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Novel dual-targeted protein specifically binding to DLL4 AND VEGf, and use thereof

A dual-targeting, specific technology, applied in the direction of hybrid immunoglobulin, anti-animal/human immunoglobulin, immunoglobulin, etc., to achieve excellent binding affinity and excellent anti-cancer effect

Active Publication Date: 2016-04-20
ABL BIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

DLL4 is known to be highly overexpressed in cancer vessels although it is also expressed in normal vessels

Method used

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  • Novel dual-targeted protein specifically binding to DLL4 AND VEGf, and use thereof
  • Novel dual-targeted protein specifically binding to DLL4 AND VEGf, and use thereof
  • Novel dual-targeted protein specifically binding to DLL4 AND VEGf, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1: Preparation of anti-DLL4 / VEGF dual targeting protein

Embodiment 1-1

[0128] Example 1-1: Preparation of DLL4 antigen

[0129] As the extracellular domain antigen of human DLL4, human DLL4 protein (catalog number: 1506-D4 / CF) purchased from R&D System was used. The DLL4 antigen includes amino acid residues 27-524 of the DLL4 amino acid sequence (accession number Q9NR61). The C-terminus of the protein has a 10-His tag.

[0130] Prepare antigens corresponding to specific regions of DLL4 extracellular domain. This specific region contains amino acid residues 27-251. This region contains a motif called "DSL(δ / Serrate) / lag-2)" domain known to bind to Noth1 receptor. A mammalian expression vector plasmid containing the CMV promoter upstream of the polynucleotide encoding the DLL4 extracellular domain deleted fragment (amino acid residues 27-251) fused to the Fc protein was prepared using standard recombinant DNA technology. Other constructs encoding DLL4 deletion fragments of human DLL4 chimera fused with Fc protein were prepared using general recombi...

Embodiment 1-2

[0131] Example 1-2: Preparation of phage library

[0132] 2.7x10 with diversity 10 Human scFv (single-chain variable fragment) library cells contain 17g2XYTCM (tryptone (CONDA, 1612.00), 10g yeast extract (CONDA, 1702.00), 5gNaCl (Sigma, S7653-5kg), 34μg / ml chloramphenicol (Sigma ,C0857)], 2% glucose (Sigma, G5400) and 5mMMgCl 2 (Sigma, M2393) culture medium (3L) at 37℃ for 2-3 hours (OD 600 =0.5-0.7), the cells were then infected with helper phage and cultured in 2XYTCMK medium (2XYTCM, 70μg / ml kanamycin (Sigma, K1876), 1mMIPTG (ELPISBIO, IPTG025)) at 30°C for 16 hours. The cultured cells were centrifuged (4500 rpm, 15 minutes, 4°C), and the supernatant was added and dissolved in 4% PEG (Fluka, 81253) 6000 and 3% NaCl (Sigma, S7653), and then incubated on ice 1 hour. Subsequently, the solution was centrifuged (8000 rpm, 20 minutes, 4°C), and the precipitate was added and dissolved in PBS and then centrifuged (12000 rpm, 10 minutes, 4°C). The supernatant containing the phage l...

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PUM

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Abstract

The present invention relates to a dual-targeted protein comprising: a novel protein specifically binding to delta like ligand 4 (DLL4); and an antibody specifically binding to a vascular endothelial cell growth factor (VEGF).

Description

Technical field [0001] The present invention relates to a novel dual-targeting protein, which comprises: a protein specifically binding to delta-like ligand 4 (DLL4) and an antibody specifically binding to vascular endothelial cell growth factor (VEGF). technical background [0002] It has been reported that Notch signaling is highly conserved in vertebrae and invertebrates and plays a very critical role in determining cell fate in the initial stages of development. Notch signal transduction is known as the main way to regulate the differentiation of nerve cells, eye cells, lymphocytes, muscle cells, hematopoietic stem cells, etc. and is also involved in the development of blood vessels. Mammals have four Notch receptors (Notch1, 2, 3 and 4), and each Notch receptor is synthesized as a protein with a size of 300-350kDa and transformed by furin-like transformation in Golgi at the S1 site The enzyme (furin-like convertase) cleaves to form heterodimers on the cell surface. In addi...

Claims

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Application Information

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IPC IPC(8): C07K16/46C07K19/00C12N15/62G01N33/574
CPCC07K16/22C07K16/28C07K2317/31C07K2317/622C07K2317/64A61K2039/505C07K16/468C07K2317/34C07K2317/76C07K2317/92A61P1/02A61P1/04A61P1/16A61P1/18A61P11/00A61P13/08A61P13/10A61P13/12A61P15/00A61P17/00A61P19/00A61P25/00A61P35/00A61P35/02A61P5/00C07K16/2803C07K16/286A61K39/00C07K1/00C07K16/00C07K16/46G01N33/57488G01N33/57492G01N2333/475G01N2333/705
Inventor 李东宪文庆德崔裕彬姜庆在金东寅安珍衡刘原圭郑镇沅
Owner ABL BIO INC
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