Abstract:
Antagonists of human proprotein convertase subtilisin-kexin type 9 (PCSK9) are disclosed. Said antagonists are effective in the inhibition of PCSK9 function and thereby provide compositions of matter useful for the treatment of conditions associated with PCSK9 activity. The present invention further discloses nucleic acids encoding PCSK9 antagonists as well as methods of making and using PCSK9 antagonists.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    Proprotein convertase subtilisin-pexin type 9 (PCSK9), also known as neural apoptosis- regulated convertase 1 (NARC-1), is a proteinase K-like subtilase which was identified as the 9 th  member of the secretory subtilase family (Seidah, N. G., et al., 2003 P ROC  N ATL  A CAD  S CI  USA 100:928-933). PCSK9 is expressed in cells capable of proliferation and differentiation such as hepatocytes, kidney mesenchymal cells, intestinal ileum, colon epithelia and embryonic brain telencephalic neurons (Seidah et al., 2003). The gene for human PCSK9 has been sequenced and is about 22-kb long, with 12 exons that encode a 692 amino acid protein (NP — 777596.2). 
         [0002]    PCSK9 has been implicated in cholesterol homeostasis, as it appears to have a specific role in cholesterol biosynthesis or uptake. In a study of cholesterol-fed rats, it was reported that PCSK9 was downregulated in a similar manner to other genes involved in cholesterol biosynthesis, (Maxwell et al., 2003 J. L IPID  R ES.  44:2109-2119), Further, the expression of PCSK9 was regulated by sterol regulatory element-binding proteins (SREBP), as seen in other genes involved in cholesterol metabolism (Maxwell, et al., 2003). 
         [0003]    PCSK9 expression has been found to be upregulated by statins in a manner attributed to the cholesterol-lowering effects of the drugs (Dubuc et al., 2004 A RTERIOSCLER . T HROMB . V ASC . B IOL.  24:1454-1459). Adenoviral expression of PCSK9 results in a time-dependent increase in circulating low density lipoprotein (LDL) (Benjannet et al., 2004 J. B IOL . C HEM.  279:48865-48875), and mice with PCSK9 gene deletions have increased levels of hepatic LDL receptors (LDLR) and clear LDL from the plasma more rapidly (Rashid et al., 2005 P ROC . N ATL . A CAD . S CI . USA 102:5374-5379). Medium from HepG2 cells which are transiently transfected with PCSK9 is found to reduce the amount of cell surface LDLRs and internalization of LDL when transferred to untransfected HepG2 cells (Cameron et al., 2006 H UMAN  M OL . G ENET.  15:1551-1558). Additionally, purified PCSK9 added to the medium of HepG2 cells reduced the number of cell-surface LDLRs in a dose- and time-dependent manner (Lagace et al., 2006 J. C LIN . I NVEST.  116:2995-3005), 
         [0004]    A number of mutations in the gene PCSK9 have been associated with autosomal dominant hypercholesterolemia (ADH), an inherited metabolism disorder which is characterized by marked elevations of LDL particles in the plasma that can lead to premature cardiovascular failure (e.g., Abifadel et al., 2003 N ATURE  G ENETICS  34:154-156; Timms et al., 2004 H UM . G ENET.  114:349-353; Leren, 2004 C LIN . G ENET.  65:419-422). 
         [0005]    PCSK9 therefore appears to play a role in the regulation of LDL production. Expression or upregulation of PCSK9 is associated with increased plasma levels of LDL cholesterol, and inhibition or the lack of expression of PCSK9 is associated with low LDL cholesterol plasma levels and lower levels of LDL cholesterol associated with sequence variations in PCSK9 confer protection against coronary heart disease (Cohen, et al., 2006 N. E NGL . J. M ED.  354:1264-1272). This is of significance as clinical trial data has demonstrated that reductions in LDL cholesterol levels are related to the rate of coronary events (Law et al., 2003 BMJ 326:1423-1427). Moderate lifelong reduction in plasma LDL cholesterol levels has been shown to be substantially correlated with a substantial reduction in the incidence of coronary events (Cohen et al., 2006), even in populations with a high prevalence of non-lipid-related cardiovascular risk factors. Accordingly, there is great benefit to be reaped from the managed control of LDL cholesterol levels. 
         [0006]    It is therefore desirable to further investigate PCSK9 as a target for the treatment of cardiovascular disease. Antibodies useful as PCSK9 antagonists have been identified and have utility as therapeutic agents. 
         [0007]    It would be further desirable to be able to identify novel PCSK9 antagonists in order to assist in the quest for compounds and/or agents effective in the treatment of cardiovascular disease. Hence, a method for measuring levels of circulating PCSK9 in a biological sample for such purposes as, e.g., assessing the effectiveness of a putative PCSK9 antagonist is of utility. 
       SUMMARY OF THE INVENTION 
       [0008]    The present invention relates to PCSK9-specific antagonists that antagonize PCSK9&#39;s inhibition of cellular LDL uptake, wherein said antagonists comprise a monoclonal antibody comprising a light chain polypeptide having the amino acid sequence of SEQ ID NO: 3 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 4. 
         [0009]    The present invention relates to PCSK9-specific antagonists that antagonize PCSK9&#39;s inhibition of cellular LDL uptake, wherein said antagonists comprise a monoclonal antibody comprising a light chain polypeptide having the amino acid sequence of SEQ ID NO: 7 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 8. 
         [0010]    The present invention further relates to a composition comprising a PCSK9-specific antagonist such as described supra and a pharmaceutically acceptable carrier. 
         [0011]    The present invention also provides a method for antagonizing PCSK9 function which comprises the step of administering a PCSK9-specific antagonist to cells, tissues, or human or animal subjects. 
         [0012]    The present invention further furnishes a use of a PCSK9-specific antagonist in the manufacture of a medicament for ameliorating a disorder, condition or disease caused and/or exacerbated by PCSK9 function. 
         [0013]    The present invention provides isolated nucleic acids coding for the heavy and light chain polypeptides of SEQ ID NOs: 1, 2, 5 and 6. 
         [0014]    The present invention also provides vectors comprising isolated nucleic acids coding for the heavy and light chain polypeptides of SEQ ID NOs: 1, 2, 5 and 6 as well as host cells comprising said vectors. 
         [0015]    The present invention also furnishes a method for producing a PCSK9-specific antagonist which comprises: (a) culturing a population of cells comprising host cells comprising vectors having isolated nucleic acids coding for the heavy and light chain polypeptides of SEQ ID NOs: 3 and 4 or 5 and 6 under conditions appropriate for production of the PCSK9-specific antagonist; and 
         [0016]    (b) isolating the PCSK9-specific antagonist produced. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]      FIG. 1  shows that the E07 Fab is a partial inhibitor of PCSK9 function. 
           [0018]      FIG. 2  shows that E07, G08 and H23 (Fab) do not compete with 1B20 IgG for PCSK9 binding. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0019]    The present invention provides antagonists of PCSK-9 function which are monoclonal antibodies. In a preferred embodiment, there is provided an antagonist of PCSK-9 function which comprises a light chain polypeptide comprising CDR1, CDR2 and CDR3 of SEQ ID NO: 3 and a heavy chain polypeptide comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 4. 
         [0020]    The present invention further furnishes a use of a PCSK9-specific antagonist in the manufacture of a medicament for ameliorating a disorder, condition or disease caused and/or exacerbated by PCSK9 function. The utility of these 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 &amp; Webb, 1981  J. Cell Biol.  90:595-604, and Stephan &amp; Yurachek, 1993  J. Lipid Res.  34:325330). In addition, means for measuring LDL cholesterol in plasma is well described in the literature (see, e.g., McNamara et al., 2006  Clinica Chimica Acta  369:158-167). 
         [0021]    The present invention provides isolated nucleic acids coding for the heavy and light chain polypeptides of SEQ ID NOs: 1, 2, 5 and 6. 
         [0022]    The present invention also provides vectors comprising isolated nucleic acids coding for the heavy and light chain polypeptides of SEQ ID NOs: 1, 2, 5 and 6 as well as host cells comprising said vectors. 
         [0023]    The present invention also furnishes a method for producing a PCSK9-specific antagonist which comprises: (a) culturing a population of cells comprising host cells comprising vectors having isolated nucleic acids coding for the heavy and light chain polypeptides of SEQ ID NOs: 3 and 4 or 7 and 8 under conditions appropriate for production of the PCSK9-specific antagonist; and 
         [0024]    (c) isolating the PCSK9-specific antagonist produced. 
         [0025]    In another aspect, the present invention provides a method for identifying, isolating, quantifying or antagonizing PCSK9 in a sample of interest using one or more PCSK9-specific antagonists of the present invention. The PCSK9-specific antagonists may be utilized as research tools in immunochemical assays, such as Western blots, ELISAs, radioimmunoassay, 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 (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. 
         [0026]    As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The term “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., (1975)  Nature,  256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). 
         [0027]    Use of the term “antagonist” herein refers to the fact that the subject molecule can antagonize the functioning of PCSK9. Use of the term “antagonizing” herein refers to the act of opposing, counteracting, 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 driven by, requires, or is exacerbated or enhanced by PCSK9. 
         [0028]    As used herein, the term “isolated” describes a property as it pertains 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 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). 
         [0029]    The CDR definitions arrived at and disclosed herein were defined using the Morphosys software program Sequence Analysis Software (SAS). Various other methods are available, however, to delineate and define start and end points of CDR sequences, e.g., most conspicuously, Kabat, E. A., Wu, T. T., Perry, H., Gottesman, K. and Foeller, C. (1991)  Sequences of Proteins of Immunological Interest , Fifth Edition. NIH Publication No. 91-3242. While the current inventors have used the SAS software to define the CDRs, the present invention encompasses different definitions around the sequences and any varying CDR delineations obtained thereby. 
         [0030]    PCSK-9 specific antagonists also have utility for various diagnostic purposes in the detection and quantification of PCSK9. 
         [0031]    The following examples are provided to illustrate the present invention without limiting the same hereto: 
       EXAMPLE 1 
       [0032]    Characterization of the PCSK9 Antagonists 
         [0033]    The PCSK9 antagonists used in this assay were antibodies E07, G08 and H23. G08 is disclosed in WO2008057459, which is incorporated in its entirety herein. 
         [0034]    The sequences of E07 and H23 are as follows: 
         [0000]    
       
         
               
               
             
           
               
                 E07 
                   
               
               
                 IgG Vl3_3 light chain nucleotide sequence PCSK9_5_CX3_E07 
               
               
                 (SEQ ID NO: 1) 
                   
               
               
                 (underlined residues are CDRs) 
                   
               
               
                 GATATCGAACTGACCCAGCCGCCTTCAGTGAGCGTTGCACCAGGTCAGACCGCGCG 
               
               
                   
               
               
                 TATCTCGTGT   AGCGGCGATTCTCTTCGTGATAAGTATGTTCAT   TGGTACCAGCAG 
               
               
                   
               
               
                 AAACCCGGGCAGGCGCCAGTT   GTTGTGATTTATTATGATACTAATCGTCCCTCA   G 
               
               
                   
               
               
                 GCATCCCGGAACGCTTTAGCGGATCCAACAGCGGCAACACCGCGACCCTGACCATT 
               
               
                   
               
               
                 AGCGGCACTCAGGCGGAAGACGAAGCGGATTATTATTGC   GCTGCTTATACTCGTTC     
               
               
                   
               
               
                     TATTTAT   GTGTTTGGCGGCGGCACGAAGTTAACCGTTCTTGGCCAGCCCAAGGCCA 
               
               
                   
               
               
                 ACCCCACCGTGACCCTGTTCCCCCCATCTTCTGAGGAGCTGCAAGCCAACAAGGCCA 
               
               
                   
               
               
                 CCCTGGTGTGCCTGATCTCTGACTTCTACCCTGGCGCTGTGACAGTGGCCTGGAAGG 
               
               
                   
               
               
                 CTGATGGCTCTCCTGTGAAGGCTGGCGTGGAGACCACCAAGCCATCTAAGCAGTCTA 
               
               
                   
               
               
                 ACAACAAGTATGCTGCCTCTTCTTACCTGTCTCTGACCCCTGAGCAGTGGAAGAGCC 
               
               
                   
               
               
                 ACCGGTCTTACTCTTGCCAGGTGACCCATGAGGGCTCTACAGTGGAGAAGACAGTG 
               
               
                   
               
               
                 GCCCCCACAGAGTGCTCT 
               
               
                   
               
               
                 IgG2M4 VH3_3 heavy chain nucleotide sequence PCSK9_5_CX3_E07 
               
               
                 (SEQ ID NO: 2) 
                   
               
               
                 (underlined residues are CDRs) 
                   
               
               
                 CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCG 
               
               
                   
               
               
                 TCTGAGCTGCGCGGCCTCC   GGATTTACCTTTTCTGATCATTGGATGCAT   TGGGTGC 
               
               
                   
               
               
                 GCCAAGCCCCTGGGAAGGGTCTCGAG   TGGGTGAGCTATATCGATTATTATGGTAG     
               
               
                   
               
               
                     CAATACCCATTATGCGGATAGCGTGAAAGGC   CGTTTTACCATTTCACGTGATAATT 
               
               
                   
               
               
                 CGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTG 
               
               
                   
               
               
                 TATTATTGCGCGCGT   ATGCTTTATGGTTGGAATTATGGTGTTTTTGATTAT   TGGGG 
               
               
                   
               
               
                 CCAAGGCACCCTGGTGACGGTTAGCTCAGCATCCACCAAGGGCCCATCCGTCTTCCC 
               
               
                   
               
               
                 CCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGT 
               
               
                   
               
               
                 CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA 
               
               
                   
               
               
                 GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA 
               
               
                   
               
               
                 GCGTGGTGACCGTGACCTCCAGCAACTTTGGCACGCAGACCTACACCTGCAACGTA 
               
               
                   
               
               
                 GATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGGAAATGCTGCGT 
               
               
                   
               
               
                 GGAGTGCCCACCATGCCCAGCACCTCCAGTGGCCGGACCATCAGTCTTCCTGTTCCC 
               
               
                   
               
               
                 CCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGT 
               
               
                   
               
               
                 GGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCG 
               
               
                   
               
               
                 TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTTC 
               
               
                   
               
               
                 CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTA 
               
               
                   
               
               
                 CAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCA 
               
               
                   
               
               
                 AAACCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCGGGAG 
               
               
                   
               
               
                 GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAG 
               
               
                   
               
               
                 CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC 
               
               
                   
               
               
                 ACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTAACCGTG 
               
               
                   
               
               
                 GACAAGAGCAGGTGGCAGCAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGC 
               
               
                   
               
               
                 TCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCTGGTAAA 
               
               
                   
               
               
                 IgG Vl3_3 light chain amino acid sequence PCSK9_5_CX3_E07 
               
               
                 (SEQ ID NO: 3) 
                   
               
               
                 (underlined residues are CDRs) 
                   
               
               
                 DIELTQPPSVSVAPGQTARISC   SGDSLRDKYVH   WYQQKPGQAPV   VVIYYDTNRPS   GIPE 
               
               
                   
               
               
                 RFSGSNSGNTATLTISGTQAEDEADYYC   AAYTRSIY   VFGGGTKLTVLGQPKANPTVTLFP 
               
               
                   
               
               
                 PSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYL 
               
               
                   
               
               
                 SLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 
               
               
                   
               
               
                 IgG2m4 VH3_3 heavy chain amino acid sequence PCSK9_5_CX3_E07 
               
               
                 (SEQ ID NO: 4) 
                   
               
               
                 (underlined residues are CDRs) 
                   
               
               
                 QVQLVESGGGLVQPGGSLRLSCAAS   GFTFSDHWM   HWVRQAPGKGLE   W   V   SYIDYYGS     
               
               
                   
               
               
                     NTHYADSVKG   RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR   MLYGWNYGVFDY   WG 
               
               
                   
               
               
                 QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH 
               
               
                   
               
               
                 TFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP 
               
               
                   
               
               
                 APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQPNWYVDGVEVHNAKTK 
               
               
                   
               
               
                 PREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYT 
               
               
                   
               
               
                 LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKL 
               
               
                   
               
               
                 TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 H23 
               
               
                 IgG Vk3_3b light chain nucleotide sequence PCSK9_6_CX1_H23 
               
               
                 (SEQ ID NO: 5) 
                   
               
               
                 (underlined residues are CDRs) 
                   
               
               
                 GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC 
               
               
                   
               
               
                 GACCCTGAGCTGC   AGAGCGAGCCAGTCTGTTAATTCTAATTATCTGGCT   TGGTAC 
               
               
                   
               
               
                 CAGCAGAAACCAGGTCAAGCACCGCGT   CTATTAATTTATGGTGCTTCTTCTCGTG     
               
               
                   
               
               
                     CAACT   GGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGA 
               
               
                   
               
               
                 CCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGC   CAGCAGTGGGGT     
               
               
                   
               
               
                     GATGTTCCTATT   ACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACGGTGGC 
               
               
                   
               
               
                 TGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC 
               
               
                   
               
               
                 CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAA 
               
               
                   
               
               
                 GGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACA 
               
               
                   
               
               
                 GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTAC 
               
               
                   
               
               
                 GAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGT 
               
               
                   
               
               
                 CACAAAGAGCTTCAACAGGGGAGAGTGT 
               
               
                   
               
               
                 IgG2M4 VH3_3 heavy chain nucleotide sequence PCSK9_6_CX1_H23 
               
               
                 (SEQ ID NO: 6) 
                   
               
               
                 (underlined residues are CDRs) 
                   
               
               
                 CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCG 
               
               
                   
               
               
                 TCTGAGCTGCGCGGCCTCC   GGATTTACCTTTTCTGATTATTATATGCAT   TGGGTGC 
               
               
                   
               
               
                 GCCAAGCCCCTGGGAAGGGTCTCGAG   TGGGTGAGCAATATCTCTGGTTCTGGTAG     
               
               
                   
               
               
                     CACTACCTATTATGCGGATAGCGTGAAAGGC   CGTTTTACCATTTCACGTGATAATT 
               
               
                   
               
               
                 CGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTG 
               
               
                   
               
               
                 TATTATTGCGCGCGT   GGTATGTTTGATTTT   TGGGGCCAAGGCACCCTGGTGACGGT 
               
               
                   
               
               
                 TAGCTCAGCATCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAG 
               
               
                   
               
               
                 CACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC 
               
               
                   
               
               
                 GGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG 
               
               
                   
               
               
                 CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGACCTCCA 
               
               
                   
               
               
                 GCAACTTTGGCACGCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACC 
               
               
                   
               
               
                 AAGGTGGACAAGACAGTTGAGCGGAAATGCTGCGTGGAGTGCCCACCATGCCCAGC 
               
               
                   
               
               
                 ACCTCCAGTGGCCGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCT 
               
               
                   
               
               
                 CATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAG 
               
               
                   
               
               
                 ACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAG 
               
               
                   
               
               
                 ACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCAC 
               
               
                   
               
               
                 CGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACA 
               
               
                   
               
               
                 AAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGA 
               
               
                   
               
               
                 GAGCCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGT 
               
               
                   
               
               
                 CAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGG 
               
               
                   
               
               
                 AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCATGCTGGACTCC 
               
               
                   
               
               
                 GACGGCTCCTTCTTCCTCTACAGCAAGCTAACCGTGGACAAGAGCAGGTGGCAGCA 
               
               
                   
               
               
                 GGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACA 
               
               
                   
               
               
                 GAAGAGCCTCTCCCTGTCTCCTGGTAAA 
               
               
                   
               
               
                 IgG Vk3_3b light chain amino acid sequence PCSK9_6_CX1_H23 
               
               
                 (SEQ ID NO: 7) 
                   
               
               
                 (underlined residues are CDRs) 
                   
               
               
                 DIVLTQSPATLSLSPGERATLSC   RASQSVNSNYLA   WYQQKPGQAPR   LLIYGASSRAT   GV 
               
               
                   
               
               
                 PARFSGSGSGTDFTLTISSLEPEDFAVYYC   QQWGDYPI   TFGQGTKVEIKRTVAAPSVFIFP 
               
               
                   
               
               
                 PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST 
               
               
                   
               
               
                 LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 IgG2m4 VH3_3 heavy chain amino acid sequence PCSK9_6_CX1_H23 
               
               
                 (SEQ ID NO: 8) 
                   
               
               
                 (underlined residues are CDRs) 
                   
               
               
                 QVQLVESGGGLVQPGGSLRLSCAAS   GFTFSDYYMH   WVRQAPGKGLE   WVSNISGSGST     
               
               
                   
               
               
                     TYYADSVKG   RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR   GMFDF   WGQGTLVTVSS 
               
               
                   
               
               
                 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS 
               
               
                   
               
               
                 GLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV 
               
               
                   
               
               
                 FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST 
               
               
                   
               
               
                 FRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMT 
               
               
                   
               
               
                 KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ 
               
               
                   
               
               
                 QGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
             
          
         
       
     
       EXAMPLE 2   
       [0035]    Production of the 3E07 and 1 H23 Antibodies 
         [0036]    DNA encoding the heavy chain variable regions were fused in-frame with DNA encoding the IgG2M4 constant region whereas DNA encoding the light chain variable regions were fused in-frame with DNA encoding either lambda or kappa light chain constant region in alignment with the corresponding variable regions. The cloning procedure is described below. The light chain vector comprises cloning sites flanked by a human CMV (HCMV) promoter and leader sequence on the 5′ end of one cloning site and the light chain constant region sequences and bovine growth hormone (BGH) pA polyadenylation signal on the 3′ side of the other cloning site. The heavy chain IgG2M4 constant region vector comprises cloning sites flanked by an HCMV promoter and leader sequence on the 5′ end of one cloning site and heavy chain IgG2M4 sequences and BGH pA polyadenylation signal on the 3′ side of the other cloning site. The expression vectors carry oriP from Epstein Barr virus (EBV) viral genome for prolonged expression in 293EBNA cells and the bacterial sequences for kanamycin selection marker and replication origin in  E. coli . The leader sequence at the amino termini of the antibodies mediated the secretion of the expressed antibodies into the culture medium. The leader sequence for heavy chain is MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 9) and light chain: MSVPTQVLGLLLLWLTDARC (SEQ ID NO:10). 
         [0037]    The respective variable regions were PCR amplified in a volume of 25 containing high fidelity PCR master mix, template volume 1 μL and forward and reverse primers: 1μL each. PCR conditions were one cycle of 94° C. for two minutes, 25 cycles of 94° C. for 1.5 minutes, 60° C. for 1.5 minutes and 72° C. for 1.5 minutes with a final extension at 72° C. for 7 minutes. The amplified light and heavy chain variable region PCR products were cloned in-frame with the appropriate leader sequence at the 5′-end and constant region at the 3′-end using In-Fusion strategy (Clontech, Palo Alto, Calif.) and cloned into  E. coil  XL10 cells from Stratagene, La Jolla, Calif.). The DNA sequences for the clones were confirmed by sequencing and the amino acid sequences were deduced from the DNA sequences. 
         [0038]    The above plasmids were transfected into 293EBNA monolayer cells using FUGENE transfection reagents (FUGENE is a trademark of Fugent LLC and is available from Roche Diagnostics, Nutley, N.J.). The transfected cells were incubated in OPTI-MEM serum free medium (Invitrogen) and the secreted antibodies were purified from the culture medium using protein A/G affinity chromatography. The concentration of purified antibodies was determined by OD at 280 nm and the purity by LABCHIP capillary SDS gel electrophoresis (Caliper Life Sciences, Hopkinton, Mass.). The antibodies purified were used for characterization described elsewhere. 
         [0039]    EXAMPLE 3 
         [0040]    Functional Analysis of E07, G08 and H23 Fab 
         [0041]    30,000 HEK293 cells/well were seeded in normal serum conditions and 24 hours later, media was changed to one lacking serum. 24 hours after that, LDL uptake was measured. 3E07 Fab was titrated with 5 ug/ml of hPCSK9 purified protein, starting at 100 ug/ml. The data in  FIG. 1  demonstrate that E07 Fab is a partial inhibitor of PCSK9 function. The E07 Fab displays about 50% inhibition on the effect of hPCSK9. 
         [0042]    EXAMPLE 4 
         [0043]    E07, H23 and G08 Do Not Compete with Binding of a Known PCSK9 Antagonist (“1B20”) 
         [0044]    As seen in  FIGS. 2 , E07, G08 and H23 (Fab) do not compete with 1B20 IgG for 
         [0045]    PCSK9 binding. The 1B20 antagonist is covered in U.S. Provisional Application No. 61/063,980 and related applications, and incorporated in its entirety herein. For this experiment, 4 nM (final concentration) of Eu-1G08 Fab was mixed with 32 nM (final concentration) of AF647-PCSK9 and various concentrations (from 1 μM to 50 pM) of unlabeled 1B20, G08, E07 and H23 Fab in 50 μl of assay buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% BSA, 100 μM CaCl 2 ) in a black U-Bottom shaped pigmented styrene 96-well microtiter plate (Dynatech). The mixtures were incubated at room temperature for 3 hours and plate was read on a Ruby Star fluorescent reader (available from BMG Technologies, Inc.) at Ex 370 mm. Signals were recorded at both 620 mm and 665 mm. The 665 mm/620 mm ratio was used to calculate the results. The experiments were performed in triplicate and repeated 3 times. The background of the assay is ˜2340 RFU.