WO2014094799A1

WO2014094799A1 - Ubiquitin moieties as a means for prolonging serum half-life

Info

Publication number: WO2014094799A1
Application number: PCT/EP2012/005281
Authority: WO
Inventors: Christian Lange; Stefanie HOFFMANN-THOMS; Una Rauchhaus; Maren MEYSING; Ulrich Haupts
Original assignee: Scil Proteins GmbH
Current assignee: Navigo Proteins GmbH
Priority date: 2012-12-19
Filing date: 2012-12-19
Publication date: 2014-06-26
Anticipated expiration: 2015-06-19

Abstract

Disclosed herein are pharmaceutically active complexes that comprise at least two ubiquitin moieties. The ubiquitin moieties confer an increased serum half-life to the complexes. The disclosure also concerns polynucleotides encoding pharmaceutically active fusion proteins comprising at least two ubiquitin moieties, vectors comprising such polynucleotides, and cells comprising such vectors. Further disclosed herein are complexes, polynucleotides, vectors, and cells for use in medicine, in particular for use in the treatment of cancer. Also disclosed are pharmaceutical compositions comprising these complexes and a method for extending the serum half-life of a pharmaceutically active moiety.

Description

UBIQUITIN MOIETIES AS A MEANS FOR PROLONGING SERUM HALF-LIFE

The present invention relates to pharmaceutically active complexes that comprise at least two ubiquitin moieties. The ubiquitin moieties confer an increased serum half-life to the complexes. The invention also concerns polynucleotides encoding pharmaceutically active fusion proteins comprising at least two ubiquitin moieties, vectors comprising such polynucleotides, and cells comprising such vectors. The invention further relates to complexes, polynucleotides, vectors, and cells for use in medicine, in particular for use in the treatment of cancer. Also disclosed are pharmaceutical compositions comprising these complexes and a method for extending the serum half-life of a pharmaceutically active moiety.

BACKGROUND OF THE INVENTION

Extending serum half-life of pharmaceutically active moieties

Pharmaceutically active drugs have often limited value because of their pharmacokinetic properties. In particular therapeutic or diagnostic agents of smaller size are rapidly eliminated from the body due to renal filtration. This affects the amount and frequency of the dosage of drugs. Administration of larger amounts of drugs often has the disadvantage of negative side effects in patients.

Some strategies for prolonging serum half-life of pharmaceutically active moieties are known in the art (see the review by Kontermann (2001) Curr Opin Biotechnol. 22: 1-9; Kontermann (2009) BioDrugs 23: 93-109; all of which are hereby incorporated by reference). For example, fusion of pharmaceutically active moieties to plasma proteins like serum albumin and the Fc part of IgG molecules (Fc fusion molecules) result in long serum half-life of several weeks. Due to specific receptor recycling mechanisms, serum albumin and IgG are not degraded in the lysosome but rather redirected to the plasma membrane where they are released into the blood plasma. By the addition of an Fc-domain to a therapeutic protein, the protein size increases to be larger than 50 kDa and thus larger than the renal filtration threshold which is in the range of 40-50 kDa.

Chemical coupling of polyethylene glycol (PEG) was established more than 20 years ago to increase the hydrodynamic volume of a protein, thereby extending its serum half-life. One or more PEG chains may be conjugated to the protein therapeutic. Examples of pegylated drugs are interferon-alpha 2b, G-CSF, human growth hormone, and erythropoietin. More recent approaches use so-called recombinant PEG mimetics for half- life extension. For example, repetitive sequences of up to 600 residues are composed of three amino acids (proline, alanine, serine = PAS) and fused to protein therapeutics.

Other strategies to prolong serum half-life include glycoengineering techniques such as introduction of additional N-glycosylation sites by genetic engineering and chemical conjugation of carbohydrates (e.g., hydroxyethyl starch or polysialic acid).

Ubiquitin

Ubiquitin is an 8.5 kDa cytosolic protein which is present in all known eukaryotic cells from protozoans to vertebrates. Ubiquitin is highly conserved in sequence. For example, in all mammals investigated up to now ubiquitin has the identical amino acid sequence (see SEQ ID NO: 1). The polypeptide chain of ubiquitin consists of 76 amino acids folded in an extraordinarily compact α/β structure (Vijay-Kumar et al. (1987) J. Mol. Biol. 194(3): 531-44; the entirety of which is herein incorporated by reference).

Because of its small size, artificial preparation of ubiquitin can be carried out both by chemical synthesis and by means of biotechnological methods. Due to its favorable folding properties, ubiquitin can be produced by genetic engineering using microorganisms such as Escherichia coli in high yields either in the cytosol or in the periplasmic space.

Specific targeting proteins based on Affilin* molecules

Affilin* (a registered trademark of Scil Proteins GmbH) molecules are artificial binding proteins on the basis of modified ubiquitin proteins (see WO 04/106368, the entirety of which is herein incorporated by reference). Affilin molecules are based on the human Ubiquitin scaffold and engineered to generate de-novo binding affinities towards disease- related targets. Affilin* molecules are created by engineering de-novo binding sites on the surface of a dimeric form of the human serum protein Ubiquitin. On the order of 15 surface- exposed amino acids are modified in order to engineer de novo binding sites. Dimeric Affilin binding proteins have molecular weights of about 17 kDa. Methods for identifying multimeric modified ubiquitins with newly generated binding capability to a pre-defined ligand are described in WO 2011/073214, the entirety of which is herein incorporated by reference. The platform allows the generation of agonistic or antagonistic binding molecules and fusion to effector molecules. Specific multimeric binding proteins based on differently modified ubiquitin monomers are described for example in WO 2011/073208, WO 2011/073209, and PCT/EP2012/061454, all of which are hereby incorporated by reference.

Compared to antibodies and fragments thereof or other artificial binding proteins (scaffolds), binding molecules on the basis of modified ubiquitin proteins have many advantages: high target affinity and specificity, high stability, low immunogenicity, and cost effective manufacturing in high yield. Furthermore, ubiquitin scaffolds are amenable to genetic and chemical modifications.

TECHNICAL PROBLEMS UNDERLYING THE PRESENT INVENTION AND THEIR SOLUTION

One disadvantage of small pharmaceutically active compounds (i.e., compounds with a molecular weight of smaller than about 50 kDa) is that such compounds are cleared rather quickly from the circulation after administration. The clearance of pharmaceutically active compounds from the blood by the kidneys restricts the availability of the drug in the body for therapeutic and/or diagnostic purposes. Thus, there is only a short time-period in which such small compounds are present in the body to exert the intended pharmaceutical activity. Larger doses or more frequent administration are required to reach a positive effect for therapy or in diagnosis.

Since renal clearance is inversely correlated to the hydrodynamic volume of a compound, attempts have been made to prolong the circulation periods in the blood by increasing the size of the pharmaceutically active compound. For example, the prior art describes the coupling of proteins or polyethylene glycol (PEG) to the pharmaceutically active compound in order to create compounds with increased size that exhibit a reduced renal clearance. However, PEG has some drawbacks. PEG is not biodegradable and may accumulate in the body. Moreover, PEG has to be added to the pharmaceutically active compound via chemical coupling in an additional production step. Other strategies used in the prior art include glycoengineering, which also has drawbacks. For example, introduction of glycosylation sites into a pharmaceutically active protein requires expression of the modified protein in eukaryotic expression systems, which is more expensive and more complicated compared to expression in prokaryotic cells.

Thus, there exists a continuous need to develop pharmaceutically active compounds having prolonged in vivo half-life.

It was therefore an objective of the present invention to identify other molecules that may be added to pharmaceutically active compounds in order to prolong the circulation time of such pharmaceutically active compounds. The inventors found that the addition of at least two ubiquitin moieties to a pharmaceutically active compound increases the serum half-life of the pharmaceutically active compound. The inventors further found that different numbers of ubiquitin moieties can be added to a pharmaceutically active compound in order to fine-tune the desired serum half-life of the pharmaceutically active compound. Ubiquitin provides several advantages: ubiquitin is biodegradable, has a low immunogenicity, and can be produced by chemical synthesis or in microorganisms.

The above-described objectives are solved and the advantages are achieved by the subject-matter of the enclosed independent claims. Preferred embodiments of the invention are included in the dependent claims as well as in the following description, examples and figures.

The above overview does not necessarily describe all problems solved by the present invention. SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a complex comprising, essentially consisting of or consisting of:

(a) at least two ubiquitin moieties; and

(b) at least one pharmaceutically active moiety,

wherein said complex exhibits an increased serum half-life as compared to the at least one pharmaceutically active moiety alone without said at least two ubiquitin moieties. In a second aspect the present invention relates to the complex according to the first aspect for use in medicine.

In a third aspect the present invention relates to the complex according to the first aspect for use in the treatment of cancer.

In a fourth aspect the present invention relates to a pharmaceutical composition comprising the complex according to the first aspect; and further comprising a pharmaceutically acceptable carrier.

In a fifth aspect the present invention relates to a use of at least two ubiquitin moieties for extending the serum half-life of a pharmaceutically active moiety.

In a sixth aspect the present invention relates to a method for extending the serum half-life of a pharmaceutically active moiety, comprising the steps:

(a) fusing a nucleic acid encoding at least two ubiquitin moieties to a nucleic acid encoding a pharmaceutically active moiety, thereby obtaining a fused nucleic acid;

(b) introducing said fused nucleic acid into an expression vector;

(c) introducing said expression vector into a host cell;

(d) cultivating the host cell;

(e) subjecting the host cell to culturing conditions under which a fusion protein is expressed from said vector, thereby producing a fusion protein comprising at least two ubiquitin moieties and a pharmaceutically active moiety, wherein said fusion protein has an extended serum half-life as compared to the pharmaceutically active moiety without ubiquitin moieties;

(f) optionally isolating the fusion protein produced in step (e).

In a seventh aspect the present invention relates to a nucleic acid comprising a sequence encoding the complex of the first aspect.

In an eighth aspect the present invention relates to a vector comprising the nucleic acid of the seventh aspect.

In a ninth aspect the present invention relates to a cell comprising the vector of the eighth aspect. This summary of the invention does not necessarily describe all features of the present invention. Other embodiments will become apparent from a review of the ensuing detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows the amino acid sequences of complexes of the invention.

a) Ubi4-Affilin^* (SEQ ID NO: 15), b) Ubi6-Affilin* (SEQ ID NO: 14), c) Ubi2-IFN (SEQ ID NO: 16), d) Ubi2-scTNF (SEQ ID NO: 17). The modified monomeric ubiquitin subunits of the Affilin^* part presented in Fig. 1A and Fig. IB are based on ubiquitin F45W, i.e., a ubiquitin mutein which differs from the wild-type sequence according to SEQ ID NO: 1 by amino acid exchanges F45W, G75A, and G76A. The exchanges F45W, G75A, and G76A are not important for binding a target. Substitutions in the ubiquitin subunits are highlighted by using bold- type. Linker regions are underlined.

FIGURE 2 gives an overview on the molecular weights of the complexes of this invention. FIGURE 3 represents an SDS-PAGE of the complexes comprising four and six ubiquitin moieties, respectively, and an Affilin" (SEQ ID NO: 14; SEQ ID NO: 15) purified as described in Example 2. Lane 1: PageRuler Unstained Broad Range Protein Ladder (Thermo Scientific); Lanes 2 and 3: 1 g and 2 respectively, of Ubi4-Affilin^* (SEQ ID NO: 15); Lane 4: PageRuler^* Prestained Protein Ladder (Thermo Scientific); Lanes 5 and 6: 1 g and 2 μg, respectively, of Ubi6-Affilin^* (SEQ ID NO: 14).

FIGURE 4 shows the production and PEGylation of an Affilin^* (SEQ ID NO: 3).

Fig. 4A: shows an SDS-PAGE of the Affilin^* purified as SUMO fusion (Lane 2) and the corresponding protein solution after SUMO cleavage (Lane 3) using SUMO Hydrolase (Example 3, step 4). Lane 1: PageRuler Unstained Broad Range Protein Ladder (Thermo Scientific).

Fig. 4B: represents an SDS-PAGE of the Affilin^* (SEQ ID NO: 3) expressed and purified as described in Example 3. Lane 1: PageRuler Prestained Protein Ladder (Thermo Scientific); Lanes 2 and 3: 1 μ§ and 2μg, respectively, of the Affilin^*.

Fig. 4C: shows an SDS-PAGE of the Affilin^* (SEQ ID NO: 3) N-terminally modified with a branched 40 kDa PEG moiety (Example 3, step 5). Lane 1: PageRuler^* Prestained Protein Ladder (Thermo Scientific); loading of 1 g (Lane 2) and 2 g (Lane 3), respectively, of the PEGylated Affilin" on the gel.

FIGURE 5 exemplarily shows the analysis of the affinity of Ubi6-Affilin^e (SEQ ID NO: 14) towards human ED-B using analytical affinity interaction chromatography as described in Example 6. Continuous line: absorption at 280 nm (mAU), dashed line: concentration of elution buffer (%).

FIGURE 6 shows the ELISA analysis of the binding of an Affilin" (Fig. 6A) and of the complex Ubi4-Affilin° (Fig. 6B), respectively, to the target domain ED-B, as described in Example 5. Black circles (A) represent binding of the Affilin^* to immobilized ED-B , black squares (B) represent Ubi4-Affilin^¾, open triangles show absence of specific binding to a control surface coated with human fibronectin without ED-B domain. All data points show mean values ± s.d. of measurements performed in triplicate. Solid lines represent fits of a one-site ligand binding model to the data, with the corresponding apparent dissociation constants (K_D) of 0.36 nM for binding of Affilin" and 0.11 nM for binding of Ubi4-Affilin^e.

FIGURE 7 (A) shows the blood concentration of radiolabeled protein variants expressed as % of injected dose per gram blood after intravenous injection at indicated time points.

Circulation half-life increases in the following order: Affilin^* (closed triangles), Ubi4-Affilin^¾ (open hash mark), Ubi6-Af ilin^* (closed circles), Ubi2-scTNF (closed squares, dotted line). There is clear evidence for prolongation of circulation half-life with an increased number of ubiquitin-monomers attached to the Affilin" molecule. Figure 7 (B) displays selected pharmacokinetic parameters calculated from the curves as described in Example 14.

FIGURE 8 shows percentage of intact labeled complex protein during circulation within the animals. Affilin* (closed triangles), Ubi4-Affilin^s (open hash mark) and Ubi6-Affilin°^> (closed circles) were injected intravenously and blood samples were taken at the indicated time points. Serum was generated from these blood samples and serum samples were analyzed via SE-HPLC with respect to protein amount and protein degradation and aggregation, respectively. Applying complexes of Affilin" and multiple ubiquitin moieties leads to increased amount of intact protein over time in line with increasing number of ubiquitin monomers included in the complex.

FIGURE 9 summarizes the activity of the pharmaceutically active moieties within the complexes. In Table 9A results from the ED-B-binding activity analysis are listed indicating target specific activity of the pharmaceutically active moiety. Table 9B displays the effector function of the pharmaceutically active moiety comprised in the complex. In both cases activity of pharmaceutically active moiety was strongly decreased by PEG-conjugation whereas the complex comprising the pharmaceutically active moiety and ubiquitin units did not show a strong reduction of complex activity.

FIGURE 10 shows the analysis of TNF receptor binding by the complex Ubi2-scTNF (Fig. 10A) in comparison to recombinant murine TNFalpha (Fig. 10B) by an ELISA assay as described in Example 12. Circles (A) represent binding of the recombinant soluble TNF-receptor I domain / Fc chimera to immobilized Ubi2-scTNF, squares (B) represent binding to recombinant murine TNFalpha, open triangles show absence of specific binding to a control surface without any bound TNFalpha construct. All data points show mean values ± s.d. of measurements performed in triplicate, Solid lines represent fits of a one-site ligand binding model to the data, with the corresponding apparent dissociation constants K_D of 0.58 nM for binding to complex Ubi2-scTNF and 0.66 nM for binding to recombinant murine TNFalpha control protein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (lUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B., and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.

Several documents (for example: patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.) are cited throughout the text of this specification. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being "incorporated by reference". In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

Sequences: All sequences referred to herein are disclosed in the attached sequence listing that, with its whole content and disclosure, is a part of this specification. The term "about" when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 10% smaller than the indicated numerical value and having an upper limit that is 10% larger than the indicated numerical value.

As used herein, the term "pharmaceutically active moiety" has to be understood in its broadest meaning as referring to any molecule, compound, or composition of matter which mediates a pharmaceutical effect in a living organism (e.g., in a mammal, particularly in a human being). A pharmaceutical effect includes but is not limited to a prophylactic, a therapeutic, and/or a diagnostic effect. It should be noted that the term "pharmaceutically active moiety" does not encompass "ubiquitin moieties", which will be defined below. However, the term "pharmaceutically active moiety" may encompass modified monomeric or dimeric "ubiquitin units" with specific binding properties to selected targets. The term "ubiquitin units" (or "ubiquitin monomer") will also be defined below. Other "pharmaceutically active moieties" that can be used in the present invention include without limitation: interferon (e.g. IFN alpha), interleukin (e.g. IL-2), tumor necrosis factor (e.g. TNFalpha), and single chain TNF (scTNF). The terms IFN, IL-2, TNFalpha, and scTNF are defined below, too. Typically, a "pharmaceutically active moiety" useable in the present invention has a molecular weight which is equal to or less than about 70 kDa. Preferably, a "pharmaceutically active moiety" useable in the present invention has a molecular weight in the range from about 10 kDa to about 60 kDa, more preferably in the range from about 15 kDa to about 55 kDa.

As used herein, a "complex" refers to a composition of matter comprising at least two components, wherein these at least two components are held together by any kind of interaction, for example by covalent bonds, by ionic bonds, by hydrogen bonds, by van der Waals interactions, or by hydrophobic interactions. As will be explained below in greater detail, the complex of the invention comprises at least two components, namely (i) at least two ubiquitin moieties [counted as one component] and (ii) at least one pharmaceutically active moiety. It is especially preferred that these two components of the complex are held together by a covalent bond. The following gives some examples on how to obtain covalent complexes according to this invention:

a) conjugation of the pharmaceutically active moiety to ubiquitin units via lysine residues;

b) conjugation via cysteine residues, which may be located C-terminally, or at any other position; conjugation with maleimide containing components;

c) peptidic or proteinogenic conjugations, e.g. contiguous genetic fusions

d) peptidic or proteinogenic conjugations, e.g. via intein-based coupling of components which are non-contiguous on the genetic level

e) "Tag"-based fusions, wherein the affinities of specific peptides located either at the C- or N-terminus of the components forming the complex are used to effect a linkage between said components.

These and other methods for covalently and non-covalently attaching a protein of interest to a support are well known in the art, and are thus not described in further detail here. The term "serum half-life" refers to the time period in which the concentration of a substance (e.g., a pharmaceutically active moiety, a fusion protein or a complex of the invention) in the serum of a subject [in vivo) is reduced by one half (50%). The half-life of a substance is increased if its physical concentration persists in vivo for a longer period than a similar molecule which has not been modified with respect to half-live extension. Typically, the half-life is increased by 10%, 20%, 30%, 40%, 50%, or more. Methods for determining the serum half-life of a given substance are detailed in the example section.

The present disclosure distinguishes between the terms (a) "ubiquitin moiety" and (b) "ubiquitin unit" or "ubiquitin protein" or "ubiquitin monomer".

As used herein, at least two "ubiquitin moieties" are obligatory parts of the complex of the present invention. Said "ubiquitin moieties" are used to increase the size of the complex of the invention so that the pharmaceutically active moiety in the complex has an increased serum half-life. As used herein, a "ubiquitin moiety" consists of an amino acid sequence that is either based on the wild-type amino acid sequence according to SEQ ID NO: 1 or on a pre-modified amino acid sequence according to SEQ ID NO: 2 (F45W, G75A, G76A) or SEQ ID NO: 23 (675A, G76A). A "ubiquitin moiety" may have a limited number of amino acid insertions, deletions, and/or exchanges as compared to the amino acid sequences according to SEQ ID NO: 1, 2, or 23. More specifically, a ubiquitin mutein is considered to be a "ubiquitin moiety" within the meaning of the present invention, if it differs from wild-type ubiquitin according to SEQ ID NO: 1 by 0, 1, 2, or 3 amino acid exchanges (substitutions), by 0, 1, 2, or 3 amino acid deletions and/or by 0, 1, 2, or 3 amino acid insertions. Likewise, a ubiquitin mutein is considered to be a "ubiquitin moiety" within the meaning of the present invention, if it differs from pre-modified ubiquitin according to SEQ ID NO: 2 or from pre- modified ubiquitin according to SEQ ID NO: 23 by 0, 1, 2, or 3 amino acid insertions, by 0, 1, 2, or 3 amino acid deletions and/or by 0, 1, 2, or 3 amino acid exchanges. According to an alternative definition, a "ubiquitin moiety" within the meaning of the present invention exhibits a sequence identity of at least 95% (preferably 96%, more preferably 97%, more preferably 98%, even more preferably 99% and most preferably 100%) to the amino acid sequence defined by SEQ ID NO: 1 or by SEQ ID NO: 2 or by SEQ ID NO: 23. It is particularly emphasized that said at least two "ubiquitin moieties" do not form a "pharmaceutically active moiety" within the meaning of the present invention. In contrast, a "ubiquitin protein" or a "ubiquitin unit" or "ubiquitin monomer" is not an obligatory part of the complex of the present invention. However, in some embodiments of the present invention the "pharmaceutically active moiety" comprises a modified dimeric ubiquitin protein which comprises two modified monomeric ubiquitin units. Such modified dimeric ubiquitin proteins are artificial binding proteins that are designed to specifically bind to a target molecule. Said modified dimeric ubiquitin based binding proteins are also referred to as Affilin" molecules.

The term "ubiquitin unit" or "ubiquitin monomer" covers the ubiquitin in accordance with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 23 and modifications thereof according to the following definition. Particularly preferred are ubiquitin molecules from humans. Additionally, ubiquitin from any other eukaryotic source can be used. For instance, ubiquitin of yeast differs only in three amino acids from the sequence of SEQ ID NO: 1. Generally, the ubiquitin proteins covered by the term "ubiquitin unit" or "ubiquitin monomer" show an amino acid identity of at least 80% (preferably at least 85%, more preferably at least 90%, or at least 94% to SEQ ID NO: 1 or to SEQ ID NO: 2 or to SEQ ID NO: 23).

The term "a modified ubiquitin unit" or "a modified ubiquitin monomer" refers to modifications of the ubiquitin protein, 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 said modified ubiquitin units (monomers) have an amino acid identity to SEQ ID NO: 1 or to SEQ ID NO: 2 or to SEQ ID NO: 23 of at least 80% (preferably at least 85%, more preferably at least 90%, or up to at least 94%). At the most, the overall number of substitutions in a monomeric unit is, therefore, limited to 15 amino acids corresponding to 80% amino acid identity. At minimum, the overall number of substitutions in a ubiquitin unit (monomer) for generating a binding property is 5 amino acids corresponding to 94% amino acid identity. The total maximum number of modified amino acids in the hetero-dimeric ubiquitin molecule is 30 amino acids corresponding to 20% amino acid modifications based on the hetero-dimeric protein. Preferably, the number of modified amino acids in a hetero-dimeric ubiquitin for generating a binding property is at least 10 amino acids, most preferred between 10 and 16 amino acids. Most preferred are substitutions. 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 or SEQ ID NO: 2 is selected from one of the group consisting of at least 80%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, and at least 90%. Preferred are substitutions in regions 2-8, 12-16, 41-45 and 62-71 of SEQ ID NO: 1, 2, or 23. More preferred are substitutions in regions 2-8 and 62-68 of SEQ ID NO: 1, 2, or 23. Preferred positions for substitutions are selected from amino acids 2, 4, 6, 8, 61, 62, 63, 64, 65, 66, 68.

For determining the extent of sequence identity between two amino acid sequences, for example, the SIM Local similarity program (Xiaoquin Huang and Webb Miller (1991), Adv Appl Math., 12: 337-357) or ClustalW can be used (Thompson et al. (1994), Nucleic Acids Res., 22(22): 4673-4680). In particular, the sequence identity percentage between a derivative of ubiquitin and the amino acid sequence of SEQ ID NO: 1 (or SEQ ID NO: 2 or SEQ ID NO: 23) can be determined with either of these programs. Preferably, the default parameters of the SIM Local similarity program or of ClustalW are used, when calculating sequence identity percentages. Preferably, the extent of the sequence identity of the modified protein to SEQ ID NO: 1 (or SEQ ID NO: 2 or SEQ ID NO: 23) is determined relative to the complete sequence of SEQ ID NO: 1 (or SEQ ID NO: 2 or SEQ ID NO: 23, respectively).

In the context of the present invention, 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.

A "dimer" is considered as a protein in this invention which comprises two monomeric ubiquitin units (also referred to herein as two ubiquitin monomers). If the dimer comprises two differently modified monomers, it is called a "heteromeric dimer" or "hetero- dimer". Thus, the "hetero-dimer" of the invention is considered as a fusion of two differently modified monomeric ubiquitin units exhibiting a combined binding property (binding domain or targeting domain) for its specific target molecule (e.g., a tumor antigens such as extra- domain B of fibronectin referred to as ED-B or any other antigens).

According to the present invention, the two monomeric modified ubiquitin units are not linked together after having screened the most potent binding ubiquitin molecules, but the screening process is performed in the presence of the hetero-dimeric ubiquitins. After having received the sequence information on the most potent binding ubiquitin molecules, these molecules may be obtained by any other method, e.g. by chemical synthesis or by genetic engineering methods, e.g. by linking the two already identified monomeric ubiquitin units together.

According to the invention, the two differently modified ubiquitin monomers which bind to one ligand are to be linked by head-to-tail fusion to each other using e.g. genetic methods. The differently modified fused ubiquitin monomers are only effective when acting together.

A "head to-tail fusion" is to be understood as fusing the C-terminus of the first protein to the N-terminus of the second protein. In a head-to-tail fusion, monomers may be connected directly without any linker, i.e., by a direct peptide bond. Alternatively, the fusion of ubiquitin monomers can be performed via linkers.

As used herein, the term "linker" generally 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.

In typical embodiments of the present invention, a "linker" is to be understood as a moiety that connects a first polypeptide with at least one further polypeptide. The second polypeptide may be the same as the first polypeptide or it may be different. Preferred in these typical embodiments are peptide linkers. This means that the peptide linker is an amino acid sequence that connects a first polypeptide with a second polypeptide. The peptide linker is connected to the first polypeptide and to the second polypeptide by a peptide bond. Typically, 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 acid sequence of the peptide linker is not immunogenic to human beings. For example, a linker having at least the amino acid sequence SG or any other linker, for example SGGGG [SEQ ID NO: 4], SGGGGSGGGG [SEQ ID NO: 5], GGGSGGGSGGGS [SEQ ID NO: 6], GGGGSGGGGSGGGGS [SEQ ID NO: 7], GIG [SEQ ID NO: 8], SGGGGIG [SEQ ID NO: 9], SGGGGSGGGGIG [SEQ ID NO: 10], GGGGS [SEQ ID NO: 11], (GGGS)_n (i.e., n repetitions of SEQ ID NO: 12, wherein n is between 1 and 10 (e.g., n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)), or (SGGG)n (i.e., n repetitions of SEQ ID NO: 13, wherein n is between 1 and 10 (e.g., n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)), or (PAS)_n wherein n is between 1 and 10 (e.g., n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or any other peptide linker can be used. Other linkers for the genetic fusion of two ubiquitin monomers are also known in the art and can be used.

As used herein, the term "PAS linker" refers to a peptide linker composed of the three amino acids proline (P), alanine (A), and serine (S), in which the tripeptide PAS occurs between once and ten times. In other words, the term "PAS linker" can be defined by the sequence (PAS)_n, wherein n is between 1 and 10 (e.g., n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

In accordance with the invention, a "dissociation constant" (abbreviated as "Kd" or alternatively as "K_D") defines the specific binding affinity which is in accordance with the invention in the range of 10^~7 - 10 ¹² M. A value of 10 ⁵ M and below can be considered as a quantifiable binding affinity. Depending on the application a value of 10^~7 M to 10^"11 M is preferred for e.g., chromatographic applications or 10^"9 to 10^"12 M for e.g., diagnostic or therapeutic applications. Further preferred 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 to the skilled person and can be selected for instance from the following methods: ELISA, Surface Plasmon Resonance (SPR) based technology (offered for instance by Biacore"), affinity interaction chromatography, fluorescence spectroscopy, isothermal titration calorimetry (ITC), analytical ultracentrifugation, or fluorescence activated cell sorting (FACS).

Preferred "target molecules" when practicing the present invention are proteins and more specifically antigenic epitopes present on proteins. More preferred "target molecules" are tumor antigens, such as proteins or epitopes that are present on the outside of a tumor cell but that are absent on normal cells of the same tissue-type or which are present in tumor tissue but absent on normal tissue from the same tissue type. Tumor antigens are also termed "tumor target molecules" in the present specification. A particularly preferred "target molecule" in the context of the present invention is extra-domain B of fibronectin.

The term "extra-domain B of fibronectin" (or briefly designated as "ED-B") comprises all proteins which show a sequence identity to SEQ ID NO: 25 of at least 70%, optionally 75%, further optionally at least 80%, 85%, 90%, 95%, 96%, or 97% or most preferably showing a sequence identity to SEQ ID NO: 25 of 100% and having the functionality of ED-B defined herein (see in particular the section below entitled "Extra-domain B of fibronectin as tumor specific protein").

As used herein, the term "IFN" encompasses interferon alpha (IFN-a) and interferon beta (IFN-β), including all subtypes thereof. The term "I F" relates to IFN-a and IFN-β from any mammalian organism. However, it is preferred when practicing the present invention to use human IFN-ct or human IFN-β. More specifically, the term "IFN" used herein includes IFN-a 2a, IFN-ct 2b, IFN-a 2c, IFN-a 6, IFN-a 14, IFN-a 4, IFN-a 5, IFN-β and biologically active muteins of any of these; especially human IFN-a 2a (SEQ ID NO: 27), human IFN-a 2b (SEQ ID NO: 28), human IFN-a 2c (SEQ ID NO: 29), human IFN-a 6 (SEQ ID NO: 30), human IFN-a 14 (SEQ ID NO: 31), human IFN-a 4 (SEQ ID NO: 32), human IFN-a 5 (SEQ ID NO: 33), human IFN-β (SEQ ID NO: 43) and biologically active muteins of any of these. The amino acid sequences shown in SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, and 43 correspond to the sequences of mature native interferon molecules after cleavage of the signal peptide. However, as used herein, the term "IFN" also covers amino acid sequences in which a start methionine or leader sequences or tags or other amino acids are added to the amino terminus of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 43.

As used herein, the term "biologically active IFN" encompasses polypeptides that are sequence variants (muteins) of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43 and exhibit the same biological functions as the naturally occurring IFN molecules according to SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43. Such "biologically active IFN" molecules can occur in nature or can be artificially created polypeptides. In the context of the present application, the term "biologically active IFN" especially refers to polypeptides that exhibit at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to the amino acid sequence set forth in SEQ ID NO: 28 and exhibit a similar physiological activity, as does naturally occurring human IFN- α 2b. A sequence variant (mutein) of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 43 is considered to be a "biologically active IFN" polypeptide for the purposes of the present invention, if said sequence variant exhibits at least 90% of the gene inducing activity of human IFN-a 2b having the amino acid sequence according to SEQ ID NO: 28, as determined by the ISRE- Reporter Gene Assay described in Example 9.

The term "scTNF" (or "scTNFalpha") refers to at least three (e.g., three, six, or nine) TNFalpha monomers that are joined by linkers, thereby forming a single chain (sc) TNFalpha (scTNF or scTNFalpha) molecule. A trimeric structure is required to be able to bind to specific TNF receptors and induce the formation of ligand/receptor complexes. Connecting linkers between the TNFalpha monomers are preferably peptide linkers.

The term "TNFalpha" (or spelling variants thereof such as "TNF-alpha", "TNFa", or "TNF alpha") covers TNFalpha molecules in accordance with SEQ ID NO: 34 (human; uniprot accession number P01375; see: http://www.uniprot.org/uniprot/P01375), SEQ ID NO: 35 (mouse; uniprot accession number P06804; http://www.uniprot.org/uniprot/P06804), SEQ ID NO: 36 (rat), or any other homologous sequences. Human TNFalpha shows 79% sequence identity to mouse TNFalpha. The amino acid sequences shown in SEQ ID NO: 34, 35, and 36 correspond to the sequences of mature native TNFalpha monomers in the respective species after cleavage of the signal peptide. However, as used herein, the term "TNFalpha" also covers amino acid sequences in which a start methionine or leader sequences or tags or other amino acids are added to the amino terminus of SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.

The term "biologically active TNFalpha" encompasses polypeptides that are sequence variants (muteins) of SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36 and exhibit the same biological functions as the naturally occurring TNFalpha molecules according to SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36. Such "biologically active TNFalpha" molecules can occur in nature or can be artificially created polypeptides. In the context of the present application, the term "biologically active TNFalpha" especially refers to polypeptides that exhibit at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to the amino acid sequence set forth in SEQ ID NO: 34 and that exhibit an apoptotic activity, as does naturally occurring TNFalpha. A sequence variant of SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36 is considered to be a "biologically active TNFalpha" polypeptide for the purposes of the present invention, if said sequence variant exhibits at least 90% of the apoptotic activity of human TNFalpha having the amino acid sequence according to SEQ ID NO: 34. The apoptotic activity can be determined by the L929 cytotoxicity assay described by Flick et al. (1984, J Immunol Methods, 68: 167-175, which is herewith incorporated by reference in its entirety).

The terms "biologically active single-chain TNFalpha", "biologically active scTNFalpha", or "biologically active scTNF" refers to at least three (e.g., three, six, or nine) monomers of biologically active TNFalpha, wherein the term "biologically active TNFalpha" is defined as above, and wherein these monomers are joined by linkers so that a biologically active single chain (sc) TNFalpha (scTNFalpha or scTNF) molecule is formed.

The term "interleukin 2" (or the abbreviation "IL-2") covers interleukin-2 molecules in accordance with SEQ ID NO: 47 (human; uniprot accession number P60568; see: http://www.uniprot.org/uniprot/P60568), SEQ ID NO: 48 (mouse; uniprot accession number P04351; see: http://www.uniprot.org/uniprot/P04351) or any other homologous sequence. The amino acid sequences shown in SEQ ID NO: 47 and 48 correspond to the sequences of mature native interleukin 2 in the respective species after cleavage of the signal peptide. The signal peptide has a length of 20 amino acids both in human IL-2 and in murine IL-2. However, as used herein, the term "interleukin 2" also covers amino acid sequences in which a start methionine or leader sequences or tags or other amino acids are added to the amino terminus of SEQ ID NO: 47 and SEQ ID NO: 48.

The term "biologically active interleukin 2" encompasses polypeptides that are sequence variants (muteins) of SEQ ID NO: 47 or SEQ ID NO: 48 and exhibit the same biological functions as the naturally occurring IL-2 molecules according to SEQ ID NO: 47 or SEQ ID NO: 48. Such "biologically active interleukin 2" molecules can occur in nature or can be artificially created polypeptides. In the context of the present application, the term "biologically active interleukin 2" especially refers to polypeptides that exhibit at least 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to the amino acid sequence set forth in SEQ ID NO: 47 and that exhibit a binding activity to the IL-2 receptor, as does naturally occurring IL-2. A sequence variant of SEQ ID NO: 47 or SEQ ID NO: 48 is considered to be a "biologically active interleukin 2" polypeptide for the purposes of the present invention, if said sequence variant exhibits a dissociation constant K_D to the IL-2 receptor, which is at most twice as high as the dissociation constant K_D of human interleukin 2 having the amino acid sequence according to SEQ ID NO: 47 to the IL-2 receptor.

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 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. An "effective amount" or "therapeutically effective amount" is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.

Embodiments of the Invention

The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous, unless clearly indicated to the contrary.

In a first aspect the present invention is directed to a complex comprising, essentially consisting of, or consisting of: (a) at least two ubiquitin moieties; and (b) at least one pharmaceutically active moiety; wherein said complex exhibits an increased serum half-life as compared to the at least one pharmaceutically active moiety without said at least two ubiquitin moieties.

In preferred embodiments of the first aspect, said complex has a molecular weight of at least 20 kDa, preferably at least 30 kDa, more preferably at least 40 kDa, more preferably at least 50 kDa. In particularly preferred embodiments, the complex of the present invention has a molecular weight of between about 30 kDa and about 200 kDa (more preferably between about 40 kDa and about 110 kDa).

In preferred embodiments of the first aspect, the complex comprises between 2 and 10 ubiquitin moieties, preferably between 2 and 8 ubiquitin moieties, more preferably 2, 3, 4, 5, 6, 7, or 8 ubiquitin moieties. In preferred embodiments of the first aspect, each one of the at least two ubiquitin moieties consists, independently from any other ubiquitin moiety, of:

- an amino acid sequence according to SEQ ID NO: 1;

- an amino acid sequence according to SEQ ID NO: 2;

- an amino acid sequence according to SEQ ID NO: 23;

- an amino acid sequence exhibiting at least 95% sequence identity to SEQ ID NO: 1;

- an amino acid sequence exhibiting at least 95% sequence identity to SEQ ID NO: 2; or

- an amino acid sequence exhibiting at least 95% sequence identity to SEQ ID NO: 23.

In some embodiments of the first aspect, the at least two ubiquitin moieties are connected to each other

via a direct covalent bond, such as a peptide bond or a disulfide bond; or

- via a linker, such as a peptide linker (e.g., SGGGG [SEQ ID NO: 4], SGGGGSGGGG [SEQ ID NO: 5], GGGSGGGSGGGS [SEQ ID NO: 6], GGGGSGGGGSGGGGS [SEQ ID NO: 7], GIG [SEQ ID NO: 8], SGGGGIG [SEQ ID NO: 9], SGGGGSGGGGIG [SEQ ID NO: 10], GGGGS [SEQ ID NO: 11], the dipeptide SG, or a PAS linker).

In particularly preferred embodiments of the first aspect, the at least two ubiquitin moieties are directly connected to each other via a peptide bond, i.e., without any linker.

In preferred embodiments of the first aspect, the at least two ubiquitin moieties are - directly connected to the pharmaceutically active moiety via a covalent bond, such as a peptide bond or a disulfide bond; or

connected to the pharmaceutically active moiety via a linker, such as a maleimide moiety or a peptide linker (e.g., GGGGS [SEQ ID NO: 11], SGGGG [SEQ ID NO: 4], SGGGGSGGGG [SEQ ID NO: 5], GGGSGGGSGGGS [SEQ ID NO: 6], GGGGSGGGGSGGGGS [SEQ ID NO: 7], GIG [SEQ ID NO: 8], SGGGGIG [SEQ ID NO: 9], SGGGGSGGGGIG [SEQ ID NO: 10], the dipeptide SG, or a PAS linker).

In particularly preferred embodiments of the first aspect, the at least two ubiquitin moieties are connected to the pharmaceutically active moiety via the peptide linker SGGG [SEQ ID NO: 4], the peptide linker GGGGSGGGGSGGGGS [SEQ ID NO: 7], or the peptide linker GGGGS [SEQ ID NO: 11]. In some embodiments of the first aspect, the complex further comprises linear or branched polyethylene glycol (PEG), preferably with a molecular weight in the range of 20 kDa to 40 kDa. The PEG may be linked to one of the ubiquitin moieties of the complex or to the pharmaceutically active moiety. Said linkage is preferably a covalent linkage and may be a direct linkage or a linkage via a linker, such as a maleimide moiety or a peptide linker.

In preferred embodiments of the first aspect, the pharmaceutically active moiety is a therapeutic or diagnostic moiety. Diagnostic moieties include fluorescent labels and radionuclides. A radionuclide as pharmaceutically active moiety is selected either from the group of gamma-emitting isotopes, preferably ⁹⁹Tc, ¹²³l, ^mln, or from the group of positron emitters, preferably ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ¹²⁴l, or from the group of beta-emitter, preferably ¹³¹l, ⁹⁰Y, ¹⁷⁷Lu, ⁶⁷Cu, or from the group of alpha-emitter, preferably ²¹³Bi, ²¹¹At. A fluorescent label as pharmaceutically active moiety is selected from the group of Alexa Fluor or Cy dyes (Berlier et al., J Histochem Cytochem. (2003), 51 (12): 1699-1712). Therapeutic moieties include proteins, small molecules, radionuclides, and toxins, e.g., selected from the group of a photosensitizer; a pro-coagulant factor, preferably tissue factor (e.g., tTF truncated tissue factor); an enzyme for pro-drug activation, preferably an enzyme selected from the group consisting of carboxy-peptidases, glucuronidases and glucosidases; and/or a functional Fc domain, preferably a human functional Fc domain. A toxic compound as pharmaceutically active moiety is preferably a small organic compound or a polypeptide, optionally selected from the group consisting of saporin, truncated Pseudomonas exotoxin A, recombinant gelonin, Ricin-A chain, calicheamicin, neocarzinostatin, esperamicin, dynemicin, kedarcidin, maduropeptin, doxorubicin, daunorubicin, auristatin, cholera toxin, modeccin, or diphtheria toxin.

In preferred embodiments of the first aspect, the pharmaceutically active moiety is a pharmaceutically active protein. In these embodiments, it is particularly preferred that the complex of the invention is a fusion protein, i.e., the fusion protein comprises at least two ubiquitin moieties and at least one pharmaceutically active protein (plus optional peptide linker sequences) which can all be encoded by one single nucleic acid molecule. However, it is also envisioned within the context of the present invention that the at least two ubiquitin moieties and the at least one pharmaceutically active protein are encoded by different nucleic acid molecules and are subsequently linked to each other, for example by a disulfide bond or by a non-peptide linker.

In some embodiments of the first aspect, the at least two ubiquitin moieties are connected to the N-terminus of the pharmaceutically active protein. In some other embodiments of the first aspect, the at least two ubiquitin moieties are connected to the C- terminus of the pharmaceutically active protein. In yet other embodiments of the first aspect, at least one ubiquitin moiety of the at least two ubiquitin moieties is connected to the N-terminus of the pharmaceutically active protein, and at least one ubiquitin moiety of the at least two ubiquitin moieties is connected to the C-terminus of the pharmaceutically active protein. In these latter embodiments in which at least one ubiquitin moiety is connected to the N-terminus and at least one ubiquitin is connected to the C-terminus, it is not required that the same number of ubiquitin moieties is positioned on both sides of the pharmaceutically active protein. For example, if four ubiquitin moieties are present, it is possible to arrange the parts of the complex as follows: U-U-U-U-P; P-U-U-U-U; U-U-U-P-U; U-U-P-U-U; or U-P-U-U-U (wherein U denotes one ubiquitin moiety, P denotes one pharmaceutically active protein, and the above arrangement of the parts of the resulting fusion protein is shown from the N-terminus to the C-terminus).

In preferred embodiments of the first aspect, said pharmaceutically active protein comprises, essentially consists of, or consists of at least one functional domain of at least one protein selected from the group consisting of an antibody mimetic, a cytokine (such as an interleukin, an interferon or a TNF), antibody fragments, a receptor fragment, and a peptide hormone. More preferably, said pharmaceutically active protein comprises, essentially consists of, or consists of at least one protein selected from the group consisting of an antibody mimetic, a cytokine (such as an interleukin, an interferon or a TNF), antibody fragments, a receptor fragment, and a peptide hormone.

In especially preferred embodiments of the first aspect, said pharmaceutically active protein is selected from the group consisting of biologically active IFN, biologically active IL- 2, biologically active TNFalpha, and biologically active single-chain TNFalpha. The terms "biologically active IFN", "biologically active IL-2", "biologically active TNFalpha", and "biologically active single-chain TNFalpha" are defined above.

In some embodiments of the present invention, the complex comprises two or more pharmaceutically active moieties, which may be the same or different. For example, the complex of the present invention may be a fusion protein comprising two different pharmaceutically active proteins, such as a cytokine and an antibody mimetic (e.g., an Affilin*). In these embodiments, it is possible that one or more ubiquitin moieties of the at least two ubiquitin moieties are positioned between the two pharmaceutically active proteins. For example, if four ubiquitin moieties are present, it is possible to arrange the parts of the complex as follows: U-U-U-U-P1-P2; P1-P2-U-U-U-U; U-U-U-P1-P2-U; U-U-P1-P2- U-U; or U-P1-P2-U-U-U; P1-U-U-U-U-P2; P1-U-P2-U-U-U; P1-U-U-P2-U-U; P1-U-U-U-P2-U (wherein U denotes one ubiquitin moiety, PI denotes one pharmaceutically active protein (for example a cytokine), P2 denotes another pharmaceutically active protein (for example Affilin), and the above arrangement of the parts of the resulting fusion protein is shown from the N-terminus to the C-terminus).

In further preferred embodiments, the antibody mimetic is selected from the group consisting of Affilin* molecules, Anticalin" molecules, DARPin^® molecules (designed ankyrin repeat proteins), Affibody" molecules, Fynomers, Nanobodies*, Maxybodies, Avimers (avidity multimers), Nanofitins, Monobody (Adnectins) or others (for a review see: Binz H.K. et al. (2005) Nat. Biotechnol. 23(10): 1257-1268; the entirety of which is herein incorporated by reference). It is further preferred that the antibody mimetic is capable of binding to a target molecule with a specific binding affinity to the target molecule of K_D < 10^"7 M, preferably < 10^"8 M, more preferably < 10^"9 M, even more preferably < 10 ¹⁰ M, and most preferably < 10^" ¹¹ M.

In further preferred embodiments, the antibody mimetic is a modified dimeric ubiquitin protein (Affilin*) that is capable of binding to a target molecule with a specific binding affinity to the target molecule of K_D < 10^"7 M, preferably < 10^~8 M, more preferably < 10^"9 M, even more preferably < 10 ¹⁰ M, and most preferably < 10 ¹¹ M.

In some embodiments of the present invention, the two ubiquitin units (monomers) of said modified dimeric ubiquitin protein have the identical amino acid sequence, i.e., the targeting domain consists of a modified homo-dimeric ubiquitin protein. However, in most embodiments of the present invention the two ubiquitin units have been differently modified so that the targeting domain consists of a modified hetero-dimeric ubiquitin protein. Thus, it is further preferred that the modified ubiquitin protein is a hetero-dimeric ubiquitin comprising two monomeric ubiquitin units linked together in a head-to-tail arrangement, wherein each monomeric ubiquitin unit in said modified hetero-dimeric ubiquitin protein is modified independently from the modifications in the other monomeric ubiquitin unit. It is further preferred that each modified monomeric ubiquitin unit exhibits an amino acid sequence identity of at least 80% and at most 94% to the amino acid sequence defined by SEQ ID NO: 1 [= wild-type ubiquitin] or to the amino acid sequence defined by SEQ ID NO: 2 [= ubiquitin mutein (F45W, G75A, G76A)] or to the amino acid sequence defined by SEQ ID NO: 23 [= ubiquitin mutein (G75A, G76A)].

In some embodiments, these two monomeric ubiquitin units are directly linked, i.e., without a linker. Alternatively, these two monomeric ubiquitin units may be linked by a linker sequence, e.g., by the linker sequences shown in SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, or by the dipeptide linker SG.

As noted above, in some embodiments of the first aspect, the pharmaceutically active moiety comprises, essentially consists of, or consists of an antibody mimetic (preferably an Affilin*) which is capable of binding to a target molecule with a specific binding affinity to the target molecule of K_D≤ 10^"7 M, preferably < 10^"8 M, more preferably < 10^"9 M, even more preferably < 10 ¹⁰ M, and most preferably < 10 ¹¹ M. In further preferred embodiments, the target molecule is a tumor target molecule, preferably extra-domain B (ED-B) of fibronectin.

In some embodiments of the first aspect, the pharmaceutically active moiety is an Affilin. In preferred embodiments, the Affilin protein is comprising at least one modified ubiquitin unit having an amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 23 of at least 80%, wherein amino acids in positions 2, 4, 6, 8, 62, 63, 64, 65, 66, and 68 of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 23 are modified by substitution in order to obtain a modified ubiquitin protein with a detectable binding to a target with a specific binding affinity of Kd = 10 ^s - 10^"12 M. In preferred embodiments, the ubiquitin monomers of the Affilin protein are differently modified by substitutions of at least 5 amino acids in positions 2, 4, 6, 8, 62, 63, 64, 65, 66, and 68 of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 23, wherein said substitutions comprise substitutions at least in amino acid positions 63, 64, 65, and 66. For example, substitutions might comprise in the first monomeric unit at least amino acid positions 2, 4, 6, 62, 63, 64, 65, and 66; and in the second monomeric unit substitutions at least in amino acid positions 6, 8, 62, 63, 64, 65, and 66. For example, substitutions might comprise in the first monomeric unit at least amino acid positions 6, 8, 62, 63, 64, 65, and 66; and in the second monomeric unit at least in amino acid positions 2, 4, 6, 62, 63, 64, 65, and 66. For example, substitutions might comprise in the first monomeric unit at least amino acid positions 6, 8, 62, 63, 64, 65, and 66; and in the second monomeric unit at least in amino acid positions 6, 8, 62, 63, 64, 65, and 66. Other combinations are possible. In preferred embodiments of the first aspect, the modified hetero-dimeric ubiquitin protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 3 and an amino acid sequence that exhibits at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 3. In a second aspect the present invention is directed to the complex of the first aspect for use in medicine.

In a third aspect the present invention is directed to the complex of the first aspect for use in the treatment of cancer.

The third aspect of the present invention can alternatively be worded as follows: In a third aspect the present invention is directed to a method for treating cancer, comprising the step: administering a therapeutic amount of the complex according to the first aspect to a subject in need thereof.

In preferred embodiments of the third aspect, the cancer is selected from the group consisting of breast cancer, colorectal cancer, hepatocellular cancer, follicular lymphoma, melanoma, osteosacroma, pancreas, prostate, lung cancer, renal cell cancer, leukaemia, multiple myeloma, cutaneous T cell lymphoma, carcinoid tumor, glioblastoma multiforme (brain), mesothelioma, squamous cell carcinoma, cell carcinoma, and Hodgkin lymphoma. In a fourth aspect the present invention is directed to a pharmaceutical composition comprising a complex as defined in the first aspect; and further comprising a pharmaceutically acceptable carrier. 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 pharmaceutical composition is preferably sterile, non-pyrogenic, and isotonic and contains the pharmaceutically conventional and acceptable additives known per se. Additionally, reference is made to the regulations of the U.S. Pharmacopoeia or Remington's Pharmaceutical Sciences, Mac Publishing Company (1990).

In the field of human and veterinary medical therapy and prophylaxis pharmaceutically effective medicaments containing at least a complex in accordance with the invention can be prepared by methods known per se. Depending on the galenic preparation, these compositions can be administered parenterally by injection or infusion, systemically, rectally, intraperitoneal^, intramuscularly, subcutaneously, transdermally, or by other conventionally employed methods of application. The type of pharmaceutical preparation depends on the type of disease to be treated, the severity of the disease, the patient to be treated and other factors known to those skilled in the art of medicine.

In a fifth aspect the present invention is directed to a use of at least two ubiquitin moieties for extending the serum half-life of a pharmaceutically active moiety. In a sixth aspect the present invention is directed to a method for extending the serum half-life of a pharmaceutically active moiety, comprising the steps:

(b) introducing said fused nucleic acid into an expression vector;

(c) introducing said expression vector into a host cell;

(d) cultivating the host cell;

(f) optionally isolating the fusion protein produced in step (e). In a seventh aspect the present invention is directed to a nucleic acid comprising a sequence encoding the complex of the first aspect. In other words, the seventh aspect of the present invention is directed to a nucleic acid comprising a sequence encoding a fusion protein, wherein said fusion protein is defined as above, when describing embodiments of the first aspect of the invention.

In a further embodiment of the seventh aspect, the polynucleotide is for use in medicine, preferably for use in the treatment of cancer. This embodiment can alternatively be worded as follows: the present invention pertains to a method for treating cancer, comprising the step: administering a therapeutic amount of the polynucleotide according to the seventh aspect to a subject in need thereof. The cancer to be treated in accordance with the seventh aspect is preferably selected from the same list of cancers as defined above for the third aspect. In an eighth aspect the present invention is directed to a vector comprising the nucleic acid of the seventh aspect. In a further embodiment of the eighth aspect, the vector is for use in medicine, e.g., for use in the treatment of cancer. This embodiment can alternatively be worded as follows: the present invention pertains to a method for treating cancer, comprising the step: administering a therapeutic amount of the vector according to the eighth aspect to a subject in need thereof. The cancer to be treated in accordance with the eighth aspect is preferably selected from the same list of cancers as defined above for the third aspect.

In a ninth aspect the present invention is directed to a cell comprising the vector of the eighth aspect. In a further embodiment of the ninth aspect, the cell is for use in medicine, e.g., for use in the treatment of cancer. This embodiment can alternatively be worded as follows: the present invention pertains to a method for treating cancer, comprising the step: administering a therapeutic amount of the cell according to the ninth aspect to a subject in need thereof. The cancer to be treated in accordance with the ninth aspect is preferably selected from the same list of cancers as defined above for the third aspect. In a tenth aspect the present invention is directed to a complex comprising (a) at least two ubiquitin moieties; and (b) a pharmaceutically active moiety, wherein said complex has a molecular weight of at least 20 kDa, preferably at least 30 kDa, more preferably at least 40 kDa, even more preferably at least 50 kDa. In particularly preferred embodiments of the tenth aspect, the complex has a molecular weight of between about 30 kDa and 200 kDa; more preferably between 40 kDa and 110 kDa.

In preferred embodiments of the tenth aspect, said complex exhibits an increased serum half-life as compared to the at least one pharmaceutically active moiety alone without said at least two ubiquitin moieties.

All preferred embodiments described above for the first aspect apply in a fully analogous manner to the tenth aspect.

Likewise, the present invention is also directed to the complex of the tenth aspect for use in medicine, preferably for use in the treatment of cancer. This aspect of the present invention can alternatively be worded as follows: the present invention is directed to a method for treating cancer, comprising the step: administering a therapeutic amount of the complex according to the tenth aspect to a subject in need thereof. The cancer to be treated in accordance with the tenth aspect is preferably selected from the same list of cancers as defined above for the third aspect. In a further aspect, the present invention is directed to a pharmaceutical composition comprising a complex as defined in the tenth aspect; and further comprising a pharmaceutically acceptable carrier. The information on different forms of pharmaceutical compositions presented above with reference to the fourth aspect applies identically to pharmaceutical compositions comprising a complex as defined in the tenth aspect.

Furthermore, the seventh, eighth, and ninth aspect of the invention have been defined above with reference to the complex of the first aspect. In analogous manner, the present invention is also directed to variations of the seventh, eighth, and ninth aspect that relate to the complex of the tenth aspect.

Extra-domain B of fibronectin as tumor specific protein The extra-domain B (ED-B) of fibronectin is a small domain inserted by alternative splicing of the primary RNA transcript into fibronectin. Fibronectins are high molecular weight extracellular matrix glycoproteins abundantly expressed in healthy tissues and body fluids. ED-B represents one of the most selective markers associated with angiogenesis and tissue remodeling. It is abundantly expressed around new blood vessels, but undetectable in virtually all normal adult tissues. ED-B is known to be involved primarily in cancer. High levels of ED-B expression were detected in primary lesions as well as metastatic sites of many human solid cancer entities. In solid cancer tissues, ED-B is either detected surrounding pro- angiogenic vessels or in a mixed mode of perivascular and stromal expression (Menrad and Menssen (2005), Expert Opin Ther Targets, 9: 491-500, the entirety of which is herein incorporated by reference). Furthermore, ED-B can be bound to diagnostic agents and used as diagnostic tool. One example is its use in molecular imaging of atherosclerotic plaques and detection of cancer, for example by immunoscintigraphy of cancer patients. Plenty of additional diagnostic uses are conceivable.

The amino acid sequence of human extra-domain B (ED-B, 91 residues) of fibronectin is shown in SEQ ID NO: 25. For expression of the protein, a start methionine has to be added. ED-B is detected in mammals, e.g., in rodents, cattle, primates, carnivore, human etc. Examples of animals in which there is a 100% sequence identity to human ED-B are Rattus norvegicus, Bos taurus, Mus musculus, Equus caballus, Macaco mulatto, Canis lupus familiaris, and Pan troglodytes.

ED-B specifically accumulates in neo-vascular structures and represents a target for molecular intervention in cancer. A number of antibodies or antibody fragments to the ED-B domain of fibronectin are known in the art as potential therapeutics for cancer and other indications (see, for example, WO 97/45544, WO 07/054120, WO 99/58570, WO 01/62800, all of which are hereby incorporated by reference). Furthermore, conjugates comprising an anti-ED-B antibody or an anti-ED-B antibody fragment with IL-12, IL-2, IL-10, IL-15, IL-24, or GM-CSF have been described for targeting drugs for inhibiting diseases such as cancer, angiogenesis, or neoplastic growth (see, for example, WO 06/119897, W0 07/128563, WO 01/62298, all of which are hereby incorporated by reference).

WO 2011/073208 and WO 2011/073209 (all of which are hereby incorporated by reference) disclose multimeric proteins based on modified ubiquitin with high affinity binding to the target ED-B. The applications describe anti-ED-B binding molecules showing a highly efficient targeting of tumor vasculature.

Interferon

Interferon alpha (abbreviations: IFN-alpha or IFN-a) is a cytokine. More than 10 different subtypes encoded by different genes exist in human. All IFN-alpha subtypes, together with IFN-beta, bind to the IFN-alpha receptor (IFNAR), which is composed of two subunits, IFNAR1 and IFNAR2. IFNAR molecules are present on most cell types, making them responsive to IFN-alpha signals. Interactions between IFN-alpha and its receptor are highly species specific. For uses in medicine, especially interferons IFN-alpha 2a and IFN-alpha 2b are of interest. Both interferons show high affinity to the IFN-alpha receptor.

The role of interferon alpha in cancer has been studied. Medicaments containing IFN- alpha 2a or 2b were used initially for indications like Hairy cell leukemia and chronic myelogenous leukemia. IFN-alpha is still used in the treatment of renal cell carcinoma and cutaneous lymphoma but other therapies show improved efficacies compared to IFN-alpha. In order to obtain therapeutic responses, high doses of IFN-alpha have to be used, leading to high toxicity. IFN-alpha has many cellular effects including an anti-cancer activity and antiviral activity. IFN-alpha therapy often has to be applied for many months in order to achieve a therapeutic result. Nevertheless, IFN-alpha is one of the very few cancer therapies that have the potential to have a curative effect on metastatic tumors in humans.

Single chain TNFalpha (scTNF)

In the prior art, single chain (sc) TNFalpha proteins of at least three monomers connected by peptide linkers are described generally. For example, Krippner-Heidenreich et al. (2008, J Immunol., 180 (12): 8176-8183; the entirety of which is herein incorporated by reference) describe polypeptides which consist of at least three monomers of a TNF family ligand which are connected by peptide linkers. Importantly, it was shown that although this construct is less toxic than wild-type TNFalpha, it shows the same bioactivity as native TNF.

EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used but some experimental errors and deviations should be accounted for. Unless indicated otherwise, molecular weight is average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.

Example 1. Production of complexes comprising four or six ubiquitin moieties and an Affilin^¾ as pharmaceutically active moiety

The complexes of the invention comprise at least two ubiquitin moieties and a pharmaceutically active moiety. As pharmaceutically active moiety, in this Example, a modified hetero-dimeric ubiquitin (Affilin^*) is comprised, which is connected to the preceding four or six ubiquitin moieties via a short peptide linker with the amino acid sequence GGGGS (SEQ ID NO:ll). The complexes were produced as soluble proteins in a suitable host, namely E. coli, and purified from the cytoplasmic fraction as described in Examples 1-3. By way of characterization, the obtained fusion protein preparations were analyzed for purity and homogeneity. Additionally, the in vitro affinity for target protein ED-B was tested as described in Examples 4-6.

Step 1: Production of vectors for cloning of ubi4-Affilin and ubi6— Affilin complexes of the invention

As vector for cloning of fusion proteins, proprietary expression vectors (Scil Proteins GmbH, pSCIL008b, see WO 05/061716, the entirety of which is herein incorporated by reference) or commercially available vectors (e.g., pET20b by Invitrogen) were modified by insertion of coding sequences for four or six wild-type Ubiquitin moieties, respectively, which were previously amplified by PCR using standard methods known to somebody skilled in the art. Unique restriction sites were introduced into all resulting expression plasmids in order to facilitate the insertion of modified ubiquitin sequences. ubi6-Affilin: Vector pET20b was modified by insertion of six wild-type ubiquitin moieties. Affilin^® (SEQ ID NO: 3) was amplified by PCR (annealing temperature 59°C, 30 cycles) and ligated into the 6 ubiquitin (ubi6) containing vector pET20b via BamH\/Xho\ restriction sites. The following primers were used:

SPW-mri-fw-BamH G CAGG G ATCCATG CGTATCTGG GTG CACACCCTG ACC (SEQ ID NO: 21) SPF-AA-Stop-Xho TG CAG CCATCTCG AGTCATTAG G CCG CACGTAAACG AAG AACTAA (SEQ ID NO: 22)

Ubi4-Affilin:

Affilin and Ubi4 were amplified separately via PCR, ligated with each other via SornHI restriction site and as fusion cloned into pET20b via Nde\/Xho\ restriction sites.

Primer for the amplification of Affilin:

SPW-mri-fw-BamH G C AG G G ATCCATG CGTATCTG G GTG CACACCCTG ACC (SEQ ID NO: 21) SPF-AA-Stop-Xho TG CAG CCATCTCG AGTCATTAG G CCG CACGTAAACG A AG AACTAA (SEQ ID NO: 22)

PCR conditions: Annealing temp 59°C, 25 cycles

Primer for the amplification of Ubi4 (four ubiquitin monomers)

Ubil-fw-Nde GGAGATATACATATGCAGATCTTTG (SEQ ID NO: 24)

Ubi4-G4S-rev-BamH CTG CG G ATCC ACCG CC ACCTG CG G C ACGT AACCG CAG G (SEQ ID NO: 38) PCR conditions: Annealing temp 55°C, 25 cycles

Step 2: Cloning of ubi4-Affilin-complexes and ubi6— Affilin- complexes

For the production of the complexes of the invention, the sequence of interest was amplified from a plasmid template by PCR according to standard procedures and inserted into the expression plasmids as described in Step 1. DNA sequence analyses confirmed the correct sequences of the expression vectors encoding the fusion proteins.

Step 3: Expression of Ubi4-Affilin-complexes and Ubi6-Affilin-complexes

Complexes were produced in E. coli and isolated from cytoplasm. For expression of the complexes, the corresponding clones were cultivated and grown by batch or fed-batch fermentation in complex media containing the appropriate antibiotics corresponding to the respective resistance gene encoded on the expression vectors. Expression was induced by adding IPTG. After 4 h of induction, microbial cells were harvested, suspended, and disrupted by high pressure dispersion in a French press. The expressed fusion proteins were purified from the soluble fraction obtained after centrifugation of the cell lysate.

Example 2: Purification of Ubi4-Affilin-complexes and Ubi6-Affilin-complexes

For purification of Ubi4-Affilin and Ubi6-Affilin complexes as fusion proteins, up to three chromatographic steps were required. These chromatographic steps included hydrophobic interaction chromatography (HIC) on e.g., Butyl 650M, ion exchange chromatography on, e.g., SP Sepharose HP and size exclusion chromatography on, e.g., Superdex gel filtration medium. In all cases, fractions were analyzed by SDS-PAGE with respect to their purity. Suitable fractions were pooled and analyzed for homogeneity and activity by a series of methods including, e.g., rpHPLC, SE-HPLC combined with light scattering detection, analytical affinity interaction chromatography, and surface plasmon resonance-based interaction analysis. For fusion protein comprising four wild-type ubiquitin moieties (Ubi4) linked to an Affilin" (SEQ ID NO: 15, MW = 52.1 kDa), yields of up to 34 mg pure and active fusion protein per liter expression culture were obtained. The yield obtained for the complex comprising six ubiquitin moieties (Ubi6) and an Affilin (SEQ ID No: 14; MW = 69.3 kDa) was in a similar range.

Example 3: Preparation of a heteromeric binding protein based on modified ubiquitin dimers (Affilin^®) Step 1: Production of vectors for cloning

As vector for cloning of Affilin⁸ (SEQ ID NO: 3), a commercially available vector (pET SUMO, Invitrogen) was modified by insertion of an oligonucleotide encompassing unique restriction sites as described in the literature (Bosse-Doenecke et al. (2008), Protein Expr Purif. 58: 114-121, the entirety of which is herein incorporated by reference). Additional unique restriction sites were introduced, in order to facilitate the subsequent insertion of modified ubiquitin (Affilin*) sequences as described in Example 3, step 2. Step 2: Cloning of Affilin molecules (modified hetero-dimeric ubiquitin-based binding proteins)

For the production of an Affilin* fused to Small Ubiquitin-like Modifier (SUMO), the sequence coding for an Affilin" (SEQ ID NO: 3) was amplified from a plasmid template by PCR according to standard procedures.

The following primers were used:

1071C12-mwi-fw-Bsa TTTTG GTCTC ATG GTATGTG G ATCTG G GTG C AC ACCCTG ACC (SEQ

ID NO: 45)

HUBI-AA-SUMO-rev A AAAG G ATCCTC ATTAG G CCG C ACGTA AACG AAG A ACT A (SEQ ID

NO: 46)

The amplified sequence was ligated into pET-SUMOadapt (already containing SUMO) via the restriction sites Bsal/BamHI.

DNA sequence analyses confirmed the correct sequences of the expression vectors encoding the SUMO-Affilin*- fusion proteins.

Step 3: Expression of SUMO-Affilin^*-fusion proteins

SUMO fusion proteins were produced in E. coli and isolated from cytoplasm. For expression of the fusion proteins, the clones were cultivated and grown by batch or fed- batch fermentation in complex media containing the appropriate antibiotics corresponding to the respective resistance gene encoded on the expression vectors. Expression was induced by adding isopropyl^-D-l-thiogalactopyranoside (IPTG). After 4 h of induction, microbial cells were harvested, suspended, and disrupted by high pressure dispersion in a French press. The expressed proteins were purified from the soluble fraction obtained after centrifugation of the cell lysate.

Step 4: Purification of Affilin* proteins

Specific binding proteins (Affilin*) were produced as cleavable SUMO fusion proteins as described in Example 3, Step 3 by a series of chromatographic steps including Immobilized Metal ion Affinity Chromatography (IMAC) on Ni-NTA Superflow and size exclusion chromatography on Superdex gel filtration medium. Before the final chromatographic purification step, the SUMO domain was removed from the Affilin* protein by cleavage with a specific SUMO hydrolase under appropriate conditions (Fig. 4A). Fractions were analyzed by SDS-PAGE to check for purity and suitable fractions were pooled. Analytical methods including rpHPLC, SE-HPLC combined with multi-angle light scattering detection, analytical affinity interaction chromatography, and surface plasmon resonance-based interaction analysis were used to analyze the protein pool with respect to homogeneity and activity. For the Affilin" (SEQ ID NO: 3), yields of up to 500 mg active protein per liter expression culture from fed-batch fermentation were obtained.

Step 5: Modification of Affilin* proteins with Polyethylene Glycol (PEG)

Pharmaceutically active moieties prepared according to Examples 3 and 8 (SEQ ID

NO: 3; SEQ ID NO: 16 and 19) were N-terminally modified with a mono-functionally activated polyethylene glycol (PEG) moiety by reductive alkylation, pursuant to established protocols. The molecular weight of the linear or branched PEG-aldehyde molecules used for protein modification was in the range of 20 to 40 kDa. Mono-PEGylated proteins were separated from unmodified and poly-PEGylated species, respectively, according to the isoelectric point of the complexes (Fig. 4B and 4C). Due to the modification described the molecular weight of the Affilin^* was increased to approx. 60 kDa. The molecular weight of Affilin^*-IFN (SEQ ID NO: 19) and Ubi2-IFN (SEQ ID NO: 16), respectively, was raised to approx. 57 kDa. Example 4: Biacore assays to determine the affinity of the Affilin fusion proteins towards human or mouse ED-B

ED-B binding activity of the complexes comprising four or six ubiquitin moieties and an Affilin^* (SEQ ID NO: 15 and SEQ ID NO: 14) as well as of the PEGylated proteins containing an Affilin^* (SEQ ID NO: 3 and SEQ ID NO: 19) was investigated using the surface plasmon resonance-based Biacore technique. Different concentrations of the proteins were analyzed (0-200 nM) for binding to human or mouse ED-B immobilized on Biacore sensor surfaces according to established methods. The obtained data were processed via Biaevaluation software and l:l-Langmuir-fitting. For the complexes, the resulting dissociation constants (K_D) as well as corresponding microscopic rate constants k_off and k_on are summarized in Table 1. _ _

Table 1. Affinity of the complexes to human ED-B

Complex microscopic rate of microscopic rate of Binding affinity of the association dissociation complex

k_on [M ¹ s ¹] [K_D value]

Affilin* 2.8 10⁶ 5.1-10^"4 0.182 nM

Ubi4- Affilin* 2.12 10⁶ 5.64-10⁴ 0.263 nM

Ubi6- Affilin* 1.45-10⁶ 3.85-10^"4 0.265 nM

PEG- Affilin* 3.42-10⁵ 1.54-10^"3 4.5 nM

PEG-Affilin*-IFN 5.02 10⁵ 8.35-10⁴ 1.66 nM

Example 5: Analysis of the ED-B-binding activity of the complexes bv enzvme-linked immunosorbent assay

The target binding activity of the complexes comprising a hetero-dimeric ubiquitin- based Affilin* (SEQ ID NO: 3) were analyzed by an ED-B-binding ELISA. For this purpose, a recombinant protein construct containing the human fibronectin exodomains 6, 7, B, 8, and 9 was coated to Nunc Medisorp microwell plates in a concentration of 100 ng/ml. Another recombinant construct containing only domains 6, 7, 8, and 9 was coated to control surfaces in an equimolar concentration (80 μ^ητιΙ). Unspecific binding sites were blocked with 3% BSA dissolved in PBST buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 8 mM disodium hydrogen phosphate and 2 mM potassium dihydrogen phosphate, supplemented with 0.1 %(w/v) polyoxyethylene(20) sorbitan monolaurate). After washing the wells with PBST buffer, the complexes were applied in appropriate concentration series and incubated for 1 h. The wells were again washed with PBST. For detection of the bound fusion proteins, a POD-conjugate of a ubiquitin-specific recombinant Fab fragment (AbD SeroTec) was applied in a dilution of 1:6500. Specific binding of the fusion proteins to the immobilized ED- B domain was monitored by the POD-catalyzed colorimetric reaction of the substrate 3,3',5,5'-tetramethylbenzidin according to the manufacturer's (KEM-EN-Tec) instructions. Reactions were stopped by adding 0.2 M H₂S0₄. The ELISA plates were read out using the TECAN Sunrise ELISA-Reader. The photometric absorbance measurements were done at 450 nm using 620 nm as a reference wavelength. Fig. 6 compares the results of an ED-B binding ELISA performed with Affilin" (SEQ ID NO: 3) and complex Ubi4-Affilin (SEQ ID NO: 15). The results for a number of complexes containing the Affilin" directed against ED-B as pharmaceutically active moiety are summarized in the Table shown in Fig. 9A.

Example 6: Binding analysis of the Ubi4-Affilin-complex and Ubi6-Affilin-complex (fusion proteins) to human ED-B by analytical affinity interaction chromatography

The affinity of the complexes of invention toward human ED-B was also investigated using analytical affinity interaction chromatography. For that purpose, a chromatographic affinity matrix was prepared by covalently coupling a construct containing human ED-B domain to Sulfolink™ resin (Pierce). Ubi4-Affilin or Ubi6-Affilin was applied to the column containing the ED-B affinity matrix (200 μg complex per ml affinity resin). Binding was performed in physiological buffer (phosphate-buffered saline (PBS)), while elution of bound protein was induced by pH shift to pH 2.5. From the ratio of bound to unbound species the fraction of active protein in the preparations could be estimated. Fig. 5 represents the affinity interaction chromatography analysis performed with complex Ubi6-Affilin (SEQ ID NO: 14); results obtained with a series of complexes (SEQ ID NO: 3, SEQ ID NO: 14; SEQ ID NO: 15) are summarized in a Table shown in Fig. 9A. Example 7. Complex composed of two ubiquitins moieties (Ubi2) and interferon-alpha as pharmaceutically active moiety

Production of the complex Ubi2-IFIM

The complex protein described consists of two ubiquitin monomers (SEQ ID NO: 2) linked by a GIG peptide linker (SEQ ID NO: 8), an SG₄-linker (SEQ ID NO: 4), and interferon- alpha 2b (SEQ ID NO: 28) as pharmaceutically active moiety. The complex was produced as inclusion bodies in f. coli and purified after in vitro refolding. By way of characterization, the obtained complex was analyzed for purity and homogeneity. The activity of the pharmaceutically active moiety IFN-alpha 2b was tested in vitro (cell culture).

Step 1: Cloning of an Ubi2-IFN complex Two ubiquitin monomers (Ubi2) and interferon IFN alpha 2b (IFN) were amplified separately via PCR.

Primers for Ubi2:

Ub2(7)-fw-EcoR GCAGGAATTCATGCAGATCTTCGTGAAAACC (SEQ ID NO: 39)

Ub2(7)-rev-Bsa G C ACG GTCTCC A ACG AAG A ACTA A ATGTA AG G (SEQ ID NO: 40)

PCR conditions: Annealing temperature 55°C, 24 cycles

Primers for IFN:

Hubi-SG4-fw-Bsa G C ACG GTCTCCCGTTTACGTG C AG C AAG CG G (SEQ ID NO: 41)

IFN-rev-Pst G CTG CCTG C AGTC ATTATTCTTTG CTACG C (SEQ ID NO: 42)

PCR conditions: Annealing temperature 54°C, 24 cycles

Ubi2 and IFN were ligated with each other by the restriction site Bsa\. The fusion of Ubi2 and IFN was inserted into the expression vector pSCIL008b (Scil Proteins, WO 05/061716) by the restriction sites EcoR\/Pst\. Standard methods known to somebody skilled in the art were applied.

Step 2. Expression of Ubi2-IFN complex

The complex was produced in E. coli and isolated in the form of inclusion bodies. For expression of the complex, the clones were cultivated and grown by fed-batch fermentation in complex medium containing the appropriate antibiotics corresponding to the respective expression vectors. Expression was induced by adding IPTG. After 2-4 h of induction, microbial cells were harvested, suspended, and disrupted by high pressure dispersion in a French press. The insoluble fraction was collected and inclusion bodies containing the expressed proteins were isolated by standard washing protocols.

Example 8: In vitro refolding and purification of Ubi2-IFN complex

Active complex was prepared by in vitro refolding at a temperature of 4 degrees centigrade after rapid dilution of inclusion body material solubilized in 6 M guanidinium chloride, and purified by a series of chromatographic steps. These chromatographic steps included at least one ion exchange chromatography on Q Sepharose HP. In all cases, fractions were analyzed by SDS-PAGE and analytical HPLC with respect to their purity. Suitable fractions were pooled and analyzed for homogeneity and activity by a series of methods including rpHPLC and SE-HPLC. For the complex, yields of up to 690 mg active protein per liter expression culture from fed-batch fermentation were obtained.

Example 9: Activity assay of the effector domain of Ubi2-IFN complex

To analyze the physiological IFN-alpha activity of the Ubi2-IFN complex, an ISRE- Reporter Gene Assay was established. IFN-alpha is capable of inducing interferon-stimulated genes (ISGs), for example ISG54. ISG54 contains a c/^'s-acting element (TAGTTTCAC 1 1 I CCC, SEQ ID NO: 26) in its promoter region, which is responsible for the inducible expression of the gene. This element is referred to as ISRE-element (IFN-stimulated response element). Five tandem copies of the ISRE element were inserted upstream of the basic promoter element (TATA box) and luciferase gene of pGL4.27-Luc2 plasmid (Promega). Hela-cells, a cervix carcinoma cell line, were transfected and a cell pool was sustained by selection with Hygromycin. To monitor the IFN-alpha activity of the Ubi2-IFN complex, the reporter cells were used for an ISRE-Reporter Gene Assay.

The cells were resuspended in suitable medium containing 10% fetal calf serum (FCS). A cell suspension with a density of 3xl0⁵ cells/ml in medium containing 5% FCS was seeded into a white 96 well cell culture plate. After 24 h, the cells were treated with different concentrations of fusion proteins (e.g., in the range of 3xl0^"10 to 4.6xl0^"14 M). The metabolic activity was measured by ONE-Glo™ Luciferase substrate (Promega). Each testing of complex of the invention was paralleled by testing a dose range of recombinant human IFN-alpha 2b (Biomol) to validate the assay. The quantitative evaluation is based on the relative potency against an IFN-alpha 2b standard by parallel line method with PLA2.0 software. Potency was determined in triplicates. The complex has a potency of 38 - 41%. The potency of IFN-alpha 2b should be in a range of 100% +/-20% (see Table in Fig. 9B). _{a n}

40

Example 10: Production of a complex composed of two ubiquitin moieties and pharmaceutically active single-chain murine TNFalpha

Production of the complex Ubi2-scTNF

The ubi2-scTNF complex protein (SEQ ID NO 17) consists of two ubiquitin moieties

(SEQ ID NO: 2) linked by a GIG peptide linker (SEQ ID NO: 8), and single-chain murine tumor necrosis factor-alpha (scTNF; SEQ ID NO: 37) as pharmaceutically active moiety, linked by a (G₄S)₃ linker (SEQ ID NO: 7). The complex was produced as inclusion bodies in E.coli and purified after in vitro refolding. By way of characterization, the obtained complex was analyzed for purity and homogeneity. The structural integrity of the pharmaceutically active scTNF moiety was tested by a receptor-binding ELISA, and its preserved biological activity was demonstrated by an in vitro cell culture assay.

Step 1: Production of a vector for cloning of the Ubi2-scTNF complex

The TNFalpha sequence was amplified via PCR using standard methods known to somebody skilled in the art. pSCIL008b was modified by insertion of the coding sequences for three murine TNFalpha subunits genetically fused head-to-tail by two (G₃S)₃ peptide linkers, thereby obtaining plasmid pSCIL008b-mscTNFa. Unique restriction sites were introduced into the resulting expression plasmids in order to facilitate an insertion of the sequence coding for the ubiquitin moieties.

Step 2: Cloning of Ubi2-scTNF

For the production of the complex of two ubiquitin moieties and murine single-chain TNFalpha, the sequence coding for two ubiquitin moieties was amplified from a plasmid template by PCR according to standard procedures, and inserted into the expression plasmids pSCIL008b-mscTNFa described in step 1. DNA sequence analyses confirmed the correct sequences of the expression vectors encoding the complex.

Ubi2 (two ubiquitin monomers) were amplified via PCR using standard procedures.

Primers used:

Ub2(7)-fw-EcoR G C AG G A ATTC ATG C AG ATCTTCGTG A A A ACC (SEQ ID NO: 39)

SPF-AA-rev2-Bsa GCAGGGTCTCACACCCGCGGCACGTAAACGAAGAAC (SEQ ID NO: 44) The PCR was performed using an annealing temperature of 52°C and 28 cycles. The amplified Ubi2 was ligated into pSCIL008b-mscTNFa using restriction sites EcoR\/Bsa\. Step 3. Expression of Ubi2-scTNF

The complex was produced in suitable E. coli host strains and isolated in the form of inclusion bodies. For expression of the complex, the clones were cultivated and grown by fed-batch fermentation in complex medium containing the appropriate antibiotics corresponding to the respective expression vectors. Expression was induced by adding IPTG. After 4 h of induction, microbial cells were harvested, suspended, and disrupted by high pressure dispersion in a French press. The insoluble fraction was collected and inclusion bodies containing the expressed proteins were isolated by standard washing protocols.

Example 11: In vitro refolding and purification of Ubi2-scTNF complex

Active complex was prepared by in vitro refolding at a temperature of 4 degrees centigrade after rapid dilution of inclusion body material solubilized in 6 M guanidinium chloride into phosphate-buffered saline (PBS) to a final protein concentration of 0.15 mg/ml, and purified by a series of chromatographic steps. These chromatographic steps included a capture step on a hydrophobic charge interaction matrix (MEP Hypercel, Pall), an intermediate anion exchange chromatography on Q. Sepharose HP (GE Healthcare) and a final size exclusion step on a Superdex 200 column (GE Healthcare). In all cases, fractions were analyzed by SDS-PAGE and analytical HPLC with respect to their purity. Suitable fractions were pooled and analyzed for homogeneity and activity by rpHPLC and SE-HPLC. Yields of up to 50 mg purified Ubi2-scTNF complex per liter expression culture from fed- batch fermentation were obtained.

Example 12: ELISA of specific TNF-receptor binding by Ubi2-scTNF complex

In order to test the structural integrity of the pharmaceutically active scTNF moiety, binding of a TNF receptor I domain to the Ubi2-scTNF complex was analyzed in an ELISA setup. For this purpose, Nunc microwell plates were coated with a commercially available polyclonal anti-TNF antiserum (PeproTech) in a concentration of 1 g/ml. Unspecific binding sites were blocked with bovine serum albumin (BSA) blocking solution in PBS. After washing the wells with PBST buffer, Ubi2-scTNF, was applied in a concentration of 1 /ητιΙ, while recombinant murine TNFalpha serving as control was applied in an equimolar concentration of 0.73 g/ml. The wells were again washed with PBST. For analysis of the receptor binding activity of the TNF moieties in the immobilized complex and the immobilized recombinant TNFalpha, respectively, a commercially available construct comprising the soluble TNF- binding domain of murine TNF receptor I and the Fc portion of human IgG (R&D systems) was employed. This chimera was applied to the wells in concentration series of 0-200 nM. After incubation and an additional washing step, a POD-conjugate of a polyclonal Fc-specific anti human IgG antibody from goat (AbD SeroTec) was applied in a dilution of 1:50000, in order to detect bound TNF receptor chimera. Specific binding of the receptor chimera to the immobilized fusion proteins was monitored by a POD-catalyzed colorimetric reaction using the substrate 3_;3',5,5'-tetramethylbenzidin (KEM-EN-Tec) according to the manufacturer's instructions. Reactions were stopped by adding 0.2 M H₂S0₄. The ELISA plates were read out using a TECAN Sunrise ELISA-Reader. The photometric absorbance measurements were done at 450 nm using 620 nm as a reference wavelength.

In this assay, the TNF receptor binding activity of Ubi2-scTNF was found to be comparable to TNFalpha, as shown in Fig. 10, indicating that the structure of the TNF moiety in the complex was fully intact. Example 13: Activity assay of the effector domain of Ubi2-scTNF complex

The physiological TNF-alpha-activity of a complex according to the invention, consisting of two ubiquitin moieties genetically fused via a (G₄S)₃-linker (SEQ ID NO: 7) to murine single-chain TNFalpha, was determined using the L929 apoptosis assay (Flick et al.(1984), J Immunol Methods. 68: 167-175, the entirety of which is herein incorporated by reference). In this assay, the effector part of the complex efficiently stimulates cell death in actinomycin D sensitized cells at EC₅₀ values in the picomolar range.

Cells were resuspended in suitable medium containing fetal bovine serum and antibiotics. A cell suspension with a density of 3.5xl0⁵ cells/ml was seeded into the wells of 96 well standard cell culture plates. After incubation, the culture medium was removed and medium containing fetal bovine serum (FBS), Actinomycin D and antibiotics was added to each well. After incubation, the complex of the invention or recombinant TNFalpha (cf. below) were added at appropriate concentration ranges (10^~7 and 10^"18 M). After further incubation, the metabolic activity as a measure of cell survival was determined using WST-1 reagent (Roche). At least three independent experiments were conducted, each of them in triplicates. Each test of the complex according to the invention was accompanied by testing a dose range of human recombinant TNF-alpha as control.

The complex was found to have a potency of 156 ± 68 % compared to the TNF-alpha control (see Table in Fig. 9B). The biological activity of the scTNF moiety in the complex was thus fully preserved.

Example 14: In vivo pharmacokinetic study in healthy mice using fusion proteins of the invention

To determine pharmacokinetic parameters of half-life prolonged fusion proteins of the invention, healthy mice were treated with a single administration of these variants after lodine-125 labeling. For treatment, groups of n=9 mice (CD-I strain) were intravenously injected into the tail vein in a total applied volume of 4.2 ml/kg. Doses of 11.4 nmol/kg unmodified affilin-equivalent were administered. Three mice per blood sampling time point were used.

Step 1: Measurement of blood and serum radioactivity levels

At the time points indicated, the blood samples were collected from saphenous vein of un-anaesthetized mouse. Each blood sample was collected in pre-weighed Microvette tubes with clotting activator (Sarstedt). The tubes were weighed and the radioactivity was measured in an automatic gamma counter (Wallace Wizard 2470 - Perkin Elmer) calibrated for lodine-125 radionuclide (efficiency: 74%). The results of this counter are expressed as cpm and the conversion in μϋ is realized as described below:

Radioactivity in μϋ = Radioactivity in cpm / 0.74 / 60 / 37 000

Explanation of this calculation:

cpm / detector efficiency (0.74) = dpm

dpm / 60 = disintegration per second = value in Bq (a Becquerel equals one radioactive disintegration per second)

Bq are then divided by 37000 to convert it to μϋ (Ιμϋ = 37 000 Bq). The concentration of radioactivity in blood is expressed as percentage of the injected dose and equivalent quantity of protein per g. Step 2: Calculation of blood pharmacokinetic parameters

The pharmacokinetic parameters were assessed using data expressed as activity per mL of blood (μθ/νηί). These data were analyzed using a macro on Excel™ software. The half- life of blood activity was calculated using two-phase exponential decay equations, which produced distribution (Ti/₂ alpha) and elimination (Ti/₂ beta) half-lives. The blood clearance and the area under curve (AUC) were also calculated. AUC and blood half-life (Ti/₂ alpha and Ti/₂ beta) are directly calculated by the program of the macro. The blood clearance is calculated from the AUC using the following equation:

Clearance = injected dose / AUC

As shown in Fig. 7 there is a clear evidence for prolongation of circulation half-life with an increased number of ubiquitin-monomers attached to the Affilin molecule.

Step 3: SE-HPLC analyses to investigate serum stability

SE-HPLC analyses of serum samples (pooled samples from three mice each time point) were conducted on a Superdex G200 10/300 GL column eluted with PBS at a flow rate of 0.8 mL/min. Between the injection of each serum sample, an injection of 70 μί. of 0.1 N NaOH followed by an injection of 70 μί of water were performed as washing steps. A total volume of 30-100 μΐ undiluted serum sample was injected. Prior to injection, serum samples were filtered with a pore diameter of 0.45 μηι.

Activity loaded onto the column was determined by calculation from the amount of the loaded radioactive protein. Eluted fractions were collected and radioactivity of each fraction was measured using a gamma counter in order to plot radio chromatogram profiles. Radioactivity of peaks corresponding to the mature protein was used for calculation of the percentage of intact labeled protein. As displayed in Fig. 8 in line with increasing numbers of ubiquitin monomers included in the complex the amount of intact protein over time in serum increases too. SEQUENCE LISTING - FREE TEXT INFORMATION

The sequences according to SEQ ID NOs: 1, 25, 27 to 36, 43, 47, and 48 shown in the attached sequence listing do not contain any free text information. Nevertheless, short explanations are presented below also for these sequences.

SEQ ID NO: 1 wild-type ubiquitin

SEQ ID NO: 2 ubiquitin mutein (F45W/G75A/G76A), start sequence for mutagenesis

SEQ ID NO: 3 Affilin

SEQ ID NO: 4 peptide linker

SEQ ID NO: 5 peptide linker

SEQ ID NO: 6 peptide linker

SEQ ID NO: 7 peptide linker

SEQ ID NO: 8 peptide linker

SEQ ID NO: 9 peptide linker

SEQ ID NO: 10: peptide linker

SEQ ID NO: 11 peptide linker

SEQ ID NO: 12: basic linker sequence

SEQ ID NO: 13: basic linker sequence

SEQ ID NO: 14: Ubi6-Affilin

SEQ ID NO: 15: Ubi4-Affilin

SEQ ID NO: 16: Ubi2-IFN

SEQ ID NO: 17: Ubi2-scTNF

SEQ ID NO: 18: Ubi2-TNF

SEQ ID NO: 19: Affilin-IFN

SEQ ID NO: 20: SUMO-Affilin

SEQ ID NO: 21: PCR primer

SEQ ID NO: 22: PCR primer

SEQ ID NO: 23: ubiquitin mutein (G75A/G76A), a ubiquitin moiety used for increasing serum half-life

SEQ ID NO: 24: PCR primer

SEQ ID NO: 25: extra-domain B (ED-B) of fibronectin

SEQ ID NO: 26 c/s-acting element

SEQ ID NO: 27: human IFN-a 2a

SEQ ID NO: 28: human IFN-a 2b

SEQ ID NO: 29: human IFN-a 2c

SEQ ID NO: 30: human IFN-a 6

SEQ ID NO: 31: human IFN-a 14

SEQ ID NO: 32: human IFN-a 4

SEQ ID NO: 33: human IFN-a 5

SEQ ID NO: 34: human TNF alpha

SEQ ID NO: 35: murine TNF alpha

SEQ ID NO: 36: rat TNF alpha

SEQ ID NO: 37: scTNF, murine

SEQ ID NO: 38: PCR primer

SEQ ID NO: 39: PCR primer

SEQ ID NO: 40: PCR primer .„

46

SEQID NO: 41: PCR primer

SEQID NO: 42: PCR primer

SEQID NO: 43: human IFN-β

SEQID NO: 44: PCR primer

SEQID NO: 45: PCR primer

SEQID NO: 46 PCR primer

SEQID NO: 47: human IL-2

SEQID NO: 48: murine IL-2

Claims

A complex comprising

(a) at least two ubiquitin moieties; and

(b) at least one pharmaceutically active moiety,

wherein said complex exhibits an increased serum half-life as compared to the at least one pharmaceutically active moiety alone without said at least two ubiquitin moieties.

The complex according to claim 1, wherein said complex has a molecular weight of at least 20 kDa.

The complex according to claim 1 or 2, comprising between 2 and 10 ubiquitin moieties.

The complex according to any one of claims 1 to 3, wherein each one of the at least two ubiquitin moieties consists, independently from any other ubiquitin moiety, of:

- an amino acid sequence according to SEQ ID NO: 1;

- an amino acid sequence according to SEQ ID NO: 2;

- an amino acid sequence according to SEQ ID NO: 23;

- an amino acid sequence exhibiting at least 95% sequence identity to SEQ ID NO:

1;

2; or

23.

The complex according to any one of claims 1 to 4, wherein the at least two ubiquitin moieties are connected to each other via a direct covalent bond or via a linker.

6. The complex according to any one of claims 1 to 5, wherein the at least two ubiquitin moieties are directly connected to the pharmaceutically active moiety via a covalent bond or connected to the pharmaceutically active moiety via a linker.

7. The complex according to any one of claims 1 to 6, wherein said complex further comprises polyethylene glycol (PEG).

8. The complex according to any one of claims 1 to 7, wherein the pharmaceutically active moiety is a therapeutic or diagnostic moiety.

9. The complex according to any one of claims 1 to 8, wherein the pharmaceutically active moiety is a protein.

10. The complex according to claim 9, wherein the at least two ubiquitin moieties are connected to the N-terminus of the pharmaceutically active moiety.

11. The complex according to claim 9, wherein the at least two ubiquitin moieties are connected to the C-terminus of the pharmaceutically active moiety.

12. The complex according to claim 9,

wherein at least one ubiquitin moiety of the at least two ubiquitin moieties is connected to the N-terminus of the pharmaceutically active moiety, and

wherein at least one ubiquitin moiety of the at least two ubiquitin moieties is connected to the C-terminus of the pharmaceutically active moiety.

13. The complex according to any one of claims 9 to 12, wherein said protein comprises at least one functional domain of at least one protein selected from the group consisting of an antibody mimetic, a cytokine, a antibody fragment, a receptor fragment, and a peptide hormone.

14. The complex according to claim 13, wherein the antibody mimetic is selected from the group consisting of Affilin" molecules, Anticalin* molecules, DARPin^® molecules, Affibody molecules, Fynomers, Nanobodies , Maxybodies, Adnectins, Avimers (avidity multimers), or Nanofitins^®.

15. The complex according to claim 14, wherein the Affilin* molecule is a modified dimeric ubiquitin protein that is capable of binding to a target molecule with a specific binding affinity to the target molecule of K_D≤ 10^"7 M.

16. The complex according to claim 15, wherein the modified ubiquitin protein is a hetero-dimeric ubiquitin comprising two monomeric ubiquitin units linked together in a head-to-tail arrangement, wherein each monomeric ubiquitin unit in said modified hetero-dimeric ubiquitin protein is modified independently from the modifications in the other monomeric ubiquitin unit.

17. The complex according to claim 16, wherein each modified monomeric ubiquitin unit has an amino acid sequence identity of at least 80% to the amino acid sequence defined by SEQ ID NO: 1 or to the amino acid sequence defined by SEQ ID NO: 2 or to the amino acid sequence defined by SEQ ID NO: 23.

18. The complex according to any one of claims 15 to 17, wherein the target molecule is a tumor target molecule.

19. The complex according to any one of claims 16 to 18, wherein the modified hetero- dimeric ubiquitin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and an amino acid sequence that exhibits at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 3.

20. The complex of claim 13, wherein the cytokine is selected from the group consisting of IFN, interleukin 2, TNFalpha, and scTNFalpha. 21. The complex of any one of claims 1 to 20 for use in medicine, preferably for use in the treatment of cancer.

22. A pharmaceutical composition comprising a complex as defined in any one of claims 1 to 20; and further comprising a pharmaceutically acceptable carrier.

23. A use of at least two ubiquitin moieties for extending the serum half-life of a pharmaceutically active moiety.

24. A method for extending the serum half-life of a pharmaceutically active moiety, comprising the steps:

(b) introducing said fused nucleic acid into an expression vector;

(c) introducing said expression vector into a host cell;

(d) cultivating the host cell;

(f) optionally isolating the fusion protein produced in step (e).

25. A nucleic acid comprising a sequence encoding the complex of any one of claims 1 to 20.

26. A vector comprising the nucleic acid of claim 25.

27. A cell comprising the vector of claim 26.