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Anti-human PCSK9 monoclonal antibody

An antibody and antigen technology, applied in the direction of antibodies, anti-enzyme immunoglobulins, biochemical equipment and methods, etc., can solve the problem of lack of anti-human PCSK9 fully human antibodies

Active Publication Date: 2016-11-09
BEIJING MABWORKS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is still a lack of anti-human PCSK9 fully human antibodies with higher affinity in the field

Method used

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  • Anti-human PCSK9 monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Example 1: Biopanning of anti-huPCSK9 single chain antibody

[0155] The immunotube was coated with huPCSK9-his (NP_777596.2) as the antigen, the antigen coating amount was 5 μg / tube, and the coating was performed overnight at 4°C. The immunotube and the naive phage antibody library (prepared by the laboratory) were respectively blocked with 4% skim milk powder / PBST, and blocked for 1 hour at 37°C. The blocked phage antibody library was added to the immune tube for antibody antigen binding, and the amount of phage input was about 10 12 , 37°C for 1h. Unbound phages were washed away with PBS (T), antibodies were eluted with 0.1M HCL-Glycine, and eluted phages were neutralized with 1.5M Tris-HCL (pH8.8).

[0156] Take 550 μl of the neutralized phage to infect about 10 mL of the TG1 bacterial solution that has grown to the logarithmic growth phase, and let it stand at 37°C for 30 minutes. Add 9 mL of 2YT medium containing ampicillin. Take out an appropriate amount of...

Embodiment 2

[0157] Example 2: Screening of anti-huPCSK9 single-chain antibody positive clones

[0158] Well-separated monoclonal colonies produced after three rounds of selection were picked, inoculated into 1 mL of 2YTAG (Ampicilline: 100 μg / ml, Glucose: 2%) medium, and cultured overnight at 37° C. and 220 rpm. The next day, transfer to a new 96-well deep-well plate, culture to its logarithmic growth phase, and add about 10 10 Helper phage VCSM13. After static infection at 37°C for 30 minutes, centrifuge at 4000 rpm at 4°C for 15 minutes, discard the supernatant, resuspend the pellet with 2YTAK (Ampicilline: 100 μg / ml, kanamycin: 70 μg / ml), culture at 28°C, 220 rpm overnight. Aspirate the amplified phage supernatant for ELISA identification (see figure 1 ). The obtained positive clones were sequenced, and a total of 4 different antibody sequences were obtained.

Embodiment 3

[0159] Example 3: Phage Determination of the affinity of anti-huPCSK9 scFv by ELISA

[0160] The clones obtained in Example 2 were subjected to display and purification of monoclonal phage, and a phage gradient dilution ELISA experiment was performed to identify the affinity of phage-abs.

[0161] huPCSK9 was coated with pH 7.4 phosphate buffer, overnight at 4°C. Wash three times with PBST, block with 4% milk-PBST at 37°C for 1h. The purified monoclonal phage was diluted proportionally in 4% skimmed milk powder, 100 μl of the diluted sample was added to each well, and stood at RT for 1 h. The ELISA plate was washed with PBST, and the HRP-anti-M13 monoclonal antibody diluted with 4% skim milk powder was added to the ELISA plate, and left at room temperature for 1 hour. TMB color development kit was used for color development at room temperature for 5 minutes. with 10%H 2 SO 4 Stop color development, 50 μl / well. The optical density value was measured with a single wave...

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Abstract

The invention belongs to the field of antibodies and particularly relates to an anti-human PCSK9 monoclonal antibody and application of the antibody in preparation of drugs for reducing lipoprotein level in blood and preventing or treating cardiovascular diseases or disorders and thrombosis-obstructive diseases or disorders. The anti-human PCSK9 monoclonal antibody has higher affinity and a good application prospect.

Description

technical field [0001] The invention belongs to the field of antibodies, and in particular relates to anti-human PCSK9 monoclonal antibodies and the use of the antibodies for preparing drugs for reducing blood lipoprotein levels, preventing or treating cardiovascular diseases or disorders, and thrombo-occlusive diseases or disorders. Background technique [0002] Proprotein Convertase Subtilisin / kexin type 9 (Proprotein Convertase Subtilisin / Kexin type 9, PCSK9) is a unique proprotein convertase belonging to the serine proteinase K subclass. The proprotein convertase (PCSK9) gene is located on human chromosome 1p32.3, with a total length of 29 kb, consisting of 12 exons, a coding region of about 2 kb, and encoding a protein comprising 692 amino acid residues. PCSK9 is mainly composed of signal peptide, prodomain, catalytic domain, and carboxy-terminal domain. PCSK9 precursor protein synthesizes a soluble zymogen in the endoplasmic reticulum, that is, PCSK9 zymogen (apo-PCSK...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13G01N33/577G01N33/573A61K39/395A61P7/02A61P9/10A61P3/06
Inventor 刘方杰耿树生李锋
Owner BEIJING MABWORKS BIOTECH
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