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Humanized monoclonal antibody against human vascular endothelial growth factor and preparation and application thereof

A monoclonal antibody, vascular endothelium technology, applied in the direction of anti-growth factor immunoglobulin, application, antibody, etc., can solve the problems of low yield, low activity, decreased antibody affinity, etc., achieve high biological activity, inhibit blood vessels. Generating, high-affinity effects

Active Publication Date: 2011-01-05
JIANGSU T MAB BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current research and development of such antibodies often encounter problems such as decreased affinity, low activity, and low yield.

Method used

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  • Humanized monoclonal antibody against human vascular endothelial growth factor and preparation and application thereof
  • Humanized monoclonal antibody against human vascular endothelial growth factor and preparation and application thereof
  • Humanized monoclonal antibody against human vascular endothelial growth factor and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The preparation of embodiment 1 TK001

[0060]1) Preparation of hybridoma cells: Rabbits were immunized with rhVEGF165 (R&D). After the rabbits produced a specific immune response, the spleen cells of the immunized rabbits were fused with the plasmacytoma line. After fusion, cells are cultured in a medium containing hypoxanthine, aminopterin, and thymidine (HAT), selected for hybridoma growth, and after 2 to 3 weeks, hybridoma cells begin to appear colony.

[0061] 2) Screening of hybridoma cells: Analyze the culture medium in which the hybridoma cells grow by enzyme-linked immunosorbent assay (ELISA) to detect whether the monoclonal antibody against the antigen is produced. A 96-well plate was coated with 20 μg / ml VEGF, and the culture supernatant to be tested was added. The culture medium was used as a negative control, and the corresponding immunized mouse serum was used as a positive control. That is, each analyte corresponds to 3 detection holes. Re-cloning of p...

Embodiment 2

[0065] Example 2 Purification of TK001

[0066] 1. Mabselect (Protein A) affinity chromatography

[0067] 1) Solution configuration:

[0068] Equilibration buffer A: 20mM PB, 0.15M NaCl, pH7.0

[0069] Elution buffer B: 20 ​​mM sodium citrate-Na 2 HPO 4 , pH3.4

[0070] 2) Purification

[0071] After equilibrating with the equilibrating buffer, the sample is loaded on the Protein A affinity chromatography column, and after equilibrating again, it is eluted with 100% elution buffer, and the eluted peaks are collected.

[0072] 2 SP Fastflow Cation Exchange Chromatography

[0073] 1) Solution configuration

[0074] Equilibration buffer A: 15mM PB, pH5.8

[0075] Elution buffer B: 20mM PB, pH7.2

[0076] 2) Purification

[0077] The elution peak obtained in the first step of purification was adjusted to pH 5.8 with 1M Tris-HCl pH 9.0, and A solution was loaded after equilibrating the filler, 100% B was eluted, and the elution peak was collected.

[0078] 3 Q Fastflow A...

Embodiment 3

[0087] Example 3 Affinity Determination of TK001

[0088] In this experiment, BIAcore-3000 was used to determine the binding affinity of TK001. According to the instructions of the instrument, VEGF was immobilized on the CM5 chip, and a series of diluted antibodies were injected into the HBS-EP buffer at a flow rate of 30ul per minute. Binding rate and (k on ) and dissociation rate (k off ) can be calculated using a 1-to-1 Langmuir binding model by simultaneously fitting the association and dissociation sensorgrams. The dissociation equilibrium constant (Kd) is given by k off / k on Calculated. Table 1 summarizes the .Kd of TK001 and the control drug Avastin. It can be seen from the table that the average affinity of TK001 is nearly 100 times higher than that of Avastin.

[0089] Table 1 Dissociation equilibrium constants (Kd) of TK001 and Avastin

[0090]

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Abstract

The invention relates to a humanized monoclonal antibody against a human vascular endothelial growth factor and preparation and application thereof. In the humanized monoclonal antibody against the human vascular endothelial growth factor, the light chain has an amino acid sequence expressed by SEQ ID No: 1, and the heavy chain has an amino acid sequence expressed by SEQ ID No: 2. The antibody of the invention can be used for treating multiple diseases such as tumor, rheumatoid arthritis, psoriasis, atherosclerosis, diabetes, other proliferative retina diseases and the like, in particular treating age-related macular degeneration (AMD).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a humanized anti-human vascular endothelial growth factor monoclonal antibody and its preparation and application. Background technique [0002] Angiogenesis is the process by which tissues use existing blood vessels to generate new blood vessels. Studies have shown that the occurrence of many diseases is closely related to angiogenesis, such as tumor, the nemesis of human health. Tumor growth and metastasis are closely related to angiogenesis. As early as 1971, Folkman proposed that tumor growth is dependent on blood vessels, and divided the growth of malignant tumors into prevascular and vascularized stages, but this view was not accepted at that time, but with the development of capillary endothelial cells The establishment of culture technology, the discovery of the first angiogenesis inhibitor, and the completion of the first angiogenesis active protein purification, etc., thi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N15/13C12N15/63C12N1/19C12N5/10C12P21/08A61K39/395A61P35/00A61P19/04A61P29/00A61P17/06A61P9/10A61P3/10A61P27/02
Inventor 郭箭吴亦亮王叶飞张伟贾艳丽贾春媛温晓芳黄岩山裘霁宛
Owner JIANGSU T MAB BIOPHARMA
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