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All-human targeted IgG sample bispecific antibody of EGFR/KDR

A bispecific antibody, fully human technology, applied in the field of bioengineering, can solve the problem of limited clinical effect of single-chain antibody

Inactive Publication Date: 2015-04-29
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the clinical effects of single chain antibodies in tumor therapy are mostly very limited

Method used

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  • All-human targeted IgG sample bispecific antibody of EGFR/KDR
  • All-human targeted IgG sample bispecific antibody of EGFR/KDR
  • All-human targeted IgG sample bispecific antibody of EGFR/KDR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of fully human IgG-like bispecific antibody targeting EGFR / KDR

[0037] Using scFv-E10, scFv-AK404R, Fc(IgG1) gene and PICZα plasmid as templates, primers were designed and synthesized for PCR amplification. The construction of a fully human IgG-like specific antibody targeting EGFR / KDR is based on first amplifying the independent variable region and Fc segment, and then performing overlapping PCR extension amplification to obtain the complete Bi-Ab gene. The PCR product was detected by 1% agarose gel electrophoresis, and the target gene fragment was recovered by the agarose gel extraction kit. The final product of PCR amplification and the plasmid PICZα were double-digested with restriction endonucleases, and the digested products were gel-recovered and ligated with T4 ligase overnight at 16°C. After ligation, transform Escherichia coli DH5α competent, smear the plate, pick out the single clone, double enzyme digestion and sequence identificatio...

Embodiment 2

[0038] Example 2 Expression, purification and identification of fully human IgG-like bispecific antibody targeting EGFR / KDR

[0039] The recombinant plasmids with correct sequencing were amplified, and then transduced into Pichia pastoris X-33 competent cells after linearization. A single colony was picked from the screening medium, inoculated in YPD (25 μg / ml Zeocin) liquid medium, and activated overnight at 30°C. Inoculate in BMGY medium with 1% inoculum on the next day and cultivate to OD 600 After about 1.5 °C, centrifuge at 3000 rpm for 10 min at 4°C to remove the supernatant, resuspend the bacteria in an equal volume of BMMY medium, add 0.5% methanol every 24 hours, and induce expression for 5 days. Centrifuge at 8000 rpm at 4°C for 30 min to collect the supernatant of the induced expression fermentation broth, slowly add ammonium sulfate powder to a saturation of 50%, and let stand overnight at 4°C. Centrifuge at 8000 rpm for 30 min at 4°C to collect the precipitate, ...

Embodiment 3

[0040] Example 3 SPR experiment of fully human IgG-like bispecific antibody targeting EGFR / KDR

[0041] In this experiment, the bispecific antibody Bi-Ab combined with each antigen was detected using Biacore X100 as an SPR-dependent biosensor

[0042] In this experiment, the bispecific antibody Bi-Ab was combined with each antigen and Biacore X100 was used as an SPR-dependent biosensor to detect the interaction between Bi-Ab and EGFR or KDR: A. EGFR was immobilized in the amino coupling method On the CM5 chip, Bi-Ab with a concentration of 0, 0.625, 1.25, 2.5, 5, 10, 20, and 40nmol / L flowed through it to obtain the binding-dissociation curve, and the equilibrium dissociation constant K was obtained by analyzing and calculating with BIA evaluation software D About 1.45nmol / L. B. KDR was immobilized on the CM5 chip by the amino coupling method, and the Bi-Ab with the concentration of 0, 0.625, 1.25, 2.5, 5, 10, 20, 40nmol / L was respectively flowed through to obtain the binding-...

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Abstract

The invention belongs to the technical field of genetically engineered antibodies and in particular relates to an IgG sample bispecific antibody (Bispecific antibody, Bi-Ab) of epidermal growth factor receptor (EGFR) and anti-vascular endothelial growth factor receptor 2 (VEGFR2 / KDR) and an application thereof. By utilizing the genetic engineering technology, heavy and light chain variable regions of anti-EGFR high-affinity single-chain antibodies E10 and anti-KDR high-affinity single-chain antibodies AK104 are cross connected and are subjected to tandem expression with Fc(IgG1) segments, the formed bispecific antibody can simultaneously act on the EGFR and KDR expressed on the surfaces of tumor cells, and the aims of simultaneously inhibiting or killing the tumor cells, inhibiting or damaging the tumor angiogenesis and remaining the ADCC effect of the antibody can be achieved.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a novel IgG-like bispecific antibody with high affinity fully human targeting EGFR / KDR that can specifically bind to human epidermal growth factor receptor and human vascular endothelial growth factor receptor 2 , can specifically inhibit the activation of epidermal growth factor receptor and vascular endothelial growth factor receptor 2. In vivo and in vitro experiments, it can significantly inhibit the growth of tumor cells (HT-29, SKOV-3) overexpressing epidermal growth factor receptor and vascular endothelial growth factor receptor 2. It is an anti-tumor and anti-angiogenesis Active specific genetically engineered antibodies. Background technique [0002] Single-chain antibodies have been widely used in the diagnosis and treatment of diseases due to their specificity, uniformity, and mass production and preparation. Because of these advantages, single-chain antibodi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C07K16/28C12N15/81C12N1/19C12N15/62A61K39/395A61P35/00C12R1/84
Inventor 张娟王旻陈治国金海珍袁锡彬赵虹
Owner CHINA PHARM UNIV
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