Synthetic cytokines derived from the il-6 family and their medical use

Abstract

The present invention is concerned with synthetic cytokines derived from the IL-6 family. The synthetic cytokines are polypeptides comprising two binding sites derived from the same cytokine of the IL-6 family, and a third binding site, derived from a further cytokine of the IL-6 family, wherein said further cytokine does not require a cytokine specific nonsignaling α-receptor subunit as part of the receptor complex to effect signaling. To effect signaling, all three binding sites of the polypeptide of the present invention need to bind their specific partners. The invention further relates to the polypeptides for use in medical applications, preferably for preventing and / or treating a condition selected from the group consisting of lymphopenia, muscular atrophy, osteoporosis, thrombocytopenia, obesity associated metabolic disorders such as type II diabetes mellitus, obesity, insulin resistance, glucose intolerance, dyslipidemia, hypertension, stroke or cardiovascular disease, neurological disorders such as paraplegia, Alzheimer's and Parkinson's disease.

Description

[0001] SYNTHETIC CYTOKINES DERIVED FROM THE IL-6 FAMILY AND THEIR MEDICAL USE

[0002] TECHNICAL FIELD

[0003] The present invention is related to the field of biomedical science and provides synthetic cytokines derived from the IL-6 family. The synthetic cytokines can be used in medical applications, preferably for preventing and / or treating a condition selected from the group consisting of lymphopenia, muscular atrophy, osteoporosis, thrombocytopenia, obesity associated metabolic disorders such as type II diabetes mellitus, obesity, insulin resistance, glucose intolerance, dyslipidemia, hypertension, stroke or cardiovascular disease, neurological disorders such as paraplegia, Alzheimer's and Parkinson's disease.

[0004] BACKGROUND OF THE INVENTON

[0005] Cytokines are a group of pharmacologically active, low molecular weight polypeptides that play an important role in the communication between cells of multicellular organisms. As intercellular mediators they regulate survival, growth, differentiation and effector functions of cells. They are key players in the regulation of the immune response, particularly during infections, inflammatory joint, kidney, vessel and bowel diseases, or neurological and endocrinological autoimmune diseases.

[0006] Typical for cytokines is their pleiotropism, which means that the cytokines act on many different target cells and frequently affect the action of other cytokines in an additive, synergistic or antagonistic manner. Besides their pleiotropic effects, cytokine actions are often redundant, i.e. similar biological responses can be achieved by several different cytokines. They exert their actions via specific cell surface receptors on their target cells. Cytokines are produced by a wide variety of cells in the body, playing an important role in many physiological responses that have a therapeutic potential.

[0007] Cytokines have, inter alia, been classified according to their three-dimensional structures. Despite the lack of amino-acid-sequence similarities, a family of cytokines has been characterized by a long-chain four-a-helix bundle structure. This family comprises interleukin (IL)-6, IL-11 , leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin (CT)-1 , erythropoietin (Epo), granulocyte colony- stimulating factor (G-CSF), IL-12, growth hormone, prolactin, IL-10, interferon (IFN) a / p, and leptin.

[0008] Among these cytokines IL-6, IL-11 , LIF, OSM, CNTF and CT-1 use the common receptor subunit gp130 for signal transduction and as a consequence elicit similar and overlapping physiological responses. Therefore this group of cytokines has been termed as ‘IL-6-type cytokines’ or as cytokines of the IL-6 family or as members of the IL-6 family of cytokines (see e.g. Heinrich, P.C., et al., lnterleukin-6-type cytokine signalling through the gp130 / Jak / STAT pathway. The Biochemical journal, 1998. 334 (Pt 2): p. 297-314).

[0009] Some IL-6 family cytokines are known to effect their signal transduction via at least two different mechanisms, namely classic signaling and trans-signaling (see e.g. Rose-John S. Interleukin-6 Family Cytokines. Cold Spring Harb Perspect Biol. 2018 Feb 1 ;10(2):a028415. doi: 10.1101 / cshperspect.a028415. PMID: 28620096; PMCID: PMC5793756).

[0010] Classic signaling occurs upon binding of the cytokine to its membrane bound receptor. In a first step the cytokine binds to its cytokine specific nonsignaling a-receptor subunit. For example, IL-6 binds to IL-6R. IL-6R is only present on a subset of cells. Upon this binding, the signaling receptors, also called p-receptors, such as gp130, are recruited, leading to signal transduction into the interior of the cell. Classic signaling thus only occurs when a cell comprises all necessary components of the signal receptor complex.

[0011] Trans-signaling however is not limited to cytokine / cytokine receptor interaction occurring on a cell surface. It is known that either by alternative splicing or by cleavage from the cell surface, soluble cytokine receptors can interact with the cytokine. This soluble cytokine / cytokine receptor complex can then interact with the called p-receptors, such as gp130, which is ubiquitously expressed. The signal transduction is in this case no longer dependent on a cell having the correct combination of components of the cytokine receptor complex. The presence of these different ways of effecting signal transduction makes it more difficult to control or predict the response to a cytokine.

[0012] IL-6 family cytokines have been implicated in many functions, including B-cell stimulation and induction of the hepatic acute phase proteins. Moreover, metabolic functions and neurotrophic functions have been ascribed to this group of cytokines. Therefore, the IL-6 family of cytokines are involved in the development and / or progression of many different conditions or diseases (see e.g. Garbers, C. and J. Scheller, Interleukin- 6 and interleukin-11: same same but different. Biological chemistry, 2013; 394(9): 1145— 1161).

[0013] IL-6 for example is a central mediator of cellular communication and is involved in the regulation of inflammatory responses as well as in the coordination of developmental, neuronal, and metabolic processes. In hepatocytes, IL-6 is a major mediator of the acutephase response. Due to this crucial role in inflammation, dysregulated IL-6-induced signaling is associated with the development of severe immunological and proliferative diseases such as rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and colon cancer.

[0014] IL-11 through its binding to its transmembrane IL-11 Ra receptor and resultant activation of downstream signaling pathways has been thought to regulate adipogenesis, osteoclastogenesis, neurogenesis and platelet maturation. More recently it has been discovered that over expression of IL-11 is associated with a variety of cancers and may provide a link between inflammation and cancer (see e.g. Ernst, M., et al., STAT3 and STAT1 mediate IL- 11 -dependent and inflammation-associated gastric tumorigenesis in gp130 receptor mutant mice. J Clin Invest, 2008. 188(5): p. 1727-38). Furthermore, it has been proposed that IL-11 may promote tissue fibrosis (see Cook SA, Schafer S. Hiding in Plain Sight: Interleukin-11 Emerges as a Master Regulator of Fibrosis, Tissue Integrity, and Stromal Inflammation. Annu Rev Med. 2020 Jan 27;71 :263-276).

[0015] In contrast to IL-6 and IL-11 , LIF and OSM lack specific a-receptors but signal via heterodimerization of the two p-receptors gp130 and either the LIF receptor (LIFR) or the OSM receptor (OSMR). Both LIF and OSM were found to improve obesity and hepatic steatosis and to have protective functions following myocardial infarction. However, there are conflicting reports on the effects of both cytokines on fibrotic processes in different tissues and in addition both cytokines have pro- and anti-tumorigenic functions. Consequently, for therapeutic purposes of OSM or LIF agonists it would be highly desirable to only target a specific subset of OSMR or LIFR expressing cells to reduce side effects including tumorigenic effects.

[0016] On principle, the IL-6 family cytokines therefore seem to present promising targets for therapy, either by blocking their receptors or by providing the patient’s body with external cytokines to induce protective or regenerative effects. However, due to their pleiotropism and their variety of signaling mechanisms, both approaches are known to elicit uncontrollable side effects.

[0017] As it is known that administration of an IL-6 family cytokine can result in beneficial and / or disadvantageous effects, depending, inter alia, on the cytokine and the cells targeted, it is desirable to decrease the pool of targetable cells by increasing the specificity of the cytokine to be able to better control the results of the administration of the cytokine.

[0018] Therefore, there is a need for a cytokine that can target only a specific subset of cells and can thereby be used to specifically effect signaling in a controlled setting without prompting severe side effects. Previously, it has been attempted to create a synthetic cytokine to overcome these problems (see e.g. Kallen, K., et al., Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6. J Biol Chem, 1999. 274(17): p. 11859-67; Findeisen, M., et al., Treatment of type 2 diabetes with the designer cytokine IC7Fc. Nature, 2019. 574(63-68); Donath, M.Y., Designer cytokine for the treatment of diabetes. Nat Metab, 2019. 1(10): p. 933-934).

[0019] This synthetic cytokine was termed IC7 and comprised binding sites I and II of IL-6 combined with binding site III derived from CNTF. CNTF requires the cytokine specific nonsignaling a-receptor subunit CNTFR to be present to effect signaling, similar to IL-6 requiring IL-6R. Instead of two gp130 receptors, CNTF requires a heterodimer of one gp130, and one LIFR subunit to induce signal transduction.

[0020] When making the present invention, it was however established that IC7 is characterized by an unexpected cross-reactivity with cells expressing OSMR, as IC7 was able to induce signal transduction in cells expressing gp130, IL-6R and OSMR (see Figure 2.1 and Example 3). An explanation may be an underlying binding affinity of the CNTF site III sequences for both LIFR and OSMR. Therefore, IC7 will activate signaling on cells which present OSMR as well as on cells which present LIFR. This reduces the specificity and suitability of this synthetic cytokine.

[0021] Therefore, there is still an urgent need for the provision of cell-specific cytokines, which do not elicit severe side effects.

[0022] This need was addressed by the present invention, which provides synthetic cytokines characterized by their specificity and better controllability, provided by their cell-type restricted activity and lessened or obviated capability of effecting trans-signaling. In contrast to IC7, in the present invention, binding site III residues from cytokines lacking the need of a specific a-receptor were engrafted onto a cytokine backbone of an a-receptor dependent IL-6 family cytokine. In this way the resulting chimeric cytokine was rendered a-receptor dependent and its activity was limited to cells expressing a specific novel receptor combination.

[0023] While earlier publications suggested that receptor binding modules of IL-6 family cytokines can be transferred from one cytokine to another, it came as a surprise that the transfer of binding site III residues from a-receptor independent cytokines to a backbone of an a-receptor dependent cytokine resulted in an a-receptor dependent biological activity of the chimeric cytokine. It was previously assumed that a combination of binding site II residues of IL-6 or IL-11 with binding site III residues would result in an a-receptor independent dimerization of gp130 and LIFR or OSMR, respectively, as occurs in natural LIF or OSM mediated signaling.

[0024] The synthetic cytokines of the present invention can be used in methods of treatment, in particular in the treatment or prevention of conditions or diseases, which can be treated or prevented by administration of the cytokine. The synthetic cytokines of the present invention are suitable for the medical use as they do not elicit the severe side effects known to be caused by administration of the naturally occurring cytokines.

[0025] SUMMARY OF THE INVENTION

[0026] The present invention is concerned with a synthetic cytokine, which can also be called a chimeric cytokine or a “cytokimera” (a portmanteau of cytokine and chimera) and its medical use.

[0027] Due to their pleiotropic character cytokines used as therapeutic agents exhibit heavy side effects in many cases. For example, many members of the IL-6 family of cytokines show numerous beneficial effects on heart regeneration after a myocardial infarction, liver regeneration or on type 2 diabetes. But unfortunately they also elicit negative effects, since they also evoke also inflammation or lead to fever and tachycardia, for example. This is due to the fact that apart from the targeted cell population, many other unwanted populations are targeted by the cytokines as well. Thus, selective targeting of a specific cell population is of magnificent importance.

[0028] To reach this aim synthetic cytokines were generated. The synthetic cytokines of the present invention need one further receptor for signal transduction compared to the natural cytokines, from which they are derived, which make them more selective, as the combination of receptors required to effect signaling is available only on a limited subset of cells. The advantage of the synthetic cytokine is that they stimulate not all cell populations, which are activated by the natural cytokine, but only a smaller subpopulation.

[0029] Some members of the IL-6 family of cytokines are able to bind the membrane anchored a-receptor followed by homo- or hetero-dimerization of the B-receptors which is called classic signaling. There is also trans-signaling in which the cytokine binds the soluble a- receptor followed by signaling via the B-receptors. Whereas the classic signaling is contributing more to reparative properties, trans-signaling rather contributes to inflammatory properties. A further advantage of the synthetic cytokines of the present invention is the fact that they are poor transducers of trans-signaling and thereby most likely cause tissue healing than inflammation. Finally, compared to many synthetic agents synthetic cytokines of the present invention have a better safety profile, since natural human cytokines are used and reassembled.

[0030] The polypeptide of the present invention comprises two binding sites derived from the same cytokine of the IL-6 family of cytokines, wherein said cytokine requires a cytokine specific nonsignaling a-receptor subunit as part of the receptor complex to effect signaling, wherein the first binding site is capable of binding the cytokine specific nonsignaling a- receptor subunit, and wherein the second binding site is capable of binding the signaling receptor gp130; and a third binding site derived from a further cytokine of the IL-6 family of cytokines, wherein said further cytokine does not require a cytokine specific nonsignaling a-receptor subunit as part of the receptor complex to effect signaling, wherein the third binding site is capable of binding the cytokine specific signaling receptor of the further cytokine.

[0031] In the present invention, the cytokine requiring a cytokine specific nonsignaling a- receptor subunit as part of the receptor complex can be selected from the group comprising IL-6 and IL-11.

[0032] In the present invention, the cytokine not requiring a cytokine specific nonsignaling a- receptor subunit as part of the receptor complex is selected from the group comprising LIF, OSM, and CT-1.

[0033] In the present invention, the cytokine requiring a cytokine specific nonsignaling a- receptor subunit as part of the receptor complex can be IL-11 , and the cytokine not requiring a cytokine specific nonsignaling receptor subunit as part of the receptor complex can be LIF.

[0034] In the present invention, the cytokine requiring a cytokine specific nonsignaling a- receptor subunit as part of the receptor complex can be IL-6, and the cytokine not requiring a cytokine specific nonsignaling receptor subunit as part of the receptor complex can be LIF or OSM.

[0035] The polypeptide of the present invention can further comprise an affinity tag, optionally at its C-terminal end, optionally an Fc constant region of an immunoglobulin, such as IgG, IgA, or IgM; and / or a signal peptide, optionally at its N-terminal end; and / or a binding site for serum albumin.

[0036] The polypeptide of the present invention can comprise an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to SEQ ID NO: 13, preferably comprising the amino acid sequence of SEQ ID NO: 13 (GIL-11); or an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to SEQ ID NO: 6, preferably comprising the amino acid sequence of SEQ ID NO: 6 (GIL-6); or an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to SEQ ID NO: 11 , preferably comprising the amino acid sequence of SEQ ID NO: 11 (GIO-6).

[0037] In the present invention, the binding sites can be derived from human cytokines.

[0038] In the present invention, the capability of the polypeptide to effect trans-signaling is significantly reduced compared to the capability of the cytokine, from which the first two binding sites are derived, to effect trans-signaling.

[0039] The present invention is also concerned with a polynucleotide comprising one or more nucleic acid sequence(s) encoding for the polypeptide of the present invention. Optionally, the polynucleotide can be an expression cassette, preferably an expression vector.

[0040] The present invention is also concerned with an expression system comprising the polynucleotide of the present invention, wherein the expression system can be of eukaryotic, prokaryotic, or archaeal origin, or synthetic in nature. For example, it can be a cell-free expression system.

[0041] The present invention is also concerned with a host cell comprising the polynucleotide of the present invention, wherein optionally the host cell is selected from eukaryotic cells, which include, but are not limited to those e.g. hamster cell lines (CHO and their derivatives), mouse cell lines (such as C127, NSO, SP2 / 0, YB2 / 0, XB2 / 09 and derivatives of all of them), or human cell lines (such as HEK and their derivatives, e.g. EXPI293, HT- 1080, PER.C6, or HuH-7). Also included are cell lines from monkeys, such as e.g. Vero cells and their derivatives and insect cells, such as SF-9 cells and their derivatives.

[0042] The present invention is also concerned with a pharmaceutical composition comprising the polypeptide of the present invention, and a pharmaceutically acceptable carrier.

[0043] The present invention is also concerned with a polypeptide of the present invention for use as a medicament.

[0044] The present invention is also concerned with a polypeptide of the present invention for use in a method of preventing and / or treating a condition in a patient, wherein the condition is selected from the group consisting of lymphopenia, muscular atrophy, osteoporosis, thrombocytopenia, obesity associated metabolic disorders such as type II diabetes mellitus, obesity, insulin resistance, glucose intolerance, dyslipidemia, hypertension, stroke or cardiovascular disease, neurological disorders such as paraplegia, Alzheimer's and Parkinson's disease. The diseases and / or conditions to be treated and / or prevented by administration of the synthetic cytokine of the present invention are associated with signaling mediated by the IL-6 family cytokine, from which binding site III of the synthetic cytokine is derived, such as LIF, OSM, or CT-1.

[0045] DETAILED DESCRIPTION OF THE INVENTION In the following, the present invention is described in detail. The features of the present invention are described in individual paragraphs. This, however, does not mean that a feature described in a paragraph stands isolated from a feature or features described in other paragraphs. Rather, a feature described in a paragraph can be combined with a feature or features described in other paragraphs.

[0046] The term “comprise / s / ing”, as used herein, is meant to include or encompass the disclosed features and further features which are not specifically mentioned. The term “comprise / es / ing” is also meant in the sense of “consist / s / ing of’ the indicated features, thus not including further features except the indicated features. Thus, the subject-matter of the present invention may be characterized by additional features in addition to the features as indicated.

[0047] The polypeptide of the present invention comprises two binding sites derived from the same cytokine of the IL-6 family of cytokines, wherein said cytokine requires a cytokine specific nonsignaling a-receptor subunit as part of the receptor complex to effect signaling, wherein the first binding site is capable of binding the cytokine specific nonsignaling a- receptor subunit, and wherein the second binding site is capable of binding the signaling receptor gp130; and a third binding site derived from a further cytokine of the IL-6 family of cytokines, wherein said further cytokine does not require a cytokine specific nonsignaling a-receptor subunit as part of the receptor complex to effect signaling, wherein the third binding site is capable of binding the cytokine specific signaling receptor of the further cytokine. In one embodiment of the present invention, the binding sites are derived from human cytokines.

[0048] As known in the art, the IL-6 family of cytokines comprises at least IL-6, IL-11 , LIF, OSM, CNTF and CT-1 , which use the common B-receptor subunit gp130 for signal transduction and as a consequence elicit similar and overlapping physiological responses. Therefore this group of cytokines has been termed as ‘IL-6-type cytokines’ or as cytokines of the IL-6 family.

[0049] There are two subgroups within the IL-6 family of cytokines. One group of cytokines requires, as a first step of signal transduction, the interaction between the cytokine and its cytokine specific nonsignaling a-receptor subunit. This group comprises IL-6, IL-11 , and CNTF. IL-6 requires the IL-6 receptor IL-6R, IL-11 requires the IL-11 receptor IL-11 R, and CNTF requires the CNTF receptor (CNTFR). The cytokine specific nonsignaling a-receptor subunit is also referred to simply as a-receptor. Its function is to interact with its cognate cytokine. Upon interaction of the cytokine with its a-receptor at a specific site called site I, the further components of the receptor complex are recruited to site II and III of the cytokines. At site II contacts between cytokine and gp130 are formed. In the case of IL-6 and IL-11 , at site III an additional gp130 molecule is recruited at site III to form gp130 homodimers to induce signal transduction. The other members of the IL-6 family recruit an additional p-receptor at site III to form heterodimers of gp130 and a cytokine specific - receptor to induce signal transduction. Examples are the LIF receptor LIFR and the OSM receptor OSMR.

[0050] In the present invention, the cytokine requiring a cytokine specific nonsignaling a- receptor subunit can be IL-11. Alternatively, it can be IL-6. These cytokines provide the backbone of the synthetic cytokine comprising binding sites I and II.

[0051] The other group of cytokines does not require a cytokine specific nonsignaling a- receptor subunit as part of the receptor complex to effect signaling. This group comprises CT-1 , LIF, and OSM. CT-1 and LIF bind directly to gp130 and LIFR. OSM binds directly to gp130 and OSMR. It is known that OSM can also bind to LIFR. This other group of cytokines provides binding site III, and the signaling pathway targeted by the synthetic cytokine of the present invention is the signaling pathway, that would be targeted by the naturally occurring member of this group of cytokines. For example, if the synthetic cytokine of the present invention comprises binding site III derived from LIF, the signaling pathway targeted by this synthetic cytokine will be the signaling pathway that would be targeted upon administration of naturally occurring LIF. The significant difference between administration of the naturally occurring cytokine and the synthetic cytokine of the present invention is that the pool of targetable cells is significantly reduced, as the signaling pathway is only addressed in cells comprising the novel combination of receptor complex components due to the a-receptor dependency of the chimeric cytokines (see Figure 2). For example, upon administration of naturally occurring LIF, all cells presenting gp130 and LIFR can be targeted. Upon administration of a synthetic cytokine of the present invention, comprising a binding site III derived from LIF, and binding sites I and II derived from IL-6 or IL-11 , only cells presenting gp130, LIFR and IL-6R or IL-11 R can be targeted.

[0052] The present invention is based on the surprising finding that the novel combination of binding sites of a cytokine requiring a cytokine specific nonsignaling a-receptor subunit, and a binding site of a cytokine, which does not require a cytokine specific nonsignaling a- receptor subunit as part of the receptor complex to effect signaling, results in a synthetic cytokine, which retains the dependency on the presence of the a-receptor, and is thereby characterized by an increased specificity. It was previously assumed that this sort of combination would result in a synthetic cytokine, which does not depend on a specific cytokine / cytokine receptor interaction. It is assumed that this is the reason why synthetic cytokines of the prior art did not comprise the novel and inventive combination provided here. In the present invention, the cytokine not requiring a cytokine specific nonsignaling a- receptor subunit can be LIF. Alternatively, it can be OSM or CT-1. Preferably, it is LIF.

[0053] A preferred embodiment of the invention is a polypeptide comprising two binding sites as defined derived from IL-11 , and a third binding site derived from LIF. This polypeptide is also referred to as GIL-11 .

[0054] Another polypeptide of the present invention comprises two binding sites as defined derived from IL-6, and a third binding site derived from LIF. This polypeptide is also referred to as GIL-6.

[0055] Another polypeptide of the present invention comprises two binding sites as defined derived from IL-6, and a third binding site derived from OSM. This polypeptide is also referred to as GIO-6.

[0056] The polypeptides of the present invention can be generated as described in the examples. The polypeptide sequences of the natural cytokines are known. For example, the amino acid sequence of human IL-11 can be as defined in SEQ ID NO: 12. For example, the amino acid sequence of human IL-6 can be as defined in SEQ ID NO: 1. For example, the amino acid sequence of human LIF can be as defined in SEQ ID NO: 2. For example, the amino acid sequence of human OSM can be as defined in SEQ ID NO: 7. The skilled person will also know how to identify the binding sites and how to design a chimera in silico.

[0057] Based on the standard techniques known to the skilled person, and the fact that cytokines of the IL-6 family show a high degree of structural homology, the regions to be exchanged can be determined on a structure-based basis. The structures of the cytokines whose binding site III should be exchanged with each other were analyzed using standard visualization systems for protein structures. When structures were not available, they were predicted using a protein structure homology server. The binding site of human LIF to LIFR contains three regions. Exemplarily, these regions are defined by SEQ ID NOS: 3 to 5. The binding site of human OSM to OSMR also contains three regions. Exemplarily, these regions are defined by SEQ ID NOS: 8 to 10. The sequences designed in silico can then be synthesized.

[0058] The resulting synthetic cytokine or cytokimera is characterized by retaining the binding capability of the first two binding sites of the cytokine, from which it is derived, which are the binding partners of the cytokine specific nonsignaling a-receptor subunit and the signaling receptor gp130, respectively, by losing the binding capability of the third binding site of the cytokine, from which it is derived, which was a binding partner of the signaling receptor gp130, and by gaining a binding capability of a third binding site derived from a different cytokine of the IL-6 family, which did not require a cytokine specific nonsignaling a-receptor subunit as part of the receptor complex to effect signaling, for example the third binding site of LIF or OSM.

[0059] The essential point is that by replacing the third binding site, the binding capability of the first two binding sites is not affected, and that the replacement results in a synthetic cytokine, which comprises a third binding site capable of binding its original receptor.

[0060] The synthetic cytokines GIL-11 , GIL-6, and GIO-6 can be defined by their amino acid sequences. GIL-11 can be defined as comprising SEQ ID NO: 13. GIL-6 can be defined as comprising SEQ ID NO: 6. GIO-6 can be defined as comprising SEQ ID NO: 11. However, as presented above, variations to these exemplary sequences are also encompassed by the present invention, as long as the binding sites can still fulfil their functions. The skilled person knows, which parts of the amino acid sequence of a cytokine of the IL-6 family are important for its binding functions. Variations in the amino acid sequence outside of the binding sites can be carried out without impacting the binding function of the synthetic cytokine. Therefore, the polypeptide of the present invention can comprise an amino acid sequence with at least 90 %, 95 %, 98 %, or 99 % sequence identity to SEQ ID NO: 13 (GIL-11); or an amino acid sequence with at least 90 %, 95 %, 98 %, or 99 % sequence identity to SEQ ID NO: 6 (GIL-6); or an amino acid sequence with at least 90 %, 95 %, 98 %, or 99 % sequence identity to SEQ ID NO: 11 (GIO-6).

[0061] A variant of GIL-11 can also be defined as a polypeptide of comprising an amino acid sequence with at least 90 %, 95 %, 98 %, or 99 % sequence identity to SEQ ID NO: 13 (GIL-11), wherein the variant comprises the three binding regions making up binding site III of LIF defined by SEQ ID NOS: 3 to 5.

[0062] A variant of GIL-6 can also be defined as a polypeptide of comprising an amino acid sequence with at least 90 %, 95 %, 98 %, or 99 % sequence identity to SEQ ID NO: 6 (GIL- 6), wherein the variant comprises the three binding regions making up binding site III of LIF defined by SEQ ID NOS: 3 to 5.

[0063] A variant of GIO-6 can also be defined as a polypeptide of comprising an amino acid sequence with at least 90 %, 95 %, 98 %, or 99 % sequence identity to SEQ ID NO: 11 (GIL-11), wherein the variant comprises the three binding regions making up binding site 111 of OSM defined by SEQ I D NOS: 8 to 10.

[0064] Sequence identity can be determined by the skilled person. For example, the sequence identity can be calculated using BLASTP as disclosed in the prior art (see e.g. Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; Altschul et al. (2005) “Protein database searches using compositionally adjusted substitution matrices.” FEBS J. 272:5101-5109), preferably using version BLASTP 2.2.29+ (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), preferably using the following settings:

[0065] • Field “Enter Query Sequence”: Query subrange: none

[0066] • Field “Choose Search Set”: Database: non-redundant protein sequences (nr); optional parameters: none

[0067] • Field “Program Selection”: Algorithm: blastp (protein-protein BLAST)

[0068] • Algorithm parameters: Field “General parameters”: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 3; Max matches in a query range: 0

[0069] • Algorithm parameters: Field “Scoring parameters”: Matrix: BLOSUM62; Gap Costs: Existence: 11 Extension: 1 ; Compositional adjustments: Conditional compositional score matrix adjustment

[0070] • Algorithm parameters: Field “Filters and Masking”: Filter: none; Mask: none.

[0071] Results are filtered for sequences with more than 35 % query coverage.

[0072] Preferably, the variants can comprise one or more conservative substitutions for amino acids comprised in the exemplary sequences SEQ ID NO: 13, 6, or 11.

[0073] A "conservative substitution" refers to the substitution of one amino acid by another, wherein the replacement results in a silent alteration. This means that one or more amino acid residues within the amino acid sequence of the present invention can be substituted by another amino acid of a similar polarity which acts as a functional equivalent. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (i.e. a conservative substitution). For example, one polar amino acid can be substituted by another polar amino acid, one positively or negatively charged amino acid, respectively, can be substituted by another positively or negatively charged amino acid, respectively, et cetera. Classes of amino acids are for example, nonpolar (hydrophobic) amino acids including alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; polar neutral amino acids including glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids including arginine, lysine and histidine; negatively charged (acidic) amino acids including aspartic acid and glutamic acid.

[0074] The polypeptide of the present invention can further comprise additional structures. The additional structures can be located at the C-terminal end, and / or the N-terminal end.

[0075] For example, the polypeptide of the present invention optionally further comprises an Fc constant region of an IgG antibody. In another embodiment, the polypeptide of the present invention optionally further comprises an Fc constant region of any other immunoglobulin classes or sub-classes, such as IgM or IgA. Adding an Fc constant region of an immunoglobulin to the polypeptide of the present invention provides the following advantages.

[0076] On the one hand, it can facilitate the purification of the polypeptide, for example by using a protein-A Sepharose matrix. On the other hand, adding an Fc constant region of an immunoglobulin to the polypeptide of the present invention improves the pharmacokinetics of the polypeptide in therapy by increasing the thermal stability of the polypeptide. The Fc constant region of an immunoglobulin can also provide a significant increase in serum half-life, which is important for the therapeutic use of the polypeptide of the present invention. This is due to the binding to the neonatal Fc receptor (FcRn) in the liver, which protects against degradation, and the increase in molecular weight can prevent renal excretion. Another option to improve the pharmacokinetics of the polypeptide of the present invention is to provide binding sites for serum albumin, as it has been shown that noncovalent association with albumin extends the half-life of otherwise short lived proteins.

[0077] These additional moieties, such as e.g. Fc constant regions, can be utilized for example for purification procedures of the polypeptide of the present invention. The term "purification", in particular purification by means of protein tags, is clearly and unambiguously known to the skilled person. Methods include all possible techniques that are known to the skilled person in art, such as methods relying on characteristics as solubility, size, charge, and specific binding affinity. Non-limiting examples are for example salting out, ammonium sulfate or ethanol precipitation, dialysis, chromatography (such as protein A purification, gel-filtration chromatography, ion-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography high-pressure, liquid chromatography (HPLC), electrophoresis, and / or centrifugation.

[0078] Moreover, the polypeptide of the present invention can additionally comprise any other moieties, for example protein tags, which are known the skilled person in art. The term “tag” as it is used herein means any naturally occurring or artificial polypeptide or another molecule structure, which allows purification and / or detection of any polynucleotide of the present invention and / or further improves the pharmacodynamics and / or pharmacokinetic properties of any polypeptide of the invention.

[0079] These protein tags can for example be used to facilitate purification of the polypeptide. Non-limiting examples related thereto are for example GFP-tags or derivatives thereof, (poly)HIS-tags, Myc-Tags, Strep-Tags, polyarginine-Tags, Flag-Tags, TAP-Tags, glutathione S-transferase (GST)-Tags, HA-Tags, calmodulin-binding peptide (CBP)-Tags, maltose-binding protein (MBP)-Tags, V5-Tags, HSV-Tags, Protein C-Tags, Luciferase- Tags, or any other common polypeptide tags. Preferably, the protein tag can be located at the C-terminal end of the polypeptide. Such tags can be removed prior to final preparation of a polypeptide.

[0080] Moreover, the polypeptide of the present invention can additionally comprise a signal peptide, optionally at its N-terminal end. Signal peptides are known to the skilled person. Preferably, the signal peptide, when used in the present invention, leads to secretion of the polypeptide, when produced in a cell line. This facilitates downstream processes such as purification of the polypeptide.

[0081] The novel combination of binding sites in the synthetic cytokines of the present invention increases the specificity, as the pool of targetable cells is significantly decreased. A further advantage is the surprising finding that the synthetic cytokines of the present invention are characterized by their severely limited, significantly reduced, or even obviated capability of effecting trans-signaling compared to the capability of effecting trans-signaling exhibited by the natural cytokines from which the synthetic cytokines of the present invention have been derived. The skilled person knows how to measure trans-signaling capability of a synthetic cytokine, and can compare the results with the level of trans- signaling obtained with a corresponding natural cytokine. The assay conditions for the synthetic cytokine and its corresponding natural cytokine will be the same to obtain comparable results. An exemplary method of determining if the trans-signaling capability of a synthetic cytokines of the present invention has been significantly reduced is shown in Example 4. A significant reduction can be determined if the decrease is statistically significant.

[0082] For example, a significant reduction can be determined if the level of trans-signaling of the synthetic cytokine compared to level of trans-signaling of its corresponding natural cytokine is about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10%, or about 5%, or even 0%. A reduction to zero is also called an obviated capability to effect trans-signaling.

[0083] An obviated capability can be determined if the EC50 in the assay cannot be determined due to the maximal cellular proliferation not being reachable even at high concentrations of the synthetic cytokines of the present invention (see Example 5, Figures 6A and 6B).

[0084] In a further aspect, the present invention also concerns a polynucleotide comprising one or more nucleic acid sequence(s) encoding for the polypeptide of the present invention. The polynucleotide can also comprise nucleic acids encoding for the additional structures, such as Fc constant regions, protein tags, or signal peptides, as described above. Optionally the polynucleotide is an expression construct or expression cassette, preferably an expression vector. An “expression vector” or an “expression construct” is usually a plasmid or virus designed for gene expression in any suitable cell. The expression vector is usually used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. In the present invention, any common expression vector, which is known to the skilled person in the art can be used for this purpose.

[0085] In a further aspect, the present invention also concerns an expression system comprising the polynucleotide of the present invention, wherein the expression system can be of eukaryotic, prokaryotic, or archaeal origin, or synthetic in nature. For example, it can be a cell-free expression system.

[0086] In a further aspect, the present invention also concerns a host cell comprising the polynucleotide of the present invention. The host cell is capable of producing the polypeptide of the present invention, and preferably allows rapid purification. The host cell is selected from eukaryotic cells, which include, but are not limited to those, hamster cell lines, such as CHO and derivatives thereof, mouse cell lines, such as C127, NSO, SP2 / 0, YB2 / 0, XB2 / 09, and derivatives thereof, or human cell lines, such as HEK and derivatives thereof, for example EXPI293, HT-1080, PER.C6, or HuH-7, monkey cell lines, such as Vero cells and derivatives thereof, and insect cell lines, such as SF-9 and derivatives thereof.

[0087] As it is used herein “derivative” and “derivatives” is to be understood as all descendant cell lines that have been derived from them or have emerged from them with modification or further development. Polypeptide expression using cellular systems can be performed by using diverse transfection systems. Non-limiting examples are for example lipid-based transfection or viral transduction techniques, which are very well known to a skilled person in the art.

[0088] In a further aspect, the present invention also concerns a pharmaceutical composition comprising the polypeptide of the present invention, and a pharmaceutically acceptable carrier.

[0089] In a further aspect, the present invention also concerns the polypeptide of the present invention for use as a medicament.

[0090] In particular, the present invention concerns the polypeptide of the present invention for use in a method of preventing and / or treating a condition in a patient, wherein the condition is selected from the group consisting of lymphopenia, muscular atrophy, osteoporosis, thrombocytopenia, obesity associated metabolic disorders such as type II diabetes mellitus, obesity, insulin resistance, glucose intolerance, dyslipidemia, hypertension, stroke or cardiovascular disease, neurological disorders such as paraplegia,

[0091] Alzheimer's and Parkinson's disease.

[0092] The patient can be a mammal, preferably a human. As is well-known in the art, IL-6 family cytokines have been implicated in many functions, including B-cell stimulation and induction of the hepatic acute phase proteins. Moreover, metabolic functions and neurotrophic functions have been ascribed to this group of cytokines.

[0093] LIF increases the angiogenic potential of mesenchymal stem cells and enhances the survival of cardiomyocytes and cardiac regeneration after myocardial infarction in mice. Several reports demonstrated protective effects of LIF against obesity and hepatic steatosis as well as LIF induced increase of glucose uptake. Cells with high levels of LIFR include kidney cells (loop of Henle), cardiomyocytes, adipocytes, thyroid glandular cells, hepatocytes, alveolar cells, colon (enteroendocrine) prostate glandular cells.

[0094] OSM signaling via gp130 and OSMR has beneficial effects on cardiac fibrosis, enhances angiogenesis and improves cardiac function following myocardial infarction. Mice injected with OSM after myocardial infarction displayed improved recovery and better cardiac function compared to untreated mice. Mice treated with OSM after spinal cord injury displayed functional recovery and neurite outgrowth. OSM was further shown to improve obesity, glucose intolerance and insulin resistance in mice. Cells with high levels of OSMR include ductal cells, Leydig cells and cholangiocytes.

[0095] For both LIF and OSM however, there are conflicting reports on their effects on fibrosis in different organs. In addition, both cytokines have pro- and antitumorogenic functions. Cell-type restricted LIF and OSM variants with reduced side-effects may thus represent promising therapeutic agents.

[0096] The synthetic cytokines of the present invention are characterized by their chimeric nature, which increases the specificity by decreasing the pool of targetable cell, as fewer cells will display all signaling complex components necessary to effect signaling.

[0097] This allows the therapeutic use of the synthetic cytokines of the present invention, as the probability of side effects is decreased. The synthetic cytokines of the present invention can be used to treat or prevent conditions associated with signaling mediated by the cytokine from which binding site III is derived, such as LIF or OSM. In addition to the specific P-receptor combination, the targetable cell also needs to express the a-receptor, such as IL-6R or IL-11 R. IL-6R is expressed with high levels on many different types of cells, including Sertoli cells, proximal tubular cells, hepatocytes, endothelial cells, ductal cells, Leydig cells and immune cells. Cells with high levels of IL-11 R include cardiomyocytes, fibroblasts, breast cells (glandular and myoepithelial), skeletal myocytes and hepatocytes.

[0098] The terms "obesity" and "obese" generally refer to individuals whose body weight is at least 20% above the average body weight for the individual's age, gender and height. An individual is also defined as "obese" if the individual is a male whose body mass index is greater than 27.8 kg / m2or a female whose body mass index is greater than 27.3 kg / m2. Those of skill in the art will recognize that individuals can be significantly above the average weight for their age, gender, and height and still technically not be "obese." Such individuals are referred to as "overweight" herein, in accordance with normal usage. The polypeptides of the present invention will be beneficial for such overweight individuals, and may also be beneficial to individuals who are prone to obesity or to being overweight and who wish to avoid a recurrence of earlier episodes of obesity or being overweight.

[0099] The term "obesity-associated metabolic disorder" means a disorder which results from, is a consequence of, is exacerbated by or is secondary to obesity. Non-limiting examples of such a disorder are osteoarthritis, Type II diabetes mellitus, increased blood pressure, stroke, and heart disease.

[0100] The polypeptide of the present invention can also be used to therapeutically or prophylactically treat a condition in a mammal, wherein the condition is characterized by inadequate insulin sensitivity.

[0101] Reference to a "condition characterized by inadequate insulin sensitivity" should be understood as a reference to any condition in which the level of insulin responsiveness of the patient is inadequate or otherwise insufficient for its physiological needs, irrespective of whether said inadequate insulin sensitivity is a cause or a symptom of said condition. Examples of such conditions include, but are not limited to diabetes mellitus, insulin resistance, glucose intolerance, obesity, dyslipidemia, liver disease, metabolic disorders, hypertension, cardiovascular disease or stroke.

[0102] These therapeutic and prophylactic aspects of the present invention are preferably achieved by administering an effective amount of the polypeptide of the present invention, or the pharmaceutical composition of the present invention, for a time and under conditions sufficient to appropriately achieve the therapeutic or prophylactic effect.

[0103] A “therapeutically effective amount” means an amount that is effective in prevention and / or therapy, or an amount sufficient to provide a preventive and / or therapeutic effect. An amount that is effective in therapy is an amount which produces a biological activity and will depend, among other things, on the individual. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0104] Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity or onset of a particular condition. "Treatment" may also reduce or retard the severity or progression of an existing condition.

[0105] Administration of the polypeptide of the present invention or of the pharmaceutical composition of the present invention may be effected by different ways of administration. Non-limiting examples include, but are not limited to, for example, intravenous, intraarterial, intraperitoneal, intramuscular, pulmonal, inhalative administration. The dosage regimen will be determined by the attending physician and other clinical factors. As well known the skilled person in art, the dosages for any one patient can vary and depend on many factors, including for example size, age, sex, time and router of administration and stage of the disease.

[0106] The invention is further explained by the attached figures and examples, which are intended to illustrate, but not to limit the present invention.

[0107] FIGURES

[0108] Figure 1 shows normalized proliferation of Ba / F3 cells with various receptors with different cytokimera and control cytokines. A) Proliferation of Ba / F3 cells with the indicated receptors without cytokine (-), with HIL-11-Ts (50 pg / ml), IL-11 (10 ng / ml), OSM (10 ng / ml), GIL-11-Ts (500 ng / ml). B) Proliferation of Ba / F3 cells with the indicated receptors without cytokine (-), with HIL-11-Ts (50 ng / ml), IL-6 (20 ng / ml), LIF (20 ng / ml), OSM 20 ng / ml), GIL- 6-Fc (20 ng / ml), GIO-6-Fc (20 ng / ml) and IC7-Fc (20 ng / ml). One representative experiment out of three is shown. Data are shown as mean ± SEM.

[0109] Figure 2 shows proliferation of Ba / F3 cells expressing various receptor combinations with different cytokimera and cytokines in mean fluorescence intensity (MFI). One representative experiment out of three is shown. Data are shown as mean ± SEM. The curves are fitted by a non-linear regression. A) Proliferation of Ba / F3 cells expressing the indicated receptors with increasing concentration of IC7-Fc (0.001-2000 ng / ml). B) Proliferation of Ba / F3 cells expressing the indicated receptors with increasing concentration of GIL-11-Ts (0.001-1000 ng / ml). C) Proliferation of Ba / F3 cells expressing the indicated receptors with increasing concentration of GIL-6-Fc (0.001-1000 ng / ml). D) Proliferation of Ba / F3 cells expressing the indicated receptors with increasing concentration of GIO-6-Fc (0.001-1000 ng / ml). E) Proliferation of Ba / F3 gp130 IL-11 R LIFR with increasing concentration of IL-11 (0.001-1000 ng / ml). F) Proliferation of Ba / F3 gp130 IL-11 R LIFR with increasing concentration of LIF (0.0001-100 ng / ml). G) Proliferation of Ba / F3 gp130 IL-6R LIFR with increasing concentration of IL-6 (0.0001-30 ng / ml). H) Proliferation of Ba / F3 gp130 IL-6R LIFR with increasing concentration of LIF (0.0001-100 ng / ml).

[0110] Figure 3 shows the results of a fluorescence based western blot against STAT3 and phosphoylated-STAT3. STAT3 activation in Ba / F3 cells with cytokines and cytokimera for 15 min. Equal amounts of protein (50 pg) were loaded each lane. One representative experiment out of three is shown. A) STAT3 activation in Ba / F3 cells expressing the indicated receptors without cytokine (-) and after stimulation with HIL-11-Ts (50 ng / ml), IL- 11 (50 ng / ml), LIF (10 ng / ml), OSM (10 ng / ml) and GIL-11-Ts (500 ng / ml). B) STAT3 activation in Ba / F3 cells expressing the indicated receptors without cytokine (-) and after stimulation with HIL-11-Ts (100 ng / ml), IL-6 (100 ng / ml), LIF (10 ng / ml), OSM (10 ng / ml) and GIO-6-Fc (100 ng / ml) und IC7-Fc (100 ng / ml)..

[0111] Figure 4 shows the result of a fluorescence based western blot against STAT3, phosphoylated-STAT3, ERK and phospho-ERK. Stimulation of Ba / F3 cells with cytokines and cytokimera for 15 min. Equal amounts of protein were loaded each lane (50 pg / lane). One representative experiment out of three is shown. A) STAT3 and ERK activation of Ba / F3 gp130 IL-11 R LIFR cells without cytokine (-), GIL-11-Ts, LIF and IL-11 in the indicated concentrations. B) STAT3 and ERK activation of Ba / F3 gp130 IL-6R LIFR cells without cytokine (-), GIL-6-Fc, GIO-6-Fc, LIF and IL-6 in the indicated concentrations.

[0112] Figure 5 shows the results of a fluorescence based western blot against phosphoylated-STAT3, STAT3, phosphoylated-STAT1 , STAT1 , phosphoylated-STAT5, STAT5, phosphoylated-STAT6, and STAT6 for HIL-11 , IL-11 , LIF, OSM, and GIL-11.

[0113] Figure 6 shows proliferation of Ba / F3 gp130 LIFR cells with constant concentration of the soluble a-receptors and increasing concentration of cytokines and cytokimera. One representative experiment out of three is shown. Data are shown as mean ± SEM. The curves are fitted by a non-linear regression. A) Proliferation of the Ba / F3 gp130 LIFR cells with (dotted line) and without (squared lines) 100 ng / ml sIL-H R and increasing concentration of IL-11 (2-2000 ng / ml). B) Proliferation of the Ba / F3 gp130 LIFR cells with (dotted line) and without (squared lines) 100 ng / ml slL-11 R and increasing concentration of GIL-11-Ts (2-2000 ng / ml). C) Proliferation of the Ba / F3 gp130 LIFR cells with (dotted line) and without (squared lines) 200 ng / ml slL-6R and increasing concentration of IL-6 (0,002-2000 ng / ml). D) Proliferation of the Ba / F3 gp130 LIFR cells with (dotted line) and without (squared lines) 200 ng / ml slL-6R and increasing concentration of IC7-Fc (2-1000 ng / ml). E) Proliferation of the Ba / F3 gp130 LIFR cells with (dotted line) and without (squared lines) 200 ng / ml slL-6R and increasing concentration of GIL-6-Fc (2-1000 ng / ml). F) Proliferation of the Ba / F3 gp130 LIFR cells with (dotted line) and without (squared lines) 200 ng / ml slL-6R and increasing concentration of GIO-6-Fc (2-1000 ng / ml).:

[0114] Figure 7 shows that GIL-11-Ts trans-signaling cannot be inhibited by sgp130-Fc. A) STAT3 activation in Ba / F3 gp130 LIFR cells without and with HIL-11-Ts (50 ng / ml), GIL- 11-Ts (1 pg / ml), GIL-11-Ts (1 pg / ml): sIL-H R (2 pg / ml), GIL-11-Ts (1 pg / ml), GIL-11-Ts (1 pg / ml):slL-11 R (2 pg / ml):sgp130-Fc (10 pg / ml) after 15 min of stimulation. Equal amounts of protein were loaded (50 pg / lane). B) Proliferation of Ba / F3 cells by adding constant concentrations of IL-11 (0.5 pg / ml):slL-11 R (1 pg / ml) or GIL-11-Ts (0.5 pg / ml) :slL11 R (1 pg / mL) and increasing concentrations of sgp130-Fc (0.01-10,000 ng / mL). The error bars reflect the standard error.

[0115] Figure 8 shows results of a fluorescence based western blot against phosphoylated- STAT3 and STAT3 in murine cells (Figure 8A, and B). C) to G) show that GIL-11 rescued IL-6R deficient mice from death following partial hepatectomy.

[0116] Figure 9 shows that GIL-6 induces JAK / STAT signaling and cellular proliferation via non-natural cytokine receptor complexes. (A) Proliferation of Ba / F3-gp130, Ba / F3-IL- 6R:gp130, Ba / F3-gp130:OSMR, Ba / F3-gp130:LIFR, Ba / F3-IL-6R:gp130:OSMR, Ba / F3-IL- 6R:gp130:OSMR, Ba / F3-IL-6R:gp130:LIFR cells without cytokine (-), with 100 ng / ml HIL- 6, 10 ng / ml IL-6, 10 ng / ml LIF, 10 ng / ml OSM, 100 ng / ml GIL-6, 100 ng / ml GIO-6 and 100 ng / ml IC7. One representative experiment out of three is shown. (B) STAT3 activation in Ba / F3, Ba / F3-gp130, Ba / F3-IL-6R:gp130, Ba / F3-gp130:OSMR, Ba / F3-gp130:LIFR, Ba / F3- IL-6R:gp130:OSMR, Ba / F3-IL-6R:gp130:LIFR cells without cytokine (-) and after stimulation with 100 ng / ml HIL-6, 10 ng / ml IL-6, 10 ng / ml LIF, 10 ng / ml OSM, 100 ng / ml GIL-6, 100 ng / ml GIO-6 and 100 ng / ml IC7 for 20 min. (C) STAT1 , STAT3, STAT5, ERK and Akt activation in Ba / F3-IL-6R:gp130:LIFR cells with the same conditions as for the STAT3 activation. (D) STAT3 activation in heart, liver and spleen after injection of 20 pg GIL-6 or GIO-6. Mice were sacrificed 30 min after intraperitoneal cytokine injection. Equal amounts of proteins (50 pg / lane) were analyzed via specific antibodies detecting phospho- STAT3 and STAT3. Western blot data shows one representative experiment out of three.

[0117] Figure 10 shows that biological activity of cytokimeras GIL-6 and GIO-6 is comparable to natural cytokines. (A) Proliferation of Ba / F3-IL-6R:gp130, Ba / F3-gp130:LIFR, Ba / F3- gp130:OSMR, Ba / F3-IL-6R:gp130:LIFR, Ba / F3-IL-6R:gp130:OSMR cells in the presence and absence of increasing concentrations of GIL-6 (0.002-1 ,000 ng / ml). The EC50 values were calculated by fitting a non-linear regression curve. One representative experiment out of three is shown. (B) Proliferation of Ba / F3-IL-6R:gp130:LIFR cells in the presence and absence of increasing concentrations LIF (0.002-50 ng / ml), IL-6 (0.002-30 ng / ml) or IC7 (0.002-1 ,000 ng / ml). One representative experiment out of three is shown. (C) Proliferation of Ba / F3-IL-6R:gp130, Ba / F3-gp130:LIFR, Ba / F3-gp130:OSMR, Ba / F3-IL- 6R:gp130:LIFR, Ba / F3-IL-6R:gp130:OSMR cells in the presence and absence of increasing concentrations of GIO-6 (0.002-1 ,000 ng / ml). One representative experiment out of three is shown. (D) Proliferation of Ba / F3-IL-6R:gp130:OSMR cells in the presence and absence of increasing concentrations of IC7 (0.002-2,000 ng / ml) and proliferation of Ba / F3-gp130:OSMR cells in the presence of OSM (0.001-100 ng / ml). One representative experiment out of three is shown. (E) STAT3 and ERK activation in Ba / F3-IL- 6R:gp130:LIFR cells without cytokine (-) and after stimulation with increasing amounts of IL-6, LIF, GIL-6 or GIO-6 (0.2, 2, 20, 200 ng / ml) for 20 min. Equal amounts of proteins (50 pg / lane) were analyzed via specific antibodies detecting phospho-STAT3, STAT3, phospho-ERK and ERK. Western blot data shows one representative experiment out of three. (F) Time-dependent STAT3 activation of Ba / F3-IL-6R:gp130:OSMR cells with OSM (10 ng / ml), IL-6 (10 ng / ml) or GIO-6 (100 ng / ml) and Ba / F3-IL-6R:gp130:LIFR cells with LIF (10 ng / ml), IL-6 (10 ng / ml) or GIL-6 (100 ng / ml) for the indicated time points. Equal amounts of proteins (50 pg / lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3.

[0118] Figure 11 shows that cytokimera GIL-6 and GIO-6 are poor inducers of trans-signaling. (A) Proliferation of Ba / F3-gp130:LIFR cells in the presence and absence of fixed concentrations of slL-6R (0 or 100 ng / ml) and increasing concentrations of IL-6 (0.002- 1 ,000 ng / ml). One representative experiment out of three is shown. (B) Proliferation of Ba / F3-gp130:LIFR cells in the presence and absence of fixed concentrations of slL-6R (0 or 100 ng / ml) and increasing concentrations of GIL-6 (0.002-2,000 ng / ml). One representative experiment out of three is shown. (C) Proliferation of Ba / F3-gp130:LIFR cells in the presence and absence of fixed concentrations of slL-6R (0 or 100 ng / ml) and increasing concentrations of GIO-6 (0.002-2,000 ng / ml). One representative experiment out of three is shown. (D) Proliferation of Ba / F3-gp130:OSMR cells in the presence and absence of fixed concentrations of slL-6R (0 or 100 ng / ml) and increasing concentrations of GIO-6 (0.002-2,000 ng / ml). One representative experiment out of three is shown. (E) Proliferation of Ba / F3-gp130:LIFR cells in the presence and absence of fixed concentrations of slL-6R (0 or 100 ng / ml) and increasing concentrations of IC7 (0.002- 1 ,000 ng / ml). One representative experiment out of three is shown.

[0119] Figure 12 shows that CNTF signals via the alternative CNTFR:gp130:OSMR complex but not via IL-6R:gp130:OSMR. (A) Proliferation of Ba / F3-CNTFR:gp130:LIFR or Ba / F3- CNTFR:gp130:OSMR cells with increasing concentrations of CNTF (0.0002-100 ng / ml). One representative experiment out of four is shown. (B) STAT3 activation Ba / F3- CNTFR:gp130:OSMR cells without cytokine (-), 10 ng / ml LIF, 10 ng / ml CNTF or 10 ng / ml OSM. Equal amounts of proteins (50 pg / lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. (C, D) CNTF dose-dependent STAT3 activation of Ba / F3- CNTFR:gp130:LIFR (c), Ba / F3-CNTFR:gp130:OSMR (d) cells without cytokine (-) or in the presence of 10 ng / ml LIF or OSM, or with 0.1, 1, 10 or 100 ng / ml CNTF for 20 min. Equal amounts of proteins (50 pg / lane) were analyzed via specific antibodies detecting phospho- STAT3 and STAT3. Western blot data shows one representative experiment out of three. (E) Proliferation of Ba / F3-gp130, Ba / F3-CNTFR:gp130:LIFR, Ba / F3- CNTFR:gp130:OSMR, Ba / F3-IL-6R:gp130:LIFR or Ba / F3-IL-6R:gp130:OSMR cells without cytokine (-) or in the presence of CNTF (0.5, 5 or 50 ng / ml), LIF (10 ng / ml), OSM (10 ng / ml) or IL-6 (10 ng / ml). One representative experiment out of three is shown. (F, G) CNTF dose-dependent STAT3 activation of Ba / F3-IL-6R:gp130:LIFR (F) or Ba / F3-IL- 6R:gp130:OSMR (G) cells without cytokine (-) or in the presence of 10 ng / ml LIF or OSM, or with 0.1 , 1, 10 or 100 ng / ml CNTF for 20 min. Equal amounts of proteins (50 pg / lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. (H) Western blotting of coimmunoprecipitation by using Protein A beads to precipitate recombinant 2 pg Hyper- CNTF-Fc, 1 pg soluble OSMR in the presence or absence of 1 pg biotinylated gp130.

[0120] Figure 13 shows receptor cell surface expression of Ba / F3-gp130:LIFR or Ba / F3-IL- 6R:gp130:LIFR cells transduced with human LIFR, Ba / F3-gp130:OSMR or Ba / F3-IL- 6R:gp130:OSMR cells transduced with human OSMR and Ba / F3-CNTFR:gp130:OSMR or Ba / F3-CNTFR:gp130:LIFR cells transduced with human CNTFR compared with nontransduced control cells by flow cytometry. Expression was demonstrated via receptorspecific antibodies.

[0121] EXAMPLES

[0122] Example 1 - Material and Methods

[0123] Cloning

[0124] The GIL-11 cDNA was ordered by BioCat GmbH. The GIL11 cDNA was then inserted into pcDNA3.1 expression vector including 5' signal peptide for human IL-11R (Q14626, aa 1-24) followed by sequences for myc tag (EQKLISEEDL; SEQ ID NO:14) and the fragment encoding for GIL-11, Gly4Ser linker, a TEV recognition site and a twin-strep-tag.

[0125] Cells, reagents and recombinant proteins How to generate Ba / F3-gp130 is known in the art. The packaging cell line Phoenix- Eco was received from Ursula Klingmuller (DKFZ, Heidelberg, Germany). HEK293T (ACC- 635) cells were purchased from the Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Culture (Braunschweig, Germany). All cells were grown at 37°C with 5% CO2 in a water-saturated atmosphere in Dulbecco's modified Eagle's medium (DMEM) high-glucose culture medium (GIBCO®, Life Technologies, Darmstadt, Germany) with 10% fetal calf serum (GIBCO®, Life Technologies) and 60 mg / l penicillin and 100 mg / l streptomycin (Genaxxon Bioscience GmbH, Ulm, Germany). Murine Ba / F3-gp130 cells were obtained from Immunex (Seattle, WA, USA) and grown in the presence of HIL-6. 0.2% (10ng / ml) conditioned medium from a stable clone of CHO-K1 cells secreting HIL-6 in the supernatant. Expi-293F™ cells (ThermoFisher Scientific) were cultured in Expi293™ expression medium without antibiotics until they reached a density of 3-5 x106c / ml in a 37°C incubator with 8% CO2 on an orbital shaker at 125 rpm. Synthetic ligands were expressed and purified as known in the art. Recombinant human OSM (catalog no.295- OM) and recombinant human LIF (catalog no. 7734-LF) were purchased from R&D Systems (Minneapolis, MN, USA).

[0126] Stimulation assay

[0127] Ba / F3-gp130 cell lines were washed three times with PBS to remove cytokines and starved in serum-free DMEM for 3 h. Inhibitor sgp130Fc or slL11 R were added 5 min prior to stimulation. Cells were stimulated for 15 min with purified protein (concentration as indicated), harvested, frozen in liquid nitrogen and then lysed. In case of the C2C12 cells, the cells were washed after the stimulation with PBS once before they were detached by 0.05% trypsin, 0.1% EDTA (Genaxxon, catalog. C4261.0100) treatment for 5 min and washed again. Cells were lysed for 45 min with buffer containing 10 mM Tris-HCI, pH 7.5, 150 mM NaCI, 0.5 mM MgCh and a complete™, EDTA-free protease inhibitor mixture tablet (Roche Diagnostics, Mannheim, Germany). Protein concentration was determined by a BCA protein assay (Thermo Fisher Scientific) according to the manufacturer’s instruction. Protein expression and pathway activation was then analysed by western blotting.

[0128] Western Blotting

[0129] 50 pg total protein were loaded on each lane and separated by SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane (Amersham Protan; Cytiva; LC, United Kingdom; catalog no. 10600016). Blocking of membrane was performed with blocking buffer (Intercept® Blocking Buffer; LI-COR; USA; catalog no. 927-60001) diluted 1 :3 in TBS (10 mM Tris-HCI pH 7.6, 150 mM NaCI) for 1 h. Primary antibodies (Phospho-STAT3; Tyr-705; D3A7; catalog no. 9145; and STAT3; 124H6; catalog no. 9139, Cell Signaling Technology, USA) were diluted 1 :1 ,000 in blocking buffer containing 0.2% Tween-20 (Sigma-Aldrich; USA; catalog no. P1379-1 L) for at least 90 min at ambient temperature or overnight at 4°C. Membranes were washed with TBS-T (0.1% Tween-20) and then incubated with secondary fluorophore-conjugated antibodies 1 :10,000 (IRDye® 800CW Donkey anti-Rabbit; catalog no. 926-32213 and IRDye® 680RD Donkey antiMouse; catalog no. 926-68072, LI-COR; USA) for 1 h. Signal detection was achieved using LI-COR Odyssey; USA; Model 2800). Secondary antibodies were detected simultaneously on different channels. Data analysis was conducted using Image Studio Lite 5.2. Liver, spleen and heart tissue were lysed in Lysis buffer (50 mM Tris HCI pH 7.5, 150 mM NaCI, 2 mM EDTA pH 8.0, 2 mM NaF, 1 mM Na3VO4, 1% NP-40, 1% Triton X-100, 1 complete™ protease inhibitor cocktail tablet). After lysis, the protein content was measured by BCA assay. 50 pg total protein amount was then loaded each line followed by Immunoblotting. Antibodies used for blotting of lysed animal organs were as follows: anti-p-STAT3 (catalog no. 9145), anti-total-STAT3 (catalog no. 9139).

[0130] Cell viability assay

[0131] Ba / F3-gp130 cell lines were washed three times with PBS to remove cytokines from the medium. Cells with a density of 5x104cells / ml were suspended in DMEM containing 10% fetal calf serum, 60 mg / l penicillin and 100 mg / ml streptomycin. Cells were cultured for 3 days in a volume of 100 pl with or without cytokines or inhibitor in the indicated concentrations. The CellTiter Blue Viability Assay (Promega, Karlsruhe, Germany) was used to determine the approximate number of viable cells by measuring the fluorescence (A 560 nm / 590 nm) using the Infinite M200 Pro plate reader (Tecan, Crailsheim, Germany). After adding 20 pl / well of CellTiter Blue reagent (time point 0), fluorescence was measured after 60 min every 20 min for up to 2 h. For each condition of an experiment, 3 wells were measured. All values were normalized by subtracting time point 0 values from the final measurement.

[0132] Transfection of cells

[0133] Ba / F3-gp130 cell lines were retrovirally transduced with the pMOWS expression plasmids as known in the art. Transduced cells were grown in DMEM medium as described above supplemented with 10 ng / ml HIL-6. Selection of transduced Ba / F3-gp130 cells was performed with puromycin (1.5 pg / ml) or hygromycin B (1 mg / ml) (Carl Roth, Karlsruhe, Germany) for at least 2 weeks. Afterwards, the generated Ba / F3-gp130 cell lines were analyzed for receptor cell surface expression via flow cytometry. HEK293T cells were transfected by 10 g pDNA with 20 pl TurboFect (Thermo Fisher, USA, catalog no. R0532) and incubated for 48 h before cell lysis and Western Blotting. C2C12 cells were transfected by 7.5 pg cDNA encoding for mlL11 R and 15 pl TurboFect and incubated for 48 h.

[0134] Cell surface detection of cytokine receptors via flow cytometry

[0135] Cell surface expression of stably transfected Ba / F3-gp130 cell lines was detected by specific antibodies. 5x105cells were washed in FACS buffer (PBS, 1 % BSA) and then incubated in 50 pl of FACS buffer containing the indicated specific primary antibody (anti- LIFR or -OSMR; 1 :20; catalog no. BAF249 and BAF4389, R&D Systems; MN, USA). After incubation of at least 1 h at room temperature, cells were washed and resuspended in 50 pl of FACS buffer containing secondary antibody (NorthernLights™ 493-conjugated antigoat IgG 1 :200) and incubated for 30 min at room temperature. Cells were washed and resuspended in 500 pl of FACS buffer and analyzed by flow cytometry (BD FACSCanto II flow cytometer using the FACSDiva software, BD Biosciences). Data analysis was conducted using FlowJo Version 10 (Tree Star Inc, US).

[0136] Animals and ethics statement

[0137] C57BL / 6 and IL-6R' / _mice were obtained from the Jackson Laboratory and the animal facility of the Heinrich-Heine University of Dusseldorf, respectively. The experiments of this study were carried out according to the requirements of LANUV-NRW, Germany with the approval number 84-02.04.2015. A462.

[0138] Animals

[0139] All mice were kept under specific pathogen-free conditions and handled according to regulations defined by FELASA and the national animal welfare body GV-SOLAS (www.gv- solas.de). All transgenic animals were on C57BL / 6N background. Mice were fed with a standard laboratory diet and given autoclaved tap water ad libitum. They were kept in an air-conditioned room with controlled temperature (20-24°C), humidity (45-65%), and day / night cycle (12 h light, 12 h dark). Laparotomy was performed predominantly on male mice at least at 10-12 weeks of age using isoflurane inhalation narcosis as known in the art. In order to perform 70% partial hepatectomy, the right upper lobe, left upper lobe and left lower lobe of liver together with the gallbladder was resected via one-step ligature using 5-0 polyester suture tie (B. Braun Surgical, S.A., Rubi, Spain). Thereafter, the abdominal cavity and outer layer of skin was closed by 5-0 polyglycolic acid (HR13, B. Braun Surgical, S.A., Rubi, Spain) and 4-0 polypropylene monofilament (DS16, B. Braun Surgical, S.A., Rubi, Spain) respectively. In order to reduce the mild pain from operation mice were treated with 5 mg / kg Carprofen (Rimadyl; Pfizer, Wurselen, Germany) after surgery. IL-6R' / _(IL-6R knock out) mice were subjected to 70% partial hepatectomy. At specific time points (0, 12, and 24 h) after surgery mice were weighed and anesthetized (100 mg / kg ketamine, 10 mg / kg xylazine; Vetoquinol GmbH, Ravensburg, Germany). Upon anesthesia mice were bled in order to generate the serum for further analysis. For the liver tissue, liver was rinsed with phosphate-buffered saline (PBS) and weighed to calculate liver weight to body weight ratio and tissue samples were stored at -80°C for histology and RNA and protein extraction.

[0140] GIL-11 expression, purification and injection into mice

[0141] GIL-11 was produced and secreted by Expi293 cells (Thermo Fisher) and purified by Strep-Tag affinity chromatography (Strep-TactinXT 4flow; IBA, catalog no. 2-5023-001) according to the manufacturer’s manual. In order to force cytokine signaling, mice were injected intraperitoneally (i.p.) with 20 pg GIL-11 24 h before and directly after surgery.

[0142] Gene expression analysis

[0143] Total RNA was extracted from liver and spleen using Trizol (Thermo Fisher Scientific, Waltham, MA, USA). RNA concentration was measured with NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA, cat. #172-5140) and adjusted to 100 ng / pl for all samples. To determine the expression of specific genes, iTaq™ Universal SYBR green One-Step Kit (BioRad, California, USA, catalog no. 1725151) was used. Master Mix was prepared according to the manufacturer’s instructions. 5 pl of iTaq universal probe reaction mix (2x), 0.125 pl of iScript advanced reverse transcriptase, 0.125 pl of primers and 200 ng of RNA was used. The total volume of the mixture was then adjusted to 10 pl by adding Nuclease-free H2O. For analysis, the expression levels of all target genes were normalized to glyceraldehyde 3-phosphate dehydrogenase (gapdh) expression (ACT). Gene expression values were the calculated based on the AACt method. Relative quantities were determined using the equation: RQ=2'AACt. The expression level of target genes was determined by ABI 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA)).

[0144] The following primer pairs were used in this study:

[0145] GAPDH fw: 5' TCCCACTCTTCCACCTTCGA (SEQ ID NO: 15)

[0146] GAPDH rev: 5' AGTTGGGATAGGGCCTCTCTT (SEQ ID NO: 16)

[0147] SAA1 fw: 5' GACACCATTGCTGAGCAGGAA (SEQ ID NO: 17)

[0148] SAA1 rev: 5' GGGAGTCCAGGAGCTCTGTAG (SEQ ID NO: 18)

[0149] Ki67 fw: 5' GCCGAGTCTGGCATTGAA (SEQ ID NO: 19)

[0150] Ki67 rev: 5' TTTTCTTTCTTCTTTTGCTGAGG (SEQ ID NQ:20) EGF fw: 5' TTCTCACAAGGAAAGAGCATCTC (SEQ ID N0:21)

[0151] EGF rev: 5' GTCCTGTCCCGTTAAGGAAAAC (SEQ ID NO:22)

[0152] Cyclin A2 fw: 5' GAGGTGGGAGAAGAATATAA (SEQ ID NO:23)

[0153] Cyclin A2 rev: 5' ACTAGGTGCTCCATTCTCAG (SEQ ID NO:24)

[0154] G0S2 fw: 5' TCTCTTCCCACTGCACCCTA (SEQ ID NO:25)

[0155] G0S2 rev: 5' TCCTGCACACTTTCCATCTG (SEQ ID NO:26) aSMA fw: 5' CTGACAGAGGCACCACTGAA (SEQ ID NO:27) aSMA rev: 5' CATCTCCAGAGTCCAGCACA (SEQ ID NO:28)

[0156] Modeling

[0157] Protein models were generated via the Phyre2 web portal (Kelley, L., et al., The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc, 2015. 10(6): p. 845- 58.). Complex models and structure based sequence alignments were generated using UCSF Chimera version 1.13.1 , developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41- GM 103311 (Pettersen, E., et al., UCSF Chimera-a visualization system for exploratory research and analysis. J Comput Chem, 2004. 25(13): p. 1605-12)

[0158] Statistical analyses

[0159] Data are provided as arithmetic means ± SEM using GraphPad Prism, Version 8. Statistically significant differences between two groups were determined with a Student’s f-test, including Welch’s correction if indicated. Statistically analysis between several groups were determined using a two-way ANOVA, including Tukey correction. Significance was calculated as follows p > 0.05: n.s.; p < 0.05: *; p < 0.01 : **; p < 0.001 : ***; p < 0.0001 :

[0160] Example 2 - Generation of synthetic cytokines based on IL-6 or IL-11

[0161] Natural IL-11 binds to the a receptor IL-11 R via site I to allow the recruitment of two gp130 p receptors via site II and site III. Similarly, natural IL-6 binds to the a receptor IL-6R via site I to allow the recruitment of two gp130 receptors via site II and site III. Of note, LIF does not require an a receptor to bind to its p receptor combination of gp130 and LIFR. Binding of LIF to LIFR is, however, also facilitated by an identically located binding site III in IL-11 or IL-6 to gp130.

[0162] We hypothesized that the structure-based exchange of the partitioned binding site III of human IL-11 or IL-6 with site III of human LIF will render the resulting chimeric cytokine into a binder of the non-natural cytokine receptor composition gp130:IL-11 R:LIFR, or gp130:IL-6R:LIFR a novel synthetic cytokine class which we called cytokimera GIL-11 and GIL-6, respectively. Since p receptor binding of LI F is a receptor independent, it was not predictable if gp130:LIFR recruitment of the chimeric cytokine GIL-11 or GIL-6, respectively, will be IL-11 R or IL-6R dependent.

[0163] Another cytokimera was generated by replacing the binding site III of IL-6 with binding site III of OSM. As LIF, OSM does not require an a receptor to bind to its receptor combination of gp130 and OSMR. Binding of OSM to OSMR is, however, also facilitated by an identically located binding site III in IL-6 to gp130.

[0164] We hypothesized that the structure-based exchange of the partitioned binding site III of human IL-6 with site III of human OSM will render the resulting chimeric cytokine into a binder of the non-natural cytokine receptor composition qp130:IL-6R:QSMR. a novel cytokine class which we called cytokimera GIO-6. Since p receptor binding of OSM is a receptor independent, it was not predictable if gp130:OSMR recruitment of the chimeric cytokine GIL-6 will be IL-6R dependent.

[0165] Cytokimera based on IL-6 were generated by replacing the binding site III of the IL-6 cytokine with the binding site III of the OSM or LIF, so that these chimeras bind gp130, IL- 6R and OSMR or LIFR, hereinafter called GIO-6 or GIL-6. For this purpose, the regions to be exchanged were determined, which led to the corresponding sequences:

[0166] SEQ ID NO:1: human IL-6 VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENN LNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKV LIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALR QM

[0167] SEQ ID NO:2: human LIF SPLPITPVNATCAIRHPCHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFPNNLDKLC GPNVTDFPPFHANGTEKAKLVELYRIVVYLGTSLGNITRDQKILNPSALSLHSKLNATADIL RGLLSNVLCRLCSKYHVGHVDVTYGPDTSGKDVFQKKKLGCQLLGKYKQIIAVLAQAF

[0168] SEQ ID NO:3: human LIF binding site III, first region ILYYTAQGEPFPNNLDKLCGPN

[0169] SEQ ID NO:4: human LIF binding site III, second region TRDQKILNPSALSLHSKL

[0170] SEQ ID NO:5: human LIF binding site III, third region

[0171] TSGKDVFQKKKLGCQLL

[0172] SEQ ID NO:6: cytokimera GIL-6 VPPGEDSKDVAAPH RQPLTSSERI DKQI RYI LDGISALRILYYTAQGEPFPNNLDKLCGPN NNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLTRDQKILNPSALSLHSKLR AVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLTSGKDVFQKKKLGCQLLRSFKEF

[0173] LQSSLRALRQM

[0174] The sequences do not comprise a signal peptide. The underlined regions show the amino acid sequences, which have been replaced to obtain the cytokimera GIL-6.

[0175] SEQ ID NO:1: human IL-6

[0176] VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENN

[0177] LNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKV LIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALR QM

[0178] SEQ ID NO:7: human OSM

[0179] AAIGSCSKEYRVLLGQLQKQTDLMQDTSRLLDPYIRIQGLDVPKLREHCRERPGAFPSEE

[0180] TLRGLGRRGFLQTLNATLGCVLHRLADLEQRLPKAQDLERSGLNIEDLEKLQMARPNILG LRNNIYCMAQLLDNSDTAEPTKAGRGASQPPTPTPASDAFQRKLEGCRFLHGYHRFMH SVGRVFSKWGESPNRSRR

[0181] SEQ ID NO:8: human OSM binding site III, first region

[0182] DPYIRIQGLDVPKLREHCRER

[0183] SEQ ID NO:9: human OSM binding site III, second region EQRLPKAQDLERSGLNIEDLEKL

[0184] SEQ ID NQ:10: human OSM binding site III, third region

[0185] TPTPASDAFQRKLEGCRFL

[0186] SEQ ID NO:11 : cytokimera GIO-6

[0187] VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRDPYIRIQGLDVPKLREHCR

[0188] ERNNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLEQRLPKAQDLERSGLNI

[0189] EDLEKLRAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLTPTPASDAFQRKLEGC RFLRSFKEFLQSSLRALRQM

[0190] The sequences do not comprise a signal peptide. The underlined regions show the amino acid sequences, which have been replaced to obtain the cytokimera GIO-6.

[0191] Cytokimera based on IL-11 were generated by replacing the binding site III of the IL- 11 cytokine with the binding site III of LIF, so that these chimeras bind gp130, IL-11R and LIFR, hereinafter called GIL-11. Structural inspection of site III in IL-11 and LIF guided the design of the cytokimera GIL-11 with framework of IL-11 and an exchange of site III from LIF. IL-11 consists of 199 amino acids, we pinpointed the partitioned site III from amino acids 58-72 for Illa, 111-128 for lllb and 162-179 for lllc. LIF has 202 amino acids with partitioned site III located from amino acids 64-88 for Illa, 119-138 for lllb and 172-189 for lllc. We decided to transfer the complete binding site from LIF to IL-11 , albeit this resulted in somewhat longer than original IL-11 cytokimera GIL-11 of 211 amino acids. Molecular modelling suggested that the transfer of the full site III amino acid stretches should not interfere with overall architecture and folding of the cytokimera GIL-11. For this purpose, the regions to be exchanged were determined, which led to the corresponding sequences:

[0192] SEQ ID NO:12: human IL-11

[0193] GPPPGPPRVSPDPRAELDSTVLLTRSLLADTRQLAAQLRDKFPADGDHNLDSLPTLAMS AGALGALQLPGVLTRLRADLLSYLRHVQWLRRAGGSSLKTLEPELGTLQARLDRLLRRL QLLMSRLALPQPPPDPPAPPLAPPSSAWGGIRAAHAILGGLHLTLDWAVRGLLLLKTRL

[0194] SEQ ID NO:2: human LIF

[0195] SPLPITPVNATCAIRHPCHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFPNNLDKLC GPNVTDFPPFHANGTEKAKLVELYRIVVYLGTSLGNITRDQKILNPSALSLHSKLNATADIL RGLLSNVLCRLCSKYHVGHVDVTYGPDTSGKDVFQKKKLGCQLLGKYKQIIAVLAQAF

[0196] SEQ ID NO:3: human LIF binding site III, first region ILYYTAQGEPFPNNLDKLCGPN

[0197] SEQ ID NO:4: human LIF binding site III, second region TRDQKILNPSALSLHSKL

[0198] SEQ ID NO:5: human LIF binding site III, third region TSGKDVFQKKKLGCQLL

[0199] SEQ ID NO:13: cytokimera GIL- 11

[0200] GPPPGPPRVSPDPRAELDSTVLLTRSLLADTRQLAILYYTAQGEPFPNNLDKLCGPNVTD SLPTLAMSAGALGALQLPGVLTRLRADLLSYLRHVQWITRDQKILNPSALSLHSKLNTLQA RLDRLLRRLQLLMSRLALPQPPPDPPAPPLATSGKDVFQKKKLGCQLLGGLHLTLDWAV RGLLLLKTRL

[0201] The sequences do not comprise a signal peptide. The underlined regions show the amino acid sequences, which have been replaced to obtain the cytokimera GIL-11.

[0202] The cDNA of the corresponding sequences were synthesized externally by BioCat GmbH before they were then cloned into the expression vectors. For the purpose of secretion and purification, a signal peptide was fused at the N-terminal and an Fc-tag (IC7, GIL-6 and GIO-6) or a TwinStrep-(Ts)-tag (GIL-11-Ts) at the C-terminal, since these tags had proven themselves in the corresponding cytokimera.

[0203] The expression of the chimera took place in Expi293 cells, since the proteins showed a high level of expression here. The Expi293 cells were transiently transfected with the plasmid DNA encoding the corresponding synthetic cytokines. The supernatants obtained were either purified immediately or stored at 80° C. for later protein purification. The successful expression was analyzed by Western blot.

[0204] Example 3 - Activity and Specificity of the cytokimera

[0205] Ba / F3 cells were used to study activities and specificity of the cytokimera. These are murine pre-B cells that proliferate with IL-3 in a cytokine-dependent manner. The cells used here also stably express at least human gp130. This allows cells with hyper-IL-11 to proliferate through the IL-11 trans signaling pathway and subsequent JAK / STAT signaling cascade. Thus, HIL-11 served as a positive control since all cells used here proliferate as a result. Cells untreated with cytokines served as a negative control. Furthermore, the receptors still required were stably introduced into the corresponding cells by means of retroviral transduction and checked for surface presentation by flow cytometry. To assess whether the cytokimera are active and specific, these and control cytokines were used to stimulate Ba / F3 cells with different receptor constellations. GIL-11-Ts shows significant activity on Ba / F3 cells expressing gp130, IL-11 R and LIFR, while all other cells do not proliferate with GIL-11-Ts (Figure 1A). As expected, Ba / F3 cells expressing gp130, IL-6R and LIFR proliferated with GIL-6-Fc, GIO-6-Fc as well as IC7-Fc. As expected, Ba / F3 gp130 IL-6R OSMR also proliferated with GIO-6-Fc, while GIL-6-Fc had no effect. Interestingly, however, IC7-Fc also shows significant activity on these cells.

[0206] In order to be able to precisely quantify the activity of the cytokimera, the proliferation of the Ba / F3 cells with various receptors was measured in concentration series and the EC50 values were determined using a non-linear regression (Figure 2, Table 1).

[0207] Cytokine / Ba / F3 EC50 EC50

[0208] Cytokimera receptors (ng / ml) (pM) gp130 IL-6R LIFR 1.12 10.8

[0209] IC7-Fc gp130 IL-6R OSMR 4.80 46.3

[0210] GIL-11-Ts gp130 IL-11 R LIFR 1.44 49.8 GIL-6-Fc gp130 IL-6R LIFR 1.21 11.6 gp130 IL-6R LIFR 6.16 57.8

[0211] GIO-6-Fc gp130 IL-6R OSMR 1.60 15.0

[0212] IL-11 gp130 IL-11 R LIFR 0.72 33.6

[0213] LIF gp130 IL-11 R LIFR 0.07 3.18

[0214] IL-6 gp130 IL-6R LIFR 0.11 4.64

[0215] LIF gp130 IL-6R LIFR 0.08 3.64

[0216] Table 1 : ECso values of the cytokimera and cytokines.

[0217] The EC50 values were determined by non-linear regression of dose-response proliferation curves. Due to the different molecular weights of the proteins, specifying the EC50 in the molar concentration is more suitable for comparability.

[0218] It was found out in proliferation assays with Ba / F3 cells expressing either gp130, IL-6R and LIFR or OSMR that IC7 is characterized by an unexpected cross-reactivity with cells expressing OSMR (Figure 2.1). An EC50 of 10.8 pM was determined for IC7 on Ba / F3- gp130-IL-6r-LIFR cells, while an EC50 of 46.3 pM was found on Ba / F3-gp130-IL-6R-OSMR cells. With an EC50 in the two digit picomolar range the interaction may be relevant in vivo. This cross-reactivity has not been described in the literature. This suggests an inherent affinity of site III residues of CNTF not only to LIFR but also to OSMR. As a consequence, CNTF may be cross-reactive with OSMR.

[0219] Despite considering the low purity in concentration-dependent proliferation, GIL-11-Ts still has an EC50 of 49.8 pM with Ba / F3 cells expressing gp130 IL-11 R LIFR. For comparison, the EC50 values of LIF and IL-11 (3.18 pM and 33.6 pM, respectively) were determined with Ba / F3 gp130 IL-11 R LIFR. At 11.6 pM, GIL-6-Fc showed a comparable EC50 value to IC7-Fc. For comparison, the EC50 values of LIF and IL-6 (3.64 pM and 4.64 pM, respectively) were determined with cells expressing Ba / F3 gp130 IL-6R LIFR. At 15 pM, GIO-6-Fc with Ba / F3 gp130 IL-6R OSMR also shows a comparable EC50 value to IC7-Fc. However, an EC50 value of 57.8 was determined with Ba / F3 gp130 IL-6R OSMR, comparable to IC7-Fc on Ba / F3 gp130 IL-6R OSMR. In summary, it can be said that the EC50 values of the cytokimera are comparable to the recombinant IL-11 , which was previously expressed and purified in previous work in this group, but not to the EC50 values of the purchased cytokines LIF and IL-6 (see Table 1 above).

[0220] Proliferation induced by the cytokimera / receptor complex is induced primarily via the JAK / STAT signal transduction pathway. Therefore, STAT3 phosphorylation in Ba / F3 cells after stimulation with the cytokimera and cytokines was subsequently analyzed to determine activity and specificity. Here, HIL-11 again served as a positive control (Figure 3).

[0221] Consistent with proliferation analysis, GIL-11-Ts shows specific STAT3 phosphorylation only on Ba / F3 gp130 IL-11 R LIFR expressing cells, while it shows no activity on cells with other receptor constellations (Figure 3A). Analogously to the proliferation analysis, GIO-6-Fc and IC7-Fc show STAT3 activation in cells expressing Ba / F3 gp130 IL-6R OSMR but also in cells expressing gp130 IL-6R LIFR. GIL-6-Fc induces STAT3 phosphorylation only in cells expressing Ba / F3 gp130 IL-6R LIFR (Figure 3B).

[0222] In order to assess a concentration dependency in signal transduction, Ba / F3 cells with the corresponding receptors were stimulated with the cytokimera. Here, GIL-11-Ts, GIL-6- Fc, GIO-6-Fc and the corresponding controls in different concentrations were used for stimulation and STAT and ERK phosphorylation were checked in the Western blot.

[0223] Concentration-dependent STAT3 and ERK activation by GIL-11-Ts, GIL-6-Fc, GIO-6- Fc, and control cytokines can be seen in Figure 4. Here, 10 ng / ml GIL-11-Ts are sufficient for STAT3 and ERK phosphorylation. Thus, the activity of the GIL-11 Ts is comparable to IL 11 in Ba / F3 gp130 IL-11 R LIFR in terms of signal transduction (Figure 4A). At 20 ng / ml, GIL-6-Fcjust shows a clear STAT3 phosphorylation and is therefore more active than GIO- 6-Fc, which at 200 ng / ml still has a solid STAT3 activation and at 20 ng / ml an ERK Activation shows (Figure 4B).

[0224] In summary, the cytokimera are active and specific but slightly less active compared to the recombinant cytokines that make up the chimera. Furthermore, it can be said that the intensity of the signal transduction runs at least via the JAK / STAT and the MAPK / ERK signaling pathway and is concentration-dependent in the Ba / F3 cells.

[0225] Furthermore, taking into account the limited receptors used here, a higher specificity of the cytokimera could be shown, since they require one more receptor for signal transduction than the original cytokines. However, promiscuity was also shown in IC7-Fc (gp130 / IL-6R / OSMR) and GIO-6-Fc (gp130 / IL-6R / LIFR). In the case of GIO-6-Fc, this cross-reactivity is as expected since the binding site-ill of the substituted human OSM can also bind to the human LIFR. In the case of IC7-Fc, this cross- reactivity was surprising as it has not been previously reported in the literature. The fact that IC7-Fc but not GIL-6-Fc activates cells expressing gp130 / IL-6R / OSMR suggests that CNTF binding site III binds to the OSMR. To demonstrate this, Ba / F3 cells expressing gp130 / CNTFR / OSMR are required.

[0226] Example 3 - GIL-11 induces Jak / STAT signaling and cellular proliferation via the non-natural gp130:IL-11 R:LIFR cytokine receptor complex.

[0227] Proliferation of the murine pre-B cell line Ba / F3 is IL-3 dependent as is known in the art. After introduction of gp130 plus additional family receptors, proliferation is shifted to the respective IL-6 type cytokine receptor combination. In case of Ba / F3-gp130 cells, proliferation is induced by IL-11 and the soluble IL-11 R or by the corresponding fusion protein Hyper IL-11 (HIL-11), additional introduction of IL-11 R renders these cells IL-11 dependent. Co-expression of gp130 and IL-6R renders these cells responsive to IL-6. Ba / F3 cells expressing gp130 and LIFR proliferate with LIF and OSM, whereas gp130 and OSMR expressing Ba / F3 cells are responsive to OSM. Using the Ba / F3 cell repertoire with the eight different receptor combinations gp130, gp130: 1 L-11 R, gp130: LI FR, gp130:OSM R, gp130:IL-11 R:OSMR, gp130:IL-6R:LIFR and gp130:IL-11 R:LIFR, the proliferation was determined after addition of IL-11 , HIL-11 , OSM, LIF and GIL-11. GIL-11 specifically induced proliferation of Ba / F3-gp130:IL-11 R:LIFR cells but not of any other tested cell line, suggesting that GIL-11 signals via gp130: 1 L-11 R:LIFR (Figure 1A). As expected, only HI L- 11 induced proliferation of all Ba / F3 cells lines, since they all express gp130. All Ba / F3 cells expressing gp130 and IL-11 R proliferated with IL-11. Expression of gp130 and LIFR resulted in LIF and OSM-induced proliferation, whereas cells expressing gp130 and OSMR cells were OSM selective. Next, phosphorylation of STAT3 which is the major hallmark of IL-6 type cytokine Jak / STAT signaling in our Ba / F3 cell portfolio, was analyzed. As expected from the cytokine-induced Ba / F3 cell proliferation assays (Figure 1A), STAT3 phosphorylation in Ba / F3 cells was induced by the following cytokine:cytokine receptor combinations: HIL-11 via gp130; IL-11 via gp130:IL-11 R; OSM via gp130:OSMR; OSM and LIF via gp130:LIFR (Figure 3A). Importantly, sustained STAT3 phosphorylation for GIL-11 was only observed in Ba / F3 cells expressing the receptor combination gp130, I L-11 R and LIFR (Figure 3B). Taken together, it was shown that GIL-11 specifically activated signaling via the non-natural receptor complex consisting of gp130:IL-11 R:LIFR.

[0228] Example 4 - Biological activity of GIL-11 is comparable to IL-11 and LIF

[0229] Next, dose-dependent proliferation were performed to determine the quality and capacity of GIL-11. Again, GIL-11 did not induce proliferation of Ba / F3 cells expressing gp130, gp130:IL-11 R, gp130:LIFR, gp130:OSMR, gp130:IL-11 R:OSMR even at the highest applied concentration of 500 ng / ml, whereas proliferation of Ba / F3-gp130:IL-11 R:LIFR cells was clearly dose-dependent with an EC50 of 1.44 ng / ml (Figure 2B). For comparison, the ECso for LIF and IL-11 was determined to be 0.074 ng / ml and 0.72 ng / ml on Ba / F3-gp130:IL- 11 R:LIFR cells (Figure 2E,F). To analyze STAT3 and ERK phosphorylation, Ba / F3- gp130: 1 L-11 R:LIFR cells were stimulated with increasing amounts of GIL-11 , IL-11 and LIF. Western blotting showed that 10 to 100 ng / ml of GIL-11 and IL-11 were needed to achieve maximal STAT3 and ERK phosphorylation, whereas biological activity of LIF was higher with 0.1 to 1 ng / ml needed for maximal STAT3 phosphorylation (Figure 4A). With respect to the activation of the other STATs in Ba / F3-gp130:IL-11 R:LIFR cells, phosphorylation of STAT1 , 3, 5 and 6 was assessed after stimulation with HIL-11 , IL-11 , OSM, LIF, and GIL- 11. Via gp130, all IL-6-type cytokines efficiently activate STAT3, but only to a minor extent STAT1 and STAT5. In the case of LIF and OSM, STAT3 and STAT1 , as well as STAT5, activation was observed. Here, all cytokines induced sustained STAT3 phosphorylation. Interestingly, HIL-11 and IL-11 did not induce STAT 1 and STAT5 phosphorylation, whereas LIF, OSM, and GIL-11 also induced STAT1 phosphorylation (Figure 5). STAT5 phosphorylation was only seen for GIL-11. As expected, none of the cytokines induced STAT6 phosphorylation. Taken together, the activity of GIL-11 is in the same concentration range as the precursor cytokine IL-11 , demonstrating that the expansion of the receptor requirement by site III transfer did not affect the overall biological cytokine activity.

[0230] Example 4 - The synthetic cytokines of the invention are poor inducers of transsignaling

[0231] While protective, regenerative, and anti-inflammatory processes tend to be ascribed to the classical signaling pathway, immune system-activating and thus pro-inflammatory processes are ascribed to the trans signaling pathway. For this reason, the behavior of the cytokimera with regard to the various signal transduction pathways was characterized. Since GIL-11-Ts is based on an IL-11 scaffold in which binding site-ill has been swapped, it was logical that it should still be able to form a complex with slL-11 via binding site-l to stimulate and Ba / F3 gp130 LIFR expressing cells. Analogous to this was the hypothesis that the IL-6-based cytokimera IC7-Fc, GIL-6-Fc and GIO-6-Fc, in which the binding site III was exchanged, also form complexes with slL-6 and subsequently bind to the membranebound gp130 and LIFR and consequently lead to trans signal transduction.

[0232] In the following, the proliferation assays were measured with Ba / F3 gp130 LIFR after adding a constant concentration of slL-6 or slL-11 depending on the cytokimera concentration (Figure 6).

[0233] Although IL- 11 forms a complex with 100 ng / ml si L-11 R and leads to proliferation via the trans signaling pathway with an EC50 of 71.6 ng / ml, GIL-11-Ts is hardly able to do so. The proliferation only begins at very high concentrations, so that saturation cannot be reached and the EC50 value cannot be determined (Figure 6A, B).

[0234] Similarly, IL-6 along with 200 ng / mL slL-6R shows solid trans-activation with an EC50 of only 1.04 ng / mL, while the cytokimera IC7-Fc and GIL-6-Fc require at least 250 ng / mL to induce a first proliferation signal (Figure 6C,D,E). GIO-6-Fc is unable to mediate the trans signaling pathway at the concentrations used here (Figure 6F).

[0235] The cytokimera's lack of a solid trans-mediated signaling pathway was further investigated in GIL-11-Ts as an example. As known in the art, olamkicept (sgp130-Fc) inhibits the IL-6 / slL-6R or I L-11 / sl L-11 R complex. Therefore, it was investigated whether the trans-signaling, which originates from GIL-11-Ts / slL-11 R, can be inhibited. Sufficiently high concentrations were chosen to force trans signaling (Figure 7).

[0236] Sufficiently high concentrations of GIL-11-Ts:slL-11 R forced the trans-signaling pathway in both the stimulation and proliferation assays. In both experiments, however, inhibition of stimulation or proliferation was not possible (Figure 7A, B). In contrast, sgp130-Fc reliably inhibited the IL-11 :slL-11 complex with an IC50 of 22.5 ng / mL (Figure 7B).

[0237] In summary, it can be said that the cytokimera only initiate signal transduction via the trans-signal path to a very limited extent in comparison to the natural cytokines (IL-6 or IL11) due to the exchange of binding site III. Furthermore, this trans signaling cannot be inhibited by sgp130-Fc.

[0238] Example 5 - GIL-11 :slL-11R complexes are poor inducer of trans-signaling via gp130:LIFR

[0239] The a receptor dependent cytokines IL-6 and IL-11 activate cells via the signal transducing receptor gp130 and the non-signaling membrane bound IL-6R or IL-11 R, respectively. Here, it was shown that GIL-11 signals via the membrane-bound IL-11 R in complex with the heterodimeric gp130:LIFR complex. Signaling via the membrane-bound IL-11 R is called classic-signaling. The IL-11 R also exists as soluble receptor that in complex IL-11 activates cells lacking membrane-bound a receptor expression in a process called trans-signaling. Here, it was analyzed to what extent GIL-11 induced trans-signaling in complex with si L-11 R on cells expressing gp130 and LI FR. Ba / F3-gp130: LI FR cells were stimulated with a fixed concentration of 100 ng / ml slL-11 R and increasing concentrations of GIL-11 (2 to 2000 ng / ml). For comparison the same fixed concentration of slL-11 R and increasing cytokine concentrations for IL-11 were used. Whereas the EC50 for IL-11 was 71.6 ng / ml for 100 ng / ml slL-11 R (Figure 6A), GIL-11 hardly induced proliferation via sIL- 11 R. It was not possible to calculate an EC50 value, because even concentrations of 2000 ng / ml GIL-11 were not sufficient to reach maximal cellular proliferation (Figure 6B). Therefore, the intracellular signal transduction in Ba / F3-gp130:LIFR cells stimulated with comparably high concentrations of 250 ng / ml GIL-11 and 500 ng / ml si L-11R was evaluated. Here, Gl L-11 :sl L-11 R complexes resulted in sustained STAT3 activation. sgp130Fc is a selective IL-6 / IL-11 trans-signaling inhibitor, which inhibits LIF signaling at least 100-1000 fold less efficient than IL-6 / IL-11 trans-signaling. Next, it was tested if sgp130Fc inhibits GIL-11 trans-signaling. Sgp130Fc is the highly effective inhibitor of IL-11 trans-signaling, but only a poor inhibitor of LIF, as is known in the art. As shown in Figure 7A, sgp130Fc (10 pg / ml) did not inhibit STAT3 phosphorylation induced by GIL-11 (1 pg / ml):slL-11 R (2 pg / ml) trans-signaling in Ba / F3-gp130:LIFR cells (Figure 7A). For Ba / F3-gp130:LIFR cells, concentrations for GIL-11 (0.5 pg / ml) or IL-11 (0.5 pg / ml) plus si L-11 R (1 pg / ml) were chosen to allow cellular proliferation via trans-signaling. Titration of increasing concentrations of sgp130Fc resulted in inhibition of IL-11 trans-signaling (IC5o=22.5 ng / ml, Figure 7B), whereas even the highest concentration of 10 pg / ml sgp130Fc was not able to inhibit GIL-11 trans-signaling. HEK293 cells express gp130 and LIFR but lack IL-11 R expression. Therefore, HEK293 cells are responsive to HIL-11 and LIF but not to IL-11 (Figure 7C). Stimulation of HEK293 cells with GIL-11 (500 ng / ml) in combination with sIL- 11 R (1 pg / ml) induced sustained STAT3 phosphorylation in HEK293 cells, whereas GIL-11 alone did not. Co-stimulation of GIL-11 and si L-11 R with sgp130Fc (3 pg / ml) did not inhibit GIL-11 trans-signaling (Figure 7C). Like LIF, GIL-11 trans-signaling is not inhibited by sgp130Fc at least under conditions that are sufficient to block IL-11 trans-signaling. As shown in Figure 1A, proliferation of Ba / F3-gp130:LIFR and Ba / F3-gp130:IL-11 R was induced by LIF and IL-11 , respectively, but not by GIL-11. It can, however, not be excluded that GIL-11 binds to I L-11 R:gp130 on Ba / F3-gp130:IL-11 R cells and blocks IL-11 signaling or to LIFR on Ba / F3-gp130:LIFR cells and blocks LIF signaling. Using cytokine coincubation for Ba / F3-gp130:IL-11 R with IL-11 and GIL-11 and for Ba / F3 gp130:LIFR with LIF and GIL-11 did not result in inhibition of cellular proliferation even at a 20fold molecular excess of GIL-11 over IL-11 and a 200 fold molar of GIL-11 excess over LIF (Figure 7D). It is therefore concluded that GIL-11 did not interfere with IL-11 and LIF signaling at least for the concentration range tested.

[0240] Example 6 - Human GIL-11 activates signal transduction via the murine gp130:IL- 11 R:LIFR cytokine receptor complex

[0241] Human IL-11 and LIF are cross-reactive between mice and men, as is known in the art. It was analyzed if human GIL-11 also activates murine cells expressing murine gp130: 1 L-11 R:LIFR chains. The murine myoblast cell line C2C12 was chosen. C2C12 cells were stimulated with human HIL-11 (200 ng / ml), human IL-11 (200 ng / ml), human LIF (10 ng / ml) and GIL-11 (200 ng / ml). HIL-11 and LIF induced sustained STAT3 phosphorylation whereas IL-11 and GIL-11 did not, suggesting that C2C12 cells express gp130 and LIFR but lack the expression of IL-11 R (Figure 8A). C2C12 cells were transfected with a cDNA coding for murine IL-11 R, making these cells responsive to IL-11 and GIL-11 as shown by STAT3 phosphorylation (Figure 8A). Next, 5, 10 and 20 pg GIL-11 were injected intraperitoneally into wild-type mice. 30 min after injection mice were sacrificed and heart, liver and spleen tissue were removed. Analysis of STAT3 phosphorylation by Western blotting showed that at least 10 pg / ml GIL-11 was sufficient to induce sustained STAT3 phosphorylation in heart, liver and spleen (Figure 8B). Taken together, it was demonstrated that human GIL-11 activates the murine receptor combination gp130:IL-11 R:LIFR.

[0242] Example 7 - GIL-11 rescued IL-6R deficient mice from death following partial hepatectomy.

[0243] Interleukin-6 (IL-6) is critically involved in liver regeneration following partial hepatectomy (PHX). IL-6 and IL-6R deficient mice have a high mortality rate of 40-80% versus 10% in wild-type mice accompanied by decreased STAT3 phosphorylation and diminished proliferation of hepatocytes, as is known in the art. It was previously shown that Hyper IL-6 (HIL-6) injection 24 h before and directly after surgery rescued mice from death following partial hepatectomy. Here, 10 pg / mouse GIL-11 was injected 24 h before and directly after PHX in IL-6R deficient mice. Overall survival rate of IL-6R deficient mice 9 days after PHX was about 40%, whereas IL-6R deficient mice injected with two doses of GIL-11 had a survival rate of 90% (Figure 8C). Body weight and liver weight to body weight ratio 9 days after PHX was not different in surviving IL-6R' / _mice, irrespective if injected with GIL-11 or PBS (Figure 8D,E). It was notable, however, that untreated IL-6R' / _mice had a bigger spleen to body weight ratio compared to GIL-11 treated IL-6R'Amice (Figure 8F). Gene expression analysis showed increase expression of fibrotic marker a-SMA and acute phase response gene SAA1 in IL-6R' / _mice treated with GIL-11 compared to untreated IL-6R' / _mice 9 days following PHX (Figure 8G). Taken together, it was shown that GIL-11 rescued IL-6R deficient mice from death following partial hepatectomy.

[0244] Our experiments define a human chimeric designer cytokine that induces family-typical Jak / STAT signaling and cellular proliferation via the non-natural gp130:IL-11 R:LIFR complex with cross-species specificity from mouse to men. The exchange of site III from LIF to IL-11 results in the cytokimera GIL-11 with receptor binding properties thus far not found in nature. Since the transfer is restricted to site III, it remains to be seen whether transfer of site I and site II will also be feasible. GIL-11 has unique features which separates it from IC7. First of all, IC7 recruits the receptor complex gp130:IL-6R:LIFR, whereas GIL- 11 assembles gp130:l L-11 R: LI FR, meaning that only those cells expressing gp130, LIFR and IL-11 R are targeted by GIL-11. In a way the chimeric cytokine can be regarded as a synthetic LIF variant restricted to targeting the pool of cells expressing both the LIFR and the IL-11 R.

[0245] Moreover, it will be interesting to compare the transcriptomic profiles of IC7, GIL-11 and LIF. It can be assumed that they are identical, since IC7, GIL-11 and LIF recruit and activate gp130 and LIFR heterodimers, whereas the transcriptomic profile should be clearly separated from IL-6 and IL-11 , which recruit gp130 homodimers. While the transcriptomic profile is expected to be similar, the pool of cells that is activated by IC7, GIL-11 and LIF is expected to be different. Secondly, the source cytokines of IC7, IL-6 and CNTF, are both dependent on a-receptor binding before binding to gp130 and LIFR, meaning that shaping of the CNTF-derived binding site III in IC7 is a direct consequence of IL-6R binding, as after binding of CNTF to CNTFR. This situation is different for GIL-11. Here, we introduced the a-receptor independent binding site III from LIF into the a-receptor dependent IL-11. As shown here, GIL-11 activates the gp130:LIFR receptor complex only after binding to the non-signal transducing IL-11 R. Moreover, GIL-11 was not able to inhibit LIF signaling on Ba / F3-gp130:LIFR cells, which should have been the case, if GIL-11 can bind to LIFR in the absence of the IL-11 R. Taken together our experiments showed that albeit the original binding site context of LIF-site III to LIFR is a-receptor independent, re-formatting of LIF- site III into the IL- 11 scaffold makes the LIF-site III binding a-receptor dependent. Therefore, this is evidence that not the exact binding site III amino acid composition but rather the interconnection mediated by a-helical shifts of site I with site III defines whether a cytokine is a-receptor dependent or independent.

[0246] With respect to biological activity, GIL-11 (EC50: 1.44 ng / ml) is comparable to IL-11 (EC50: 0.72 ng / ml) but less effective than LIF (EC50: 0.074 ng / ml). This might be based on the dominant scaffold effect of IL-11 rather than the minor site III exchange effect from LIF. Biological activity of GIL-11 might, however, be increased by site I D186A mutation, since this amino acid exchange is known to increase the affinity of IL-11 to IL-11 R. Improving cytokine binding to the a-receptor is a common strategy to improve overall activity in this cytokine family, as has also been shown for CNTF to CNTFR and IL-6 to IL-6R.

[0247] After having characterized the biological activity and the unique receptor composition of GIL-11 , we have investigated the ability of GIL-11 to functionally substitute IL-6 during liver regeneration following PHX in IL-6R deficient mice. We and others have previously shown that IL-6 and the IL-6R are critically involved in liver regeneration after PHX, resulting in higher mortality in IL-6R deficient mice. Importantly, HIL-6 rescued mice from death following partial hepatectomy. Moreover, in wild-type mice the combined injection of IL-6 and slL-6R, but not of IL-6 alone, accelerates liver regeneration after PHX. Likely because hepatocytes express much more gp130 than IL-6R, the increased presence of IL-6 and slL6R result in more gp130 activation and stronger IL-6 signaling compared to IL-6 alone.

[0248] Finally, blockade of IL-6 trans-signaling by sgp130Fc results in increased mortality following PHX. Mechanistically, IL-6 trans-signaling induced hepatocyte growth factor (HGF) production by hepatic stellate cells directly contributed to liver regeneration following PHX. Since, the IL-6R deficient mice used in this work lack in the expression of IL-6R, both classic- and trans-signaling are disabled. Thus, they exhibit a mortality rate of 90% versus 10% in wild-type mice. A crucial difference between GIL-11 treatment and HIL-6 is that GIL- 11 targets only cells expressing gp130:IL-11 R:LIFR, which limits the scope of potential targeted cells in the body, whereas HIL-6 targets almost all cells, because unlike IL-11 R and LIFR, gp130 is considered to be ubiquitously expressed. However, since IL-11 R, LIFR as well as gp130 are expressed on hepatocytes, GIL-11 was able to compensate for IL-6 trans-signaling and rescued IL-6R deficient mice from death following PHX. Interestingly, most parameters including body, liver and spleen weight were not changed in surviving GIL- 11 -treated and untreated mice. GIL-11 , however, increases SAA more than 300-fold following PHX. SAA induce proliferation of hepatic stellate cells, which might contribute to the liver regeneration following GIL-11 application.

[0249] Having shown the general in vitro and in vivo activity, we believe that GIL-11 will be of common interest for conditions, for which IL-6 type cytokines including IC7 show beneficial effects. In mice, IC7 selectively activates metabolic pathways resulting in increased fatty acid oxidation accompanied by prevented steatosis, increased energy dissipation accompanied by weight loss, muscle hypertrophy and preserved lean mass with improved bone stability. In contrast to IL-6 and CNTF, IC7 injection has proven to be safe in both, mice and non-human primate macaques without promoting excess inflammatory responses.

[0250] Recently, the originally described beneficial effects of human IL-11 in murine models of human diseases has been challenged. It was stated that the mode of action of injected human IL-11 into mice actually relies on competitive inhibition of endogenous IL-11 signaling. It was concluded that IL-11 has rather detrimental than beneficial effects in a variety of murine disease models, including non-alcoholic steatohepatitis, cardiovascular fibrosis, idiopathic pulmonary fibrosis and fibrotic lung disease.

[0251] Interestingly, IL-11 was considered to preferentially induce ERK signaling and not, like IL-6, which acts via the same gp130 homodimer, STAT3 phosphorylation. Therefore, we treated murine myoblasts (C2C12 cells) with recombinant HIL-11 , IL-11 and GIL-11 in the presence and absence of murine IL-11 R, which, however, resulted in sustained IL-11 R- dependent STAT3 phosphorylation. Injection of GIL-11 into mice also resulted in sustained STAT3 phosphorylation in heart, liver and spleen tissue, demonstrating that our human cytokines and GIL-11 activate canonical gp130 and gp130:LIFR signaling pathways characterized by STAT3 phosphorylation. For unknown reasons and unlike IL-11 , GIL-11 is only poorly inducing trans-signaling via GIL-11 :slL11 R complexes and is not inhibited by sgp130Fc.

[0252] Although LIF is related to fertility and a series of neurological disorders including multiple sclerosis, recombinant LIF is not used as therapeutic. GIL-11s ability to induce LIF- like signaling via gp130:LIFR complexes might open new LIF-like applications as a as potential surrogate for recombinant hLIF. Since GIL-11 activity need cells not only expressing LIFR but also the IL-11 R, GIL-11s activity is restricted to a lower limited number of target cells compared to LIF which might alleviate potential unwanted negative side effects.

[0253] In conclusion, our study defines GIL-11 as a novel promising cytokimera with specific high-affinity activation of the non-natural receptor gp130:LIFR:IL-11 R complex. The modular architecture of cytokimeras in general enables a wide range of targeted receptor combinations and directed cell targeting.

[0254] Example 8 - Structural design of the cytokimeras GIL-6 and GIO-6.

[0255] Using structure-based modelling with IL-6 as a backbone, two novel cytokimera GIL- 6, and GIO-6 were designed with an exchanged site 3 from LIF, and OSM, respectively (see also Example 1). The site 3 is partitioned and consists of amino acids residues of the C-terminal a-helix and a N-terminal loop connecting helices A and B (site 3-1), the BC loop (site 3-2) and the C-terminal loop connecting helices C and D in addition the N-terminal part of helix D (site 3-3), which mediates binding of IL-6 to gp130, of LIF to LIFR and of OSM to LIFR or OSMR. Human IL-6 consists of 212 amino acids including signal peptide. The partitioned site 3 of IL-6 was defined from amino acids R68-N88 as site 3-1 , from L129- R141 as site 3-2 and from L179-R196 as site 3-3. LIF consists of 202 amino acids with site 3 located from amino acids F63-V86 for site 3-1 , I119-N138 for site 3-2 and D171-G189 for site 3-3. OSM has 221 amino acids with site 3 from amino acids L56-P78 for site 3-1 , LUS- QI 37 for site 3-2 and P176-H196 for site 3-3. The novel cytokimeras were larger than the natural cytokines with 240 amino acids for GIL-6, and 229 amino acids for GIO-6.

[0256] In silico modelling using structure-based alignment suggested that the transfer of the complete site 3 should not interfere with overall architecture. While IL-6 forms a hexameric receptor complex with 2xlL-6:2xlL-6R:2xgp130, the a-receptor-independent signaling complexes for LIF and OSM, on the other hand, are trimeric, with LIF:gp130:LIFR, OSM:gp130:OSMR and OSM:gp130:LIFR. The cytokimeras GIL-6 and GIO-6 will, however, specifically form tetrameric complexes with IL-6R as a-receptor consisting of GIL- 6:IL-6R:gp130:LIFR, GIO-6:IL-6R:gp130:LIFR and GIO-6:IL-6R:gp130:LIFR.

[0257] Example 9 - Via non-natural cytokine receptor complexes, cytokimera GIL-6 and GIL- 6 promote signal transduction and cellular proliferation.

[0258] IL-3 is required to induce proliferation of the murine pre-B cell line Ba / F3, as is known in the art. After the introduction of the coding cDNA for human gp130 and additional receptors of the family, including IL-6R, IL-11 R, OSMR, LIFR, and CNTFR (Figure 13), proliferation of Ba / F3 cells became responsive to the respective IL-6-type cytokine receptor combinations.

[0259] To determine signaling properties of the cytokimeras GIL-6 and GIO-6, a Ba / F3 cell collection expressing the human IL-6-type receptors gp130, IL-6R:gp130, gp130:OSMR, gp130:LIFR, IL-6R:gp130:OSMR, IL-6R:gp130:LIFR, CNTFR:gp130:LIFR, and CNTFR:gp130:OSMR was used. GIO-6 induced proliferation of cells expressing IL- 6R:gp130:LIFR as well as IL-6R:gp130:OSMR, whereas GIL-6 exclusively induced proliferation of Ba / F3-IL-6R:gp130:LIFR cells (Figure 9A). Hyper IL-6 (soluble IL-6RJL-6 fusion protein), IL-6, LIF, OSM and the cytokimera IC7 were used as controls to verify receptor specificity of the Ba / F3 cell repertoire.

[0260] As expected, Hyper IL-6 induced proliferation of all cell lines because of the general expression of gp130. After the addition of IL-6R, the derivative cell lines proliferated with IL-6. LIFR:gp130-expressing cell lines proliferated with LIF, and OSM, while OSM induced additionally proliferation of OSMR:gp130 cells. The IL-6 / CNTF-hybrid cytokine IC7 induced proliferation of Ba / F3-IL-6R:gp130:LIFR cells but unexpectedly also of Ba / F3-IL- 6R:gp130:OSMR cells, suggesting a novel receptor complex for IC7 (Figure 9A). Next, the STAT3 phosphorylation induced by cytokimeras GIL-6, GIO-6 and IC7 and the cytokines HIL-6, IL-6, LIF and OSM in the Ba / F3 cell collection were analyzed. Importantly, STAT3 phosphorylation in Ba / F3 cells (Figure 9B) after cytokine stimulation mirrored the data obtained in proliferation assays (Figure 9A). STAT3 phosphorylation was observed in Ba / F3 cells expressing gp130 after stimulation with HIL-6; IL-6R:gp130 with IL-6; gp130:LIFR with LIF and gp130:LIFR or gp130:OSMR with OSM. IC7 and GIO-6 induced STAT3 phosphorylation in IL-6R:gp130:LIFR and IL-6R:gp130:OSMR expressing Ba / F3 cells, whereas GIL-6 induced STAT3 phosphorylation solely in Ba / F3-IL-6R:gp130:LIFR cells (Figure 9B). To characterize the primary signaling pathways in more detail, Ba / F3-IL- 6R:gp130:LIFR cells were used, because every cytokine and cytokimera that was tested had an effect on this cell line. Phosphorylation of STAT 1 , 3, and 5, as well as ERK, and Akt phosphorylation was assessed after stimulation with HIL-6, IL-6, LIF, OSM, GIL-6, GIO-6, and IC7 (Figure 9C). As expected, all cytokines and cytokimeras induced STAT3, ERK, and Akt phosphorylation. The strongest STAT1 phosphorylation was seen for IL-6, LIF, GIO-6, and IC7 but also for HIL-6, OSM and GIL-6, albeit to a lesser extent. Weak STAT5 phosphorylation was seen for LIF, GIO-6, GIL-6, and IC7, largely pointing to a LIFR- mediated STAT1 and STAT5 phosphorylation in comparison to signaling solely through gp130 (Figure 9C). Human IL-6, LIF, CNTF, OSM and IC7 are species cross-reactive in mice. Therefore, 10 pg GIL-6 or GIO-6 were injected intraperitoneally into wild-type C57BL / 6N mice. Heart, spleen, and liver were analyzed for STAT3 phosphorylation. GIL-6 as well as GIO-6 led to STAT3-phosphorylation in the liver, whereas only weak STAT3 phosphorylation was observed in the spleen and no signal in the heart (Figure 9D).

[0261] Taken together, our data demonstrate that GIL-6, and GIO-6 are biologically active in mice as well as on human receptors and induce signal transduction in cells expressing IL- 6R:gp130:LIFR, and IL-6R:gp130:OSMR, respectively. Moreover, the previously described cytokimera IC7 also induced signaling via IL-6R:gp130:OSMR complexes. These novel findings make GIL-6 the first IL-6R:LIFR-selective cytokimera.

[0262] Example 10 - GIL-6 and GIO-6 are cytokimera with biological activity similar to that of natural cytokines

[0263] To unravel the biological properties of GIL-6, and GIO-6, the dose-dependent proliferation was determined. Consistent with previous activity assays (Figure 9A,B), GIL- 6 did not induce proliferation of Ba / F3 cells expressing any receptor combination, even with the highest applied concentration of 1000 ng / ml, except for IL-6R:gp130:LIFR. For Ba / F3- IL-6R:gp130:LIFR cells, an EC50 of 1.21 ng / ml was determined for GIL-6, which is above the commercially purchased cytokines IL-6 (EC50: 0.11 ng / ml), and LIF (EC50: 0.08 ng / ml) but in the same range of IC7 (EC50: 1.11 ng / ml) (Figure 10A,B).

[0264] GIO-6 efficiently induced proliferation of Ba / F3 cells expressing IL-6R:gp130:OSMR with an EC50 of 1.60 ng / ml and of Ba / F3-IL-6R:gp130:LIFR with an EC50 of 6.16 ng / ml, which is less efficient compared to IL-6, OSM and LIF (EC50: 0.11 , 0.12 ng / ml and 0.08 ng / ml, respectively) but also in the same range as IC7 (EC50: 1.21 ng / ml for IL- 6R:gp130:LIFR and 4.8 ng / ml for IL-6R:gp130:OSMR) (Figure 10A,C,D).

[0265] Next, STAT3 and ERK phosphorylation were analyzed after stimulation of Ba / F3-IL- 6R:gp130:LIFR cells with increasing concentrations of IL-6, LIF, GIL-6 and GIO-6 (0.2, 2, 20 and 200 ng / ml). Western blotting showed that 20 ng / ml GIL-6, and IL-6 were sufficient to induce STAT3 and ERK phosphorylation; whereas 2 ng / ml LIF and 200 ng / ml GIO-6 were needed for sustained signaling (Figure 10E).

[0266] In order to determine the time response of signal transduction, Ba / F3-IL- 6R:gp130:OSMR and Ba / F3-IL-6R:gp130:LIFR cells were stimulated in intervals up to 240 min with the indicated cytokines including LIF, OSM, IL-6, GIO-6, and GIL-6. As expected, STAT3 phosphorylation was typically induced as early as 5-10 min after cytokine addition and decreased after 120-240 min (Figure 10F), which is likely due to negative feedback by SOCS3.

[0267] Taken together, our data showed that the activity of GIL-6, and GIO-6 is comparable to that of IC7 but weaker than the natural cytokines IL-6, LIF, and OSM. Both GIO-6, and IC7 showed cross-reactivity for LIFR and OSMR.

[0268] Example 11 -The cytokimera GIL-6 and GIO-6 are ineffective trans-signaling inducers.

[0269] IL-6 requires the non-signaling, membrane-anchored IL-6R prior to signal transduction via homodimerization of gp130, which is named classic-signaling. IL-6 can also bind to the soluble IL-6R prior to forming a complex with gp130, which is called trans-signaling. It should be noted that the primary reaction in chronic inflammatory illnesses is IL-6 trans- signaling. So the cytokimeras’ capacity to promote trans-signaling was investigated. Ba / F3 cells expressing gp130:LIFR or gp130:OSMR were stimulated with increasing concentrations of GIL-6 or GIO-6 (0.0005-2000 ng / ml) in the presence and absence of 100 ng / ml soluble IL-6R (slL-6R). For comparison, increasing concentrations of IL-6 were also used. Whereas IL-6:slL-6R complexes induced proliferation of Ba / F3-gp130-LIFR cells in a dose-dependent manner with an EC50 of 5.3 ng / ml for IL-6 and 100 ng / ml slL-6R, GIL- 6 and GIO-6 were not able to induce proliferation of Ba / F3-gp130:LIFR in presence of the 100 ng / ml slL-6R at any concentration tested (Figure 11A, B, C). Interestingly, GIO-6, and IC7 led to a weak proliferation of Ba / F3-gp130:OSMR and Ba / F3-gp130:LIFR cells, respectively, in the presence of the slL-6R at high concentration of GIO-6 above 500 ng / ml (Figure 11 D, E). Our data showed that GIL-6, GIO-6, and IC7 are ineffective trans-signaling inducers.

[0270] Example 12 - CNTF signals via the alternative CNTFR:gp130:OSMR complex.

[0271] IC7 was described to signal via the receptor complex consisting of IL-6R:gp130:LIFR. Here, it was identified that IC7 also signals via the receptor complex consisting of IL- 6R:gp130:OSMR (Figure 9D). It was conceivable to investigate if CNTF additionally interacts with the OSMR via site 3 given that site 3 was transferred from CNTF to IL-6 in IC7. The Ba / F3 cells expressing CNTFR:gp130:LIFR or CNTFR:gp130:OSMR were used to analyze the ability of CNTF to induce cellular proliferation in a dose-dependent manner (0.0002-100 ng / ml). Of note, CNTF induced cellular proliferation of Ba / F3- CNTFR:gp130:LIFR and of Ba / F3-CNTFR:gp130:OSMR cells (Figure 12A). Moreover, a lower EC50 value for CNTF to induce proliferation of Ba / F3 cells expressing CNTFR:gp130:OSMR (EC50: 9.09 pg / ml) was found compared to Ba / F3 cells expressing CNTFR:gp130:LIFR (EC50: 50.15 pg / ml, (Figure 12A). Western blotting revealed STAT3 phosphorylation in Ba / F3-CNTFR:gp130:OSMR cells stimulated with 10 ng / ml CNTF and OSM, but not with LIF (Figure 12B). As depicted in Figure 12C, D, 1 ng / ml CNTF was sufficient to induce STAT3 phosphorylation in Ba / F3-CNTFR:gp130:LIFR and Ba / F3- CNTFR:gp130:OSMR, supporting our finding that CNTF signaling is comparably efficient via OSMR, and LIFR. CNTF is also able recruit the IL-6R as an alternative low-affinity a- receptor in CNTF:IL-6R:gp130:LIFR complexes. Therefore, we tested if CNTF also signals via the IL-6R:gp130:OSMR receptor complex. Ba / F3 cells expressing gp130, CNTFR:gp130:LIFR, CNTFR:gp130:OSMR, IL-6R:gp130:LIFR, or IL-6R:gp130:OSMR were stimulated with 0.5, 5 and 50 ng / ml CNTF, 10 ng / ml LIF, OSM, IL-6 or left untreated. As expected, proliferation of Ba / F3-gp130 cells was not induced by these cytokines (Figure 12E). Proliferation of Ba / F3- CNTFR:gp130:LIFR and Ba / F3-IL-6R:gp130:LIFR cells were, however, observed after stimulation with CNTF, demonstrating the IL-6R low-affinity crosstalk of CNTF. Albeit Ba / F3-IL-6R:gp130:OSMR proliferated after stimulation with OSM via gp130:OSMR complexes and IL-6 via IL-6R:gp130 receptor complex, no proliferation was induced by CNTF even for the highest concentration applied (50 ng / ml) (Figure 12E). STAT3 phosphorylation in Ba / F3-IL-6R:gp130:LIFR and Ba / F3-IL-6R:gp130:OSMR cells was determined after stimulation with 0.1 , 1 , 10 and 100 ng / ml CNTF. Whereas 10 ng / ml CNTF was sufficient to induce STAT3 phosphorylation in Ba / F3-IL-6R:gp130:LIFR cells, also 100 ng / ml CNTF failed to induce STAT3 phosphorylation in Ba / F3-IL-6R:gp130:OSMR cells (Figure 12G,H). Finally, we investigated whether CNTF directly binds to the recombinant OSMR in a cell-free manner via co-immunoprecipitation using purified soluble recombinant proteins. Hyper CNTF-Fc (CNTF fused to the soluble CNTFR and an I gGi Fc- part) was precipitated by Protein A beads after incubation with the soluble OSMR in the presence and absence of soluble gp130. Since, unlike LIF, OSM needs to bind to gp130 via site 2 prior to the recruitment of the OSMR via site 3, it was assumed CNTF binding to the OSMR could also be gp130 dependent. As shown in Figure 12H, OSMR was precipitated by Hyper CNTF-Fc only in the presence of soluble gp130. This indicates that the recruitment of CNTF to OSMR via site 3 is dependent on the previous binding of CNTF to site 2 of gp130. When viewed collectively, our findings show that CNTF can employ OSMR as a different receptor after binding to CNTFR but not after binding to IL-6R. Example 13 - Discussion of experimental results

[0272] IL-6, IL-11 , and CNTF cannot bind to their p-receptors gp130 and / or LIFR in the absence of the a-receptor, suggesting that interaction of the cytokine and the a-receptor pushes binding site 2 and / or 3 into the right conformation to enable p-receptor binding. It was far from clear that the transfer of site 3 from an a-receptor-independent cytokine such as LIF to GIL-11 and GIL-6, or OSM to GIO-6 resulted in cytokimeras that are still a- receptor-dependent because we introduced the a-receptor-independent binding sites 3 from LIF / OSM into the a-receptor-dependent IL-6 and IL-11 backbones. As shown here, GIL-6 and GIO-6 activate the gp130:LIFR and gp130:OSMR receptor complexes only after binding to the non-signaling IL-6R. This adds to evidence that albeit the original LIF / OSM- site 3 to LIFR / OSMR is a-receptor-independent, reformatting of LIF / OSM-site 3 into the IL- 6 scaffold makes the site 3 of LIF and OSM a-receptor-dependent. This suggests that the interconnection mediated by a-helical shifts of site 1 with site 3 determines if a cytokine is a-receptor-dependent.

[0273] With respect to biological activity, GIL-6 (EC50: 1.21 ng / ml for IL-6R:gp130:LIFR) and GIO-6 (EC50: 6.16 ng / ml for IL-6R:gp130:LIFR and EC50: 1.6 ng / ml for IL-6R:gp130:OSMR) are comparable to IC7 (EC50: 1.11 ng / ml for IL-6R:gp130:LIFR and 4.8 ng / ml for IL- 6R:gp130:OSMR) but less effective than the native cytokines IL-6, LIF and OSM (EC50: 0.11 ng / ml IL-6R:gp130 and EC50: 0.08 ng / ml for gp130:LIFR, EC50: 0.12 ng / ml for gp130:OSMR, respectively). The biological activity of GIL-6 and GIO-6 might, however, be increased by the introduction of affinity-enhancing mutations in site 1 to increase the affinity towards IL-6R.

[0274] To directly compare IC7 and GIL-6 activity, the biological properties of IC7 were revisited. IC7 was previously not tested on cells expressing IL-6R, gp130 and OSMR. It was verified here that GIO-6 signals via IL-6R:gp130:OSMR, therefore, IC7 originally considered as negative control was found to also bind and activate cells via IL- 6R:gp130:OSMR, defining a second high-affinity non-natural receptor complex for IC7.

[0275] Consequently, we also tested and verified that CNTF uses CNTFR:gp130:OSMR as the second high-affinity natural receptor complex. According to the human protein atlas, CNTFR is limited to some cells and largely not expressed in immune cells and cardiomyocytes, whereas OSMR and gp130 are more generally expressed. Of note, coexpression of all three receptors is found in breast glandular cells, cholangiocytes, neurons, astrocytes, skeletal myocytes, smooth muscle cells, hepatocytes and fibroblasts. With respect to cancer cell lines, expression of LIFR and OSMR is overlapping and found in almost all cells, whereas CNTFR expression is restricted to only some cell lines. Therefore, -M- it comes as no surprise that OSMR was not discovered as alternative CNTFR previously, because only Ba / F3 cells lack the expression of all IL-6 type cytokine receptors with the exception of endogenous IL-27RA expression. Heterologous expression receptor chains in Ba / F3 cells, therefore, is a valid approach to identify and validate receptor complexes of IL- 6 type cytokines and cytokimera.

[0276] Treatment with the human CNTF derivative Axokine induced enduring weight loss in clinical trials, but it later therapeutically failed due to the rapid development of anti- Axokine / CNTF antibodies for unknown reasons. Recently, IC7 was shown to have CNTF- like effects on weight regulation in mice. However, it remains to be seen if also IC7 provokes anti-IC7 antibodies in men. Whereas CNTF also possessed inflammatory properties, and its administration triggered inflammatory responses including fever and the induction of the acute phase response, proinflammatory effects were not described for IC7 to date.

[0277] Now the question emerged whether the beneficial effects of CNTF and IC7 on weight maintenance is mediated via LIFR or OSMR signaling or both. The cytokimeras GIL-6 and GIL-11 differentiate between LIFR and OSMR signaling and are therefore promising therapeutic candidate proteins. In case the OSMR instead of the LIFR is responsible for the beneficial effects of IC7 / CNTF, an OSMR selective cytokimera could be generated by the transfer of site 3 AB-loop from murine OSM to CNTF / IL-6, which likely will prevent LIFR cross-talk.

[0278] In conclusion, our study showed the development of the two cytokimeras GIL-6 and GIO-6 with specific high-affinity activation of the non-natural receptor IL-6R:gp130:LIFR and IL-6R:gp130:OSMR complexes. Moreover, we showed that the previously described cytokine IC7 and also the natural cytokine CNTF uses OSMR as an alternative p-receptor.

Claims

CLAIMS1. Polypeptide, comprising a) two binding sites derived from the same cytokine of the IL-6 family of cytokines, wherein said cytokine requires a cytokine specific nonsignaling a-receptor subunit as part of the receptor complex to effect signaling, wherein the first binding site is capable of binding the cytokine specific nonsignaling a-receptor subunit, and wherein the second binding site is capable of binding the signaling receptor gp130; and b) a third binding site derived from a further cytokine of the IL-6 family of cytokines, wherein said further cytokine does not require a cytokine specific nonsignaling a- receptor subunit as part of the receptor complex to effect signaling, wherein the third binding site is capable of binding the cytokine specific signaling receptor of the further cytokine.

2. The polypeptide of claim 1 , wherein the cytokine requiring a cytokine specific nonsignaling a-receptor subunit as part of the receptor complex is selected from the group comprising IL-6 and IL-11.

3. The polypeptide of claim 1 or 2, wherein the cytokine not requiring a cytokine specific nonsignaling a-receptor subunit as part of the receptor complex is selected from the group comprising LIF, OSM, and CT-1.

4. The polypeptide of any one of claims 1 to 3, wherein the cytokine requiring a cytokine specific nonsignaling a-receptor subunit as part of the receptor complex is IL-11, and wherein the cytokine not requiring a cytokine specific nonsignaling receptor subunit as part of the receptor complex is LIF.

5. The polypeptide of any one of claims 1 to 3, wherein the cytokine requiring a cytokine specific nonsignaling a-receptor subunit as part of the receptor complex is IL-6, and wherein the cytokine not requiring a cytokine specific nonsignaling receptor subunit as part of the receptor complex is LIF or OSM.

6. The polypeptide of any one of claims 1 to 5, further comprising an affinity tag, optionally at its C-terminal end, optionally an Fc constant region of an immunoglobulin,such as IgG, IgA, or IgM; and / or a signal peptide, optionally at its N-terminal end; and / or a binding site for serum albumin.

7. The polypeptide of any one of claims 1 to 6, comprising an amino acid sequence with at least 90 %, 95 %, 98 %, or 99 % sequence identity to SEQ ID NO: 13, preferably comprising the amino acid sequence of SEQ ID NO: 13; or comprising an amino acid sequence with at least 90 %, 95 %, 98, % or 99 % sequence identity to SEQ ID NO: 6, preferably comprising the amino acid sequence of SEQ ID NO: 6; or comprising an amino acid sequence with at least 90 %, 95 %, 98, % or 99 % sequence identity to SEQ ID NO:11 , preferably comprising the amino acid sequence of SEQ ID NO: 11.

8. The polypeptide of any one of claims 1 to 6, wherein the binding sites are derived from human cytokines.

9. The polypeptide of any one of claims 1 to 8, wherein the capability of the polypeptide to effect trans-signaling is significantly reduced compared to the capability of the cytokine, from which the first two binding sites are derived, to effect trans-signaling.

10. A polynucleotide comprising one or more nucleic acid sequence(s) encoding the polypeptide of any one of claims 1 to 9, wherein optionally the polynucleotide is an expression cassette, preferably an expression vector.

11. An expression system comprising the polynucleotide of claim 10, wherein the expression system can be of eukaryotic, prokaryotic, or archaeal origin, or synthetic in nature, such as a cell-free expression system.

12. A host cell comprising the polynucleotide of claim 10 or the expression system of claim 11, wherein optionally the host cell is selected from eukaryotic cells including hamster cell lines, such as CHO and derivatives thereof, mouse cell lines, such as C127, NS0, SP2 / 0, YB2 / 0, XB2 / 09, and derivatives thereof, or human cell lines, such as HEK and derivatives thereof, for example EXPI293, HT-1080, PER.C6, or HuH-7, monkey cell lines, such as Vero cells and derivatives thereof, and insect cell lines, such as SF-9 and derivatives thereof.

13. Pharmaceutical composition comprising the polypeptide of any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

14. Polypeptide of any one of claims 1 to 9 or pharmaceutical composition of claim 13 for use as a medicament.

15. Polypeptide of any one of claims 1 to 9 or pharmaceutical composition of claim 13 for use in a method of preventing and / or treating a condition in a patient, wherein the condition is selected from the group consisting of lymphopenia, muscular atrophy, osteoporosis, thrombocytopenia, obesity associated metabolic disorders such as type II diabetes mellitus, obesity, insulin resistance, glucose intolerance, dyslipidemia, hypertension, stroke or cardiovascular disease, neurological disorders such as paraplegia, Alzheimer's and Parkinson's disease.

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