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Anti-human ErbB2 bispecific antibody as well as preparation method and application thereof

A bispecific antibody, antibody technology, applied in the biological field, can solve problems such as drug resistance of patients, and achieve excellent endocytosis effect and good anti-tumor effect.

Active Publication Date: 2017-12-01
程联胜
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2011, the United States approved the combination of Pertuzumab and Trastuzumab to overcome the problem of drug resistance caused by Trastuzumab alone, but some patients still develop drug resistance. Therefore, finding more effective anti-HER2 antibody drugs has become a top priority

Method used

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  • Anti-human ErbB2 bispecific antibody as well as preparation method and application thereof
  • Anti-human ErbB2 bispecific antibody as well as preparation method and application thereof
  • Anti-human ErbB2 bispecific antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction of recombinant vectors for expressing anti-human ErbB2 bispecific antibodies

[0037] The sequence of the anti-human ErbB2 monoclonal antibody Trastuzumab (IgG1, κ) comes from the patent US5821337 of Genentech. Due to the differences in the tandem sequence and the number of amino acids of the connecting short peptides between the two antibodies, the following six bispecific antibodies were constructed: A 1L H,A 2L H,A 3L H and H 1L A, H 2L A and H 3L a.

[0038] A 1L H,A 2L H,A 3L H is the ScFv formed by connecting the variable regions of the light and heavy chains of HuA21 in series to the N-terminus of the heavy chain of Herceptin through linkers of different lengths. In the same way, H 1L A, H 2L A and H 3L A is the N-terminus of the heavy chain of HuA21, in which the ScFv formed by linking the variable regions of the light and heavy chains of Herceptin is connected in series through linkers of different lengths. To put it simply,...

Embodiment 2

[0043] Example 2. Expression and purification of bispecific antibodies

[0044] with A 3L H as an example, pcDNA3.4 (A 3L HVHCH) and pcDNA3.4 (A 3L HVLCL) were simultaneously transferred to the human kidney epithelial cell line HEK293F (ATCC American Cell Bank), and after three days of culture, the antibody protein was purified from the culture supernatant using a Protein A affinity chromatography column. Purified antibody proteins were quantified by the BCA method. The molecular weight and purity of the purified antibody were tested by polyacrylamide gel electrophoresis under reducing and non-reducing conditions respectively. Additionally, A was detected by capillary electrophoresis 3L The isoelectric point of H.

[0045] The results showed that under reducing conditions (10%), the heavy chains of the six bispecific antibodies were all in the range of 75-80KD, and the size of the light chains was slightly different, but also in the range of 25-30KD ( figure 2 ). The r...

Embodiment 3

[0049] Example 3, ELISA method to determine the binding properties of bispecific antibodies and antigens

[0050] T6-17 cells (donated by Professor Mark I Greene, School of Medicine, University of Pennsylvania, USA, recorded in "Xian Wang, Xiaoqiang Xie, Liang Cao et al. Expression of tumor protein P185 in the culture supernatant of T6-17 cells. Journal of Cellular and Molecular Immunology , 2008,24(10):1018-1019", the public can obtain from the applicant, and can only be used to repeat the experiment of the present invention) is the stable transfer of mouse epithelial fibroblast NIH3T3 after being transferred into the human Her2 / ErbB2 gene A cell line that highly expresses HER2 protein on the surface of its cell membrane. After the T6-17 cells in the logarithmic growth phase were resuspended in the trypsinization medium, centrifuge at 1000rpm / min for 5min, take the supernatant, and divide according to 1×10 7 Add cell lysate (recipe: 50mM Tris-HCl pH7.5, 150mMNaCl, volume fra...

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Abstract

The invention discloses an anti-human ErbB2 bispecific antibody as well as a preparation method and application thereof. The anti-human ErbB2 bispecific antibody provided by the invention is prepared by linking a single chain antibody of anti-human ErbB2 to the N-terminal of the heavy chain of the Trastuzumab antibody through linker peptides; the heavy chain variable region in the single-chain antibody is a heavy chain variable region of an HuA21 antibody; a light chain variable region in the single chain antibody is a light chain variable region of an HuA21 antibody; the amino acid sequence of the linker peptides is the 275th to 289th sites in the sequence I of the sequence table. The anti-human ErbB2 bispecific antibody provided by the invention has a good antitumor effect and has a more excellent endocytosis effect than the original Trastuzumab antibody and the HuA21 antibody, so that the anti-human ErbB2 bispecific antibody is of great significance as used for preparing antibody targeting drugs as a drug carrying tool.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an anti-human ErbB2 bispecific antibody, its preparation method and application. Background technique [0002] ErbB2 receptor, also known as HER2 or P185, is a glycoprotein that belongs to the epidermal growth factor receptor (ErbB / HER) family in the type I receptor tyrosine kinase family. This family also has the following members: ErbB1 / HER1 / EGFR, ErbB3 / HER3, and ErbB4 / HER4. ErbB family receptors generally exist in the form of inactive monomers when they are not bound to ligands, and the binding of extracellular ligands will promote receptor dimerization, leading to the activation of signaling pathways such as MAPK and PI3K downstream of the receptors, and then perform biological functions. Although the ligand that can bind to ErbB2 has not been reported so far, it plays a central role in the activation process of the entire ErbB family receptors, so its abnormal expression will h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61K47/68A61P35/00
CPCC07K16/32C07K2317/31C07K2317/622C07K2317/73C07K2317/77
Inventor 程联胜刘兢徐兴赵群方晴刘雯婷
Owner 程联胜
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