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Anti-tie2 antibody and use thereof

A TIE-2 and antibody technology, applied in the direction of antibodies, antibody medical components, applications, etc., can solve the problems of reduced drug accessibility and reduced therapeutic effect

Pending Publication Date: 2022-01-28
PHARMABCINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, even when a therapeutic agent is used to treat a tumor, the therapeutic effect is reduced due to the reduced accessibility of the drug to the interior of the tissue (Gianfranco Baronzio et al., Frontiers in Oncology. 5:165, 2015)

Method used

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  • Anti-tie2 antibody and use thereof
  • Anti-tie2 antibody and use thereof
  • Anti-tie2 antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1 Screening of Antibodies Binding to TIE2

[0123] A human primary ScFv (scFv) library disclosed in Korean Patent Laid-Open Publication No. 10-2008-0109417 was used to screen for antibodies binding to TIE2 and to prepare the library. Spread 100 μl of antigen (hTIE2-his: Sino Biological, 10700-H08H) on 96-well Ni at 2 μl / ml + plate and incubate overnight at 4°C. The next day, the antigen-coated plate was washed three times with 0.1% TBST, and then reacted with 200 μl of 2% BSA blocking buffer for 2 hours at room temperature. Add 50 μl of XL1-Blue stock solution to 2 ml of 2xYT-TET (tetracycline 10 μg / ml) growth medium, and incubate at 37°C at 200 rpm for 2 hours, then add 13 ml of 2xYT-TET growth medium, and Incubate until OD 600 reach 0.5. After blocking for 2 hours, the wells were washed 3 times with 1XPBS. The phage library group was combined with each washed well, the phage library was mixed with 4% BSA in equal amounts, and then 200 µl of the mixture wa...

Embodiment 2

[0124] Example 2 Screening of monoclonal ScFv phage that specifically binds to TIE2 and neutralizes the binding to TIE2 (combination ELISA)

[0125] After completing the panning process, the final circular cell stock solution was diluted to form 200 to 500 colonies, plated on CM agar plates, and then incubated overnight at 37 °C. The next day, when the colony grows, add 200 μl of 2xYT medium (CM 34 μg / ml+1% glucose) into a 96-well deep-well plate, insert one colony into each well, and then place the plate at 37°C and 3,000rpm Incubate overnight. The next day, spread 200 μl of 2xYT medium (CM 34 μg / ml+1% glucose) on a fresh 96-well deep-well plate, inject 20 ml of the cells grown the day before into each well, and incubate at 37°C and 3,000 Grow for 1 hour and 10 minutes at rpm. Store the remaining cells in 100 μl of 50% glycerol at -70 °C. While the cells were growing, mix 1 μl of helper phage with 19 μl of 2xYT medium, inject 20 μl of the resulting mixture into each well, ...

Embodiment 3

[0141] Example 3 Selection of antibodies with binding ability to cells expressing TIE2

[0142] To determine binding to TIE2-expressing cells, binding to self-established human / mouse TIE2-overexpressing CHO-K1 cell lines (hTIE2 / CHO-K1, mTIE2 / CHO-K1) was measured using flow cytometry. Each cell line was maintained and cultured, hTIE2 CHO-K1 was washed with PBS, 5 mM EDTA was added thereto to obtain a cell suspension, and each cell was recovered by centrifugation. Prepare 5×10 cells in FACS-specific buffer (PBS containing 2% FBS, 0.05% sodium azide) 6 cells / mL of cell suspension, and dispense 100 μL of cell suspension into each tube. Add 100 µL of 2 µg / mL clones to each tube and let stand at 4 °C for 30 min to induce cell binding. The cells were washed with 2 mL of FACS buffer, 100 μL of secondary antibody dilution (1 / 500, anti-human IgG-Fc polyclonal antibody fragment conjugated with PE, #A80-248PE, BethylLaboratories Inc.US) was added to it, and the resulting The mixture wa...

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Abstract

The present invention relates to an antibody, which specifically binds to tyrosine-protein kinase receptor TIE-2 (TIE2) so as to possess functions of blood vessel normalization and stabilization through receptor phosphorylation, and relates to: an anti-TIE2 antibody; a nucleic acid encoding same; a vector comprising the nucleic acid; a cell transformed with the vector; a method for preparing the antibody; an agent for stabilizing blood vessels and a composition for treating angiogenesis-associated diseases, both of which comprise the antibody; and a composition for co-administration with a pharmaceutical composition for tumor or cancer treatment and with a composition other than an antibody binding to TIE2.

Description

technical field [0001] The present invention relates to an antibody that specifically binds to TIE2 (tyrosine kinase receptor) and has a function of normalizing and stabilizing blood vessels through phosphorylation of the receptor. More specifically, the present invention relates to an anti-TIE2 antibody, a nucleic acid encoding the antibody, a vector comprising the nucleic acid, a cell transformed with the vector, a blood vessel stabilizer and a , a composition of the blood vessel stabilizer for treating an angiogenesis-related disease, a pharmaceutical composition for treating tumor or cancer, and a composition for administration in combination with a drug other than an antibody that binds to TIE2. Background technique [0002] Angiogenesis is the mechanism by which existing blood vessels form new blood vessels through the growth, division, migration, etc. of endothelial cells, and plays a key role in normal growth processes including wound healing and the female menstrual...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28G01N33/574C12N15/13A61K39/395A61P35/00A61P35/04A61P3/10A61P9/10A61P27/02A61P37/06A61P27/06A61P17/00A61P17/06A61P19/02A61P17/02A61P9/14A61P29/00A61P37/02A61P1/00A61P1/16A61P13/12A61P11/00A61P31/04A61P25/00
CPCC07K16/2863G01N33/574A61K39/3955A61P35/00A61P35/04A61P3/10A61P9/10A61P27/02A61P37/06A61P27/06A61P17/00A61P17/06A61P19/02A61P17/02A61P9/14A61P29/00A61P37/02A61P1/00A61P1/16A61P13/12A61P11/00A61P31/04A61P25/00C07K2317/56C07K2317/565C07K2317/92C07K2317/33C07K2317/76G01N2333/71A61K2039/505A61K2300/00A61K45/06G01N2800/7014G01N2333/515G01N33/6872C07K2317/622A61K2039/55C07K16/005
Inventor 李恩雅朴范用姜努日朴千虎李英爱金度玧李元燮柳珍山
Owner PHARMABCINE
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