Anti-epidemic growth factor receptor (EGFR) humanized antibody L1-H3 and coded gene and application thereof

A technology encoding genes and antibodies, applied in applications, antibodies, gene therapy, etc., can solve the problems of prolonging antibody half-life, murine glycosylation pattern, hypersensitivity reactions, etc., and achieve the effect of ensuring anti-tumor effect and broad application prospects.

Inactive Publication Date: 2011-08-17
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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  • Anti-epidemic growth factor receptor (EGFR) humanized antibody L1-H3 and coded gene and application thereof
  • Anti-epidemic growth factor receptor (EGFR) humanized antibody L1-H3 and coded gene and application thereof
  • Anti-epidemic growth factor receptor (EGFR) humanized antibody L1-H3 and coded gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Acquisition of genes encoding light chain and heavy chain variable regions of antibodies

[0049] 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 amino acid sequence of the light chain and the "variable region + heavy chain" of the heavy chain Amino acid sequence of constant region 1".

[0050] The antibody of the present invention consists of light chain L1 and heavy chain H3; heavy chain H3 consists of heavy chain variable region (VH), heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2) and heavy chain constant Region 3 (CH3) consists of (H3=VH+CH1+hinge+CH2+CH3). The antibodies of the present invention are referred to as L1-H3.

[0051] Light chain L1: the amino acid sequence is shown in SEQ ID NO: 1; the coding gene sequence is shown in positions 85-726 of SEQ ID NO: 2;

[00...

Embodiment 2

[0073] Example 2, Expression and Purification of Antibodies

[0074] 1. Construction of recombinant expression vectors:

[0075] Recombinant vector pMD18-T / L1 and pIRES double expression vector were respectively digested with corresponding restriction endonucleases (Nhe I and EcoR I), and after agarose gel electrophoresis, the target fragment was recovered and purified; the light chain gene fragment L1 Mix well with the carrier fragment, and react at 16°C for 12h under the action of the ligation reagent. Transform Escherichia coli DH5a, pick a single clone, extract the plasmid, and sequence and identify it. The result: between the Nhe I and EcoR I restriction sites of the vector pIRES (along the direction from the Nhe I restriction site to the EcoR I restriction site ) was inserted into the light chain coding gene shown in nucleotides 9-729 of SEQ ID NO: 2, indicating that the constructed recombinant vector was correct, which was designated as the recombinant expression vecto...

Embodiment 3

[0113] Embodiment 3, the function of antibody

[0114] 1. Biacore detects the binding ability of antibody and antigen

[0115] Sensor Chip CM5 was purchased from BD Company, the product catalog number is Br-1000-14; BD BioCoat TM Matrigel TM Invasion Chamber is purchased from BD Company, and the product catalog number is 354480.

[0116] EGFR protein was purchased from Sigma Company, catalog number E2645-500UN.

[0117] The affinity of the antibody 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 antibodies (0, 10.55, 21.1, 42.2 and ...

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Abstract

The invention discloses an anti-epidemic growth factor receptor (EGFR) humanized antibody L1-H3 and a coded gene and application thereof. The antibody consists of a light chain and a heavy chain, wherein the heavy chain is formed by connecting a heavy chain variable region and a heavy chain constant region; the amino acid sequence of the heavy chain variable region is shown as the 1st-145th sites in SEQ ID NO: 3; the heavy chain constant region is the heavy chain constant region of a humanized antibody IgG1; and the amino acid sequence of the light chain is shown as SEQ ID NO: 1. Proved by experimental results, the antibody has good bonding activity and capacity of inhibiting tumor cell growth migration; and the affinity of an anti-EGFR human-mouse nested cetuximab antibody common in foreign markets is 1.1*10<-9>M. The humanized antibody can be better combined with the EGFR so as to ensure an anti-tumor effect. A method for preparing the antibody can simultaneously express the light chain and the heavy chain, so that the expression ratio of the light chain to the heavy chain can be closer to 1:1, and a mutually matched double-chain antibody with higher ratio is generated. In sum, the antibody and the preparation method thereof have broad application prospects in the field of prevention and/or treatment of tumors.

Description

technical field [0001] The present invention relates to an anti-EGFR humanized antibody L1-H3 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. , especially head and neck squamous cell carcinoma (80% to 100%), colorectal cancer (25% to 77%), non-small cell lung cancer (40% to 80%), etc. have good curative effect. 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 h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/66C12P21/00A61K39/395A61K48/00A61P35/00A61P43/00
Inventor 靳彦文戴维・威孚米歇尔・瑞奇韦兹曹诚
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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