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Endoglin polypeptides and uses thereof

An endoglin and immunoglobulin technology, applied in the field of endoglin polypeptides

Pending Publication Date: 2018-07-31
ACCELERON PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While many anti-angiogenic agents have an effect on angiogenesis regardless of the affected tissue, others may tend to have tissue-selective effects

Method used

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  • Endoglin polypeptides and uses thereof
  • Endoglin polypeptides and uses thereof
  • Endoglin polypeptides and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0175] Example 1: Expression of a fusion protein comprising the full-length extracellular domain of human ENG

[0176] Applicants constructed a soluble endoglin (ENG) fusion protein (hENG(26 to 586)-hFc) in which the full-length extracellular domain (ECD) of human ENG was (Figure 9, SEQ ID NO: 9) linked to the human IgG1 Fc domain (Figure 11, SEQ ID NO: 11). hENG(26 to 586)-hFc was expressed by transient transfection into HEK 293 cells. Briefly, HEK 293 cells were set up in a 500ml rotator in 6×10 5cells / mi in Freestyle medium (Invitrogen) in a volume of 250 ml and cultured overnight. The next day, these cells were treated with a DNA:PEI (1:1) mixture (at a final DNA concentration of 0.5 ug / ml). After 4 hours, 250 ml of medium was added, and the cells were cultured for 7 days. Conditioned media was harvested by spinning down the cells and concentrated. For expression in CHO cells, the ENG polypeptide construct was transfected into the CHO DUKX B11 cell line. Clones are t...

Embodiment 2

[0184] Example 2: Expression of Fusion Proteins Including the Full-Length Extracellular Domain of Murine ENG

[0185] Applicants constructed a soluble murine ENG fusion protein (mENG(27-581)-mFc), in which the full-length extracellular domain of murine ENG ( Figure 10 , SEQ ID NO: 10) with mouse IgG via the smallest possible linker between these domains 2a The Fc domain is fused. mENG(27 to 581)-mFc was expressed by transient transfection into HEK 293 cells.

[0186] Selected versions of mENG(27 to 581)-mFc use a TPA leader sequence with Figure 16 The unprocessed amino acid sequence (SEQ ID NO: 19) is shown and encoded by the nucleotide sequence (SEQ ID NO: 20) shown in Figure 17 . Purification was achieved by purification of conditioned medium from transfected HEK 293 cells followed by protein A chromatography. The purity of the samples was assessed by analytical size exclusion chromatography, SDS-PAGE, silver staining and Western blotting.

Embodiment 3

[0187] Example 3: Selective binding of BMP-9 / BMP-10 to proteins comprising a full-length extracellular ENG domain

[0188] ENG is considered a co-receptor and is generally believed to function by facilitating the association of TGF-β1 and TGF-3 to form a multiprotein complex of type I and type II receptors. To investigate the possibility of isolated ENG directly binding ligands, applicants used the surface plasmon resonance (SPR) method (Biacore TM instrument) to screen captured proteins including the full-length extracellular domain of ENG for binding to various soluble human TGF-β family ligands.

[0189]

[0190] *[hBMP-9], [hBMP-10] = 2.5nM; all other ligands detected at 100nM

[0191] **[hBMP-9], [hBMP-10] = 2.5nM; all other ligands detected at 25nM

[0192] ***[hBMP-9], [hBMP-10] = 0.5 nM; [hTGF-β1], [hTGF-β2], [hTGF-β3] = 10 nM; all other ligands detected at 25 nM

[0193] As shown in this table, the binding affinity of hENG(26 to 586)-hFc for hBMP-9 and hBMP-10 ...

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Abstract

Endoglin polypeptides and uses thereof. In certain aspects, the present disclosure relates to the insight that a polypeptide comprising a truncated, ligand-binding portion of the extracellular domainof endoglin (ENG) polypeptide may be used to inhibit angiogenesis in vivo, particularly in mammals suffering angiogenesis-related disorders.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201280030070.0. The original application is the PCT international application PCT / US2012 / 034295 filed on April 19, 2012, which entered the Chinese national phase on December 18, 2013. [0002] related application [0003] This application claims the benefit of the filing date of U.S. Provisional Patent Application Serial No. US 61 / 477,585, filed April 20, 2011, entitled "Endoglin Polypeptides And Uses Thereof," under 35 U.S.C. §119(e), the entire contents of which are incorporated herein by reference. into this article. technical field [0004] The present invention relates to endoglin polypeptides and uses thereof. Background technique [0005] Angiogenesis (the process of forming new blood vessels) is crucial in many normal and abnormal physiological states. Under normal physiological conditions, humans and animals undergo angiogenesis under specific and restrict...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705
CPCC07K14/70596A61K38/00C07K2319/24C07K2319/23C07K2319/21C07K2319/30C07K2319/02C07K2319/705C07K2319/50C07K2319/31C07K2319/41A61P27/02A61P35/00A61P43/00A61P9/00A61K38/179
Inventor 阿斯亚·格林贝格罗斯莱因·卡斯顿盖埃里克·维尔纳拉温德拉·库玛
Owner ACCELERON PHARMA INC
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