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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • G—PHYSICS
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  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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  • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

• the invention relates to novel scaffolds with high affinity binding properties to targets due to modifications extending the binding site.
• the present invention relates to dimeric modified ubiquitin-based proteins with high binding capability to non-natural target proteins of human ubiquitin.
• the invention refers to fusion proteins or conjugates comprising said dimeric binding protein and diagnostically or therapeutically active components.
• the invention also relates to a multimer of dimeric modified ubiquitins and to multimers of fusion proteins or conjugates thereof and to pharmaceutical compositions containing these multimers.
• the invention is directed to polynucleotides coding for said novel binding protein or fusion protein or conjugate, vectors comprising said polynucleotide and host cells comprising said protein, fusion protein, conjugate, multimer and/or polynucleotide.
• said dimeric binding protein or fusion protein or multimer is included in a medicament or a diagnostic agent.
• WO 04/106368 (Scil Proteins GmbH) describes the generation of artificial binding proteins on the basis of ubiquitin.
• Ubiquitin is a small, monomeric, and cytosolic protein which is highly conserved among Eucaryota. In the organism, it plays a crucial role in an enormous range of physiological processes and attachment of ubiquitin is the second-most common posttranslational modification following phosphorylation.
• Ubiquitin is particularly characterized by beta sheets arranged in an antiparallel manner and subdivided into a and ⁇ segments.
• a characteristic of ubiquitin protein is an antiparallel beta sheet exposed to one surface of the protein onto the back side of which a a helix is packed which lies perpendicularly on top of it. This ubiquitin-like folding motif clearly distinguishes ubiquitin from other proteins.
• ubiquitin proteins also referred to as Affilin ® , a registered trademark of Scil Proteins GmbH
• Affilin ® a registered trademark of Scil Proteins GmbH
• WO 05/05730 generally describes the use of ubiquitin scaffolds in order to obtain artificial binding proteins
• no solution is provided on dimeric ubiquitin proteins in order to obtain a specific and high affinity binding to protein targets.
• novel binding proteins have the potential as effective therapeutics in medicine.
• the object of the present invention is to provide alternative ubiquitin-based binding proteins with high binding specificities for non-natural target proteins of human ubiquitin.
• the problem is solved by extending the binding site for such target proteins.
• the solution to this problem is to insert structures in the modified ubiquitin protein that allow for an extended binding site.
• the structures described by the embodiments of claim 1 allow the formation of an extended structure, optionally a loop, thereby evolving new functionalities, for example by forming an extending binding site for non-natural targets, while preserving the overall structure and function of the ubiquitin scaffold.
• Still further objects are the provision of methods on how to obtain said binding proteins, uses of these novel binding proteins as well as fusion proteins and conjugates comprising said novel binding proteins.
• the present invention relates to a fusion protein or a conjugate comprising a hetero-dimeric modified ubiquitin protein according to the first aspect fused with or conjugated to a pharmaceutically or diagnostically active component, wherein said
• pharmaceutically active component is optionally a cytokine, a chemokine, a cytotoxic compound, a ubiquitin based binding protein or an enzyme, or wherein said diagnostically active component is selected from a fluorescent compound, a photosensitizer, or a
• the present invention relates to a multimer of a hetero-dimeric modified ubiquitin protein according to the first aspect or a fusion protein or conjugate thereof according to the second aspect.
• the present invention relates to a pharmaceutical composition containing a hetero-dimeric modified ubiquitin protein according to the first aspect or a modified ubiquitin fusion protein or a conjugate according to the second aspect or a multimer according to the third aspect or a combination thereof and a pharmaceutically acceptable carrier.
• the present invention relates a diagnostic agent comprising a hetero-dimeric modified ubiquitin protein according to the first aspect or a modified ubiquitin fusion protein or a conjugate according to the second aspect or a multimer according to the third aspectwith a diagnostically acceptable carrier.
• the present invention relates to a polynucleotide encoding the protein as according to the first aspect or a modified ubiquitin fusion protein or a conjugate according to the second aspect or a multimer according to the third aspect.
• the present invention relates to a vector comprising the polynucleotide of the sixth aspect.
• the present invention relates to a host cell the protein as according to the first aspect or a modified ubiquitin fusion protein or a conjugate according to the second aspect or or a multimer according to the third aspect or a polynucleotide as defined in the sixth aspect; or a vector as defined in the seventh aspect.
• the present invention relates to a method for the generation of a hetero- dimeric modified ubiquitin protein according to the first aspect comprising the following steps:
• the present invention relates to a protein according to the first aspect, a fusion protein or conjugate according to the second aspect, or a multimer according to the third aspect for use in a method of medical treatment or diagnosis.
• ubiquitin protein covers the ubiquitin in accordance with SEQ ID NO: 1 and modifications thereof according to the following definition.
• Ubiquitin is highly conserved in eukaryotic organisms. For example, in all mammals investigated up to now ubiquitin has the identical amino acid sequence. Particularly preferred are ubiquitin molecules from humans, rodents, pigs, and primates. Additionally, ubiquitin from any other eukaryotic source can be used. For instance ubiquitin of yeast differs only in three amino acids from the wild-type human ubiquitin.
• the unmodified monomeric ubiquitin proteins covered by said term "ubiquitin protein” show an amino acid identity of more than 70%, preferably more than 75% or more than 80%, of more than 85%, of more than 90%, of more than 95%, of more than 96% or up to a sequence identity of 97% to SEQ ID NO: 1.
• the polypeptide chain of ubiquitin consists of 76 amino acids (SEQ ID NO: 1) and corresponding to a monomer and is folded in an extraordinary compact ⁇ / ⁇ structure (Vij ay- Kumar, 1987): almost 87% of the polypeptide chain is involved in the formation of the secondary structural elements by means of hydrogen bonds. Secondary structures are three and a half alpha-helical turns as well as an antiparallel ⁇ sheet consisting of four strands. The characteristic arrangement of these elements is generally considered as so-called ubiquitin- like folding motif. A further structural feature is a marked hydrophobic region in the protein interior between the alpha helix and the ⁇ sheet.
• the amino acids of the four beta strands which contribute to the formation of the antiparallel beta sheet are according to the invention and according to the structure 1UBQ in the following amino acid positions of SEQ ID NO: 1: First strand (amino-terminal): 2 to 7; second beta sheet strand: 12 to 16; third strand: 41 to 45; fourth strand carboxy- terminal): 65 to 71.
• the position of the strands if the sheet is viewed from the top (amino terminus at the bottom, carboxy terminus on top) from left to right is: 2nd, 1st, 4th, 3rd strand wherein the polypeptide chain between the 1st and 4th strand forms the alpha helix.
• a modified ubiquitin protein refers to modifications of the ubiquitin protein of any one of substitutions, insertions or deletions of amino acids or a combination thereof while substitutions are the most preferred modifications which may be supplemented by any one of the modifications described above.
• the number of modifications is strictly limited as each of said modified monomeric ubiquitin units has an amino acid identity to SEQ ID NO: 1 of at least one of the group of 75%, at least 83%, at least 85%%, at least 87% and at least 90%.
• the overall number of substitutions in a monomeric unit related to a novel binding property to a non-natural target is, therefore, limited to 16 amino acids corresponding to 80% amino acid identity taking into account only substitutions and deletions.
• said identity to SEQ ID NO: 1 may be between 75% and 80%.
• the total number of substituted or deleted amino acids in the dimeric ubiquitin molecule may be up to 32 amino acids. This is corresponding to about 20% amino acids that are substituted based on the unmodified dimeric ubiquitin protein.
• the amino acid identity of the dimeric modified ubiquitin protein compared to a dimeric unmodified ubiquitin protein with a basic monomeric sequence of SEQ ID NO: 1 is selected from at least one of the group of at least 75%, at least 80%, at least 83%, at least 85%%, at least 86%, at least 87%, at least 88%, at least 89% and at least 90%.
• loop or "loop region” refers to regions of non-repetitive conformations connecting regular secondary structure elements such as alpha-helix or beta-strands of ubiquitin.
• the structure of human ubiquitin reveals 7 reverse turns (loops) which connect secondary structure elements: 7-11, 18-21, 37-40, 45-48, 51-54, 57-60, 62-65 (Vijay-Kumar et al. 1987 J Mol Biol.;194(3):531-44).
• insertions comprises the addition of amino acids to the original amino acid sequence of a protein. In this invention, the additional amino acids to a ubiquitin monomer are described wherein the ubiquitin remains stable without significant structural change. Location of the insertion in the dimeric ubiquitin
• Said monomeric ubiquitin units contain an insertion in one or in both ubiquitin monomers, preferably in one monomer. It is also possible that the two ubiquitin monomers contain each identical or different insertions. In a still further embodiment, two or three or four insertions are included in one or in both ubiquitin monomers.
• the insertion may comprise 6 - 10 amino acids or 7 - 9 amino acids or 8 amino acids in one or both monomeric ubiquitin proteins or any other number (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) of amino acid insertions covered by the range of 2 to 15 amino acids. At the most insertions of 15 amino acids may be tolerated by an monomeric ubiquitin, preferably 6 - 10 amino acids, most preferred 8 amino acids.
• An insertion of 6 tolO (6, 7, 8, 9, or 10) amino acids, preferably 8 amino acids, is extending the natural loop region and thereby extending the binding site for targets which is beneficial for the binding interaction between the target and the modified ubiquitin (Affilin®).
• the insertion of amino acids is closely adjacent, optionally 0, 1, 2, 3, 4, or 5 amino acids, distant from beta sheet strands, preferably distant from the fourth (C- terminal) or the first (N-terminal) beta- strand, optionally wherein said insertion is located in the N-terminal (first) ubiquitin monomer.
• the insertion is generally not located in a beta sheet but adjacent to beta sheets, optionally 0, 1, 2, 3, 4, or 5 amino acids, distant from beta sheets thereby forming an extended structure close to substituted amino acids in beta sheets. It is preferred that the insertion is 0, 1, 2, 3, 4, or 5 amino acids distant from the fourth (C- terminal) or the first (N-terminal) beta- strand.
• a preferred insertion of amino acids is in one loop region of said modified monomeric ubiquitin.
• a loop region in ubiquitin refers to residues 7 - 11, 18 - 21, 37 - 40, 45 - 48, 51 - 54, 57 - 60, 62 - 65 of SEQ ID NO: 1, as further defined below.
• an insertion of additional amino acids is before (between positions 61 and 62) or within the loop region (amino acids 62 to 64) which is adjacent to the C-terminal beta sheet, thereby extending the existing loop region and forming a larger binding site.
• the insertion is located in the loop region of residues 61 to 65, between amino acid residues corresponding to amino acids 61 - 62 or 62 - 63 or 63 - 64 or 64 - 65 of SEQ ID NO: 1, most preferred between amino acids corresponding to positions 61 and 62 of SEQ ID NO: 1. It is most preferred that an insertion of amino acids is C-terminal to substitutions of amino acids in the fourth beta-strand or in other words, that the insertion of amino acids is close to or within the loop region of amino acids 62 to 65.
• the inserted sequence does not affect the global structure, but the insertion site does.
• the insertion of amino acids is preferred between amino acids corresponding to 61 - 62 or 62 - 63 or 63 - 64 or 64 - 65 of human ubiquitin of SEQ ID NO: 1, most preferred between amino acids corresponding to positions 61 and 62 of SEQ ID NO: 1.
• Such an insertion in the adjacent region to the fourth beta strand extends the natural loop region significantly, thereby forming an extended binding site for non-natural targets.
• insertion in one monomeric ubiquitin unit there is only one insertion in one monomeric ubiquitin unit.
• an insertion in the N-terminal (first) monomeric ubiquitin unit of the dimer Said insertion may participate in the newly generated binding of the modified ubiquitin to a non-natural target protein, for example to VEGF-A and its isoforms.
• a further positive effect of the insertion is an increase of the number of amino acids which may be substituted and may therefore participate in binding to the target.
• the insert may optionally form an extended structure, for example, a loop structure. The elongation of the ubiquitin structure by adding additional amino acids has no significant effects on the overall conformation and stability of the protein.
• the modified hetero-dimeric ubiquitin scaffold having substitutions and insertions remains soluble.
• the invention provides for engineered, novel high affinity binding alternative scaffolds with extended interaction interface for non-natural targets.
• the formation of an extended or elongated structure, e.g. a loop structure, in close proximity to the substituted amino acids has several advantages.
• First, the target binding site is extended by the additional amino acids without disrupting the overall structure of the protein.
• the overall structure and function of the ubiquitin scaffold is preserved although supplementary amino acids are added to the protein. This results in binding to the non-natural targets with high affinity and specificity.
• Second, by forming an extended structure the conformation might be changed in such a way that special targets or epitopes could be reached.
• the structural flexibility has the positive effect that the binding site is not fixed and therefore, the binding of targets to such a binding site formed by a combination of substituted amino acids and insertions is stronger. The binding would not be hindered by changes to the conformation of the target.
• the insertion site close to the C-terminal beta-sheet is thus more important for the binding to targets than the sequence of the insert itself. The insertion site is determining the target binding.
• the SIM Local similarity program (Xiaoquin Huang and Webb Miller, " Advances in Applied Mathematics, vol. 12: 337- 357, 1991) or Clustal, W. can be used (Thompson et al., Nucleic Acids Res., 22(22): 4673-4680, 1994.).
• the extent of the sequence identity of the modified protein to SEQ ID NO: 1 as defined herein is determined relative to the complete sequence of SEQ ID NO: 1.
• the extent of sequence identity between a modified sequence and the sequence from which it is derived (also termed: "parent sequence") is generally calculated with respect to the total length of the unmodified sequence, if not explicitly stated otherwise.
• target is a protein and more specifically an antigenic epitope present on proteins.
• a target, ligand and binding partner as understood in this invention is any protein capable of binding with an affinity as defined herein to the hetero-dimeric modified ubiquitin protein.
• the target or ligand or binding partner of the invention is a non- natural target for human ubiquitin or for ubiquitin dimers. This implies that the binding property is de novo generated and did not exist before creating hetero-dimeric modified ubiquitin binding proteins.
• VEGF-A or briefly designated as “VEGF” comprises all proteins which show a sequence identity to SEQ ID NO: 13 (accession number P15692) of at least 70%, optionally 75%, further optionally 80%, 85%, 90%, 95%, 96% or 97% or more, or 100% and having the above defined functionality of VEGF.
• VEGF-A or briefly designated as “VEGF” also comprises isoforms of VEGF-A; well-known isoforms of VEGF-A are VEGF 121 und VEGF 165.
• Ubiquitin dimers A "dimer” is considered as a protein in this invention which comprises two monomeric ubiquitin proteins (ubiquitin monomers).
• the dimer comprises two differently modified monomers, it is called a "heteromeric-dimer” or “hetero-dimer”.
• the "hetero-dimeric fusion protein” or “hetero-dimeric protein” of the invention is considered as a protein which comprises at least two differently modified monomeric ubiquitin proteins with binding regions providing together a specific binding property for a non-natural target as the specific binding partner.
• a hetero-dimer is accomplished by fusing two monomeric ubiquitin molecules wherein both of these molecules are differently modified as described herein.
• the "homo-dimeric fusion protein” or “homo-dimeric protein” of the invention is considered as a protein which comprises two identically modified monomeric ubiquitin proteins with binding regions.
• A-A Homo-Dimer
• A-B Hetero-Dimer
• A-B-A-B A-A-A-B
• A-B-A-A A-A-A-A
• A-B-C-D A-A-C-D.
• the constructs A-B-C-D and A-A-C-D can be bi-specific binding proteins with specificity for two different targets.
• the two modified ubiquitin monomers which bind to one protein ligand are to be linked by head-to-tail fusion to each other using e.g. genetic methods.
• a "head to-tail fusion" is to be understood in this invention as fusing two proteins together by connecting them in the direction N-terminus to C-terminus.
• the ubiquitin monomers may be connected directly without any linker.
• the fusion of ubiquitin monomers can be performed via linkers, for example, a polypeptide linker.
• linker refers to a molecule that joins at least two other molecules either covalently or non-covalently, e.g., through hydrogen bonds, ionic or van der Waals interactions, e.g., a nucleic acid molecule that hybridizes to one complementary sequence at the 5' end and to another complementary sequence at the 3' end, thus joining two non- complementary sequences.
• a “linker” is to be understood in the context of the present application as a moiety that connects a first polypeptide with at least a further polypeptide.
• the second polypeptide may be the same as the first polypeptide or it may be different.
• peptide linkers are peptide linkers.
• the peptide linker is an amino acid sequence that connects a first polypeptide with a second polypeptide.
• the peptide linker is an amino acid sequence which is able to link two ubiquitin monomers.
• a peptide linker has a length of between 1 and 20 amino acids; e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. It is preferred that the amino sequence of the peptide linker is not immunogenic to human beings.
• linker is a glycine-serine-linker of variable length, for example, having at least the amino acid sequence GIG (SEQ ID NO: 14) or having at least the amino acid sequence SGGGG, for example GIG (SEQ ID NO: 14), SGGGG (SEQ ID NO: 15), SGGGGIG (SEQ ID NO: 16), SGGGGSGGGGIG (SEQ ID NO: 17) or SGGGGSGGGG (SEQ ID NO: 18) or (SGGG)n wherein n is any number between 1 to 4
• the linkers may have a length between 2 and 16 amino acids. Also other linkers for the genetic fusion of two ubiquitin monomers are known in the art and can be used.
• the two monomeric proteins are not linked together after having screened the most potent binding ubiquitin molecules but already the screening process is performed in the presence of the dimeric ubiquitins.
• these molecules may be obtained by any other method, for example by chemical synthesis or by genetic engineering methods, e.g. by linking the two already identified monomeric ubiquitin units (ubiquitin monomers) together.
• the aforesaid also applies to homomers of the modified ubiquitin protein which provides a more efficient binding to a non-natural ligand protein.
• modified novel binding proteins having an insertion in at least one monomer and substitutions bind to VEGF, preferably VEGF-A and isoforms as non- natural ligand protein.
• VEGF vascular endothelial growth factor
• isoforms as non- natural ligand protein.
• multiple spliced isoforms of VEGF-A have been identified. The most common isoforms are composed of 121, 165 and 189 amino acids, and the murine homologues lack one amino acid per isoform.
• the longer splice isoforms of VEGF-A, including VEGF165 contain a highly basic heparin-binding domain.
• modified ubiquitin binding proteins with insertions and substitutions could bind to any other non- natural target.
• substitution of amino acids for the generation of the novel binding domain specific to a given target, for example VEGF-A can be performed according to the invention with any desired amino acid, i.e. for the modification to generate the novel binding property to a target, e.g. VEGF-A; it is not mandatory to take care that the amino acids have a particular chemical property or a side chain, respectively, which is similar to that of the amino acids substituted so that any generally amino acid desired can be used for this purpose provided it enhances the binding affinity to VEGF-A and does not deteriorate the structural integrity of the ubiquitin binding molecule.
• the step of modification of the selected amino acids is performed according to the invention preferably by mutagenesis on the genetic level by random mutagenesis, i.e. a random substitution of the selected amino acids.
• the modification of ubiquitin is carried out by means of methods of genetic engineering for the alteration of a DNA belonging to the respective protein.
• expression of the ubiquitin protein is then carried out in prokaryotic or eukaryotic organisms.
• the amino acid residues are altered by the combination of amino acid substitutions and by insertions at least in one monomer.
• the number of amino acids which may be inserted is limited to 2 to 15 amino acids in a ubiquitin monomer, and accordingly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids with respect to the dimeric ubiquitin protein.
• amino acid insertions are made in one monomeric subunit of the dimeric ubiquitin based binding protein, preferably in the N-terminal (first) monomeric subunit. Preferred positions for insertion are differently or identically modified and/or wherein 6 - 10 amino acids or 7 - 9 amino acids or 8 amino acids are inserted in one or both monomeric ubiquitin monomer.
• insertion of amino acids is in one loop region of said modified monomeric ubiquitin or within or in close proximity of the amino acid substitutions specified in claim 1, optionally 0, 1, 2, or 3 amino acids in direction of the N- or C-terminus of the substituted positions specified in claim 1.
• the insertion of amino acids is closely adjacent, optionally 0, 1, 2, 3, 4, or 5 amino acids, distant from beta sheet strands, preferably from the fourth (C-terminal) or the first (N-terminal) strand. Preferred are insertions in the N-terminal (first) monomer.
• Optional positions for insertion of amino acids are for example positions corresponding to human ubiquitin positions 61 - 62 or 62 - 63 or 63 - 64 or 64 - 65 which are closely adjacent to the substituted amino acid positions. Specifically the following positions for insertion of amino acids are chosen corresponding to human ubiquitin positions: 9 - 10 (1st beta-strand corresponding to amino acids 2- 7), 35 - 36 (3rd beta-strand corresponding to amino acids 41- 45), and most preferred before position 65 in close proximity to the 4 th beta-strand (4th beta- strand corresponding to amino acids 65 - 71). Provided that e.g.
• non-human ubiquitin is used as starting protein (for example ubiquitin of yeast), the amino acid positions given for wild type human ubiquitin (SEQ ID NO: 1) have to be aligned with the modified ubiquitin in order to allot the corresponding proteins and amino acid positions to each other.
• the numbering (and alignment) of each of the monomeric ubiquitin subunits is done in the same way, i.e. an alignment of, for example, a dimer is started at amino acid position 1 for each respective subunit.
• the modifications of a monomeric ubiquitin according to the invention used as building unit for a hetero-dimer accounts for in total up to about 25% of amino acids if the modifications comprise the combination of both substitutions and insertions.
• the modifications of a monomeric ubiquitin according to the invention used as building unit for a hetero- dimer accounts for in total up to about 15%, preferably 10%, of amino acids if the modifications comprise only substitutions. Considering this, there is a sequence identity to SEQ ID NO: 1 of the modified monomeric ubiquitin protein of at least 75% if modifications comprise substitutions and insertions.
• sequence identity to SEQ ID NO: 1 of the modified monomeric ubiquitin protein of at least 80%, at least 83%, at least 85%, at least 87% if modifications comprise only substitutions.
• sequence identity on amino acid level is at least 80%, at least 83%, at least 85%, at least 87% and furthermore at least 90% or at least 93% sequence identity to the amino acid sequence of SEQ ID NO: 1. If only substitutions are considered that are involved in generating a new binding to a target (and not substitutions related to biochemical properties, e.g.
• sequence identity on amino acid level is at least 88%, at least 91%, at least 92%, at least 93% to the amino acid sequence of SEQ ID NO: 1. If substitutions and insertions are considered that are involved in generating a new binding to a target (and not substitutions related to biochemical properties, e.g. changes in Position 45, 75, 76) then the sequence identity on amino acid level is at least 80% to the amino acid sequence of SEQ ID NO: 1. Thus, for example, in one embodiment, 7 amino acids are substituted and 8 amino acids are inserted to generate a new binding property, accounting to a sequence identity of at least 80% to SEQ ID NO: 1.
• each of two ubiquitin monomers is substituted in 5 or 6 or 7 amino acids selected from regions 2 - 8 and 62 - 68, preferably selected from positions 6, 8, 62, 63, 64, 65, 66 of SEQ ID NO: 1 and additionally 2 - 15 amino acids are inserted at region 61 - 65, preferably in position 61 - 62 or 62 - 63 or 63 - 64 or 64 - 65 of the first (N-terminal) monomer, thus within close proximity to said substituted amino acids.
• substitutions in region 2 to 8 of SEQ ID NO: 1 and a combination of substitutions and an insertion of 2 to 15 amino acids in at least one monomer of ubiquitin in amino acid region 61 to 68 of SEQ ID NO: l are preferred.
• the ubiquitin monomers to be modified in these positions were already pre-modified which does not influence the binding of targets.
• further modifications could comprise substitutions at amino acids 75 and 76 or at amino acid 45 to generate better stability or protein-chemical properties.
• a modified ubiquitin monomer is obtainable wherein at least 5 amino acids, but in total up to 9, 10, 11, 12, 13, 14, 15 and a maximum of 16 amino acids, most preferred 5 to 9 amino acids, of the monomeric ubiquitin of SEQ ID NO: 1 are substituted. Additional 2 to 15 amino acids are inserted into the sequence.
• a modified monomeric ubiquitin could be obtained having 8 substitutions being involved in novel binding to a target and an insertion of 8 amino acids (for example, further 3 amino acids can be modified that does not affect the binding). Based on the total number of amino acids of ubiquitin this corresponds to a percentage of all modifications of about 26% (modifications involved in binding: about 22%). This was extraordinarily surprising and could not be expected since usually a much lower percentage is already sufficient to disturb the folding of the protein.
• mutagenesis of surface exposed amino acids these can be identified with respect to the available X-ray crystallographic structure. If no crystal structure is available attempts can be made by means of computer analysis to predict surface-exposed amino acids and the accessibility of individual amino acid positions with respect to the available primary structure or to model the 3D protein structure and to obtain information about potential surface- exposed amino acids in this manner. Further disclosure thereof can be taken e.g. from Vijay- Kumar et al. 1987 J. Mol. Biol. 194(3):531-44.
• Surface exposed amino acid positions to be mutagenized, preferably substituted, are subjected to random mutagenesis and are afterwards re-integrated into the DNA coding for the protein from which they were removed previously. This is followed by a selection process for mutants with the desired binding properties.
• "Surface-exposed amino acids” are amino acids that are accessible to the surrounding solvent. If the accessibility of the amino acids in the protein is more than 8% compared to the accessibility of the amino acid in the model tripeptide Gly-X-Gly, the amino acids are called "surface-exposed".
• amino acid positions to be mutagenized within these selected regions are identified.
• the amino acid positions selected in this way can then be mutagenized on the DNA level either by site-directed mutagenesis, i.e. a codon coding for a specific amino acid is substituted by a codon encoding another previously selected specific amino acid, or this substitution is carried out in the context of a random mutagenesis wherein the amino acid position to be substituted is defined but not the codon encoding the novel, not yet determined amino acid.
• Variations of ubiquitin protein differing by amino acid substitutions or/and amino acid insertions in the region of the de novo generated artificial binding site from the parental protein and from each other can be generated by a targeted mutagenesis of the respective sequence segments.
• amino acids having certain properties such as polarity, charge, solubility, hydrophobicity or hydrophilicity can be replaced or substituted, respectively, by amino acids with respective other properties.
• mutagenesis and “modified” and “replaced” comprise also insertions.
• modifications can also be carried out by chemical alteration of the amino acid side chains according to methods known to those skilled in the art.
• the cDNA of ubiquitin which can be prepared, altered, and amplified by methods known to those skilled in the art can be used.
• site-specific alteration of ubiquitin in relatively small regions of the primary sequence about 1-3 amino acids
• commercially available reagents and methods are on hand ("Quik Change", Agilent; "Mutagene Phagemid in vitro Mutagenesis Kit", Bio-Rad).
• site-directed mutagenesis of larger regions specific embodiments of e.g. the polymerase chain reaction (PCR) are available to those skilled in the art.
• a mixture of synthetic oligodeoxynucleotides having degenerated base pair compositions at the desired positions can be used for example for the introduction of the mutation.
• This can also be achieved by using base pair analogs which do not naturally occur in genomic DNA, such as e.g. inosine.
• Starting point for the mutagenesis of can be for example the cDNA of ubiquitin or also the genomic DNA.
• the gene coding for the ubiquitin protein can also be prepared synthetically.
• mutagenesis comprising inter alia methods for site-specific mutagenesis, methods for random mutagenesis, mutagenesis using PCR or similar methods.
• the amino acid positions to be mutagenized are predetermined.
• the selection of amino acids to be modified is carried out to meet the limitations of present claim 1 with respect to those amino acids which have to be modified.
• a library of different mutants is generally established which is screened using methods known per se.
• a pre- selection of the amino acids to be modified can be particularly easily performed as sufficient structural information is available for the ubiquitin protein to be modified.
• Random mutagenesis can be introduced by propagation of the DNA in mutator strains or by PCR amplification (error-prone PCR) (e.g. Pannekoek et al., 1993 Gene 128, 135 140). For this purpose, a polymerase with an increased error rate is used.
• the mutations in the PCR fragments can be combined by means of DNA shuffling (Stemmer, 1994 Nature 370,
• Random modification is performed by methods well-established and well-known in the art.
• a "randomly modified nucleotide or amino acid sequence” is a nucleotide or amino acid sequence which in a number of positions has been subjected to insertion, deletion or substitution by nucleotides or amino acids, the nature of which cannot be predicted.
• the random nucleotides (amino acids) or nucleotide (amino acid) sequences inserted will be” completely random” (e. g. as a consequence of randomized synthesis or PCR-mediated mutagenesis).
• the random sequences can also include sequences which have a common functional feature (e. g. reactivity with a ligand of the expression product) or the random sequences can be random in the sense that the ultimate expression product is of completely random sequence with e. g. an even distribution of the different amino acids.
• the random nucleotides are introduced into the expression vector by the principle of site directed PCR-mediated mutagenesis.
• other options are known to the skilled person, and it is e. g. possible to insert synthetic random sequence libraries into the vectors as well.
• To generate mutants or libraries by fusion PCR for example three PCR reactions may carried out. Two PCR reactions are performed to generate partially overlapping intermediate fragments. A third PCR reaction is carried out to fuse the intermediate fragments.
• the method for construction the library or mutant variants may include constructing a first set of primers around a desired restriction site (restriction site primer), a forward and reverse restriction primer and a second set of primers around, e.
• the cohesive ends of the linear product are fused by use of a DNA ligase to produce a circular product, e. g. a circular polynucleotide sequence.
• a DNA ligase to produce a circular product, e. g. a circular polynucleotide sequence.
• the design and synthesis of two sets of forward and reverse primers are performed, a first set containing a restriction enzymes digestion site together with its flanking nucleotide sequence, and the second set contains at least one variant codon of interest (mutagenic primers).
• mutagenic primers variant codon of interest
• the amplification product obtained can be added to another polymerase chain reaction using flanking oligodeoxynucleotides which introduce for example recognition sequences for restriction endonucleases. It is preferred according to the invention to introduce the gene cassette obtained into a vector system suitable for use in the subsequent selection procedure for the isolation of ubiquitin variations having binding properties to a
• the regions for modification can be basically selected as to whether they can be accessible for said non-natural target protein of human ubiquitin, e.g. VEGF, in particular VEGF-A or its isoforms, as binding partner and whether the overall structure of the protein will presumably show tolerance to a modification.
• VEGF vascular endothelial growth factor
• VEGF-A vascular endothelial growth factor-A
• VEGF-A vascular endoforms
• a substitution of one or more of the surface-exposed amino acids of regions 2 - 8 and 62 - 68 of a ubiquitin monomer Prefered are substitutions selected from amino acids of the following positions of a ubiquitin monomer, preferably mammalian (human) ubiquitin: 2, 4, 6, 8, 62, 63, 64, 65, 66, 68 of SEQ ID NO: 1 or of those amino acids corresponding to these positions.
• the dimer of ubiquitin according to the invention binding to a non-natural protein target with
• the fusion protein is a genetically fused hetero-dimer of said ubiquitin monomer having substitutions in positions 2, 4, 6, 8, 62-66, 68 of the first ubiquitin monomer and 2 - 15 amino acids inserted, for example between the following amino acid residues corresponding to wild-type human ubiquitin 61 - 62, 62 - 63, 63 - 64 and/or 64 - 65, and substitutions in amino acid residues in positions 2, 4, 6, 8, 62-66, 68 of the second ubiquitin monomer, preferably those shown in Figure 1.
• insertions of up to 8 amino acids in the c-terminal region of the monomeric ubiquitin are more preferred. More preferred are insertions in those positions that are closely adjacent to the fourth beta sheet (before amino acid position 65). Most preferred are insertions in close proximity to said substitutions, preferably between amino acids 61 and 62, 62 and 63, 63 and 64, 64 and 65, most preferred between amino acids 61 and 62.
• Substitutions in the first monomeric unit at least K6Y, L8D, Q62S, K63W, E64M, S65P, and T66A;
• Table 1 shows preferred amino acid substitutions in hetero-dimeric ubiquitin-based VEGF-A binding proteins with 8 amino acid insertion in the first monomer (insertion not shown in this table).
• positions 6, 8, 62, 63, 64, 65, 66 substitutions in the N-terminal (first) ubiquitin monomer
• positions 6', 8', 62', 63', 64', 65', 66' substitutions in the C-terminal (second) ubiquitin monomer of the binding protein.
• Further substitutions in other positions are not shown but are possible.
• substitutions that are not relating to the binding to a non-natural target such as substitutions in position 45, 75, and 76 are not shown.
• the "-" indicates that there is no substitution in this position; rather the wild-type amino acid remains. Please refer to Figure 1 a-i for the complete sequence information. Table 1. Preferred substitutions of modified ubiquitin proteins required for binding
• consensus substitutions are the following: K6Y, L8D, Q62S, K63W, E64M, S65P, T66A.
• the insertion is located within the first monomer, closely adjacent to the 4 th (C-terminal) beta sheet and thus closely adjacent the substituted amino acids in positions 62 - 66 (in direction of the N-terminus), preferably between amino acids 61 and 62, 62 and 63, 63 and 64, 64 and 65, most preferred between amino acids 61 and 62.
• the second monomer unit for the dimeric ubiquitin shows variable substitutions, wherein in these embodiments 5- 7 amino acids are substituted in positions 6, 8, 62, 63, 64, 65, and/or 66 of SEQ ID NO: 1.
• the present invention covers also polynucleotides which encode for a protein or a modified ubiquitin fusion protein or conjugate as described further below. Additionally, vectors comprising said polynucleotides are covered by the invention. In an additional aspect of the present invention, host cells are covered which comprise a dimeric modified ubiquitin protein or a fusion protein or conjugate described herein and/or a polynucleotide coding for said recombinant protein or fusion protein or conjugate of the invention or a vector containing said polynucleotide.
• Binding specificities (Dissociation constants)
• Kd Binding specificities
• 10 " 7' - 10 " 1 1 2" M A value of 10 " 5 J M and below can be considered as a quantifiable binding affinity.
• a value of 10 - " 7 M to 10 - " 11 M is preferred for e.g.
• binding affinities are in the range of 10 "7 to 10 "10 M, preferably to 10 "11 M.
• the methods for determining the binding affinities are known per se and can be selected for instance from the following methods: ELISA, Surface Plasmon Resonance (SPR) based technology (offered for instance by Biacore®), fluorescence spectroscopy, isothermal titration calorimetry (ITC), analytical ultracentrifugation, FACS.
• SPR Surface Plasmon Resonance
• ITC isothermal titration calorimetry
• FACS analytical ultracentrifugation
• the hetero-ubiquitin-based binding molecule is covalently or non-covalently fused with or conjugated to a protein or peptide or chemical compound having therapeutically or diagnostically relevant properties.
• fusion protein relates to a fusion protein comprising a binding or non-binding protein of the invention fused to a functional or an effector component.
• the invention relates to a fusion protein comprising a hetero -dimeric binding protein of the invention as targeting moiety fused to a functional or an effector domain.
• a fusion protein of the invention may further comprise non-polypeptide components, e.g. non-peptidic linkers, non-peptidic ligands, e.g. for therapeutically or diagnostically relevant radionuclides. It may also comprise small organic or non-amino acid based compounds, e.g. a sugar, oligo- or polysaccharide, fatty acid, etc.
• fusion describes a multimeric modified ubiquitin which is fused either by covalent bonds or by molecular interactions with a therapeutically or diagnostically molecule, e.g. a protein or a non-protein chemical substance. Fusion with other protein or peptide molecules preferably takes place by genetic means. However, there is no sharp border line limiting the term “fusion” and “conjugate” so that both may overlap; for these reasons, both terms are used interchangeably.
• ubiquitin-based fusion proteins or conjugates with binding capacity to a given target e.g. VEGF-A or its isoforms: a) conjugation of the protein via Lysine residues present in ubiquitin;
• Cysteine residues - can be located C-terminal, or at any other position (e.g. amino acid residue 24 or 57); conjugation with maleimid selectable components;
• hetero-dimeric ubiquitin-based binding protein according to the invention may contain artificial amino acids.
• said active component is preferably a component selected from the groups of a radionuclide either from the group of gamma-emitting isotopes, preferably 99T c , 123I, l l li n , or from the group of positron emitters, preferably 18F, 64C u , 68o a , 86 ⁇ ,124 ⁇ 5 or from the group of beta-emitter, preferably 13 li, 90 ⁇ , 177 Lu , 67 Cu , or from the group of alpha-emitter, preferably 213BI, 211 At ; or a fluorescent dye, preferably Alexa Fluor or Cy dyes (Berlier et al., J. Histochem.
• a further embodiment relates to fusion proteins according to the invention, further comprising a component modulating serum half-life, preferably a component selected from the group consisting of polyethylene glycol, albumin-binding peptides, and immunoglobulin or immunoglobulin fragments.
• the modified ubiquitin binding proteins of the invention are to be used for instance for preparing diagnostic means for in vitro or in vivo use as well as therapeutic means.
• the proteins according to the invention can be used e.g. as direct effector molecules (modulator, antagonist, agonist) or antigen-recognizing domains.
• the pharmaceutical composition of the invention can be used for treatment of cancer, e.g. breast or colon cancers, or any other tumor diseases in which VEGF-A is abundant.
• VEGF-A binding proteins can be used for eye diseases, such as age-related macular degeneration (AMD) or diabetic macular edema (DME).
• AMD age-related macular degeneration
• DME diabetic macular edema
• the compositions are adapted to contain a therapeutically effective dose.
• the quantity of the dose to be administered depends on the organism to be treated, the type of disease, the age and weight of the patient and further factors known per se.
• the invention covers a pharmaceutical composition containing a dimeric modified ubiquitin protein or a modified ubiquitin fusion protein or a conjugate or a combination thereof and a pharmaceutically acceptable carrier.
• the invention further covers a diagnostic agent comprising a dimeric modified ubiquitin protein or a fusion protein or conjugate with a diagnostically acceptable carrier.
• the compositions contain a pharmaceutically or
• the pharmaceutical composition can be in the form of a liquid preparation, a cream, a lotion for topical application, an aerosol, in the form of powders, granules, tablets, suppositories, or capsules, in the form of an emulsion or a liposomal preparation.
• the compositions are preferably sterile, non-pyrogenic and isotonic and contain the pharmaceutically conventional and acceptable additives known per se. Further reference is made to the regulations of the U.S. Pharmacopoeia or Remington's Pharmaceutical Sciences, Mac Publishing Company (1990).
• medicaments containing at least one dimeric VEGF-A binding ubiquitin protein modified in accordance with the invention can be prepared by methods known per se.
• compositions can be administered parentally by injection or infusion, systemically, rectally, intraperitoneally, intramuscularly,
• a "pharmaceutical composition” according to the invention may be present in the form of a composition, wherein the different active ingredients and diluents and/or carriers are in admixed with each other, or may take the form of a combined preparation, where the active ingredients are present in partially or totally distinct form.
• An example for such a combination or combined preparation is a kit-of-parts.
• the pharmaceutical composition is in the form of a kit of parts, providing separated entities for the recombinant ubiquitin protein/fusion protein of the invention and for the one or more chemo therapeutic agents.
• the invention discloses diagnostic compositions comprising modified ubiquitins according to the invention specifically binding e.g. tumor- associated proteins, e.g. VEGF-A or its isoforms together with diagnostically acceptable carriers. Since enhanced expression of e.g. tumor associated molecules is correlated with tumor malignancy, the modified ubiquitins with binding capacity to said tumor- specific target molecules can also be used as a diagnostic agent for non-invasive imaging in order to gain information about e.g. VEGF in patients.
• the modified ubiquitins with binding capacity to e.g. VEGF- A and its isoforms can be used for the assessment of the response of a patient to an anti- angiogenic therapy. Due to their small size and high affinity, radiolabeled proteins based on a ubiquitin scaffold are of high importance for use as an e.g. VEGF imaging diagnostic.
• a recombinant protein and/or a fusion protein or conjugate is covered for use in a method of medical treatment or diagnosis.
• binding proteins according to the invention may be prepared by any of the many conventional and well known techniques such as plain organic synthetic strategies, solid phase-assisted synthesis techniques or by commercially available automated synthesizers. On the other hand, they may also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques.
• a method for generating a recombinant modified ubiquitin protein with novel binding property is provided. The method comprises at least the following steps:
• a method for identifying a modified ubiquitin protein comprises at least the following steps:
• a further embodiment covers a method for generating a hetero-dimeric fusion protein or conjugate, comprising the following steps:
• a further embodiment covers a method of preparation of a protein as defined in the first aspect, said method comprising the following steps:
• Example 1 outlines the production of a complex library.
• Quality of a library in scaffold technology is in the first place dependent from its complexity (number of individual variants) as well as functionality (structural and protein-chemical integrity of the resulting candidates). Both characteristics, however, may exert negative influences on each other: enhancing the complexity of a library by increasing the number of modified positions on the scaffold might lead to a deterioration of the protein-chemical characteristics of the variants. This might result in a decreased solubility, aggregation and/or low yields. A reason for this is the larger deviation from native scaffolds having an energetically favourable protein packaging.
• these libraries are genetically fused by e.g. linker technology to obtain DNA molecules encoding for hetero-dimeric modified ubiquitin proteins.
• the DNA of these libraries is expressed into proteins and the modified dimeric proteins obtained thereby are contacted according to the invention with the ligand VEGF-A to optionally enable binding of the partners to each other if a binding affinity does exist.
• the contacting and screening process is performed already with respect to the homo- or hetero-dimeric ubiquitin protein. This process enables screening on those ubiquitin proteins which provide a binding activity to a given target.
• Contacting according to the invention is preferably performed by means of a suitable presentation and selection method such as the phage display, ribosomal display, mRNA display or cell surface display, yeast surface display or bacterial surface display methods, preferably by means of the phage display method.
• a suitable presentation and selection method such as the phage display, ribosomal display, mRNA display or cell surface display, yeast surface display or bacterial surface display methods, preferably by means of the phage display method.
• a suitable presentation and selection method such as the phage display, ribosomal display, mRNA display or cell surface display, yeast surface display or bacterial surface display methods, preferably by means of the phage display method.
• the determination whether the modified protein has a quantifiable binding affinity with respect to a predetermined binding partner can be performed according to the invention preferably by one or more of the following methods: ELISA, plasmon surface resonance spectroscopy, fluorescence spectroscopy, FACS, isothermal titration calorimetry and analytical ultracentrifugation.
• the protein modified according to the invention can be fused to other peptide sequences having an increased affinity to separation materials.
• such fusions are selected that do not have a detrimental effect on the functionality of the ubiquitin protein or can be separated after the purification due to the introduction of specific protease cleavage sites.
• Such methods are also known per se to those skilled in the art. Vectors, host cells and methods of production of proteins
• Vectors may be expression and cloning vectors containing a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host
• Expression and cloning vectors may contain a selection gene, also termed a selectable marker.
• Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
• Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the modified ubiquitin scaffold protein.
• Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide.
• N may be any nucleotide.
• AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
• Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells.
• Suitable bacteria include gram negative or gram positive organisms, for example, E. coli or Bacillus spp. Yeast, preferably from the Saccharomyces species, such as S. cerevisiae, may also be used for production of polypeptides.
• Various mammalian or insect cell culture systems can also be employed to express recombinant proteins. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers,
• Figure 1 shows different dimeric modified ubiquitin binding proteins with inserts.
• the linker sequence here: GIG
• GIG linker sequence
• Not substituted amino acids of the ubiquitin monomers and of the linker are shown in blue (highlighted); substituted amino acids are not highlighted and appear with white background.
• the modifications account to at least 15 amino acids that are substituted and inserted to generate a new binding property to a non-natural protein target (about at least 20% of all amino acids of the first monomer).
• Further 3 amino acids e.g. at positions 45, 75, 76 are substituted to improve biochemical properties, but not being related to the binding to a target.
• Figure la shows the amino acid sequence of VEGF-A binding protein 40401 (SEQ ID NO:
• Figure lc shows the amino acid sequence of VEGF-A binding protein 59649 (SEQ ID NO:
• the clone is substituted in positions 6, 8, 62-66 in the first ubiquitin unit and positions 6, 8, 62, 63, 65, and 66 of the second ubiquitin unit.
• the protein shows an additional insertion of 8 amino acids.
• 6 amino acids (8% of all amino acids) are modified that are involved in a novel binding property to a given protein target.
• Figure Id shows the amino acid sequence of VEGF-A binding protein 60423 (SEQ ID NO:
• the clone is substituted in positions 6, 8, 62-66 in the first ubiquitin unit and positions 6, 8, 63-66 of the second ubiquitin unit.
• the protein shows an additional insertion of 8 amino acids.
• 6 amino acids (8% of all amino acids) are modified that are involved in a novel binding property to a given protein target..
• Figure lg shows the amino acid sequence of VEGF-A binding protein 59507 (SEQ ID NO:
• Figure lh shows the amino acid sequence of VEGF-A binding protein 59987 (SEQ ID NO:
• the clone is substituted in positions 6, 8, 62-66 in the first ubiquitin unit and positions 6, 8, 62, 64, and 65 of the second ubiquitin unit.
• the protein shows an additional insertion of 8 amino acids.
• 5 amino acids (6,5% of all amino acids) are modified that are involved in a novel binding property to a given protein target.
• Figure li shows the amino acid sequence of VEGF-A binding protein 59603 (SEQ ID NO:
• Figure lj shows the amino acid sequence of dimeric ubiquitin used as basis for substitutions to generate high specific binding proteins (SEQ ID NO: 11). Positions 6, 8, 62-66 in both ubiquitin units are marked with an X. An amino acid sequence for a linker is not shown since any possible linker known in the art could be used or even no linker could be used. Note that compared to wildtype the ubiquitin is further modified in position 45 in both monomers to Tryptophan. This modification does not influence the binding. At the last C-terminal amino acids in the first monomer, there is either Glycine or an exchange from Glycine to Alanine at the last C-terminal amino acids of the first monomer.
• FIG. 2 shows high affinity binding of binding protein 40401 (SEQ ID NO: 2) to VEGF-A. The binding is shown by closed circles connected by a fitted line. The figure shows a concentration dependent ELISA of the binding of the hetero-dimeric ubiquitin variant to human VEGF-A, in particular to isoform 121 and to isoform 165. As negative control, NGF was used (symbol -star- connected by a broken line).
• the binding affinities to VEGF-A 121 and to VEGF-A 165 are very similar.
• Figure 3 shows results of an analysis of the modified hetero-dimeric ubiquitin molecule 40401 via label-free interaction assays using Surface Plasmon Resonance (Biacore®).
• VEGF165 and a Kd of 1.5 x 10 "8 M (k off rate of 1.76 x 10 "3 s "1 ) to VEGF121 which indicates a long half time of a complex of 40401 and VEGF-A.
• Figure 4 shows inhibition of VEGF-A-induced proliferation of HUVEC by the binding protein 40401.
• Different concentrations of 40401 were preincubated with VEGF-A in medium together with a fixed VEGF-A concentration and the mixture applied to growing HUVEC. After three days the proliferation of cells was quantified with WST reagent. The dose response curve was fitted and an IC50 of 8 nM was calculated.
• modified amino acids which were substituted by NNK triplets, comprised amino acids selected from positions 6, 8, 62, 63, 64, 65, 66 within the proximal / N-terminal (first) ubiquitin monomer and amino acids selected from positions 6, 8, 62, 63, 64, 65, 66 within the distal / C-terminal (second) ubiquitin monomer.
• Both ubiquitin monomers were genetically linked (head to tail) by a Glycine/Serine linker with at least the sequence GIG or at least the sequence SGGGG, for example GIG, SGGGG, SGGGGIG, SGGGGSGGGGIG or SGGGGSGGGG, but any other or no linker is possible.
• the target protein VEGF-A The target protein VEGF-A
• the heterodimeric ubiquitin library was enriched against VEGF-A using, for example, TAT phage display as selection system. Other selection methods known in the art can be used.
• the target can be immobilized non- specifically onto protein binding surfaces or via biotinylated residues which were covalently coupled to the protein. The immobilization via biotin onto streptavidin beads or neutravidin strips is preferred.
• the target-binding phages are selected either in solution or on immobilized target; for example, the biotinylated and immobilized target with phage was incubated followed by washing of the phages bound to the matrix and by elution of matrix -bound phages.
• the beads were magnetically separated from solution and washed several times.
• the biotinylated target was immobilized to neutravidin strips whereas in cycles two to four selections in solution were performed followed by immobilization of target-phage complexes on Streptavidin-coated Dynabeads® (Invitrogen).
• the phages of target-binding modified ubiquitin molecules were released by elution with acidic solution.
• selection cycles three and four elution of phages was carried out by competitive elution with excess target. The eluted phages were reamplified.
• a protein similar to the target can be included during selection.
• TAT phage display selection Ribosome Display Selection
• the ubiquitin library was enriched against the target using, for example, ribosome display as selection system. Other selection methods known in the art can be used.
• the target was biotinylated according to standard methods and immobilized on Streptavidin-coated
• Dynabeads® (Invitrogen). Ternary complexes comprising ribosomes, mRNA and nascent ubiquitin polypeptide were assembled using the PURExpressTM In Vitro Protein Synthesis Kit (NEB). Up to four primary rounds of selection were performed, wherein ternary complexes were incubated followed by two similar rounds of selection. In each cycle following target incubation, the beads were magnetically separated from solution and washed with ribosome display buffer with increasing stringency. After washing in the first two selection cycles, the beads were again magnetically separated from solution and mRNA of target-binding modified ubiquitin molecules was released from ribosomes by addition of 50 mM EDTA.
• RNA purification and cDNA synthesis were performed using RNeasy MinElute Cleanup Kit (Qiagen, Germany), Turbo DNA-free Kit (Applied Biosystems, USA) and Transcriptor Reverse Transcriptase (Roche, Germany). Cloning of Enriched Pools
• ubiquitin building units (monomers) of a dimer of selected binding proteins were fused to naive monomelic ubiquitin libraries.
• ubiquitin building units monomers
• VEGF-A binding molecules were selected and the N-terminal ubiquitin monomer with substitutions in positions 6, 8, 62, 63, 64, 65, and/or 66, and optionally an insertion at position 61- 62, was fused to naive monomelic ubiquitin libraries with randomized amino acid positions 6, 8, 62, 63, 64, 65, and / or 66 via a suitable amino acid linker, for example GIG.
• ubiquitin monomers of the C-terminal region of a hetero-dimeric binding protein having substitutions in positions 6, 8, 62, 63, 64, 65, and/or 66 were fused to naive monomeric ubiquitin libraries with randomized amino acid positions 6, 8, 62, 63, 64, 65 and/or 66 and/or 42, 44, 68, 70, and 72-74 via a suitable amino acid linker, for example GIG.
• the resulting dimeric ubiquitin libraries with up to 7 randomized positions were pooled and exhibited a theoretical number of around 1,5 x 10 10 different variants which could be fully displayed in a ribosome display with up to 10-fold presentation of each variant using methods known to somebody skilled in the art.
• the mixed library was applied to 4 rounds of ribosome display including 3 rounds competitive elution of VEGF-A binding molecules with soluble VEGF121.
• VEGF-A binders with high affinity to VEGF-A do not always form complexes which are stable for a longer period of time.
• Some complexes ubiquitin-dimer and VEGF-A
• Some complexes have high off-rates, meaning that the binding is strong but the complex differentiates quickly.
• a lower off-rate as determined e.g. by Biacore assays is desirable.
• Biacore assays e.g. by Biacore assays.
• one round with 16 hrs off -rate selection was performed. The elution is performed under competitive conditions with 1000 x non bound target protein (compared to the target protein which is bound to e.g. streptavidin-beads).
• binders which are still bound to the immobilizied target protein after 16h are further analyzed. After this selection, pools with VEGF-A binding molecules were subcloned to an expression vector using standard methods known to a skilled person and probed for binding to different types of VEGF-A in hit-screening (e.g. ELISA) as described below.
• hit-screening e.g. ELISA
• exemplary VEGF-A binding proteins having inserts for example 40401, are shown in Figure 1 and in Tables 1, 2 and 3. The binding proteins were analysed as described in Example 2.
• Binding of ubiquitin-based variants to human VEGF-A was assayed by a concentration dependent ELISA. Increasing amounts of purified protein applied to NUNC-medisorp plates coated with human VEGF-A 121 or VEGF-A 165 and NGF as negative control. Antigen coating with 1 to 2.5 ⁇ g/ml per well was performed at 4°C overnight. After washing the plates with PBS, 0.1 % Tween 20 pH 7.4 (PBST) the wells were blocked using blocking solution (PBS pH 7.4; 3 % BSA; 0.5% Tween 20) at room temperature for 2 h. Wells were washed again three times with PBST.
• a-Ubi-Fab POD conjugate was applied in an appropriate dilution (for example, 1:6500) in PBST.
• the plate was washed three times with 300 ⁇ buffer PBST/well. 50 ⁇ TMB substrate solution (KEM-EN-Tec) were added to each well and was incubated. The reaction was stopped by adding 0.2 M H 2 SO 4 per well.
• the ELISA plates were read out using the TEC AN Sunrise ELISA-Reader.
• FIG. 3a shows clearly the very high affinity binding of 40401 (SEQ ID NO: 2) to VEGF-A with an apparent KD value of 2.2 to 2.5 nM. Further examples are shown in Figure 3. Thus, only very few modifications (up to 6 substitutions in each monomer) in the ubiquitin-wildtype result in a high affinity binding to VEGF-A.
• Figure 2 shows clearly the very high affinity binding of 40401 (SEQ ID NO: 2) to VEGF-A with an apparent KD value of 2.2 to 2.5 nM. No variant showed binding to the control (NGF). Further results of other VEGF-A binding proteins are shown in Table 2 (above).
• Example 2B Binding analysis of modified ubiquitin-based VEGF binding variants by Biacore assays.
• CM5-chip CM5-chip
• the K D of 40401 for VEGF165 was 2.2 x
• Example 3 Inhibition of VEGF stimulated cell proliferation by modified hetero-dimeric ubiquitin based binding proteins of the invention
• HUVEC cells Promocell
• Hams F-12 Nutrient Mixture (Kaighn's Modification, Gibco) with 10 % FCS , 0.1 mg/ml Heparin, 10 ng/ml b-FGF and passages 5 and 6 were used.
• 6000 cells were seeded in complete medium in collagen coated 96 well plates.
• cells were preincubated with 100% Hams F12 Nutrient Mixture for 6 h.
• the medium was exchanged for the preincubation mix, prepared of medium containing 5% FCS, 0.1 mg/ml Heparin and gentamycin supplemented with dilution series of the VEGF- specific binding protein premixed with 15 ng/ml VEGF 121 (Biomol/Humanzyme).
• the dilution series were prepared in 1:3 steps (starting from 1.5 ⁇ as indicated and incubated 1 h at room temperature. Each concentration was run in triplicate.
• VEGF-specific therapeutic monoclonal antibody Avastin® (Roche) was used as control (not shown). Viability of the cells was assessed after 3 days with WST reagent (Roche) according to the manufacturer's instructions. Results of this inhibition assay are shown in Figure 4 and Table 2. Further results of other VEGF-A binding proteins are shown in Table 2 (above).
• the binding protein of the invention clearly shows a significant inhibition of VEGF-A induced proliferation of HUVEC cells.

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