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Recombinant monoclonal antibody against EGFR

A monoclonal antibody and receptor technology, applied in the field of recombinant human anti-epidermal growth factor receptor monoclonal antibodies, can solve problems such as limited application

Active Publication Date: 2006-09-06
SHANGHAI ZHANGJIANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This antibody proved to be clinically better than the murine antibody, but the mouse V region of the chimeric antibody still contains components that make patients immunogenic, limiting its application

Method used

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  • Recombinant monoclonal antibody against EGFR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Construction and screening of human antibody library

[0040] According to Marks et al. J. Mol. Biol., 222, 581-597; Hoogenboom and Winte, J. Mol. Biol., 227, 381-388; Haidaris CG et al., J Immunol Methods. 2001 Nov 1; 257 (1- 2): 185-202; Griffiths, A.D. et al. EMBO J., 13, 3245-3260 (1994); Nissim, A. et al. The method described in EMBO J., 13, 692-698 (1994) to construct a human antibody library .

[0041] The starting material is the peripheral blood of 2028 healthy people. According to the method provided in the above literature, mRNA is prepared, and the variable region genes of immunoglobulin heavy chain and light chain are amplified in vitro by PCR method, and cloned into bacteriophage The carrier utilizes the characteristics of antibody molecular fragments presented on the surface of phages, and uses EGFR protein (purchased from Jingmei Biological Co., Ltd.) as an antigen to screen specific antibodies to EGFR protein.

[0042] Add 1 ml of the recov...

Embodiment 2

[0050] Example 2. Cloning of Expression Vectors for Antibody Variable Region Coding Sequences

[0051] The cloned bacterial strain obtained in Example 1 was amplified in 100 ml of LB medium, and the plasmid DNA was purified with a plasmid DNA extraction and purification kit from Promega Company.

[0052] The above plasmid DNA was digested with XbaI and NheI, and the digested fragments were separated on 1.5% agarose gel electrophoresis, and a band of about 360 bp was taken for gel recovery, and the obtained fragment was the heavy chain variable region coding sequence.

[0053] The above plasmid DNA was digested with HindIII and BsiWI, and the digested fragments were separated on 1.5% agarose gel electrophoresis, and a band of about 320 bp was taken for gel recovery, and the resulting fragment was the light chain variable region coding sequence.

[0054]Then first insert the above heavy chain variable region coding sequence into the expression vector pMG18 (see book DEVELOPMENT ...

Embodiment 3

[0055] Example 3. Transfection of CHO cells and screening of recombinant clones

[0056] The expression vector with the antibody gene constructed in the above-mentioned Example 2 was transferred into the Escherichia coli DH5α strain, and then inoculated in 100 milliliters of LB medium for amplification, and the ultrapure plasmid DNA purification kit (Ultrapure Plasmid DNA Purification Kit) to extract and purify plasmid DNA. The above-mentioned purified plasmid DNA was transfected into CHO cells using the liposome method kit of Invitrogen Company, and the operation was performed according to the manufacturer's instructions.

[0057] The transformed CHO cells were continuously selected on the selection medium for 9 weeks, and finally cultured in extreme dilution on a 96-well plate for 3 consecutive times for monoclonalization.

[0058] The selected monoclonal cell lines were cultured on RPM1641 medium, and the supernatant was subjected to Western blot experiments, and the expre...

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Abstract

The invention provides a monoclonal antibody of human source anti-epidermal growth factor receptor comprising heavy-chain variable area with amino acid sequence displayed by SEQ ID NO three and light-chain variable area with amino acid sequence displayed by SEQ ID NO four. The invention also provides methods for preparation of DNA molecule used for coding the said amino acid sequence, expression carrier which comprises the said DNA molecules, host cell and antibody, the usage and drug composition comprising the said antibody.

Description

field of invention [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a recombinant human anti-epidermal growth factor receptor monoclonal antibody. In addition, the present invention also relates to the preparation method of the monoclonal antibody, the pharmaceutical composition containing the monoclonal antibody and the use of the monoclonal antibody. Background technique [0002] Cancer is the second leading cause of death in humans after heart disease, and major advances have been made in new treatments for this deadly disease. [0003] Proliferation of normal cells is strictly controlled by activation of their growth factor receptors, such as growth factor receptor tyrosine kinases, by their respective ligands. Cancer cells also proliferate under the activation of growth factor receptors, but lose the tight control of normal proliferation. This loss of control can be caused by many factors, such as over...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/12C12N15/63
Inventor 王皓侯盛
Owner SHANGHAI ZHANGJIANG BIOTECH
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