Antihuman PCSK9 monoclonal antibody and application thereof

A monoclonal antibody and fully human antibody technology, which is applied in the direction of antibodies, antibody medical components, anti-tumor drugs, etc., can solve the problems of low output, long time, low antibody purity, etc., and achieve the effect of high output and short cycle

Active Publication Date: 2019-05-21
ZHEJIANG BLUE SHIELD PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of hybridoma technology for antibody screening include long time, low yield, low antibody purity, and antibodies produced by this technology are murine antibodies
Regarding the preparation method of anti-PCSK9 monoclonal antibody, most of the patents currently screen out PCSK9

Method used

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  • Antihuman PCSK9 monoclonal antibody and application thereof
  • Antihuman PCSK9 monoclonal antibody and application thereof
  • Antihuman PCSK9 monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Screening Fab phage library to obtain anti-human PCSK9 positive monoclonal

[0030]1. Dilute the PCSK9 protein (GenScript Biotechnology Co., Ltd.) to a concentration of 100ug / ml, add 1.5ml to the immunotube, and coat overnight at 4°C. On the next day, the coating solution in the immunotube was discarded and washed 3 times with PBS. Prepare 2% skimmed milk powder to seal the immunotube for 3-4 hours, and at the same time display the natural human Fab phage constructed by us according to the report of Hans]. W.de Haard et al. (Antibody PhageDisplay, 2002, p87-100) The library was also blocked for 1 hour, and then the phages were added to the immune tube, turned over repeatedly for 2 hours, and the phages bound to the immune tube were eluted with 0.1% Tween 20, and neutralized. Infect TG1 with the neutralized phages to prepare for the next round of screening.

[0031] 2. After 3-5 rounds of screening, the eluted phages were infected with Escherichia coli TG1 (B...

Embodiment 2

[0044] Example 2 Expression and Purification of Positive Monoclonal PA6 Antibody Fab Fragment

[0045] 1. The screened PA6 antibody with high affinity was infected with TG1 and amplified, then the bacterial liquid was collected, the plasmid was extracted using a plasmid extraction kit (omega, D6950-01), and Gene III of the plasmid was excised.

[0046] 2. Transform the obtained GeneIII excised plasmid into Escherichia coli TG 1, 2YTA medium, culture to logarithmic phase at 37°C, add 1mM IPTG, induce expression overnight at 37°C.

[0047] 3. The supernatant was collected by centrifugation, and purified using Protein A (Kangwei Century, CW0894S) to obtain PA6Fab protein.

Embodiment 3

[0048] Example 3 Expression and purification of positive clones in the form of full-length antibody IgG1

[0049] 1. Construction of the heavy chain expression plasmid: Amplify the CH1-VH part of the Fab fragment of the PA6 antibody by PCR and combine the CH1-VH part with the Fc fragment stored in the laboratory (see fully human monoclonal antibody against PCSK9 for details). The variable region gene and its application, Patent No. CN 104861071A) was ligated using T4 ligase. A Kozak sequence (AAG CTT GCCACC), signal peptide and restriction enzyme sites were then added to the N-terminus of the heavy chain variable region VH. The amplified heavy chain fragment and the expression vector UCOE-Mu-P were digested with restriction endonucleases NgoMIV and NheI, and the fragment was connected to the vector after digestion.

[0050] 2. Construction of the light chain expression plasmid: the complete light chain part of the Fab fragment of the PA6 antibody was amplified by PCR. A Koza...

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PUM

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Abstract

The invention discloses a brand-new PCSK9 antibody. The PCSK9 antibody is sieved through a phage display technology, then, through a gene engineering technology, a full-length gene sequence is obtained and applied to preparation of the antibody, and the prepared antibody is high in yield and short in period and is a humanized antibody. The invention further discloses an anti-tumor effect of the antihuman PCSK9 antibody. The way of thinking is expanded for research on occurrence and development of tumors in the future, and foundations are laid when the antihuman PCSK9 antibody is prepared intomonoclonal antibody medicine used for treating tumors in the later period.

Description

technical field [0001] The invention relates to genetic engineering antibody technology, in particular to a preparation method of fully human monoclonal antibody and full-length antibody against PCSK9 and its anti-tumor application. Background technique [0002] PCSK9, formerly known as Neural Apoptosis-Regulated Convertase 1 (NARC-1), is the ninth member of the subtilisin family of proprotein convertases. It was discovered by Canadian biochemist Nabil Seidah and his team for the first time. reports. The main physiological function of PCSK9 is to mediate the degradation of low-density lipoprotein receptor (LowDensity Lipoprotein Receptor, LDLR) in liver cells, reduce the uptake of plasma low-density lipoprotein cholesterol (LDL-cholesterol, LDL-c) by LDLR, and lead to LDL- c levels increased. Targeted inhibition of PCSK9 helps reduce LDL-c levels. Therefore, PCSK9 has become a hot new target for lipid-lowering, and PCSK9 inhibitors have emerged as the times require. At p...

Claims

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Application Information

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IPC IPC(8): C07K16/40A61K39/395A61P35/00
CPCC07K16/40A61K39/395A61P35/00A61K39/39583A61K2039/505C07K2317/24C07K2317/55C07K2317/565
Inventor 郭志刚景丽瓦基德·阿里
Owner ZHEJIANG BLUE SHIELD PHARM CO LTD
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