Modified ubiquitin proteins having a specific binding activity for the extradomain b of fibronectin

Inactive Publication Date: 2012-11-29
SCIL PROTEINS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although they can be produced quite easily and may be directed to almost any target, they have a quite complex molecular structure.
This overall flexibility of the interaction site by which antibodies bind the epitope is a mainly enthalpically driven process; this process, however, leads to an unfavorable entropic

Method used

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  • Modified ubiquitin proteins having a specific binding activity for the extradomain b of fibronectin
  • Modified ubiquitin proteins having a specific binding activity for the extradomain b of fibronectin
  • Modified ubiquitin proteins having a specific binding activity for the extradomain b of fibronectin

Examples

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example 1

Identification of ED-B Binding Proteins Based on Modified Ubiquitin Proteins

Library Construction for Monomeric Binding Proteins and Cloning

[0240]Unless otherwise indicated, established recombinant genetic methods were used, for example as described in Sambrook et al. A random library of human ubiquitin monomers with high complexity was prepared by concerted mutagenesis of in total up to 10 selected amino acid positions. The modified amino acids, which were substituted by NNK triplets, comprised at least 3 amino acids selected from positions 2, 4, 6, 8, 62, 63, 64, 65, 66, 68 within the ubiquitin monomer.

Library Construction for Hetero-Dimeric Binding Proteins and Cloning

[0241]Unless otherwise indicated, established recombinant genetic methods were used, for example as described in Sambrook et al. A random library of human ubiquitin hetero-dimers with high complexity was prepared by concerted mutagenesis of in total 15 selected amino acid positions. The modified amino acids, which we...

example 2

Production of Fusion Proteins from ED-B-Binding Modified Ubiquitin Variants and TNFalpha (for Example, Human TNFα, Referred to as TNFα)

[0251]The variants can be expressed as fusion proteins between the modified ubiquitins, for example heterodimeric variant1041-D11, and mammalian, for example mouse or human, TNFα in E. coli. Protein analysis of the fusion protein includes: protein expression and purity, no aggregation potential, TNFα activity in cell culture, affinity for target protein ED-B, Selectivity, specific binding in cell culture can be anaylzed. A prerequisite for an animal experiment to induce tumor shrinkage in F9 tumor bearing mice is a fusion with mouse TNFα

Step 1: Production of a Vector for Cloning of Fusion Proteins (pETSUMO— TNFα)

[0252]pETSUMOadapt is a modified vector pETSUMO (Invitrogen), which was modified by insertion of an additional multiple cloning site (MCS). Starting from TNFα cloned in pETSUMOadapt, restriction sites for the insertion of modified ubiquitin v...

example 3

Expression and Purification of Ubiquitin-Based-TNFα Fusion Proteins

[0275]DNA sequence analysis showed the correct sequences of the SUMO-TNFα fusion proteins. For expression of the variants the clones were cultivated in a shaker flask by diluting a preculture 1:100 with LB / Kanamycin and agitating the culture at 200 rpm and 37° C. up to an optical density at 600 nm (OD600) of 0.5. Expression was induced by adding IPTG (final concentration 1 mM). Culturing was continued for 4 hours at 30° C. and 200 rpm. The bacteria cells were harvested by centrifugation at 4° C., 6000×g for 20 min. The cell pellet was suspended in 30 ml of NPI-20 buffer including benzonase and lysozyme. Cells were disrupted by ultrasonication (3×20 sec) on ice. The supernatant containing the soluble proteins was obtained after centrifugation of the suspension at 4° C. and 40000×g for 30 min. Both proteins were purified by affinity chromatography at room temperature. One column of Ni-Agarose (5 ml, GE Healthcare) was ...

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Abstract

The present invention refers to novel recombinant proteins obtained from modified ubiquitin capable of binding the extradomain B of fibronectin (ED-B). Furthermore, the invention refers to fusion proteins comprising said recombinant protein fused to a pharmaceutically and/or diagnostically active component.

Description

FIELD OF THE INVENTION[0001]The present invention refers to novel proteins, in particular hetero-multimeric proteins, capable of binding the extradomain B of fibronectin (ED-B). Furthermore, the invention refers to fusion proteins comprising said binding protein fused to a pharmaceutically and / or diagnostically active component. The invention is further directed to a method for the generation of such binding protein or fusion protein and to pharmaceutical / diagnostic compositions containing the same. In addition, the invention refers to libraries which are based on a scaffold protein comprising linear polyubiquitin chains with at least two interacting binding determining regions (BDR).[0002]In further embodiments, the invention is directed polynucleotides coding for said binding protein or fusion protein, vectors comprising said polynucleotide and host cells comprising said protein, fusion protein, vector and / or polynucleotide. In a preferred embodiment, said binding protein or fusio...

Claims

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Application Information

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IPC IPC(8): A61K38/16C12N9/96A61K33/24G01N33/53C12N15/62C12N15/63C12N1/21A61P35/00C07K14/00A61K51/00
CPCC07K14/525C07K2319/31C07K2319/95C12N15/1044C07K14/435G01N33/6878C12N15/1034C12N15/1037C12N15/1041G01N33/6845C07K14/47A61P35/00C07K14/00C07K19/00C12N15/11C12N15/62
Inventor STEUERNAGEL, ARNDFIEDLER, ERIKFIEDLER, MARKUSKUNERT, ANJANERKAMP, JOERGGOETTLER, THOMASGLOSER, MANJAHAENSSGEN, ILKA
Owner SCIL PROTEINS
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