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Preparation and application of anti-human PCSK9 (pro-protein convertase subtilisin/kexin 9) antibody

An antibody and chimeric antibody technology, applied in the field of genetic engineering, can solve the problem that the lipid level cannot reach the expected target, etc.

Active Publication Date: 2015-11-11
成都金洛克锶生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Statins are currently the most successful drugs for the treatment of hypercholesterolemia, but some patients who cannot tolerate statins and have high LDL levels, their lipid levels can never reach the expected target

Method used

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  • Preparation and application of anti-human PCSK9 (pro-protein convertase subtilisin/kexin 9) antibody
  • Preparation and application of anti-human PCSK9 (pro-protein convertase subtilisin/kexin 9) antibody
  • Preparation and application of anti-human PCSK9 (pro-protein convertase subtilisin/kexin 9) antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of anti-hPCSK9 chimeric antibody

[0033] 1. Obtaining the sequence of anti-hPCSK9 monoclonal antibody

[0034] 1.1 hPCSK9 immunized mouse hybridoma cell line was obtained

[0035] Mix equal volumes of hPCSK9 and quickantibody-mouse3W immune adjuvant (KX0210041, Beijing Kang Biquan Biotechnology Co., Ltd.), the final concentration of PCSK9 is 200 μg / ml, immunize Balb / c cells, and use intramuscular injection, 100 μl / mouse, to obtain immune spleen cells . The immune splenocytes were fused with Sp / 20 (mouse myeloma cells), selectively cultured by HAT, cultured in a semi-solid medium, and anti-hPCSK9 positive hybridoma cell lines were screened out, and then a subcloning screening was performed to finally obtain a cell line capable of Monoclonal hybridoma cell line (4-2B4-4 cell line) secreting anti-hPCSK9 antibody protein.

[0036] 1.2 Obtaining the variable region sequence of anti-hPCSK9 antibody

[0037] Using the anti-PCSK9 antibody monoclonal c...

Embodiment 2

[0057] Example 2 Chimeric Antibody Binding Affinity to Human PCSK9

[0058] The binding affinity of the chimeric antibody shown in the present invention to human PCSK9 is determined by biomembrane interference technology. First, human PCSK9 (R&D Corporation) was biotinylated to obtain biotinylated human PCSK9. Then in OtectQK e (PallFortebio) protein molecular interaction instrument, immobilize it on the SAbiosensor sensor (Pall company), and after equilibrating in the same buffer, transfer to samples containing chimeric antibody in serial three-fold dilution (600nM to 0.82nM) Binding-dissociation reactions are carried out in the wells. Using Octet software to record the response signal and calculate the binding kinetic parameters, summarized in Table 1, the data are expressed as Mean±SD.

[0059] Table 1. Kinetic parameters of chimeric antibody binding to PCSK9

[0060]

Embodiment 3

[0061] Example 3 Chimeric Antibody Biological Activity Determination

[0062] 1. Effect of chimeric antibody on PCKS9 degradation of hepatic LDLR

[0063] Human liver cancer HepG2 cells were cultured to the logarithmic growth phase, the cells were washed once with PBS, digested with trypsin to make a cell suspension, and the cell density was adjusted to 1×10 6 cells / ml. The cells were seeded in a 96-well culture plate (100 μl / well), and 200 μl of medium was added to the edge wells. Place the culture plate at 37°C, 5% CO 2 Incubate for 24 hours in the incubator. Remove the cell culture supernatant, add 100 μl serum-free cell culture medium to each well, and continue culturing for 16 hours; human PCSK9 (25 μg / ml) and 100 nM, 75 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM chimeric antibody were added in serum-free h -Pre-incubated in DMEM medium for 2 hours, added to the cell wells cultured without serum, placed in 37°C, 5% CO2 incubator and incubated for 6 hours, used for LDLR immuno...

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Abstract

The invention discloses an anti-human PCSK9 (pro-protein convertase subtilisin / kexin 9) chimeric antibody, and preparation and application thereof. The preparation method comprises the following steps: respectively amplifying mouse light chain and heavy chain variable region genes from mouse hybridoma cells, respectively carrying out chimerism with light chain and heavy chain constant region genes of human IgG, and carrying out recombination expression to obtain the human-mouse chimeric antibody. The human-mouse chimeric antibody has favorable affinity with human PCSK9, obviously inhibits the degradation activity of the PCSK9 for liver cell low-density lipoprotein receptors (LDLR), enhances the ingestion of liver cells for LDL-cholesterol (LDL-C), and lowers the cholesterol level in blood.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the preparation and application of a novel anti-human PCSK9 antibody. Background technique [0002] Proprotein convertase subtilisin 9 (PCSK9) is the ninth member of the kexin-like proprotein convertase subtilisin family, consisting of 692 amino acids. PCSK9 is mainly derived from the liver, kidney and small intestine. The synthesis of PCSK9 occurs in the endoplasmic reticulum, and then it is transported to the Golgi apparatus through the endoplasmic reticulum, and secreted into the blood after a series of modifications in the Golgi apparatus. At present, it is believed that only PCSK9 secreted by the liver can be secreted into the blood, and bind to the low-density lipoprotein receptor (LDLR) on the surface of liver cells, mediate the degradation of LDLR, and regulate plasma cholesterol levels. As a negative regulator of the low-density lipoprotein receptor (LDL-R), excess P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13C12N15/85A61K39/395A61P3/06A61P3/04A61P9/10A61P9/00
Inventor 罗弟祥付伟李生伟张晟高小平
Owner 成都金洛克锶生物技术有限公司
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