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Monoclonal antibody that antagonizes and inhibits binding of vascular endothelial cell growth factor to its receptor, hybridoma cell line and use for secreting it

A hybridoma cell line, monoclonal antibody technology, applied in the direction of anti-growth factor immunoglobulins, antibodies, anti-tumor drugs, etc., can solve the problem of inability to achieve neutralization, inhibition of pro-angiogenesis, inability to bind or cover VEGF epitopes, etc. site, etc.

Active Publication Date: 2016-03-16
ACROIMMUNE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] 1) Like most monoclonal antibodies, the mouse monoclonal antibody A4.6.1 and its humanized antibody Bevacizumab can only bind to some sites in the VEGF epitope, and cannot bind or cover the VEGF epitope Numerous other loci
[0010] 2) Previous animal experiments and clinical studies in recent years have found that only administration of mouse monoclonal antibody A4.6.1 or Bevacizumab antibody alone cannot achieve complete neutralization and inhibition of VEGF and its mediated angiogenesis in vivo

Method used

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  • Monoclonal antibody that antagonizes and inhibits binding of vascular endothelial cell growth factor to its receptor, hybridoma cell line and use for secreting it
  • Monoclonal antibody that antagonizes and inhibits binding of vascular endothelial cell growth factor to its receptor, hybridoma cell line and use for secreting it
  • Monoclonal antibody that antagonizes and inhibits binding of vascular endothelial cell growth factor to its receptor, hybridoma cell line and use for secreting it

Examples

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Embodiment 1

[0052] The following examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way. Embodiment 1: establishment and screening identification of hybridoma cell lines stably secreting anti-VEGF monoclonal antibody

[0053] Step 1. Preparation of recombinant human VEGF165 protein (immunization antigen)

[0054] Preparation of recombinant human VEGF165 protein (immune antigen) (prepared by the method recorded in the Chinese invention patent application No. 2007100473522, titled "Isolation and purification of recombinant human vascular endothelial cell growth factor, in vitro chemical labeling and its application") :

[0055] The gene fragment encoding the complete open-reading frame (ORF) of human VEGF165 was amplified from the cDNA library of human lung tissue cells by PCR technology, and was identified correctly by sequence determination. After treatment with restriction endonuclease, the It was c...

Embodiment 2

[0064] Example 2. In vitro preparation and purification of anti-VEGF monoclonal antibody

[0065] In this example, the separation and purification of anti-VEGF monoclonal antibody (PV19) adopts affinity chromatography. The purification steps are as follows: after the PV19 hybridoma cells are amplified, they are inoculated in 200ml of serum-free 1640 medium, cultured at 37°C for 5 days, and then the supernatant filtrate is loaded into a sample containing ProteinG-SepharoseFastFlow (purchased from General Electric Company ) affinity chromatography; the affinity chromatography column is eluted with PBS to remove impurity proteins, and then the adsorbed antibody protein is eluted with low pH (2.7) glycine (0.1M) solution. Adjust the pH of the eluent to 7.0 with 1mol / LTris (pH9.0), and then dialyze against 10 times the volume of 1xPBS for 12 to 16 hours (change the liquid 2-3 times during the period), and then pass the dialyzed sample through a 0.45μm filter membrane Purified PV19...

Embodiment 3

[0067] Example 3. Western blot analysis and identification of PV19 monoclonal antibody binding to human VEGF protein and other related proteins

[0068] image 3 The pattern of experimental results for Western blot analysis to identify the specific binding of PV19 monoclonal antibody to human VEGF165 protein. The VEGF protein in the experiment was derived from the recombinant human VEGF165 protein expressed in Escherichia coli (product of R&D System, USA). The VEGF165 protein was first dissolved in PBS-1%BSA solution (50 μg / ml), and 10 μl of each sample was loaded for 15% SDS-PAGA electrophoresis, and the gel protein band was transferred to PVDF fiber membrane (Millipore company product) according to the conventional method. , blocked overnight at 4°C with 5% skimmed milk powder, added the PV19 monoclonal antibody prepared in Example 2, and reacted at room temperature for 2 hours. 0.05% Tween-20) was washed 3 times, then horseradish peroxidase-(HRP)-labeled goat anti-mouse I...

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Abstract

The invention belongs to the field of biotechnology-monoclonal antibody. The present invention discloses a monoclonal antibody for antagonistic inhibition of combination of vascular endothelial cell growth factor (VEGF) and a receptor of the VEGF, and a hybridoma cell line secreting the monoclonal antibody, wherein the preservation number of the hybridoma cell line is CGMCC No.5889. The present invention further discloses a preparation method and a use of the monoclonal antibody, wherein the use comprises application of the monoclonal antibody in VEGF protein detection and application of the monoclonal antibody in preparation of a drug preparation for treatment of angiogenesis-related diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology-monoclonal antibody. The invention relates to a monoclonal antibody capable of antagonizing and inhibiting the combination of vascular endothelial cell growth factor (VEGF) and its receptor (VEGF-R) and its application. Background technique [0002] Angiogenesis or hyperplasia (angiogenesis) biologically refers to the process in which existing blood vessels (such as capillaries and tiny arteries and veins) in the body produce new blood vessels through budding or division. Angiogenesis is beneficial and necessary to maintain many normal physiological processes of the body, such as tissue embryonic development, wound healing and repair. However, excessive angiogenesis or hyperplasia is also closely related to many pathological changes (such as tumor proliferation, inflammation, etc.). The key to the continuous proliferation and growth of blood vessels in the body is that the vascular endothelial cells...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/22G01N33/68A61K39/395A61P35/00C12R1/91
Inventor 胡红群罗师平孙亚男宋晓琦蔡明文徐一清周群敏
Owner ACROIMMUNE BIOTECH CO LTD