Anti-PCSK9 monoclonal antibody

A monoclonal antibody, antibody technology, applied in the direction of antibodies, antibody medical components, anti-enzyme immunoglobulins, etc., can solve the problem of lack of fully human antibodies and achieve good functions

Active Publication Date: 2017-05-31
BEIJING DONGFANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is still a lack of self-developed fully human antibodies against PCSK9 with high affinity in this field in China

Method used

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  • Anti-PCSK9 monoclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Embodiment 1, biopanning of anti-PCSK9 single chain antibody

[0069] The method of gene cloning was used to transform the pCom3 vector, and the transformed vector was named pScFvDisb-S1 ( figure 1 ). A fully synthetic phage antibody library was constructed based on this vector.

[0070] Antigen PCSK9-His 10μg / 1ml / tube was coated in the immunotube, and coated overnight at 4°C. Block the immune tube and the phage antibody library with PBST-4% milk (the input amount of phage is about 10 9 -10 12 ), blocked at 37°C for 1h. The blocked phage antibody library was added to the immunotube for antigen-antibody binding, and reacted at 37°C for 1 hour. Unbound phages were washed away with PBST-PBS, eluted with Glycine-HCl of 0.1M pH2.2, and the eluate was neutralized to about pH7.0 with 1.5M Tris-HCl of pH8.8. The eluate was used to infect 10ml of XL1-Blue bacterial solution grown to an OD value of about 0.5-0.8, and then allowed to stand at 37°C for 30 minutes and then sha...

Embodiment 2

[0071] Example 2, Screening of Anti-PCSK9 Single Chain Antibody Positive Clones

[0072] After three rounds of screening, well-separated monoclonal colonies were picked and inoculated in a 96-well deep-well plate supplemented with 2YTATG liquid medium, cultured at 37°C and 220rpm for about 5 hours to its logarithmic growth phase, and about 10 10 The helper phage M13KO7 was allowed to stand at 37°C for 30 minutes and then cultured with shaking at 150 rpm for 1 hour. Centrifuge at 4000rpm for 15min, resuspend the pellet in 2YTATKA liquid medium, and culture overnight at 220rpm at 28°C. Centrifuge at 4000rpm for 15min at 4°C, and absorb the supernatant containing phages for monoclonal ELISA identification. The single-chain antibody DFSK9-1 with higher affinity was screened, and its heavy chain variable region was named DFSK9-H1, and its amino acid sequence was shown in SEQ NO.1; its light chain variable region was named DFSK9-L1, and its amino acid sequence was shown in SEQ NO. ...

Embodiment 3

[0075] Example 3. In vitro affinity maturation of the single-chain antibody DFSK9-1

[0076] 3.1. Construction of DFSK9-1 light chain CDR123 mutation library

[0077] The pScFvDisb-DFSK9-1 plasmid was double digested with NheI and NotI, and after agarose gel electrophoresis of the digested product, a band with a size of 5.5kb was recovered by gel cutting; the synthetic light chain mutant library gene VLCDR123M was digested with NheI and NotI Double enzyme digestion, the product was recovered by the universal product recovery kit. The mutant library gene and the vector were ligated at a molar ratio of 3:1 by T4 DNA ligase at 16°C for 4 hours. The ligation product was transformed into XL1-Blue electroporation competent by electric shock method. 37 ° C, 150rpm shaking culture 1h recovery. Take 1% bacterial solution, spread it on a small plate after dilution, and calculate the library capacity. After the rest of the bacterial liquid was centrifuged at 4000rpm for 15min, the pr...

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Abstract

The invention relates to the technical field of antibody engineering and specifically discloses an anti-PCSK9 monoclonal antibody. The invention comprises an amino acid sequence coding an antibody variable region and a CDR region as well as an acquisition method and an application of the monoclonal antibody. The monoclonal antibody disclosed by the invention is prepared by the following steps: screening out anti-PCSK9 monoclonal antibodies from a phage library; performing affinity maturation by a method of establishing phage library through strand displacement; screening the mutant libraries of light-chain CDR1, 2, and 3 regions of the monoclonal antibody obtained by primary screening, and selecting the monoclonal antibodies with relatively high affinity; and screening the mutant libraries of heavy-chain CDR1, 2, and 3 to finally obtain the anti-PCSK9 monoclonal antibody with high affinity. In the invention, the obtained PCSK9 antibody has good affinity on PCSK9 and can inhibit combination of the PCSK9 with ligand thereof and can be used for treating dyslipidemia, cardiovascular and cerebrovascular diseases and thrombosis-obstructive diseases.

Description

technical field [0001] The present invention relates to the technical field of antibody engineering, in particular to a fully human anti-PCSK9 monoclonal antibody, its obtaining method and application. Background technique [0002] PCSK9 (Proprotein convertase subtilisin / kexintype 9) belongs to the proteinase K subfamily of the proprotein convertase family. The human PCSK9 gene is located on chromosome 1p32.3, is about 22kb long, has 12 exons in total, and encodes a protein with 692 amino acid residues. PCSK9 protein is composed of signal peptide, prodomain, catalytic domain and carboxy-terminal domain (V domain), which is synthesized as a soluble 74kDa precursor, and undergoes autocatalytic cleavage in the endoplasmic reticulum to produce a 14kDa propeptide and 60kDa mature protease. PCSK9 is mainly expressed in the liver, intestines and kidneys, and a small amount is also expressed in the skin and nervous system, but only PCSK9 in the liver can be secreted into the blood...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13A61K39/395A61P3/06A61P9/00A61P9/10A61P7/02G01N33/577G01N33/573
CPCC07K16/40A61K39/00A61K2039/505C07K2317/92C07K2317/565C07K2317/24A61P9/00A61K35/17C07K2317/14
Inventor 周海平李晓敏张稳文圣梅白义
Owner BEIJING DONGFANG BIOTECH
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