Human anti-human VEGF monoclonal antibody molecule and application thereof

A fully human, antibody technology, applied in applications, anti-growth factor immunoglobulins, antibodies, etc., can solve problems such as increased risk

Inactive Publication Date: 2013-01-16
DANYANG ZHENGYUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The risk increases significantly when the drusen becomes larger and more numerous and interferes with the pigmented cell layer beneath the macula

Method used

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  • Human anti-human VEGF monoclonal antibody molecule and application thereof
  • Human anti-human VEGF monoclonal antibody molecule and application thereof
  • Human anti-human VEGF monoclonal antibody molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1. Construction of a large-scale fully human antibody library

[0127] This example describes how to construct a very large-scale fully human natural antibody library in Fab format. The Fab antibody library of the present invention was constructed by using blood samples from more than 3,000 healthy adults from different regions and different ethnicities, referring to the following documents, and the detailed construction process is described after the bibliography.

[0128] 1. Dantas, BC, et al, 2005, Construction of a human Fab phage display library from antibody repertoires of osteosarcoma patients. Gene. Mo. Res, 4(2): 126-140.

[0129]2. Hiroshi, T, et al, 1999, Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica, Clinical and Diagnostic Labor Immunol, May 1999, 383-387.

[0130] 3. Wu, BP, et al, 2001, Construction and selection of the natural immune Fab antibody phage display library from patients with col...

Embodiment 2

[0148] Example 2. Using human VEGF to screen the fully human antibody library

[0149] The self-prepared Avastin-Fab was used as a positive control, and the recombinant human VEGF was used as an antigen to screen the above HuLib. The panning process is as follows:

[0150] Add 1 small portion of HuLib to a 25ml cell culture square bottle pre-coated with recombinant human VEGF protein, and incubate at 37°C for 1 hour. Then, after washing 20 times with PBS containing 1% Tween-20, 1 ml of TG1 cells in logarithmic phase was added, and cultured with shaking at 37C for 16 hours. The supernatant was transferred to a new 50ml test tube and centrifuged at 12000rpm for 10 minutes. Take 500 μl of supernatant, and pan again according to the above method, for a total of 4 rounds. After the final round, the obtained bacterial cell suspension was diluted to 100,000 cells / ml and screened on 1.5% agar culture plates containing 0.1% ampicillin to obtain single spots. Inoculate ten 96-well d...

Embodiment 3

[0159] Example 3 Molecular Evolution

[0160]All CDRs of the 7F2 clone were analyzed by the method of Cunningham and Wells (Science, 244: 1081-1085, 1989), and residues sensitive to glycine substitution were identified, which were preferred mutation sites. The following table shows the preferred amino acid positions in the 3D7 clone, where the underlined ones are extremely sensitive to substitutions, and the dotted ones are relatively sensitive to substitutions, and they are all preferred mutation positions.

[0161] Table 1. Preferred mutated amino acid positions for 3D7 clones

[0162]

CDR1

CDR2

CDR3

[0163] light chain

R A TQDA TV ASAW

Y AS SF I YS

Q N S FL NA A T

heavy chain

AASGF TIG Dy A M H

G PA TP Y G AT TYY A DS V KG

f S f DA Sy S m D Y

[0164] The above-mentioned preferred sites are mutated by oligonucleotide-mediated random mutation (Kunkel metho...

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Abstract

The invention relates to an anti-human VEGF monoclonal antibody and a coding nucleic acid sequence thereof, and especially relates to an anti-human VEGF monoclonal antibody having a high affinity and a low dissociation rate. The preferable anti-human VEGF monoclonal antibody comprises a human Fc region, a framework region, and a human light-chain and/or heavy-chain variable region obtained through molecular evolution. The invention also discloses medicinal compositions composed of the human antibody, an application of the human antibody in human disease treatment, and a method for using the human antibody in the treatment.

Description

field of invention [0001] The present invention relates to anti-human VEGF monoclonal antibody and its encoding nucleic acid sequence. The present invention is particularly concerned with high affinity low off-rate anti-human VEGF monoclonal antibodies. The preferred anti-human VEGF monoclonal antibody of the present invention comprises a fully human Fc region and framework region, and a fully human light chain and / or heavy chain variable region obtained through molecular evolution. The invention also discloses the application of the pharmaceutical composition composed of the fully human antibody in the treatment of human diseases and the method of using the fully human antibody in the treatment. Background technique [0002] The Biological Function of Vascular Endothelial Growth Factor [0003] Vascular endothelial growth factor (abbreviated as VEGF) is a signaling protein produced by cells that stimulate the growth of new blood vessels. It is a component of the mechanism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N1/15C12N1/19C12N1/21C12N5/10A01K67/027A01K67/033A01H5/00A61K39/395A61P35/00A61P27/02A61P9/00
CPCC07K2317/565C07K2317/92C07K2317/21C07K16/22A61K2039/505C07K2317/76C07K2317/55C07K2317/73A61P9/00A61P27/02A61P35/00A61P37/00
Inventor 刘庆法
Owner DANYANG ZHENGYUAN BIOTECH
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