Human monoclonal antibody against EGFR, preparation method and purpose thereof

A fully human, antibody technology, applied in the biological field, can solve the problems of failing to achieve humanization and low affinity

Active Publication Date: 2014-05-14
越海百奥药业(绍兴)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Cetuximab is a chimeric antibody, which has not yet been humanized and has the problem of low affinity

Method used

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  • Human monoclonal antibody against EGFR, preparation method and purpose thereof
  • Human monoclonal antibody against EGFR, preparation method and purpose thereof
  • Human monoclonal antibody against EGFR, preparation method and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0020] Preparation of Example Antibodies

[0021] (1) Cloning of human antibody light and heavy chain constant region genes

[0022] Lymphocyte separation fluid (product of Dingguo Biotechnology Development Co., Ltd.) was used to separate healthy human lymphocytes, and total RNA was extracted with Trizol reagent (product of Invitrogen Company). , 1982, 10: 4071-4079) reported sequences, respectively designed primers and used RT-PCR reaction to amplify the antibody heavy chain and light chain constant region genes. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector (promega company product). After sequencing verification, it was confirmed that the correct clone was obtained. SEQ ID NO: 1 and SEQ ID NO: 2 show the heavy chain constant region (C H ) nucleotide sequence and amino acid sequence. SEQ ID NO: 3 and SEQ ID NO: 4 show the light chain constant region (C L ) nucleotide sequence and amino acid sequence. The corr...

experiment example 1

[0035] Experimental example 1. Apoptosis experiment of anti-EGFR antibody

[0036] Human breast cancer cells A431 (high expression of EGFR, ATCC number: CRL 1555), MDA-MB-231 (expression of EGFR, ATCC number: HTB-27) and MCF-7 (low expression of EGFR, ATCC number: HTB-22) ) were incubated with different dilutions of anti-EGFR antibodies (including 15H8, C225, 2F8, 3C6) at 37°C for 20 hours, and after washing the cells, use the AnnexinV / PI kit (BD company product) to detect the percentage of early apoptotic cells according to the product instructions . Anti-apoptosis assays such as figure 1 As shown: the ability of 15H8 antibody to kill A431 cells is significantly stronger than that of C225, 2F8 and 3C6 (when the antibody concentration is ≥0.5nM, P<0.05, t test, the same result is also obtained in MDA-MB-231 cells Confirmed (when the concentration of antibody concentration ≥ 0.5nM, P<0.05, t test). However, in MCF-7 cells with low expression of EGFR, the killing ability of 15...

experiment example 2

[0037] Experimental Example 2. Growth Inhibition Experiment of Anti-EGFR Antibody

[0038] Incubate human breast cancer cells A431, MDA-MB-231 and MCF-7 with different dilutions of anti-EGFR antibodies (including 15H8, C225, 2F8, 3C6) at 37°C, and stain with MTT staining on the fifth day Growth inhibition rate was calculated after reading. Growth inhibition assays such as figure 2 Shown: 15H8 antibody's ability to inhibit the growth of A431 cells was significantly stronger than that of C225, 2F8 and 3C6 (when the antibody concentration was ≥2.5nM, P<0.05, t test), the same result was also found in MDA-MB-231 Confirmed in cells (P<0.05, t-test at antibody concentration ≥ 2.5 nM concentration). However, in MCF-7 cells with low EGFR expression, the inhibitory ability of 15H8 antibody was similar to that of C225, 2F8 and 3C6. These results show that 15H8 antibody is EGFR specific for cell growth inhibition, and its ability to inhibit cells with high expression of EGFR is stron...

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Abstract

The invention relates to the biotechnology field, and more specifically, the invention discloses a human monoclonal antibody against EGFR, a preparation method and a purpose thereof. According to the invention, a natural human phage antibody library of large capacity is constructed; a human antibody 15H8 strain against EGFR is obtained through screening from the library; an amino acid sequence of a heavy chain variable region is expressed as SEQ ID NO: 6; and the amino acid sequence of a light chain variable region is expressed as SEQ ID NO: 8. Besides, the invention also discloses the preparation method of the antibody 15H8, as well as a nucleotide sequence for encoding the antibody 15H8 and an expression vector and a host cell comprising the nucleotide sequence. The antibody 15H8 of the invention, compared with other antibodies against EGFR, has a higher antibody affinity, as well as a stronger proliferation inhibition effect and an apoptosis induction active effect of EGFR high expression tumor cell, so as to be able to obviously inhibit increase of the tumour and be used for the preparation of antitumor medicine.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically, the invention discloses a fully human monoclonal antibody, its preparation method and application. Background technique [0002] Malignant tumor is a disease that seriously endangers human health in today's society, ranking second among the deaths caused by various diseases. In recent years, the incidence of malignant tumors has shown an obvious upward trend, and the treatment effect of malignant tumors is poor, the rate of advanced metastasis is high, and the prognosis is often poor. Although the current clinical conventional treatment methods such as radiotherapy, chemotherapy and surgery can alleviate the pain and prolong the survival time to a large extent, these methods have great limitations, and the curative effect is difficult to further improve. [0003] The signaling pathway of epidermal growth factor receptor (EGFR) is an important factor affecting the occurrence an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00
Inventor 仝昕李川聂丽
Owner 越海百奥药业(绍兴)有限公司
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