Improved anti-VEGFR (vascular endothelial growth factor receptor)-2 monoclonal antibody

A VEGFR-2, monoclonal antibody technology, applied in the direction of antibodies, antibody mimics/scaffolds, anti-tumor drugs, etc., to achieve the effects of high affinity, good biological activity, and broad development prospects

Active Publication Date: 2017-05-17
BEIJING DONGFANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our company's patent No. CN103333247 B is a series of anti-VEGFR-2 antibodies obtained through computer-aided design and phage antibody library technology screening, which lays the foundation for obtaining anti-VEGFR-2 antibody drugs. Our company is fu

Method used

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  • Improved anti-VEGFR (vascular endothelial growth factor receptor)-2 monoclonal antibody
  • Improved anti-VEGFR (vascular endothelial growth factor receptor)-2 monoclonal antibody
  • Improved anti-VEGFR (vascular endothelial growth factor receptor)-2 monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, Biopanning of single-chain antibody against VEGFR-2

[0051] A series of gene cloning methods were used to transform the vector pCom3 vector (purchased from China Plasmid Vector Strain Cell Line Gene Collection Center) to be used for the construction and expression of phage single-chain antibody library. The transformed vector was named pScFvDisb-S, and its plasmid map is as follows: figure 1 As shown, and based on this vector, a new fully synthetic phage antibody library based on the antibody sequence in the patent CN10333247B was constructed.

[0052] The immunization tube was coated with the extracellular region of VEGFR-2 as the antigen, the amount of antigen coating was 2ug / 500ul / tube, and the tube was coated overnight at 4°C. The immunotube and the fully synthetic phage antibody library were then blocked with 4% skimmed milk powder / PBST for 1 hour at room temperature. The blocked phage antibody library was added to the immunotube for antigen-antibody ...

Embodiment 2

[0055] Example 2, Identification of Anti-VEGFR2 Phage Single-Chain Antibody Positive Clones

[0056] After three rounds of screening, well-separated monoclonal colonies were picked and inoculated in a 96-well deep-well plate supplemented with 2YTAG liquid medium, cultivated at 37°C and 220 rpm until the logarithmic growth phase, and about 10 10 The helper phage M13KO7 was statically infected at 37°C for 30 minutes. Centrifuge at 4000rpm at 4°C for 15 minutes, discard the supernatant, resuspend the pellet with 2YTAK, and culture overnight at 28°C at 220rpm. The supernatant containing the phage was taken for ELISA identification, and the phage of the biological antibody sequence of CN10333247B (heavy chain is CN10333247B sequence No. 3, and the light chain is CN10333247B sequence No. 9) with the same carrier and the same construction method was used as a positive control (named as O-1). Monoclonal antibodies N-1, N-2 and N-3 with higher affinity were screened, and gene sequenc...

Embodiment 3

[0057] Example 3, Gradual dilution of phage ELISA to compare the affinity of anti-VEGFR2 single-chain antibody

[0058] The clones obtained in Example 2 were displayed and purified by monoclonal phages, and the affinity was identified by phage serial dilution ELISA experiments.

[0059] Coat the extracellular region of VEGFR-2 with pH 9.6 carbonate buffer, 20ng / well / 100ul, overnight at 4°C. Washed three times with PBST, blocked with 4% milk-PBST at 37°C for 1 hour. The purified phage was diluted five-fold with 4% milk-PBST, and 100 ul of the diluted sample was added to each well, and allowed to stand at room temperature for 1 hour. The ELISA plate was washed with PBST, the HRP-anti-M13 monoclonal antibody diluted in 4% milk-PBST was added to the ELISA plate, and left at room temperature for 1 hour. TMB color development kit was used for color development at room temperature for 10 minutes. with 2M H 2 SO 4 After termination, read at 450nm / 630nm. The result is as figure...

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Abstract

The invention relates to the technical field of biomedicine, in particular to an improved anti-VEGFR (vascular endothelial growth factor receptor)-2 monoclonal antibody and application. On the basis of the original research, a new phage antibody library is designed by computer-aided simulation by taking two of them with the highest affinity as templates, and improved heavy chain variable region and light chain variable region amino acid sequences are obtained by multi-round screening; the affinity and biological activity of the anti-VEGFR 2 monoclonal antibody are higher than those of the original antibody. The anti-VEGFR 2 monoclonal antibody obtained by the invention has relatively high affinity, can inhibit the binding of VEGFR-2 and a ligand VEGF thereof in vitro and has good biological activity, and can be used for the treatment of tumors, macular degeneration and other diseases caused by neovascularization.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to an improved anti-VEGFR-2 monoclonal antibody. Background technique [0002] The dependence of tumor growth on the formation of new blood vessels has been thoroughly studied in tumor biology. Angiogenesis can provide oxygen, nutrients, growth factors, hormones and proteolytic enzymes, thereby promoting the spread and metastasis of tumor cells to distant places, and accelerating the growth and deterioration of tumors. Angiogenesis is a highly complex dynamic process regulated by many pro / anti-angiogenic molecules. The angiogenic switch is considered a malignant hallmark of pro-angiogenic over anti-angiogenic. The VEGF / VEGFR axis triggers multiple signaling networks leading to epithelial cell survival, mitosis, metastasis and differentiation, and vascular penetration, and VEGF and its receptors play a central role in normal and pathological angiogenesis. In many human ...

Claims

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Application Information

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IPC IPC(8): C07K16/22C12N15/12G01N33/577A61K39/395A61P35/00A61P27/02
CPCG01N33/577C07K16/22A61K2039/505C07K2317/56C07K2317/24C07K2317/31C07K16/2863C07K2317/622C07K2317/92C07K2317/52C07K2319/00C07K2317/76A61P35/00A61P27/02A61K47/6803
Inventor 周海平谷香果白义
Owner BEIJING DONGFANG BIOTECH
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