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Full humanized HER2 antibody as well as coding gene and application thereof

A fully human, antibody technology, applied in applications, antibodies, genetic engineering, etc., can solve problems such as the loss of phosphorylated PTEN, abnormal signal transmission pathways, and incomplete understanding of the resistance mechanism of Herceptin.

Active Publication Date: 2015-05-27
GENOR BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The drug resistance mechanism of Herceptin is not yet fully understood. There are abnormalities in the signaling pathways below the HER2 receptor, such as the continuously activated PI3K pathway and the loss of phosphorylated PTEN, etc.

Method used

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  • Full humanized HER2 antibody as well as coding gene and application thereof
  • Full humanized HER2 antibody as well as coding gene and application thereof
  • Full humanized HER2 antibody as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Screening the positive clones of scFv from the fully human source scFv phage library

[0043]Human HER2 (extracellular domain)-Fc fusion protein (hereinafter referred to as hHER2-Fc) antigen (purchased from Sino Biological Company, product number: 10004-H02H) was washed with phosphate buffered saline PBS (0.01M Na 2 HPO 4 12H 2 O+0.002M KH 2 PO 4 +0.14M NaCl+0.002M KCl, pH=8.6) was diluted to 5 μg / ml, added to a microtiter plate at 100 μl / well, and coated overnight at 4°C. After the plate was washed 4 times with PBST (PBS buffer containing 0.05% Tween 20), 300 μl / well of 5% BSA (bovine serum albumin, product number: A7030, purchased from Sigma) was added, and blocked at 37° C. for 1 hour. Then wash the plate twice with PBST. will contain 7×10 10 An independently cloned fully human scFv phage antibody library (this antibody library was constructed by Eureka (Beijing) Biotechnology Co., Ltd. by combining multiple antibody variable region genes of healthy h...

Embodiment 2

[0044] Enzyme-linked immunosorbent assay (ELISA) identification of embodiment 2 scFv phage positive clone

[0045] The hHER2-Fc antigen was diluted to 2 μg / ml with PBS (pH=8.6), added to the microtiter plate at 100 μl / well, and coated overnight at 4°C. After washing the plate 4 times with PBST, add 300 μl / well of 5% BSA, and block for 1 hour at 37°C. Then wash the plate twice with PBST, add 100 μl / well phage-positive clone suspension, and incubate at 37° C. for 2 hours. Wash the plate 4 times with PBST, add HRP (horseradish peroxidase)-labeled anti-M13 phage antibody (purchased from GE, product number: 27-9421-01, PBST diluted 1:5000, 100 μl / well), and incubate at room temperature 1 hour. Wash the plate 4 times with PBST, add 100 μl / well color developing solution (soluble one-component TMB substrate solution, purchased from Tiangen Company, article number: PA107-01), incubate at room temperature for 15 minutes to develop color, add 50 μl / well stop solution (1M sulfuric acid...

Embodiment 3

[0049] Example 3 ELISA method detected 102 scFv phage positive clones and monkey HER2-Fc (hereinafter referred to as mkHER2-Fc, purchased from Sino Biological Company, article number: 90295-C02H), mouse HER2-Fc (hereinafter referred to as moHER2-Fc, purchased from Sino Biological Company, product number: 50714-M02H), human HER1-Fc (hereinafter referred to as hHER1-Fc, purchased from Sino Biological Company, product number: 10001-H02H), human HER3-Fc (hereinafter referred to as hHER3-Fc, Purchased from Sino Biological Company, product number: 10201-H05H) and human HER4-Fc (hereinafter referred to as hHER4-Fc, purchased from Sino Biological Company, product number: 10363-H02H) antigen cross-reaction.

[0050] The method is the same as in Example 2, only the coated hHER2-Fc antigens are replaced with mkHER2-Fc, moHER2-Fc, hHER1-Fc, hHER3-Fc and hHER4-Fc antigens respectively.

[0051] The results showed that 96 positive clones of scFv phage had cross-reaction with mkHER2-Fc antig...

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Abstract

The invention relates to the field of medicinal chemistry and particularly relates to a full humanized HER2 antibody as well as a coding gene and an application thereof. According to the full humanized HER2 antibody, the amino acid sequence of the heavy chain variable region is SEQ ID NO:1 and the amino acid sequence of the light chain variable region is SEQ ID NO:2. By adopting the full humanized HER2 antibody, the infusion reaction and the immunogenicity can be reduced, the safety of the medicine can be increased and the better pharmacodynamic characteristics can be obtained.

Description

technical field [0001] The invention relates to the technical field of antibodies, in particular to a fully human HER2 antibody, its coding gene and its application. Background technique [0002] HER2 / neu (Human Epidermal Growth Factor Receptor 2, human epidermal growth factor receptor 2), also known as erbB-2, is a member of the growth factor receptor family. The receptor protein is usually only expressed in the fetal period, and it is only expressed at a low level in a few normal tissues after adulthood. However, it is expressed in a variety of human tumor tissues (such as breast cancer, gastric cancer, ovarian cancer, lung cancer, primary renal cell carcinoma). , endometrial cancer, etc.) are overexpressed, and suggest a poor prognosis. Overexpression of HER2 / neu can lead to excessive proliferation of tumor cells and formation of new blood vessels, resulting in high tumor recurrence rate, high metastasis rate and high mortality. Studies have shown that overexpression of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63A61K39/395A61P35/00A61P35/02G01N33/68
Inventor 周清舒孟军石姝言苏谭涛超
Owner GENOR BIOPHARMA
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