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Fusion proteins binding to growth factors

A fusion polypeptide and simultaneous combination technology, which is applied in the direction of growth factors/growth regulators, fusion polypeptides, peptide/protein components, etc., can solve problems such as low pharmacokinetic performance

Active Publication Date: 2010-03-24
APLOGEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the Ig-like domain 2 of VEGFR1 itself cannot be used as a therapeutic protein with decoy receptor properties due to its low pharmacokinetic properties

Method used

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  • Fusion proteins binding to growth factors
  • Fusion proteins binding to growth factors
  • Fusion proteins binding to growth factors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0166] Embodiment 1-experimental scheme

[0167] The code consists of the Ig-like domain 1 of human Tie2, the Ig-like domain 2 of human Tie2, the three EGF-like domains of human Tie2, the Ig-like domain of human VEGFR1 and the Fc domain of human IgG Four different assembled fusion proteins (DAAP#1, DAAP#2, DAAP#3 and DAAP#4) ( image 3 A and 3B) gene constructs were cloned into the pCMV-dhfr vector (Hwang SJ, et al., Protein Express Purif.2005; 39:175-183) ( Figure 4 ).

[0168] Since only DAAP#1 exhibited simultaneous binding to VEGF-A and Ang2, seven modified forms (DAAP#11, DAAP#12, DAAP# 13, DAAP #13, DAAP #14, DAAP #15, DAAP #16 and DAAP #17) ( Figure 5 with Image 6 ).

[0169] All DAAP recombinant proteins were obtained by transient expression in HEK293 cells (American Culture Collection of Type Microorganisms, Manassas, VA) using Effectene liposome transfection according to the manufacturer's instructions (Qiagen, Inc., Hilden, Germany) . Supernatants were col...

Embodiment 2

[0173] ELISA analysis showed that DAAP#1, DAAP#14, DAAP#16 and DAAP#17 could bind VEGF-A and Ang2( Figure 10 ). DAAP#2, DAAP#3, DAAP#4, DAAP#11 and Fc cannot bind to VEGF-A and Ang2 ( Figure 10 ). DAAP#12 and VEGF-trap can preferentially and selectively bind to VEGF-A, while DAAP#13 and Tie2-Fc can preferentially and selectively bind to VEGF-A ( Figure 10 ). Since DAAP#1 and DAAP#14 bind VEGF-A and / or Ang2 comparable to VEGF-trap or Tie2-Fc, DAAP#1 and DAAP#14 may be the strongest candidates for further development as therapeutic proteins candidate.

[0174] In order to verify whether the DAAP recombinant protein can simultaneously bind to VEGF and Ang2, 100 ng of each DAAP recombinant protein (DAAP#1, DAAP#12, DAAP#13, DAAP#14, DAAP#15, DAAP#16 and DAAP#17 ), with serial amounts (0 ng, 10 ng, 100 ng, 1,000 ng) of Ang2 or VEGF in 100 μl of blocking solution at 37 °C before they were added to the plates coated with VEGF-A or Ang2 used in the above ELISA -A pre-incubate...

Embodiment 3

[0177] Based on the above findings, DAAP#1 was further investigated for use as a therapeutic protein. Recombinant Chinese hamster ovary (rCHO) cells expressing DAAP#1 (CHO-DAAP#1) were established according to the method described previously (Hwang SJ, et al., Protein Express Purif. 2005; 39:175-183). Briefly, CHO-DAAP#1 cells were transfected into dhfr-deficient CHO cells (CRL-9096, American Culture Collection of Type Microorganisms) by transfecting a vector containing dihydrofolate reductase (dhfr) and a DAAP#1 gene construct. , Manassas, Virginia). This is followed by dhfr / methhotrexate (MTX)-mediated gene amplification. Stable rCHO cells secreting DAAP#1 were selected using sequentially expanded concentrations of MTX (0.02-1.0 [mu]M, Sigma-Aldrich). For recombinant DAAP#1 protein production, CHO-DAAP#1 cells were incubated at 37°C in a humidified 5% CO 2 In an incubator, on an orbital shaker (Vision, Bucheon, Korea) at 110 rpm, 2 × 10 5 cells / mL were inoculated. After...

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Abstract

The present application discloses an isolated nucleic acid molecule encoding a polypeptide capable of synchronously binding two different growth factors polypeptide components. Such a polypeptide is capable of synchronously binding VEGF polypeptide and Angiopoietin polypeptide.

Description

technical field [0001] The present invention relates to the field of anti-angiogenic compositions. The invention also relates to a composition that simultaneously binds two different growth factors or cytokines. Background technique [0002] There are three known VEGF receptors in humans, VEGFR1, VEGFR2 and VEGFR3. Human VEGFR1 consists of 1338 amino acids and is divided into three main domains: an extracellular domain consisting of seven immunoglobulin (Ig)-like domains, a transmembrane domain, and an intracellular tyrosine kinase domain (UniProKB / Swiss-Prot entryP17948)( figure 1 ). VEGFR2 and VEGFR3 have a similar organization and display about 80% identity with VEGFRl on the tyrosine kinase domain. [0003] The activation of VEGFR1 and VEGFR2 by binding to VEGF-A plays a key role in the growth, migration and survival of vascular endothelial cells (which are the basic processes of angiogenesis and angiogenesis), while the activation of VEGFR3 by binding to VEGF-C and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K14/71C07K14/715A61K38/16A61P35/00A61P27/02A61P29/00A61P9/10
CPCC07K14/71C07K2319/00A61K38/00A61P19/02A61P27/02A61P29/00A61P35/00A61P43/00A61P7/10A61P9/00A61P9/10C12N15/62C07K19/00A61K38/16
Inventor 金学祖高永俊金昊民郑基勋全春周高古泳
Owner APLOGEN CO LTD
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