TNFR2 agonists with improved stability

Abstract

The present invention relates to polypeptides consisting of three TNF homology domains (THDs) of TNF ligand family member proteins that specifically bind to the extracellular portion of TNFR2, where the C- and N-terminal reference points are defined by consensus sequences. The THDs are linked by additional C- and / or N-terminal amino acids of short sections of THD or its variants and a peptide linker. These peptides have improved stability. Furthermore, the present invention relates to polypeptide multimers comprising several of the polypeptides of the present invention.

Description

[Technical field]

[0001] The present invention relates to a polypeptide consisting of three TNF homology domains (THDs) of TNF ligand family member proteins that specifically bind to the extracellular portion of TNFR2, where the C-terminal and N-terminal reference points are defined by consensus sequences. The THDs are linked by additional C-terminal and / or N-terminal amino acids of a short section of the THD or its variants and a peptide linker. These polypeptides have improved stability. Furthermore, the present invention relates to polypeptide multimers comprising several of the polypeptides of the present invention. Furthermore, the present invention relates to nucleic acid molecules encoding said polypeptides or polypeptide multimers, vectors comprising said nucleic acid molecules, and pharmaceutical compositions comprising said polypeptides, polypeptide multimers, nucleic acid molecules, or vectors. Furthermore, the present invention relates to said polypeptides, polypeptide multimers, nucleic acid molecules, or vectors for use as medicines or for use in the prevention or treatment of hyperproliferative, inflammatory, or metabolic disorders. [Background technology]

[0002] The tumor necrosis factor (TNF) superfamily is a family of structurally related cytokines with diverse functions. The structural feature that defines the TNF ligand family is the carboxy-terminal TNF homology domain (THD), which is composed of two stacked β-pleated sheets that adopt a conserved jelly-roll-like folded tertiary structure (Bodmer et al., 2000, Trends Biochem. Sci. 27, 19-26; Fesik, 2000, Cell 103, 273-282; Locksley et al., 2001, Cell 104, 487-501). This structural composition leads to self-association of THD monomers into trimers, which is necessary for receptor binding. Due to the localization of the THD to the carboxy terminus, both transmembrane and soluble TNF ligands assemble into trimers.

[0003] Tumor necrosis factor (TNF) itself is a multifunctional cytokine with pleiotropic functions. TNF is a master regulator of the immune system and plays a key role in the initiation and regulation of inflammation and immunity. Like most ligands of the superfamily, TNF is synthesized as a trimeric type 2 transmembrane protein (tmTNF), which can be proteolytically cleaved to a soluble, circulating TNF homotrimer (sTNF). Interestingly, sTNF and tmTNF differ in their ability to activate two distinct TNF receptors (TNFRs), TNFR1 and TNFR2. TNFR1 is activated by both sTNF and tmTNF, whereas TNFR2 is robustly activated in a tmTNF-dependent manner (Muhlenbeck et al., 2000, J. Biol., Chem. 275, 32208-32213; Wajant et al., 2001, Oncogene 20, 4101-4106).

[0004] Deregulation of TNF expression and signaling can cause chronic inflammation, leading to the development of autoimmune diseases and tissue damage (Fischer et al., 2015, Antibodies 4, 48-70; Kalliolias & Ivashkiv, 2016, Nat. Rev. Rheumatol. 12, 49-62). Indeed, elevated TNF levels are associated with several inflammatory diseases, such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease: therapeutic agents that neutralize TNF have been successfully used to treat these diseases (Monaco et al., 2015, Int. Immunol. 27, 55-62). Surprisingly, however, clinical trials with anti-TNF drugs that block both sTNF and tmTNF in multiple sclerosis patients led to disease exacerbation and had to be discontinued. Furthermore, approved TNF inhibitors can cause severe side effects, including opportunistic infections, reactivation of tuberculosis, development of autoimmune diseases, increased susceptibility to lymphoma development, and demyelinating diseases (Fischer et al., 2015, Antibodies 4, 48-70; Monaco et al., 2015, Int. Immunol. 27, 55-62). These undesirable clinical reactions are likely dependent on the different biological actions of TNF mediated through the two receptors.

[0005] Recent studies have revealed that TNF receptors induce opposing biological responses. TNFR1 signaling promotes inflammation and tissue degeneration, whereas TNFR2 contributes to immunosuppression and tissue homeostasis and regeneration (Probert et al., 2015, Neuroscience 302, 2-22). Therefore, next-generation therapeutic approaches targeting the TNF system have been developed, including blocking sTNF-TNFR1 interaction or signaling, and selective activation of TNFR2 (Shibata et al., 2009, Biomaterials 30, 6638-6647; Steed et al., 2003, Science 301, 1895-1898; Dong et al., 2016, PNAS 113, 12304-12309). TNFR2-mediated immunosuppressive activity is of particular interest for its potential therapeutic application in autoimmune diseases. The immunosuppressive properties of TNFR2 are due to its prominent role in the proliferation and stabilization of Treg cells, a highly specialized subpopulation of T cells that function to suppress immune responses (Chen et al., 2007, J. Immunol. 179, 154-161; Chen et al., 2013, J. Immunol. 190, 1076-1084). According to the prevailing view, Treg cells control the self-tolerance of the immune system and help prevent the development of autoimmune diseases. In addition to CD4+ Treg cells, there exists a further T cell subpopulation with regulatory activity (i.e., CD8+ Treg cells). Similar to CD4+ Treg cells, the most potent CD8+ suppressors are characterized by the expression of TNFR2 (Ablamunits et al., 2010, Eur. J. Immunol. 40, 2891-2901).

[0006] To investigate the effect of selective activation of TNFR2 on Treg cell proliferation and function, we recently developed soluble, multivalent, TNFR2-selective TNF derivatives that mimic the activity of tmTNF. These molecules are based on single-chain derivatives of TNF (scTNF) (Krippner-Heidenreich et al., 2008, J. Immunol. 180, 8176-8183) that combine mutations in the THD of TNF that confer selectivity for TNFR2, and fusion of scTNF to dimerization or multimerization modules. These modules include the trimerizing tenascin domain resulting in nonavalent molecules (binding up to nine TNFR2s) (Fischer et al., 2011, PLoS ONE 6:e27621; Fischer et al., 2014, Glia 62, 272-283), the dimerizing heavy domain 2 (EHD2) of IgE (Dong et al., 2016, PNAS 113, 12304-12309), the homotetramerization domains of p53, GCN4 (Fischer et al., 2017, Sci. Rep. 7, 6607), VASP, and fusions with the N- and C-termini of the immunoglobulin Fc region of scTNF (PCT / EP2018 / 058786).

[0007] In all these studies, we applied TNF subunits (domains) consisting of amino acids 80-233 containing the TNF homology domain (THD), linking the three subunits with a flexible linker of 12 or 16 residues (L1: (GGGS)3 (SEQ ID NO: 53); L2: (GGGS)4 (SEQ ID NO: 54); Krippner-Heidenreich et al., 2008, J. Immunol. 180, 8176-8183). In a subsequent study, we reduced the linker to a GGGGS (SEQ ID NO: 26) sequence linking the C-terminus of the first and second THD to the N-terminus of the second and third THD, respectively (all THDs consist of aa80-233 of human TNF) (Fischer et al., 2011, PLoS ONE 6:e27621).

[0008] Additionally, mutations were introduced that conferred receptor selectivity for human TNFR2 (TNFR2-selective scTNF D143N / A145R) (Loetscher et al., 1993, J. Biol. Chem. 268, 26350-26357) or human TNFR1 (TNFR1-selective scTNF R32W / S86T) (van Ostade et al., 1993, Nature 361, 266-269) with corresponding substitutions in all three subunits (Krippner-Heidenreich et al., 2008, J. Immunol. 180, 8176-8183). Also, functionally corresponding mutations (D221N / A223R) were introduced into mouse TNF for selective binding to TNFR2 (Fischer et al., 2014, Glia 62, 272-283). Mutations could be introduced into only one or two of the three THD subunits (Boschert et al., 2010, Cell Signal. 22, 1088-1096).

[0009] TNFR2-selective TNF muteins were selected by phage display from a library of TNF mutants (Abe et al., 2011, Biomaterials 32, 5498-5504; Ando et al., 2016, Biochem. Biophys. Reports 7, 309-315). Furthermore, a TNF molecule with improved TNFR2 signaling was generated by mutating two residues at the THD interface to cysteines (S95C / G148C) and introducing an internal covalent crosslink (Ban et al., 2015, Mol. Cell. Ther. 3:7).

[0010] Previously, we demonstrated that oligomerized, covalently stabilized, TNFR2-selective scTNF mimics tmTNF and efficiently activates TNFR2. These TNFR2-selective TNF mutant proteins were shown to induce anti-inflammatory responses, reduce symptoms of experimental arthritis, rescue neurons and oligodendrocytes from oxidative stress, and be protective in a mouse model of NMDA-induced acute neurodegeneration (Fischer et al., 2011, PLoS One 6, e27621; Maier et al., 2013, Biochem. Biophys. Res. Commun. 440, 336-341; Fischer et al., 2018, Arthritis Reumatol. 70, 722-735; Dong et al., 2016, PNAS 113, 12304-12309).

[0011] Recently, the inventors have demonstrated that the stability of single-chain members of the TNF superfamily can be improved by shortening the linkers between the three subunits and reducing the subunit sequence to the minimal THD domain (WO 2016 / 146818).

[0012] By applying this strategy to scTNF (i.e., direct linkage of the C-terminus of the 1st / 2nd domain to the N-terminus of the 2nd / 3rd domain), we found that stability was improved by shortening the THD domain to positions (84-233), but further shortening (86-233) reduced stability. The scTNF derivative with the THD formed by aa 84-233 directly linked to each other showed a 10°C increase in thermal stability as determined by dynamic light scattering, but an approximately 6-fold decrease in bioactivity. The present invention surprisingly demonstrated that for molecules composed of either three THDs (aa80-233) directly linked without a linker or three truncated THDs (aa85-233) linked with a four amino acid linker (GGGG; SEQ ID NO: 16), an increased thermostability (67°C vs. 62°C) with full retention of biological activity was observed compared to a reference scTNF molecule composed of three THDs (aa80-233) linked with a GGGGS (SEQ ID NO: 26) linker. Summary of the Invention

[0013] In a first aspect, the present invention provides a polypeptide comprising a binding domain consisting of three peptide TNF homology domains (THDs) of a TNF ligand family member protein which specifically binds to the extracellular portion of TNFR2, wherein the C-terminus of the first and second THDs (which in each case are defined by the C-terminal consensus sequence VF / YFGA / I-X1 (SEQ ID NO:1)) is linked to the N-terminus of the second and third THDs (which in each case are defined by the N-terminal consensus sequence PV / AAHV / L (SEQ ID NO:2)) by peptide X a (which are in each case independently selected and have a length of 9 to 12 amino acids, preferably 9 to 11, more preferably 9 to 10), and preferably a does not contain the amino acid sequence SSRTPSDK (SEQ ID NO: 10); X1 is a non-polar / hydrophobic or polar / neutral amino acid, preferably selected from the group consisting of F and I.

[0014] In a second aspect, the present invention provides a polypeptide multimer comprising at least two polypeptides according to the first aspect of the invention, (a) linked together, preferably linked together by an amino acid linker having a length of 1 to 30 amino acids, preferably 7 to 15 amino acids; or (b) is linked to a protein, preferably a multimerization domain (preferably selected from the group consisting of a multimerization domain, a serum protein, a cytokine, a targeting moiety, or a toxin); Optionally, wherein said polypeptide is linked to said protein by an amino acid linker having a length of 1 to 30 amino acids, preferably 7 to 15 amino acids.

[0015] In a third aspect, the present invention provides a nucleic acid molecule encoding a polypeptide according to the first aspect of the invention or a polypeptide multimer according to the second aspect of the invention.

[0016] In a fourth aspect, the present invention provides a vector encoding a nucleic acid molecule according to the third aspect of the invention.

[0017] In a fifth aspect, the present invention provides a polypeptide according to the first aspect of the invention, a polypeptide multimer according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, for use as a medicament.

[0018] In a sixth aspect, the present invention provides a pharmaceutical composition comprising as an active substance a polypeptide according to the first aspect of the invention, a polypeptide multimer according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, or a vector according to the fourth aspect of the invention.

[0019] In a seventh aspect, the present invention provides a polypeptide according to the first aspect of the invention, a polypeptide multimer according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, a vector according to the fourth aspect of the invention or a pharmaceutical composition according to the fifth aspect of the invention for use in the diagnosis, prevention or treatment of a hyperproliferative or inflammatory disorder (preferably cancer or hematological malignancies), an autoimmune disorder and a metabolic disease, a cardiovascular disease, a neuropathic disease and a neurological injury. [Brief description of the drawings]

[0020] The contents of the drawings contained in this specification are described below, in which context reference is also made to the above and / or following detailed description of the invention.

[0021] [Figure 1] Schematic representation of the scTNFR2 mutant protein of the invention and its dimerized complex. (A) Schematic polypeptide chains of scTNFR2 and its dimeric complex scTNFR2-Fc. TNFR2 subunits were genetically fused with or without peptide linker L1 to obtain scTNFR2. Dimerization domain Fc was genetically fused to the C-terminus of scTNFR2 using peptide linker L2. (B) Schematic representation of the tertiary / quaternary structure of scTNFR2 and scTNFR2-Fc. (C) Schematic representation of an example embodiment of (1) polypeptide and (3)-(6) polypeptide multimers. Optionally, the polypeptide comprises an additional module 2 (6), which allows, for example, organ- or tissue-specific delivery and / or transport through tissue barriers (such as the blood-brain barrier). (2) shows a schematic representation of the linker polypeptide Xa. (D) Top panel: exemplary polypeptides (127, 139 and 138) according to the invention and reference polypeptides. Bottom panel: exemplary polypeptide multimers of the invention (742, 744) and a reference polypeptide multimer.

[0022] [Diagram 2]Comparative SDS-PAGE analysis of the scTNFR2 mutant proteins of the present invention and their dimerized complexes. Fusion proteins were produced in HEK293-6E cells and purified by affinity chromatography. (A) scTNFR2 mutants were separated on 12% SDS-PAGE under non-reducing and reducing conditions and stained with Coomassie. 1, scTNFR2(118); 2, scTNFR2(127); 3, scTNFR2(129); 4, scTNFR2(130); 5, scTNFR2(131); 6, scTNFR2(138); 7, scTNFR2(139), M, molecular weight marker. (B) ScTNFR2-Fc(Δab) complexes were separated on 10% SDS-PAGE under non-reducing and reducing conditions and stained with Coomassie. 1, scTNFR2(127)-Fc(Δab)742; 2, scTNFR2(129)-Fc(Δab)743; 3, scTNFR2(139)-Fc(Δab)744; 4, scTNFR2(118)-Fc(Δab)745; M, molecular weight marker.

[0023] [Diagram 3] Native structure of the scTNFR2 mutant of the present invention and its dimerized complex. Proteins were analyzed by size-exclusion chromatography using a SuperSW mAb HR, 7.8×300 mm column (Tosoh Bioscience). The positions of the standard proteins used are indicated in the figure.

[0024] [Figure 4] Thermal stability of the scTNFR2 mutant of the present invention and its dimerized complex. The protein was analyzed for its denaturation temperature by dynamic light scattering. The detected melting point (aggregation point) is indicated by the dotted line.

[0025] [Diagram 5]In vitro bioactivity of scTNFR2 mutants on Kym-1 cells. ScTNFR2 mutants were analyzed for bioactivity on Kym-1 cells with respect to cell death induction. Mechanistically, activation of TNFR2 by scTNFR2 mutants (which requires cross-linking of TNFR2 by antibody 80M2) leads to expression of trimeric TNF, which in turn induces apoptotic cell death via activation of TNFR1. Kym-1 cells were cultured in the presence of serially diluted and purified scTNFR2 for 24 h, and then cell viability was measured using crystal violet staining. Additionally, TNF receptor 2 molecules on Kym-1 cells were cross-linked with 80M2 antibody (1 μg / ml) before incubation with scTNFR2 mutants (n=1).

[0026] [Figure 6] Binding of scTNFR2-Fc(Δab) complexes to TNF-R2. Binding of scTNFR2-Fc(Δab) complexes to TNF-R2-Fc (etanercept) was tested by ELISA. For comparison, the binding curves of the state-of-the-art molecule scTNFR2-Fc(Δab)745 were plotted in separate diagrams in combination with the proteins of the invention scTNFR2-Fc(Δab)742, 743 and 744 (mean ± SD, n=3).

[0027] [Figure 7] TNF-R2-selective binding of scTNFR2-Fc(Δab) complex to MEF-TNF-R2. Mouse embryonic fibroblasts stably transfected with either human TNF-R2 (MEF-TNF-R2) or human TNF-R1 (MEF-TNF-R1) were tested for binding of scTNFR2-Fc(Δab) complex by flow cytometry. The scTNFR2-Fc(Δab) complex selectively bound to MEF-TNF-R2, with no detectable binding to MEF-TNF-R1 (n=1).

[0028] [Figure 8]In vitro bioactivity of scTNFR2-Fc(Δab) complex on Kym-1 cells. The in vitro bioactivity of scTNFR2-Fc(Δab) complex was analyzed on Kym-1 cells. Cells were cultured in the presence of serially diluted and purified scTNFR2-Fc(Δab) complex for 24 h, and then cell viability was measured using crystal violet staining (mean ± SD, n=3).

[0029] [Figure 9] NF-κB activation by scTNFR2-Fc(Δab) complex. In vitro NF-κB activation by scTNFR2-Fc(Δab) complex was analyzed using a luciferase reporter assay in HeLa cells stably transfected with human TNF receptor 2 (HeLa-TNF-R2). 16 hours after transfection with experimental and control reporter plasmids, cells were stimulated for 6 hours with serial titrations of scTNFR2-Fc(Δab) complex, and then cells were lysed and luciferase activity was measured (mean ± SD, n = 4).

[0030] [Figure 10] Stability of scTNFR2-Fc(Δab) complexes in human plasma. The integrity of scTNFR2-Fc(Δab) complexes after 0 days (control), 3 days or 8 days of incubation in 50% human plasma at 37°C was tested by their ability to bind to HeLa-TNF-R2 cells in flow cytometry. The percentage of intact protein (mean ± SD, n=4) was calculated from the reciprocal of the EC50 value normalized to the unincubated control (100%). Statistical analysis was performed by two-way ANOVA with Dunnett's post-hoc test. Results were summarized as ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns=not significant.

[0031] [Figure 11]Comparative SDS-PAGE analysis of scTNFR2 variants of Example 11. Fusion proteins were produced in HEK293-6E cells and purified by affinity chromatography. ScTNFR2 variants were separated on 10% SDS-PAGE under non-reducing (B) and reducing (A) conditions and stained with Coomassie. M, molecular weight marker.

[0032] [Figure 12] Comparative SDS-PAGE analysis of scTNFR2-Fc proteins from Example 11. Fusion proteins were produced in HEK293-6E cells and purified by affinity chromatography. ScTNFR2-Fc variants were separated on 10% SDS-PAGE under non-reducing and reducing conditions and stained with Coomassie. M, molecular weight marker.

[0033] [Figure 13] Native structure of scTNFR2-Fc protein of Example 11. The protein was analyzed by size-exclusion chromatography using a SuperSW mAb HR, 7.8×300 mm column (Tosoh Bioscience). The positions of the standard proteins used are indicated in the figure.

[0034] [Figure 14] Thermal stability of scTNFR2 variants of Example 11. The proteins were analyzed for their denaturation temperatures by dynamic light scattering. The detected melting points (aggregation points) are indicated by dotted lines (n=2).

[0035] [Figure 15] Thermal stability of the dimeric scTNFR2-Fc protein of Example 11. The protein was analyzed for its denaturation temperature by dynamic light scattering. The detected melting point (aggregation point) is indicated by the dotted line (n=1).

[0036] [Figure 16]Binding of scTNFR2 variants of Example 11 to TNF-R2 Binding of scTNFR2 mutant proteins to TNF-R2-Fc (etanercept) was tested by ELISA (mean values ​​± range of technical duplicates, n=1).

[0037] [Figure 17] Binding of scTNFR2-Fc proteins to TNF-R2 of Example 11. Binding of scTNFR2-Fc mutant proteins to TNF-R2-Fc (etanercept) was tested by ELISA (mean values ​​± range of technical duplicates, n=1-2).

[0038] [Figure 18] In vitro bioactivity of scTNFR2 variants of Example 11 on Kym-1 cells. scTNFR2 variants were analyzed for bioactivity on Kym-1 cells in terms of cell death induction. Mechanistically, activation of TNFR2 by scTNFR2 variants (which requires cross-linking of TNFR2 by antibody 80M2) leads to expression of trimeric TNF, which then induces apoptotic cell death via activation of TNFR1. Kym-1 cells were cultured in the presence of serially diluted and purified scTNFR2 for 24 hours, and then cell viability was measured using crystal violet staining. In addition, TNF receptor 2 molecules on Kym-1 cells were cross-linked with 80M2 antibody (1 μg / ml) before incubation with scTNFR2 variants (n=3-8±SD).

[0039] [Figure 19]In vitro bioactivity of the dimeric scTNFR2-Fc protein of Example 11 on Kym-1 cells. The scTNFR2Fc mutants were analyzed for bioactivity on Kym-1 cells in terms of cell death induction. Mechanistically, activation of TNFR2 by scTNFR2-Fc mutants leads to the expression of trimeric TNF, which in turn induces apoptotic cell death via activation of TNFR1. Kym-1 cells were cultured in the presence of serially diluted and purified scTNFR2 for 24 hours, and then cell viability was measured using crystal violet staining. In addition, TNF receptor 2 molecules on Kym-1 cells were crosslinked with 80M2 antibody (1 μg / ml) before incubation with scTNFR2 mutants (n=3-4±SD). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, which may vary.It should also be understood that the terms used herein are only for describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0041] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0042] It is understood that everywhere in this specification and the claims that follow, unless the context requires otherwise, the words "comprise" and variations such as "comprise" 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 integers or steps. In the following sections, various aspects of the invention are defined in more detail. Each aspect thus defined may be combined with any other aspect, unless clearly indicated to the contrary. In particular, any feature indicated as optional, preferred or advantageous may be combined with any other feature indicated as optional, preferred or advantageous.

[0043] Several documents are cited throughout the text of this specification. Each document cited herein (including any patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein in its entirety by reference. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by prior invention. Some of the documents cited herein are characterized as "incorporated by reference." In the event of a conflict between a definition or teaching of such incorporated reference and a definition or teaching set forth herein, the text of this specification shall control.

[0044] In the following, the elements of the present invention are described. These elements are described with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be interpreted to limit the present invention to only the embodiments explicitly described. The description should be understood to support and encompass embodiments that combine any number of the disclosed elements and / or preferred elements with the embodiments explicitly described. Furthermore, any permutation and combination of any elements described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0045] definition Below are provided definitions of some of the terms frequently used herein, which, in each instance of their use, have their respective defined and preferred meanings in the remainder of the specification.

[0046] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0047] When used in connection with numerical values, the term "about" is meant to encompass numerical values ​​in a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0048] In the context of the present invention, the term "peptide" refers to a short polymer of amino acids linked by peptide bonds. Peptides have the same chemical (peptide) bonds as proteins, but are generally shorter in length. The shortest peptides are dipeptides, consisting of two amino acids linked by peptide bonds. Tripeptides, tetrapeptides, pentapeptides, etc. can also exist. Unless a peptide is a cyclic peptide, it has an amino terminus and a carboxy terminus. Peptides usable in the present invention (including peptide derivatives, peptide variants, peptide fragments, peptide segments, peptide epitopes, and peptide domains) can be further modified by chemical modification. This means that such chemically modified peptides can contain other chemical groups other than the 20 naturally occurring proteinogenic amino acids. Examples of such other chemical groups include, but are not limited to, glycosylated amino acids and phosphorylated amino acids. Chemical modification of peptides can confer advantageous properties (e.g., one or more of enhanced stability, increased biological half-life, or increased solubility) compared to the parent peptide.

[0049] The term "polypeptide" refers to any polymer of amino acids linked by peptide bonds. A polypeptide may be a single chain or may be composed of two or more chains linked by covalent (e.g., disulfide) and / or non-covalent bonds. Modifications of the peptide bonds or side chain residues are possible as long as the activity of the resulting chemical entity (e.g., component A linked to component B) is not completely lost. The term is not to be construed as limiting the length of the polypeptide.

[0050] The term "protein" as used in the present context refers to a molecule comprising one or more polypeptides that adopt secondary and tertiary structures, and also to a protein that is composed of several polypeptides (i.e. several subunits) and forms a quaternary structure. Proteins may have non-peptidic groups attached, which may be called prosthetic groups or cofactors.

[0051] The term "C-terminus" (also known as the carboxyl terminus, carboxy terminus, C-terminal chain, C-terminus or COOH terminus) described in the context of the present invention is the end of an amino acid chain (protein or polypeptide) that is terminated by a free carboxy group (-COOH). When a protein is translated from messenger RNA, it is made from the N-terminus to the C-terminus. The term "N-terminus" (also known as the amino terminus, NH2-terminus, N-terminus or amine terminus) refers to the beginning of a protein or polypeptide that is terminated by an amino acid that has a free amine group (-NH2). The convention for writing peptide sequences is to place the N-terminus on the left and write the sequence from the N-terminus to the C-terminus.

[0052] The term "TNF homology domain of a TNF ligand family member protein" (THD) in the present specification refers to a protein domain shared by all tumor necrosis factor (TNF, previously known as TNFα or TNF alpha) ligand family members. Homology refers to evolutionary descent from a common ancestor. A homology domain is a conserved part of the sequence and (tertiary) structure of a given protein that can evolve, function and exist independently of other parts of the protein chain. It is a structural feature shared by all members of a particular protein family. Each domain forms a compact three-dimensional structure, which in many cases can be independently stable, can fold and can be important for biological activity. The C-terminus of a THD in the sense of the present invention is defined by the C-terminus consensus sequence: VF / YFGA / I-X1 (SEQ ID NO: 1) and the N-terminus is defined by the N-terminus consensus sequence: PV / AAHV / L (SEQ ID NO: 2), where X1 is a non-polar / hydrophobic or polar / neutral amino acid, preferably selected from the group consisting of F and I. Based on the sequence of a given TNF ligand family member protein, and using the C-terminal and N-terminal homology sequences defined above, one skilled in the art can determine the THD for a given TNF ligand family member protein. The location and length of individual THDs among members of the TNF family vary significantly, but can be defined by the presence of conserved amino acid residues identified by multiple sequence alignment using appropriate software tools (Bodmer et al., 2002). More importantly, crystal structures can reveal distinct interactions between amino acid residues (e.g., involved in homotrimerization of TNF family ligands). Such kind of information can be useful to improve the THD of a given member of the TNF superfamily as described in Bodmer et al., 2002. Furthermore, functional aspects such as protein solubility or bioactivity (e.g., receptor binding and activation) of designed protein variants can provide important hints regarding the critical amino acid residues or minimal length of individual THDs.The term THD includes polypeptides based on the sequences of naturally occurring TNF ligand family member proteins and variants thereof that retain the ability to specifically bind to the respective TNF ligand family member receptors. Preferably, such THD variants have at least 50% of the affinity of wild-type THD, more preferably at least 60%, 70%, 80%, 90%, and most preferably at least 99% of the affinity.

[0053] TNF ligand family member proteins comprise a group of multifunctional cytokines that can, for example, cause programmed cell death (apoptosis), differentiation, cell survival, and immune regulation. TNF is a monocyte-derived cytokine that is recognized by specific receptors and is associated with tumor regression, septic shock, and cachexia. Nineteen proteins have been identified as part of the TNF ligand family based on sequence, functional, and structural similarities. All of these cytokines appear to form homotrimeric (or heterotrimeric, in the case of LT alpha / beta) complexes that are recognized by specific receptors. The following proteins are members of the TNF ligand family: TNF-related apoptosis-inducing ligand (TRAIL; TNFSF10), a cytokine that induces apoptosis; CD40L (TNFSF5 = tumor necrosis factor superfamily member 5), a cytokine thought to be important for B cell development and activation; CD27L (TNFSF7), a cytokine that plays a role in T cell activation inducing proliferation of costimulated T cells and enhancing the generation of cytolytic T cells; CD30L (TNFSF8), a cytokine that induces T cell proliferation; FasL (TNFSF6), a cell surface protein involved in cell death; 4-1BBL (TNFSF9), an inducible T cell surface molecule that contributes to the stimulation of T cells; OX40L (TNFSF4), a cell surface protein that costimulates T cell proliferation and cytokine production; and LTA (TNFSF1), a protein that has antiproliferative activity and plays an important role in immune regulation. Further members of the TNF ligand family include EDA; LTB (TNFSF3); CD153 (TNFSF8); RANKL (TNFSF11); TWEAK (TNFSF12); APRIL (TNFSF13); BAFF (TNFSF13B); LIGHT (TNFSF14); VEGI (TNFSF15); GITRL (TNFSF18). Further information regarding the sequences of TNF ligand family members can be obtained from publicly accessible databases, such as Genbank.TNF ligand family members interact with their cognate receptors (e.g., TNF with TNFR1 and TNFR2, TRAIL with TRAILR1 (DR4), TRAILR2 (DR5), TRAILR3 (DcR1), TRAILR4 (DcR2) and OPG). Ligands mediate oligomerization and activation of each receptor. The interaction of TNF receptor family members with their ligands is characterized by the binding of the receptor in the space between two of the three TNF ligand family member protein monomers of the TNF ligand family member protein homotrimer, which is the biologically active form of TNF and other members of the TNF ligand family.

[0054] The term "consensus sequence" as used herein refers to the calculated order of the most frequent residues (either nucleotides or amino acids) found at each position in a sequence alignment between two or more sequences. It represents the result of a multiple sequence alignment in which related sequences are compared to each other and similar sequence motifs are calculated. Conserved sequence motifs are shown as consensus sequences, which show identical amino acids (i.e., identical amino acids between the compared sequences), conserved amino acids (i.e., amino acids that vary between the compared amino acid sequences but all belong to a particular functional or structural group of amino acids (e.g., polar or neutral)), and variable amino acids (i.e., amino acids that show no obvious relationship between the compared sequences).

[0055] The consensus sequences of the C-terminus and N-terminus of THD are located within the sequence of TNF ligand family members, respectively, and are sequences that are particularly conserved among TNF ligand family members. These sequences represent the parts of the TNF ligand family members that are involved in trimerization. Thus, the two consensus sequences serve as reference points for the C-terminus and N-terminus within a given TNF ligand family member, which may contain additional N-terminus or C-terminus amino acids that may not be present in other TNF ligand family members. Thus, the use of consensus sequences allows the same regions of various TNF ligand family members to be referenced without referring to specific positions as the N-terminus and C-terminus of the fragments of the TNF ligand family members present in the polypeptide of the present invention.

[0056] The term "multimerization domain" as used herein refers to a protein or polypeptide, a fragment or a portion of a protein or polypeptide, that mediates close proximity between at least two identical or different protein or polypeptide molecules (monomers) of the present invention, thus allowing protein-protein interactions that allow multimerization of multiple structurally similar or different monomers that are non-covalently or covalently linked. Multimerization results in the formation of a macromolecular complex formed by multiple covalently or non-covalently linked macromolecules (such as proteins). Multimerization domains that allow multimerization of two, three or four polypeptide molecules of the present invention are called dimerization, trimerization or tetramerization domains, respectively.

[0057] An "amino acid linker" in the context of the present invention refers to an amino acid sequence that sterically separates two parts or components of a complex (eg, a polypeptide and a multimerization domain). Typically, such linkers consist of 1 to 100 amino acids, having a minimum length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, and a maximum length of at least 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids or less. The indicated preferred minimum and maximum lengths of peptide linkers in the present invention may be combined if such combinations make mathematical sense, for example such linkers may consist of 1-15, 1-30, 1-60, 6-30, 7-15, 12-40 or 25-75 or 1-100 amino acids. Amino acid linkers may also provide flexibility between the two proteins they are linking. Such flexibility is generally increased when the amino acids are small. Thus, flexible amino acid linkers include an increased content of small amino acids (especially glycine and / or alanine) and / or hydrophilic amino acids (such as serine, threonine, asparagine and glutamine). Preferably, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the amino acids of the peptide linker are small amino acids. Amino acid linkers may also include N-glycosylation consensus sequences. Glycosylation of an amino acid linker is known to increase the stability of the linker (Imperialia and O'Connor, Curr Opin Chem Biol. 1999 Dec;3(6):643-9). Preferably, the consensus sequence is Asn-X-Ser / Thr or Asn-X-Cys, where X is in each case any amino acid except Pro, more preferably the consensus sequence is Asn-X-Ser / Thr. Further amino acid linkers that can be used in the polypeptide multimers of the present invention are known in the art.Such suitable linkers can be found in Chen et al (Adv Drug Deliv Rev. 2013 Oct; 65(10):1357-69) and Klein et al (Protein Eng Des Sel. 2014 Oct;27(10):325-30).

[0058] In the context of the present invention, the term "half-life prolonging domain" refers to a binding moiety that prolongs the serum / plasma half-life of a pharma- ceutical active moiety (i.e., when part of a half-life prolonging domain, the pharma- ceutical active moiety exhibits prolonged serum / plasma half-life). A binding moiety can be, but is not limited to, a polypeptide or a protein.

[0059] The term "target" or "target molecule" as used herein refers to a naturally occurring cellular or molecular structure to which other molecules have a particular binding affinity or to which other molecules specifically bind. "Specifically binds" refers to a binding moiety (e.g., a polypeptide or polypeptide multimer of the invention, or an antibody) that binds to a second target with a dissociation constant (K d By "binding" we mean binding to a target (such as a receptor or epitope) more tightly in a manner that is specific when the binding moiety binds to a first target with a dissociation constant lower than the K ) compared to binding to another target. A target can be recognized by a ligand that binds to that target with a particular affinity, such that ligand binding to each target produces a biological effect. Preferably, the dissociation constant (K ) of the target to which the binding moiety specifically binds is less than the K ). d ) is the dissociation constant (K d ) is more than 10-fold, preferably more than 20-fold, more preferably more than 50-fold, even more preferably more than 100-fold, 200-fold, 500-fold, 1000-fold, 5000-fold or more than 10.000-fold lower.

[0060] As used herein, the term "K" (measured in "mol / L", sometimes abbreviated as "M") d" is intended to refer to the dissociation equilibrium constant of a particular interaction between a binding moiety (e.g., a polypeptide or polypeptide multimer of the invention) and a target molecule (e.g., a receptor). Such affinities are preferably measured at 37°C. Suitable assays include surface plasmon resonance measurements (e.g., Biacore), quartz crystal microbalance measurements (e.g., Attana), biolayer interferometry (e.g., Octet) and competition assays.

[0061] As used herein, the term "variant" should be understood as a peptide or protein that differs from the peptide or protein from which it is derived by one or more changes in its length or sequence. The polypeptide from which a protein variant is derived is also known as the parent polypeptide. The term "variant" includes the "fragment" or "derivative" of the parent molecule. Usually, a "fragment" is smaller in length or size than the parent molecule, and a "derivative" shows one or more differences in sequence compared to the parent molecule. Also included are post-translational modifications of the parent protein (e.g., glycosylation, biotinylation, phosphorylation, ubiquitination, palmitoylation, or proteolysis). Usually, the variant is artificially created (preferably by means of genetic technology), while the parent polypeptide or polynucleotide is a wild-type protein or polynucleotide. However, it should be understood that naturally occurring variants are also included in the term "variant" herein. Furthermore, variants that can be used in the present invention can be derived from homologs, orthologs or paralogs of a parent molecule, or artificially created variants, provided that the variant exhibits at least one biological activity of the parent molecule (i.e., is functionally active).

[0062] The term "antibody" generally refers to a glycoprotein belonging to the immunoglobulin superfamily, which comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein as CH or CH). The heavy chain constant region can be further subdivided into three parts called CH1, CH2 and CH3 (or CH1, CH2 and CH3). The Fc (fragment crystallizable) region contains two heavy chain constant regions, and the Fab (fragment, antigen-binding) region contains one constant domain and one variable domain from each heavy and light chain of the antibody. Each light chain is composed of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region (abbreviated herein as CL or CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0063] The term "antibody fragment" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antibody fragment" include antigen-binding fragment (Fab) fragments, Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single domain antibodies (sdAbs), single chain variable fragments (scFv), variable fragments (Fv), V, and VF. H Domain, V LThese include domains, single domain antibodies, nanobodies, IgNARs (immunoglobulin novel antigen receptors), di-scFvs, bispecific T cell-inducing antibodies (BITEs), dual affinity retargeting (DART) molecules, triple bodies, alternative scaffold proteins, and their fusion proteins.

[0064] The term “V L Area" and "V H The "region" is the V L Area and V H The individual V L Area and V H Each region is composed of three hypervariable regions (complementarity determining region (CDR) 1, CDR2 and CDR3) and four framework regions (framework (FR) region 1, FR2, FR3, FR4). Identifying each subregion within a given sequence is routine in the art and can be achieved, for example, by IgBlast at NCBI. The variable regions of the heavy and light chains together form the binding region of the antibody. In immunoglobulins, the V L Area and V H The regions are located on different polypeptide chains, but in recombinant antibody derivatives they may be located on the same chain. L Area and V H The interaction of the domains enables the polypeptide of the invention to interact with its respective target antigen.

[0065] The term "bispecific antibody" as used herein refers to a fusion protein or bivalent antibody capable of binding to different antigens. Bispecific antibodies are composed of two single protein chains that contain fragments of antibodies (i.e., variable fragments). Bispecific antibodies are antibodies that combine light chain variable domains (V) on the same polypeptide chain. L ) linked to a heavy chain variable domain (V H ) (V H -V L or V L -V H). By using a short peptide linking the two variable domains, the domains are forced to pair with the complementary domains of another chain, thus creating two antigen-binding sites. Bispecific antibodies can target the same antigen (monospecific) or different antigens (bispecific).

[0066] The term "nucleic acid" as used herein includes all known forms of polymeric or oligomeric macromolecules or large biomolecules important to life. Nucleic acids, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. Most naturally occurring DNA molecules consist of two complementary biopolymeric chains that wrap around each other to form a double helix. DNA chains are also known as polynucleotides made of nucleotides. Each nucleotide is composed of a nitrogenous nucleobase and a monosaccharide called deoxyribose or ribose and a phosphate group. Naturally occurring nucleobases include guanine (G), adenine (A), thymine (T), uracil (U), or cytosine (C). Nucleotides are linked to each other in the chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next nucleotide, resulting in an alternating sugar-phosphate backbone. When the sugar is deoxyribose, the polymer is DNA. When the sugar is ribose, the polymer is RNA. Usually, polynucleotides are formed by phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, the term "nucleic acid" includes, but is not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof (such as, for example, RNA-DNA hybrids (within one strand)), as well as cDNA, genomic DNA, recombinant DNA, cRNA, and mRNA. The nucleic acid may consist of an entire gene or a portion thereof, and the nucleic acid may be miRNA, siRNA, or piRNA.

[0067] The term "vector" as used herein is also called an expression construct and is usually a plasmid or virus designed for protein expression in cells. Vectors are used to introduce a specific gene into a target cell and can use the cell's machinery for protein synthesis to produce the protein encoded by that gene. Expression vectors are designed to contain control sequences that function as enhancer and promoter regions and result in efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the production of substantial amounts of stable messenger RNA and protein. Examples of suitable vectors include, but are not limited to, plasmids, cosmids, phages, viruses or artificial chromosomes.

[0068] As used herein, the term "pharmaceutical composition" refers to a substance and / or combination of substances used for the identification, prevention or treatment of a tissue condition or disease. A pharmaceutical composition is formulated to be suitable for administration to a patient to prevent and / or treat a disease. Moreover, a pharmaceutical composition refers to a combination of an active substance and an inert or active carrier that makes the composition suitable for therapeutic use. A pharmaceutical composition may be formulated for oral, parenteral, topical, inhalation, rectal, sublingual, transdermal, subcutaneous or vaginal application routes, depending on its chemical and physical properties. Pharmaceutical compositions include solids, semi-solids, liquids, transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powders, granules, pellets, capsules, effervescent tablets or transdermal therapeutic systems. Also included are liquid compositions selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous administration, solutions for injection or solutions of the carrier systems of the present invention. Semi-solid compositions that may be used in the context of the present invention include emulsions, suspensions, creams, lotions, gels, globules, buccal tablets and suppositories.

[0069] The term "active agent" refers to a substance in a pharmaceutical composition or formulation that is biologically active (i.e., provides pharmaceutical value). A pharmaceutical composition may contain one or more active agents, which may act in combination with each other or independently. The active agent may be formulated as a neutral or salt form. Pharmaceutically acceptable salts include salts formed with free amino groups (such as salts derived from hydrochloric, phosphoric, acetic, oxalic, and tartaric acids, etc.) and salts formed with free carboxy groups (such as, but not limited to, salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, and procaine, etc.).

[0070] The terms "disease" and "disorder" are used interchangeably herein and refer to an abnormal condition (especially an abnormal medical condition such as a disease or injury) in which a cell, tissue, organ or individual can no longer perform its function efficiently. A disease is usually, but not necessarily, associated with certain symptoms or signs that indicate the presence of such a disease. Thus, the presence of such symptoms or signs may indicate a cell, tissue, organ or individual afflicted with a disease. A change in these symptoms or signs may indicate the progression of such a disease. The progression of a disease is usually characterized by an increase or decrease in such symptoms or signs, which may indicate a "worsening" or "improvement" of the disease. A "worsening" of a disease is characterized by a decrease in the ability of a cell, tissue, organ or individual / patient to perform its function efficiently, and an "improvement" of a disease is usually characterized by an increase in the ability of a cell, tissue, organ or individual / patient to perform its function efficiently.

[0071] The term "hyperproliferative disorder" in this application refers to a disorder in which cell division is increased compared to normal tissue. Such disorders are characterized by abnormal proliferation (production) of cells (i.e., overproduction). Proliferative disorders include neoplastic diseases. Neoplastic diseases can include benign or malignant tumors, and malignant neoplastic diseases are called cancers. The term hyperproliferative disorder includes cancer and precancerous disorders. Cancer includes proliferative disorders of mesenchymal origin (i.e., connective tissue (sarcoma)) and epithelial tissue (carcinoma). Common examples of sarcomas are osteosarcoma, chondrosarcoma, liposarcoma, leiomyosarcoma, angiosarcoma, and fibrosarcoma, as well as sarcoma of the gastrointestinal tract (GIST). Examples of cancers are cancers of the skin, testes, liver, gastrointestinal tract (such as esophagus, stomach, pancreas and colon), nasopharynx, bladder, cervix, ovaries, urethra and bladder; prostate and other genitourinary cancers, lung, kidney, endocrine system tissues (such as thyroid and pituitary), teratocarcinomas, and brain. Hematological malignancies are classified as lymphomas or leukemias. Inflammation modulates the microenvironment around the tumor and contributes to the proliferation, survival and migration of cancer cells, which may promote malignancy.

[0072] Inflammation is essentially a protective immunovascular response involving immune cells, blood vessels and a number of molecular mediators. The purpose of inflammation is to eliminate the initial cause of cellular injury, clear necrotic cells and damaged tissue from the original injury and inflammatory process, and initiate tissue repair. The term "inflammatory disorder" as used in the context of the present invention refers to a situation in which the physiological inflammatory response is transformed into a potentially harmful effect on the body. Inflammatory disorders that cause damage to normal tissue include, but are not limited to, autoimmune disorders and neurodegenerative diseases.

[0073] As used herein, the term "metabolic disorder" refers to a disease or disorder that affects the way the body processes (i.e., metabolizes) substances necessary to carry out physiological functions. Examples of metabolic disorders include, but are not limited to, diabetes, obesity, metabolic syndrome, and cardiovascular disease.

[0074] The terms "diabetes" or "diabetes mellitus", used interchangeably herein, refer to a disease characterized by elevated levels of sugar (glucose) in the blood. Diabetes can be caused by too little insulin (a chemical produced by the pancreas to regulate blood sugar), resistance to insulin, or both. In a preferred embodiment, the diabetes is type 2 diabetes (i.e., resistance to insulin).

[0075] As used herein, the term "obesity" refers to a condition in which a subject has excess body fat relative to lean body mass. In a preferred embodiment, obesity is defined as a weight gain or weight of 30 kg / m 2 is defined as a BMI (body mass index) exceeding .

[0076] As used herein, the term "metabolic syndrome" is defined as a condition that occurs in individuals with impaired glucose tolerance (IGT) or diabetes mellitus (DM), and / or insulin resistance, in combination with two or more of the following listed factors: 1. Elevated arterial pressure, i.e. ≥ 140 / 90mm Hg 2. Elevated plasma triglycerides (≥150 mg / dl) and / or low HDL-C (<35 mg / dl in men and <39 mg / dl in women) 3. Central obesity (i.e., waist-to-hip ratio (WHR) >0.9 in men and >0.85 in women) and / or body mass index (BMI) >30 kg / m 2 4. Microalbuminuria, i.e., urinary albumin excretion rate ≥ 20 μgm / min or albumin / creatine ratio ≥ 30 μgm / mg.

[0077] The terms "circulatory disorder", "circulatory disease" and / or "cardiovascular disease" are used interchangeably herein and, as defined herein, include systemic (or essential) hypertension, pulmonary hypertension (e.g., pulmonary arterial hypertension, pulmonary hypertension of the newborn), congestive heart failure, coronary artery disease, atherosclerosis, stroke, thrombosis, conditions of reduced vascular patency (e.g., following percutaneous transluminal coronary angioplasty), peripheral vascular disease, renal disease (particularly occurring with diabetes), angina pectoris (including stable angina, unstable angina, and variant (Prinzmetal) angina), hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, mixed dyslipidemia, and any condition in which improved blood flow leads to improved end-organ function.

[0078] The term "neurodegenerative disease" is used interchangeably herein and includes, as defined herein, Alzheimer's disease, HIV-associated dementia, migraine, progressive supranuclear palsy, corticobasal degeneration, tauopathy, Pick's disease, Parkinson's disease, neuropathy, dementia with Lewy bodies, multiple system atrophy, Huntington's disease, spinal-bulbar muscular atrophy, Friedreich's ataxia, spinocerebellar ataxia, Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinke disease, and other conditions that may be caused by neurodegenerative diseases. Syndrome, Fatal Familial Insomnia, Kuru, Amyotrophic Lateral Sclerosis, Spinal Muscular Atrophy, and Batten Disease, Spinal Cord Injury, Traumatic Brain Injury, Neuropathic Pain, Multiple Sclerosis, Acute Disseminated Encephalomyelitis, Barro's Disease, Charcot-Marie-Tooth Disease, Guillain-Barré Syndrome, HTLV-I Associated Myelopathy, Neuromyelitis Optica, Optic Nerve Atrophy, Nonarteritic Anterior Ischemic Optic Neuropathy, Schilder's Disease, Transverse Myelitis, Stroke, Epilepsy, and Diabetic Neuropathy.

[0079] "EC 50 The EC value refers to the half maximal effective concentration of a substance and is therefore a measure of the concentration of said substance that induces a response halfway between the baseline and the maximum after a specific exposure time. Thus, the EC 50represents the concentration of a substance at which 50% of the maximum effect is observed. Typically, the polypeptides and polypeptide multimers of the invention have an EC for binding to the TNFR2 receptor of 50 nM to 1 pM, more preferably 10 nM to 10 pM, and even more preferably 1 nM to 50 pM (i.e., 50 nM, 10 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, or 1 pM). 50 Indicates the value.

[0080] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia (Ph.Eur.), or other generally recognized pharmacopoeias for use in animals, especially humans.

[0081] The term "carrier" herein refers to a diluent, adjuvant, excipient, or solvent administered with a therapeutic substance. Such pharmaceutical carriers can be sterile liquids such as saline in water and oil (including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil). Saline is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline, aqueous dextrose, and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, fat milk powder, glycerol, propylene, glycol, water, and ethanol, and the like. If necessary, the composition can contain small amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, and sustained release formulations. The compositions may be formulated as suppositories with traditional binders and carriers, such as triglycerides. The compounds of the present invention may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with free amino groups, such as salts derived from hydrochloric, phosphoric, acetic, oxalic, and tartaric acids, and salts formed with free carboxy groups, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, and procaine. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions will contain a therapeutically effective amount of the compound (preferably in purified form) together with an appropriate amount of carrier, so as to provide the form for proper administration to the patient. The formulation should be compatible with the method of administration.

[0082] Implementation In the following sections, the various aspects of the invention are defined in more detail. Each aspect thus defined may be combined with any other aspect, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature indicated as being preferred or advantageous.

[0083] In a first aspect, the present invention provides a polypeptide comprising a binding domain consisting of three peptide TNF homology domains (THDs) of a TNF ligand family member protein which specifically binds to the extracellular portion of TNFR2, wherein the C-terminus of the first and second THDs (which in each case are defined by the C-terminal consensus sequence VF / YFGA / I-X1 (SEQ ID NO:1)) is linked to the N-terminus of the second and third THDs (which in each case are defined by the N-terminal consensus sequence PV / AAHV / L (SEQ ID NO:2)) by peptide X a (which are in each case independently selected and have a length of 9 to 12 amino acids, preferably 9 to 11, more preferably 9 to 10), and preferably a does not contain the amino acid sequence SSRTPSDK (SEQ ID NO: 10); X1 is a non-polar / hydrophobic or polar / neutral amino acid, preferably selected from the group consisting of F and I.

[0084] In the research that led to the present invention, the linking peptide X a It was shown that truncation of peptide X significantly improved the thermal stability but at the same time significantly reduced the biological activity. Surprisingly, the inventors have found peptide X, which provides significantly improved stability while maintaining biological activity (such as binding to the respective receptor, preferably TNFR2). a A narrow range of sizes was identified.

[0085] Thus, it is preferred that the polypeptides of the present invention have a thermal stability of at least 63°C, at least 64°C, at least 65°C, at least 66°C, at least 67°C, more preferably at least 65°C, at least 66°C, and most preferably at least 66°C, as measured by the dynamic light scattering method disclosed herein.

[0086] It is further preferred that the polypeptide of the present invention has a specific biological activity with respect to the activation of the TNFR2 receptor. A preferred example of this biological activity is the activation of the TNFR2 receptor on Kym-1 cells as described in Example 5. Preferably, the polypeptide of the present invention has an EC value of 100 or more for the activation of the TNFR2 receptor on Kym-1 cells, preferably involving TNFR2 cross-linking with the antibody 80M2. 50 Values ​​are less than 400 pM, less than 350 pM, less than 300 pM, less than 250 pM, more preferably less than 300 pM.

[0087] The C-terminal and N-terminal consensus sequences serve the purpose of providing a reference point for the ends of the regions of the THD of TNF family members necessarily included in the polypeptide of the present invention. This is illustrated by the preferred example of the TNF family members TNF alpha and LT alpha. In SEQ ID NO:5, the sequence of human TNF alpha is disclosed. The C-terminal sequence is VYFGII (SEQ ID NO:3), which corresponds to amino acids 226-231 of SEQ ID NO:5, and the N-terminal sequence is PVAHV (SEQ ID NO:4), which corresponds to amino acids 88-92 of SEQ ID NO:5. In SEQ ID NO:55, the sequence of human LT alpha is disclosed. The C-terminal sequence is VFFGAF (SEQ ID NO:56), which corresponds to amino acids 198-203 of SEQ ID NO:55, and the N-terminal sequence is PAAHL (SEQ ID NO:57), which corresponds to amino acids 63-67 of SEQ ID NO:55.

[0088] In a preferred embodiment of the first aspect of the invention, peptide X a isX C -X L -X N where X Cis selected from the group consisting of A, AL, L, preferably A and AL, more preferably AL; X L is absent or is an amino acid linker consisting of 1 to 11 amino acids, preferably 1 to 10 amino acids, more preferably 1 to 9 amino acids, and most preferably 4 to 8 amino acids; X N is absent or is selected from the group consisting of K, DK, SDK, PSDK (SEQ ID NO: 6), TPSDK (SEQ ID NO: 7), RTPSDK (SEQ ID NO: 8), SRTPSDK (SEQ ID NO: 9), SSRTPSDK (SEQ ID NO: 10), TK, STK, HSTK (SEQ ID NO: 11), AHSTK (SEQ ID NO: 12), LAHSTK (SEQ ID NO: 13), HLAHSTK (SEQ ID NO: 14), LHLAHSTK (SEQ ID NO: 15), preferably SSRTPSDK (SEQ ID NO: 10), SDK.

[0089] In a more preferred embodiment of the first aspect of the invention, peptide X a isX C -X L -X N where X C is selected from the group consisting of A, AL, L, preferably A and AL, more preferably AL; X L is absent or is an amino acid linker consisting of 1 to 11 amino acids, preferably 1 to 10 amino acids, more preferably 1 to 9 amino acids, and most preferably 4 to 8 amino acids; X Nis absent or is selected from the group consisting of K, DK, SDK, PSDK (SEQ ID NO:6), TPSDK (SEQ ID NO:7), RTPSDK (SEQ ID NO:8), SRTPSDK (SEQ ID NO:9), TK, STK, HSTK (SEQ ID NO:11), AHSTK (SEQ ID NO:12), LAHSTK (SEQ ID NO:13), HLAHSTK (SEQ ID NO:14), LHLAHSTK (SEQ ID NO:15), preferably SRTPSDK (SEQ ID NO:9), RTPSDK (SEQ ID NO:8), TPSDK (SEQ ID NO:7), PSDK (SEQ ID NO:6), SDK, more preferably RTPSDK (SEQ ID NO:8) and SRTPSDK (SEQ ID NO:9).

[0090] Peptide X a is three components X C , X L and X N However, component X L and X N X, provided that there is at least one of L and X N X may or may not be present. C contains amino acids derived from the C-terminus of a TNF ligand family member. L is an amino acid linker. Preferably, the amino acid linker is a glycine-serine linker. X N comprises amino acids derived from the N-terminus of the THD of a TNF ligand family member, preferably X N does not contain the amino acid sequence SSRTPSDK (SEQ ID NO: 10).

[0091] In a preferred embodiment of the first aspect of the invention, the three THDs of the polypeptide of the invention are identical in their amino acid sequences.

[0092] In a preferred embodiment of the first aspect of the invention, the C-terminus of the first and second THD is in each case defined by the C-terminal sequence VYFGII (SEQ ID NO: 3) and the N-terminus of the second and third THD is in each case defined by the N-terminal sequence PVAHV (SEQ ID NO: 4). These C-terminal and N-terminal sequences are derived from human TNF alpha.

[0093] In a preferred embodiment of the first aspect of the invention, the THD comprises a contiguous amino acid sequence consisting of amino acids 88 to 231 of SEQ ID NO:5, optionally comprising at least one mutation selected from the group consisting of: D143Y, D143F, D143E, D143N, D143T, D143S, E146Q, E146H, E146K, A145R / S147T, Q88N / T89S / A145S / E146A / S147D ... 88N / A145I / E146G / S147D, A145H / E146S / S147D, A145H / S147D, L29V / A145D / E146D / S147D, A145N / E146D / S147D, A14 5T / E146S / S147D, A145Q / E146D / S147D, A145T / E146D / S147D, A145D / E146G / S147D, A145D / S147D, A145K / E146D / S14 7T, A145R / E146T / S147D, A145R / S147T, E146D / S147D, E146N / S147, S95C / G148C, K65A, K65W, Q67K, Q67T, Q67Y, L75H , L75W, D143W, D143V, D143V / F144L / A145S, D143N / A145R, D143V / A145S, L29V, L29T, L29S, L29A, L29G, R31H, R31I, R 31L, R32G, R32E, S147L, S147R, S147P, S147T, S147A, Q149E, Q149N, E146D, E146N, E146S, E146G, A145R, A145S, A145T, A145H, A145K, A145F, A145D, A145G, A145N, A145P, A145Q, A145Y, A145V, and A145W, preferably selected from D143N and A145R.

[0094] The mutations disclosed above increase the specificity of binding to the extracellular portion of TNFR2. Preferably, the mutations reduce the binding affinity to TNFR1 but essentially maintain the affinity to TNFR2, thereby increasing the specificity for TNFR2 (i.e., the Kd for binding to TNFR2 is at least 10-fold, at least 100-fold, at least 1.000-fold, preferably at least 5.000-fold higher than the Kd for binding to TNFR1).

[0095] These mutations are known in the art and are disclosed in Loetscher et al (JBC, vol 268, no 35, pp. 26350-26357, 1993; see Table 1), Abe et al (Biomaterials 32 (2011) 5498-5504; see Table 1), Ando et al (Biochemistry and Biophysics Reports, 7; 2016; 309-315; see Table 2), and Ban et al (Molecular and Cellular Therapies (2015) 3:7). Preferably, the polypeptide contains 5, 4, 3, 2 or 1 of these mutations, more preferably 2 or 1, most preferably 1.

[0096] In a preferred embodiment of the first aspect of the present invention, X C is selected from A or AL, X L is absent or is a glycine and / or serine linker having a length of 1 to 11 amino acids, preferably 1 to 10 amino acids, more preferably 1 to 9 amino acids, and most preferably 4 to 8 amino acids; X Nis absent or selected from K, DK, SDK, PSDK (SEQ ID NO:6), TPSDK (SEQ ID NO:7), RTPSDK (SEQ ID NO:8), SRTPSDK (SEQ ID NO:9), and SSRTPSDK (SEQ ID NO:10); more preferably selected from K, DK, SDK, PSDK (SEQ ID NO:6), TPSDK (SEQ ID NO:7), RTPSDK (SEQ ID NO:8), and SRTPSDK (SEQ ID NO:9).

[0097] In a preferred embodiment of the first aspect of the present invention, X C is selected from A or AL, X Lare G, S, GG, SG, GS, GGG, SGG, GSG, GGS, GGGG (SEQ ID NO: 16), GGGS (SEQ ID NO: 17), GGSG (SEQ ID NO: 18), GSGG (SEQ ID NO: 19), SGGG (SEQ ID NO: 20), GGGGG (SEQ ID NO: 21), SGGGG (SEQ ID NO: 22), GSGGG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GGGSG (SEQ ID NO: 25), GGGGS (SEQ ID NO: 26), GGGGGG (SEQ ID NO: 27), SGGGGG (SEQ ID NO: 28), GSGGGG (SEQ ID NO: 29), GGSGGG (SEQ ID NO: 30), GGGSGG (SEQ ID NO: 31), GGGGSG (SEQ ID NO: 32), GGGGGS (SEQ ID NO: 33), GGGSGGGS (SEQ ID NO: 34), SGGGSGGG (SEQ ID NO: 35), GGGGGGGG (SEQ ID NO: 36), GSGGGSGG (SEQ ID NO: 37), 7), GGSGGGSG (SEQ ID NO: 38), SGGGSGGGS (SEQ ID NO: 39), GSGGGGSGG (SEQ ID NO: 40), GGSGGGSGG (SEQ ID NO: 41), GGGSGGGSG (SEQ ID NO: 42), SGGGSGGGSG (SEQ ID NO: 43), GSGGGSGGGS (SEQ ID NO: 44), GGSGGGSGGG (SEQ ID NO: 45), GGGSGGGSGG (SEQ ID NO: 46), SGGGSGGGSGG (SEQ ID NO: 47), GSGGGGSGGGSG (SEQ ID NO: 48), GGSGGGSGGGS (SEQ ID NO: 49), and GGGSGGSGGG (SEQ ID NO: 50), preferably selected from GGGG (SEQ ID NO: 16), GGGGS (SEQ ID NO: 26), and GGGSGGGS (SEQ ID NO: 34), more preferably GGGG (SEQ ID NO: 16) and GGGSGGGS (SEQ ID NO: 34); X Nis absent or selected from K, DK, SDK, PSDK (SEQ ID NO:6), TPSDK (SEQ ID NO:7), RTPSDK (SEQ ID NO:8), SRTPSDK (SEQ ID NO:9), and SSRTPSDK (SEQ ID NO:10); more preferably selected from K, DK, SDK, PSDK (SEQ ID NO:6), TPSDK (SEQ ID NO:7), RTPSDK (SEQ ID NO:8), and SRTPSDK (SEQ ID NO:9).

[0098] In a preferred embodiment, XL is a glycine / serine linker having a length of 1-11 amino acids, preferably 1-10 amino acids, more preferably 1-9 amino acids, most preferably 4-8 amino acids, and a glycine to serine content of 3:1. In other words, for every 3 glycines, there is one serine. Preferably, each serine is separated from another serine by 3 glycines.

[0099] In a preferred embodiment of the first aspect of the present invention, X C is AL, and X L does not exist, and X N is selected from SRTPSDK (SEQ ID NO: 9), SSRTPSDK (SEQ ID NO: 10), SSSRTPSDK (SEQ ID NO: 51), and RSSSRTPSDK (SEQ ID NO: 52), preferably SRTPSDK (SEQ ID NO: 9) and SSRTPSDK (SEQ ID NO: 10); more preferably, selected from SRTPSDK (SEQ ID NO: 9).

[0100] In a preferred embodiment of the first aspect of the present invention, X C is AL, and X L is GGGG (SEQ ID NO: 16), and X N is selected from SDK, PSDK (SEQ ID NO: 6), TPSDK (SEQ ID NO: 7), and RTPSDK (SEQ ID NO: 8).

[0101] In a preferred embodiment of the first aspect of the present invention, X C is AL, and X L is GGGSGGGS (SEQ ID NO: 34), and X N is selected from K and DK.

[0102] In a preferred embodiment of the first aspect of the present invention, X C is AL, and X L is G and X N is selected from RTPSDK (SEQ ID NO: 8), SRTPSDK (SEQ ID NO: 9), preferably SRTPSDK (SEQ ID NO: 9).

[0103] In a preferred embodiment of the first aspect of the present invention, X C is AL, and X L is GG and X N is selected from TPSDK (SEQ ID NO: 7), RTPSDK (SEQ ID NO: 8), SRTPSDK (SEQ ID NO: 9), preferably RTPSDK (SEQ ID NO: 8).

[0104] In a preferred embodiment of the first aspect of the present invention, X C is AL, and X L is GGG and X N is selected from PSDK (SEQ ID NO: 6), TPSDK (SEQ ID NO: 7), RTPSDK (SEQ ID NO: 8), SRTPSDK (SEQ ID NO: 9), preferably TPSDK (SEQ ID NO: 7).

[0105] In a preferred embodiment of the first aspect of the present invention, X C is AL, and X L is GGGG (SEQ ID NO: 16), and X Nis selected from SDK, PSDK (SEQ ID NO: 6), TPSDK (SEQ ID NO: 7), RTPSDK (SEQ ID NO: 8), preferably PSDK (SEQ ID NO: 6).

[0106] In a preferred embodiment of the first aspect of the invention, the polypeptide has an aggregation onset temperature (T) determined by dynamic light scattering of greater than 62° C., greater than 63° C., greater than 64° C., greater than 65° C., greater than 66° C., greater than 67° C., greater than 68° C., preferably greater than 65° C., greater than 66° C., or greater than 67° C., most preferably greater than 66° C. or greater than 67° C. m ) The aggregation onset temperature (T m ) is preferably determined by dynamic light scattering as disclosed in Example 4 herein. The onset of aggregation as a function of increasing temperature is indicative of protein denaturation. The higher the onset aggregation temperature of a protein (such as the polypeptide of the invention), the more thermally stable the protein is. The terms "denaturation temperature" and "aggregation temperature" are used interchangeably herein.

[0107] In a second aspect, the present invention provides a polypeptide multimer comprising at least two polypeptides according to the first aspect of the invention, (a) linked together, preferably linked together by an amino acid linker having a length of 1 to 30 amino acids, preferably 7 to 15 amino acids; or (b) is linked to a protein, preferably a multimerization domain (preferably selected from the group consisting of a multimerization domain, a serum protein, a cytokine, a targeting moiety, or a toxin); Optionally, wherein said polypeptide is linked to said protein by an amino acid linker having a length of 1 to 30 amino acids, preferably 7 to 15 amino acids.

[0108] In a preferred embodiment of the second aspect of the present invention, the polypeptides of the first aspect of the present invention are linked together to form a chain structure, in which the polypeptides are directly linked together by their amino or carboxy termini.If an amino acid linker is present, the linker is attached to the amino or carboxy termini of the polypeptides.In a preferred embodiment, the chain structure further comprises a protein attached to at least one polypeptide of the first aspect of the present invention.Preferred examples of such proteins are multimerization domains, serum proteins, cytokines, targeting moieties or toxins.

[0109] The polypeptide multimer of the present invention has improved stability (particularly thermal stability) while retaining biological activity, similar to the polypeptide of the present invention. When no linker is present, the polypeptide of the first aspect of the present invention is directly linked to the multimerization domain.

[0110] Thus, the polypeptide multimers of the present invention have a thermal stability (T) of greater than 71° C., at least 72° C., at least 73° C., at least 74° C., preferably at least 72° C., at least 73° C., at least 74° C., at least 75° C., at least 76° C., at least 77° C., or at least 78° C., more preferably at least 74° C., as measured by dynamic light scattering as disclosed herein. m ) (i.e., aggregation onset temperature). The higher the aggregation onset temperature, the more thermally stable the polypeptide multimer is.

[0111] Another preferred example of the stability of the polypeptide multimer of the present invention is the stability after 3 days of incubation in human plasma at 37° C., as disclosed in Example 10. Preferably, the EC of the polypeptide multimer of the present invention for binding to TNFR2 in HeLa-TNF-R2 cells in Example 10 after 3 days of incubation in human plasma at 37° C. is 50 is the EC before incubation in human plasma 50is not reduced by more than 15%, 12%, 10%, preferably 10% compared to

[0112] Another preferred example of the stability of the polypeptide multimer of the present invention is the stability after 8 days of incubation in human plasma at 37° C., as disclosed in Example 10. Preferably, the EC of the polypeptide multimer of the present invention for binding to TNFR2 in HeLa-TNF-R2 cells in Example 10 after 3 days of incubation in human plasma at 37° C. is 50 is the EC before incubation in human plasma 50 is not reduced by more than 15%, 12%, 10%, preferably 10% compared to

[0113] It is further preferred that the polypeptide multimer of the present invention has a specific biological activity with respect to activation of the TNFR2 receptor.

[0114] Thus, the polypeptide multimers of the present invention have an EC50 of less than 100 pM, preferably less than 80 pM, and more preferably less than 70 pM, as assessed by binding to TNFR2 expressed on mouse embryonic fibroblasts (MEFs), as disclosed in Example 7. 50 Preferably, the polypeptide multimer of the present invention does not bind to TNFR1 under the conditions of Example 7.

[0115] Another preferred example of this biological activity is the binding to TNFR2 on Kym-1 cells, as disclosed in Example 8, where EC 50 is less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM, preferably less than 100 pM or less than 75 pM, more preferably less than 75 pM.

[0116] Another preferred example of this biological activity is the activation of NF-κB in HeLa-TNF-R2 cells, as disclosed in Example 9, where EC 50is less than 30 pM, less than 20 pM, less than 10 pM, less than 5 pM, preferably less than 10 pM or less than 5 pM, more preferably less than 5 pM.

[0117] In a preferred embodiment of the second aspect of the present invention, the amino acid linker linking the polypeptide to the protein (preferably the multimerization domain) has a length of 5 to 50, 5 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, 7 to 20, 7 to 15, 7 to 12, or 9 to 11 amino acids, preferably 7 to 15, 7 to 12, or 9 to 11, more preferably 7 to 12 or 9 to 11, and most preferably 9 to 11 amino acids.

[0118] In a preferred embodiment of the second aspect of the invention, the amino acid linker linking the polypeptide of the invention to the protein (preferably the multimerization domain) is a glycine-serine linker.

[0119] In a preferred embodiment of the second aspect of the invention, the amino acid linker linking the polypeptide of the invention to the protein (preferably the multimerization domain) is GGSGGGGSGG (SEQ ID NO:5).

[0120] In a preferred embodiment of the second aspect of the invention, the amino acid linker linking the polypeptide of the invention to the protein (preferably the multimerization domain) comprises a consensus sequence for N-glycosylation.

[0121] In a preferred embodiment, the polypeptide of the invention is linked (optionally by an amino acid linker according to the second aspect of the invention) to the N-terminus of a protein (preferably a multimerisation domain).

[0122] In a preferred embodiment, the polypeptide of the invention is linked (optionally by an amino acid linker according to the second aspect of the invention) to the C-terminus of a protein (preferably a multimerisation domain).

[0123] In a preferred embodiment, at least one polypeptide of the invention is linked to the N-terminus of a protein (preferably a multimerization domain) and at least one polypeptide is linked to the C-terminus of a protein (preferably a multimerization domain), optionally each of these links separately from each other comprising an amino acid linker according to the second aspect of the invention.

[0124] In a preferred embodiment of the second aspect of the invention, the multimerization domain is a dimerization domain.

[0125] Preferred dimerization domains are those derived from antibodies (including but not limited to antibodies, antibody heavy chains, Fc regions, heavy chain domain 2 (CH2) of IgM (MHD2), heavy chain domain 2 (CH2) of IgE (EHD2), heavy chain domain 3 (CH3) of IgG, heavy chain domain 3 (CH3) of IgA, heavy chain domain 3 (CH3) of IgD, heavy chain domain 4 (CH4) of IgM, heavy chain domain 4 (CH4) of IgE, Fab, Fab2, and CH1 and CL domains). Preferred dimerization domains derived from antibodies are Fc regions, variants or fragments thereof. Fc regions that can be used as dimerization domains are preferably derived from the following isotypes IgA, IgD, IgE, IgG, and IgM.

[0126] More preferred dimerization domains are immunoglobulin Fc region mutants that do not have FcR and / or C1q binding. Preferred examples of immunoglobulin Fc region mutants that do not have FcR and / or C1q binding are FcΔab, LALA, LALA-GP, IgG2, IgG2σ, aglycosylated IgG1, IgG1(L234F / L235E / LP331S), IgG2m4, and IgG4 ProAlaAla. Even more preferred examples of Fc region mutants are FcΔab that do not have Fcγ receptor I binding and C1q binding (Armour et al; Eur. J. Immunol. 1999, 29:2613-2624).

[0127] Other dimerization or multimerization domains include barnase-barstar, C4bp, CD59, collagen-derived peptides, leucine zipper motifs, miniantibodies and ZIP miniantibodies, GST, the α and β subunits of inactive human chorionic gonadotropin, maltose binding protein (MBP), p53 and its fragments, phosphatases, streptavidin, surfactant protein D, tenascin, tetranectin, dock-and-lock (DNL) motifs, and uteroglobin.

[0128] In a preferred embodiment of the second aspect of the invention, the multimerization domain is a trimerization domain.

[0129] Preferred trimerization domains are tenascin-C (TNC), the trimerization region of the C-terminal non-collagenous domain of collagen XVIII (NC1), Fab3-like molecules, and TriBi minibodies, more preferably TNC.

[0130] In a preferred embodiment of the second aspect of the invention, the multimerization domain is a tetramerization domain.

[0131] Preferred tetramerization domains are the tetramerization domain of p53, the tetramerization domain of general control protein 4 (GCN4), the tetramerization domain of VASP (vasodilator stimulated phosphoprotein), tandem bispecific antibodies, and di-diabodies.

[0132] In a preferred embodiment of the second aspect of the present invention, the protein that the polypeptide is linked to is a ligand that is specific for tissue, organ or cell type.Preferably, the ligand is a targeting moiety that is specific for organ, tissue or cell type.More preferably, the targeting moiety is specific for cells of the immune system (e.g., regulatory T cells (Treg); costimulatory ligand), cells of the central nervous system (e.g., microglial cells), cardiac muscle (including cardiac progenitor cells), colon, skin, inflamed tissue or pancreatic cells.

[0133] In a preferred embodiment of the second aspect of the present invention, the polypeptide multimer further comprises a ligand specific for a tissue, an organ or a cell type.Preferably, the ligand is a targeting moiety specific for an organ, a tissue or a cell type.More preferably, the targeting moiety is specific for a cell of the immune system (e.g., regulatory T cells (Treg); costimulatory ligand), a cell of the central nervous system (e.g., microglial cells), myocardium (including cardiac progenitor cells), colon, skin, inflamed tissue or pancreatic cells.Preferably, the targeting moiety is present in the polypeptide multimer in addition to the protein to which the polypeptide is linked.

[0134] In a preferred embodiment of the second aspect of the invention, the targeting moiety binds to a target selected from the following receptors: transferrin receptor, CD98, IGF1R, LRP1, insulin receptor, low density lipoprotein receptor (LDLR), diphtheria toxin receptor, efflux pumps, CD25, CD28, GLUT1, LAT1, TMEM119, PDGFR, VEGFR1, VEGFR3, and RVG-29.

[0135] In a preferred embodiment of the second aspect of the invention, the protein to which the polypeptide is linked is a cytokine (preferably IL-2 or TGFβ), or a half-life prolonging domain (preferably an albumin binding moiety, an immunoglobulin binding moiety, a PEG mimetic polypeptide, PEGylation or HESylation).

[0136] In a preferred embodiment of the second aspect of the invention, the polypeptide multimer further comprises a cytokine (preferably IL-2 or TGFβ), or a half-life prolonging domain (preferably an albumin binding moiety, an immunoglobulin binding moiety, a PEG-mimetic polypeptide, PEGylated or HESylated). Preferably, the cytokine, half-life prolonging domain or immunoglobulin binding moiety is present in the polypeptide multimer in addition to the protein to which the polypeptide is linked.

[0137] In a third aspect, the present invention provides a nucleic acid molecule encoding a polypeptide according to the first aspect of the present invention or a polypeptide multimer according to the second aspect of the present invention. The nucleic acid may be RNA or DNA or a hybrid thereof. Preferably, the nucleic acid also comprises a sequence that allows the expression of the polypeptide according to the first and second aspects of the present invention in a suitable expression system. The nucleic acid may be codon-optimized for each expression system.

[0138] In a fourth aspect, the present invention provides a vector encoding a nucleic acid molecule according to the third aspect of the invention. The polypeptide or polypeptide multimer of the present invention is preferably encoded by an introduced nucleic acid molecule according to the third aspect of the invention and expressed in a cell by introduction of a vector. Preferably, the vector provides for transcription and expression of the polypeptide encoded by the nucleic acid in a suitable host cell system. Preferably, the expression vector is selected from the group consisting of bacterial, yeast, baculovirus, plant, viral and mammalian expression vectors, more preferably the expression vector is a bacterial expression vector or a cell-free expression vector.

[0139] In a fifth aspect, the present invention provides a polypeptide according to the first aspect of the invention, a polypeptide multimer according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, for use as a medicament.

[0140] In a sixth aspect, the present invention provides a pharmaceutical composition comprising as an active substance a polypeptide according to the first aspect of the invention, a polypeptide multimer according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, or a vector according to the fourth aspect of the invention. The pharmaceutical composition preferably further comprises a pharma- ceutically acceptable carrier and / or a suitable excipient. The pharmaceutical composition is selected from the group consisting of a solid, liquid, semi-solid or transdermal therapeutic system. It is envisaged that the pharmaceutical composition of the present invention comprises one or more of the polypeptide of the first aspect of the invention and / or the polypeptide multimer of the second aspect of the invention.

[0141] In a seventh aspect, the present invention provides a polypeptide according to the first aspect of the invention, a polypeptide multimer according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, a vector according to the fourth aspect of the invention or a pharmaceutical composition according to the fifth aspect of the invention for use in the prevention or treatment of hyperproliferative disorders, inflammatory disorders, autoimmune disorders and metabolic diseases, cardiovascular diseases, neuropathic diseases and neurological injuries.

[0142] A preferred hyperproliferative disorder is cancer or hematological malignancies.

[0143] Particularly preferred cancers to be prevented or treated by the polypeptides or polypeptide multimers of the invention are cancers of the gastrointestinal tract, liver, kidney, bladder, prostate, endometrium, ovary, testis, skin, invasive oral cancer, small cell and non-small cell lung cancer, hormone-dependent breast cancer, hormone-independent breast cancer, transitional cell carcinoma, squamous cell carcinoma, neurological malignancies (including neuroblastoma, glioma), astrocytoma, osteosarcoma, soft tissue sarcoma, hemangioma, endocrinological tumors, hematological tumors (including leukemia, lymphoma, and other myeloproliferative and lymphoproliferative disorders), intraepithelial carcinoma, hyperplastic lesions, adenomas, fibromas, histiocytosis, chronic inflammatory proliferative disorders, vascular proliferative disorders and virally induced proliferative disorders, skin disorders characterized by hyperproliferation of keratinocytes and / or T cells. Particularly preferred diseases which may be treated with the compounds of the invention are solid tumours, especially lung, breast, pancreatic, colorectal, ovarian, prostate and gastric carcinoma and adenocarcinoma.

[0144] Preferred inflammatory diseases to be prevented or treated by the polypeptides or polypeptide multimers of the invention include, but are not limited to, acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis (also Lou Gehrig's disease; motor neuron disease), ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune heart disease, and the like. Myopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune pancreatitis, autoimmune peripheral neuropathy, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Baro's disease / Baro's concentric sclerosis, Behçet's disease, Berger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, , Castleman's disease, celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, chronic obstructive pulmonary disease, chronic relapsing multifocal osteomyelitis, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, capitis arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Degos' disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes Diarrhea, diffuse cutaneous systemic sclerosis, discoid lupus erythematosus, Dressler's syndrome, drug-induced lupus, eczema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evans' syndrome, fibrodysplasia ossificans progressiva, fibrous alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoidpemphigoid), glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalopathy, Hashimoto's thyroiditis, Henoch-Schönlein purpura, herpes gestationis (also known as gestational pemphigoid), sweat gland abscess, Hughes-Stovin syndrome, hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (see autoimmune thrombocytopenic purpura), IgA nephropathy, inclusion body myositis, interstitial cystitis, juvenile idiopathic arthritis (also known as juvenile rheumatoid arthritis), Kawasaki disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear immunoglobulin A disease (LAD), lupoid hepatitis (also known as autoimmune hepatitis), lupus erythematosus, Majeed syndrome, microscopic colitis, microscopic polyangiitis, Miller Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease (also known as acute pityriasis lichenoidis), multiple sclerosis, myasthenia gravis, myositis, Meniere's disease, narcolepsy, neuromyelitis optica (also known as Devic's disease), neuromyotonia, ophthalmic cicatricial pemphigoid, opsoclonus, myoclonus syndrome, Ord's thyroiditis, relapsing rheumatism, PANDAS (Streptococcus associated pediatric autoimmune neuropsychiatric disorders), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry's disease, Romberg syndrome, pars planitis, Parsonage-Turner syndrome, pemphigus vulgaris, perivenous encephalomyelitisencephalomyelitis), pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, Rasmussen encephalitis, Raynaud's phenomenon, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, schizophrenia, Schmidt's syndrome, another form of APS, Schnitzler's syndrome, scleritis, scleroderma, serum sickness, Sjögren's syndrome, spondyloarthropathy, systemic These include stiff joint syndrome, Still's disease (see Juvenile Rheumatoid Arthritis), subacute bacterial endocarditis (SBE), Susac syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus (see Lupus Erythematosus), Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, Tolosa–Hunt syndrome, transverse myelitis, ulcerative colitis (one of two types of idiopathic inflammatory bowel disease "IBD"), undifferentiated connective tissue disease distinct from mixed connective tissue disease, undifferentiated spondyloarthropathy, urticarial vasculitis, vasculitis, vitiligo, and Wegener's granulomatosis. Hypersensitivity disorders include, but are not limited to, allergies (such as asthma, anaphylaxis or atopy); cytotoxic antibody-dependent diseases (such as autoimmune hemolytic anemia, thrombocytopenia, rheumatic heart disease, erythroblastosis fetalis, Goodpasture's syndrome, membranous nephropathy, Graves' disease, myasthenia gravis); immune complex diseases (such as serum sickness, Arthus reaction, rheumatoid arthritis, poststreptococcal glomerulonephritis, lupus nephritis, systemic lupus erythematosus, extrinsic allergic alveolitis (hypersensitivity pneumonitis)), cellular immune responses such as contact dermatitis, tuberculin reactions, chronic graft rejection, and multiple sclerosis.

[0145] Particularly preferred neurodegenerative diseases to be prevented or treated by the polypeptides or polypeptide multimers of the invention include Alzheimer's disease, HIV-associated dementia, migraine, progressive supranuclear palsy, corticobasal degeneration, tauopathy, Pick's disease, Parkinson's disease, neuropathy, dementia with Lewy bodies, multiple system atrophy, Huntington's disease, spinal-bulbar muscular atrophy, Friedreich's ataxia, spinocerebellar ataxia, Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Human Neuropathy (2019) 21:1311–1326 1 3 . These conditions include Scheinker's syndrome, fatal familial insomnia, kuru, amyotrophic lateral sclerosis, spinal muscular atrophy, and Batten disease, spinal cord injury, traumatic brain injury, neuropathic pain, multiple sclerosis, acute disseminated encephalomyelitis, Barro's disease, Charcot-Marie-Tooth disease, Guillain-Barré syndrome, HTLV-I-associated myelopathy, neuromyelitis optica, optic nerve atrophy, non-arteritic anterior ischemic optic neuropathy, Schilder's disease, transverse myelitis, stroke, epilepsy, and diabetic neuropathy.

[0146] Particularly preferred cardiovascular diseases prevented or treated by the polypeptides or polypeptide multimers of the invention include, but are not limited to, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, mixed dyslipidemia, coronary heart disease, atherosclerosis, peripheral vascular disease, cardiomyopathy, vasculitis, inflammatory heart disease, ischemic heart disease, congestive heart failure, valvular heart disease, hypertension, myocardial infarction, diabetic heart disease, embolism, aneurysm, hypertensive heart disease, pseudoaneurysm, stroke, and arrhythmias.

[0147] Particularly preferred metabolic diseases prevented or treated by the polypeptides or polypeptide multimers of the present invention include, but are not limited to, diabetes, obesity, metabolic syndrome, and insulin resistance. EXAMPLES

[0148] Example 1: Genetic Engineering of Proteins of the Invention Human TNFR2-selective TNF (TNF R2) mutant domain was designed from the ectodomain of human TNF containing the mutations D143N / A145R, restricting the biological activity of the molecule to TNF receptor 2, to show exclusive specificity for TNFR2 (Loetscher et al., 1993, J. Biol. Chem. 268, 26350-26357). The TNFR2-selective TNF (TNF R2 The mutant domains are characterized by different lengths due to variable N-terminal start positions. In particular, the proteins of the invention contain human TNFα domains having N-terminal start positions at aa residues 80, 82, 84, 85, 86 and 88, respectively, and ending at aa 233 (C-terminus). R2 The mutant domains are included (see Table 1; human TNF sequence from UniPRotKB entry P01375). Single-chain derivatives of these domains (scTNF R2 ) and three TNF R2 The mutant domains were generated by fusing them into one polypeptide chain. The gene fusion was achieved either by linking the three TNF domains using two peptide linkers or by fusing the TNF domains directly without a peptide linker. The TNF domains at aa position 80 were combined with the glycine-serine peptide linker L1 (GGGGS) (SEQ ID NO: 26) consisting of 5 aa residues, as present in the reference molecule 118. R2 The N-terminal start position of the mutant was considered to be the leading edge (Fischer et al., 2011, PLoS One, e27621). R2 The mutant TNF domains were fused to a peptide linker L1 consisting of GGGGS (SEQ ID NO:26) (starting at aa 80 of TNF, variant 118 [SEQ ID NO:65]), GGGG (SEQ ID NO:16) (starting at aa 85 of TNF, variant 139 [SEQ ID NO:68]), or GGGSGGGS (SEQ ID NO:34) (starting at aa 88 of TNF, variant 138 [SEQ ID NO:69]). In contrast, scTNF R2In variants 127 [SEQ ID NO: 66], 130 [SEQ ID NO: 70], 129 [SEQ ID NO: 67] and 131 [SEQ ID NO: 71], three TNFR2-specific TNF domains of various lengths (see Table 1, FIG. 1) were directly fused without a connecting peptide linker (variant 127 starting at aa 80 of TNF, variant 130 starting at aa 82 of TNF, variant 129 starting at aa 84 of TNF, variant 131 starting at aa 86 of TNF). R2 The mutant exhibits a trivalent arrangement of the three TNF THDs (ie, forming three TNFR2 binding sites).

[0149] Cutting edge scTNF R2 Mutant variant 118 and selected scTNF R2 Mutant variants (127, 129, 139) were linked to the N-terminus of the Fc(Δab) dimerization region via a peptide linker L2 consisting of GGSGGGGSGG (SEQ ID NO:5) (the Fc region contains mutations for the deletion of Fc effector functions, such as binding to Fcγ receptors and complement component C1; Armour et al., 1999, Eur. J. Immunol. 29, 2613-2624). These hexavalent fusion proteins (i.e., proteins displaying six TNFR2 binding sites) were expressed as scTNFα. R2 (118)-Fc(Δab)(745) [SEQ ID NO: 72], scTNF R2 (127)-Fc(Δab)(742) [SEQ ID NO: 73], scTNF R2 (129)-Fc(Δab)(743) [SEQ ID NO: 74], and scTNF R2 This is designated (139)-Fc(Δab)(744) [SEQ ID NO: 75] (see Table 2, FIG. 1).

[0150] scTNF R2 The overall codon usage of all the complexes was adapted for expression in mammalian cells. To facilitate secretion of the protein into the supernatant, an Igκ leader sequence was fused to the N-terminus of the construct. To facilitate protein purification, scTNF R2The mutants were tagged with His at the N-terminus, but R2 -Fc(Δab) complex. R2 Mutant and scTNF R2 The coding DNA sequence of the -Fc(Δab) mutant was cloned into a mammalian expression vector and expressed as a single 6×His-tagged single-chain protein, 6×His-scTNF R2 (pTT5 vector) or untagged Fc fusion protein scTNF R2 -Fc(Δab) (pSecTag vector) to allow recombinant production. Table 1: State-of-the-art molecule scTNF R2 118 and the scTNF of the present invention R2 protein. [Table 1] Table 2: scTNF-Fc(Δab) fusion proteins containing the reference scTNF module (#118) and the scTNF of the invention R2 -Nomenclature of the Fc molecule. [Table 2]

[0151] Example 2: Production and purification of the protein of the invention All proteins (see Example 1) were produced in HEK293-6E cells (NRC-BRI) grown under shaking conditions at 37°C and 5% CO2 in F17 medium (Life Technologies), and the cells were transiently transfected with plasmid DNA using polyethyleneimine (Polysciences). The next day, 0.5% tryptone N1 (Organotechnie) was added to the cell culture, and the cells were cultivated for another 5 days. The supernatant was then collected, centrifuged cell-free, and the recombinant proteins were isolated therefrom.

[0152] 6×His-scTNF R2The mutants were purified by immobilized metal ion chromatography (IMAC). Briefly, the supernatant was batch-incubated with Ni-NTA agarose (Macherey-Nagel) on a roller mixer for 16 h at 4° C. and then collected on a chromatography column. Unbound proteins were removed using IMAC wash buffer (50 mM sodium phosphate buffer, pH 7.5). Bound proteins were eluted using IMAC elution buffer (50 mM sodium phosphate buffer, 250 mM imidazole, pH 7.5) and dialyzed (membrane cut-off 14 kDa, Roth) against PBS buffer (pH 7.4) overnight at 4° C.

[0153] ScTNF R2 -Fc(Δab) complexes were purified by Protein A affinity chromatography. The supernatants were batch incubated with Protein A Sepharose 4 Fast Flow (GE Healthcare) or Toyopearl AF-rProtein A-650F (Tosoh) on a roller mixer for 16 h at 4° C. and collected on a chromatography column. Unbound proteins were removed with PBS, pH 7.4. Bound proteins were eluted with Protein A elution buffer (100 mM glycine-HCl, pH 3.5), immediately neutralized by adding 1 M Tris-HCl, pH 9.0, and dialyzed overnight at 4° C. against PBS buffer (pH 7.4) (membrane cut-off 14 kDa, Roth). Table 3 shows examples of protein amounts of the proteins described in Example 1 obtained after the affinity chromatography step.

[0154] The dialyzed proteins were further purified by gel filtration (size exclusion chromatography). Protein preparations were separated on a Superdex 200 10 / 300 GL column (GE Healthcare) using an AKTA FPLC instrument (GE Healthcare) and eluted with PBS, pH 7.4. Protein concentrations were determined spectrophotometrically at 280 nm and calculated using the individual extinction coefficients.

[0155] Protein preparations were analyzed by SDS-PAGE followed by Coomassie staining (Figure 2). 5 μg or 3 μg of purified protein in Example 1 were denatured in Laemmli buffer (50 mM Tris pH 6.8, 4 M urea, 1% SDS, 15% glycerol, 0.01% bromophenol blue) under reducing conditions (with 5% 2-mercaptoethanol) and non-reducing conditions (without 2-mercaptoethanol) and separated by 10% or 12% SDS-PAGE. For protein visualization, SDS-PAGE gels were incubated in InstantBlue stain (Expedion). Table 3: scTNF after recombinant expression and affinity purification. R2 Mutant and scTNF R2 -Yield of Fc(Δab) molecules. [Table 3] 1) After Ni-NTA IMAC 2) After Protein A affinity chromatography

[0156] Example 3: Molecular integrity and purity of the protein of the invention under native conditions The purity and oligomerization state of the protein in Example 1 were further characterized by HPLC size exclusion chromatography (SEC). Approximately 20 μg of protein was applied to a SuperSW mAb HR, 7.8×300 mm column (Tosoh Bioscience) equilibrated with SEC buffer (0.1 M Na2HPO4 / NaH2PO4, 0.1 M Na2SO4, pH 6.7) and eluted at a flow rate of 0.5 ml / min. ScTNF R2 and the complex eluted as a single major peak at the expected size, indicating correct assembly and high purity of the protein (see Figure 3).

[0157] Example 4: Thermal stability of the protein of the present invention The thermal stability of the protein in Example 1 was analyzed by dynamic light scattering using a Malvern Zetasizer instrument. The protein was diluted to 150 μg / ml in PBS (total volume 1.1 ml) and transferred to a quartz cuvette. The previously reported reference variant scTNF R2 118 showed a denaturation (onset of aggregation) temperature of 62°C (see Figure 4, Table 4). Moreover, the initial partial denaturation of this protein was already observed at a temperature of 49°C. In contrast, the modified scTNF R2 Mutants 127, 129 and 139 showed significantly elevated melting temperatures of 67°C (127, 139) and 72°C (129), respectively. R2 The melting temperature of mutant 138 remained unchanged (62°C) compared to the reference variant, and the scTNF R2 Mutants 130 and 131 showed slightly reduced thermostability.

[0158] scTNF R2 The variants were fused to the Fc(Δab) region and then to the reference scTNF R2 Variant 745, which contains mutant 118, showed a melting point of 71°C (Figure 4, Table 5). Notably, the thermal stability of scTNF was improved. R2 scTNF containing mutants (127, 129, and 139) R2 -Fc(Δab) complexes 742, 743 and 744 showed significantly higher melting temperatures compared to variant 745. For example, scTNF R2 scTNF containing variant 127 R2 -Fc(Δab) variant 742 and scTNF R2 scTNF containing variant 139 R2 Both the -Fc(Δab) variant and the 744 variant showed a melting temperature of 74°C. R2 The higher thermostability of the mutants (e.g., variants 127 and 139) was consistent with the scTNF R2 -Fc(Δab) complex.

[0159] protein: scTNF R2Molecules 118, 127, 129, 130, 131, 138 and 139 were purified by Ni-NTA-IMAC and gel filtration and eluted in 1xPBS buffer (8 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4). Proteins were present in 1xPBS at the following concentrations: 230 μg / ml (118), 300 μg / ml (127), 480 μg / ml (129), 230 μg / ml (130), 260 μg / ml (131), 110 μg / ml (138) and 450 μg / ml (139).

[0160] scTNF R2 -Fc(Δab) fusion proteins 745, 742, 743 and 744 were purified by protein A affinity chromatography and gel filtration and eluted in 1x PBS buffer. Proteins were present in 1x PBS at the following concentrations: 800 μg / ml (745), 1200 μg / ml (742), 320 μg / ml (743) and 1700 μg / ml (744).

[0161] DLS measurement: For analysis of aggregation temperature by dynamic light scattering, proteins were diluted to a concentration of 150 μg / ml in calcium- and magnesium-free DPBS (Gibco, catalog number 14190144; 8.06 mM Na2HPO4x7H2O, 1.47 mM KH2PO4, 2.67 mM KCl, 137.9 mM NaCl, pH 7.0-7.3). Proteins 138 were analyzed undiluted. 1.1 ml of the diluted protein solution was filtered free of particles through an Acrodisc 13 mm syringe filter, 0.2 μm (Pall Corporation, part number 4602) previously equilibrated with 5x1 ml DPBS and transferred to a PCS8501 glass cuvette with a circular opening (Malvern Panalytical) previously washed with 1 M NaOH and extensively washed with deionized water and DPBS. The cuvette was then placed in the measurement chamber of a pre-heated Zetasizer Nano-ZS ZEN3600, serial number MAL501015 (Malvern Panalytical), controlled by Dispersion Technology software 5.00. Measurements were performed in manual mode with the following software settings: Material: Protein, RI 1.45; Absorption, 0.00 Dispersant: ICN PBS tablets; Temperature, 25°C; Viscosity, 0.8882cP; RI, 1.33 Cell type: PCS8501 Trend sequence: Start temperature, 25°C; End temperature, 85°C; Temperature interval, 1.0°C; No melting point check Size measurement: Equilibration time, 2 min; Number of measurements, 2; Delay between measurements, 0 sec; No optimization of measurement settings; Measurement time, automatic; Advanced, Positioning method automatic attenuation selection; Data processing, Analysis model multiple narrow modes (high resolution).

[0162] The average of the two kcps values ​​measured at each temperature was calculated and plotted against temperature using GraphPad Prism 4.0 (GraphPad Software Inc.). The aggregation temperature was calculated as the quotient kcps T / kcps (T-5) is defined as the temperature T at which the coefficient reaches at least 2.0. Table 4: scTNF determined by dynamic light scattering R2 Denaturation temperature of mutants. [Table 4] Table 5: scTNF determined by dynamic light scattering R2 -Denaturation temperature of the Fc(Δab) complex. [Table 5] Table 6: 6xHis-scTNF R2 118 DLS measurements [Table 6] Table 7: 6xHis-scTNF R2 127 DLS measurements [Table 7] Table 8: 6xHis-scTNF R2 129 DLS measurements [Table 8] Table 9: 6xHis-scTNF R2 130 DLS measurements [Table 9] Table 10: 6xHis-scTNF R2 131 DLS measurements [Table 10] Table 11: 6xHis-scTNF R2 138 DLS measurements [Table 11] Table 12: 6xHis-scTNF R2 139 DLS measurements [Table 12] Table 13: scTNF R2 DLS measurement of -Fc(Δab)745 [Table 13] Table 14: scTNF R2 DLS measurement of -Fc(Δab)742 [Table 14] Table 15: scTNF R2 DLS measurement of -Fc(Δab)743 [Table 15] Table 16: scTNF R2 DLS measurement of -Fc(Δab)744 [Table 16]

[0163] Example 5: scTNF-R2 coactivation on Kym-1 cells with and without TNF-R2 coactivation using 80M2 antibody R2 In vitro bioactivity of mutants scTNF R2 The basal bioactivity of the mutants was analyzed in an in vitro assay using Kym-1 cells. Stimulation of TNFR2 on Kym-1 leads to the expression of endogenous TNF, which induces cell apoptosis by activating signaling through TNFR1. Notably, pure trivalent scTNF R2has been shown to be largely inactive in activating TNFR2 and further requires TNFR2 cross-linking (e.g., by using the anti-TNFR2 antibody 80M2, which is itself a non-agonist) for bioactivity. For the experiments, 15,000 Kym-1 cells / well were seeded in 96-well plates, cultured at 37°C and 5% CO2 for 24 h, and incubated with serially diluted proteins in triplicate for an additional 24 h. Under TNFR2 cross-linking conditions, 1 μg / ml 80M2 antibody (Hycult Biotech) was added to the cells 30 min prior to the addition of duplicate titrated proteins. Cell viability was determined by crystal violet staining. Data were normalized to untreated controls and positive controls (1% TritonX-100). In combination with 80M2, all scTNF R2 The variants induced cell death in Kym-1 cells. However, some variants (e.g., 129) were more potent than the reference scTNF. R2 118 showed reduced activity compared to 118 (Figure 5, Table 17). Table 17: scTNF in combination with TNF-R2 cross-linking by antibody 80M2 R2 EC2 of biological activity of mutants on Kym-1 cells 50 value. [Table 17]

[0164] Example 6: scTNF R2 Binding of -Fc(Δab) complexes to immobilized TNF-R2 scTNF R2 Binding of TNFR2-Fc(Δab) complexes to TNFR2-Fc (etanercept) was analyzed by ELISA. 96-well ELISA plates were coated with 200 ng / well etanercept in coating buffer (0.1 M sodium carbonate, pH 9.5) overnight at 4° C., blocked with 2% skim milk in PBS (MPBS), and washed with washing buffer PBST (PBS, 0.05% Tween 20). R2The scTNFα-Fc(Δab) complexes were titrated in duplicate and incubated on the plates for 2 h at room temperature, followed by washing with PBST. Receptor-bound complexes were detected with mouse anti-huTNFα F6C5 (Novus, 1 μg / ml) and goat anti-mouse IgG(Fc)-HRP (Sigma-Aldrich, 1:10,000), followed by extensive washing with PBST, respectively. R2 -Fc(Δab) complexes have EC 50 The results showed dose-dependent binding to TNF-R2-Fc with the sigma-independent binding values ​​(Figure 6, Table 18). ScTNF R2 -Fc(Δab)742, 743 and 744 are state-of-the-art scTNF R2 scTNF containing molecule 118 R2 -Fc(Δab)745 variant showed binding behavior equivalent to that of the Fc(Δab)745 variant. Table 18: scTNF R2 EC binding of -Fc(Δab) complex to TNF-R2-Fc 50 Values ​​(mean ± SD, n = 3). [Table 18]

[0165] Example 7: scTNF on cells R2 -Fc(Δab) complex selectively binds to TNF-R2 scTNF R2 Binding of the -Fc(Δab) complex to mouse embryonic fibroblasts (MEFs) stably transfected with human TNFR1 (MEF-TNFR1) or human TNFR2 (MEF-TNFR2) was analyzed by flow cytometry (Krippner-Heidenreich et al., 2002, J. Biol. Chem. 277, 44155-44163). To this end, cells were trypsinized and washed once with ice-cold FACS buffer (PBS, 2% FBS, 0.05% sodium azide). 200,000 cells per sample were incubated with serially diluted scTNFα-Fc(Δab) complexes. R2The cells were incubated with -Fc(Δab) complex in 100 μl FACS buffer for 2 h at 4° C. Unbound proteins were then removed by two washing steps with FACS buffer and then detected with anti-human IgG (γ-chain specific)-R-phycoerythrin antibody (Sigma-Aldrich, 1:500) for 1 h at 4° C. After two final washing steps with FACS buffer, the cells were analyzed on a MACSQuant® Analyzer 10 equipped with a 585 / 40 nm filter. All scTNF R2 The -Fc(Δab) molecules (745, 742, 743 and 744) showed dose-dependent binding to MEF-TNFR2 (Figure 7, Table 19). However, the scTNF R2 -Fc(Δab) complexes 742 and 744 have an EC 50 Value) state-of-the-art scTNF R2 scTNF containing molecule 118 R2 - Same EC as Fc(Δab)745 variant 50 Values ​​indicate the molecule scTNF R2 -Fc(Δab)743 showed weaker binding (approximately 2.8-fold reduction) to MEF-TNFR2 (EC 50 Furthermore, scTNFα was also shown to inhibit MEF-TNFR1. R2 No binding of the -Fc(Δab) complex was detectable, indicating the selectivity of the molecule for TNFR2. Table 19: scTNF R2 EC binding of -Fc(Δab) complex to MEF-TNFR2-Fc 50 value. [Table 19]

[0166] Example 8: scTNF on Kym-1 cells R2 In vitro bioactivity of -Fc(Δab) complex scTNF R2The in vitro bioactivity of the -Fc(Δab) complex was analyzed on Kym-1 cells in an experimental set-up similar to that described in Example 5, except that TNFR2 cross-linking by addition of antibody 80M2 was omitted (hexavalent scTNF R2 -Fc(Δab) protein does not require TNFR2 cross-linking (Figure 8, Table 20). R2 -Fc(Δab)742 is a state-of-the-art scTNF R2 Reference molecule scTNF containing variant 118 R2 -Fc(Δab)745 showed the same biological activity. R2 -Fc(Δab)744 showed reduced (approximately 4-fold) biological activity. R2 The biological activity of -Fc(Δab)743 was similar to that of scTNF R2 -Fc(Δab)745 was significantly (approximately 228-fold) reduced compared to -Fc(Δab)745. These findings indicate that structural properties due to too short peptide linkers negatively affect protein folding and receptor activation. Table 20: scTNF against Kym-1 cells R2 EC of bioactivity of -Fc(Δab) complex 50 Values ​​(mean ± SD, n = 3). [Table 20]

[0167] Example 9: scTNF in HeLa-TNF-R2 cells R2 NF-κB activation by -Fc(Δab) complex scTNF R2In vitro NF-κB activation by -Fc(Δab) complexes was analyzed using a luciferase reporter assay in HeLa cells stably transfected with human TNF receptor 2 (HeLa-TNF-R2). For this, 15,000 cells were seeded per 96-well and cultured at 37 °C and 5% CO2. After 24 h, the medium (RPMI / 10% FBS, PenStrep) was changed and cells were transiently transfected with pNF-κB Luc firefly luciferase experimental reporter plasmid (66 ng / well) (Agilent Technologies) and pRL-TK Renilla luciferase control plasmid (33 ng / well) (Promega) using Lipofectamine 2000 (Thermo, 4 μl / 1 μg DNA). After 16 h of culture, transfected cells were incubated with duplicate titrated scTNF R2 HeLa-TNF-R2 cells were stimulated with NF-κB-Fc(Δab) protein. After 6 h of stimulation, the medium was replaced with phenol red-free RPMI / 5% FBS (35 μl / well) and luciferase activity was measured by the Dual-Glo® Luciferase Assay System (Promega) in combination with luminescence detection using a Spark® microplate reader (Tecan). pNF-κB-regulated firefly luciferase activity was normalized to the control Renilla luciferase activity in individual wells. NF-κB activity in HeLa-TNF-R2 cells was normalized to the control Renilla luciferase activity in individual wells. R2 The dose-response curve for scTNF-Fc(Δab) complex was sigmoidal (Fig. 9). R2 EC calculated from four independent experiments reflecting the bioactivity of -Fc(Δab) complexes 50 The values ​​(Table 21) showed no statistically significant differences for proteins 745, 742 and 744 (P>0.05, one-way ANOVA with Tukey's post-hoc test). However, scTNF R2 -Fc(Δab)743 showed a statistically significant (p<0.01) lower (approximately 95-fold reduced) bioactivity compared to the state-of-the-art protein 745 with respect to NF-κB activation. Table 21: scTNF R2EC20 of -Fc(Δab) complex for NF-κB activation in HeLa-TNF-R2 cells 50 Values ​​(mean ± SD, n = 3). [Table 21]

[0168] Example 10: scTNF in human plasma R2 -Fc(Δab) protein stability scTNF in human plasma after 3-day and 8-day incubation periods at 37°C R2 The stability of the -Fc(Δab) protein was evaluated by binding of protein samples to HeLa cells stably overexpressing TNF-R2 (HeLa-TNF-R2, Richter et al., 2012, Mol. Cell Biol. 32, 2515-2529). In particular, proteins at stock concentrations of 5.17 μM (745), 8.05 μM (742), 2.11 μM (743) and 11.25 μM (744) in 1xPBS (8 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4) were diluted to a concentration of 400 nM in PBS and incubated in 50% human plasma (final protein concentration 200 nM) at 37 °C for 0 days (control), 3 days or 8 days. After incubation, samples were stored at -80 °C and thawed before testing protein integrity by binding to HeLa-TNF-R2 in flow cytometry. For this, HeLa-TNF-R2 cells with a plate density of 50-70% were trypsinized and washed once with ice-cold FACS buffer (1x PBS, 2% FBS, 0.05% sodium azide). 150,000 cells per sample were incubated with scTNF, serially diluted 1:3 with FACS buffer starting at 30 nM. R2The cells were incubated with -Fc(Δab) protein in 100 μl FACS buffer in a V-bottom 96-well plate for 1.5 h at 4 °C. Unbound proteins were then removed by two washing steps with FACS buffer. Thus, the V-bottom 96-well plate was centrifuged at 500 × g and the liquid in the wells was removed with an aspirator. 180 μl of FACS buffer was added per well and the cell pellet was resuspended by gently pipetting up and down. Bound proteins were detected with a goat anti-human IgG Fcγ fragment-specific-R-phycoerythrin antibody (Jackson ImmunoResearch, 1:500) for 1 h at 4 °C. After two final washing steps with FACS buffer, the cells were analyzed on a MACSQuant® VYB flow cytometer equipped with a 586 / 15 nm filter for detection of phycoerythrin. Binding curves were fitted using GraphPad Prism and EC values ​​of binding were calculated from four independent experiments. 50 Values ​​were calculated. EC 50 The percentage of intact protein was calculated for each single experiment from the reciprocal of the values ​​obtained by normalization.

[0169] scTNF R2 No reduction in the amount of intact protein was observed for -Fc(Δab) proteins 742 and 744, whereas the protein scTNF R2 -Fc(Δab) protein 745 showed a slight decrease in residual activity to approximately 80% after 3 and 8 days. In contrast, scTNF R2 -Fc(Δab)743 showed a large time-dependent decline in intact protein with approximately 40% remaining after 8 days. These data confirm that variants 742 and 744 exhibit statistically significantly improved stability under physiological conditions compared to the parent version 745 and engineered version 743. Table 22: scTNF incubated in 50% human plasma R2 EC binding of -Fc(Δab) protein to HeLa-TNF-R2 cells 50 Value (pM). [Table 22]

[0170] Example 11: Further variants of the proteins of the invention Variable N-terminal start position (X N ) and linker X L Further human scTNF derivatives selective for TNFR2 were generated that are characterized by different linker compositions with different lengths. In detail, the proteins contain human TNFR2 variant domains with N-terminal initiation at aa residues 81, 82, 83 and 84, respectively, and ending at aa 233 (C-terminus) (see Tables 1 and 2; human TNF sequence from UniPRotKB entry P01375). Single chain derivatives of these domains (scTNF R2 ) was generated by fusing three TNFR2 mutant domains onto one polypeptide chain. This gene fusion was achieved either by linking the three TNF domains with two peptide linkers or by fusing the TNF domains directly without a peptide linker. R2 The mutant TNF domains were either fused directly (variant 140, SEQ ID NO: 76) or with a peptide linker consisting of one glycine (variant 141, SEQ ID NO: 77; variant 142, SEQ ID NO: 78), two glycines (variant 144, SEQ ID NO: 80 variant 145, SEQ ID NO: 81), three glycines (variant 143, SEQ ID NO: 79; variant 146, SEQ ID NO: 82) or four glycines (variant 147, SEQ ID NO: 83) (Table 23). R2 The mutant exhibits a trivalent arrangement of the three TNF THDs (ie, forming three TNFR2 binding sites).

[0171] In addition, scTNF R2 The mutant variants were linked to the N-terminus of the Fc(Δab) dimerization region via a peptide linker L2 consisting of GGSGGGGSGG (SEQ ID NO: 92) to form the hexavalent scTNF R2TNFR2 binding sites were expressed in the form of scTNF-Fc fusion proteins (the Fc region contains mutations for the deletion of Fc effector functions, such as binding to Fcγ receptors and complement component C1; Armour et al., 1999, Eur. J. Immunol. 29, 2613-2624). These hexavalent fusion proteins (i.e., proteins displaying six TNFR2 binding sites) were then expressed in the form of scTNF-Fc fusion proteins (the Fc region contains mutations for the deletion of Fc effector functions, such as binding to Fcγ receptors and complement component C1; Armour et al., 1999, Eur. J. Immunol. 29, 2613-2624). R2 (140)-Fc(Δab) (protein 148, SEQ ID NO: 84), scTNF R2 (141)-Fc(Δab) (protein 149, SEQ ID NO: 85), scTNF R2 (143)-Fc(Δab) (protein 151, SEQ ID NO: 87), scTNF R2 (144)-Fc(Δab) (protein 152, SEQ ID NO: 88), and scTNF R2 This is designated (145)-Fc(Δab) (protein 153, SEQ ID NO: 89) (Table 24).

[0172] scTNF R2 The overall codon usage of all Fc fusion proteins was adapted for expression in mammalian cells. To facilitate secretion of the proteins into the supernatant, an Igκ leader sequence was fused to the N-terminus of the construct. To facilitate protein purification, scTNF R2 The mutants were tagged with His at the N-terminus, but R2 -Fc(Δab) fusion protein. R2 Mutant and scTNF R2 The coding DNA sequence of the -Fc(Δab) mutant was cloned into a mammalian expression vector and expressed as a single 6×His-tagged single-chain protein, 6×His-scTNF R2 (pTT5 vector) or untagged Fc fusion protein scTNF R2 -Fc(Δab) (pSecTag vector) to allow recombinant production. Table 23: scTNF of Example 11 R2 variant. [Table 23] Table 24: scTNF of Example 11 R2 -Fc variants [Table 24]

[0173] Example 12: Production and purification of the protein of Example 11 All proteins in Example 11 were produced in HEK293-6E cells (NRC-BRI) grown under shaking conditions at 37°C and 5% CO2 in F17 medium (Life Technologies), and the cells were transiently transfected with plasmid DNA using polyethyleneimine (Polysciences). The next day, 0.5% tryptone N1 (Organotechnie) was added to the cell culture, and the cells were cultured for another 5 days. The supernatant was then collected and centrifuged cell-free, from which recombinant proteins were isolated.

[0174] 6×His-scTNF R2 The mutants were purified by immobilized metal ion chromatography (IMAC). Briefly, the supernatant was batch-incubated with Ni-NTA agarose (Macherey-Nagel) on a roller mixer for 16 h at 4° C. and then collected on a chromatography column. Unbound proteins were removed using IMAC wash buffer (50 mM sodium phosphate buffer, pH 7.5). Bound proteins were eluted using IMAC elution buffer (50 mM sodium phosphate buffer, 250 mM imidazole, pH 7.5) and dialyzed (membrane cut-off 14 kDa, Roth) against PBS buffer (pH 7.4) overnight at 4° C.

[0175] ScTNF R2The -Fc(Δab) fusion protein was purified by Protein A affinity chromatography. The supernatant was batch-incubated with Protein A Sepharose 4 Fast Flow (GE Healthcare) or Toyopearl AF-rProtein A-650F (Tosoh) on a roller mixer for 16 h at 4°C and collected on a chromatography column. Unbound proteins were removed with PBS, pH 7.4. Bound proteins were eluted with Protein A elution buffer (100 mM glycine-HCl, pH 3.5), immediately neutralized by adding 1 M Tris-HCl, pH 9.0, and dialyzed overnight at 4°C against PBS buffer (pH 7.4) (membrane cut-off 14 kDa, Roth).

[0176] The dialyzed proteins were further purified by preparative size-exclusion chromatography (SEC). Protein preparations were separated on a Superdex 200 10 / 300 GL column (GE Healthcare) using an AKTA FPLC instrument (GE Healthcare) and eluted with PBS, pH 7.4. Protein concentrations were determined spectrophotometrically at 280 nm and calculated using the individual extinction coefficients.

[0177] Protein preparations were analyzed by SDS-PAGE followed by Coomassie staining (Figures 11 and 12). 2.5 μg of purified protein in Example 11 was denatured in Laemmli buffer (50 mM Tris pH 6.8, 4 M urea, 1% SDS, 15% glycerol, 0.01% bromophenol blue) under reducing conditions (with 5% 2-mercaptoethanol) and non-reducing conditions (without 2-mercaptoethanol) and separated by 10% or 12% SDS-PAGE. For protein visualization, SDS-PAGE gels were incubated in InstantBlue stain (Expedion).

[0178] Example 13: Molecular integrity and purity of the protein of Example 11 under native conditions scTNF in Example 11 R2 The purity and oligomerization state of the -Fc(Δab) fusion protein were further characterized by analytical HPLC size-exclusion chromatography (SEC). Approximately 20 μg of protein was applied to a SuperSW mAb HR, 7.8×300 mm column (Tosoh Bioscience) equilibrated with SEC buffer (0.1 M Na2HPO4 / NaH2PO4, 0.1 M Na2SO4, pH 6.7) and eluted at a flow rate of 0.5 ml / min. The fusion protein eluted as a single peak at the expected size (with an apparent molecular weight of approximately 160 kDa), indicating the correct assembly and high purity of the protein (see FIG. 13).

[0179] Example 14: Thermal stability of the protein of Example 11 The thermal stability of the protein in Example 11 was analyzed by dynamic light scattering using a Malvern Zetasizer instrument.

[0180] scTNF R2 Molecules 140, 141, 142, 143, 144, 145, 146, and 147 were present in 1xPBS (8 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4) at the following concentrations: 370 μg / ml (140), 350 μg / ml (141), 450 μg / ml (142), 1.1 mg / ml (143), 430 μg / ml (144), 170 μg / ml (145), 350 μg / ml (146), and 470 μg / ml (147). R2 -Fc(Δab) fusion proteins 148, 149, 150, 151, 152, 153, 154, and 155 were present in 1x PBS at the following concentrations: 1.59 mg / ml (148), 2.35 mg / ml (149), 1.59 mg / ml (151), 1.59 mg / ml (152), and 220 μg / ml (153).

[0181] Proteins were diluted to 100 μg / ml in a total volume of 1.1 ml with calcium- and magnesium-free DPBS (Gibco, cat. no. 14190144; 8.06 mM Na2HPO4x7H2O, 1.47 mM KH2PO4, 2.67 mM KCl, 137.9 mM NaCl, pH 7.0-7.3), transferred to a quartz cuvette and analyzed by dynamic light scattering (DLS) as follows: 1.1 ml of the diluted protein solution was filtered free of particles through an Acrodisc 13 mm syringe filter, 0.2 μm (Pall Corporation, part number 4602) previously equilibrated with 5×1 ml DPBS and transferred to a PCS8501 glass cuvette with a circular opening (Malvern Panalytical) previously washed with 1 M NaOH and extensively washed with deionized water and DPBS. The cuvette was then placed in the measurement chamber of a pre-heated Zetasizer Nano-ZS ZEN3600, serial number MAL501015 (Malvern Panalytical), controlled by Dispersion Technology software 5.00. Measurements were performed in manual mode with the following software settings: Material: Protein, RI 1.45; Absorption, 0.00 Dispersant: ICN PBS tablets; Temperature, 25°C; Viscosity, 0.8882cP; RI, 1.33 Cell type: PCS8501 Trend sequence: Start temperature, 25°C; End temperature, 85°C; Temperature interval, 1.0°C; No melting point check Size measurement: equilibration time, 2 min; number of measurements, 2; delay between measurements, 0 sec; no optimization of measurement settings; measurement time, automatic; advanced positioning methods automatic attenuation selection; data processing, multiple limited modes of analysis models (high resolution).

[0182] The average of the two kcps values ​​measured at each temperature was calculated and plotted against temperature using GraphPad Prism8 (GraphPad Software Inc.). The aggregation temperature was calculated as the quotient kcps T / kcps(T-5) is defined as the temperature T at which the coefficient reaches at least 2.0.

[0183] Compared to the original variant 118 (see Figure 1), the modified scTNF R2 Variants 140, 141, 142, 143, 144, 145, 146 and 147 exhibited significantly elevated melting points of 67 °C (variants 145, 146), 68 °C (variants 141, 143, 144, 147), and 69 °C (variants 140, 142), respectively (see Figure 14, Tables 25-33).

[0184] scTNF with Xa length of 10 aa R2 The -Fc variants showed melting temperatures of 74 °C (variants 149, 152) and 75 °C (variant 154) (Figure 15, Tables 34-41), comparable to variant 742 (see Figure 1). Of note, the scTNF-Fc variants, whose Xa is 9 aa in length, R2 The -Fc(Δab) fusion proteins showed increased thermostability of 78° C. (variants 148, 153), 76° C. (variant 151) and 75° C. (variant 150). R2 The variants showed increased thermostability relative to variant 118 (Figure 1). R2 The thermal stability of the -Fc variants was significantly increased compared to variant 745 (Figure 1), with variants containing an Xa linker length of 9 aa being more stable than variants containing an Xa linker length of 10 aa. Table 25: scTNF determined by dynamic light scattering R2 Denaturation temperature of mutants. [Table 25] Table 26: 6xHis-scTNF R2 140 DLS measurements [Table 26-1] [Table 26-2] Table 27: 6xHis-scTNF R2 141 DLS measurements [Table 27-1] [Table 27-2] Table 28: 6xHis-scTNF R2 142 DLS measurements [Table 28-1] [Table 28-2] Table 29: 6xHis-scTNF R2 143 DLS measurements [Table 29-1] [Table 29-2] Table 30: 6xHis-scTNF R2 144 DLS measurements [Table 30-1] [Table 30-2] Table 31: 6xHis-scTNF R2 145 DLS measurements [Table 31-1] [Table 31-2] Table 32: 6xHis-scTNF R2 146 DLS measurements [Table 32-1] [Table 32-2] Table 33: 6xHis-scTNF R2 147 DLS measurements [Table 33-1] [Table 33-2] Table 34: scTNF determined by dynamic light scattering R2 - Denaturation temperature of the Fc protein. [Table 34] Table 35: scTNF R2 -DLS measurement of Fc148 [Table 35-1] [Table 35-2] Table 36: scTNF R2 -DLS measurement of Fc149 [Table 36-1] [Table 36-2] Table 37: scTNF R2 -DLS measurement of Fc150 [Table 37-1] [Table 37-2] Table 38: scTNF R2 -DLS measurement of Fc151 [Table 38-1] [Table 38-2] Table 39: scTNF R2 -DLS measurement of Fc152 [Table 39-1] [Table 39-2] Table 40: scTNF R2 -DLS measurement of Fc153 [Table 40-1] [Table 40-2] Table 41: scTNF R2 -DLS measurement of Fc154 [Table 41-1] [Table 41-2]

[0185] Example 15: scTNF R2 and scTNF R2 Binding of -Fc(Δab) protein to immobilized TNF-R2 scTNF R2 and scTNF R2 Binding of ScTNF-Fc(Δab) protein to human TNFR2-Fc (etanercept) was analyzed by ELISA. 96-well ELISA plates were coated with 200 ng / well etanercept in coating buffer (0.1 M sodium carbonate, pH 9.5) overnight at 4° C., blocked with 2% skim milk in PBS (MPBS) and washed with washing buffer PBST (PBS, 0.05% Tween 20). R2 and scTNF R2-Fc(Δab) proteins were titrated in duplicate and incubated on the plates for 2 h at room temperature followed by washing with PBST. Receptor-bound complexes were detected with mouse anti-huTNFα F6C5 (Novus, 1 μg / ml) and goat anti-mouse IgG(Fc)-HRP (Sigma-Aldrich, 1:10,000), followed by extensive washing with PBST, respectively, before incubation with HRP substrate.

[0186] scTNF R2 The variant has an EC 50 TNF with values R2 -Fc showed similar dose-dependent binding (Figure 16, Table 42). R2 -Fc(Δab) fusion proteins have a lower EC 50 Values ​​were shown (Figure 17, Table 43) indicating increased binding of the hexavalent Fc fusion protein due to an avidity effect. Table 42: scTNF R2 EC of variant binding to human TNFR2-Fc 50 value [Table 42] Table 43: scTNF R2 EC of binding of -Fc fusion protein variants to TNFR2-Fc 50 value [Table 43]

[0187] Example 16: scTNF of Example 11 on Kym-1 cells with TNF-R2 coactivation using 80M2 antibody R2 In vitro bioactivity of variants Example 11 scTNF R2The basal bioactivity of the variants was analyzed in an in vitro assay using Kym-1 cells. Stimulation of TNFR2 on Kym-1 leads to the expression of endogenous TNF, which induces cell apoptosis by activating signaling through TNFR1. Notably, pure trivalent scTNF R2 has been shown to be largely inactive in activating TNFR2 and further requires TNFR2 cross-linking (e.g., by using the anti-TNFR2 antibody 80M2, which is itself a non-agonist) for bioactivity. For the experiments, 10,000 Kym-1 cells / well were seeded in 96-well plates, cultured at 37°C and 5% CO2 for 24 h, and incubated with serially diluted proteins in triplicate for an additional 24 h. For TNFR2 cross-linking, 1 μg / ml 80M2 antibody (Hycult Biotech) was added to the cells 30 min prior to the addition of triplicate titrated proteins. Cell viability was determined by crystal violet staining. Data were normalized to untreated controls and positive controls (1% TritonX-100). In combination with 80M2, all scTNF R2 The variants induced cell death in Kym-1 cells in the sub-nanomolar concentration range (Figure 18, Table 44). Table 44: scTNF R2 EC of variants for bioactivity against Kym-1 50 value. [Table 44]

[0188] Example 17: scTNF of Example 11 on Kym-1 cells R2 In vitro biological activity of -Fc(Δab) protein Example 11 scTNF R2 The in vitro bioactivity of the -Fc(Δab) protein was analyzed on Kym-1 cells in an experimental set-up similar to that described in Example 16, except that TNFR2 cross-linking by addition of antibody 80M2 was omitted (hexavalent scTNF R2-Fc(Δab) protein does not require TNFR2 cross-linking (Figure 19, Table 45). R2 -Fc(Δab) variants 148, 149, 150, 151, 152, 153 and 154 are identical to the reference molecule scTNF R2 -scTNF-Fc(Δab)742 alone or in the presence of TNFR2 cross-linking antibody 80M2 R2 It showed similar biological activity to 140. Table 45: scTNF against Kym-1 R2 EC of bioactivity of -Fc mutants 50 value [Table 45]

[0189] The present invention also relates to the following items: 1. A polypeptide comprising a binding domain consisting of three peptide TNF homology domains (THDs) of a TNF ligand family member protein that specifically binds to the extracellular portion of TNFR2, wherein the C-terminus of the first and second THDs (which in each case are defined by the C-terminal consensus sequence VF / YFGA / I-X1 (SEQ ID NO:1)) is linked to the N-terminus of the second and third THDs (which in each case are defined by the N-terminal consensus sequence PV / AAHV / L (SEQ ID NO:2)) by peptide X a (which are independently selected in each case and have a length of 9 to 12 amino acids, preferably 9 to 11, more preferably 9 to 10), a does not contain the amino acid sequence SSRTPSDK (SEQ ID NO: 10), and X1 is a non-polar / hydrophobic or polar / neutral amino acid, preferably selected from the group consisting of F and I.

[0190] 2. Peptide X a X C -X L -X N 2. The polypeptide according to item 1, comprising XC is selected from the group consisting of A, AL, L, preferably A and AL; X L is absent or is an amino acid linker consisting of 1 to 11, preferably 1 to 10, more preferably 1 to 9 amino acids; X N is absent or is selected from the group consisting of K, DK, SDK, PSDK (SEQ ID NO:6), TPSDK (SEQ ID NO:7), RTPSDK (SEQ ID NO:8), SRTPSDK (SEQ ID NO:9), TK, STK, HSTK (SEQ ID NO:11), AHSTK (SEQ ID NO:12), LAHSTK (SEQ ID NO:13), HLAHSTK (SEQ ID NO:14), LHLAHSTK (SEQ ID NO:15).

[0191] 3. The polypeptide of any of the preceding claims, wherein the three THDs are identical.

[0192] 4. A polypeptide according to any of the preceding items, wherein the C-terminus of the first and second THD is defined in each case by the C-terminal sequence VYFGII (sequence number 3), and the N-terminus of the second and third THD is defined in each case by the N-terminal sequence PVAHV (sequence number 4).

[0193] 5. The polypeptide of any of the preceding items, wherein THD comprises a contiguous amino acid sequence consisting of amino acids 88 to 231 of SEQ ID NO:5, optionally comprising at least one mutation selected from the group consisting of: D143Y, D143F, D143E, D143N, D143T, D143S, E146Q, E146H, E146K, A145R / S147T, Q88N / T89S / A145S / E146A / S147D, Q88 N / A145I / E146G / S147D, A145H / E146S / S147D, A145H / S147D, L29V / A145D / E146D / S147D, A145N / E146D / S147D, A145T / E146S / S147D, A145Q / E146D / S147D, A145T / E146D / S147D, A145D / E146G / S147D, A145D / S147D, A145K / E146D / S147T , A145R / E146T / S147D, A145R / S147T, E146D / S147D, E146N / S147, S95C / G148C, K65A, K65W, Q67K, Q67T, Q67Y, L75H, L 75W, D143W, D143V, D143V / F144L / A145S, D143N / A145R, D143V / A145S, L29V, L29T, L29S, L29A, L29G, R31H, R31I, R31 L, R32G, R32E, S147L, S147R, S147P, S147T, S147A, Q149E, Q149N, E146D, E146N, E146S, E146G, A145R, A145S, A145T, A145H, A145K, A145F, A145D, A145G, A145N, A145P, A145Q, A145Y, A145V, and A145W (preferably selected from D143N and A145R).

[0194] 6.X C is selected from A or AL, X Lis absent or is a glycine and / or serine linker having a length of 1 to 11 amino acids, preferably G, S, GG, SG, GS, GGG, SGG, GSG, GGS, GGGG (SEQ ID NO: 16), GGGS (SEQ ID NO: 17), GGSG (SEQ ID NO: 18), GSGG (SEQ ID NO: 19), SGGG (SEQ ID NO: 20), GGGGG (SEQ ID NO: 21), SGGGG (SEQ ID NO: 22), GSGGG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GGGSG (SEQ ID NO: 25), GGGGS (SEQ ID NO: 26), GGGGGG (SEQ ID NO: 27), SGGGGG (SEQ ID NO: 28), GSGGGG (SEQ ID NO: 29), GGSGGG (SEQ ID NO: 30), GGGSGG (SEQ ID NO: 31), GGGGSG (SEQ ID NO: 32), GGGGGS (SEQ ID NO: 33), GGGSGGGS (SEQ ID NO: 34), SGG GSGGG (SEQ ID NO:35), GGGGGGGG (SEQ ID NO:36), GSGGGSGG (SEQ ID NO:37), GGSGGGSG (SEQ ID NO:38), SGGGSGGGS (SEQ ID NO:39), GSGGGGSGG (SEQ ID NO:40), GGSGGGSGG (SEQ ID NO:41), GGGSGGGSG (SEQ ID NO:42), SGGGSGGGSG (SEQ ID NO:43), GSGGGSGGGS (SEQ ID NO:44), GGSGGGSGGG (SEQ ID NO:45), GGGSGGGSGG (SEQ ID NO:46), SGGGSGGGSGG (SEQ ID NO:47), GSGGGSGGGSSG (SEQ ID NO:48), GGSGGGSGGGS (SEQ ID NO:49), and GGGSGGSGGG (SEQ ID NO:50), more preferably selected from GGGG (SEQ ID NO:16), GGGGS (SEQ ID NO:26), and GGGSGGGS (SEQ ID NO:34); X Nis absent or is selected from K, DK, SDK, PSDK (SEQ ID NO:6), TPSDK (SEQ ID NO:7), RTPSDK (SEQ ID NO:8), and SRTPSDK (SEQ ID NO:9).

[0195] 7.(i)X C is AL and X L does not exist and X N is selected from SRTPSDK (SEQ ID NO:9); (ii) X C is AL and X L is GGGG (SEQ ID NO: 16), and X N is selected from SDK, PSDK (SEQ ID NO: 6), TPSDK (SEQ ID NO: 7), RTPSDK (SEQ ID NO: 8), preferably PSDK (SEQ ID NO: 6); (iii) X C is AL and X L is GGGSGGGS (SEQ ID NO: 34), and X N is selected from K and DK; (iv) X C is AL and X L is G and X N is selected from RTPSDK (SEQ ID NO: 8), SRTPSDK (SEQ ID NO: 9), preferably SRTPSDK (SEQ ID NO: 9); (v) X C is AL and X L is GG and X N is selected from TPSDK (SEQ ID NO: 7), RTPSDK (SEQ ID NO: 8), SRTPSDK (SEQ ID NO: 9), preferably RTPSDK (SEQ ID NO: 8); or (vi) X C is AL and X L is GGG and X N5. The polypeptide of any of the preceding items, wherein is selected from PSDK (SEQ ID NO: 6), TPSDK (SEQ ID NO: 7), RTPSDK (SEQ ID NO: 8), SRTPSDK (SEQ ID NO: 9), preferably TPSDK (SEQ ID NO: 7).

[0196] 8. Aggregation onset temperature (T) greater than 62 °C as determined by dynamic light scattering m 2. The polypeptide of any of the preceding claims, having the following structure:

[0197] 9. A polypeptide multimer comprising at least two polypeptides according to any of the preceding claims, (a) linked together, preferably linked together by an amino acid linker having a length of 1 to 30 amino acids, preferably 7 to 15 amino acids; or (b) is linked to a protein (preferably selected from the group consisting of a multimerization domain, a serum protein, a cytokine, a targeting moiety or a toxin), optionally wherein the polypeptide is linked to the protein by an amino acid linker having a length of 1 to 30 amino acids, preferably 7 to 15 amino acids.

[0198] 10.A. The polypeptide multimer has at least one of the following properties: An aggregation onset temperature (T m ); The EC of binding to TNFR2 in HeLa-TNF-R2 cells is not decreased by more than 15%, 12%, 10%, preferably 10%, after 8 days of incubation in human plasma at 37° C. 50 ; EC for binding to TNFR2 expressed on MEFs less than 100 pM, preferably less than 80 pM 50 ; EC of binding to TNFR2 on Kym-1 cells less than 200 pM, preferably less than 100 pM 50 ; EC for activation of NF-κB in HeLa-TNF-R2 cells less than 30 pM, preferably less than 10 pM 50 ; and / or B. The multimerization domain is a dimerization domain, a trimerization domain or a tetramerization domain, preferably (i) the dimerization domain is selected from the group consisting of an antibody, an antibody heavy chain, an immunoglobulin Fc region, heavy chain domain 2 (CH2) of IgM (MHD2), heavy chain domain 2 (CH2) of IgE (EHD2), heavy chain domain 3 (CH3) of IgG, heavy chain domain 3 (CH3) of IgA, heavy chain domain 3 (CH3) of IgD, heavy chain domain 4 (CH4) of IgM, heavy chain domain 4 (CH4) of IgE, Fab, Fab2, leucine zipper motif, barnase-barstar dimer, miniantibody and ZIP miniantibody, preferably an immunoglobulin Fc region variant without FcR and / or C1q binding, more preferably FcΔab, LALA, LALA-GP, IgG2, IgG2σ, aglycosylated IgG1, IgG1(L234F / L235E / LP331S), IgG2m4, IgG4 ProAlaAla, most preferably FcΔab; (ii) the trimerization domain is selected from the group consisting of tenascin-C (TNC), the trimerization region of the C-terminal non-collagenous domain of collagen XVIII (NC1), a Fab3-like molecule, and a TriBi minibody; or (iii) the tetramerization domain is selected from the group consisting of a tetramerization domain of p53, a tetramerization domain of general control protein 4 (GCN4), a tetramerization domain of VASP (vasodilator-stimulated phosphoprotein), a tandem bispecific antibody, and a di-diabody; and / or C. The polypeptide multimer of paragraph 9, wherein the polypeptide multimer further comprises a targeting moiety that binds to an organ, tissue or cell type specific ligand, more preferably a target selected from the group consisting of transferrin receptor, insulin receptor, low density lipoprotein receptor (LDLR), diphtheria toxin receptor, efflux pumps, CD25, CD28, GLUT1, LAT1, TMEM119, PDGFR, VEGFR1, VEGFR3, and RVG-29 receptors.

[0199] 11. A nucleic acid molecule encoding the polypeptide according to any one of items 1 to 8 or the polypeptide multimer according to item 9 or 10.

[0200] 12. A vector encoding the nucleic acid molecule according to item 11.

[0201] 13. A polypeptide according to any one of items 1 to 8, a polypeptide multimer according to item 9 or 10, a nucleic acid according to item 11, or a vector according to item 12, for use as a pharmaceutical.

[0202] 14. A pharmaceutical composition comprising as an active substance the polypeptide according to any one of items 1 to 8, the polypeptide multimer according to item 9 or 10, the nucleic acid according to item 11, or the vector according to item 12.

[0203] 15. The polypeptide according to any one of items 1 to 8, the polypeptide multimer according to item 9 or 10, the nucleic acid according to item 11, the vector according to item 12, or the pharmaceutical composition according to item 14 for use in the diagnosis, prevention or treatment of a hyperproliferative disorder, an inflammatory disorder, a neurodegenerative disease or a metabolic disorder (preferably cancer or a hematological malignancy), an autoimmune disorder, a metabolic syndrome, a cardiovascular disease, a neuropathic disease, and a nerve injury.

Claims

1. A polypeptide comprising a binding domain consisting of three peptide TNF homology domains (THDs) of a TNF ligand family member protein which specifically binds to the extracellular portion of TNFR2, wherein the C-terminus of the first and second THDs (which in each case are defined by the C-terminal sequence V-Y-F-G-I-I (SEQ ID NO:3)) is linked to the N-terminus of the second and third THDs (which in each case are defined by the N-terminal sequence P-V-A-H-V (SEQ ID NO:4)) and the N-terminus of the second and third THDs (which in each case are defined by the N-terminal sequence P-V-A-H-V (SEQ ID NO:4)). ａ (which are independently selected in each case and have a length of 9 to 10 amino acids), wherein peptide X a consists of X C -X L -X N , Xc is A-L; X L is absent or selected from the group consisting of G, GG, GGG, and GGGGG (SEQ ID NO:16); X N is selected from the group consisting of P-S-D-K (SEQ ID NO:6), T-P-S-D-K (SEQ ID NO:7), R-T-P-S-D-K (SEQ ID NO:8) and S-R-T-P-S-D-K (SEQ ID NO:9); wherein the THD comprises a contiguous amino acid sequence consisting of amino acids 88 to 231 of SEQ ID NO:5, and includes the mutations D143N and A145R.

2. The polypeptide of claim 1 , wherein the three THDs are identical.

3. (i) X Ｌ does not exist, and X Ｎ is S-RT-P-S-D-K (SEQ ID NO: 9); (ii) X Ｌ is G-G-G-G (SEQ ID NO: 16), and X Ｎ is P-S-D-K (SEQ ID NO:6); (iii) X L is G and X N is selected from R-T-P-S-D-K (SEQ ID NO:8) and S-R-T-P-S-D-K (SEQ ID NO:9); (iv) X L is GG and X N is selected from TPSDK (SEQ ID NO:7) and RTPSDK (SEQ ID NO:8); or (v) The polypeptide of claim 1 or 2, wherein X L is GGG and X N is selected from P-S-D-K (SEQ ID NO: 6) and T-P-S-D-K (SEQ ID NO: 7).

4. Aggregation onset temperature (T) greater than 62° C. as determined by dynamic light scattering ｍ The polypeptide according to any one of claims 1 to 3,

5. A polypeptide described in any one of claims 1 to 4, wherein the mutations D143N and A145R are D219N and A221R, respectively, relative to sequence number 5.

6. A polypeptide multimer comprising at least two polypeptides according to any one of claims 1 to 5, wherein: (a) linked together; or (b) a polypeptide multimer linked to a protein selected from the group consisting of a multimerization domain, a serum protein, a cytokine, a targeting moiety, and a toxin.

7. The polypeptide, (a) are linked together by an amino acid linker having a length of 1 to 30 amino acids, or 7 to 15 amino acids; or (b) the polypeptide multimer of claim 6, which is linked to the protein by an amino acid linker having a length of 1 to 30 amino acids.

8. A. The polypeptide multimer has at least one of the following properties: Agglomeration onset temperature (T ｍ ). The EC50 value for binding to TNFR2 in HeLa-TNF-R2 cells was not reduced by more than 15%, 12%, or 10% after 8 days of incubation in human plasma at 37°C. ５０ ; EC50 for binding to TNFR2 expressed on MEFs less than 100 pM ５０ ; EC for binding to TNFR2 on Kym-1 cells less than 200 pM ５０ ; EC of NF-κB activation in HeLa-TNF-R2 cells was less than 30 pM. ５０ ; and / or B. The polypeptide multimer further comprises an organ, tissue or cell type specific ligand; A polypeptide multimer according to claim 6 or 7.

9. A polypeptide multimer described in claim 6 or 7, wherein the multimerization domain is a dimerization domain, a trimerization domain or a tetramerization domain.

10. The polypeptide multimer of claim 9, wherein the dimerization domain is selected from the group consisting of an antibody, an antibody heavy chain, an immunoglobulin Fc region, heavy chain domain 2 (CH2) of IgM (MHD2), heavy chain domain 2 (CH2) of IgE (EHD2), heavy chain domain 3 (CH3) of IgG, heavy chain domain 3 (CH3) of IgA, heavy chain domain 3 (CH3) of IgD, heavy chain domain 4 (CH4) of IgM, heavy chain domain 4 (CH4) of IgE, Fab, Fab2, leucine zipper motif, barnase-barstar dimer, miniantibody and ZIP miniantibody. (i) the trimerization domain is selected from the group consisting of tenascin-C (TNC), the trimerization region of the C-terminal non-collagenous domain (NC1) of collagen XVIII, a Fab3-like molecule, and a TriBi minibody; or (ii) the tetramerization domain is selected from the group consisting of a tetramerization domain of p53, a tetramerization domain of general control protein 4 (GCN4), a tetramerization domain of VASP (vasodilator stimulated phosphoprotein), a tandem bispecific antibody, and a di-diabody; The polypeptide multimer according to claim 9.

12. The polypeptide multimer of claim 10, wherein the dimerization domain is selected from the group consisting of FcΔab, LALA, LALA-GP, IgG2, IgG2σ, aglycosylated IgG1, IgG1(L234F / L235E / LP331S), IgG2m4, and IgG4 ProAlaAla.

13. A nucleic acid molecule encoding the polypeptide according to any one of claims 1 to 5 or the polypeptide multimer according to any one of claims 6 to 12.

14. A vector comprising the nucleic acid molecule of claim 13.

15. A polypeptide according to any one of claims 1 to 5, a polypeptide multimer according to any one of claims 6 to 12, a nucleic acid molecule according to claim 13, or a vector according to claim 14, for use as a pharmaceutical.

16. A pharmaceutical composition comprising as an active substance the polypeptide according to any one of claims 1 to 5, the polypeptide multimer according to any one of claims 6 to 12, the nucleic acid molecule according to claim 13, or the vector according to claim 14.

17. A polypeptide according to any one of claims 1 to 5, a polypeptide multimer according to any one of claims 6 to 12, a nucleic acid molecule according to claim 13, a vector according to claim 14, or a pharmaceutical composition according to claim 16, for use in the diagnosis, prevention or treatment of a hyperproliferative disorder, an inflammatory disorder, a neurodegenerative or metabolic disorder, an autoimmune disorder, a metabolic syndrome, a cardiovascular disease, a neuropathic disease, and a neurological injury.

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