Humanized antibody against epidermal growth factor receptor and application thereof

An epidermal growth factor and antibody technology, which is used in the preparation of drugs for the prevention or treatment of diseases related to epidermal growth factor high activity or high expression. Reduced biological activity, no obvious advantages, etc.

Active Publication Date: 2014-05-07
SHANGHAI JMT BIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, panitumumab is an IgG2 subtype antibody. Compared with IgG1, IgG2’s CDC activity and ADCC and other biological activities are significantly reduced; in addition, IgG2 subtype antibodies are less stable, which may be due to the clinical effect of fully human antibodies. The main reason why nimumab has no obvious advantage compared with the chimeric antibody Erbitux

Method used

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  • Humanized antibody against epidermal growth factor receptor and application thereof
  • Humanized antibody against epidermal growth factor receptor and application thereof
  • Humanized antibody against epidermal growth factor receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1. Design of heavy chain variable region sequence of anti-surface epidermal growth factor receptor humanized monoclonal antibody

[0083] The following is the sequence of the heavy chain variable region of mouse anti-M225, the underlined parts are FR1, FR2, FR3 and FR4 framework sequences: QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWL G VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYDYEFAY WGQGTLVTVSA (SEQ ID NO: 17).

[0084] According to its heavy chain variable region FR1, FR2, FR3 and FR4 sequence, compare the human antibody gene sequence library to find the human germline (germline) similar to the mouse anti-M225 heavy chain variable region FR1, FR2, FR3 and FR4 sequence ) corresponding sequence of antibody variable region. Then use the NetMHCIIpan software or self-compiled software to analyze the binding affinity of similar sequences to HLA-DR molecules through the in silicon method, and select the framework sequence with the lowest a...

Embodiment 2

[0104] Example 2. Design of light chain variable region sequence of anti-epidermal growth factor receptor humanized monoclonal antibody

[0105] The following is the sequence of the light chain variable region of mouse anti-M225, the underlined parts are FR1, FR2, FR3 and FR4 framework sequences:

[0106] DILLTQSPVILSVSPGERVSFSC RASQSIG TNIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAG TKLELK (SEQ ID NO: 18).

[0107]Also analyze the sequence of the light chain variable region of the mouse anti-225 antibody, compare the human antibody gene sequence library according to its light chain variable region FR1, FR2, FR3 and FR4 sequences, and find out the light chain variable region of the mouse anti-M225 antibody FR1, FR2, FR3 and FR4 sequences are similar to the human germline (germline) antibody variable region corresponding sequence, and then use NetMHCIIpan software or self-compiled software to analyze the binding affinity of the selected si...

Embodiment 3

[0125] Example 3, Preparation of anti-epidermal growth factor receptor humanized monoclonal antibody

[0126] (1) According to the antibody heavy chain variable region sequence and light chain variable region sequence determined in Examples 1 and 2, respectively design and synthesize PCR primer oligonucleotide fragments encoding heavy chain and light chain variable region sequences, synthesize Introduce the necessary enzyme cutting sites. PCR primer oligonucleotide fragments are generally about 54 bases in length, and adjacent oligonucleotide fragments overlap by about 18 bases. Mix equal amounts of each PCR primer fragment and perform overlap extension PCR reaction.

[0127] PCR reaction system: dNTPs 0.2 μM (final concentration); each PCR primer fragment 1 μl; 10×buffer 3 μl; cloned pfu (Invitrogen) 1 μl; add water to 30 μl.

[0128] PCR reaction conditions: 94°C for 3min→(94°C for 30s→56°C for 30s→72°C for 1min)×30→72°C for 10min

[0129] The PCR product was separated by...

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Abstract

The invention relates to a humanized antibody against an epidermal growth factor receptor. The invention further relates to a nucleic acid molecule coding the antibody, a vector, a host cell and a composition containing the nucleic acid molecule, a detection reagent and a kit. The invention also relates to application of the antibody in preparation of drugs used for prevention/treatment of diseases related to an epidermal growth factor or the epidermal growth factor acceptor and preparation of the detection reagent or kit. The molecule of the antibody provided by the invention has lower immunogenicity and higher target binding activity, so the antibody has stronger biological activity and in-vitro and in-vivo antineoplastic activity, and it is expected that the antibody has a smaller side-effect and a better curative effect in clinic application.

Description

technical field [0001] The present invention relates to a humanized antibody, in particular to a humanized anti-epidermal growth factor receptor antibody. The present invention also relates to the use of the antibody in the preparation of drugs for preventing or treating diseases related to high activity or high expression of epidermal growth factor. Compared with the human-mouse chimeric antibody, the humanized anti-epidermal growth factor antibody involved in the present invention further reduces the immunogenicity of the antibody, and at the same time enhances its antigen recognition activity and anti-tumor function in vivo and in vitro, and has better clinical efficacy. Application prospect. Background technique [0002] Epidermal Growth Factor Receptor (EGFR, also known as HER1, c-ErbB1) is a cell surface receptor of the epidermal growth factor family, which is composed of 1186 amino acid residues and has a molecular weight of 170kD. Glycoproteins (Jorissen RN, Walker ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K39/395A61K48/00A61P35/00G01N33/68G01N33/574
Inventor 刘劼
Owner SHANGHAI JMT BIO INC
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