WO2014094799A1 - Fragments d'ubiquitine en tant que moyens pour prolonger la demi-vie sérique - Google Patents

Fragments d'ubiquitine en tant que moyens pour prolonger la demi-vie sérique Download PDF

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WO2014094799A1
WO2014094799A1 PCT/EP2012/005281 EP2012005281W WO2014094799A1 WO 2014094799 A1 WO2014094799 A1 WO 2014094799A1 EP 2012005281 W EP2012005281 W EP 2012005281W WO 2014094799 A1 WO2014094799 A1 WO 2014094799A1
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seq
ubiquitin
pharmaceutically active
complex
amino acid
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PCT/EP2012/005281
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Christian Lange
Stefanie HOFFMANN-THOMS
Una Rauchhaus
Maren MEYSING
Ulrich Haupts
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Scil-Proteins-Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • 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.
  • Pharmaceutically active drugs have often limited value because of their pharmacokinetic properties.
  • 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.
  • PEG polyethylene glycol
  • 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 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 ⁇ / ⁇ structure (Vijay-Kumar et al. (1987) J. Mol. Biol. 194(3): 531-44; the entirety of which is herein incorporated by reference).
  • ubiquitin 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.
  • 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.
  • WO 2011/073214 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.
  • 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.
  • small pharmaceutically active compounds i.e., compounds with a molecular weight of smaller than about 50 kDa
  • 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.
  • ubiquitin is biodegradable, has a low immunogenicity, and can be produced by chemical synthesis or in microorganisms.
  • the present invention relates to a complex comprising, essentially consisting of or consisting of:
  • the present invention relates to the complex according to the first aspect for use in medicine.
  • the present invention relates to the complex according to the first aspect for use in the treatment of cancer.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the complex according to the first aspect; and further comprising a pharmaceutically acceptable carrier.
  • the present invention relates to a use of at least two ubiquitin moieties for extending the serum half-life of a pharmaceutically active moiety.
  • the present invention relates to a method for extending the serum half-life of a pharmaceutically active moiety, comprising the steps:
  • step (f) optionally isolating the fusion protein produced in step (e).
  • the present invention relates to a nucleic acid comprising a sequence encoding the complex of the first aspect.
  • the present invention relates to a vector comprising the nucleic acid of the seventh aspect.
  • the present invention relates to a cell comprising the vector of the eighth aspect.
  • FIGURE 1 shows the amino acid sequences of complexes of the invention.
  • 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 ⁇ g, 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 ⁇ and 2 ⁇ g, 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 3 ⁇ 4
  • 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 3 ⁇ 4 (open hash mark), Ubi6-Af ilin * (closed circles), Ubi2-scTNF (closed squares, dotted line).
  • 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.
  • 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.
  • 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.
  • 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.
  • pharmaceutically active moiety does not encompass “ubiquitin moieties”, which will be defined below.
  • 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.
  • 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).
  • interferon e.g. IFN alpha
  • interleukin e.g. IL-2
  • tumor necrosis factor e.g. TNFalpha
  • scTNF single chain TNF
  • a "pharmaceutically active moiety” useable in the present invention has a molecular weight which is equal to or less than about 70 kDa.
  • 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.
  • 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.
  • 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.
  • peptidic or proteinogenic conjugations e.g. via intein-based coupling of components which are non-contiguous on the genetic level
  • 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%).
  • concentration of a substance e.g., a pharmaceutically active moiety, a fusion protein or a complex of the invention
  • 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.
  • the half-life is increased by 10%, 20%, 30%, 40%, 50%, or more.
  • the present disclosure distinguishes between the terms (a) “ubiquitin moiety” and (b) “ubiquitin unit” or “ubiquitin protein” or “ubiquitin monomer”.
  • 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.
  • 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.
  • 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.
  • 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.
  • the "pharmaceutically active moiety” comprises a modified dimeric ubiquitin protein which comprises two modified monomeric ubiquitin units.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the default parameters of the SIM Local similarity program or of ClustalW are used, when calculating sequence identity percentages.
  • the extent of the sequence identity of the modified protein to SEQ ID NO: 1 is determined relative to the complete sequence of SEQ ID NO: 1 (or SEQ ID NO: 2 or SEQ ID NO: 23, respectively).
  • sequence identity between a modified sequence and the sequence from which it is derived 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).
  • a tumor antigens such as extra- domain B of fibronectin referred to as ED-B or any other antigens.
  • 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.
  • 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.
  • 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.
  • monomers may be connected directly without any linker, i.e., by a direct peptide bond.
  • the fusion of ubiquitin monomers can be performed via linkers.
  • 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.
  • 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.
  • 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.
  • 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.
  • the amino acid sequence of the peptide linker is not immunogenic to human beings.
  • 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
  • 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.
  • P proline
  • A alanine
  • S serine
  • 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).
  • 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.
  • Kd dissociation constant
  • a value of 10 5 M and below can be considered as a quantifiable binding affinity.
  • a value of 10 ⁇ 7 M to 10 "11 M is preferred for e.g., 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).
  • SPR Surface Plasmon Resonance
  • ITC isothermal titration calorimetry
  • FACS fluorescence activated cell sorting
  • 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.
  • extra-domain B of fibronectin 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”).
  • IFN interferon alpha
  • IFN- ⁇ interferon beta
  • I F relates to IFN-a and IFN- ⁇ from any mammalian organism. However, it is preferred when practicing the present invention to use human IFN-ct or human IFN- ⁇ .
  • IFN includes IFN-a 2a, IFN-ct 2b, IFN-a 2c, IFN-a 6, IFN-a 14, IFN-a 4, IFN-a 5, IFN- ⁇ 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- ⁇ (SEQ ID NO: 43) and biologically active muteins of any of these.
  • 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
  • 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.
  • 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.
  • 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.
  • 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- ⁇ 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.
  • scTNF 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.
  • TNFalpha 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.
  • SEQ ID NO: 34 human; uniprot accession number P01375; see: http://www.uniprot.org/uniprot/P01375
  • SEQ ID NO: 35 mimouse; uniprot accession number P06804; http://www.uniprot.org/uniprot/P06804
  • SEQ ID NO: 36 rat
  • Human TNFalpha shows 79% sequence identity to mouse TNFalpha.
  • 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.
  • 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.
  • 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.
  • 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).
  • biologically active single-chain TNFalpha 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.
  • interleukin 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • each one of the at least two ubiquitin moieties consists, independently from any other ubiquitin moiety, of:
  • the at least two ubiquitin moieties are connected to each other
  • a direct covalent bond such as a peptide bond or a disulfide bond
  • 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).
  • 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
  • the at least two ubiquitin moieties are directly connected to each other via a peptide bond, i.e., without any linker.
  • 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
  • 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).
  • a linker such as a maleimide moiety or a peptide linker
  • a linker such as a maleimide moiety or a peptide linker
  • a linker such as a maleimide moiety or a peptide linker
  • 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].
  • 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.
  • 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 99 Tc, 123 l, m ln, or from the group of positron emitters, preferably 18 F, 64 Cu, 68 Ga, 86 Y, 124 l, or from the group of beta-emitter, preferably 131 l, 90 Y, 177 Lu, 67 Cu, or from the group of alpha-emitter, preferably 213 Bi, 211 At.
  • 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.
  • the pharmaceutically active moiety is a pharmaceutically active protein.
  • 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.
  • 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.
  • 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.
  • U-U-U-U-P 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).
  • 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.
  • 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.
  • biologically active IFN biologically active IL-2
  • biologically active TNFalpha biologically active single-chain TNFalpha
  • the complex comprises two or more pharmaceutically active moieties, which may be the same or different.
  • 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*).
  • a cytokine e.g., a cytokine
  • an antibody mimetic e.g., an Affilin*
  • U-U-U-U-P1-P2 P1-P2-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-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).
  • PI denotes one pharmaceutically active protein
  • P2 denotes another pharmaceutically active protein (for example Affilin)
  • 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).
  • 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.
  • 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.
  • Adfilin* modified dimeric ubiquitin protein
  • 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.
  • the two ubiquitin units have been differently modified so that the targeting domain consists of a modified hetero-dimeric ubiquitin protein.
  • 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.
  • these two monomeric ubiquitin units are directly linked, i.e., without a linker.
  • 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.
  • 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.
  • the target molecule is a tumor target molecule, preferably extra-domain B (ED-B) of fibronectin.
  • the pharmaceutically active moiety is an Affilin.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention is directed to the complex of the first aspect for use in medicine.
  • 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:
  • 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.
  • 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.
  • 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).
  • compositions 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.
  • 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.
  • the present invention is directed to a method for extending the serum half-life of a pharmaceutically active moiety, comprising the steps:
  • the present invention is directed to a nucleic acid comprising a sequence encoding the complex of the first aspect.
  • 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.
  • the polynucleotide is for use in medicine, preferably for use in the treatment of cancer.
  • 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.
  • the present invention is directed to a vector comprising the nucleic acid of the seventh aspect.
  • the vector is for use in medicine, e.g., for use in the treatment of cancer.
  • 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.
  • the present invention is directed to a cell comprising the vector of the eighth 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.
  • 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.
  • the complex has a molecular weight of between about 30 kDa and 200 kDa; more preferably between 40 kDa and 110 kDa.
  • 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 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.
  • 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.
  • the seventh, eighth, and ninth aspect of the invention have been defined above with reference to the complex of the first aspect.
  • 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.
  • ED-B 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.
  • diagnostic agents One example is its use in molecular imaging of atherosclerotic plaques and detection of cancer, for example by immunoscintigraphy of cancer patients.
  • ED-B extra-domain B
  • SEQ ID NO: 25 The amino acid sequence of human extra-domain B (ED-B, 91 residues) of fibronectin is shown in SEQ ID NO: 25.
  • ED-B is detected in mammals, e.g., in rodents, cattle, primates, carnivore, human etc.
  • mammals e.g., in rodents, cattle, primates, carnivore, human etc.
  • 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).
  • 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 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 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.
  • IFN-alpha 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 (sc) TNFalpha proteins of at least three monomers connected by peptide linkers are described generally.
  • Krippner-Heidenreich et al. 2008, J Immunol., 180 (12): 8176-8183; the entirety of which is herein incorporated by reference
  • polypeptides which consist of at least three monomers of a TNF family ligand which are connected by peptide linkers.
  • this construct was shown that although this construct is less toxic than wild-type TNFalpha, it shows the same bioactivity as native TNF.
  • Example 1 Production of complexes comprising four or six ubiquitin moieties and an Affilin 3 ⁇ 4 as pharmaceutically active moiety
  • the complexes of the invention comprise at least two ubiquitin moieties and a pharmaceutically active moiety.
  • 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
  • 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:
  • 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.
  • Step 2 Cloning of ubi4-Affilin-complexes and ubi6— Affilin- complexes
  • 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.
  • 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.
  • 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.
  • HIC hydrophobic interaction chromatography
  • 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.
  • yields of up to 34 mg pure and active fusion protein per liter expression culture were obtained.
  • Example 3 Preparation of a heteromeric binding protein based on modified ubiquitin dimers (Affilin ® ) Step 1: Production of vectors for cloning
  • Affilin 8 As vector for cloning of Affilin 8 (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)
  • sequence coding for an Affilin (SEQ ID NO: 3) was amplified from a plasmid template by PCR according to standard procedures.
  • the amplified sequence was ligated into pET-SUMOadapt (already containing SUMO) via the restriction sites Bsal/BamHI.
  • Step 3 Expression of SUMO-Affilin * -fusion proteins
  • SUMO fusion proteins were produced in E. coli and isolated from cytoplasm.
  • 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).
  • IPTG isopropyl ⁇ -D-l-thiogalactopyranoside
  • 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.
  • 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.
  • 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)
  • 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.
  • K D dissociation constants
  • 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* were analyzed by an ED-B-binding ELISA.
  • 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 ⁇ ).
  • 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.
  • a chromatographic affinity matrix was prepared by covalently coupling a construct containing human ED-B domain to SulfolinkTM resin (Pierce).
  • SulfolinkTM resin Pieris-B domain
  • Ubi4-Affilin or Ubi6-Affilin was applied to the column containing the ED-B affinity matrix (200 ⁇ g 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.
  • PBS phosphate-buffered saline
  • the complex protein described consists of two ubiquitin monomers (SEQ ID NO: 2) linked by a GIG peptide linker (SEQ ID NO: 8), an SG 4 -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.
  • Hubi-SG4-fw-Bsa G C ACG GTCTCCCGTTTACGTG C AG C AAG CG G (SEQ ID NO: 41)
  • 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.
  • the complex was produced in E. coli and isolated in the form of inclusion bodies.
  • 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.
  • 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.
  • 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).
  • TATA box basic promoter element
  • 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.
  • the reporter cells were used for an ISRE-Reporter Gene Assay.
  • the cells were resuspended in suitable medium containing 10% fetal calf serum (FCS).
  • FCS fetal calf serum
  • a cell suspension with a density of 3xl0 5 cells/ml in medium containing 5% FCS was seeded into a white 96 well cell culture plate.
  • 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-GloTM 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
  • Example 10 Production of a complex composed of two ubiquitin moieties and pharmaceutically active single-chain murine TNFalpha
  • 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 (G 4 S) 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 (G 3 S) 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.
  • 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.
  • the complex was produced in suitable E. coli host strains and isolated in the form of inclusion bodies.
  • 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.
  • 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.
  • PBS phosphate-buffered
  • Example 12 ELISA of specific TNF-receptor binding by Ubi2-scTNF complex
  • TNF receptor I domain binding of a TNF receptor I domain to the Ubi2-scTNF complex was analyzed in an ELISA setup.
  • 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 / ⁇ , while recombinant murine TNFalpha serving as control was applied in an equimolar concentration of 0.73 g/ml.
  • BSA bovine serum albumin
  • the wells were again washed with PBST.
  • 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.
  • a POD-conjugate of a polyclonal Fc-specific anti human IgG antibody from goat was applied in a dilution of 1:50000, in order to detect bound TNF receptor chimera.
  • the physiological TNF-alpha-activity of a complex according to the invention consisting of two ubiquitin moieties genetically fused via a (G 4 S) 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 EC 50 values in the picomolar range.
  • 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
  • mice healthy mice were treated with a single administration of these variants after lodine-125 labeling.
  • 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
  • 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 ⁇ is realized as described below:
  • Radioactivity in ⁇ Radioactivity in cpm / 0.74 / 60 / 37 000
  • Step 2 Calculation of blood pharmacokinetic parameters
  • the pharmacokinetic parameters were assessed using data expressed as activity per mL of blood ( ⁇ / ⁇ ). These data were analyzed using a macro on ExcelTM 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:
  • Step 3 SE-HPLC analyses to investigate serum stability
  • SE-HPLC analyses of serum samples 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 ⁇ . of 0.1 N NaOH followed by an injection of 70 ⁇ of water were performed as washing steps. A total volume of 30-100 ⁇ undiluted serum sample was injected. Prior to injection, serum samples were filtered with a pore diameter of 0.45 ⁇ .
  • 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.
  • 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: 2 ubiquitin mutein (F45W/G75A/G76A), start sequence for mutagenesis
  • SEQ ID NO: 10 peptide linker
  • SEQ ID NO: 12 basic linker sequence
  • SEQ ID NO: 14 Ubi6-Affilin
  • SEQ ID NO: 15 Ubi4-Affilin
  • SEQ ID NO: 18 Ubi2-TNF
  • 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: 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 .
  • SEQID NO: 41 PCR primer
  • SEQID NO: 42 PCR primer
  • SEQID NO: 43 human IFN- ⁇
  • SEQID NO: 44 PCR primer
  • SEQID NO: 45 PCR primer
  • SEQID NO: 47 human IL-2
  • SEQID NO: 48 murine IL-2

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Abstract

La présente invention concerne des complexes pharmaceutiquement actifs qui comprennent au moins deux fragments d'ubiquitine. Les fragments d'ubiquitine confèrent une demi-vie sérique accrue aux complexes. La présente invention concerne en outre des polynucléotides codant pour des protéines de fusion pharmaceutiquement actives comprenant au moins deux fragments d'ubiquitine, des vecteurs comprenant de tels polynucléotides, et des cellules comprenant de tels vecteurs. La présente invention concerne en outre des complexes, des polynucléotides, des vecteurs, et des cellules pour utilisation en médecine, en particulier pour utilisation dans le traitement du cancer. La présente invention concerne en outre des compositions pharmaceutiques comprenant ces complexes, et un procédé pour prolonger la demi-vie sérique d'un fragment pharmaceutiquement actif.
PCT/EP2012/005281 2012-12-19 2012-12-19 Fragments d'ubiquitine en tant que moyens pour prolonger la demi-vie sérique WO2014094799A1 (fr)

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JP2018529310A (ja) * 2015-07-20 2018-10-11 ナフィゴ プロテインズ ゲゼルシャフト ミット ベシュレンクテル ハフツングＮａｖｉｇｏ Ｐｒｏｔｅｉｎｓ ＧｍｂＨ ジユビキチン変異タンパク質に基づくＨｅｒ２結合タンパク質
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WO2017013136A1 (fr) * 2015-07-20 2017-01-26 Scil Proteins Gmbh Nouvelles protéines de liaison à base de mutéines de di-ubiquitine et procédés de production
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