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Anti-human PCSK9 monoclonal antibody

A monoclonal antibody and antibody technology, applied in the treatment of diseases mediated by human PCSK9, in the field of human-derived functional anti-human PCSK9 monoclonal antibodies, can solve the problems of patients' intolerance and failure of blood lipid levels to reach the standard

Active Publication Date: 2016-02-24
BEIJING WISDOMAB BIOTECHNOLOGY CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Statins are currently the most effective drugs for lowering LDL-C, but there are still some patients whose blood lipid levels fail to reach the target, and some patients cannot tolerate statins

Method used

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  • Anti-human PCSK9 monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of various recombinant proteins.

[0030] A variety of different recombinant proteins are used in the preparation of anti-PCSK9 monoclonal antibodies, including human PCSK9 (huPCSK9, SeqIDNo: 1), mouse PCSK9 (moPCSK9, SeqIDNo: 2) and macaque PCSK9 (mmPCSK9, SeqIDNo: 3), Human LDLR extracellular region (EGF-AB, SeqIDNo:4), and recombinant antibody. These proteins have a large number of post-translational modifications (such as: glycosylation or disulfide bonds, etc.), so the use of mammalian cell expression systems will be more conducive to maintaining the structure and function of recombinant proteins. In addition, for the convenience of purification, a His-tag (SeqIDNo:5) or the Fc fragment of a mouse antibody (mFc, SeqIDNo:6) was added to the C-terminus of the non-antibody recombinant protein. When preparing recombinant antibodies, the heavy chain constant region of the antibody can be of IgG1 subtype (SeqIDNo: 7), IgG2 subtype (SeqIDNo: 8) o...

Embodiment 2

[0033] Example 2: Using phage display antibody library technology to screen and optimize anti-human PCSK9 monoclonal antibody.

[0034] Using the prepared recombinant huPCSK9-his (hereinafter abbreviated as PCSK9-His) as the antigen, refer to the literature (anti-human IL-17 monoclonal antibody, patent application number 201510097117.0), and use the solid-phase screening strategy to screen the phages presenting the human single-chain antibody library , obtained multiple human antibodies with different sequences but specific binding to human PCSK9, including clones: S2B8 (SeqIDNo: 12), S2F5 (SeqIDNo: 13), S2G1 (SeqIDNo: 14), S3A12 (SeqIDNo: 15). Among them, the recombinant full antibody S3A12 can effectively inhibit the binding of PCSK9 to the EGF-AB domain of the extracellular region of recombinant human LDLR ( figure 1 ).

[0035] Then referring to the literature (anti-human IL-17 monoclonal antibody, patent application number 201510097117.0), the antibody was subjected to...

Embodiment 3

[0037] Example 3: BIAcore3000 measures the affinity of whole antibodies.

[0038] Aminecoulingkit and humanantibodycapturekit as well as CM5 chip and pH7.4 10×HBS-EP were purchased from GE Healthcare.

[0039] According to the instructions in the kit, anti-human F C The antibody of the segment is coupled to the surface of the chip CM5, and the antibody protein is diluted to an appropriate concentration to ensure that about 200RU of the antibody is captured by the anti-human Fc antibody. Set a series of concentration gradients (ex.100nm, 50nm, 25nm, 12.5nm, 6.25nm, 3.125nm, 1.5625nm, 0nm) of huPCSK9 to flow through the surface of the stationary phase, and measure the affinity of each monoclonal antibody, and use the same method to determine The affinity of evolocumab (SeqIDNo:21, SeqIDNo:22) and alirocumab (SeqIDNo:23, SeqIDNo:24) was used as a control. The results are shown in Table 2.

[0040] Antibody Kon (1 / MS) Koff(1 / S) KD H4H4-IgG4 7.14E+05 4.7...

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Abstract

The invention discloses a novel anti-human PCSK9 monoclonal antibody or its fragment prepared by using a bacteriophage antibody library technology for screening and using a gene engineering method. The novel anti-human PCSK9 monoclonal antibody has high affinity to human PCSK9. The antibody can be a full-length antibody, a basically complete antibody, a Fab fragment, a F(ab')2 fragment or a single-chain Fv fragment. The monoclonal antibody or its fragment is used for treating disease mediated by human PCSK9, and the disease comprises, but not limited to, primary hypercholesterolemia, combined hyperlipidemia and familial hypercholesterolemia.

Description

technical field [0001] The invention belongs to the field of genetic engineering antibody medicine, especially the field of monoclonal antibody against human PCSK9. The present invention relates to a humanized functional anti-human PCSK9 monoclonal antibody and the use of this type of antibody in the treatment of diseases mediated by human PCSK9. Background technique [0002] Cardiovascular disease is currently the number one cause of death in my country, among which heart disease, stroke and peripheral arterial disease caused by atherosclerosis are the main causes of death, and elevated low-density lipoprotein cholesterol (LDL-C) levels are the main cause of death in arteries. The greatest risk factor for atherosclerotic cardiovascular disease. Statins are currently the most effective drugs for lowering LDL-C, but there are still some patients whose blood lipid levels fail to reach the target, and some patients cannot tolerate statins. Therefore, there is a need to find po...

Claims

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Application Information

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IPC IPC(8): C07K16/40A61K39/395A61P3/06
CPCA61K39/395C07K16/40
Inventor 刘志刚刘玉兰郭晶晶郝小勃
Owner BEIJING WISDOMAB BIOTECHNOLOGY CO LTD
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