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Design of novel anti-EGFR humanized antibody TGM10 and application thereof

A TGM10, human antibody technology, applied in the direction of anti-animal/human immunoglobulin, antibody, application, etc., can solve the problem of high cost, limited use of anti-EGFR monoclonal antibody, and insufficient clinical application of anti-EGFR monoclonal antibody, etc. question

Inactive Publication Date: 2010-11-03
BEIJING MABWORKS BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the clinical application of anti-EGFR monoclonal antibody in my country is not widespread enough, and the cost is relatively high
Anti-EGFR monoclonal antibodies cannot be used alone in the treatment of tumors, and need to be used in combination with radiotherapy or chemotherapy; and there are fewer types of tumors used in clinical treatment, which limits the clinical application of anti-EGFR monoclonal antibodies to a certain extent. use on

Method used

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  • Design of novel anti-EGFR humanized antibody TGM10 and application thereof
  • Design of novel anti-EGFR humanized antibody TGM10 and application thereof
  • Design of novel anti-EGFR humanized antibody TGM10 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Human Anti-EGFR Antibody Obtained by Computer Aided Molecular Design

[0050] 1. Materials:

[0051] InsightII 2005 program package (MSI molecular simulation, San Diego), which includes:

[0052] Homology homology modeling;

[0053] Discover mechanical optimization;

[0054] Discover_3 normal temperature dynamics simulation;

[0055] Docking molecular docking;

[0056] Delphi apparent electrostatic potential energy analysis.

[0057] Ludi molecular design program (1995);

[0058] IBM graphics workstation;

[0059] PDB 2008 database (download from www.rcsb.org);

[0060] SwissProt database (2008, online download);

[0061] Kabat database (2001, online download);

[0062] IMGT database (2008, online download).

[0063] 2. Method results:

[0064] Using the determined structural features of the functional epitope in the extracellular region of EGFR, a short peptide sequence that can recognize the characteristic epitope was designed by the Ludi program...

Embodiment 2

[0067] Example 2. Expression and Identification of Anti-EGFR Human Antibody TGM10

[0068] 1. Materials:

[0069] The primer design software was biosun software, and the primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.; Pyrobest DNA polymerase was a product of TAKARA Company; dNTP was a product of TAKARA Company; pGEM-T Easy vector system was a product of Invitrogen Company; T4DNA ligase was a product of NEB Company ;Gene sequencing was completed by Beijing Nuosai Genome Research Center Co., Ltd.; the antibody eukaryotic expression vector pTGS-FRT-DHFR was constructed by our company and applied for a national patent (patent authorization number: ZL200510064335.0); liposome and MTT are products of Invitrogen , goat anti-human IgG, horseradish-labeled goat anti-human IgG and human IgG were prepared by our company; endonuclease was a product of NEB Company; other reagents were commercially available.

[0070] 2. Method results

[0071] 1. Synthesis of EGFR a...

Embodiment 3

[0081] Example 3. Functional test of anti-EGFR antibody TGM10-1

[0082] Taking the EGFR human antibody TGM10-1 whose heavy chain variable region gene is SEQ ID NO: 2 and light chain variable region gene as SEQ ID NO: 6 as an example, the biological function of the antibody was further verified.

[0083] 1. Materials

[0084] The breast cancer cell line SKVO3, the liver cancer cell line HepG2 and the lung cancer cell line A549 were purchased from ATCC; the DELFIA EuTDA cytotoxicity kit was a product of PE Company; other related reagents refer to Example 2.

[0085] 2. Method results

[0086] 1) Flow cytometry analysis of the binding of TGM10-1 to cell surface EGFR

[0087] EGFR-positive breast cancer cell line SKVO3, liver cancer cell line HepG2 and lung cancer cell line A549 were used as target cells to detect the ability of different concentrations of TGM10-1 antibody to bind to EGFR antigen on the cell surface; Erbitux antibody was used as a positive control. The results...

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Abstract

The invention discloses a computer-assisted design of novel anti-EGFR humanized antibody TGM10 and application thereof. The antibody can be used for inhibiting the growth of human tumor cell for expressing EGFR. The invention obtains the novel anti-EGFR humanized antibody TGM10 by virtual screening and designing of a computer on the basis of the structural characteristic that an EGFR protein cell outer region interacts with the functional antibody. The PCR method is applied to carry out the total synthesis of antibody gene to build into a eukaryon expression carrier, and 9 types of TGM10 antibodies capable of specifically recognizing EGFR can be expressed. The antibody TGM10-1 can effectively kill the positive EGFR tumor cells comprising breast cancer cell lines SKOV3, liver cancer cell lines HepG2 and lung cancer cell lines A549.

Description

technical field [0001] The present invention relates to novel antibodies and uses thereof. Based on the spatial structure of the interaction between EGFR and functional antibodies, use computer-aided methods to reasonably evaluate the dynamic mode of interaction between EGFR protein and functional antibodies and the epitope structural characteristics and physical and chemical properties of functional antibodies that specifically recognize the EGFR extracellular region domain. Through virtual screening Combining with auxiliary molecular design, rationally design and obtain a new anti-EGFR human antibody TGM10. Apply molecular biology techniques for gene synthesis and protein expression, and evaluate the function of new antibodies through immunology experiments and cell biology experiments. Background technique [0002] Epidermal growth factor receptor (EGFR) is one of the important molecules of cell signal transduction and cell differentiation [N.Prenzel, O.M.Fischer, S.Stre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21G06F17/50G01N33/53A61K39/395A61P35/00
Inventor 沈倍奋冯健男黎燕刘旭吕明叶伟亮朱康勤
Owner BEIJING MABWORKS BIOTECH
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