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Antibody, and coding gene and application thereof

A technology that encodes genes and antibodies, applied in applications, antibodies, gene therapy, etc., can solve problems such as mouse glycosylation patterns, decreased affinity of antigen-antibody, prolong antibody half-life, etc., achieve broad application prospects, combined with the effect of activity inhibition

Inactive Publication Date: 2010-11-03
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mouse monoclonal antibody can produce human anti-mouse antibody reaction in human application, thus affecting its function
The mouse-human chimeric antibody modified by genetic engineering technology can greatly reduce the immunogenicity of the mouse monoclonal antibody, prolong the half-life of the antibody in vivo, and can mediate immune opsonization and ADCC effects with the help of the Fc segment of human immunoglobulin, thereby enhancing the antibody Biological effects, but the ability of the chimeric antibody to bind antigen is lower than that of the mouse antibody 98.7%
A large number of preclinical and clinical trials have confirmed that cetuximab alone and combined with chemotherapy / radiotherapy has a good curative effect, but simple CDR transplantation often causes a decrease in antigen-antibody affinity; panitumumab was developed using transgenic mouse technology The prepared fully human antibody has nearly 100% human sequence compared with chimeric antibody and humanized antibody, which greatly enhances the antibody target affinity, but the antibody has a murine glycosylation pattern, short half-life and more hypersensitivity reactions and other shortcomings
Nimotizumab obtained the humanized antibody by humanizing the anti-EGFR mouse monoclonal antibody, and linked the light and heavy chain genes of the antibody to different expression vectors for expression. Large variance, often resulting in very low expression levels of intact antibody molecules

Method used

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  • Antibody, and coding gene and application thereof
  • Antibody, and coding gene and application thereof
  • Antibody, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Acquisition of genes encoding light chain and heavy chain variable regions of antibodies

[0036] According to computer simulation, using the amino acid sequence of the mouse-human chimeric antibody cetuximab as a template, the mouse FR surface gene was humanized to design and synthesize the light chain amino acid sequence L1 and the heavy chain variable region amino acid sequence H1 ;

[0037] Heavy chain variable region H1: the amino acid sequence is shown in sequence 1 in the sequence listing; the coding gene sequence is shown in sequence 4 in the sequence listing;

[0038] Heavy chain variable region H2: the amino acid sequence is shown in sequence 2 in the sequence listing; the coding gene sequence is shown in sequence 5 in the sequence listing;

[0039] Light chain L1: the amino acid sequence is shown in sequence 3 in the sequence listing; the coding gene sequence is shown in sequence 6 in the sequence listing;

[0040] The antibody of the present inv...

Embodiment 2

[0051] Embodiment 2, the preparation of antibody

[0052] 1. Construction of recombinant expression vectors:

[0053] The pIRES double expression vector was purchased from Clontech Company, the product catalog number is 631605; the pMD18-T expression vector was purchased from Takara Bio Company , the product catalog number is: D504 CA. The pIRES vector itself contains the heavy chain constant region gene.

[0054] Recombinant vector pMD18-T / L1 and pIRES double expression vector were respectively digested with corresponding restriction endonucleases (Nhe I and EcoRI), and after agarose gel electrophoresis, the target fragment was recovered and purified; the light chain gene fragment L1 was combined with The carrier fragments were mixed well, and reacted at 16° C. for 12 h under the action of the ligation reagent. Escherichia coli DH5a was transformed, the clone was picked, the plasmid was extracted and identified by sequencing. The results showed that the insertion direction...

Embodiment 3

[0090] Example 3, Functional Detection of Antibodies

[0091] EGFR protein was purchased from (Sigma), catalog number (E2645-500UN).

[0092] 1. Biacore detects the binding ability of antibody and antigen

[0093] The affinity of antibody C2 to EGFR was determined with Biacore3000 equipment. Prepare 10mmol / L NaAc diluted EGFR protein with different pH values ​​(4.0, 4.5, 5.0 and 5.5), pre-concentrate on the CM5 chip, and select the NaAc diluted protein with the optimal pH value. The purified antibody (i.e. the eluate obtained in step 2 in Example 2) was covalently coupled to the CM5 sensor chip, the mobile phase was HBS-EP (pH7.4), the flow rate was 20 μl / min, and five concentrations were taken Detection of binding affinity of antibody C2 (0, 10.55, 21.1, 42.2 and 84.4nmol / L) to EGFR protein. Affinity was calculated with Biacore3000 accompanying software. At the same time, cetuximab was used as a control.

[0094] The experiment was repeated 3 times, and the results were ...

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Abstract

The invention discloses an antibody, and a coding gene and application thereof. An amino acid sequence of a heavy chain variable region of the antibody is shown in the sequence 2 in a sequence table, while the amino acid sequence of a light chain is shown in the sequence 3 in the sequence table. Experimental results prove that the antibody of the invention has high binding activity (affinity is 2.7*10-8 mol / L) and high tumor cell growth and migration suppression capacity; and the affinity of an anti-epidermal growth factor receptor (EGFR) human-mouse chimeric antibody Cetuximab in foreign markets is 1.1*10-9 M. The humanized antibody of the invention can be better bound with an EGFR so as to ensure anti-tumor effect thereof. An anti-body preparation method of the invention has the advantage of simultaneously expressing the light chain and the heavy chain variable region. After all, the antibody and the preparation method thereof have vast application prospect in the field of the prevention and / or treatment of tumors.

Description

technical field [0001] The present invention relates to an antibody and its coding gene and application. Background technique [0002] Epidermal growth factor receptor (EGFR), a member of the epidermal growth factor gene (erbB) family, is overexpressed in about 30% of human tumors, especially non-small cell lung cancer, head and neck squamous cell cancer and colorectal cancer. Many studies at home and abroad have shown that antibodies against EGFR can effectively inhibit the EGFR signal transduction pathway by blocking the binding of ligands outside the cell. Tumors, especially squamous cell carcinoma of the head and neck (80% to 100%), colorectal cancer (25% to 77%), and non-small cell lung cancer (40% to 80%) have good curative effects. Epidermal growth factor receptor (EGFR) has become one of the most in-depth studies and one of the most concerned targets for tumor therapy. The application of genetic engineering to develop anti-EGFR monoclonal antibodies has become one ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K39/395A61K48/00A61P35/00
CPCC07K16/2863A61P35/00
Inventor 戴维·威孚米歇尔·瑞奇韦兹曹诚靳彦文
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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