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

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...

AI Technical Summary

Benefits of technology

[0033]An advantage of multimerization of differently modified ubiquitin monomers in order to generate hetero-multimeric binding proteins (here: hetero-dimeric proteins) with monovalent binding activity lies in the increase of the total number of amino acid residues that can be modified to generate a new high affinity binding property to ED-B. The main advantage is that while even more amino acids are modified, the protein-chemical integrity is maintained without decreasing the overall stability of the scaffold of said newly created binding protein to ED-B. The total number of residues which can be modified in order to generate a novel binding site for ED-B is increased as the modified residues can be allocated to two monomeric ubiquitin proteins. The number of modifications can so be two corresponding to the number of modified monomeric ubiquitin molecules. A modular structure of the ubiquitin-based ED-B binding protein allows increasing the overall number of modified amino acids as said modified amino acids are included on two monomeric ubiquitin molecules. The present method provides for the identification of hetero-dimeric ubiquitin molecules having one monovalent specificity for ED-B.

[0171]Therefore, it is a balancing act to construct such a scaffold library suitably between the extreme positions of introducing as many variations as possible into the original sequence in order to optimize it for a target and, on the other hand, of conserving the original primary sequence as much as possible in order to avoid negative protein-chemical effects.

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 contribution by loss of mobility upon association of the flexible complementarity determining region.

Current chemotherapeutic agents and radiation treatment suffer from poor selectivity and most chemotherapeutic agents do not accumulate at the tumor site and thus fail to achieve adequate levels within the tumor.

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