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Anti-human vascular endothelial growth factor antibody, preparation method and application thereof

A vascular endothelium and growth factor technology, applied in the biological field, can solve the problems of the small number of positive clones, affecting the biological function of the signaling pathway, and difficult to obtain a better immune response serum titer of heavy chain antibody transgenic mice, etc., to reduce proliferation. , the effect of inhibiting the proliferation of vascular endothelial cells

Active Publication Date: 2018-12-18
SHANGHAI CHEMPARTNER CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional binding experiments, such as flow cytometry (FACS) and enzyme-linked immunoassay (ELISA), can only screen out antibodies with affinity, but these antibodies will not necessarily affect subsequent signaling pathways and corresponding biological processes after binding to VEGFA. biological function, therefore not biologically active
At present, there is no VEGFA heavy chain antibody in China, and the difficulty in its development is that it is difficult to obtain a good immune response and serum titer of heavy chain antibody transgenic mice, resulting in a small number of positive clones after fusion

Method used

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  • Anti-human vascular endothelial growth factor antibody, preparation method and application thereof
  • Anti-human vascular endothelial growth factor antibody, preparation method and application thereof
  • Anti-human vascular endothelial growth factor antibody, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1V

[0049] The preparation of embodiment 1VEGFA antibody

[0050](1) Preparation of immunogen (hVEGFA165-hFc protein) and hVEGFA165-his protein

[0051] VEGFA is a soluble ligand, we designed the product of VEGFA and human constant region,

[0052] See Table 1 for the specific sequence.

[0053] Table 1 Immunogen sequence

[0054] serial number

Sequence (5'→3')

hVEGFA165-hFc

VEGFA165 (P15692-4)

hVEGFA165-his

VEGFA165(P15692-4)-6×his

[0055] The nucleotide sequence (number NM_001171626 in Genebank) containing the amino acid sequence of the extracellular region encoding the human source VEGFA165 protein as shown in the above table at position 1-191 (methionine 1 to arginine 191) was cloned into human IgG Fc The pCpC vector of the fragment (hFc) (purchased from Invitrogen, V044-50), or the nucleotides from position 1 to position 191 (methionine 1 to arginine 191) of the amino acid sequence encoding human VEGFA165 protein extracellular regio...

Embodiment 2

[0070] Example 2 Production and Purification of Antibodies

[0071] The concentration of antibodies produced by hybridoma cells is low, only about 1-10 μg / ml, and the concentration varies greatly. Moreover, various proteins produced by cell culture in the culture medium and fetal calf serum components contained in the culture medium have varying degrees of interference with many biological activity analysis methods, so small-scale (1-5 mg) antibody production and purification are required.

[0072] The hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and acclimatized and passaged for 3 generations with a production medium (Hybridomaserum free medium, purchased from Thermo & Fisher, Life technologies company). When it grows well, inoculate the cell culture spinner bottle. Add 500ml of production medium to each 2-liter culture spinner bottle, and inoculate the cell density at 1.0×10 5 / ml. Close the bottle cap tightly, and place the spinner ...

Embodiment 3

[0077] The assay of embodiment 3 antibody

[0078] (1) Detection of antigen-antibody binding sites by enzyme-linked immunosorbent assay (ELISA)

[0079] The purified VEGFA antibody obtained in Example 2 was subjected to a binding reaction with hVEGFA165-his.

[0080] The hVEGFA165-his prepared in Example 1 was diluted with PBS to a final concentration of 5.0 μg / mL, and then added to a 96-well ELISA plate at 100 μl per well. Seal with plastic film and incubate overnight at 4°C, wash the plate twice with plate washing solution [PBS+0.01% (v / v) Tween20] the next day, add blocking solution [PBS+0.01% (v / v) Tween20+1 %(w / w) BSA] for blocking at room temperature for 2 hours. The blocking solution was discarded, and 100 μl of the purified VEGFA antibody obtained in Example 2 was added to each well. After incubation at 37° C. for 2 hours, the plate was washed 3 times with plate washing solution [PBS+0.01% (v / v) Tween20]. Add HRP (horseradish peroxidase)-labeled secondary antibody ...

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Abstract

The invention discloses an anti-human vascular endothelial growth factor (VEGFA) antibody, a preparation method and an application thereof. The heavy chain variable region of the VEGFA antibody comprises one or more of CDR1, CDR2 and CDR3, and an amino acid sequence and a nucleic acid sequence thereof are as described in the present invention respectively. the VEGFA antibody has high affinity andhigh biological activity, can be better combined with VEGF, and inhibit or block the binding of VEGF and VEGFR, so that a subsequent signal pathway and corresponding biological functions can be influenced, and the anti-human vascular endothelial growth factor can be well applied in anti-tumor, age-related macular degeneration, diabetic retinopathy, chronic eczema, rheumatoid arthritis and chronicinflammation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antibody against human vascular endothelial growth factor and its preparation method and application. Background technique [0002] Angiogenesis refers to the mechanism by which new blood vessels are formed from pre-existing blood vessels through the growth, differentiation and migration of endothelial cells. It is well known that angiogenesis plays an important role in normal growth processes such as wound healing and women's menstrual cycle (Risau, Nature, 1997, 386:671), abnormal excessive angiogenesis in tumor growth and metastasis, and age-related macular It plays a key role in the onset of diseases such as ARMD, diabetic retinopathy, psoriasis, rheumatoid arthritis, and chronic inflammation (Carmeliet and Jain, Nature, 2000, 407:249). [0003] Vascular endothelial growth factor (VEGF), also known as vascular endothelial growth factor A (vascular endothelial growth factor a,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N15/13C12N15/63A61K39/395A61P35/00A61P27/02A61P3/10A61P9/14A61P29/00A61P17/00
CPCC07K16/22A61K2039/505C07K2317/565C07K2317/24
Inventor 何姗段清邓长静金磊刘礼乐孙志武邵晓慧张洁胡少平徐辉杨欣秀张瑜
Owner SHANGHAI CHEMPARTNER CO LTD
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