoproteases will cleave only the primary cleavage sites of the protein substrate (i.e., sites that are on the surface of the protein substrate and - 62 - WO 2009/100297 PCT/US2009/033341 exposed to the solvent), The binding of ID05 Fab to wt-hPCSK9 will mask some surface residues normally exposed to the solvent in both proteins. Therefore the primary sites cleaved in wt-hPCSK9 and not in the 1D05/wt-hPCSK9 complex correspond to residues of PCSK9 protected by ID05 in the complex. Some of these residues are likely to be directly involved in 5 ID05 binding. The proteolytic peptides generated by wt-hPCSK9 and 1D05/wt-hPCSK9 limited proteolysis are identified and characterized by analysis of the digest by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), Finally, the use of endoproteases with different specificity helps to more accurately define the residues involved in binding. The amount of proteolytic enzyme normally used in limited proteolysis 10 experiments had to be considerably reduced to avoid excess hydrolysis of wt-hPCSK9 and loss of the primary binding sites (exposed residues). Incubation of wt-hPCSK9, ID05 and 1D05/wt hPCSK9 with endoproteases was done in 25 mM HEPES pH 7.5, 150 mM NaCl at room temperature. The endoproteases used were AspN added at a 2500/1 (w/w) excess of protein compared to proteolytic enzyme, and Trypsin and GluC added at a 1000/1 (w/w) protein to 15 endoprotease ratio. At periods of time 5, 15 and 30 minutes after endoprotease addition, an aliquot of sample was deposited onto the MALDI target and subjected to direct MALDI-MS analysis in the presence of sinapinic acid (SA) as matrix. The fragment peptides originated from wt-PCSK9 after incubation with the proteolytic enzyme at various time were compared with those originated from wt-hPCSK9 in the 1D05/wt-hPCSK9 complex sample to identify the 20 residues protected from proteolysis in the 1D05/wt-hPCSK9 interaction. The Figures and Tables provided herein report only the most relevant fragment peptides. Limited proteolysis with endoprotease AsyN: The wt-hPCSK9 protein, 1 D05/wt hPCSK9 complex and ID05 Fab were incubated for 5, 15 and 30 minutes in the presence of AspN, which cleaves N-terminally to Asp residues, at a 2500/1 (w/w) ratio between the protein 25 and the proteolytic enzyme. MALDI-MS analysis of the digests revealed the primary wt hPCSK9 and 1D05/wt-hPCSK9 complex cleavage sites (see Figures 7A and 7B and Table 5 below). Table 5 illustrates the wt-hPCSK9 fragment peptides obtained after AspN incubation for 5 minutes with wt-PCSK9 and 1D05/wt-hPCSK9 complex. In italics are peptides formed only when wt-hPCSK9 hydrolyzes. 30 Table 5 Measured m/z Expected MW Peptide Cleaved AA 1969.1 1969.1 153-168 Asp 169 2222.0 2222.0 31-49 Asp49 4412.9 4411.2 698-737 Asp698 -63- WO 2009/100297 PCT/US2009/033341 The species at m/z 1969.1, originated from the cleavage at Asp169 and matching the theoretical mass of peptide 153-168 of the catalytic domain of wt-hPCSK9, was formed only in the wt-hPCSK9 sample indicating that this residue is protected from proteolysis by I D05 Fab binding in the 1D05/wt-hPCSK9 complex. Several species were formed in both wt-hPCSK9 and 5 1D05/wt-hPCSK9 hydrolyses. In particular the ions at m/z 2222.0 and 4412.9 corresponding to peptides 31-49 of the prodomain and 698-737 of the catalytic domain of wt-hPCSK9 were originated from cleavage at Asp49 and Asp698. At longer endoprotease AspN incubation time (i.e., 15, 30 minutes) the peptide profile shown in the MALDI-MS spectra did not change significantly compared to the one at 5 10 minutes shown in Figures 7A and 7B confirming that Asp 169 is protected from hydrolysis in the I D05/wt-PCSK9 interaction. It is important to note that the observed degree of agreement between the expected and measured mass values is within the norm for this type of experiment, since mass calibration must be made with an external standard. 15 Limited proteolysis with endoprotease GluC: Endoprotease GluC cleaves C terminally Glu residues. Incubation of GluC with wt-hPCSK9, 1D05/wt-hPCSK9 complex and 1 D05 was conducted at a 1000/1 and 100/1 (w/w) ratio between protein and proteolytic enzyme. To detect the primary cleavage sites, the MALDI-MS analysis of the samples was conducted after 5, 15 and 30 minutes of incubation. The wt-hPCSK9 residues cleaved in the wt-hPCSK9 20 protein and protected in the 1D05/wt-hPCSK9 complex, and the corresponding peptides detected in the MS spectrum, are shown in Figures 8A-H and Table 6 below. Table 6 illustrates peptide fragments obtained upon GluC incubation for 15 minutes with wt-PCSK9 and 1 D05/wt-PCSK9 complex. In italics are the peptides originating from wt-PCSK9 and not from the complex. 25 Table 6 Measured m/z Ex pected MW PpieCleaved AA Glu]81 1675.9 1675.8 182-195 Gu] 95 1918.0 191&0 182-197 Glul97 2213.2 2213.1 153-170 Glu 70 3357.4 3357.7 153-181 Glu181 With endoprotease GluC, protection is shown in the wt-hPCSK9 surface area including residues Glul70, Glu197 and Glul95. The specie at m/z 3357.4, corresponding to peptide 153-181, and obtained in the incubation with GluC of both wt-hPCSK9 and 1 D05/wt 30 hPCSK9, indicates that Glul 81 is not protected by the Fab 1 D05 binding to wt-hPCSK9. - 64 - WO 2009/100297 PCT/US2009/033341 Limited proteolysis with Trvpsin: Trypsin cleaves C-terminally Arg and Lys residues. The enzyme was added at 1000 (w/w) ratio to wt-PCSK9, 1D05/wt-PCSK9 complex and ID05 for 5, 15 and 30 minutes. MALDI-MS analysis of the wt-PCSK9 and 1D05/wt PCSK9 complex is shown in Figures 9A-D and the most relevant peptide fragments are reported 5 in Table 7. Table 7 illustrates peptide fragments obtained upon Trypsin incubation for 5 minutes with wt-PCSK9 and 1 D05/wt-PCSK9 complex. In italics are the peptides originating from wt PCSK9 and not from the complex. Table 7 Measured m/z nExpected MW Peptide Cleaved AA 1877.9 1878.0 31-46 Arg46 2279.3 2279.5 200-218 Argl99 Arg218 2581.9 2581.9 706-729 Arg7O5 Arg729 3562.9 3562.9 706-737 Arg705 3909.9 3910.2 166-199 ArgI6S Arg199 4474.6 4474.9 161-199 Arg]60 Arg199 Argi167 5410.5 5410.8 168-215 ArgJl5 Arg215 5729.7 5730.2 166-215 Argl65 .. .. ... .... . A rg215 5850.8 5851.3 168-218 Argl67 Arg218 6170.2 61707 166-218 Arg16S Arg218 7290.7 7291.0 153-215 Arg215 7731.5 7731.5 153-218 Arg218 10 The primary cleavage sites at 5 minutes were Arg46 on the prodomain and Arg160, Arg165, Argl67, Arg199, Arg215, Arg218, Arg705 and Arg729 on the catalytic domain of wt-hPCSK9. The species at m/z 2279.3, 3909.9 and 4474.6 corresponding to peptides 200 218, 166-199 and 161-199 are detected only in the wt-hPCSK9 hydrolysis and indicate that 15 residue Arg199 is protected by 1D05 binding. These peptide fragments together with the species - 65 - WO 2009/100297 PCT/US2009/033341 at m/z 5410.5, 5729.7, 5850.8, 6170.2 correspond to peptides 168-215, 166-215, 168-218, 166 218, detected only in wt-PCSK9 thus indicating protection also on residues Arg165 and Argl67. At 15 minutes of Trypsin incubation, protection on residue Arg199 is confirmed. In fact the species at m/z 2280.0, 3591.4, 3910.9 and 4475.6, all originated from cleavage at 5 Arg199, are present only in the wt-PCSK9 hydrolysis and become more abundant. In addition, protection at Arg194 is detected (as shown by the presence of the specie at m/z 3325.7 in the wt PCSK9 spectrum). The species originated from cleavage at Arg165 and Arg167 (m/z 5411.5, 5730.9, 5851.8 and 6171.6) are present in the wt-hPCSK9 hydrolysis and start to appear with much lower intensity also in the 1D05/wt-hPCSK9 complex proteolysis. This may indicate that 10 the protection from proteolysis on such residues is due to steric hindrance of the Fab rather than to primary contacts between 1 DO5 and wt-PCSK9 residues. Results: With LP-MS using three enzymes of different specificity, we identified the surface area of wt-hPCSK9 protected by the ID05 Fab in the 1D05-wt-hPCSK9. Arg165, Arg167, Asp169, Glul70, Argl94, Glul97 and Arg199 are the residues of wt-hPCSK9 protected 15 upon binding to the 1 D05 Fab (see Figure 10). These residues belong to the catalytic domain of wt-hPCSK9 and are exposed on the surface of the molecule (see Figure 11). In addition, the limited proteolysis with trypsin shows the possibility that residues 194-199 are directly involved in ID05 binding whereas protection from proteolysis on residues Arg165 and Argl67 may be due to steric hindrance of the Fab instead of direct contacts between 1 D05 and wt-PCSK9 20 residues. Residues in peptides R1 94-RI 99 are conserved in human and mouse PCSK9. 1 DO5 Fab recognizes human and mouse protein. As illustrated by the sequence alignment between human and mouse PCSK9 (see Figure 12), the residues included in the peptide 194-199 of wt-PCSK9 and protected by 1D05 are conserved in both human and mouse PCSK9 while 25 residues in peptide 165-169 (also protected by 1D05 binding) are not, This would support a hypothesis that only residues 194-199 are directly interacting with 1D05 while the others (165 169) are protected from proteolysis by steric hindrance. EXAMPLE 13 30 PCSK9/1DO5 TR-FRET ASSAY Anti-V5 antibody (QED Biosciences) was labeled and purified as described previously (see Fisher et al., 2007 J Biol. Chem. 282 (28): 20502-20512) using 4 equivalents of AlexaFluor 647 (Invitrogen). 1D05 IgG was labeled in a similar manner using 5 equivalents of Eu(W8044)-DTA (Perkin-Elmer). Materials were protected from light and stored at 4*C prior to 35 use. V5/lis-PCSK9 was generated as described previously (see Fisher et al., Id.). TR-FRET assays were carried out in black Microfluor 2 96 well plates (Dynex Technologies) in 10 mM Hepes pH 7.4, 150 mM NaCl, 100 uM CaCl 2 and 0.05% BSA. To 25 - 66 - WO 2009/100297 PCT/US2009/033341 tL of 20 nM each AF647 labeled anti-V5 antibody and V5/His-PCSK9 was added a serial dilution of the unlabeled candidate antibody (i.e., ID05 and 1B20), either Fab or IgG. Reagents were equilibrated for ~15 minutes at room temperature and then Eu(W8044)-1 D05 IgG was added to give a final concentration of 1.5 nM Eu labeled antibody (18000 counts at F1 620 nm; 5 S/B=12) and a total volume of 50 uL. After, equilibration assays were read in a BMG LabTech Rubystar Reader as described previously (Fisher et al., Id.). Data are reported as F1 665 /F1 6 20 x 10000. ICos were determined using data fitted to a sigmoidal dose response curve using non linear regression analysis (Kaleidagraph 4.03, Synergy Software). Figure 13 illustrates an analysis of ID05 and a distinct antibody 1B20 in a 10 PCSK9-1 D05 interaction TR-FRET assay. Both Fabs are potent and inhibit the interaction fully. Figures 14A-D illustrates 1B20's inhibition of PCSK9 in the Exopolar assay described, e.g., in Example 9. 1B20 Fab inhibited murine PCSK9 at an IC50 of 152 nM (n=5); and human PCSK9 at an IC50 of 145 nM (n=5). IB20 IgG inhibited murine PCSK9 at an IC50 of 13 nM; and human PCSK9 at an IC50 of 22 nM. The binding particulars of 1B20 Fab are illustrated in the 15 following Table. Table 8 ______________ hPCSK9vS His mPCSK9vSHiS ka (1/Ms) 6.6E+04 ± 6.1E +03 1.41E+05 ± 1.2E+04 kd (/s) 4.8E-05 ± 7.4E-06 7.18E-05 + 2.9E-06 KA (1/M) 1.5E+09± 3,OE+08 2.0E+09 1.5E+08 KD (M) 7.4E-10 ± 1.6E-10 5.1E-10 3.8E-11 EXAMPLE 14 20 1D05 RHESUS PK/PD STUDY To characterize pharmacokinetics, pharmacodynamics and target engagement of 1 D05, a single dose IV study was conducted in male Rhesus monkeys at 3 mg/kg (7.0-9,0kg, n=3). All Rhesus monkeys used in the study were naive to biologics. Monkeys were given an IV bolus dose of ID05 via the cephalic vein. Blood 25 samples were collected from the saphenous/femoral vessel at designated time points post dosing and the resulting plasma/serum was stored at -70 *C until analysis. The dosing solutions of ID05 were prepared at 47.2 mg/mL in 100mM Histidine, 100mM Arginine, 6% sucrose, pH 6.0. The dosing solutions were stored at 4*C and kept on wet ice during dosing. 30 The lipoprotein analysis of the serum samples were carried out as described below. An anti-human IgG ELISA using commercially available reagents was used to quantify ID05 levels. - 67 - WO 2009/100297 PCT/US2009/033341 As shown in FIGURE 15, 1D05 lowered LDL-C by - 50% at 3 mpk and > 25% LDL-C lowering was observed for - 16 days. The t 1
/
2 of iD05 (FIGURE 16) was 77 hr. EXAMPLE 15 5 LIPOPROTEIN ANALYSIS OF PLASMA/SERUM SAMPLES FROM ID05 RHESUS PK/PD STUDY To generate lipoprotein profiles, plasma or serum was fractionated by chromatography over Superose-6 size exclusion column (GE LifeSciences, Inc.). Total cholesterol levels in the column effluent were continuously measured via in-line mixture with a 10 commercially available enzymatic colorimetric cholesterol detection reagent (Total Cholesterol E, Wako USA) followed by downstream spectrophotometric detection of the reaction products at 600nm absorbance. The first peak of cholesterol eluted from the column was attributed to VLDL, the second peak to LDL and the third to HDL; the area under each peak was calculated using software provided with the HPLC. To calculate the cholesterol concentration for each 15 lipoprotein fraction, the ratio of the corresponding peak area to total peak area was multiplied by the total cholesterol concentration measured in the sample. EXAMPLE 16 FORMULATION 20 Monoclonal antibodies comprising a light chain comprising SEQ ID NO: 26 and a heavy chain comprising SEQ ID NO: 25) were dialyzed into the appropriate formulations and concentrated. Solutions were then dispensed into 3 mL glass vials for stability studies. Studies carried out in liquid form were immediately placed on stability at 2-8*C or 25 0 C. Analytical methods included Size Exclusion Chromatography (SEC-HPLC) to 25 measure aggregation and fragmentation. Below is a table of Time 0 and 6M SEC data. The formulations containing 3/50/50 or 6/100/100 (sucrose/His/Arg) form fewer aggregates and fragments after storage for 6 months at 2-8*C or 254C. All formulations are at 6.0 except for the standard - that is frozen at I mg/mL in Phosphate buffered saline (pH ~7). Table 9 Sample Name % High Order % Dimer % Monomer % Clipped Aggregates I D05 standard TO 0.32% 1.59% 98,09% 0.00% 1OHis/ 150 NaCl Time 0 0.26% 1.59% 98.15% 0.00% 3/50/50 Time 0 0.30% 1.59% 98.11% 0.00% . . . - ...- ,-.. 1D05 standard -70C 6M 0.60% 2.79% 96.61% 0.00% His/NaCI 4C 6M 0.98% 3.18% 95.83% 0.01% 6/100/100 4C 6M 0.95% 2.90% 96.13% 0.02% 3/50/50 4C 6M 0.92% 3.00% 96.07% 0.01% 100mg/mL 4C 6M 0.97% 3.13% 95.85% 0.05% - 68 - WO 2009/100297 PCT/US2009/033341 His/NaCI 25C 6M 1.71% 4,45% 93.45% 0.40% 6/100/100 25C 6M 1.16% 3.69% 94,74% 0.39% 3/50/50 25C 6M 1.31% 3.70% 94.62% 0.37% EXAMPLE 17 VARIANTS Site-directed mutant variants of IDO5 were generated and are disclosed herein as 5 SEQ ID NOs: 51-60. Kds of site-directed mutant variants of 1 D05 Fabs were determined using a Bio-Rad ProteOn; with affinity being measured against human PCSK9-V5-His, The methodologies for measuring Fab affinities are essentially the same as previously described for Biacore@. Table 10 Ab ID Qhain Mutated Comp .risingV KD:(nM) H32Y HEAVY SEQ ID NO: 51 2.01 M48AQ HEAVY SEQ ID NO: 52 2.06 M48L HEAVY SEQ ID NO: 53 1.52 H99Y HEAVY SEQ ID NO: 54 1.45 M48L/M109L/MI15L HEAVY SEQ ID NO: 55 1.13 M48V HEAVY SEQ ID NO: 56 1.95 N50D LIGHT SEQ ID NO: 57 3.42 N50Q LIGHT SEQ ID NO: 58 0.615 N50T LIGHT SEQ ID NO: 59 2.13 N50Y LIGHT SEQ ID NO: 60 2.58 10 * Amino acid numbering begins with the first residue of FRI, immediately following signal peptide. - 69 -

权利要求:
Claims (33)
[1] 2. The PCSK9-specific antagonist of claim 1 wherein the CDR3 domain(s) 15 are in a human germline region in the CDR3 region thereof.
[2] 3. The PCSK9-specific antagonist of claim I that binds to human PCSK9 with an equilibrium dissociation constant (KD) of less than 1200 nM. 20 4. The PCSK9-specific antagonist of claim 1 that binds to human PCSK9 with a KD of less than 500 nM.
[3] 5. The PCSK9-specific antagonist of claim I that binds to human PCSK9 with a KD of less than 100 nM. 25
[4] 6. The PCSK9-specific antagonist of claim 1 that binds to human PCSK9 with a KD of less than 5 nM.
[5] 7. The PCSK9-specific antagonist of claim 1 that antagonizes PCSK9's 30 inhibition of cellular LDL uptake at an IC50 of less than 500 nM.
[6] 8. The PCSK9-specific antagonist of claim I that antagonizes PCSK9's inhibition of cellular LDL uptake at an IC50 of less than 200 nM. 35 9. The PCSK9-specific antagonist of claim 1 that antagonizes PCSK9's inhibition of cellular LDL uptake at an IC50 of less than 100 nM. -70- WO 2009/100297 PCT/US2009/033341
[7] 10. The PCSK9-specific antagonist of claim 1 that antagonizes PCSK9's inhibition of cellular uptake by at least 20%.
[8] 11. The PCSK9-specific antagonist of claim 1 which is an antibody molecule. 5
[9] 12. The PCSK9-specific antagonist of claim 1 which further comprises: (a) a heavy chain variable CDR1 sequence comprising SEQ ID NO: 13; (b) a heavy chain variable CDR2 sequence comprising SEQ ID NO: 15; (c) a light chain variable CDR1 sequence comprising SEQ ID NO: 3; and/or 10 (d) a light chain variable CDR2 sequence comprising SEQ ID NO: 5,
[10] 13. The PCSK9-specific antagonist of claim 12 wherein the CDRI, CDR2 and/or CDR3 domain(s) are in a human germline region in the respective CDRl, CDR2 and/or CDR3 regions thereof, 15
[11] 14. The PCSK9-specific antagonist of claim 1 comprising: (a) a heavy chain variable region comprising a CDR3 domain comprising SEQ ID NO: 17; (b) a light chain variable region comprising a CDR3 domain comprising SEQ 20 ID NO: 7; (c) a heavy chain variable CDRl sequence comprising SEQ ID NO: 13; (d) a light chain variable CDRl sequence comprising SEQ ID NO: 3; (e) a heavy chain variable CDR2 sequence comprising SEQ ID NO: 15; and (f) a light chain variable CDR2 sequence comprising SEQ ID NO: 5. 25
[12] 15. The PCSK9-specific antagonist of claim 14 wherein the CDR1, CDR2 and/or CDR3 domain(s) are in a human germline variable region in the respective CDR1, CDR2 and/or CDR3 regions thereof. 30 16. The PCSK9-specific antagonist of claim 12 which comprises a heavy chain variable region comprising SEQ ID NO: 11 and/or a light chain variable region comprising SEQ ID NO: 27.
[13] 17. The PCSK9-specific antagonist of claim 12 which comprises a heavy 35 chain having constant sequence comprising: SEQ ID NO: 24. -71 - WO 2009/100297 PCT/US2009/033341
[14] 18. The PCSK9-specific antagonist of claim 16 which comprises a heavy chain having constant sequence comprising: SEQ ID NO: 24.
[15] 19. A PCSK9-specific antagonist which comprises: 5 (a) a light chain comprising SEQ ID NO: 1; and (b) a heavy chain comprising SEQ ID NO: 11; wherein said PCSK9-specific antagonist is an antibody molecule that antagonizes PCSK9's inhibition of cellular LDL uptake. 10 20. A PCSK9-specific antagonist of claim 19 wherein SEQ ID NO: 11 is followed in sequence by an amino acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.
[16] 21. An isolated PCSK9-specific antagonist which comprises: 15 (a) a light chain comprising SEQ ID NO: 26; and (b) a heavy chain comprising SEQ ID NO: 25; wherein said PCSK9-specific antagonist is an antibody molecule that antagonizes PCSK9's inhibition of cellular LDL uptake. 20 22. An isolated PCSK9-specific antagonist that: (a) inhibits the binding of a ID05 Fab to PCSK9 by at least 50%; said ID05 Fab characterized as comprising a light chain comprising SEQ ID NO: 1 and an Fd chain comprising amino acids 1-233 of SEQ ID NO: 9; and (b) antagonizes (i) PCSK9 binding to the LDL receptor and/or (ii) PCSK9 25 internalization into cells.
[17] 23. A PCSK9-specific antagonist that: (a) inhibits the binding of a 1D05 IgG to PCSK9 by at least 50%; said 1D05 IgG characterized as comprising (i) a light chain comprising SEQ ID NO: 1 and (ii) a heavy chain 30 comprising SEQ ID NO: 11; and (b) antagonizes (i) PCSK9 binding to the LDL receptor and/or (ii) PCSK9 internalization into cells.
[18] 24. A PCSK9-specific antagonist of claim 23 wherein SEQ ID NO: 11 is 35 followed in sequence by an amino acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. - 72 - WO 2009/100297 PCT/US2009/033341
[19] 25. An isolated PCSK9-specific antagonist which comprises: (a) heavy chain variable region CDR3 sequence of SEQ ID NO: 45; (b) heavy chain variable region CDR3 sequence of SEQ ID NO: 45; heavy chain 5 CDRI sequence of SEQ ID NO: 43 and heavy chain CDR2 sequence of SEQ ID NO: 44; (c) light chain variable region CDR3 sequence of SEQ ID NO: 48; (d) light chain variable region CDR3 sequence of SEQ ID NO: 48; light chain CDR1 sequence of SEQ ID NO: 46 and light chain CDR2 sequence of SEQ ID NO: 47; (e) both (a) and (c); 10 (f) both (b) and (d); (g) heavy and/or light chain variable regions comprising SEQ ID NOs: 50 and 49, respectively; (h) a heavy chain variable region comprising any one of SEQ ID NOs: 51-56 and optionally a light chain variable region comprising SEQ ID NO: 27; or 15 (i) a light chain variable region comprising any one of SEQ ID NOs: 57-60 and optionally a heavy chain variable region comprising SEQ ID NO: 11; wherein said PCSK9-specific antagonist is an antibody molecule that antagonizes PCSK9's inhibition of cellular LDL uptake. 20 26. A composition comprising the PCSK9-specific antagonist of any of claims 1-25 and a pharmaceutically acceptable carrier.
[20] 27. A composition in accordance with claim 26 which comprises: (a) about 50 mg/mL to about 200 mg/mL of the PCSK9-specific antagonist; 25 (b) a polyhydroxy hydrocarbon (including but not limited to sorbitol, mannitol, glycerol and dulcitol) and/or a disaccharide (including but not limited to sucrose, lactose, maltose and trehalose); the total of said polyhydroxy hydrocarbon and/or disaccharide being about 1% to about 6% w/v of the formulation; (c) about 5 mM to about 200 mM of histidine, imidazole, phosphate or acetic 30 acid; (d) about 5 mM to about 200 mM of arginine, proline, phenylalanine, alanine, glycine, lysine, glutamic acid, aspartic acid or methionine; (e) about 0.01 M to about 0.1 M of hydrochloric acid ("HCl")in an amount sufficient to achieve a pH in the range of about 5.5 to about 7.5; and 35 (f) a liquid carrier including but not limited to sterile water, petroleum, animal oil, vegetable oil, mineral oil, synthetic oil, physiological saline solution, dextrose or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol; - 73 - WO 2009/100297 PCT/US2009/033341 wherein said pharmaceutical composition has a pH in the range of about 5,5 to about 7.5; and wherein said pharmaceutical composition optionally comprises about 0.0 1% to about 1% w/v of the formulation of a non-ionic surfactant (including but not limited to Polysorbate-80 (Tween 8OTM), Polysorbate-60 (Tween 60TM), Polysorbate-40 (Tween 40TM), and 5 Polysorbate-20 (Tween 20TM), polyoxyethylene alkyl ethers, including but not limited to Brij 58TM, Brij35TM, as well as others such as Triton X-100TM, Triton X-l 14TM, NP4OTM, Span 85 and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121)).
[21] 28. The composition of claim 27 which comprises: 10 (a) about 50 mg/mL to about 200 mg/mL of the PCSK9-specific antagonist; (b) about 1% to about 6% w/v of mannitol, trehalose or sucrose; (c) about 10 mM to about 150 mM of histidine; (d) about 10 mM to about 150 mM of arginine or proline; (e) about 0.003 M to about 0.005 M of hydrochloric acid ("HCl")in an amount 15 sufficient to achieve a pH in the range of about 5.8 to about 6.5; and (f) a liquid carrier including but not limited to sterile water; petroleum, animal oil, vegetable oil, mineral oil, synthetic oil, physiological saline solution dextrose, or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol; wherein said pharmaceutical composition has a pH in the range of about 5.8 to 20 about 6.5; and wherein said pharmaceutical composition optionally comprises about 0.0 1% to about 1% w/v of Polysorbate-80 (Tween 80TM) or Polysorbate-20 (Tween 20TM).
[22] 29. The composition of claim 28 which comprises: (a) about 50 mg/mL to about 200 mg/mL of the PCSK9-specific antagonist; 25 (b) about 2% to about 6% w/v of sucrose; (c) about 25 mM to about 100 mM of histidine; (d) about 25 mM to about 100 mM of arginine; (e) about 0.0040 M to about 0.0045 M of hydrochloric acid ("HCI")in an amount sufficient to achieve a pH in the range of about 6; and 30 (f) sterile water; wherein said pharmaceutical composition has a pH in the range of about 6; and wherein said pharmaceutical composition optionally comprises about 0.01% to about 1% w/v of Polysorbate-80 (Tween 8OTM) or Polysorbate-20 (Tween 20TM). 35 30. The composition of claim 29 which comprises: (a) about 50 mg/mL to about 200 mg/mL of the PCSK9-specific antagonist; - 74 - WO 2009/100297 PCT/US2009/033341 (b) sucrose, histidine and arginine in one of the following amounts: (i) about 3% w/v sucrose, about 50 mM histidine and about 50 mM arginine; or (ii) about 6% w/v sucrose, about 100 mM histidine and about 100 mM arginine; (c) about 0.0040 M to about 0.0045 M of hydrochloric acid ("HC1")in an amount 5 sufficient to achieve a pH in the range of about 6; and (d) sterile water; wherein said pharmaceutical composition has a pH in the range of about 6; and wherein said pharmaceutical composition optionally comprises about 0.0 1% to about 1% w/v of Polysorbate-80 (Tween 8OTM) or Polysorbate-20 (Tween 20TM). 10
[23] 31. A method for antagonizing PCSK9 function which comprises administering a PCSK9-specific antagonist of any of claims 1-25.
[24] 32. Use of a PCSK9-specific antagonist of any of claims 1-25 in the 15 manufacture of a medicament for ameliorating a disorder, condition or disease caused and/or exacerbated by PCSK9 function.
[25] 33. Isolated nucleic acid encoding a PCSK9-specific antagonist of any of claims 1-25. 20
[26] 34. Isolated nucleic acid which encodes a PCSK9-specific antagonist of claim 1; wherein the CDR3 domain of the heavy chain variable region is encoded by a nucleotide sequence comprising SEQ ID NO: 18; and/or wherein the CDR3 domain of the light chain variable region is encoded by a nucleotide sequence comprising SEQ ID NO: 8. 25
[27] 35. The isolated nucleic acid of claim 34 which further comprises: (a) CDR1 and/or CDR2 domains in the heavy chain variable region that are encoded, respectively, by a nucleotide sequence comprising SEQ ID NO: 14 and SEQ ID NO: 16; and/or 30 (b) CDR1 and/ or CDR2 domains in the light chain variable region that are encoded, respectively, by a nucleotide sequence comprising SEQ ID NO: 4 and SEQ ID NO: 6.
[28] 36. Isolated nucleic acid which encodes a PCSK9-specific antagonist of claim 1; wherein said PCSK9-specific antagonist comprises: 35 (a) a heavy chain variable region encoded by a nucleotide sequence comprising SEQ ID NO: 12; and/or - 75 - WO 2009/100297 PCT/US2009/033341 (b) a light chain variable region encoded by a nucleotide sequence comprising SEQ ID NO: 28.
[29] 37. Isolated nucleic acid which encodes a PCSK9-specific antagonist of claim 5 1; wherein said PCSK9-specific antagonist comprises: (a) a heavy chain region encoded by a nucleotide sequence comprising SEQ ID NO: 29; and/or (b) a light chain region encoded by a nucleotide sequence comprising SEQ ID NO: 30. 10
[30] 38. A vector comprising nucleic acid of any of claims 33-37.
[31] 39. An isolated host cell or population of host cells in vitro or in situ comprising nucleic acid of any of claims 33-37. 15
[32] 40. A method for producing a PCSK9-specific antagonist which comprises: (a) culturing the cell(s) of claim 39 under conditions appropriate for production of the PCSK9-specific antagonist; and (b) isolating the PCSK9-specific antagonist produced. 20
[33] 41. An isolated host cell or population of host cells in vitro or in situ comprising a PCSK9-specific antagonist of claim 1. -76-

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

EP1067182A3|1999-07-08|2001-11-21|Helix Research Institute|Secretory protein or membrane protein|

---

US20030119038A1|1999-09-09|2003-06-26|Bingham Brendan William|NARC1, novel subtilase-like homologs|

---

US7029895B2|1999-09-27|2006-04-18|Millennium Pharmaceuticals, Inc.|27411, a novel human PGP synthase|

---

EP1226243A2|1999-10-22|2002-07-31|Millennium Pharmaceuticals, Inc.|Nucleic acid molecules derived from rat brain and programmed cell death models|

---

EP1230360A4|1999-11-09|2003-04-02|Human Genome Sciences Inc|15 human secreted proteins|

---

AU4146101A|2000-02-07|2001-08-14|Millennium Pharm Inc|Narc-1, novel subtilase-like homologs|

---

JP2003530847A|2000-04-12|2003-10-21|ヒューマンゲノムサイエンシズインコーポレイテッド|Albumin fusion protein|

---

EP1292684A2|2000-06-16|2003-03-19|Incyte Genomics, Inc.|Proteases|

---

EP1309614A2|2000-08-11|2003-05-14|Eli Lilly And Company|Novel secreted proteins and their uses|

---

CA2423227C|2000-10-12|2011-11-29|Genentech, Inc.|Reduced-viscosity concentrated protein formulations|

---

JP2005500005A|2000-12-08|2005-01-06|インサイト・ゲノミックス・インコーポレイテッド|Protein modifying and maintenance molecules|

---

AU2002332391A1|2001-03-21|2003-01-02|Human Genome Sciences, Inc.|Human secreted proteins|

---

EP1409982A4|2001-06-14|2006-05-24|Anadys Pharmaceuticals Inc|Methods of screening for ligands of target molecules|

---

JP2005500034A|2001-06-20|2005-01-06|プロションバイオテクリミテッド|Antibody that blocks activation of receptor protein tyrosine kinase, screening method thereof, and use thereof|

---

US6875433B2|2002-08-23|2005-04-05|The United States Of America As Represented By The Secretary Of The Army|Monoclonal antibodies and complementarity-determining regions binding to Ebola glycoprotein|

---

EP1440981A3|2003-01-21|2005-11-23|Research Association for Biotechnology|Full-length human cdna|

---

US20060234242A1|2003-04-07|2006-10-19|Ribonomics, Inc.|Methods for identifying therapeutic targets involved in glucose and lipid metabolism|

---

EP1471152A1|2003-04-25|2004-10-27|Institut National De La Sante Et De La Recherche Medicale |Mutations in the human PCSK9 gene associated to hypercholesterolemia|

---

JP5570806B2|2006-05-11|2014-08-13|アルナイラムファーマシューティカルズ，インコーポレイテッド|Compositions and methods for inhibiting the expression of the PCSK9 gene|

---

EP2106261A4|2006-11-07|2010-05-26|Merck Sharp & Dohme|Antagonists of pcsk9|

---

EP2083861A4|2006-11-07|2010-11-24|Merck Sharp & Dohme|Antagonists of pcsk9|

---

EP2083860A4|2006-11-07|2010-05-26|Merck Sharp & Dohme|Antagonists of pcsk9|

---

CA2668131A1|2006-11-07|2008-05-15|Merck & Co., Inc.|Antagonists of pcsk9|

---

US20090060906A1|2007-01-09|2009-03-05|Wyeth|Anti-IL-13 antibody formulations and uses thereof|

---

CA2680832A1|2007-03-27|2008-10-02|Merck & Co., Inc.|Method for detecting autoprocessed, secreted pcsk9|

---

US7947134B2|2007-04-04|2011-05-24|California Institute Of Technology|Process for joining materials using bulk metallic glasses|

---

EA200901376A1|2007-04-13|2010-12-30|Новартис Аг|MOLECULES AND METHODS OF MODULATION PROPROTEINKONVERTASY SUBTILYZIN / KEKINOVYY TYPE 9 |

---

EP2215124B1|2007-08-23|2016-02-24|Amgen Inc.|Antigen binding proteins to proprotein convertase subtilisin kexin type 9 |

---

KR20100091191A|2007-10-26|2010-08-18|쉐링 코포레이션|Anti-pcsk9 and methods for treating lipid and cholesterol disorders|

---

AR070315A1|2008-02-07|2010-03-31|Merck & Co Inc|ANTIBODIES 1B20 ANTAGONISTS OF PCSK9|

---

TWI516501B|2008-09-12|2016-01-11|禮納特神經系統科學公司|Pcsk9 antagonists|JP5781762B2|2007-08-10|2015-09-24|プロテリックス、インク|Universal type III fibronectin binding domain library|

---

US8680019B2|2007-08-10|2014-03-25|Protelica, Inc.|Universal fibronectin Type III binding-domain libraries|

---

US8470966B2|2007-08-10|2013-06-25|Protelica, Inc.|Universal fibronectin type III binding-domain libraries|

---

EP2215124B1|2007-08-23|2016-02-24|Amgen Inc.|Antigen binding proteins to proprotein convertase subtilisin kexin type 9 |

---

AR070315A1|2008-02-07|2010-03-31|Merck & Co Inc|ANTIBODIES 1B20 ANTAGONISTS OF PCSK9|

---

US8206943B1|2008-06-30|2012-06-26|Schering Corporation|Assay for PCSK9 inhibitors|

---

TWI516501B|2008-09-12|2016-01-11|禮納特神經系統科學公司|Pcsk9 antagonists|

---

US20130064834A1|2008-12-15|2013-03-14|Regeneron Pharmaceuticals, Inc.|Methods for treating hypercholesterolemia using antibodies to pcsk9|

---

US8357371B2|2008-12-15|2013-01-22|Regeneron Pharmaceuticals, Inc.|Methods for treating hypercholesterolemia using antibodies to PCSK9|

---

JO3672B1|2008-12-15|2020-08-27|Regeneron Pharma|High Affinity Human Antibodies to PCSK9|

---

EP2865752A1|2009-09-03|2015-04-29|Pfizer Vaccines LLC|PCSK9 vaccine|

---

AU2013204732B2|2009-10-01|2016-02-18|Cadila Healthcare Limited|Compounds for the treatment of dyslipidemia and related diseases|

---

ES2537652T3|2009-10-01|2015-06-10|Cadila Healthcare Limited|Compounds for the treatment of dyslipidemia and related diseases|

---

CA2777542A1|2009-10-30|2011-05-05|Merck Sharp & Dohme Corp.|Ax213 and ax132 pcsk9 antagonists and variants|

---

EP2493505A4|2009-10-30|2013-06-12|Merck Sharp & Dohme|Ax1 and ax189 pcsk9 antagonists and variants|

---

AR079336A1|2009-12-11|2012-01-18|Irm Llc|ANTAGONISTS OF THE PRO-PROTEIN CONVERTASE-SUBTILISINE / TYPE 9 QUEXINE |

---

EP2450382A1|2010-11-04|2012-05-09|Affiris AG|Immunogenic peptide|

---

JOP20200202A1|2010-11-23|2017-06-16|Regeneron Pharma|Human antibodies to the glucagon receptor|

---

KR20130118925A|2010-12-22|2013-10-30|제넨테크, 인크.|Anti-pcsk9 antibodies and methods of use|

---

AU2012210481B2|2011-01-28|2017-05-18|Sanofi Biotechnology|Pharmaceutical compositions comprising human antibodies to PCSK9|

---

BR112013020402A2|2011-02-11|2018-09-25|Irm Llc|pcsk9 antagonists|

---

JOP20200043A1|2011-05-10|2017-06-16|Amgen Inc|Methods of treating or preventing cholesterol related disorders|

---

WO2012168491A1|2011-06-10|2012-12-13|Novartis Ag|Pharmaceutical formulations of pcsk9 antagonists|

---

WO2012170607A2|2011-06-10|2012-12-13|Novartis Ag|Use of pcsk9 antagonists|

---

AR087305A1|2011-07-28|2014-03-12|Regeneron Pharma|STABILIZED FORMULATIONS CONTAINING ANTI-PCSK9 ANTIBODIES, PREPARATION METHOD AND KIT|

---

PL2570135T3|2011-09-13|2016-07-29|Affiris Ag|PCSK9 Vaccine|

---

BR112014005522A2|2011-09-16|2017-03-21|Regeneron Pharma|methods for reducing lipoproteinlevels by administering a subtilisin proprotein inhibitor quexin-9 convertase |

---

AR087715A1|2011-09-16|2014-04-09|Lilly Co Eli|ANTI PCSK9 ANTIBODIES AND USES OF THE SAME|

---

JP6431372B2|2011-10-25|2018-11-28|プロシーナ バイオサイエンシーズ リミテッド|Antibody formulations and methods|

---

CA2858572A1|2011-12-08|2013-06-13|Amgen Inc.|Human lcat antigen binding proteins and their use in therapy|

---

US20140030270A1|2012-05-03|2014-01-30|Amgen Inc.|Methods of treating or preventing cholesterol related disorders|

---

US9255154B2|2012-05-08|2016-02-09|Alderbio Holdings, Llc|Anti-PCSK9 antibodies and use thereof|

---

EP2667197B1|2012-05-25|2015-09-16|Zora Biosciences OY|Sensitive efficacy and specificity biomarkers for proprotein convertase subtilisin/kexin type 9inhibition|

---

US9266961B2|2012-06-15|2016-02-23|Genentech, Inc.|Anti-PCSK9 antibodies, formulations, dosing, and methods of use|

---

WO2014028354A1|2012-08-13|2014-02-20|Regeneron Pharmaceuticals, Inc.|Anti-pcsk9 antibodies with ph-dependent binding characteristics|

---

WO2014031174A1|2012-08-24|2014-02-27|The Regents Of The University Of California|Antibodies and vaccines for use in treating ror1 cancers and inhibiting metastasis|

---

CN104780940B|2012-11-06|2017-11-07|拜耳制药股份公司|Preparation for bispecific T cell convergence body |

---

EP3081249B1|2012-11-21|2020-12-30|Amgen Inc.|Drug delivery device|

---

TWI580451B|2013-03-15|2017-05-01|安美基公司|Cassette for an injector and method of using an autoinjector apparatus having an autoinjector and a cassette|

---

JP6768501B2|2013-03-15|2020-10-14|アムゲン・インコーポレーテッド|Drug cassettes, automatic injection machines, and automatic injection machine systems|

---

WO2014149699A1|2013-03-15|2014-09-25|Eli Lilly And Company|Bifunctional protein|

---

US10850037B2|2013-03-22|2020-12-01|Amgen Inc.|Injector and method of assembly|

---

US10111953B2|2013-05-30|2018-10-30|Regeneron Pharmaceuticals, Inc.|Methods for reducing remnant cholesterol and other lipoprotein fractions by administering an inhibitor of proprotein convertase subtilisin kexin-9 |

---

WO2014197752A1|2013-06-07|2014-12-11|Regeneron Pharmaceuticals, Inc.|Methods fo inhibting atherosclerosis by administering an inhibitor of pcsk9|

---

EP3013422A1|2013-06-28|2016-05-04|Amgen Inc.|Methods for treating homozygous familial hypercholesterolemia|

---

WO2015061386A1|2013-10-24|2015-04-30|Amgen Inc.|Injector and method of assembly|

---

US10758683B2|2013-10-24|2020-09-01|Amgen Inc.|Drug delivery system with temperature-sensitive control|

---

KR20160081978A|2013-11-12|2016-07-08|사노피 바이오테크놀로지|Dosing regimens for use with pcsk9 inhibitors|

---

WO2016023916A1|2014-08-12|2016-02-18|Kymab Limited|Treatment of disease using ligand binding to targets of interest|

---

US8883157B1|2013-12-17|2014-11-11|Kymab Limited|Targeting rare human PCSK9 variants for cholesterol treatment|

---

WO2016071701A1|2014-11-07|2016-05-12|Kymab Limited|Treatment of disease using ligand binding to targets of interest|

---

DE202014010499U1|2013-12-17|2015-10-20|Kymab Limited|Targeting of human PCSK9 for cholesterol treatment|

---

DE202015009002U1|2014-07-15|2016-08-18|Kymab Limited|Targeting of human PCSK9 for cholesterol treatment|

---

DE202015008988U1|2014-07-15|2016-06-30|Kymab Limited|Targeting of human PCSK9 for cholesterol treatment|

---

EP3332790A1|2014-07-15|2018-06-13|Kymab Limited|Antibodies for use in treating conditions related to specific pcsk9 variants in specific patients populations|

---

WO2015119906A1|2014-02-05|2015-08-13|Amgen Inc.|Drug delivery system with electromagnetic field generator|

---

SG11201609219QA|2014-05-07|2016-12-29|Amgen Inc|Autoinjector with shock reducing elements|

---

CN105085684B|2014-05-14|2020-04-17|上海亨臻实业有限公司|Design and application of PCSK9 targeted recombinant vaccine|

---

SG11201609963PA|2014-06-03|2016-12-29|Amgen Inc|Devices and methods for assisting a user of a drug delivery device|

---

US9051378B1|2014-07-15|2015-06-09|Kymab Limited|Targeting rare human PCSK9 variants for cholesterol treatment|

---

US8992927B1|2014-07-15|2015-03-31|Kymab Limited|Targeting human NAV1.7 variants for treatment of pain|

---

US9045545B1|2014-07-15|2015-06-02|Kymab Limited|Precision medicine by targeting PD-L1 variants for treatment of cancer|

---

US8986694B1|2014-07-15|2015-03-24|Kymab Limited|Targeting human nav1.7 variants for treatment of pain|

---

US9045548B1|2014-07-15|2015-06-02|Kymab Limited|Precision Medicine by targeting rare human PCSK9 variants for cholesterol treatment|

---

US8980273B1|2014-07-15|2015-03-17|Kymab Limited|Method of treating atopic dermatitis or asthma using antibody to IL4RA|

---

US8945560B1|2014-07-15|2015-02-03|Kymab Limited|Method of treating rheumatoid arthritis using antibody to IL6R|

---

US9023359B1|2014-07-15|2015-05-05|Kymab Limited|Targeting rare human PCSK9 variants for cholesterol treatment|

---

US9914769B2|2014-07-15|2018-03-13|Kymab Limited|Precision medicine for cholesterol treatment|

---

US9150660B1|2014-07-15|2015-10-06|Kymab Limited|Precision Medicine by targeting human NAV1.8 variants for treatment of pain|

---

US9017678B1|2014-07-15|2015-04-28|Kymab Limited|Method of treating rheumatoid arthritis using antibody to IL6R|

---

US9067998B1|2014-07-15|2015-06-30|Kymab Limited|Targeting PD-1 variants for treatment of cancer|

---

US9034332B1|2014-07-15|2015-05-19|Kymab Limited|Precision medicine by targeting rare human PCSK9 variants for cholesterol treatment|

---

US8986691B1|2014-07-15|2015-03-24|Kymab Limited|Method of treating atopic dermatitis or asthma using antibody to IL4RA|

---

US9139648B1|2014-07-15|2015-09-22|Kymab Limited|Precision medicine by targeting human NAV1.9 variants for treatment of pain|

---

MX2017000627A|2014-07-16|2017-04-27|Sanofi Biotechnology|METHODS FOR TREATING PATIENTS WITH HETEROZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA .|

---

WO2016020798A1|2014-08-06|2016-02-11|Rinat Neuroscience Corp.|Methods for reducing ldl-cholesterol|

---

WO2016020799A1|2014-08-06|2016-02-11|Rinat Neuroscience Corp.|Methods for reducing ldl-cholesterol|

---

MX2017003535A|2014-09-16|2017-10-27|Regeneron Pharma|Anti-glucagon antibodies and uses thereof.|

---

US10472424B2|2014-09-23|2019-11-12|Pfizer Inc.|Treatment with anti-PCSK9 antibodies|

---

MX2017004836A|2014-10-14|2017-07-20|Amgen Inc|Drug injection device with visual and audio indicators.|

---

US10799630B2|2014-12-19|2020-10-13|Amgen Inc.|Drug delivery device with proximity sensor|

---

EP3233159B1|2014-12-19|2020-03-04|Amgen Inc.|Drug delivery device with live button or user interface field|

---

CA3069716C|2015-02-17|2021-11-09|Amgen Inc.|Drug delivery device with vacuum assisted securement and/or feedback|

---

EP3261690B1|2015-02-27|2021-12-15|Amgen Inc.|Drug delivery device having a needle guard mechanism with a tunable threshold of resistance to needle guard movement|

---

AU2016308111A1|2015-08-18|2018-03-01|Regeneron Pharmaceuticals, Inc.|Anti-PCSK9 inhibitory antibodies for treating patients with hyperlipidemia undergoing lipoprotein apheresis|

---

WO2017039786A1|2015-09-02|2017-03-09|Amgen Inc.|Syringe assembly adapter for a syringe|

---

WO2017055966A1|2015-10-01|2017-04-06|Pfizer Inc.|Low viscosity antibody compositions|

---

US20180318526A1|2015-12-09|2018-11-08|Amgen Inc.|Auto-injector with signaling cap|

---

US11154661B2|2016-01-06|2021-10-26|Amgen Inc.|Auto-injector with signaling electronics|

---

ES2814287T3|2016-03-15|2021-03-26|Amgen Inc|Reduce the likelihood of glass breakage in drug delivery devices|

---

CN105801701B|2016-03-31|2019-03-29|河北仁博科技有限公司|The heavy chain and light chain variable region of a kind of PCSK9 antibody and its application|

---

US20190192766A1|2016-04-29|2019-06-27|Amgen Inc.|Drug Delivery Device with Messaging Label|

---

WO2017192287A1|2016-05-02|2017-11-09|Amgen Inc.|Syringe adapter and guide for filling an on-body injector|

---

GB201608323D0|2016-05-12|2016-06-29|Ucb Biopharma Sprl|Pharmaceutical compositions|

---

EP3455142A1|2016-05-13|2019-03-20|Amgen Inc.|Vial sleeve assembly|

---

WO2017200989A1|2016-05-16|2017-11-23|Amgen Inc.|Data encryption in medical devices with limited computational capability|

---

WO2017209899A1|2016-06-03|2017-12-07|Amgen Inc.|Impact testing apparatuses and methods for drug delivery devices|

---

SG11201811559WA|2016-06-27|2019-01-30|Univ California|Cancer treatment combinations|

---

US20190167908A1|2016-07-01|2019-06-06|Amgen Inc.|Drug delivery device having minimized risk of component fracture upon impact events|

---

US20190328965A1|2016-08-17|2019-10-31|Amgen Inc.|Drug delivery device with placement detection|

---

TW201825524A|2016-09-20|2018-07-16|中國大陸商上海藥明生物技術有限公司|Novel anti-PCSK9 antibody|

---

US20190248888A1|2016-10-20|2019-08-15|Regeneron Pharmaceuticals, Inc.|Methods of lowering blood glucose levels|

---

EP3532127A1|2016-10-25|2019-09-04|Amgen Inc.|On-body injector|

---

MA46758A|2016-11-14|2019-09-18|Amgen Inc|COMBINATION TREATMENTS FOR ATHEROSCLEROSIS, INCLUDING ATHEROSCLEROTIC CARDIOVASCULAR DISEASE|

---

CN108239150A|2016-12-24|2018-07-03|信达生物制药有限公司|Anti- PCSK9 antibody and application thereof|

---

MX2019008432A|2017-01-17|2019-11-18|Amgen Inc|Injection devices and related methods of use and assembly.|

---

AU2018220538A1|2017-02-17|2019-06-27|Amgen Inc.|Drug delivery device with sterile fluid flowpath and related method of assembly|

---

AU2018221351A1|2017-02-17|2019-08-22|Amgen Inc.|Insertion mechanism for drug delivery device|

---

AU2018231107A1|2017-03-06|2019-08-01|Amgen Inc.|Drug delivery device with activation prevention feature|

---

AU2018230546A1|2017-03-07|2019-08-22|Amgen Inc.|Needle insertion by overpressure|

---

JP2020509837A|2017-03-09|2020-04-02|アムジエン・インコーポレーテツド|Insertion mechanism for drug delivery device|

---

JP2020512111A|2017-03-28|2020-04-23|アムジエン・インコーポレーテツド|Plunger rod and syringe assembly system and method|

---

EP3634546A1|2017-06-08|2020-04-15|Amgen Inc.|Torque driven drug delivery device|

---

CA3061982A1|2017-06-08|2018-12-13|Amgen Inc.|Syringe assembly for a drug delivery device and method of assembly|

---

US20200206429A1|2017-06-22|2020-07-02|Amgen Inc|Device activation impact/shock reduction|

---

WO2018237225A1|2017-06-23|2018-12-27|Amgen Inc.|Electronic drug delivery device comprising a cap activated by a switch assembly|

---

EP3651832A1|2017-07-14|2020-05-20|Amgen Inc.|Needle insertion-retraction system having dual torsion spring system|

---

EP3655063A1|2017-07-21|2020-05-27|Amgen Inc.|Gas permeable sealing member for drug container and methods of assembly|

---

US20210154410A1|2017-07-25|2021-05-27|Amgen Inc.|Drug delivery device with gear module and related method of assembly|

---

MA49676A|2017-07-25|2020-06-03|Amgen Inc|DRUG ADMINISTRATION DEVICE EQUIPPED WITH A CONTAINER ACCESS SYSTEM AND ASSOCIATED ASSEMBLY PROCEDURE|

---

WO2019032482A2|2017-08-09|2019-02-14|Amgen Inc.|Hydraulic-pneumatic pressurized chamber drug delivery system|

---

WO2019036181A1|2017-08-18|2019-02-21|Amgen Inc.|Wearable injector with sterile adhesive patch|

---

US11103636B2|2017-08-22|2021-08-31|Amgen Inc.|Needle insertion mechanism for drug delivery device|

---

US20200297927A1|2017-10-04|2020-09-24|Amgen Inc.|Flow adapter for drug delivery device|

---

MA50614A|2017-10-06|2020-08-12|Amgen Inc|DRUG DELIVERY DEVICE INCLUDING A LOCKING ASSEMBLY AND ASSEMBLY METHOD|

---

WO2019074579A1|2017-10-09|2019-04-18|Amgen Inc.|Drug delivery device with drive assembly and related method of assembly|

---

WO2019090086A1|2017-11-03|2019-05-09|Amgen Inc.|Systems and approaches for sterilizing a drug delivery device|

---

MA50569A|2017-11-06|2020-09-16|Amgen Inc|FILLING-FINISHING UNITS AND ASSOCIATED PROCESSES|

---

MA50553A|2017-11-06|2020-09-16|Amgen Inc|DRUG ADMINISTRATION DEVICE WITH POSITIONING AND FLOW DETECTION|

---

EP3706826A1|2017-11-10|2020-09-16|Amgen Inc.|Plungers for drug delivery devices|

---

MA50904A|2017-11-16|2020-09-23|Amgen Inc|NEEDLE INSERTION MECHANISM FOR DRUG DELIVERY DEVICE|

---

JP2021503311A|2017-11-16|2021-02-12|アムジエン・インコーポレーテツド|Auto-injector with stall and end point detection|

---

US10835685B2|2018-05-30|2020-11-17|Amgen Inc.|Thermal spring release mechanism for a drug delivery device|

---

US11083840B2|2018-06-01|2021-08-10|Amgen Inc.|Modular fluid path assemblies for drug delivery devices|

---

CA3103682A1|2018-07-24|2020-01-30|Amgen Inc.|Delivery devices for administering drugs|

---

US20210228815A1|2018-07-24|2021-07-29|Amgen Inc.|Hybrid drug delivery devices with grip portion|

---

CA3103681A1|2018-07-24|2020-01-30|Amgen Inc.|Delivery devices for administering drugs|

---

US20210260279A1|2018-07-24|2021-08-26|Amgen Inc.|Hybrid drug delivery devices with optional grip portion and related method of preparation|

---

CA3103105A1|2018-07-31|2020-02-06|Amgen Inc.|Fluid path assembly for a drug delivery device|

---

CA3106452A1|2018-09-24|2020-04-02|Amgen Inc.|Interventional dosing systems and methods|

---

EP3856283A1|2018-09-28|2021-08-04|Amgen Inc.|Muscle wire escapement activation assembly for a drug delivery device|

---

TW202027806A|2018-10-02|2020-08-01|美商安進公司|Injection systems for drug delivery with internal force transmission|

---

AR116607A1|2018-10-05|2021-05-26|Amgen Inc|DRUG ADMINISTRATION DEVICE WITH DOSE INDICATOR|

---

TW202031306A|2018-10-15|2020-09-01|美商安進公司|Platform assembly process for drug delivery device|

---

WO2020081479A1|2018-10-15|2020-04-23|Amgen Inc.|Drug delivery device having damping mechanism|

---

TW202100191A|2018-11-01|2021-01-01|美商安進公司|Drug delivery devices with partial needle retraction|

---

US11213620B2|2018-11-01|2022-01-04|Amgen Inc.|Drug delivery devices with partial drug delivery member retraction|

---

AU2019370159A1|2018-11-01|2021-04-22|Amgen Inc.|Drug delivery devices with partial drug delivery member retraction|

---

AU2020263289A1|2019-04-24|2021-09-16|Amgen Inc.|Syringe sterilization verification assemblies and methods|

---

WO2021041067A2|2019-08-23|2021-03-04|Amgen Inc.|Drug delivery device with configurable needle shield engagement components and related methods|

---

WO2021058597A1|2019-09-24|2021-04-01|INSERM |Methods of determining whether a subject is at risk of developing arterial plaques|

法律状态:
2014-07-10| PC1| Assignment before grant (sect. 113)|Owner name: MERCK SHARP & DOHME CORP.; MSD ITALIA S.R.L. Free format text: FORMER APPLICANT(S): ISTITUTO DI RICERCHE DI BIOLOGIA MOLECOLARE S.P.A.; MERCK SHARP & DOHME CORP. |

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2015-08-06| FGA| Letters patent sealed or granted (standard patent)|

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2017-08-31| MK14| Patent ceased section 143(a) (annual fees not paid) or expired|

优先权:

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申请号 | 申请日 | 专利标题

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US6394908P| true| 2008-02-07|2008-02-07||

---

US61/063,949||2008-02-07||

---

US6657708P| true| 2008-02-21|2008-02-21||

---

US61/066,577||2008-02-21||

---

PCT/US2009/033341|WO2009100297A1|2008-02-07|2009-02-06|1d05 pcsk9 antagonists|

---