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Antibody fusion protein targeting VEGFR2 as well as preparation and use thereof

A fusion protein and antibody technology, which is applied in the fields of peptide preparation methods, antibodies, peptide/protein components, etc.

Inactive Publication Date: 2015-05-20
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, tumor cells with positive expression of MIC-A are difficult to escape from the immune mechanism of the body, and the shedding of MICA makes the above tumor cells escape immune surveillance even though they are positive for MICA.

Method used

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  • Antibody fusion protein targeting VEGFR2 as well as preparation and use thereof
  • Antibody fusion protein targeting VEGFR2 as well as preparation and use thereof
  • Antibody fusion protein targeting VEGFR2 as well as preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The construction of embodiment 1 fusion protein mAb04-MICA

[0036] Using mAb04 heavy and light chain genes, MICA gene, pCA puro, and pMH3 plasmids as templates, primers were designed and synthesized for PCR amplification. The fusion protein mAb04-MICA was constructed based on the heavy chain gene H' of mAb04, and the complete H'-MICA gene was obtained by overlapping PCR extension amplification. The PCR product was detected by 1.0% agarose gel electrophoresis, and the target gene was recovered by the agarose gel recovery kit. The final products of PCR amplification and plasmids pCApuro and pMH3 were double-digested with restriction endonucleases, and the digested products were gel-recovered and then ligated with T4 ligase overnight at 16°C. After ligation, transform Escherichia coli HB2151 competent, smear the plate, pick out the single clone, double enzyme digestion and sequencing identification the next day.

Embodiment 2

[0037] Expression, purification and identification of embodiment 2 fusion protein mAb04-MICA

[0038] The recombinant plasmids H'-MICA-pCA puro, H'-MICA-pMH3, L-pCA puro, and L-pMH3 were electrotransformed into CHOs cell lines, and single clones were selected through three rounds to obtain high-yielding cell lines stably expressing the fusion protein. The high-yield cell line was expanded and cultured, the supernatant was taken, centrifuged at 8000rpm for 15min, the sample was filtered with a 0.22μm filter membrane and purified with a Protein A column, and eluted with the eluent to obtain the purified protein. A. 8% (12%) SDS-PAGE analysis collected samples, and selected high-purity proteins for identification. B. Perform Western Blot identification on high-purity protein samples. 4°C, 250mA constant current transfer for 1.5h, transfer protein to PVDF membrane (purchased from Millipore); after transfer, seal the membrane in 5% skimmed milk at room temperature for 2h; wash 3 t...

Embodiment 3

[0039] The SPR experiment of embodiment 3 fusion protein mAb04-MICA

[0040] In this experiment, the fusion protein mAb04-MICA and the respective antigens were combined using Biacore X100 as an SPR-dependent biosensor to detect the interaction between the fusion protein and VEGFR2 or NKG2D: A. Using the CM5 chip, the recombinant fusion with the Fc fragment was attached protein molecule. The VEGFR2 antigens with concentrations of 0.84375, 1.6875, 3.375, 6.25, 12.5, 25, 50, 100, and 200nmol / L were detected respectively, and the binding and dissociation curves were obtained. The ka (1 / Ms) was analyzed by BIA evaluation software to be 6.18E+05 , kd(1 / s) is 8.00E-04 and the equilibrium dissociation constant KD(M) is calculated to be 1.29E-09. B. Using the CM5 chip, the recombinant fusion protein molecules with Fc fragments were ligated. 3.906, 7.8125, 15.625, 31.25, 62.5, 125, 250nmol / L of NKG2D were detected respectively, and the binding and dissociation curves were obtained. Th...

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Abstract

The invention belongs to the technical field of genetically engineered antibody, in particular relates to a fusion protein of tumor vascular endothelial growth factor receptor 2 (VEGFR2 / KDR3) antibody and MICA as well as a preparation method and use thereof. In the invention, a full-length antibody of VEGFR2 is linked with a ligand MHC I molecule associated protein A (MICA) of activated receptor of NK cell (NKG2D) through a flexible linker and expressed by CHO cells by utilizing the genetic engineering technology, and the formed fusion protein can act on VEGFR on the surface of a tumor cell to inhibit or kill tumor and inhibit or destroy tumor angiogenesis, and on the other hand, the formed fusion protein can increase the level of MICA on the surface of the tumor cell and stimulate the NK cells to kill tumor cells through identification of NKG2D on the surfaces of the NK cells, thereby reestablishing the immunological surveillance of an body activated by the NKG2D pathway, and enhancing effects of antibody-dependent cell-mediated cytotoxicity (ADCC) and the like mediated by the Fc region of the antibody to kill tumor cells.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a high-affinity fully human fusion protein that can specifically bind to the tumor vascular endothelial growth factor receptor (VEGFR / KDR) and the activated receptor (NKG2D) of NK cells, and can inhibit Activation of vascular endothelial growth factor (KDR) receptor can inhibit the growth of human vascular endothelial cells HUVEC with high expression of vascular endothelial growth factor receptor, and can enhance the antibody-dependent cell-mediated activation of NK cells to human breast cancer cells ADCC is a highly specific genetically engineered fusion protein with anti-tumor and angiogenesis activities targeting VEGFR and NKG2D. Background technique [0002] How to kill tumor cells without harming patients is a difficult problem we have been facing. At present, the key to the design of anti-tumor drugs is not only to kill tumor cells, but also to restore the immune s...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10A61K39/395A61K38/17A61P35/00
CPCA61K39/395C07K1/22C07K19/00C12N15/62C12N15/63
Inventor 张娟王旻王佑富解伟柳芳任学艳
Owner CHINA PHARM UNIV
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