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Antagonists of pcsk9

An antagonist, antagonism technology, applied in the direction of antibodies, anti-enzyme immunoglobulins, metabolic diseases, etc.

Inactive Publication Date: 2010-01-27
SCHERING AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it would be beneficial to manage LDL cholesterol levels

Method used

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  • Antagonists of pcsk9
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  • Antagonists of pcsk9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Isolation of recombinant Fab displaying phage

[0114] A recombinant Fab phage display library was panned against immobilized recombinant human PCSK9 (see, for example, Knappik et al., 2000 J. Mol. Biol. 296:57-86), and the method used was briefly described as follows: first, the phage Fab display library was divided into three pools ( pool): One pool is VH2+VH4+VH5, another pool is VH1+VH6, and the third pool is VH3. Phage libraries and immobilized PCSK9 protein were blocked with nonfat dry milk.

[0115] For the first round of panning, each phage pool was independently bound to V5-, histidine-tagged PCSK9 protein in wells immobilized on Nunc Maxisorp plates. The immobilized phage-PCSK9 complex was sequentially washed with the following reagents: (1) PBS / 0.5% Tween TM 20 (three quick washes); (2) PBS / 0.5% Tween TM 20 (one 5 minute incubation with moderate shaking); (3) PBS (three quick washes); and (4) PBS (two 5 minute incubations with moderate shaking). Bound pha...

Embodiment 2

[0120] ELISA screening of bacterially expressed FABS

[0121] Cultures of each transformant were induced with IPTG and grown overnight to express the Fab. Culture supernatants (candidate Fabs) were incubated with purified V5-, histidine-tagged PCSK9 protein immobilized in wells of a 96-well Nunc Maxisorp plate, prepared in PBS with a plate washer 0.1% of Tween20 TM Wash, incubate with HRP-conjugated anti-Fab antibody, again with PBS / Tween20 TM washing. Bound HRP was detected by adding TMP substrate, and the A of each well was read with a plate reader. 450 value.

[0122] Include the following negative controls:

[0123] A control for non-specific Fab binding on each plate was incubated with a parallel expressed anti-EsB (an unrelated Fab) preparation.

[0124] growth medium only.

[0125] Include positive controls for ELISA and Fab expression as follows;

[0126] EsB antigen was allowed to bind to three wells of the plate and then incubated with anti-EsB Fab. For a co...

Embodiment 3

[0128] DNA Sequencing of PCSK9 ELISA-Positive FAB Clones

[0129] Bacterial cultures for DNA preparation were obtained by inoculating 1.2 ml of 2xYT broth containing chloramphenicol in a master glycerol stock of positive Fabs and growing overnight. DNA was prepared from cell pellets obtained by centrifugation from overnight cultures using a Qiagen Turbo Mini prep procedure performed on a BioRobot 9600. ABI dye terminator (DyeTerminator) cycle sequencing was performed on the DNA on the ABI 3100 Genetic Analyzer (Genetic Analyzer) with the sequencing primers determined by Morphosys to obtain the DNA sequence of the Fab clone. The DNA sequences were compared to each other to determine unique clone sequences and to determine the light and heavy chain subtypes of the Fab clones.

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Abstract

Antagonists of human proprotein convertase subtilisin-kexin type 9 ('PCSK9') are disclosed. The disclosed antagonists are effective in the inhibition of PCSK9 function and, accordingly, present desirable antagonists for the use in the treatment of conditions associated with PCSK9 activity. The present invention also discloses nucleic acid encoding said antagonists, vectors, host cells, and compositions comprising the antagonists. Methods of making PCSK9-specific antagonists as well as methods of using the antagonists for inhibiting or antagonizing PCSK9 function are also disclosed and form important additional aspects of the present disclosure.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Applications 60 / 857,293 and 60 / 857,248, filed November 7,2006. [0003] Statement Regarding Federally Funded Research and Development [0004] Not applicable. [0005] Reference to Microfiche Addendum [0006] Not applicable. Background of the invention [0007] Proprotein convertase subtilisin-kexin type 9 (hereinafter referred to as "PCSK9"), also known as neuroapoptosis-regulated converting enzyme 1 ("NARC-1"), is a Proteinase K-like subtilase, identified as the ninth member of the secreted subtilase family; see Seidah et al., 2003 PNAS 100:928-933. The gene encoding PCSK9 is mapped to human chromosome 1p33-p34.3; Seidah et al., supra. PCSK9 is expressed in cells capable of proliferation and differentiation including, for example, hepatocytes, renal mesenchymal cells, intestinal ileum and colon epithelium, and embryonic telencephalic neurons; Seidah et al., su...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/40
CPCC07K2317/92C07K16/40C07K2317/55C07K2317/56C07K2316/96A61P3/00A61P3/04A61P3/06A61P7/00A61P9/00A61P9/10A61P9/12A61P13/12A61P43/00C07K2317/76
Inventor A·西特拉尼C·P·斯帕罗S·潘迪特J·H·康德拉H·A·哈蒙
Owner SCHERING AG
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