Targeted EGFR/KDR (Epidermal Growth Factor Receptor/Kinase Insert Domain Receptor) specific diabody

A double-chain antibody, bispecific technology, applied in the field of targeting EGFR/KDR-specific double-chain antibody, can solve the problem of limited clinical efficacy of antibodies, and achieve the effect of inhibiting tumor growth

Inactive Publication Date: 2014-06-18
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although antibodies have powerful natural effector functions, the clinical ...

Method used

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  • Targeted EGFR/KDR (Epidermal Growth Factor Receptor/Kinase Insert Domain Receptor) specific diabody
  • Targeted EGFR/KDR (Epidermal Growth Factor Receptor/Kinase Insert Domain Receptor) specific diabody
  • Targeted EGFR/KDR (Epidermal Growth Factor Receptor/Kinase Insert Domain Receptor) specific diabody

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Construction of fully human anti-EGFG / KDR diabody Db

[0037] Using scFv-E10, scFv-AK404R gene and pHEN2 plasmid as templates, primers were designed and synthesized for PCR amplification. The construction of the anti-EGFR / KDR diabody Db was based on first amplifying the independent variable region, and then performing overlapping PCR extension amplification to obtain the complete Db gene. 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 product of PCR amplification and plasmid pHEN2 were double-digested with restriction endonucleases, and after the digested products were gel-cut and recovered, they were 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

[0038] Example 2 Expression, purification and identification of fully human anti-EGFR / KDR diabody Db

[0039] The recombinant plasmid was transformed into Escherichia coli HB2151 strain, and a single transformed colony was picked, inoculated in 2×TY (100 μg / ml Amp) liquid medium, and activated overnight at 37°C. The next day, inoculate fresh 2×TY medium with 1% inoculum, add 0.4M sucrose to the medium, when OD 600 When it reaches 0.8, add final concentration of 0.8mM IPTG, and induce expression at 25°C for 18-24h. 4°C, centrifuge at 8000rpm for 15min to collect the induced expression fermentation broth cells, resuspend the cells in ice-cold 5% initial fermentation volume hypertonic solution (50mM Tris-HCl, 20% sucrose, 1mM EDTA, pH8.0), Stir gently for 10 min. Centrifuge at 12000rpm for 30min, collect the supernatant, resuspend the pellet in an equal volume of ice-cold 5mM MgSO4, and stir gently on ice for 15min. The supernatant was mixed with the supernatant of the hyperto...

Embodiment 3

[0040] Example 3 SPR experiment of fully human anti-EGFR / KDR diabody Db

[0041] In this experiment, the diabody Db and the respective antigens were combined to use Biacore X100 as an SPR-dependent biosensor to detect the interaction between Db and EGFR or KDR: A. Using an NTA chip with a nitrilotriacetic acid surface, connecting with His-Tag-tagged recombinant antibody Db molecules. The concentration of KDR antigen at 6.25, 12.5, 25, 50, 100, 200nmol / L was detected respectively, and the binding and dissociation curves were obtained. The equilibrium dissociation constant Kd was about 34nmol / L through analysis and calculation with BIA evaluation software. B. Using a CM5 chip labeled with an anti-Fc antibody, the EGFR antigen molecule with the Fc fragment was attached. The Db antibodies at 1.56, 3.125, 6.25, 12.5, 25, 50, and 100nmol / L were detected respectively, and the binding and dissociation curves were obtained. The equilibrium dissociation constant Kd was about 11nmol / L t...

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Abstract

The invention belongs to the technical field of gene engineering antibodies, and in particular relates to a preparation method and an application of a bispecific diabody of an anti-tumor epidermal growth factor receptor (EGFR) and an anti-tumor vascular endothelial growth factor (VEGFR/KDR). The bispecific diabody formed by cross-linking and expressing heavy/light chain variable areas of an anti-EGFR high-affinity single-chain antibody E10 and an anti-KDR single-chain antibody AK404 by using genetic engineering technology can simultaneously act on EGFR with high expression on the surfaces of tumor cells and KDR with high expression on the surfaces of tumor angiogenesis vascular endothelial cells, and can achieve the purposes of simultaneously inhibiting or killing tumor cells or inhibiting or damaging tumor angiogenesis.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a high-affinity fully human bispecific diabody that can specifically bind to human epidermal growth factor receptor (EGFR) / human vascular endothelial growth factor receptor (KDR), which can inhibit The activation of epidermal growth factor receptor (EGFR) / vascular endothelial growth factor (KDR) receptor can inhibit the growth of tumor cell A431 with high expression of EGFR and the growth of human vascular endothelial cell HUVEC with high expression of vascular endothelial growth factor receptor Growth, is a specific double-chain genetically engineered antibody with targeting anti-tumor and angiogenesis activities. Background technique [0002] Monoclonal antibodies have been widely used in the diagnosis and treatment of diseases due to their specificity, uniformity, and mass production. Although antibodies have powerful natural effector functions, the clinical efficacy ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/70C12N1/21A61K39/395A61P35/00
Inventor 张娟王旻金海珍徐孟怀周雅琼
Owner CHINA PHARM UNIV
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