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Human vascular endothelial growth factor specifically-binding antibody or antigen-binding fragment and application thereof

A technology that combines fragments and vascular endothelium, applied in the field of biomedicine, can solve problems such as antibodies that need to be improved

Active Publication Date: 2016-02-17
WATERSTONE PHARMA WUHAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the current antibodies that specifically recognize VEGF still need to be improved

Method used

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  • Human vascular endothelial growth factor specifically-binding antibody or antigen-binding fragment and application thereof
  • Human vascular endothelial growth factor specifically-binding antibody or antigen-binding fragment and application thereof
  • Human vascular endothelial growth factor specifically-binding antibody or antigen-binding fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Preparation and Screening of Mouse Monoclonal Antibody

[0043] (1) Immunization of mice

[0044] HuVEGF 165 The amino acid sequence is:

[0045] MGWSCIILFLVATGVHSAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRRHHHHHH

[0046] huVEGF 165 The protein was dissolved in 1×PBS to obtain a protein solution. An appropriate amount of protein solution was taken, mixed and emulsified with complete Freund's adjuvant (Sigma), and 8-week-old BALB / c female mice were taken, and the antigen was injected subcutaneously at multiple points. Two weeks later, an appropriate amount of protein solution was mixed and emulsified with incomplete Freund's adjuvant (Sigma), and subcutaneously administered to the mice for the second immunization. Thereafter, the third and fourth immunizations were carried out every 3 weeks. Three days after the fourth immunization, sp...

Embodiment 2

[0066] Embodiment 2 Mouse monoclonal antibody characteristic determination

[0067] 1) Determination of the relative affinity of monoclonal antibodies by indirect ELISA

[0068] Coat the microtiter plate with 0.5 μg / ml huVEGF standard, 100 μl per well. Incubate at 37°C for 2 hours, wash with PBS containing 1% Tween-20, and block for 2 hours. The monoclonal antibody NevegiMab was diluted threefold with an initial concentration of 1 μg / ml, and commercialized bevacizumab (avastin) was used as a comparison, and incubated at 37 degrees Celsius for 1 hour. After 5 washes, 100 μl of HRP-labeled corresponding secondary antibodies were added and incubated at 37°C for 1 hour. After 5 washes, add 50 μl of chromogenic solution, incubate at 37°C for 15 minutes, stop the reaction with 50 μl of 2M sulfuric acid, OD 450 reading.

[0069] After four-parameter fitting (OriginPro8), the EC of NevegiMab 50 20pM, EC of Avastin 50 is 190pM. EC 50 The value refers to the concentration of hal...

Embodiment 3

[0089] Example 3 Activity Research of Mouse Monoclonal Antibody

[0090] 1) HuVEC proliferation consistent method to determine the ability of antibodies to antagonize the biological activity of VEGF

[0091] Human umbilical vein endothelial cells (HuVEC) in good growth state were taken, and the cell concentration was adjusted to 5×10 4 / ml, inoculated in 96-well cell culture plate, 100 μl / well, cultured in the incubator for 24 hours, changed the medium with 0.5% serum content, continued to cultivate for 72 hours, added VEGF antibodies with different concentration gradients, and took 3 for each concentration Parallel wells, add huVEGF at a final concentration of 100ng / ml 165 , After culturing for 24 hours, it was detected with a chemiluminescence kit. The results are attached Figure 4 shown.

[0092] as attached Figure 4 As shown, the lower the Lum reading, the more obvious the inhibition of cell proliferation by the monoclonal antibody. The results showed that at the s...

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Abstract

The invention brings forward a human vascular endothelial growth factor specifically-binding antibody or its antigen-binding fragment and an application thereof. The antibody or its antigen-binding fragment comprises a heavy-chain variable region having at least one of the following amino acid sequences: (1) an amino acid sequence as shown in SEQ ID NO:1; (2) an amino acid sequence as shown in SEQ ID NO:2; (3) an amino acid sequence as shown in SEQ ID NO:3; and (4) an amino acid sequence having more than one conserved amino acid mutations in comparison with the amino acid sequences in (1)-(3). According to the antibody or its antigen-binding fragment of the embodiment, human vascular endothelial growth factor can be specifically recognized, and binding of human vascular endothelial growth factor to its receptor can be effectively blocked so as to further block correlated signal pathways of the human vascular endothelial growth factor receptor such as VEGFR1 or 2. Thus, growth of cancer can be effectively inhibited.

Description

technical field [0001] The present invention relates to the field of biomedicine, and more specifically, the present invention relates to an antibody specifically binding to human vascular endothelial growth factor or an antigen-binding fragment thereof and use thereof. Further, the present invention relates to an antibody specifically binding to human vascular endothelial growth factor or an antigen-binding fragment thereof, an isolated polynucleotide, a method for preparing the antibody or an antigen-binding fragment thereof, hybridomas, and hybridomas in the preparation of monoclonal antibodies The use of the antibody or its antigen-binding fragment, the pharmaceutical composition comprising the polynucleotide or the hybridoma, the method for identifying a drug capable of binding to human vascular endothelial growth factor, and drug combination. Background technique [0002] In 1971, Folkman first observed that when the tumor volume exceeds 1-2mm 3 , requires the formati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N15/13C12N5/20A61K48/00A61K39/395A61P35/00G01N33/68G01N33/577
Inventor 张发明席甘黄莺吴炯林峰
Owner WATERSTONE PHARMA WUHAN
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