Preparation of novel anti-EGFR human source antibody MIL27 and application thereof

A technology of human antibodies and antibodies, which is applied in the direction of anti-animal/human immunoglobulins, antibodies, anti-receptors/cell surface antigens/cell surface determinant immunoglobulins, etc., which can solve the problem of limiting the use of anti-EGFR monoclonal antibodies , high cost, and insufficient clinical application of anti-EGFR monoclonal antibodies

Inactive Publication Date: 2011-01-19
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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  • Preparation of novel anti-EGFR human source antibody MIL27 and application thereof
  • Preparation of novel anti-EGFR human source antibody MIL27 and application thereof
  • Preparation of novel anti-EGFR human source antibody MIL27 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Human Anti-EGFR Antibody Obtained by Computer Aided Molecular Design

[0040] 1. Materials:

[0041] InsightII 2005 program package (MSI molecular simulation, San Diego), which includes:

[0042] Hornology homology modeling;

[0043] Discover mechanical optimization;

[0044] Discover 3 room temperature dynamics simulation;

[0045] Docking molecular docking;

[0046] Delphi apparent electrostatic potential energy analysis.

[0047] Ludi molecular design program (1995);

[0048] IBM graphics workstation;

[0049] PDB 2008 database (download from www.rcsb.org);

[0050] SwissProt database (2008, online download);

[0051] Kabat database (2001, online download);

[0052] IMGT database (2008, online download).

[0053] 2. Method results:

[0054] 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

[0057] Example 2. Expression and Identification of Anti-EGFR Human Antibody MIL27

[0058] 1. Materials:

[0059] 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.

[0060] 2. Method results

[0061] 1. Synthesis of EGFR a...

Embodiment 3

[0071] Example 3. Functional test of anti-EGFR antibody MIL27

[0072] EGFR human antibodies MIL27-1, MIL27-2 and MIL27-3 were taken as examples to further verify the biological functions of the antibodies.

[0073] 1. Materials

[0074] The breast cancer cell line SKVO3 and the liver cancer cell line HepG2 were purchased from ATCC; the DELFIAEuTDA cytotoxicity kit was a product of PE Company; other related reagents refer to Example 2.

[0075] 2. Method results

[0076] 1. Flow cytometry analysis of the binding of MIL27 to cell surface EGFR

[0077] 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 MIL27 antibodies to bind to cell surface EGFR antigens; Erbitux antibody was used as a positive control. The results showed that MIL27 can specifically recognize target cells, and its binding positive rate is similar to that of the control antibod...

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PUM

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Abstract

The invention discloses an anti-EGFR human source antibody MIL27 based on computer aided design and application thereof. The antibody can be used for obviously inhibiting and killing human cancer cell expressing EGFR. The invention is based on the interaction structure characteristic of EGFR protein extracellular region and functional antibody, computer virtual screening and design are adopted to obtain the anti-EGFR functional human source antibody MIL27, PCR method is applied to complete synthesis of antibody gene, eukaryotic expression vector is constructed, and MIL27 antibody capable of specifically identifying EGFR is expressed. The antibody MIL27 can effectively kill EGFR positive tumour cells, including blaster cancer cell and liver cancer cell. The antibody identifies EGFR new epitope, and competitive experiment verifies that the new epitope is different from EGFR epitope identified by Erbitux.

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 MIL27. 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] The human epidermal growth factor receptor (EGFR) gene is located in the p13-q22 region of chromosome 7, with a total length of 200kb, composed of 28 exons, e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N5/09A61K39/395A61P35/00
Inventor 吕明冯健男黎燕沈倍奋耿树生朱康勤
Owner BEIJING MABWORKS BIOTECH
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