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Preparation and application of cytokine mutant fusion antibody

A technology of fusion antibodies and mutants, applied in the field of bioengineering, can solve the problems of limited function and low affinity, and achieve strong inhibitory effect and growth inhibitory effect.

Inactive Publication Date: 2017-06-16
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although wild-type interferon has strong biological functions, its function is limited due to its low affinity with receptors

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of Fully Human Anti-VEGFR2 Fusion Antibody Gene

[0034] Our research group has successfully obtained the recombinant plasmid of anti-VEGFR2 fusion antibody JZA01. JZA01 is at the C-terminus of antibody Fc segment through G 4S Linker is conjugated to the fusion antibody of IFNα. In its heavy chain expression plasmid, the two ends of the IFNα expression part contain restriction sites BamHI and PmeI. Based on this, first obtain the sequence expressing IFNα by double enzyme digestion of the plasmid, then use the obtained IFNα as a template, design mutation primers, use overlap PCR to obtain IFNαmut, and then replace the JZA01-H chain with the restriction sites at both ends. The part of wild-type IFNα was obtained by expression vector JZA02-H chain. A large amount of the obtained mutant expression vector and the common light chain L chain were extracted and prepared to be transfected into CHO cells.

Embodiment 2

[0035] Example 2 Expression, purification and SDS identification of anti-VEGFR2 fusion antibody

[0036] The light and heavy chains were transiently transfected into CHO cells, and stable and high-yielding monoclonal cell lines were selected by pressure selection and two limited dilution methods. The stable high-yielding cell line was expanded and cultured, the supernatant was collected, centrifuged at 6000 rpm, 4°C for 30 min, filtered with a 0.22 μm filter membrane, purified with a Protein A affinity chromatography column, and the eluent was used to elute the protein. The purified JZA02 and JZA01 were mixed with non-reducing SDS-PAGE loading buffer at a ratio of 4:1 (20μl:5μl) in an Eppendorf tube, placed in a water bath at 100°C for 5 minutes, then centrifuged at 3000rpm for 5 minutes, and the protein samples were prepared. stand-by. Configure 10% SDS-PAGE gel for electrophoresis, stop electrophoresis after 80mA constant current for 30min, 120min constant current for 30min...

Embodiment 3

[0037] Western Blot identification of embodiment 3 anti-VEGFR2 fusion antibody

[0038] Purified JZA01, JZA02, and JZA00 were subjected to SDS-PAGE electrophoresis, 80V constant voltage for 30min, 120V constant voltage for 30min, and transfer for 2h. The protein was transferred to PVDF membrane (purchased from Millipore). (TBS containing 5% skimmed milk) at room temperature for 2 hours; dilute the primary antibody with 5% MTBS at 1:2000 (A&B picture is HRP-coupled goat anti-human Fc antibody without secondary antibody; C&D picture is rabbit antibody Human κ chain antibody; E&F picture is mouse anti-human IFNα antibody) (purchased from abcam), incubated at room temperature for 1.5h, washed three times with TBST, washed three times with TBS, each 10min; diluted 1:5000 with 5% MTBS Secondary antibody (C&D picture is HRP-conjugated goat anti-rabbit secondary antibody; E&F picture is goat anti-mouse secondary antibody) (purchased from Lianke Biology), incubated at room temperature ...

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Abstract

Belonging to the technical field of genetic engineering antibodies, the invention specifically discloses a human vascular endothelial growth factor receptor (VEGFR2 or KDR) targeted antibody JZB00 and a fusion antibody JZA02 of an interferon alpha (IFN alpha) mutant, a preparation method and application thereof. The invention also discloses amino acid sequences of the heavy chain and light chain immunoglobulin molecules of JZA02. The invention also provides a construction method of JZA02 heavy chain and light chain gene, CHO cells are transfected, monoclone is selected by means of limiting dilution method, and then eukaryotic cell secretory expression and affinity chromatography purification are carried out to obtain the fusion antobody. The fusion antibody provided by the invention can specifically bound to VEGFR2, and can inhibit tumor angiogenesis, also a coupling mutation interferon part can play a direct part of tumor killing and immunoregulation, and no toxic or side effect is generated, thus better inhibiting tumor growth. The fusion antibody can inhibit human umbilical vein endothelial cells (HUVEC) in vitro and the proliferation of part of tumor cells, and the inhibition degree is higher than that of a fusion antibody JZA01 of wild type IFN alpha and JZB00.

Description

[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. The antibody is based on JZA01, and its affinity with interferon receptors IFNAR1 and IFNAR2 is improved by mutating interferon , so as to exert a better antitumor effect. The antibody can not only inhibit the activation of human vascular endothelial growth factor receptor 2, thereby inhibiting the formation of tumor angiogenesis, but also enrich IFN in tumors and tumor neovascularization sites through targeting, without toxic side effects; more importantly Yes, by mutating interferon, under the premise of ensuring that its activity does not change, its affinity with interferon receptors IFNAR1 and IFNAR2 is greatly improved, and it exerts a stronger tumor killing effect and immune regulation effect. Genetically engineered antibodies with anti-angiogenic activity and anti-tumor activity. Background tech...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/395A61K38/21A61P31/12A61P35/00A61P37/00
CPCA61K2039/505C07K14/56C07K16/2863C07K2317/51C07K2317/515C07K2317/55C07K2317/622C07K2317/626C07K2319/00
Inventor 张娟朱怡佳李晨晨雪莉·莫里森王旻
Owner CHINA PHARM UNIV
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