# Patent

## Title
PCSK9 immunoassay

## Abstract

Methods of using PCSK9 antagonists More specifically, methods for measuring circulating PCSK9 levels in a biological sample by means of an immunoassay The immunoassay used can be a solid phase immunoassay, such as a dissociation-enhanced lanthanide fluorescence immunoassay utilizing an E07 capture antibody or coating and a G08 or H23 detecting antibody.

## Background
BACKGROUND OF THE INVENTION
Proprotein convertase subtilisin-pexin type 9 (PCSK9), also known as neural apoptosis-regulated convertase 1 (NARC-1), is a proteinase K-like subtilase identified as the 9th member of the secretory subtilase family (Seidah, N. G., et al., 2003 PROC NATL ACAD SCI 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 found to be about 22-kb long with 12 exons that encode a 692 amino acid protein (NP—777596.2). PCSK9 is disclosed and/or claimed in several patent publications, including: 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 Publication Nos. EP 1 440 981, EP 1 067 182, and EP 1 471 152.
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, Maxwell et al. found that PCSK9 was downregulated in a similar manner to other genes involved in cholesterol biosynthesis, (Maxwell et al., 2003 J. LIPID RES. 44:2109-2119). The expression of PCSK9 was regulated by sterol regulatory element-binding proteins (SREBP), which is seen in other genes involved in cholesterol metabolism (Maxwell, et al., 2003).
Additionally, PCSK9 expression is upregulated by statins in a manner attributed to the cholesterol-lowering effects of the drugs (Dubuc et al., 2004 ARTERTOSCLER. THROMB. VASC. BIOL. 24:1454-1459). Adenoviral expression of PCSK9 has been shown to lead to a notable time-dependent increase in circulating low density lipoprotein (LDL) (Benjannet et al., 2004 J. BIOL. CHEM. 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 PROC. NATL. ACAD. SCI. USA 102:5374-5379). Medium from HepG2 cells transiently transfected with PCSK9 reduce the amount of cell surface LDLRs and internalization of LDL when transferred to untransfected HepG2 cells (Cameron et al., 2006 HUMAN MOL. GENET. 15:1551-1558). It has been further demonstrated that purified PCSK9 added to the medium of HepG2 cells had the effect of reducing the number of cell-surface LDLRs in a dose- and time-dependent manner (Lagace et al., 2006 J. CLIN. INVEST. 116:2995-3005).
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 (e.g., Abifadel et al., 2003 NATURE GENETICS 34:154-156; Timms et al., 2004 HUM. GENET. 114:349-353; Leren, 2004 CLIN. GENET. 65:419-422).
It therefore appears that PCSK9 plays 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. Significantly, lower levels of LDL cholesterol associated with sequence variations in PCSK9 confer protection against coronary heart disease (Cohen, et al., 2006 N. ENGL. J. MED. 354:1264-1272).
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.
Accordingly, it would be 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. In support of such investigations, it would be useful to have a method for measuring levels of circulating PCSK9 in a biological sample which has been exposed to a PCSK9 antagonist, such as an antibody.
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 desirable.
Additionally, it would be of use to provide kits to assay levels of circulating PCSK9 in biological samples.
SUMMARY OF THE INVENTION
The present invention relates to a method of measuring circulating PCSK9 levels in a biological sample. Said method comprises the steps of performing an immunoassay on a biological sample obtained from a subject and comparing the level of PCSK9 in said sample against a standard having a known concentration of PCSK9.
The present invention further relates to a method for identifying novel PCSK9 antagonists, comprising the steps of performing an immunoassay on a biological sample which has been contacted with a putative PCSK9 antagonist and comparing the level of PCSK9 in said sample against a standard having a known concentration of PCSK9.
A further aspect of the present invention relates to a kit for measuring circulating PCSK9 levels in a biological sample, wherein said kit comprises:
a) a biological sample collection device;
b) a composition comprising an immunoassay, comprising a coating or capture antibody and a detection antibody;
and c) a means for detecting a reaction between PCSK antigen in the sample and antibodies in the immunoassay.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a human PCSK9 DELFIA assay standard curve using the E07-G08 and E07-H23 formats.
FIG. 2 shows the DELFIA assay tolerance for human serum/plasma.
FIG. 3 gives a comparison of human plasma PCSK9 levels measured by PCSK9 DELFIA assays using both antibody pairs.
FIG. 4 illustrates a human PCSK9 MESO assay standard curve using the E07-G08 format.
FIG. 5 depicts human serum or plasma PCSK9 levels measured by PCSK9 mesoscale assays.
FIG. 6 shows that the E07 Fab is a partial inhibitor of PCSK9 function.
FIG. 7 shows that E07, G08 and H23 (Fab) do not compete with 1B20 IgG for PCSK9 binding.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of measuring circulating PCSK9 levels in a biological sample, comprising the steps of performing an immunoassay on a biological sample obtained from a subject and comparing the level of PCSK9 in said sample against a standard having a known concentration of PCSK9.
In a preferred embodiment, the immunoassay is a solid phase immunoassay. In a more preferred embodiment, the solid phase immunoassay is a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). However, it is within the scope of the current invention to use any solution-based or solid phase immunoassay as will be well familiar to those of skill in the art. Such assays include, without limitation, assays using magnetic beads as labels in lieu of enzymes, ELISAs, radioisotopes, or fluorescent moieties (fluorescent immunoassays).
The biological sample is selected from the group consisting of blood, plasma and serum.
The present invention further relates to a method for measuring PCSK9 in the presence of a putative PCSK9 antagonist. Said method comprises the steps of performing an immunoassay on a biological sample which has been contacted with a putative PCSK9 antagonist and comparing the level of PCSK9 in said sample against a standard having a known concentration of PCSK9. In a preferred embodiment, the immunoassay is a solid phase immunoassay. In a more preferred embodiment, the solid phase immunoassay is a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA).
The biological sample is selected from the group consisting of blood, plasma and serum.
The present invention additionally relates to a kit for measuring circulating PCSK9 levels in a biological sample, comprising:
a) a biological sample collection device;
b) a composition comprising an immunoassay, comprising a coating or capture antibody and a detection antibody;
and c) a means for detecting a reaction between PCSK antigen in the sample and antibodies in the immunoassay.
Kits typically but need not include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
The following examples are provided to illustrate the present invention without limiting the same hereto:
Example 1
Characterization of the PCSK9 Antagonists
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.
E07 and H23 are characterized as follows (complementarity-determining regions (CDRs) are designated in bold and underlined):








E07



Sequences of PCSK9_5_CX3_E07 Fab as expressed from Morphosys library in E. coli


Fab Vl3_3 light chain nucleotide sequence PCSK9_5_CX3_E07


(SEQ ID NO: 1)



                                                                  CDR1



GATATCGAACTGACCCAGCCGCCTTCAGTGAGCGTTGCACCAGGTCAGACCGCGCGTATCTCGTGTAGCGGCGATTCTCTTCGTGATAAGTAT


 


                                       CDR2


GTTCATTGGTACCAGCAGAAACCCGGGCAGGCGCCAGTTGTTGTGATTTATTATGATACTAATCGTCCCTCAGGCATCCCGGAACGCTTTAGC


 


                                                                           CDR3


GGATCCAACAGCGGCAACACCGCGACCCTGACCATTAGCGGCACTCAGGCGGAAGACGAAGCGGATTATTATTGCGCTGCTTATACTCGTTCT


 


ATTTATGTGTTTGGCGGCGGCACGAAGTTAACCGTTCTTGGCCAGCCGAAAGCCGCACCGAGTGTGACGCTGTTTCCGCCGAGCAGCGAAGAA


 


TTGCAGGCGAACAAAGCGACCCTGGTGTGCCTGATTAGCGACTTTTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTC


 


AAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAG


 


TCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAGGGGAGCACCGTGGAAAAAACCGTTGCGCCGACTGAGGCC


 


Fab VH3_3 heavy chain nucleotide sequence PCSK9_5_CX3_E07


(SEQ ID NO: 2)



                                                                           CDR1



CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTTCTGAT


 


                                             CDR2


CATTGGATGCATTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCTATATCGATTATTATGGTAGCAATACCCATTATGCGGAT


 


AGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTG


 


               CDR3


TATTATTGCGCGCGTATGCTTTATGGTTGGAATTATGGTGTTTTTGATTATTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCAgcgtcgacc


 


aaaggtccaagcgtgtttccgctggctccgagcagcaaaagcaccagcggcggcacggctgccctgggctgcctggttaaagattatttcccgg


 


aaccagtcaccgtgagctggaacagcggggcgctgaccagcggcgtgcatacctttccggcggtgctgcaaagcagcggcctgtatagcctga


 


gcagcgttgtgaccgtgccgagcagcagcttaggcactcagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaa


 


aagtggaaccgaaaagcgaattcgagcagaagctgatctctgaggaggatctgaacggcgcgccgcaccatcatcaccatcac


 


Fab Vl3_3 light chain amino acid sequence PCSK9_5_CX3_E07


(SEQ ID NO: 3)



                      CDR1                  CDR2                                       CDR3



DIELTQPPSVSVAPGQTARISCSGDSLRDKYVHWYQQKPGQAPVVVIYYDTNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCAAYTRS


 


IYVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK


 


SHRSYSCQVTHEGSTVEKTVAPTEA


 


Fab VH3_3 heavy chain amino acid sequence PCSK9_5_CX3_E07


(SEQ ID NO: 4)



                         CDR1                 CDR2



QVQLVESGGGLVQPGGSLRLSCAASGFTFSDHWMHWVRQAPGKGLEWVSYIDYYGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV


 


     CDR3


YYCARMLYGWNYGVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL


 


SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSEFEQKLISEEDLNGAPHHHHHH


 


Sequences of PCSK9_5_CX3_E07 IgG2M4 as expressed in Mammalian cell culture


IgG Vl3_3 light chain nucleotide sequence PCSK9_5_CX3_E07


(SEQ ID NO: 5)



                                                                  CDR1



GATATCGAACTGACCCAGCCGCCTTCAGTGAGCGTTGCACCAGGTCAGACCGCGCGTATCTCGTGTAGCGGCGATTCTCTTCGTGATAAGTAT


 


                                       CDR2


GTTCATTGGTACCAGCAGAAACCCGGGCAGGCGCCAGTTGTTGTGATTTATTATGATACTAATCGTCCCTCAGGCATCCCGGAACGCTTTAGC


                                                                           CDR3


GGATCCAACAGCGGCAACACCGCGACCCTGACCATTAGCGGCACTCAGGCGGAAGACGAAGCGGATTATTATTGCGCTGCTTATACTCGTTCT


 


ATTTATGTGTTTGGCGGCGGCACGAAGTTAACCGTTCTTGGCCAGCCCAAGGCCAACCCCACCGTGACCCTGTTCCCCCCATCTTCTGAGGAG


 


CTGCAAGCCAACAAGGCCACCCTGGTGTGCCTGATCTCTGACTTCTACCCTGGCGCTGTGACAGTGGCCTGGAAGGCTGATGGCTCTCCTGTG


 


AAGGCTGGCGTGGAGACCACCAAGCCATCTAAGCAGTCTAACAACAAGTATGCTGCCTCTTCTTACCTGTCTCTGACCCCTGAGCAGTGGAAG


 


AGCCACCGGTCTTACTCTTGCCAGGTGACCCATGAGGGCTCTACAGTGGAGAAGACAGTGGCCCCCACAGAGTGCTCT


 


IgG2M4 VH3_3 heavy chain nucleotide sequence PCSK9_5_CX3_E07


(SEQ ID NO: 6)



                                                                           CDR1



CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTTCTGAT


 


                                             CDR2


CATTGGATGCATTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCTATATCGATTATTATGGTAGCAATACCCATTATGCGGAT


 


AGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTG


 


               CDR3


TATTATTGCGCGCGTATGCTTTATGGTTGGAATTATGGTGTTTTTGATTATTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCAGCATCCACC


 


AAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC


 


GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC


 


AGCAGCGTGGTGACCGTGACCTCCAGCAACTTTGGCACGCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG


 


ACAGTTGAGCGGAAATGCTGCGTGGAGTGCCCACCATGCCCAGCACCTCCAGTGGCCGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAG


 


GACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTG


 


GATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCAC


 


CAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAACCAAAGGG


 


CAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC


 


TACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCATGCTGGACTCCGACGGCTCC


 


TTCTTCCTCTACAGCAAGCTAACCGTGGACAAGAGCAGGTGGCAGCAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC


 


CACTACACACAGAAGAGCCTCTCCCTGTCTCCTGGTAAA


 


IgG Vl3_3 light chain amino acid sequence PCSK9_5_CX3_E07


(SEQ ID NO: 7)



                      CDR1                  CDR2                                       CDR3



DIELTQPPSVSVAPGQTARISCSGDSLRDKYVHWYQQKPGQAPVVVIYYDTNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCAAYTRS


 


IYVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWK


 


SHRSYSCQVTHEGSTVEKTVAPTECS


 


IgG2m4 VH3_3 heavy chain amino acid sequence PCSK9_5_CX3_E07


(SEQ ID NO: 8)



                         CDR1                 CDR2



QVQLVESGGGLVQPGGSLRLSCAASGFTFSDHWMHWVRQAPGKGLEWVSYIDYYGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV


 


     CDR3


YYCARMLYGWNYGVFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL


 


SSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV


 


DGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF


 


YPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK


 


H23


Sequences of PCSK9_6_CX1_H23 Fab as expressed from Morphosys library in E. coli


Fab Vk3_3b light chain nucleotide sequence PCSK9_6_CX1_H23


(SEQ ID NO: 9)



                                                                     CDR1



GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGTCTGTTAATTCT


 


                                             CDR2


AATTATCTGGCTTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGTGCTTCTTCTCGTGCAACTGGGGTCCCGGCGCGT


 


                                                                                 CDR3


TTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGTGGGGT


 


GATGTTCCTATTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACGgtggctgctccgagcgtgtttatttttccgccgagcgatgaa


 


caactgaaaagcggcacggcgagcgtggtgtgcctgctgaacaacttttatccgcgtgaagcgaaagttcagtggaaagtagacaacgcgctg


 


caaagcggcaacagccaggaaagcgtgaccgaacaggatagcaaagatagcacctattctctgagcagcaccctgaccctgagcaaagcggat


 


tatgaaaaacataaagtgtatgcgtgcgaagtgacccatcaaggtctgagcagcccggtgactaaatcttttaatcgtggcgaggcc


 


Fab VH3_3 heavy chain nucleotide sequence PCSK9_6_CX1_H23


(SEQ ID NO: 10)



                                                                           CDR1



CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTTCTGAT


 


                                             CDR2


TATTATATGCATTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCAATATCTCTGGTTCTGGTAGCACTACCTATTATGCGGAT


 


AGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTG


 


               CDR3


TATTATTGCGCGCGTGGTATGTTTGATTTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCAgcgtcgaccaaaggtccaagcgtgtttccg


 


ctggctccgagcagcaaaagcaccagcggcggcacggctgccctgggctgcctggttaaagattatttcccggaaccagtcaccgtgagctgg


 


aacagcggggcgctgaccagcggcgtgcatacctttccggcggtgctgcaaagcagcggcctgtatagcctgagcagcgttgtgaccgtgccg


 


agcagcagcttaggcactcagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagcgaa


 


ttcgagcagaagctgatctctgaggaggatctgaacggcgcgccgcaccatcatcaccatcac


 


Fab Vk3_3b light chain amino acid sequence PCSK9_6_CX1_H23


(SEQ ID NO: 11)



                       CDR1                   CDR2                                       CDR3



DIVLTQSPATLSLSPGERATLSCRASQSVNSNYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWG


 


DVPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD


 


YEKHKVYACEVTHQGLSSPVTKSFNRGEA


 


Fab VH3_3 amino acid sequence PCSK9_6_CX1_H23


(SEQ ID NO: 12)



                         CDR1                 CDR2



QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMHWVRQAPGKGLEWVSNISGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY


 


    CDR3


YCARGMFDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS


 


SSLGTQTYICNVNHKPSNTKVDKKVEPKSEFEQKLISEEDLNGAPHHHHHH


 


Sequences of PCSK9_6_CX1_H23 IgG2M4 as expressed in Mammalian cell culture


IgG Vk3_3b light chain nucleotide sequence PCSK9_6_CX1_H23


(SEQ ID NO: 13)



                                                                     CDR1



GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGTCTGTTAATTCT


 


                                             CDR2


AATTATCTGGCTTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGTGCTTCTTCTCGTGCAACTGGGGTCCCGGCGCGT


 


                                                                                 CDR3


TTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGTGGGGT


 


GATGTTCCTATTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG


 


CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTC


 


CAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGAC


 


TACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT


 


IgG2M4 VH3_3 heavy chain nucleotide sequence PCSK9_6_CX1_H23


(SEQ ID NO: 14)



                                                                           CDR1



CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTTCTGAT


 


                                             CDR2


TATTATATGCATTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCAATATCTCTGGTTCTGGTAGCACTACCTATTATGCGGAT


 


AGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTG


 


               CDR3


TATTATTGCGCGCGTGGTATGTTTGATTTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCAGCATCCACCAAGGGCCCATCCGTCTTCCCC


 


CTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG


 


AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGACC


 


TCCAGCAACTTTGGCACGCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGGAAATGCTGC


 


GTGGAGTGCCCACCATGCCCAGCACCTCCAGTGGCCGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGG


 


ACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAAT


 


GCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAG


 


GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAGCCACAGGTG


 


TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTG


 


GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTA


 


ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTC


 


TCCCTGTCTCCTGGTAAA


 


IgG Vk3_3b light chain amino acid sequence PCSK9_6_CX1_H23


(SEQ ID NO: 15)



                       CDR1                   CDR2                                       CDR3



DIVLTQSPATLSLSPGERATLSCRASQSVNSNYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWG


 


DVPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD


 


YEKHKVYACEVTHQGLSSPVTKSFNRGEC


 


IgG2m4 VH3_3 heavy chain amino acid sequence PCSK9_6_CX1_H23


(SEQ ID NO: 16)



                         CDR1                 CDR2



QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMHWVRQAPGKGLEWVSNISGSGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV


 


     CDR3


YYCARGMFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVT


 


SSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN


 


AKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV


 


EWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK






Example 2
Solid Phase Immunoassay (DELFIA)
96-well plates (high-binding 4HBX plates from ThermoLabsystems, part #3855) were coated overnight at 4° with 50 μl of 10 μg/ml of anti-PCSK9 antibody (E07), the coating/capture antibody. The next day, the wells were blocked with 250 μl of blocking solution (1% BSA in TBS with 0.05% Tween-20) for 1 hour at room temperature. Plates were washed in a plate-washer with wash buffer (imidazole buffered saline with Tween 20 (KPL)). For the standard, purified human PCSK9 protein was titrated starting at 1 μg/ml. Purified human PCSK9 protein was diluted in assay buffer (1% BSA in PBS) and 100 μl of dilute protein was added on the plate as standard. Plates were incubated at 37° for 1 hour. Plates were again washed in a plate-washer with wash buffer.
Subsequently, the detection step was carried out. 1 μg/ml of biotinylated G08 or H23 was used as detecting antibody. 100 μl of 1 μg/ml biotinylated anti-PCSK9 IgG (H23 or G08) was added on the plates. After the plates were washed, 75 μl of 1:1000 Streptavidin/Europium (Perkin Elmer, part #1244-360) (diluted in assay buffer) was added. The plates were then incubated at room temperature for 20 minutes. The plates were washed again followed by the addition of 100 μl of DELFIA Enhance solution (Perkin Elmer part #1244-105) in order to enhance the fluorescence. The europium fluorescence was measured using a plate reader after one hour.
As mentioned in Example 1, all the anti-PCSK9 antibodies used are specific for human PCSK9 and were generated at Merck. The sensitivity of this assay, with either E07-G08 or E07-H23 paired format, is  ̃100 pM with a signal to noise ratio of >2.
As shown in FIG. 1, PCSK9 levels rang from 100-400 ng/ml in these samples and there is excellent agreement between the two DELFIA formats. Over 90 additional antibody pairs have been tested and none were as sensitive as these two antibody pairs (data not shown),
FIG. 2 illustrates the DELFIA human plasma assay tolerance. Here, PCSK9 levels from healthy individuals were tested in human PCSK9 DELFIA using E07-H23 and E07-G08 format. Human plasma samples were diluted 4 fold before testing. Results are mean±SD, n=3. Human plasma sample was diluted with assay buffer (1% BSA in PBS) and then assayed in PCSK9 ELISA using either E07-G08 or E07-H23 format. As shown in FIG. 2, this assay can tolerate up to 25% of human plasma.
As shown in FIG. 3, PCSK9 levels were assessed in ten human subjects and range from 100-400 ng/ml in these samples. Again, there is excellent agreement between the two DELFIA formats.
In the study depicted in FIG. 4, 400 nM of anti-PCSK9 antibody, E07, was used as coating or capture antibody. Purified human PCSK9 protein was titrated starting at 100 nM. 2 ug/ml of biotinylated G08 or H23 IgG was used as detection antibody. As shown in FIG. 4, sensitivity of this assay is greater than 25 pM.
Example 3
Solid Phase Immunoassay
This assay was carried out using an electrochemical-based immunoassay system sold by Meso Scale Discovery (MSD, Gaithersburg, Md., USA, affiliated with IGEN International, Inc.). The wells of a 96 well standard Meso Scale Discovery plate were coated overnight at 4° C. with 504 of E07 antibody solution, at a concentration of 10 μg/mL. The following day, the wells were washed three times with wash buffer, and blocked for 1 hour at room temperature with 150 μL 3% BSA in wash buffer, with shaking. Next, 50 μL of recombinant human PCSK9 standards (varying concentrations of diluted recombinant protein) were added to the wells as a standard curve. Plasma samples were then diluted 1:4 in sample diluent and added to their respective wells. The plate was allowed to incubate for 1 hour at 37° C., 100% humidity, with shaking. Following incubation, the wells were washed three times with wash buffer; and 25 μL of a 2 μg/mL 5 equimolar biotinylated-G08 or H23 antibody was added for a 60 minute incubation at room temperature, with shaking.
After decanting, wells were washed three times with wash buffer to remove the unbound G08 antibody, and 25 μL of a 2 μg/mL Streptavidin-RU solution was added to the wells for hour incubation at room temperature, with shaking. Subsequently, after decanting, the wells were washed three times with wash buffer and 150 μL of 1× Read buffer T (without surfactant) was then added to all wells. The plate was read immediately on the Sector imager and Prism, version 4, was used to fit the standard curves.
PCSK9 levels from healthy individuals were tested in human PCSK9 mesoscale assay using E07-G08 format and E07-H23 format. Plasma samples were diluted 4 fold before testing. Results are mean±SD, n=3. As shown in FIG. 5, PCSK9 levels rang from 100-400 ng/ml in these samples. There is excellent agreement between the two mesoscale formats, as well as between the DELFIA (FIG. 4) and mesoscale methods.
Example 4
Functional Analysis of E07, G08 and H23 Fab
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. 6 demonstrate that E07 Fab is a partial inhibitor of PCSK9 function. The E07 Fab displays about 50% inhibition on the effect of hPCSK9.
Example 5
E07, H23 and G08 do not Compete with Binding of a Known PCSK9 Antagonist (“1B20”)
As seen in FIG. 7, E07, G08 and H23 (Fab) do not compete with 1B20 IgG for PCSK9 binding. 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 CaCl2) 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.

## Claims

1. A method of measuring circulating Proprotein Convertase Subtilisin-Kexin 9 (PCSK9) levels in a biological sample obtained from a subject, comprising the steps of performing a solid phase immunoassay on the biological sample to determine a level of PCSK9 therein and comparing the level of PCSK9 in the biological sample against a standard having a known concentration of PCSK9, wherein the immunoassay is performed using a coating or capture antibody that is E07 and a detecting antibody that is H23; and wherein:
a) the E07 antibody comprises a light chain consisting of the amino acid sequence of SEQ ID NO: 3 or 7, and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 4 or 8; and
b) the H23 antibody comprises a light chain consisting of the amino acid sequence of SEQ ID NO: 11 or 15, and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 12 or 16.

2. The method of claim 1, wherein the solid phase immunoassay is a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA).

3. The method of claim 1, wherein the sample is selected from the group consisting of blood, plasma and serum.