Vascular endothelial growth factor fusion protein

A growth factor and fusion protein technology, applied in the direction of anti-growth factor immunoglobulin, growth factor/growth regulator, fusion polypeptide, etc., can solve the problems of VEGF abnormal angiogenesis, etc., to achieve enhanced binding affinity, excellent migration and invasion, Inhibition of growth and proliferation

Active Publication Date: 2017-11-03
KOREA PRIME PHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, increased amounts of VEGF may cause abnormal angiogenesis

Method used

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  • Vascular endothelial growth factor fusion protein
  • Vascular endothelial growth factor fusion protein
  • Vascular endothelial growth factor fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0054] Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood that these examples are not to be construed as limiting the invention in any way.

Embodiment 1

[0056] Example 1: Production of VEGFR fusion protein KP-VR2

[0057] human VEGFR2 domain 3 (KP-VR1) fused to a human IgG1 Fc domain; human VEGFR1 domain 2 (SEQ ID NO: 1) (KP-VR2) fused to a human IgG1 Fc domain (SEQ ID NO: 2); Human VEGFR2 domain 3 (SEQ ID NO:3) and VEGFR1 domain 2 (KP-VR3) fused to a human IgG1 Fc domain; human VEGFR1 domain 2 and VEGFR2 domain 3 fused to a human IgG1 Fc domain (KP-VR3) VR4, aflibercept) were cloned into pCHO 1.0 vector (Invitrogen) respectively.

[0058] CHO-S cells (Invitrogen) were transfected with KP-VR1, KP-VR2, KP-VR3 and KP-VR4 constructs, respectively, and transfected cells were selected by using methotrexate and puromycin. KP-VR2 and KP-VR4 were purified by protein A-agarose affinity chromatography (VEGF-Trap; Aflibercept; Eylea). Purified proteins were quantified by HPLC analysis followed by SDS-PAGE and Western blotting sequentially. figure 1 Each construct is shown. image 3 Indicates the results of western blotting. Such ...

Embodiment 2

[0059] Example 2: Determination of the binding affinity of KP-VR2, aflibercept and bevacizumab of the present invention to VEGF-A

[0060] In this experiment, the binding affinities of KP-VR2, aflibercept and bevacizumab of the present invention to VEGF-A were compared. The ELISA assay was performed according to the BEVACIZUMAB Summary Validation Report (February 28, 2014). Coat 96-well plates (petri dishes) with 50ng / mL VEGFA165(I) or VEGFA121(III) (R&D Systems), and then KP-VR2, KP-VR4 (aflibercept) or Avastin (bevacizumab) Antibody) was added to the wells in increasing amounts from 0.0122 to 1,000 ng / mL. Next, the plate was washed and reacted with a peroxidase-conjugated anti-human Fc antibody. Then, 3,3,5,5-tetramethylbenzidine (TMB) solution (Roche) was added, and then the absorbance was measured at 450 nm using an ELISA reader (spectrophotometer, Biorad). Figure 5 (I and III) show the results.

[0061] In addition, 96-well plates were coated with 2 nM of KP-VR2 or...

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Abstract

The present invention relates to a vascular endothelial growth factor fusion protein. The present invention relates to a fusion protein binding to a vascular endothelial growth factor (VEGFR) and / or a placental growth factor (PLGF). The fusion protein of the present invention comprises (a) a Fc domain of IgG1, wherein two heavy chains are linked by disulfide bond, and (b) four immunoglobulin domain2s of the VEGFR1, wherein two immunoglobulin domain2s are sequentially fused to each heavy chain of the Fc domain of (a). The present fusion protein has excellent activities of inhibiting cell migration and cell invasion, and has highly enhanced growth inhibition effects to various carcinomas and fibroblasts. Therefore, the fusion protein of the present invention can be used in the preparation of an agent for treating cancers or ocular diseases.

Description

technical field [0001] The invention relates to a vascular endothelial growth factor receptor (VEGFR) fusion protein, which is used for preparing a medicament (agent) for treating cancer or eye diseases. The fusion protein of the present invention is a multivalent bispecific antibody in which the extracellular domain of VEGFR is fused to the Fc region of an immunoglobulin-like domain. Background technique [0002] Vascular endothelial growth factor (VEGF) is a signaling protein that plays an important role in promoting both angiogenesis and angiogenesis. VEGF functions as part of a system that restores and supplies oxygen to tissues when blood circulation is poor. A well-known function of VEGF is to generate new blood vessels to bypass blocked blood vessels during embryonic development, at the site of injury, in muscle after exercise, and in vessel formation. [0003] However, increased amounts of VEGF may cause abnormal angiogenesis. In particular, angiogenesis is associ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/395A61P35/00A61P35/02A61P27/02A61P9/10A61P3/10A61P7/10A61P27/10
CPCA61K2039/505C07K16/22C07K16/2863C07K2317/31C07K2317/35C07K2319/30A61K38/00A61K38/18A61P1/04A61P1/18A61P3/10A61P7/10A61P9/08A61P9/10A61P13/08A61P13/12A61P15/00A61P17/00A61P27/02A61P27/10A61P35/00A61P35/02C07K14/705A61K38/177A61K47/6811C07K14/71C12N15/62C12N15/85C12P21/00A61K39/395C07K14/00C07K2319/00
Inventor 金大翼徐仁罗
Owner KOREA PRIME PHARM
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