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Preparation method of anti-erbB2 functional antibody based on erbB2 protein extracellular C-terminal functional epitope characteristics

An antibody and human antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the toxicity, limited efficacy and expensive treatment fees, etc.

Inactive Publication Date: 2012-12-12
BEIJING MABWORKS BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, a series of clinical trials showed that Herceptin The curative effect of Herceptin is limited, and its effective rate is only 11.6-16% when used alone, and the effective rate of combined treatment with chemotherapy drugs can reach 50%, but the combined drug can cause obvious cardiotoxicity; at the same time, Herceptin Expensive treatment costs are also unaffordable for ordinary patients in third world countries

Method used

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  • Preparation method of anti-erbB2 functional antibody based on erbB2 protein extracellular C-terminal functional epitope characteristics
  • Preparation method of anti-erbB2 functional antibody based on erbB2 protein extracellular C-terminal functional epitope characteristics
  • Preparation method of anti-erbB2 functional antibody based on erbB2 protein extracellular C-terminal functional epitope characteristics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Rational design of anti-erbB2 antibody

[0059] 1. Materials:

[0060] InsightII 2005 program package (MSI molecular simulation, San Diego), which includes:

[0061] Homology homology modeling;

[0062] Discover mechanical optimization;

[0063] Discover_3 normal temperature dynamics simulation;

[0064] Docking molecular docking;

[0065] Delphi apparent electrostatic potential energy analysis.

[0066] Ludi molecular design program (1995);

[0067] IBM graphics workstation;

[0068] PDB 2008 database (download from www.rcsb.org);

[0069] SwissProt database (2008, online download);

[0070] Kabat database (2001, online download);

[0071] IMGT database (2008, online download).

[0072] 2. Method results:

[0073] 1. Simulation of the space conformation of the extracellular region of the antigen erbB2

[0074] Using the existing crystal structure of the erbB2 extracellular region (PDB code: 1n8y), on the basis of the homology modeling method, hydr...

Embodiment 2

[0083] Preparation and functional verification of the second antibody erbB2 antibody HF1

[0084] 1. Materials:

[0085] 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, and horseradish-labeled goat anti-human IgG and human IgG were prepared by our company; the pEGFP-N1 vector was a product of Clonetech; the endonuclease was a product of NEB; SKVO3, MCF7, and SKBR3 were purchased from ATCC; T...

Embodiment 3

[0151] Example 3 Preparation of erbB2 antibodies HF2, HF3, HF4, HF5, HF6 and their functional verification

[0152] According to the method described in Example 2, SEQ_ID NO: 2 and SEQ_ID NO: 8 were randomly selected as the heavy chain and light chain variable region amino acid sequences of antibody HF2 respectively; SEQ_ID NO: 3 and SEQ_ID NO: 9 were respectively used as heavy chains of antibody HF3 and light chain variable region amino acid sequences; SEQ_ID NO: 4 and SEQ_ID NO: 10 are respectively the heavy chain and light chain variable region amino acid sequences of antibody HF4; SEQ_ID NO: 5 and SEQ_ID NO: 11 are respectively the heavy chain of antibody HF5 and light chain variable region amino acid sequences; SEQ_ID NO: 6 and SEQ_ID NO: 12 are respectively the heavy chain and light chain variable region amino acid sequences of antibody HF6; use computer-aided analysis to investigate the complex effect of these antibodies and target antigen erbB2 mode of rationality. Us...

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Abstract

The invention provides a preparation method of anti-erbB2 functional antibody based on erbB2 protein extracellular C-terminal functional epitope characteristics, characterized in that the VH amino acid sequence is selected from SEQ_ID NO: 1, SEQ_ID NO: 2, SEQ_ID NO: 3, SEQ_ID NO: 4, SEQ_ID NO: 5 or SEQ_ID NO: 6; and the VL amino acid sequence is selected from SEQ_ID NO: 7, SEQ_ID NO: 8, SEQ_ID NO: 9, SEQ_ID NO: 10, SEQ_ID NO: 11 or SEQ_ID NO: 12. The antibody provided by the invention is capable of specifically identifying target antigens and target cells, inhibiting the proliferation of target cells in vitro and mediating ADCC activity to kill the target cells.

Description

Technical field [0001] The present invention involves the design of the structure characteristics of the C-end function recipe position based on the ERBB2 protein sarcinometer.Specifically, using the ERBB2 crystal structure model and its functional antibody HERCEPTIN The characteristics of the identification table (ERBB2 protein perfection area), through the computer-assisted molecular design method to obtain an anti-ERBB2 human source antibody; reverse translation through biological informatics to determine its appropriate genetic structure, application moleculesBiological technology is performed for genes and evaluates the functions of antibodies in cell biology experimental antibodies. Background technique [0002] As a member of the epidermal growth factor receptor (ERBB family), ERBB2 (HER2, HER2 / Neu) is a cross -membrane protein with tyrosine kinase activity.The molecular structure of ERBB2 can be divided into three parts: extracellular domain, cross -membrane domain, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30G01N33/577A61K39/395A61P35/00
Inventor 吕明乔春霞冯健男黎燕沈倍奋白敏姿
Owner BEIJING MABWORKS BIOTECH