Preparation and application of cytokine fusion antibody

A technology of fusing antibodies and growth factor receptors, applied in the field of bioengineering, can solve the problems of strong systemic toxic and side effects, inability to reduce systemic toxic and side effects well, and short interferon half-life.

Inactive Publication Date: 2016-11-02
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the shortcomings of interferon, such as short half-life and strong systemic side effects, limit its clinical application.
At present, some long-acting interferons have been listed on the market. Although they can increase the half-life of interferons in vivo, they cannot reduce their systemic side effects very well.

Method used

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  • Preparation and application of cytokine fusion antibody
  • Preparation and application of cytokine fusion antibody
  • Preparation and application of cytokine fusion antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 4

[0050] Example 4 Flow cytometric binding of anti-VEGFR2 fusion antibody to HUVEC cells

[0051] Digest HUVEC cells and divide into 1 x 10 6 Each group was centrifuged at 1500rpm at 4°C for 5min, washed three times with PBS+2%, and reacted with 100nM JZA01, 100nM JZB00 and skimmed milk solution at 4°C for 1h. Centrifuge at 1500 rpm at 4°C for 5 minutes, wash with PBS+2% FBS three times, and react with Donkey Anti-Human IgG (H+L)-FITC antibody (Life Sciences) at 4°C for 1 hour. Centrifuge at 1500rpm at 4°C for 5min, wash with PBS+2% for three times, resuspend the cells with PBS after the last centrifugation, and detect by flow cytometry.

Embodiment 5

[0052] Example 5 The growth inhibitory effect of anti-VEGFR2 fusion antibody on HUVEC and some tumor cells

[0053] Cells were prepared as 3 x 10 4 cells / ml cell suspension, inoculate 96-well cell culture plate, 100μl / well, at 37℃5%CO 2 Cultivate in the incubator for 24h. The drug was formulated with 2% fetal bovine serum medium, and there were three groups of JZA01, JZB00 and IFNα. Each group was set with 8 concentrations of 2.5nM, 5nM, 10nM, 20nM, 40nM, 80nM, 160nM, 320nM (0.01nM, 0.1nM, 1nM, 10nM, 100nM, 200nM total 6 concentrations). Discard the supernatant of the cultured cells, add drugs according to grouping, 100 μL per well, set up three parallel wells for each drug concentration in each group, continue to culture for 72 hours, add 11 μl of MTT to each well, continue to culture at 37 ° C for 4 hours, carefully pour off the upper 150 μl DMSO was added to each well, and the light absorption value (OD value) was measured at a wavelength of 570 nm / 630 nm on a microplate...

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Abstract

Belonging to the technical field of genetic engineering antibodies, the invention specifically discloses a vascular endothelial growth factor receptor 2 (VEGFR2 or KDR) targeted fusion antibody JZA01of antibody JZB00 and interferon alpha (IFN alpha), a preparation method and application thereof. The invention also discloses the amino acid sequences of heavy chain and light chain immunoglobulin molecules of JZA01. The invention also provides a construction method of JZA01 heavy chain and light chain gene, CHO cells are transfected, monoclone is picked by limited dilution method, then by means of eukaryotic cell secretory expression and affinity chromatography purification, the fusion antibody can be obtained. The fusion antibody provided by the invention can specifically bind VEGFR2 and inhibit tumor angiogenesis, the coupled interferon part also can also play a direct tumor killing and immunoregulation role, and no toxic or side effect is generated at the same time, thereby better inhibiting tumor growth. The fusion antibody can inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) and some tumor cells in vitro.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a high-affinity fully human fusion antibody that can specifically bind to human VEGFR2. Genes, using flexible peptides to connect two pieces of protein. On the one hand, the antibody can inhibit the activation of human vascular endothelial growth factor receptor 2, thereby inhibiting the formation of tumor angiogenesis; It has immunoregulatory effect and does not produce toxic and side effects. It is a genetically engineered antibody with anti-angiogenesis and anti-tumor activities. Background technique [0002] The occurrence and development of tumor cells is inseparable from the nutrient supply of new blood vessels, and the growth of new blood vessels is inseparable from the interaction of various cytokines and their receptors. Existing studies have shown that the vascular endothelial growth factor / vascular endothelial growth factor receptor (VEGF / VEGFR) signaling pat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/21A61P35/00
CPCC07K14/56A61K38/00C07K16/2863C07K2319/33
Inventor 张娟雪莉·莫里森任学艳王旻李晨晨李昭廷朱怡佳
Owner CHINA PHARM UNIV
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