Synthetic cytokines from the IL-6 family and their medical uses

Abstract

The present invention relates to a synthetic cytokine from the IL-6 family. The synthetic cytokine is a polypeptide comprising two binding sites from the same cytokine of the IL-6 family and a third binding site from an additional cytokine of the IL-6 family, wherein the additional cytokine does not require a cytokine-specific non-signaling alpha receptor subunit as part of the receptor complex to trigger signal transduction. All three binding sites of the polypeptide of the present invention must bind to their specific partners to trigger signal transduction. The present invention further relates to a polypeptide for use in medical applications, preferably for the prevention and / or treatment of conditions selected from the group consisting of lymphopenia, muscle atrophy, osteoporosis, thrombocytopenia, obesity-related metabolic disorders such as type II diabetes, obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, hypertension, stroke, or cardiovascular disease, neurological disorders such as paraplegia, Alzheimer's disease, and Parkinson's disease.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine and provides synthetic cytokines from the IL-6 family, which may be used in medical applications, preferably for the prevention and / or treatment of conditions selected from the group consisting of lymphopenia, muscle atrophy, osteoporosis, thrombocytopenia, obesity-related metabolic disorders such as type II diabetes, obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, hypertension, stroke, or cardiovascular disease, neurological disorders such as paraplegia, Alzheimer's disease, and Parkinson's disease. [Background technology]

[0002] Background of the Invention Cytokines are a group of small, pharmacologically active polypeptides that play an important role in intercellular communication in multicellular organisms. As intercellular mediators, cytokines regulate cell survival, growth, differentiation, and effector functions. They are key players in regulating immune responses, particularly in infectious diseases, inflammatory diseases of the joints, kidneys, blood vessels, and intestines, and autoimmune diseases of the nervous and endocrine systems.

[0003] Typical of cytokines is pleiotropy, meaning that they act on many different target cells and often affect the actions of other cytokines in an additive, synergistic, or antagonistic manner. In addition to pleiotropic effects, cytokine actions are often overlapping, i.e., similar biological responses can be achieved by several different cytokines. Cytokines exert their effects through specific cell surface receptors on target cells. Cytokines are produced by a wide variety of cells in the body and play important roles in many physiological responses with therapeutic potential.

[0004] Cytokines are classified according to, among other things, their three-dimensional structure. Despite the lack of amino acid sequence similarity, the cytokine family is characterized by a long, four-α-helical bundle structure. This family includes interleukin (IL)-6, IL-11, leukemia 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) α / β, and leptin.

[0005] Among these cytokines, IL-6, IL-11, LIF, OSM, CNTF, and CT-1 share a common receptor subunit, gp130, for signal transduction, resulting in similar and overlapping physiological responses. Therefore, this group of cytokines has been designated "IL-6-type cytokines," or cytokines of the IL-6 family, or members of the IL-6 cytokine family (see, e.g., Heinrich, PC, et al., Interleukin-6-type cytokine signaling through the gp130 / Jak / STAT pathway. The Biochemical Journal, 1998, 334(Pt 2):297-314).

[0006] Several IL-6 family cytokines are known to signal via at least two different mechanisms: canonical 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 (Non-Patent Document 2)).

[0007] Classical signaling occurs when a cytokine binds to a membrane-bound receptor. In the first step, the cytokine binds to a non-signaling α receptor subunit specific to that cytokine. For example, IL-6 binds to the IL-6R, which is present only on a subset of cells. This binding recruits a signaling receptor, also known as a β receptor, such as gp130, which leads to signal transduction into the cell. Therefore, classical signaling can occur only when a cell contains all the necessary components of the signal-receptor complex.

[0008] However, trans-signal transduction is not limited to cytokine / cytokine receptor interactions that occur on the cell surface.It is known that soluble cytokine receptors, either by alternative splicing or cleavage from the cell surface, can interact with cytokines.The soluble cytokine / cytokine receptor complex can then interact with the so-called ubiquitously expressed beta receptor, such as gp130.In this case, signal transduction no longer depends on cells having the correct combination of components of the cytokine receptor complex.The existence of these different ways of causing signal transduction makes it more difficult to regulate or predict responses to cytokines.

[0009] IL-6 family cytokines have been implicated in many functions, including B cell stimulation and induction of hepatic acute phase proteins. Furthermore, metabolic and neurotrophic functions have been attributed to this group of cytokines. Thus, the IL-6 cytokine family is involved in the development and / or progression of many different conditions or diseases (see, for example, Garbers, C. and J. Scheller, Interleukin-6 and interleukin-11: same but different. Biological chemistry, 2013; 394(9): 1145-1161 (Non-Patent Document 3)).

[0010] For example, IL-6 is a central mediator of intercellular communication and is involved in regulating inflammatory responses and coordinating developmental, neuronal, and metabolic processes. In hepatocytes, IL-6 is a major mediator of the acute phase response. Due to its critical role in inflammation, dysregulation of IL-6-induced signaling has been linked to the development of severe immune and proliferative diseases, such as rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and colon cancer.

[0011] IL-11 is thought to regulate adipogenesis, osteoclastogenesis, neurogenesis, and platelet maturation through binding to the transmembrane IL-11Rα receptor and the resulting activation of downstream signaling pathways. More recently, it has been discovered that overexpression of IL-11 is associated with various cancers and may provide a link between inflammation and cancer (see, for example, 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): pp. 1727-38 (Non-Patent Document 4)). Furthermore, it has been proposed that IL-11 can 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 (Non-Patent Document 5)).

[0012] In contrast to IL-6 and IL-11, LIF and OSM lack specific α receptors but signal through heterodimerization with two β receptors, gp130 and either the LIF receptor (LIFR) or the OSM receptor (OSMR). Both LIF and OSM have been shown to improve obesity and hepatic steatosis and to have protective functions after myocardial infarction. However, there are conflicting reports regarding the effects of these cytokines on fibrotic processes in various tissues. Furthermore, both cytokines have pro- and anti-tumorigenic functions. Therefore, for therapeutic purposes, targeting only specific subsets of cells expressing OSMR or LIFR would be highly desirable to reduce side effects, including tumorigenic effects.

[0013] Therefore, in principle, IL-6 family cytokines may represent promising targets for treatment by either receptor blockade or exogenous cytokine delivery to the patient's body to induce protective or regenerative effects. However, due to the pleiotropy and diversity of signaling mechanisms, both approaches are known to induce uncontrollable side effects.

[0014] Since it is known that administration of IL-6 family cytokines can have beneficial and / or detrimental effects depending, inter alia, on the cytokine and the cells targeted, it is desirable to reduce the pool of cells that can be targeted by increasing the specificity of the cytokine in order to be able to better control the outcome of cytokine administration.

[0015] Therefore, there is a need for cytokines that can target only specific subsets of cells and thus can be used to specifically trigger signaling in regulated situations without inducing severe side effects.

[0016] Previous attempts have been made to overcome these problems by creating synthetic cytokines (see, for example, 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): pp. 11859-67 (Non-Patent Document 6); Findeisen, M., et al., Treatment of type 2 diabetes with the designer cytokine IC7Fc. Nature, 2019. 574(63-68) (Non-Patent Document 7); Donath, M.Y., Designer cytokine for the treatment of diabetes. Nat Metab, 2019. 1(10): pp. 933-934 (Non-Patent Document 8)).

[0017] This synthetic cytokine, designated IC7, contained IL-6 binding sites I and II combined with binding site III from CNTF. Like IL-6, which requires the IL-6R, CNTF requires the presence of the cytokine-specific non-signaling α-receptor subunit CNTFR to initiate signaling. CNTF requires a heterodimer of one gp130 and one LIFR subunit, rather than two gp130 receptors, to induce signaling.

[0018] However, during the creation of the present invention, it was confirmed that IC7 was characterized by unexpected cross-reactivity with cells expressing OSMR, because IC7 was able to induce signaling in cells expressing gp130, IL-6R, and OSMR (see Figure 2.1 and Example 3). This may be explained by the underlying binding affinity of the CNTF site III sequence for both LIFR and OSMR. Therefore, IC7 activates signaling in both cells expressing OSMR and cells expressing LIFR. This reduces the specificity and suitability of this synthetic cytokine.

[0019] Therefore, there remains an urgent need to provide cell-specific cytokines that do not induce severe side effects.

[0020] This need is addressed by the present invention, which provides a synthetic cytokine characterized by specificity and greater regulatability afforded by cell-type-restricted activity and reduced or eliminated ability to induce trans-signaling. In contrast to IC7, in the present invention, binding site III residues from a cytokine lacking the requirement for a specific α receptor were grafted onto the cytokine backbone of an α receptor-dependent IL-6 family cytokine. The resulting chimeric cytokine became α receptor-dependent, and its activity was restricted to cells expressing a specific novel receptor combination.

[0021] Previous publications have suggested that the receptor-binding modules of IL-6 family cytokines can be transferred from one cytokine to another, but it was surprising that transferring binding site III residues from an α-receptor-independent cytokine to the backbone of an α-receptor-dependent cytokine resulted in α-receptor-dependent biological activity of the chimeric cytokine. It was previously assumed that the combination of binding site II and binding site III residues of IL-6 or IL-11 would result in α-receptor-independent dimerization of gp130 with LIFR or OSMR, respectively, as occurs in signaling mediated by native LIF or OSM.

[0022] The synthetic cytokines of the present invention can be used in methods of treatment, particularly in the treatment or prevention of conditions or diseases that can be treated or prevented by the administration of cytokines. The synthetic cytokines of the present invention are suitable for medical applications because they do not induce the serious side effects known to be caused by the administration of naturally occurring cytokines. [Prior art documents] [Non-patent literature]

[0023] [Non-Patent Document 1] Heinrich, PC, et al., Interleukin-6-type cytokine signaling through the gp130 / Jak / STAT pathway. The Biochemical journal, 1998.334(Pt 2):p.297-314 [Non-patent document 2] 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 [Non-patent document 3] Garbers, C. and J. Scheller, Interleukin-6 and interleukin-11: same but different. Biological chemistry, 2013;394(9):1145-1161 [Non-patent document 4] 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 [Non-patent document 5] 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 [Non-patent document 6] 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 [Non-Patent Document 7] Findeisen,M.,et al.,Treatment of type 2 diabetes with the designer cytokine IC7Fc.Nature,2019.574(63-68) [Non-patent document 8] Donath, MY, Designer cytokine for the treatment of diabetes.Nat Metab,2019.1(10):p.933-934 Summary of the Invention

[0024] The present invention relates to synthetic cytokines, also called chimeric cytokines or "cytochimeras" (a portmanteau of cytokine and chimera), and their medical uses.

[0025] Due to their pleiotropic properties, cytokines used as therapeutic agents often exhibit severe side effects. For example, many members of the IL-6 cytokine family exhibit many beneficial effects on cardiac regeneration after myocardial infarction, liver regeneration, or type 2 diabetes. Unfortunately, however, they also induce negative effects, for example, by inducing inflammation or by causing fever and tachycardia. This is due to the fact that in addition to the targeted cell population, many other unwanted populations are also targeted by cytokines. Therefore, selective targeting of specific cell populations is very important.

[0026] To achieve this goal, synthetic cytokines have been produced.Compared to the natural cytokines from which they are derived, the synthetic cytokines of the present invention require one additional receptor for signal transduction, and are therefore more selective because the receptor combination required to trigger signal transduction is only available in a limited subset of cells.The advantage of synthetic cytokines is that they stimulate only a smaller subpopulation of cells, rather than the entire cell population activated by natural cytokines.

[0027] Some members of the IL-6 cytokine family can bind to membrane-anchored α receptors, followed by homodimerization or heterodimerization of β receptors, a process known as classical signaling. Trans-signaling also exists, in which cytokines bind to soluble α receptors, followed by signal transduction via β receptors. While classical signaling is more conducive to repair, trans-signaling is more conducive to inflammatory properties. A further advantage of the synthetic cytokines of the present invention is that they are poor transducers of trans-signaling and are therefore most likely to induce tissue healing rather than inflammation. Finally, because natural human cytokines are used and reconstituted, the synthetic cytokines of the present invention have a superior safety profile compared to many synthetic drugs.

[0028] The polypeptides of the invention comprise two binding sites derived from the same cytokine of the IL-6 cytokine family, where the cytokine requires a cytokine-specific non-signaling alpha receptor subunit as part of a receptor complex to cause signal transduction, wherein a first binding site is capable of binding to the cytokine-specific non-signaling alpha receptor subunit and a second binding site is capable of binding to the signaling receptor gp130, and a third binding site derived from an additional cytokine of the IL-6 cytokine family, where the additional cytokine does not require a cytokine-specific non-signaling alpha receptor subunit as part of a receptor complex to cause signal transduction, wherein the third binding site is capable of binding to the cytokine-specific signaling receptor of the additional cytokine.

[0029] In the present invention, cytokines that require a cytokine-specific non-signaling alpha receptor subunit as part of a receptor complex may be selected from the group including IL-6 and IL-11.

[0030] In the present invention, cytokines that do not require a cytokine-specific non-signaling alpha receptor subunit as part of a receptor complex are selected from the group including LIF, OSM, and CT-1.

[0031] In the present invention, a cytokine that requires a cytokine-specific, non-signaling alpha receptor subunit as part of its receptor complex may be IL-11, and a cytokine that does not require a cytokine-specific, non-signaling receptor subunit as part of its receptor complex may be LIF.

[0032] In the present invention, a cytokine that requires a cytokine-specific non-signaling alpha receptor subunit as part of its receptor complex may be IL-6, and a cytokine that does not require a cytokine-specific non-signaling receptor subunit as part of its receptor complex may be LIF or OSM.

[0033] The polypeptides of the present invention may further optionally comprise an affinity tag at the C-terminus, and may optionally comprise the Fc constant region of an immunoglobulin such as IgG, IgA, or IgM; and / or may optionally comprise a signal peptide at the N-terminus; and / or may comprise a binding site for serum albumin.

[0034] The polypeptide of the present invention may comprise an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:13, preferably the amino acid sequence of SEQ ID NO:13 (GIL-11); or an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:6, preferably the amino acid sequence of SEQ ID NO:6 (GIL-6); or an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:11, preferably the amino acid sequence of SEQ ID NO:11 (G10-6).

[0035] In the present invention, the binding site may be derived from a human cytokine.

[0036] In the present invention, the ability of the polypeptide to cause trans-signalling is significantly reduced compared to the ability of the cytokine from which the first two binding sites are derived to cause trans-signalling.

[0037] The present invention also relates to polynucleotides comprising one or more nucleic acid sequences encoding the polypeptides of the present invention. Optionally, the polynucleotide may be an expression cassette, preferably an expression vector.

[0038] The present invention also relates to expression systems comprising the polynucleotides of the present invention, wherein the expression system may be of eukaryotic, prokaryotic, or archaeal origin, or synthetic in nature, e.g., may be a cell-free expression system.

[0039] The present invention also relates to host cells comprising a polynucleotide of the present invention, wherein optionally the host cell is selected from eukaryotic cells, including, but not limited to, hamster cell lines (CHO and their derivatives), mouse cell lines (e.g., C127, NS0, SP2 / 0, YB2 / 0, XB2 / 09, and all their derivatives), or human cell lines (e.g., HEK and its derivatives, e.g., EXPI293, HT-1080, PER.C6, or HuH-7). Also included are cell lines derived from monkeys, such as Vero cells and their derivatives, and insect cells, such as SF-9 cells and their derivatives.

[0040] The present invention also relates to pharmaceutical compositions comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0041] The present invention also relates to a polypeptide of the invention for use as a pharmaceutical.

[0042] The present invention also relates to a polypeptide of the invention for use in a method for preventing and / or treating a condition in a patient, wherein the condition is selected from the group consisting of lymphopenia, muscle atrophy, osteoporosis, thrombocytopenia, obesity-related metabolic disorders such as type II diabetes, obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, hypertension, stroke, or cardiovascular disease, neurological disorders such as paraplegia, Alzheimer's disease, and Parkinson's disease. The diseases and / or conditions treated and / or prevented by administration of a synthetic cytokine of the invention are those associated with signal transduction mediated by IL-6 family cytokines, such as LIF, OSM, or CT-1, from which binding site III of the synthetic cytokine is derived. DETAILED DESCRIPTION OF THE INVENTION

[0043] Detailed Description of the Invention The present invention is described in detail below. Features of the present invention are described in individual paragraphs. However, this does not mean that a feature described in one paragraph is isolated from a feature described in another paragraph. Rather, a feature described in one paragraph can be combined with a feature described in another paragraph.

[0044] The term "comprising," as used herein, means including or encompassing the features disclosed as well as additional features not specifically mentioned. The term "comprising" is meant in the sense of "consisting of" the features indicated and, therefore, does not include additional features other than the indicated features. Thus, the subject matter of the present invention may be characterized by additional features in addition to the features indicated.

[0045] The polypeptides of the invention comprise two binding sites derived from the same cytokine of the IL-6 cytokine family, where the cytokine requires a cytokine-specific, non-signaling alpha receptor subunit as part of a receptor complex to initiate signal transduction, the first binding site being capable of binding to the cytokine-specific, non-signaling alpha receptor subunit and the second binding site being capable of binding to the signaling receptor gp130, and a third binding site derived from an additional cytokine of the IL-6 cytokine family, where the additional cytokine does not require a cytokine-specific, non-signaling alpha receptor subunit as part of a receptor complex to initiate signal transduction, the third binding site being capable of binding to the cytokine-specific, signaling receptor of the additional cytokine. In one embodiment of the invention, the binding sites are derived from a human cytokine.

[0046] As is known in the art, the IL-6 cytokine family includes at least IL-6, IL-11, LIF, OSM, CNTF, and CT-1, which use a common β receptor subunit, gp130, for signal transduction and, as a result, induce similar and overlapping physiological responses. Therefore, this group of cytokines has been termed "IL-6-type cytokines" or IL-6 family cytokines.

[0047] The IL-6 cytokine family consists of two subgroups. One group of cytokines requires interaction between the cytokine and its specific non-signaling α-receptor subunit as the first step in signal transduction. This group includes IL-6, IL-11, and CNTF. IL-6 requires the IL-6 receptor IL-6R, IL-11 requires the IL-11 receptor IL-11R, and CNTF requires the CNTF receptor (CNTFR). The cytokine-specific non-signaling α-receptor subunit is also referred to as the α-receptor. Its function is to interact with its cognate cytokine. When a cytokine interacts with the α-receptor at a specific site called site I, additional components of the receptor complex are recruited to sites II and III of the cytokine. At site II, contact is made between the cytokine and gp130. In the case of IL-6 and IL-11, additional gp130 molecules are recruited to site III, forming gp130 homodimers and inducing signal transduction. Other members of the IL-6 family recruit additional β-receptors to site III, resulting in the formation of heterodimers between gp130 and cytokine-specific β-receptors, leading to signal transduction, such as the LIF receptor LIFR and the OSM receptor OSMR.

[0048] In the present invention, the cytokine requiring a cytokine-specific non-signaling alpha receptor subunit may be IL-11 or IL-6. These cytokines provide the framework for a synthetic cytokine containing binding sites I and II.

[0049] The other group of cytokines does not require a cytokine-specific non-signaling alpha receptor subunit as part of the receptor complex to trigger signal transduction. This group includes CT-1, LIF, and OSM. CT-1 and LIF bind directly to gp130 and LIFR. OSM binds directly to gp130 and OSMR. OSM is known to also bind to LIFR. This other group of cytokines provides binding site III, and the signaling pathway targeted by the synthetic cytokines of the present invention is the signaling pathway targeted by naturally occurring members of this group of cytokines. For example, if a synthetic cytokine of the present invention contains binding site III derived from LIF, the signaling pathway targeted by this synthetic cytokine is the signaling pathway targeted by administration of naturally occurring LIF. A significant difference between administration of naturally occurring cytokines and administration of the synthetic cytokines of the present invention is that the alpha receptor dependence of chimeric cytokines significantly reduces the pool of cells that can be targeted, as the signaling pathway operates only in cells containing the novel combination of receptor complex components (see Figure 2). For example, upon administration of naturally occurring LIF, all cells presenting gp130 and LIFR can be targeted, whereas upon administration of a synthetic cytokine of the present invention containing binding site III from LIF and binding sites I and II from IL-6 or IL-11, only cells presenting gp130, LIFR, and IL-6R or IL-11R can be targeted.

[0050] The present invention is based on the surprising discovery that a novel combination of a cytokine binding site that requires a cytokine-specific, non-signaling α-receptor subunit as part of the receptor complex to trigger signal transduction, and a cytokine binding site that does not require a cytokine-specific, non-signaling α-receptor subunit, results in a synthetic cytokine that retains its dependence on the presence of the α-receptor and is therefore characterized by increased specificity. It was previously assumed that this type of combination would result in a synthetic cytokine that is independent of a specific cytokine / cytokine receptor interaction. It is therefore postulated that prior art synthetic cytokines did not include the novel and inventive combination provided herein.

[0051] In the present invention, the cytokine that does not require a cytokine-specific non-signaling α-receptor subunit may be LIF, or may be OSM or CT-1. Preferably, it is LIF.

[0052] A preferred embodiment of the present invention is a polypeptide comprising two defined binding sites derived from IL-11 and a third binding site derived from LIF, also referred to as GIL-11.

[0053] Another polypeptide of the invention contains two defined binding sites derived from IL-6 and a third binding site derived from LIF, this polypeptide is also referred to as GIL-6.

[0054] Another polypeptide of the invention contains two defined binding sites derived from IL-6 and a third binding site derived from OSM, this polypeptide is also referred to as GIO-6.

[0055] The polypeptides of the present invention can be produced as described in the Examples. The polypeptide sequences of 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. Methods for identifying binding sites and designing chimeras in silico are also known to those skilled in the art.

[0056] Based on standard techniques known to those skilled in the art and the fact that cytokines of the IL-6 family exhibit a high degree of structural homology, the regions to be exchanged can be determined on a structure-based basis. The structures of cytokines whose binding site III is exchanged with each other were analyzed using standard protein structure visualization systems. When structures were not available, they were predicted using a protein structure homology server. The binding site of human LIF for LIFR contains three regions. Exemplarily, these regions are defined by SEQ ID NOs: 3-5. The binding site of human OSM for OSMR also contains three regions. Exemplarily, these regions are defined by SEQ ID NOs: 8-10. The in silico designed sequences can then be synthesized.

[0057] The resulting synthetic cytokine or cytochimera is characterized by retention of the binding capacity of the first two binding sites of the cytokine from which it is derived, which are binding partners of the cytokine-specific non-signaling alpha receptor subunit and the signaling receptor gp130, respectively; loss of binding capacity of the third binding site of the cytokine from which it is derived, which is a binding partner of the signaling receptor gp130; and gain of binding capacity of a third binding site from a different cytokine of the IL-6 family that does not require the cytokine-specific non-signaling alpha receptor subunit as part of the receptor complex to trigger signal transduction, e.g., LIF or OSM.

[0058] The key point is that by substituting the third binding site, the binding ability of the first two binding sites is not affected, and the substitution results in a synthetic cytokine containing a third binding site that is capable of binding to the original receptor.

[0059] 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 noted above, variations of these exemplary sequences are encompassed by the present invention as long as the binding site remains functional. Those skilled in the art know which portions of the amino acid sequence of cytokines in the IL-6 family are important for binding function. Variations in the amino acid sequence outside the binding site can be made without affecting the binding function of the synthetic cytokines. Thus, a polypeptide of the present invention may comprise an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:13 (GIL-11); or an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:6 (GIL-6); or an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:11 (GI0-6).

[0060] A variant of GIL-11 may also be defined as a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:13 (GIL-11), wherein the variant comprises the three binding regions that constitute binding site III of LIF as defined by SEQ ID NOs:3-5.

[0061] A variant of GIL-6 may also be defined as a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:6 (GIL-6), wherein the variant comprises the three binding regions that constitute binding site III of LIF as defined by SEQ ID NOs:3-5.

[0062] A variant of G1O-6 may also be defined as a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:11 (G1O-11), wherein the variant comprises the three binding regions that make up binding site III of OSM as defined by SEQ ID NOs:8-10.

[0063] Sequence identity can be determined by those skilled in the art.For example, sequence identity can be calculated using BLASTP disclosed in the prior art (see, for example, 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: - Field "Enter Query Sequence": Query subrange specification: None. Field "Choose Search Set": Database: non-redundant protein sequences (nr); Optional parameters: none. Field "Program Selection": Algorithm: blastp (protein-protein BLAST). Algorithm parameters: Field "General parameters": Maximum target sequence: 100; Short query: Automatically adjust parameters for short input sequences; E-value threshold (Expect threshold): 10; Word size: 3; Maximum number of matches in query range: 0. Algorithm parameters: Field "Scoring parameters": matrix: BLOSUM62; gap costs: presence: 11; elongation: 1; Compositional adjustments: Conditional compositional score matrix adjustment. · Algorithm · parameters: Field "Filter and Masking": filter: none; mask: none.

[0064] Results are filtered for sequences with query coverage greater than 35%.

[0065] Preferably, the variant may contain one or more conservative substitutions with respect to the amino acids contained in the exemplary sequences, SEQ ID NO:13, 6, or 11.

[0066] "Conservative substitution" refers to the substitution of one amino acid for another, resulting in a silent change. This means that one or more amino acid residues in the amino acid sequences of the present invention can be replaced with another amino acid of similar polarity, which acts as a functional equivalent. The amino acid that replaces an amino acid in the sequence can 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 replaced with another polar amino acid, one positively or negatively charged amino acid can be replaced with the other positively or negatively charged amino acid, respectively, and so on. 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; and negatively charged (acidic) amino acids, including aspartic acid and glutamic acid.

[0067] The polypeptide of the present invention may further comprise an additional structure, which may be located at the C-terminus and / or N-terminus.

[0068] For example, the polypeptides of the present invention optionally further comprise an Fc constant region of an IgG antibody. In another embodiment, the polypeptides of the present invention optionally further comprise an Fc constant region of another immunoglobulin class or subclass, for example, IgM or IgA. The addition of an immunoglobulin Fc constant region to the polypeptides of the present invention provides the following advantages:

[0069] On the one hand, it can facilitate polypeptide purification, for example, by using a protein A Sepharose matrix. On the other hand, adding an immunoglobulin Fc constant region to the polypeptide of the present invention improves the pharmacokinetics of the polypeptide in therapy by increasing the thermal stability of the polypeptide. The immunoglobulin Fc constant region 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 binding to the neonatal Fc receptor (FcRn) in the liver, which prevents degradation, and the increased molecular weight can prevent renal excretion. Another option for improving the pharmacokinetics of the polypeptide of the present invention is to provide a binding site for serum albumin, as non-covalent association with albumin has been shown to extend the half-life of otherwise short-lived proteins.

[0070] These additional moieties, such as the Fc constant region, can be utilized, for example, for purification techniques of the polypeptides of the present invention. The term "purification", in particular purification by protein tag, is clearly and unambiguously known to those skilled in the art. Methods include all possible techniques known to those skilled in the art, such as methods relying on characteristics such as solubility, size, charge, and specific binding affinity. Non-limiting examples are, for example, salting out, precipitation with ammonium sulfate or ethanol, dialysis, chromatography (e.g., 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.

[0071] Furthermore, the polypeptides of the present invention may additionally comprise any other moiety known to those skilled in the art, such as a protein tag. As used herein, the term "tag" refers to a naturally occurring or artificial polypeptide or other molecular structure that allows for the purification and / or detection of the polynucleotides of the present invention and / or further improves the pharmacodynamic and / or pharmacokinetic properties of the polypeptides of the present invention.

[0072] These protein tags can be used, for example, to facilitate the purification of polypeptides.Non-limiting examples in this regard include, for example, GFP tag or its derivatives, (poly)HIS tag, Myc tag, Strep tag, polyarginine tag, Flag tag, TAP tag, glutathione S-transferase (GST) tag, HA tag, calmodulin-binding peptide (CBP) tag, maltose-binding protein (MBP) tag, V5 tag, HSV tag, protein C tag, luciferase tag, or any other common polypeptide tag.Preferably, the protein tag can be located at the C-terminus of the polypeptide.Such tags can be removed before the final preparation of the polypeptide.

[0073] Furthermore, the polypeptide of the present invention may additionally contain a signal peptide, optionally at the N-terminus. Signal peptides are known to those skilled in the art. Preferably, the signal peptide used in the present invention results in the secretion of the polypeptide when produced in a cell line. This facilitates downstream processes, such as polypeptide purification.

[0074] The novel combination of binding sites in the synthetic cytokines of the present invention increases specificity, since the pool of cells that can be targeted is significantly reduced. A further advantage is the surprising discovery that the synthetic cytokines of the present invention are characterized by a significantly limited, significantly reduced, or even eliminated ability to induce trans-signaling compared to the ability exhibited by the natural cytokines from which the synthetic cytokines of the present invention are derived. Methods for measuring the trans-signaling ability of a synthetic cytokine are known to those skilled in the art, and they can compare the results with the level of trans-signaling obtained by the corresponding natural cytokine. The assay conditions for the synthetic cytokine and its corresponding natural cytokine are the same to obtain comparable results. An exemplary method for determining whether the trans-signaling ability of a synthetic cytokine of the present invention is significantly reduced is shown in Example 4. A significant reduction can be determined if the reduction is statistically significant.

[0075] For example, a significant reduction can be determined when the level of trans-signaling of a synthetic 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%, compared to the level of trans-signaling of the corresponding naturally occurring cytokine. A reduction to zero is also referred to as elimination of the ability to cause trans-signaling.

[0076] The elimination of this ability is due to the inability to reach maximum cell proliferation even at high concentrations of the synthetic cytokines of the present invention, resulting in a decrease in EC in the assay. 50 If Λ cannot be determined, it can be determined (see Example 5, Figures 6A and 6B).

[0077] In a further aspect, the present invention also relates to a polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide of the present invention. The polynucleotide may also comprise the aforementioned additional structures, such as nucleic acids encoding an Fc constant region, a protein tag, or a signal peptide. Optionally, the polynucleotide is an expression construct or expression cassette, preferably an expression vector. An "expression vector" or "expression construct" is generally a plasmid or virus designed for gene expression in appropriate cells. An expression vector is generally used to introduce a specific gene into a target cell, and can instruct the cell's protein synthesis mechanism to produce the protein encoded by the gene. In the present invention, any common expression vector known to those skilled in the art can be used for this purpose.

[0078] In a further aspect, the present invention also relates to an expression system comprising a polynucleotide of the present invention, wherein the expression system may be of eukaryotic, prokaryotic, or archaeal origin, or synthetic in nature, e.g., may be a cell-free expression system.

[0079] In a further aspect, the present invention also relates to a host cell containing the polynucleotide of the present invention. The host cell is capable of producing the polypeptide of the present invention and preferably allows for rapid purification. The host cell is selected from eukaryotic cells, including, but not limited to, hamster cell lines, such as CHO and their derivatives, mouse cell lines, such as C127, NS0, SP2 / 0, YB2 / 0, XB2 / 09, and their derivatives, or human cell lines, such as HEK and their derivatives, e.g., EXPI293, HT-1080, PER.C6, or HuH-7, monkey cell lines, such as Vero cells and their derivatives, and insect cell lines, such as SF-9 and their derivatives.

[0080] As used herein, "derivative" is understood to mean all descendant cell lines derived from or emerging from, including modifications or further development. Polypeptide expression using cell lines can be carried out by using a variety of transfection systems. Non-limiting examples are, for example, lipid-based transfection or viral transduction techniques, which are well known to those skilled in the art.

[0081] In a further aspect, the present invention also relates to a pharmaceutical composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0082] In a further aspect, the present invention also relates to a polypeptide of the invention for use as a medicament.

[0083] In particular, the present invention relates to a polypeptide of the invention for use in a method for the prevention and / or treatment of a condition in a patient, wherein the condition is selected from the group consisting of lymphopenia, muscle atrophy, osteoporosis, thrombocytopenia, obesity-related metabolic disorders such as type II diabetes, obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, hypertension, stroke, or cardiovascular disease, neurological disorders such as paraplegia, Alzheimer's disease, and Parkinson's disease.

[0084] The patient may be a mammal, preferably a human.

[0085] As is well known in the art, IL-6 family cytokines have been implicated in many functions, including B cell stimulation and induction of hepatic acute phase proteins. Furthermore, metabolic and neurotrophic functions have been attributed to this group of cytokines.

[0086] LIF increases the angiogenic potential of mesenchymal stem cells and enhances cardiomyocyte survival and cardiac regeneration after myocardial infarction in mice. Several reports have demonstrated the protective effects of LIF against obesity and fatty liver, as well as LIF-induced increases in glucose uptake. Cells with high levels of LIF include kidney cells (loop of Henle), cardiomyocytes, adipocytes, thyroid cells, hepatocytes, alveolar cells, colon (enteroendocrine) cells, and prostate cells.

[0087] OSM signaling via gp130 and OSMR has beneficial effects on myocardial fibrosis, enhances angiogenesis, and improves cardiac function after myocardial infarction. Mice injected with OSM after myocardial infarction showed improved recovery and better cardiac function compared with untreated mice. Mice treated with OSM after spinal cord injury showed functional recovery and neurite outgrowth. OSM has also been shown to improve obesity, glucose intolerance, and insulin resistance in mice. Cells with high levels of OSMR include ductal cells, Leydig cells, and bile duct cells.

[0088] However, there are conflicting reports regarding the effects of both LIF and OSM on fibrosis in various organs. Furthermore, both cytokines have pro- and anti-tumorigenic functions. Therefore, cell-type-restricted variants of LIF and OSM with reduced side effects may be promising therapeutic agents.

[0089] The synthetic cytokines of the present invention are characterized by their chimeric nature, which increases specificity by reducing the pool of cells that can be targeted, allowing fewer cells to display all signaling complex components necessary to trigger signal transduction.

[0090] This allows for therapeutic use of the synthetic cytokines of the present invention, as the probability of side effects is reduced. The synthetic cytokines of the present invention can be used to treat or prevent conditions associated with signal transduction mediated by cytokines from which binding site III is derived, such as LIF or OSM. In addition to a specific combination of beta receptors, cells that can be targeted must also express an alpha receptor, such as IL-6R or IL-11R. IL-6R is expressed at high levels in many different cell types, including Sertoli cells, proximal tubule cells, hepatocytes, endothelial cells, ductal cells, Leydig cells, and immune cells. Cells with high levels of IL-11R include cardiomyocytes, fibroblasts, breast cells (glandular and myoepithelial), skeletal muscle cells, and hepatocytes.

[0091] The terms "obesity" and "obese" generally refer to an individual whose weight is at least 20% above the average weight for the individual's age, sex, and height. 2 Men over 27.3 kg / m 2 An individual is also defined as "obese" if the individual is a woman who weighs more than 100g. Those skilled in the art will recognize that an individual may be significantly above average weight for their age, sex, and height and yet not technically be "obese." Such individuals are referred to herein, in accordance with common usage, as "overweight." The polypeptides of the present invention may be beneficial to such overweight individuals, and may also be beneficial to individuals prone to obesity or overweight, and individuals who wish to avoid a recurrence of a past episode of obesity or overweight.

[0092] The term "obesity-related metabolic disorder" refers to a disorder caused by, resulting from, exacerbated by, or secondary to obesity. Non-limiting examples of such disorders are osteoarthritis, type II diabetes, elevated blood pressure, stroke, and heart disease.

[0093] The polypeptides of the present invention may also be used to therapeutically or prophylactically treat mammalian conditions characterized by inadequate insulin sensitivity.

[0094] Reference to a "condition characterized by inadequate insulin sensitivity" should be understood to refer to a condition in which a patient's level of insulin responsiveness is inadequate or insufficient relative to physiological needs, regardless of whether inadequate insulin sensitivity is a cause or symptom of the condition. Examples of such conditions include, but are not limited to, diabetes, insulin resistance, impaired glucose tolerance, obesity, dyslipidemia, liver disease, metabolic disorders, hypertension, cardiovascular disease, or stroke.

[0095] These therapeutic and prophylactic aspects of the present invention are preferably achieved by administering an effective amount of a polypeptide of the present invention or a pharmaceutical composition of the present invention for a time and under conditions sufficient to achieve the appropriate therapeutic or prophylactic effect.

[0096] A "therapeutically effective amount" refers to an amount that is effective in prevention and / or treatment, or an amount sufficient to provide a preventive and / or therapeutic effect. A therapeutically effective amount is an amount that produces biological activity and depends, inter alia, on the individual. The amount will vary depending on the health and physical condition of the individual receiving treatment, the taxonomic group of the individual receiving treatment, the degree of protection desired, the formulation of the composition, evaluation of the medical condition, and other relevant factors. It is expected that the amount will fall within a relatively broad range that can be determined through routine trials.

[0097] The references to "treatment" and "prevention" herein should be considered in the broadest sense. The term "treatment" does not necessarily mean that a subject is treated until complete recovery. Similarly, "prevention" does not necessarily mean that a subject will not eventually contract a disease state. Thus, treatment and prevention include ameliorating the symptoms of a particular condition, or preventing or reducing the risk of developing a particular condition. The term "prevention" can be considered as reducing the severity or onset of a particular condition. "Treatment" can also reduce or delay the severity or progression of an existing condition.

[0098] The administration of the polypeptide of the present invention or the pharmaceutical composition of the present invention can be carried out by various administration methods. Non-limiting examples include, but are not limited to, intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, pulmonary administration, and inhalation administration. The administration schedule is determined by the attending physician and other clinical factors. As is well known to those skilled in the art, the dosage for any single patient can vary and depends on many factors, including, for example, size, age, sex, time and route of administration, and stage of disease.

[0099] The present invention is further illustrated by the accompanying figures and examples, which are intended to illustrate but not to limit the invention. [Brief explanation of the drawings]

[0100] [Figure 1]Figure 1 shows the normalized proliferation of Ba / F3 cells bearing various receptors in response to different cytochimeras and control cytokines. (A) Proliferation of Ba / F3 cells bearing the indicated receptors in response to no cytokine (-), HIL-11-Ts (50 μg / ml), IL-11 (10 ng / ml), OSM (10 ng / ml), and GIL-11-Ts (500 ng / ml). (B) Proliferation of Ba / F3 cells bearing the indicated receptors in response to no cytokine (-), 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. [Figure 2A] Figure 2 shows the mean fluorescence intensity (MFI) of proliferation of Ba / F3 cells expressing various receptor combinations in response to different cytochimeras and cytokines. One representative experiment out of three is shown. Data are presented as mean ± SEM. Curves are fitted by nonlinear regression. (A) Proliferation of Ba / F3 cells expressing the indicated receptors in response to increasing concentrations of IC7-Fc (0.001-2000 ng / ml). [Figure 2B] Figure 2 shows the mean fluorescence intensity (MFI) of proliferation of Ba / F3 cells expressing various receptor combinations in response to different cytochimeras and cytokines. One representative experiment out of three is shown. Data are presented as mean ± SEM. Curves are fitted by nonlinear regression. (B) Proliferation of Ba / F3 cells expressing the indicated receptors in response to increasing concentrations of GIL-11-Ts (0.001–1000 ng / ml). [Figure 2C]Figure 2 shows the mean fluorescence intensity (MFI) of proliferation of Ba / F3 cells expressing various receptor combinations in response to different cytochimeras and cytokines. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (C) Proliferation of Ba / F3 cells expressing the indicated receptors in response to increasing concentrations of GIL-6-Fc (0.001–1000 ng / ml). [Figure 2D] Figure 2 shows the mean fluorescence intensity (MFI) of proliferation of Ba / F3 cells expressing various receptor combinations in response to different cytochimeras and cytokines. One representative experiment out of three is shown. Data are presented as mean ± SEM. Curves are fitted by nonlinear regression. (D) Proliferation of Ba / F3 cells expressing the indicated receptors in response to increasing concentrations of GIO-6-Fc (0.001–1000 ng / ml). [Figure 2E] Figure 2 shows the mean fluorescence intensity (MFI) of proliferation of Ba / F3 cells expressing various receptor combinations with different cytochimeras and cytokines. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (E) Proliferation of Ba / F3 gp130 IL-11R LIFR cells with increasing concentrations of IL-11 (0.001–1000 ng / ml). [Figure 2F] Figure 2 shows the mean fluorescence intensity (MFI) of proliferation of Ba / F3 cells expressing various receptor combinations with different cytochimeras and cytokines. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (F) Proliferation of Ba / F3 gp130 IL-11R LIFR cells with increasing concentrations of LIF (0.0001–100 ng / ml). [Figure 2G]Figure 2 shows the mean fluorescence intensity (MFI) of proliferation of Ba / F3 cells expressing various receptor combinations with different cytochimeras and cytokines. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (G) Proliferation of Ba / F3 gp130 IL-6R LIFR cells with increasing concentrations of IL-6 (0.0001–30 ng / ml). [Figure 2H] Figure 2 shows the mean fluorescence intensity (MFI) of proliferation of Ba / F3 cells expressing various receptor combinations with different cytochimeras and cytokines. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (H) Proliferation of Ba / F3 gp130 IL-6R LIFR cells with increasing concentrations of LIF (0.0001–100 ng / ml). [Figure 3A] Figure 3 shows the results of a fluorescence-based Western blot for STAT3 and phosphorylated STAT3. STAT3 activation in Ba / F3 cells by cytokines and cytochimeras for 15 minutes. Equal amounts of protein (50 μg) were loaded in each lane. One representative experiment out of three is shown. (A) STAT3 activation in Ba / F3 cells expressing the indicated receptors after stimulation with no cytokine (-), 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). [Figure 3B]Figure 3 shows the results of a fluorescence-based Western blot for STAT3 and phosphorylated STAT3. STAT3 activation in Ba / F3 cells by cytokines and cytochimeras for 15 minutes. Equal amounts of protein (50 μg) were loaded in each lane. One representative experiment out of three is shown. (B) STAT3 activation in Ba / F3 cells expressing the indicated receptors after stimulation with no cytokine (-), 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), and IC7-Fc (100 ng / ml). [Figure 4A] Figure 4 shows the results of a fluorescence-based Western blot for STAT3, phosphorylated STAT3, ERK, and phosphorylated ERK. Stimulation of Ba / F3 cells with cytokines and cytochimeras for 15 minutes. Equal amounts of protein were loaded in each lane (50 μg / lane). A representative experiment out of three is shown. (A) Activation of STAT3 and ERK in Ba / F3 gp130 IL-11R LIFR cells by no cytokines (-), GIL-11-Ts, LIF, and IL-11 at the indicated concentrations. [Figure 4B] Figure 4 shows the results of a fluorescence-based Western blot for STAT3, phosphorylated STAT3, ERK, and phosphorylated ERK. Stimulation of Ba / F3 cells with cytokines and cytochimeras for 15 minutes. Equal amounts of protein were loaded in each lane (50 μg / lane). A representative experiment out of three is shown. (B) Activation of STAT3 and ERK in Ba / F3 gp130 IL-6R LIFR cells by no cytokine (-), GIL-6-Fc, GIO-6-Fc, LIF, and IL-6 at the indicated concentrations. [Figure 5] FIG. 5 shows the results of fluorescence-based Western blots for phosphorylated STAT3, STAT3, phosphorylated STAT1, STAT1, phosphorylated STAT5, STAT5, phosphorylated STAT6, and STAT6 for HIL-11, IL-11, LIF, OSM, and GIL-11. [Figure 6A] Figure 6 shows proliferation of Ba / F3 gp130 LIFR cells with a constant concentration of soluble α receptor and increasing concentrations of cytokines and cytochimeras. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (A) Proliferation of Ba / F3 gp130 LIFR cells with increasing concentrations of IL-11 (2-2000 ng / ml) in the presence (circle line) and absence (square line) of 100 ng / ml sIL-11R. [Figure 6B] Figure 6 shows proliferation of Ba / F3 gp130 LIFR cells with a constant concentration of soluble α receptor and increasing concentrations of cytokines and cytochimeras. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (B) Proliferation of Ba / F3 gp130 LIFR cells with increasing concentrations of sIL-11-Ts (2-2000 ng / ml) in the presence (circle line) and absence (square line) of 100 ng / ml sIL-11R. [Figure 6C] Figure 6 shows proliferation of Ba / F3 gp130 LIFR cells with a constant concentration of soluble α-receptor and increasing concentrations of cytokines and cytochimeras. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (C) Proliferation of Ba / F3 gp130 LIFR cells with increasing concentrations of IL-6 (0.002-2000 ng / ml) in the presence (circle line) and absence (square line) of 200 ng / ml sIL-6R. [Figure 6D]Figure 6 shows proliferation of Ba / F3 gp130 LIFR cells with a constant concentration of soluble α receptor and increasing concentrations of cytokines and cytochimeras. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (D) Proliferation of Ba / F3 gp130 LIFR cells with increasing concentrations of IC7-Fc (2-1000 ng / ml) in the presence (circle line) and absence (square line) of 200 ng / ml sIL-6R. [Figure 6E] Figure 6 shows proliferation of Ba / F3 gp130 LIFR cells with a constant concentration of soluble α receptor and increasing concentrations of cytokines and cytochimeras. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (E) Proliferation of Ba / F3 gp130 LIFR cells with increasing concentrations of sIL-6-Fc (2-1000 ng / ml) in the presence (circle line) and absence (square line) of 200 ng / ml sIL-6R. [Figure 6F] Figure 6 shows proliferation of Ba / F3 gp130 LIFR cells with a constant concentration of soluble α receptor and increasing concentrations of cytokines and cytochimeras. One representative experiment out of three is shown. Data are shown as mean ± SEM. Curves are fitted by nonlinear regression. (F) Proliferation of Ba / F3 gp130 LIFR cells with increasing concentrations of GIO-6-Fc (2-1000 ng / ml) in the presence (circle line) and absence (square line) of 200 ng / ml sIL-6R. [Figure 7A]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 stimulation and after 15 min of stimulation with GIL-11-Ts (50 ng / ml), GIL-11-Ts (1 μg / ml), GIL-11-Ts (1 μg / ml):sIL-11R (2 μg / ml), GIL-11-Ts (1 μg / ml), GIL-11-Ts (1 μg / ml), GIL-11-Ts (1 μg / ml):sIL-11R (2 μg / ml):sgp130-Fc (10 μg / ml). Equal amounts of protein were loaded (50 μg / lane). (B) Proliferation of Ba / F3 cells by adding a constant concentration of IL-11 (0.5 μg / ml):sIL-11R (1 μg / ml) or IL-11-Ts (0.5 μg / ml):sIL11R (1 μg / ml) and increasing concentrations of sgp130-Fc (0.01–10,000 ng / ml). Error bars reflect standard error. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 8A] Figure 8 shows the results of fluorescent-based Western blot analysis of phosphorylated STAT3 and STAT3 in mouse cells (Figures 8A and B). (C)-(G) show that GIL-11 rescued IL-6R-deficient mice from death after partial hepatectomy. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 8F] See legend to Figure 8A. [Figure 8G] See legend to Figure 8A. [Figure 9A]Figure 9 shows that GIL-6 induces JAK / STAT signaling and cell proliferation through a non-native cytokine receptor complex. (A) Proliferation of Ba / F3-gp130 cells, Ba / F3-IL-6R:gp130 cells, Ba / F3-gp130:OSMR cells, Ba / F3-gp130:LIFR cells, Ba / F3-IL-6R:gp130:OSMR cells, Ba / F3-IL-6R:gp130:OSMR cells, and Ba / F3-IL-6R:gp130:LIFR cells in the presence of no cytokine (-), 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-IL-6R:OSMR, Ba / F3-gp130:LIFR, Ba / F3-IL-6R:gp130:OSMR, and Ba / F3-IL-6R:gp130:LIFR cells after 20 min of stimulation with no cytokine (-), 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. (C) Activation of STAT1, STAT3, STAT5, ERK, and Akt in Ba / F3-IL-6R:gp130:LIFR cells under the same conditions as for STAT3 activation. (D) STAT3 activation in the heart, liver, and spleen after injection of 20 μg of GIL-6 or GIO-6. Mice were sacrificed 30 min after intraperitoneal cytokine injection. Equal amounts of protein (50 μg / lane) were analyzed via specific antibodies detecting phosphorylated STAT3 and STAT3. Western blot data show one representative experiment out of three. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10A]Figure 10 shows that the biological activity of the cytochimeric GIL-6 and GIO-6 is comparable to that of the native cytokine. (A) Proliferation of Ba / F3-IL-6R:gp130 cells, Ba / F3-gp130:LIFR cells, Ba / F3-gp130:OSMR cells, Ba / F3-IL-6R:gp130:LIFR cells, and Ba / F3-IL-6R:gp130:OSMR cells in the presence and absence of increasing concentrations of GIL-6 (0.002–1,000 ng / ml). EC50 values ​​were calculated by nonlinear regression curve fitting. 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 of 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 cells, Ba / F3-gp130:LIFR cells, Ba / F3-gp130:OSMR cells, Ba / F3-IL-6R:gp130:LIFR cells, and Ba / F3-IL-6R:gp130:OSMR cells in the presence and absence of increasing concentrations of GI0-6 (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 in the presence of OSM (0.001–100 ng / ml). A representative experiment out of three is shown. (E) Activation of STAT3 and ERK in Ba / F3-IL-6R:gp130:LIFR cells after 20 minutes of stimulation with no cytokines (-), increasing amounts of IL-6, LIF, GI IL-6, or GI IO-6 (0.2, 2, 20, or 200 ng / ml). Equal amounts of protein (50 μg / lane) were analyzed using specific antibodies detecting phosphorylated STAT3, STAT3, phosphorylated ERK, and ERK. Western blot data show a representative experiment out of three.(F) Time-dependent STAT3 activation in Ba / F3-IL-6R:gp130:OSMR cells by OSM (10 ng / ml), IL-6 (10 ng / ml), or G1O-6 (100 ng / ml) and in Ba / F3-IL-6R:gp130:LIFR cells by LIF (10 ng / ml), IL-6 (10 ng / ml), or G1O-6 (100 ng / ml) for the indicated time points. Equal amounts of protein (50 μg / lane) were analyzed using specific antibodies detecting phosphorylated STAT3 and STAT3. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 10E] See legend to Figure 10A. [Figure 10F] See legend to Figure 10A. [Figure 11]Figure 11 shows that the cytochimeric 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 a fixed concentration of sIL-6R (0 or 100 ng / ml) and increasing concentrations of IL-6 (0.002 to 1,000 ng / ml). A representative experiment out of three is shown. (B) Proliferation of Ba / F3-gp130:LIFR cells in the presence and absence of a fixed concentration of sIL-6R (0 or 100 ng / ml) and increasing concentrations of GIL-6 (0.002 to 2,000 ng / ml). A representative experiment out of three is shown. (C) Proliferation of Ba / F3-gp130:LIFR cells in the presence and absence of a fixed concentration of sIL-6R (0 or 100 ng / ml) and increasing concentrations of GIO-6 (0.002–2,000 ng / ml). A representative experiment out of three is shown. (D) Proliferation of Ba / F3-gp130:OSMR cells in the presence and absence of a fixed concentration of sIL-6R (0 or 100 ng / ml) and increasing concentrations of GIO-6 (0.002–2,000 ng / ml). A representative experiment out of three is shown. (E) Proliferation of Ba / F3-gp130:LIFR cells in the presence and absence of a fixed concentration of sIL-6R (0 or 100 ng / ml) and increasing concentrations of IC7 (0.002–1,000 ng / ml). A representative experiment out of three is shown. [Figure 12A]Figure 12 shows that CNTF signals through the alternative CNTFR:gp130:OSMR complex but not through 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). A representative experiment out of four is shown. (B) STAT3 activation in Ba / F3-CNTFR:gp130:OSMR cells with no cytokine (-), 10 ng / ml LIF, 10 ng / ml CNTF, or 10 ng / ml OSM. Equal amounts of protein (50 μg / lane) were analyzed with phosphorylated STAT3 and specific antibodies detecting STAT3. Western blot data show a representative experiment out of three. (C, D) CNTF dose-dependent STAT3 activation in Ba / F3-CNTFR:gp130:LIFR cells (c) and Ba / F3-CNTFR:gp130:OSMR cells (d) in the presence of 0.1, 1, 10, or 100 ng / ml CNTF, or in the absence (-) or presence of 10 ng / ml LIF or OSM for 20 minutes. Equal amounts of protein (50 μg / lane) were analyzed using specific antibodies detecting phosphorylated STAT3 and STAT3. Western blot data show 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 in the absence (-) or 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 in Ba / F3-IL-6R:gp130:LIFR cells (F) or Ba / F3-IL-6R:gp130:OSMR cells (G) in the presence of no cytokines (-), 10 ng / ml LIF or OSM, or 0.1, 1, 10, or 100 ng / ml CNTF for 20 minutes. Equal amounts of protein (50 μg / lane) were analyzed using specific antibodies detecting phosphorylated STAT3 and STAT3. Western blot data show one representative experiment out of three. (H) Western blotting of co-immunoprecipitations using protein A beads to precipitate 2 μg of recombinant HyperCNTF-Fc and 1 μg of soluble OSMR in the presence or absence of 1 μg of biotinylated gp130. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 12E] See legend to Figure 12A. [Figure 12F] See legend to Figure 12A. [Figure 12G] See legend to Figure 12A. [Figure 12H] See legend to Figure 12A. [Figure 13] Figure 13 shows receptor cell surface expression by flow cytometry in 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 untransduced with human CNTFR, compared to untransduced control cells. Expression was verified via receptor-specific antibodies. [Example]

[0101] Example 1 – Materials and Methods Cloning GIL-11 cDNA was ordered from BioCat GmbH, and then inserted into the pcDNA3.1 expression vector, which contains the 5' signal peptide of human IL-11R (Q14626, aa 1-24), followed by the sequence for a myc tag (EQKLISEEDL; SEQ ID NO: 14), as well as a fragment encoding GIL-11, a Gly4Ser linker, a TEV recognition site, and a twin-strep tag.

[0102] Cells, reagents, and recombinant proteins Methods for producing Ba / F3-gp130 are 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 in Dulbecco's Modified Eagle's Medium (DMEM) high-glucose culture medium (GIBCO®, Life Technologies, Darmstadt, Germany) containing 10% fetal bovine serum (GIBCO®, Life Technologies) and 60 mg / L penicillin and 100 mg / L streptomycin (Genaxxon Bioscience GmbH, Ulm, Germany) at 37°C, 5% CO2, in a water-saturated atmosphere. Murine Ba / F3-gp130 cells were obtained from Immunex (Seattle, WA, USA) and grown in the presence of HIL-6 (0.2% (10 ng / ml) conditioned medium from a stable clone of CHO-K1 cells secreting HIL-6 in the supernatant). Expi-293F™ cells (ThermoFisher Scientific) were grown at 3–5 × 10 cells / well in a 37°C incubator containing 8% CO2 on an orbital shaker at 125 rpm. 6 The cells were cultured in antibiotic-free Expi293™ expression medium until they reached a density of 1000 c / ml. 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).

[0103] Stimulation assay The Ba / F3-gp130 cell line was washed three times with PBS and starved in serum-free DMEM for 3 hours to remove cytokines. The inhibitors sgp130Fc or sIL11R were added 5 minutes before stimulation. Cells were stimulated with purified proteins (at the indicated concentrations) for 15 minutes, harvested, frozen in liquid nitrogen, and then lysed. For C2C12 cells, after stimulation, cells were washed once with PBS, detached by treatment with 0.05% trypsin, 0.1% EDTA (Genaxxon, catalog C4261.0100) for 5 minutes, and washed again. Cells were lysed for 45 minutes with a buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM MgCl2, and cOmplete™, an EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). Protein concentrations were determined by BCA protein assay (Thermo Fisher Scientific) according to the manufacturer's instructions. Protein expression and pathway activation were then analyzed by Western blotting.

[0104] Western blotting 50 μg of total protein was loaded per lane, separated by SDS-PAGE under reducing conditions, and transferred to a nitrocellulose membrane (Amersham Protan; Cytiva; LC, United Kingdom; catalog no. 10600016). The membrane was blocked for 1 h with blocking buffer (Intercept® Blocking Buffer; LI COR, USA; catalog no. 927-60001) diluted 1:3 with TBS (10 mM Tris-HCl pH 7.6, 150 mM NaCl). Primary antibodies (phosphorylated STAT3; Tyr-705; D3A7; Catalog No. 9145; and STAT3; 124H6; Catalog No. 9139, Cell Signaling Technology, USA) were diluted 1:10,000 in blocking buffer containing 0.2% Tween-20 (Sigma-Aldrich, USA; Catalog No. P1379-1L) for at least 90 minutes at ambient temperature or overnight at 4°C. The membrane was washed with TBS-T (0.1% Tween-20) and then incubated with secondary fluorochrome-conjugated antibodies 1:10,000 (IRDye® 800CW donkey anti-rabbit; Catalog No. 926-32213 and IRDye® 680RD donkey anti-mouse; Catalog No. 926-68072, LI-COR, USA) for 1 hour. Signal detection was achieved using a LI-COR Odyssey (USA; Model 2800). Secondary antibodies were detected simultaneously in different channels. Data analysis was performed using Image Studio Lite 5.2. Liver, spleen, and heart tissues were lysed in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 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, protein content was measured by BCA assay. 50 μg of total protein was then loaded onto each line, followed by immunoblotting.Antibodies used for blotting of lysed animal organs were as follows: anti-p-STAT3 (cat. no. 9145), anti-total STAT3 (cat. no. 9139).

[0105] Cell viability assay To remove cytokines from the medium, the Ba / F3-gp130 cell line was washed three times with PBS. 5 × 10 cells were cultured in DMEM containing 10% fetal bovine serum, 60 mg / l penicillin, and 100 mg / ml streptomycin. 4 Cells were suspended at a density of 1000 cells / ml. Cells were cultured for 3 days in the presence or absence of the indicated concentrations of cytokines or inhibitors in a volume of 100 μl. Fluorescence (λ) was measured using an Infinite M200 Pro plate reader (Tecan, Crailsheim, Germany). ex 560nm / λ em The CellTiter Blue Viability Assay (Promega, Karlsruhe, Germany) was used to determine the approximate number of viable cells by measuring the fluorescence intensity (590 nm). Fluorescence was measured 60 minutes after the addition of 20 μl / well of CellTiter Blue reagent (time point 0), and then every 20 minutes for up to 2 hours. For each experimental condition, three wells were measured. All values ​​were normalized by subtracting the value at time point 0 from the final measurement.

[0106] Cell transfection The Ba / F3-gp130 cell line was retrovirally transduced with the pMOWS expression plasmid as known in the art. The transduced cells were grown in the above-mentioned DMEM medium supplemented with 10 ng / ml HIL-6. The transduced Ba / F3-gp130 cells were selected for at least 2 weeks with puromycin (1.5 μg / ml) or hygromycin B (1 mg / ml) (Carl Roth, Karlsruhe, Germany). The resulting Ba / F3-gp130 cell line was then analyzed for receptor cell surface expression via flow cytometry. HEK293T cells were transfected with 10 μg pDNA using 20 μl of TurboFect (Thermo Fisher, USA, catalog number R0532) and incubated for 48 hours, after which cell lysis and Western blotting were performed. C2C12 cells were transfected with 7.5 μg of cDNA encoding mIL11R and 15 μl of TurboFect and incubated for 48 hours.

[0107] Cell surface detection of cytokine receptors via flow cytometry Cell surface expression of stably transfected Ba / F3-gp130 cell lines was detected by specific antibodies. 5The cells were washed with FACS buffer (PBS, 1% BSA) and then incubated in 50 μl of FACS buffer containing the indicated specific primary antibody (anti-LIFR or anti-OSMR; 1:20; catalog numbers BAF249 and BAF4389, R&D Systems; MN, USA). After at least 1 hour of incubation at room temperature, the cells were washed and resuspended in 50 μl of FACS buffer containing the secondary antibody (NorthernLights™ 493-conjugated anti-goat IgG 1:200) and incubated at room temperature for 30 minutes. The cells were washed, resuspended in 500 μl of FACS buffer, and analyzed by flow cytometry (BD FACSCanto II flow cytometer, BD Biosciences, using FACSDiva software). Data analysis was performed using FlowJo version 10 (Tree Star Inc, US).

[0108] Animal and ethical statements C57BL / 6 mice and IL-6R - / - Mice were obtained from the Jackson Laboratory and the animal facilities of the Heinrich-Heine University of Düsseldorf, respectively. Experiments in this study were performed in accordance with the requirements of LANUV-NRW (Germany) under approval number 84-02.04.2015.A462.

[0109] animal All mice were maintained under specific pathogen-free conditions and handled in accordance with regulations established by FELASA and the national animal welfare body GV-SOLAS (www.gv-solas.de). All transgenic animals were on a C57BL / 6N background. Mice were fed standard laboratory chow and provided autoclaved tap water ad libitum. They were maintained in an air-conditioned room with controlled temperature (20–24°C), humidity (45–65%), and a day-night cycle (12 h light, 12 h dark). Laparotomy was primarily performed on male mice at least 10–12 weeks old using isoflurane inhalation anesthesia as known in the art. To perform a 70% partial hepatectomy, the right upper, left upper, and left lower lobes of the liver, along with the gallbladder, were resected via one-step ligation using 5-0 polyester suture ligatures (B. Braun Surgical, SA, Rubi, Spain). The abdominal cavity and outer layer of the skin were then closed with 5-0 polyglycolic acid (HR13, B. Braun Surgical, SA, Rubi, Spain) and 4-0 polypropylene monofilament (DS16, B. Braun Surgical, SA, Rubi, Spain), respectively. To reduce mild surgical pain, mice were treated with 5 mg / kg carprofen (Rimadyl; Pfizer, Wurselen, Germany) after surgery. IL-6R - / - (IL-6R knockout) mice underwent 70% partial hepatectomy. At specific postoperative time points (0, 12, and 24 h), mice were weighed and anesthetized (100 mg / kg ketamine, 10 mg / kg xylazine; Vetoquinol GmbH, Ravensburg, Germany). After anesthesia, mice were bled to generate serum for further analysis. For liver tissue, livers were rinsed with phosphate-buffered saline (PBS) and weighed to calculate the liver weight to body weight ratio. Tissue samples were stored at -80°C for histology and RNA and protein extraction.

[0110] GIL-11 expression, purification, and injection into mice GIL-11 was produced and secreted by Expi293 cells (Thermo Fisher Scientific) and purified by Strep-tag affinity chromatography (Strep-TactinXT 4flow; IBA, catalog no. 2-5023-001) according to the manufacturer's instructions. 24 hours before and immediately after surgery, mice were injected intraperitoneally (ip) with 20 μg of GIL-11 to enforce cytokine signaling.

[0111] Gene expression analysis Total RNA was extracted from liver and spleen using Trizol (Thermo Fisher Scientific, Waltham, MA, USA). RNA concentration was measured using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA, catalog number 172-5140) and adjusted to 100 ng / μl for all samples. To determine the expression of specific genes, the iTaq™ Universal SYBR green One-Step Kit (BioRad, California, USA, catalog number 1725151) was used. A master mix was prepared according to the manufacturer's instructions: 5 μl of iTaq Universal Probe Reaction Mix (2x), 0.125 μl of iScript Advanced Reverse Transcriptase, 0.125 μl of primers, and 200 ng of RNA. The total volume of the mixture was then adjusted to 10 μl by adding nuclease-free HO. For analysis, the expression levels of all target genes were normalized to glyceraldehyde 3-phosphate dehydrogenase (gapdh) expression (ΔCT). Gene expression values ​​were calculated based on the ΔΔCt method. Relative quantities were calculated using the formula: RQ = 2 -ΔΔCt The expression levels of the target genes were determined using an ABI 7500 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA).

[0112] The following primer pairs were used in this study: TIFF2026500337000001.tif91134

[0113] modeling 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): pp. 845-58). Complex models and structure-based sequence alignments were generated using UCSF Chimera version 1.13.1, developed by the University of California (San Francisco) Resource for Biocomputing, Visualization, and Informatics with support from NIH P41-GM103311 (Pettersen, E., et al., UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem, 2004. 25(13): pp. 1605-12).

[0114] statistical analysis Data are presented as mean ± SEM using GraphPad Prism version 8. Statistically significant differences between two groups were determined by Student's t-test, including Welch's correction, where appropriate. Statistical analysis between several groups was determined using two-way ANOVA with Tukey's correction. Significance was calculated as follows: p>0.05: ns; p<0.05: * ;p<0.01: ** ;p<0.001: *** ;p<0.0001: **** .

[0115] Example 2 – Production of IL-6- or IL-11-based synthetic cytokines Native IL-11 binds to the α receptor IL-11R via site I to enable recruitment of two gp130β receptors via sites II and III. Similarly, native IL-6 binds to the α receptor IL-6R via site I to enable recruitment of two gp130β receptors via sites II and III. Notably, LIF does not require an α receptor to bind to the β receptor combination of gp130 and LIFR. However, LIF binding to LIFR is also facilitated by the identically located binding site III for gp130 on IL-11 or IL-6.

[0116] The inventors have demonstrated that by structure-based exchange of the split binding site III of human IL-11 or IL-6 with site III of human LIF, the resulting chimeric cytokines bind to non-natural cytokine receptor compositions. gp 130: I L- 11 R: L IFR or gp 130: I L- 6 R: L We hypothesized that these novel synthetic cytokine classes would be IL-11R- and IL-6R-binding proteins, respectively. Because β-receptor binding of LIF is α-receptor-independent, it was unpredictable whether gp130:LIFR recruitment by the chimeric cytokines GIL-11 and GIL-6 would be dependent on IL-11R or IL-6R, respectively.

[0117] Another chimera was generated by substituting binding site III of IL-6 for binding site III of OSM. Like LIF, OSM does not require α-receptors to bind to the β-receptor combination of gp130 and OSMR. However, binding of OSM to OSMR is also facilitated by the identically located binding site III of IL-6 for gp130.

[0118] The inventors have demonstrated that by structure-based exchange of the split binding site III of human IL-6 with site III of human OSM, the resulting chimeric cytokine binds to non-natural cytokine receptor compositions. gp 130: I L- 6 R: O We hypothesized that these cytokines would act as binders of the SMR, and termed this novel cytokine class the cytochimeric GIL-6. Because the β-receptor binding of OSM is α-receptor independent, it was unpredictable whether the gp130:OSMR recruitment of the chimeric cytokine GIL-6 would be IL-6R dependent.

[0119] IL-6-based cytochimeras were generated by replacing the binding site III of the IL-6 cytokine with that of OSM or LIF, henceforth referred to as GIO-6 or GIL-6. These chimeras bind to gp130, IL-6R, and OSMR or LIFR. For this purpose, the regions to be exchanged were determined, and the corresponding sequences were obtained.

[0120] SEQ ID NO: 1: Human IL-6 TIFF2026500337000002.tif24154SEQ ID NO:2:Human LIF TIFF2026500337000003.tif18154SEQ ID NO: 3: Human LIF binding site III, domain 1 TIFF2026500337000004.tif4128SEQ ID NO: 4: Human LIF binding site III, second domain TIFF2026500337000005.tif4128SEQ ID NO: 5: Human LIF binding site III, third region TIFF2026500337000006.tif4128SEQ ID NO:6: Cytochimeric GIL-6 The TIFF2026500337000007.tif24154 sequence does not contain the signal peptide. The underlined regions indicate the amino acid sequences that were substituted to obtain the cytochimeric GIL-6.

[0121] SEQ ID NO: 1: Human IL-6 TIFF2026500337000008.tif24154SEQ ID NO:7:Human OSM TIFF2026500337000009.tif24154SEQ ID NO:8: Human OSM binding site III, domain 1 TIFF2026500337000010.tif4128SEQ ID NO:9: Human OSM binding site III, second domain TIFF2026500337000011.tif4128SEQ ID NO: 10: Human OSM binding site III. Third domain TIFF2026500337000012.tif4128SEQ ID NO:11: Cytochimera GIO-6 The TIFF2026500337000013.tif24155 sequence does not contain the signal peptide. The underlined regions indicate the amino acid sequences that were substituted to obtain the cytochimeric GIO-6.

[0122] IL-11-based cytochimeras were generated by substituting the binding site III of the IL-11 cytokine with that of LIF, henceforth referred to as GIL-11. These chimeras bind to gp130, IL-11R, and LIFR. Structural examination of the IL-11 and LIF sites III led to the design of the cytochimera GIL-11, which has an exchange of the IL-11 framework and LIF-derived site III. IL-11 consists of 199 amino acids, and the inventors identified the split site III at amino acids 58-72 for IIIa, 111-128 for IIIb, and 162-179 for IIIc. LIF has 202 amino acids, with the split site III located at amino acids 64-88 for IIIa, 119-138 for IIIb, and 172-189 for IIIc. Although somewhat longer than the original IL-11 site chimera GIL-11, which was 211 amino acids, we decided to transfer the entire LIF-derived binding site to IL-11. Molecular modeling suggested that the transfer of the complete site III amino acid stretch would not interfere with the overall architecture and folding of the site chimera GIL-11. To this end, we determined the regions to be exchanged and obtained the corresponding sequences.

[0123] SEQ ID NO: 12: Human IL-11 TIFF2026500337000014.tif18154SEQ ID NO:2:Human LIF TIFF2026500337000015.tif18154SEQ ID NO: 3: Human LIF binding site III, domain 1 TIFF2026500337000016.tif4128SEQ ID NO: 4: Human LIF binding site III, second domain TIFF2026500337000017.tif4128SEQ ID NO: 5: Human LIF binding site III, third region TIFF2026500337000018.tif4128SEQ ID NO:13: Cytochimera GIL-11 The TIFF2026500337000019.tif24155 sequence does not contain the signal peptide. The underlined regions indicate the amino acid sequences that were substituted to obtain the cytochimeric GIL-11.

[0124] The corresponding cDNA sequences were synthesized externally by BioCat GmbH and then cloned into expression vectors. For secretion and purification purposes, a signal peptide was fused to the N-terminus, and an Fc tag (for IC7, GIL-6, and GIO-6) or a TwinStrep (Ts) tag (for GIL-11-Ts) was fused to the C-terminus, as these tags functioned in the corresponding cytochimeras.

[0125] Because the protein showed high levels of expression in Expi293 cells, chimeras were expressed in Expi293 cells. Expi293 cells were transiently transfected with plasmid DNA encoding the corresponding synthetic cytokine. The resulting supernatant was either purified immediately or stored at 80°C for later protein purification. Successful expression was analyzed by Western blot.

[0126] Example 3 - Activity and specificity of cytochimeras To study the activity and specificity of the cytochimeras, Ba / F3 cells were used. These are mouse pre-B cells that proliferate in a cytokine-dependent manner with IL-3. The cells used here also stably express at least human gp130. This allows the cells to proliferate through hyper-IL-11 via the IL-11 transduction pathway and the subsequent JAK / STAT signaling cascade. Therefore, hyper-IL-11 was used as a positive control because all cells used here proliferate as a result. Cells untreated with cytokines were used as a negative control. Furthermore, the required receptors were stably introduced into the corresponding cells by retroviral transduction, and surface expression was checked by flow cytometry. To evaluate the activity and specificity of the cytochimeras, these cytokines and control cytokines were used to stimulate Ba / F3 cells bearing different receptors. GIL-11-Ts showed significant activity against Ba / F3 cells expressing gp130, IL-11R, and LIFR, whereas all other cells were not proliferated by GIL-11-Ts (Figure 1A). As expected, Ba / F3 cells expressing gp130, IL-6R, and LIFR were proliferated by GIL-6-Fc, GIO-6-Fc, and IC7-Fc. As expected, Ba / F3 gp130 IL-6R OSMR also proliferated by GIO-6-Fc, but GIL-6-Fc had no effect. Interestingly, however, IC7-Fc also showed significant activity against these cells.

[0127] To accurately quantify the activity of the cytochimeras, the proliferation of Ba / F3 cells bearing various receptors was measured at a range of concentrations, and EC 50 Values ​​were determined using nonlinear regression (Figure 2, Table 1).

[0128] Table 1. Cytochimera and cytokine EC 50 value TIFF2026500337000020.tif143148

[0129] EC 50 The EC values ​​were determined by nonlinear regression of dose-response growth curves. Due to the different molecular weights of the proteins, the EC values ​​were calculated in molar concentrations. 50 Identifying the relevant data is more suitable for comparability.

[0130] In a proliferation assay with Ba / F3 cells expressing gp130, IL-6R, and either LIFR or OSMR, IC7 was found to feature unexpected cross-reactivity with cells expressing OSMR (Figure 2.1). For IC7, an EC of 10.8 pM was observed for Ba / F3-gp130-IL-6r-LIFR cells. 50 The EC value of 46.3 pM was determined for Ba / F3-gp130-IL-6R-OSMR cells. 50 was found. 50 Interactions with binding activators in the double-digit picomolar range may be relevant in vivo. This cross-reactivity has not been described in the literature. This suggests that site III residues of CNTF have an inherent affinity not only for LIFR but also for OSMR. As a result, CNTF may be cross-reactive with OSMR.

[0131] Even considering the low purity, GIL-11-Ts still exhibited an EC of 49.8 pM against Ba / F3 cells expressing gp130 IL-11R LIFR in a concentration-dependent proliferation. 50 For comparison, the EC of LIF and IL-11 50 Values ​​(3.18 pM and 33.6 pM, respectively) were determined for Ba / F3 gp130 IL-11R LIFR. At 11.6 pM, GIL-6-Fc had a comparable EC 50 For comparison, the EC values ​​for LIF and IL-6 are shown. 50 IC values ​​(3.64 pM and 4.64 pM, respectively) were determined for cells expressing Ba / F3 gp130 IL-6R LIFR. At 15 pM, GIO-6-Fc also showed comparable EC values ​​to IC7-Fc for Ba / F3 gp130 IL-6R OSMR. 50However, the EC value of 57.8 was comparable to the IC7-Fc for Ba / F3 gp130 IL-6R OSMR. 50 Values ​​were determined for Ba / F3 gp130 IL-6R OSMR. In summary, the EC 50 The values ​​are comparable to those of recombinant IL-11 previously expressed and purified in previous studies from our group, but not the EC values ​​of the purchased cytokines LIF and IL-6. 50 values ​​are not comparable (see Table 1 above).

[0132] The proliferation induced by the cytochimera / receptor complex is primarily mediated through the JAK / STAT signaling pathway. Therefore, STAT3 phosphorylation in Ba / F3 cells after stimulation with the cytochimera and cytokines was then analyzed to determine activity and specificity. Again, HIL-11 was used as a positive control (Figure 3).

[0133] Consistent with the proliferation assay, GIL-11-Ts specifically induced STAT3 phosphorylation only in Ba / F3 gp130 IL-11R LIFR-expressing cells, but not in cells bearing other receptors (Figure 3A). Similarly to the proliferation assay, GIO-6-Fc and IC7-Fc induced STAT3 activation in cells expressing Ba / F3 gp130 IL-6R OSMR, but also in cells expressing gp130 IL-6R LIFR. GIL-6-Fc induced STAT3 phosphorylation only in cells expressing Ba / F3 gp130 IL-6R LIFR (Figure 3B).

[0134] To evaluate the concentration-dependence of signal transduction, Ba / F3 cells bearing the corresponding receptors were stimulated with the cytochimeras. Different concentrations of GIL-11-Ts, GIL-6-Fc, GIO-6-Fc, and corresponding controls were used for stimulation, and the phosphorylation of STATs and ERKs was checked by Western blot.

[0135] Figure 4 shows concentration-dependent activation of STAT3 and ERK by GIL-11-Ts, GIL-6-Fc, GIO-6-Fc, and control cytokines. Here, 10 ng / ml GIL-11-Ts is sufficient for STAT3 and ERK phosphorylation. Thus, with respect to signal transduction, the activity of GIL-11-Ts at Ba / F3 gp130 IL-11R LIFR is comparable to that of IL-11 (Figure 4A). At 20 ng / ml, only GIL-6-Fc showed pronounced STAT3 phosphorylation and was therefore more active than GIO-6-Fc, still with substantial STAT3 activation at 200 ng / ml and ERK activation at 20 ng / ml (Figure 4B).

[0136] In summary, the cytokine chimeras are active and specific, but slightly less active than the recombinant cytokines that comprise them. Furthermore, in Ba / F3 cells, signaling intensity occurs via at least the JAK / STAT and MAPK / ERK signaling pathways and is concentration-dependent.

[0137] Furthermore, given the limited receptors used here, the cytokine chimeras may have higher specificity because they require one more receptor than the original cytokine for signaling. However, promiscuity was also observed 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 expected because the substituted binding site III of human OSM can bind to human LIFR. In the case of IC7-Fc, this cross-reactivity was surprising because it had not been previously reported in the literature. The fact that IC7-Fc activates cells expressing gp130 / IL-6R / OSMR, but not GIL-6-Fc, suggests that binding site III of CNTF binds to OSMR. To prove this, Ba / F3 cells expressing gp130 / CNTFR / OSMR are required.

[0138] Example 3 - GIL-11 induces Jak / STAT signaling and cell proliferation via the non-native gp130:IL-11R:LIFR cytokine receptor complex As is known in the art, the proliferation of the murine pre-B cell line Ba / F3 is IL-3 dependent. After the introduction of gp130 plus additional family receptors, proliferation shifts to the respective IL-6-type cytokine receptor combinations. In the case of Ba / F3-gp130 cells, proliferation is induced by IL-11 and soluble IL-11R or the corresponding fusion protein hyper-IL-11 (HIL-11), and the additional introduction of IL-11R makes these cells IL-11 dependent. Coexpression of gp130 and IL-6R makes these cells responsive to IL-6. Ba / F3 cells expressing gp130 and LIFR proliferate with LIF and OSM, whereas Ba / F3 cells expressing gp130 and OSMR are responsive to OSM. Using a Ba / F3 cell repertoire harboring eight different receptor combinations—gp130, gp130:IL-11R, gp130:LIFR, gp130:OSMR, gp130:IL-11R:OSMR, gp130:IL-6R:LIFR, and gp130:IL-11R:LIFR—we determined proliferation following the addition of IL-11, HIL-11, OSM, LIF, and GIL-11. GIL-11 specifically induced proliferation of Ba / F3-gp130:IL-11R:LIFR cells but not of the other cell lines tested, suggesting that GIL-11 signals via gp130:IL-11R:LIFR (Figure 1A). As expected, because all Ba / F3 cell lines express gp130, only HIL-11 induced proliferation of all of them. All Ba / F3 cells expressing gp130 and IL-11R proliferated with IL-11. Expression of gp130 and LIFR conferred proliferation induced by LIF and OSM, whereas cells expressing gp130 and OSMR were OSM-selective. We next analyzed STAT3 phosphorylation, a key feature of IL-6-type cytokine Jak / STAT signaling, in the Ba / F3 cell portfolio.As expected from the cytokine-induced Ba / F3 cell proliferation assay (Fig. 1A), STAT3 phosphorylation in Ba / F3 cells was induced by the following cytokine:cytokine receptor combinations: IL-11 via gp130; IL-11 via p130:IL-11R; OSM via gp130:OSMR; and OSM and LIF via gp130:LIFR (Fig. 3A). Importantly, sustained STAT3 phosphorylation for GIL-11 was observed only in Ba / F3 cells expressing the receptor combination of gp130, IL-11R, and LIFR (Fig. 3B). Taken together, these findings demonstrate that GIL-11 specifically activates signaling through a non-native receptor complex consisting of gp130:IL-11R:LIFR.

[0139] Example 4 - The biological activity of IL-11 is comparable to IL-11 and LIF Next, dose-dependent proliferation was performed to determine the quality and potency of GIL-11. Again, GIL-11 failed to induce proliferation of Ba / F3 cells expressing gp130, gp130:IL-11R, gp130:LIFR, gp130:OSMR, or gp130:IL-11R:OSMR, even at the highest applied concentration of 500 ng / ml, whereas proliferation of Ba / F3-gp130:IL-11R:LIFR cells was clearly dose-dependent, with an EC 50 For comparison, the EC values ​​for LIF and IL-11 were 50The concentrations of GIL-11 and IL-11R were determined to be 0.074 and 0.72 ng / ml, respectively, for Ba / F3-gp130:IL-11R:LIFR cells (Figure 2E,F). To analyze STAT3 and ERK phosphorylation, Ba / F3-gp130:IL-11R:LIFR cells were stimulated with increasing amounts of GIL-11, IL-11, and LIF. Western blotting showed that 10–100 ng / ml of GIL-11 and IL-11 were required to achieve maximal STAT3 and ERK phosphorylation, whereas the biological activity of LIF was higher, requiring 0.1–1 ng / ml for maximal STAT3 phosphorylation (Figure 4A). Regarding activation of other STATs in Ba / F3-gp130:IL-11R:LIFR cells, we assessed phosphorylation of STATs 1, 3, 5, and 6 after stimulation with HIL-11, IL-11, OSM, LIF, and GIL-11. All IL-6-type cytokines efficiently activate STAT3 via gp130, but to a lesser extent STAT1 and STAT5. In the case of LIF and OSM, activation of STAT3 and STAT1, as well as STAT5, was observed. Here, all cytokines induced sustained STAT3 phosphorylation. Interestingly, HIL-11 and IL-11 did not induce phosphorylation of STAT1 or STAT5, whereas LIF, OSM, and GIL-11 also induced phosphorylation of STAT1 (Figure 5). STAT5 phosphorylation was only observed with GIL-11. As expected, none of the cytokines induced phosphorylation of STAT6. Taken together, the activity of GIL-11 was within the same concentration range as the precursor cytokine IL-11, demonstrating that the expansion of receptor requirements by site III translocation did not affect overall biological cytokine activity.

[0140] Example 4 - Synthetic cytokines of the present invention are poor inducers of trans-signaling Protective, regenerative, and anti-inflammatory processes tend to be attributable to classical signaling, while immune system activation and, therefore, proinflammatory processes are attributable to trans-signaling pathways. Therefore, we characterized the behavior of the cytochimeras with respect to various signaling pathways. Because GIL-11-Ts is based on an IL-11 scaffold with an exchanged binding site III, it should still be able to complex with sIL-11 via binding site I and stimulate Ba / F3 gp130 LIFR-expressing cells. Similarly, we hypothesized that the IL-6-based cytochimeras IC7-Fc, GIL-6-Fc, and GIO-6-Fc with exchanged binding site III would also complex with sIL-6 and subsequently bind to membrane-bound gp130 and LIFR, resulting in trans-signaling.

[0141] In the following, a proliferation assay dependent on the cytochimera concentration was measured by Ba / F3 gp130 LIFR after adding a fixed concentration of sIL-6 or sIL-11 (FIG. 6).

[0142] IL-11 complexed with 100 ng / ml sIL-11R and had an EC of 71.6 ng / ml. 50 GIL-11-Ts induces proliferation through trans-signaling pathways, whereas GIL-11-Ts does little to stimulate proliferation. Because proliferation begins only at extremely high concentrations, saturation cannot be reached and EC 50 values ​​could not be determined (Figure 6A, B).

[0143] Similarly, IL-6 had an EC of only 1.04 ng / mL with 200 ng / mL sIL-6R. 50 Although the chimeric IC7-Fc and GIL-6-Fc showed substantial transactivation at 100 ng / mL, they required at least 250 ng / mL to induce the initial growth signal (Figure 6C, D, E). GIL-6-Fc was unable to mediate the transactivation pathway at the concentrations used here (Figure 6F).

[0144] The lack of substantial trans-mediated signaling pathways in the cell chimeras was further investigated using GIL-11-Ts as an example. As known in the art, olamkicept (sgp130-Fc) inhibits the IL-6 / sIL-6R or IL-11 / sIL-11R complex. Therefore, we investigated whether trans-signaling caused by GIL-11-Ts / sIL-11R could be inhibited. A sufficiently high concentration was selected to force trans-signaling (Figure 7).

[0145] Sufficiently high concentrations of IL-11-Ts:sIL-11R forced the trans-signaling pathway in both stimulation and proliferation assays, but failed to inhibit stimulation or proliferation in either experiment (Fig. 7A, B). In contrast, sgp130-Fc robustly inhibited the IL-11:sIL-11 complex with an IC50 of 22.5 ng / mL (Fig. 7B).

[0146] In summary, the chimeras initiate signaling through the trans-signaling pathway to a much lesser extent than the native cytokines (IL-6 or IL11) due to the exchange of binding site III, and this trans-signaling cannot be inhibited by sgp130-Fc.

[0147] Example 5 - The GIL-11:sIL-11R complex is a poor inducer of gp130:LIFR-mediated trans-signaling The α receptor-dependent cytokines IL-6 and IL-11 activate cells through the signaling receptor gp130 and the non-signaling membrane-bound IL-6R or IL-11R. Here, we have shown that GIL-11 signals through membrane-bound IL-11R in complex with the heterodimeric gp130:LIFR complex. Signaling through membrane-bound IL-11R is called classical signaling. IL-11R also exists as a soluble receptor and activates cells lacking membrane-bound α receptor expression in complex with IL-11 in a process called trans-signaling. Here, we analyzed the extent to which GIL-11 induces trans-signaling in cells expressing gp130 and LIFR in complex with sIL-11R. Ba / F3-gp130:LIFR cells were stimulated with a fixed concentration of sIL-11R at 100 ng / ml and increasing concentrations of GIL-11 (2–2000 ng / ml). For comparison, the same fixed concentration of sIL-11R and increasing cytokine concentrations of IL-11 were used. For 100 ng / ml sIL-11R, the EC 50 GIL-11 hardly induced sIL-11R-mediated proliferation, whereas the sIL-11R-mediated proliferation was 71.6 ng / ml (Fig. 6A). Even a concentration of 2000 ng / ml GIL-11 was not sufficient to reach maximum cell proliferation, and EC 50It was not possible to calculate the values ​​(Figure 6B). Therefore, intracellular signaling was evaluated in Ba / F3-gp130:LIFR cells stimulated with a comparatively high concentration of 250 ng / ml GIL-11 and 500 ng / ml sIL-11R. Here, the GIL-11:sIL-11R complex led to sustained STAT3 activation. sgp130Fc is a selective IL-6 / IL-11 trans-signaling inhibitor, inhibiting LIF signaling with at least 100- to 1000-fold less efficiency than IL-6 / IL-11 trans-signaling. Next, we tested whether sgp130Fc inhibits GIL-11 trans-signaling. As is known in the art, sgp130Fc is a highly effective inhibitor of IL-11 trans-signaling but a poor inhibitor of LIF. As shown in Figure 7A, sgp130Fc (10 μg / ml) did not inhibit STAT3 phosphorylation induced by GIL-11 (1 μg / ml):sIL-11R (2 μg / ml) trans-signaling in Ba / F3-gp130:LIFR cells (Figure 7A). For Ba / F3-gp130:LIFR cells, the concentrations of GIL-11 (0.5 μg / ml) or IL-11 (0.5 μg / ml) + sIL-11R (1 μg / ml) were selected to allow cell proliferation via trans-signaling. Titration of increasing concentrations of sgp130Fc significantly reduced the inhibition of IL-11 trans-signaling (IC). 50Although the highest concentration of sgp130Fc (10 μg / ml) resulted in a significant increase in GIL-11 trans-signaling (=22.5 ng / ml, Figure 7B), even the highest concentration of 10 μg / ml failed to inhibit GIL-11 trans-signaling. HEK293 cells express gp130 and LIFR but lack IL-11R expression. Thus, 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-11R (1 μg / ml) induced sustained STAT3 phosphorylation in HEK293 cells, but GIL-11 alone did not. Co-stimulation of GIL-11 and sIL-11R with sgp130Fc (3 μg / ml) did not inhibit GIL-11 trans-signaling (Figure 7C). Similar to LIF, GIL-11 trans-signaling is not inhibited by sgp130Fc, at least under conditions sufficient to block IL-11 trans-signaling. As shown in Figure 1A, proliferation of Ba / F3-gp130:LIFR and Ba / F3-gp130:IL-11R was induced by LIF and IL-11, respectively, but not by GIL-11. However, it is possible that GIL-11 binds to IL-11R:gp130 on Ba / F3-gp130:IL-11R cells and blocks IL-11 signaling, or binds to LIFR on Ba / F3-gp130:LIFR cells and blocks LIF signaling. The use of cytokine co-incubation with IL-11 and GIL-11 for Ba / F3-gp130:IL-11R and with LIF and GIL-11 for Ba / F3 gp130:LIFR did not result in inhibition of cell proliferation, even at a 20-fold molar excess of GIL-11 over IL-11 and a 200-fold molar excess of GIL-11 over LIF (Fig. 7D). Thus, it can be concluded that GIL-11 did not interfere with IL-11 and LIF signaling, at least in the concentration range tested.

[0148] Example 6 - Human IL-11 activates signaling through the murine gp130:IL-11R:LIFR cytokine receptor complex As is known in the art, human IL-11 and LIF are cross-reactive between mice and humans. We analyzed whether human GIL-11 also activates mouse cells expressing mouse gp130:IL-11R:LIFR chains. The mouse myoblast cell line C2C12 was selected. 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, but not IL-11 or GIL-11, induced sustained STAT3 phosphorylation, suggesting that C2C12 cells express gp130 and LIFR but lack IL-11R expression (FIG. 8A). When C2C12 cells were transfected with a cDNA encoding the mouse IL-11R, these cells became responsive to IL-11 and GIL-11, as indicated by STAT3 phosphorylation (Figure 8A). Next, 5, 10, and 20 μg of GIL-11 were intraperitoneally injected into wild-type mice. Thirty minutes after injection, the mice were sacrificed, and heart, liver, and spleen tissues were removed. Analysis of STAT3 phosphorylation by Western blotting showed that at least 10 μg / ml GIL-11 was sufficient to induce sustained STAT3 phosphorylation in the heart, liver, and spleen (Figure 8B). Taken together, these findings demonstrate that human GIL-11 activates the mouse receptor combination gp130:IL-11R:LIFR.

[0149] Example 7 - GIL-11 rescued IL-6R-deficient mice from death after partial hepatectomy Interleukin-6 (IL-6) is critically involved in liver regeneration after partial hepatectomy (PHX). As is known in the art, mice deficient in IL-6 and IL-6R have a high mortality rate of 40-80% compared to 10% in wild-type mice, and concurrently, have reduced STAT3 phosphorylation and attenuated hepatocyte proliferation. Hyper-IL-6 (HIL-6) injection 24 h before and immediately after surgery was previously shown to rescue mice from death after partial hepatectomy. Here, IL-6R-deficient mice were injected with 10 μg / mouse of GIL-11 24 h before and immediately after PHX. Nine days after PHX, the overall survival rate of IL-6R-deficient mice was approximately 40%, whereas IL-6R-deficient mice injected with two doses of GIL-11 had a 90% survival rate (Figure 8C). Nine days after PHX, body weight and liver weight to body weight ratios were significantly higher in the viable IL-6R mice, regardless of whether they were injected with GIL-11 or PBS. - / - There was no difference in the IL-6R expression in untreated mice (Fig. 8D, E). - / - Mice were treated with IL-11 and showed IL-6R - / - It was notable that the spleen-to-body weight ratio was greater in the naive IL-6R mice compared to the control mice (Figure 8F). Gene expression analysis revealed that 9 days after PHX, the naive IL-6R - / - IL-6R in mice treated with GIL-11 compared to - / - We demonstrated that the expression of the fibrotic marker αSMA and the acute-phase response gene SAA1 was increased in IL-6R-deficient mice (Figure 8G). Taken together, we demonstrated that GIL-11 rescues IL-6R-deficient mice from death after partial hepatectomy.

[0150] Our experiments define a human chimeric designer cytokine with cross-species specificity from mouse to human, which induces family-typical Jak / STAT signaling and cell proliferation via the non-native gp130:IL-11R:LIFR complex. The exchange of site III from LIF to IL-11 results in the cytochimeric GIL-11, which possesses receptor-binding properties not previously found in nature. Because transfer is restricted to site III, it remains unclear whether site I and site II transfer is also feasible. GIL-11 possesses unique features that distinguish it from IC7. First, IC7 recruits the receptor complex gp130:IL-6R:LIFR, whereas GIL-11 assembles the gp130:IL-11R:LIFR complex. This means that only cells expressing gp130, LIFR, and IL-11R are targeted by GIL-11. In a sense, this chimeric cytokine can be viewed as a synthetic LIF variant restricted to targeting the pool of cells expressing both LIFR and IL-11R.

[0151] Furthermore, it is interesting to compare the transcriptome profiles of IC7, GIL-11, and LIF. Because IC7, GIL-11, and LIF recruit and activate the gp130 / LIFR heterodimer, their transcriptome profiles are expected to be identical, but their transcriptome profiles should be distinctly different from those of IL-6 and IL-11, which recruit the gp130 homodimer. Although the transcriptome profiles are expected to be similar, the pools of cells activated by IC7, GIL-11, and LIF are expected to be different. Second, both IL-6 and CNTF, the cytokines from which IC7 is derived, depend on α-receptor binding before binding to gp130 and LIFR. Thus, the formation of CNTF-derived binding site III in IC7, as occurs after CNTF binding to CNTFR, is a direct result of IL-6R binding. This situation is different for GIL-11. Here, we introduced the α-receptor-independent binding site III from LIF into α-receptor-dependent IL-11. As shown here, GIL-11 activates the gp130:LIFR receptor complex only after binding to non-signaling IL-11R. Furthermore, GIL-11 failed to inhibit LIF signaling in Ba / F3-gp130:LIFR cells, whereas it would have done so if it could bind to LIFR in the absence of IL-11R. Taken together, our experiments demonstrated that although the original binding site context of LIF site III for LIFR is α-receptor-independent, reorganization of LIF site III into the IL-11 scaffold renders LIF site III binding α-receptor-dependent. Thus, this provides evidence that the interconnection with site III, mediated by the α-helix shift of site I, rather than the precise amino acid composition of binding site III, determines whether a cytokine is α-receptor-dependent or -independent.

[0152] Regarding biological activity, GIL-11 (EC 50 :1.44ng / ml) is a IL-11 (EC 50:0.72ng / ml), but it is comparable to LIF (EC 50 The effect of IL-11 was less than that of IL-11 (0.074 ng / ml). This may be due to the influence of the dominant scaffold of IL-11 rather than the minor effect of the site III exchange from LIF. However, since the D186A mutation in site I is known to increase the affinity of IL-11 for the IL-11R, this amino acid exchange may increase the biological activity of IL-11. Improving the binding of cytokines to α-receptors is a general strategy to improve the overall activity of this cytokine family, as has been shown for CNTF and CNTFR, and IL-6 and IL-6R.

[0153] After characterizing the biological activity and unique receptor composition of GIL-11, we investigated its ability to functionally substitute for IL-6 in liver regeneration after PHX in IL-6R-deficient mice. We previously showed that IL-6 and IL-6R are critically involved in liver regeneration after PHX, which explains the higher mortality rate of IL-6R-deficient mice. Importantly, GIL-6 rescued mice from death after partial hepatectomy. Furthermore, in wild-type mice, combined injection of IL-6 and sIL-6R accelerated liver regeneration after PHX, whereas injection of IL-6 alone did not. The increased presence of IL-6 and sIL-6R resulted in greater gp130 activation and stronger IL-6 signaling compared with IL-6 alone, likely because hepatocytes express far more gp130 than IL-6R.

[0154] Finally, blockade of IL-6 trans-signaling by sgp130Fc resulted in increased mortality after PHX. Mechanistically, hepatocyte growth factor (HGF) production by hepatic stellate cells, induced by IL-6 trans-signaling, directly contributed to liver regeneration after PHX. The IL-6R-deficient mice used in this study lack IL-6R expression, resulting in ineffectiveness of both canonical and trans-signaling. Therefore, they exhibit a 90% mortality rate compared with 10% in wild-type mice. A key difference between GIL-11 treatment and HIL-6 is that GIL-11 targets only cells expressing gp130:IL-11R:LIFR, limiting the range of cells that can be targeted in the body. HIL-6 targets virtually all cells because gp130, unlike IL-11R and LIFR, is thought to be ubiquitously expressed. However, because hepatocytes express IL-11R, LIFR, and gp130, GIL-11 can compensate for IL-6 trans-signaling and rescue IL-6R-deficient mice from death after PHX. Interestingly, most parameters, including body weight, liver weight, and spleen weight, remained unchanged between surviving GIL-11-treated and untreated mice. However, GIL-11 increased SAA by more than 300-fold after PHX. SAA induces hepatic stellate cell proliferation, which may contribute to liver regeneration after GIL-11 administration.

[0155] Given its overall in vitro and in vivo activity, GIL-11 appears to be of interest for conditions in which IL-6-type cytokines, including IC7, exert beneficial effects. In mice, IC7 selectively activates metabolic pathways that result in increased fatty acid oxidation with prevention of adiposity, increased energy dissipation with weight loss, muscle hypertrophy, and preservation of lean body mass with improved bone stability. In contrast to IL-6 and CNTF, injection of IC7 has been shown to be safe in both mice and non-human primates (macrocephalus monkeys) without promoting excessive inflammatory responses.

[0156] Recently, the originally described beneficial effects of human IL-11 in mouse models of human disease have been questioned. It has been shown that the mode of action of injected human IL-11 in mice actually relies on competitive inhibition of endogenous IL-11 signaling. It was concluded that IL-11 has detrimental, rather than beneficial, effects in a variety of mouse disease models, including nonalcoholic steatohepatitis, cardiovascular fibrosis, idiopathic pulmonary fibrosis, and fibrotic lung disease.

[0157] Interestingly, IL-11, like IL-6, which acts through the same gp130 homodimer, appears to preferentially induce ERK signaling rather than STAT3 phosphorylation. Therefore, we treated mouse myoblasts (C2C12 cells) with recombinant HIL-11, IL-11, and GIL-11 in the presence and absence of mouse IL-11R. However, this resulted in sustained IL-11R-dependent STAT3 phosphorylation. Injection of GIL-11 into mice also resulted in sustained STAT3 phosphorylation in heart, liver, and spleen tissues, demonstrating that human cytokines and GIL-11 activate the canonical gp130 and gp130:LIFR signaling pathways, which are characterized by STAT3 phosphorylation. For unknown reasons, GIL-11, unlike IL-11, poorly induces trans-signaling through the GIL-11:sIL11R complex and is not inhibited by sgp130Fc.

[0158] Although LIF has been implicated in fertility and a range of neurological disorders, including multiple sclerosis, recombinant LIF has not been used as a therapeutic agent. The ability of GIL-11 to induce LIF-like signaling through the gp130:LIFR complex may open up new LIF-like applications as a potential alternative to recombinant hLIF. Because GIL-11 activity requires cells that express not only LIFR but also IL-11R, its activity is restricted to a select few target cells compared to LIF, which may reduce unwanted negative side effects.

[0159] In conclusion, our study defines GIL-11 as a novel and promising cytochimera with specific, high-affinity activation of the non-native receptor gp130:LIFR:IL-11R complex. In general, the modular architecture of the cytochimera allows for a wide range of targeted receptor combinations and directional cell targeting.

[0160] Example 8 - Structural design of chimeric GIL-6 and GIO-6 Using structure-based modeling with IL-6 as the scaffold, we designed two novel site chimeras, GIL-6 and GIO-6, which contain site 3 exchanges from LIF and OSM, respectively (see also Example 1). Site 3 is split and consists of amino acid residues from the C-terminal α-helix and the 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 (site 3-3), which are added to the N-terminal portion of helix D, mediating the binding of IL-6 to gp130, LIF to LIFR, and OSM to LIFR or OSMR. Human IL-6 consists of 212 amino acids, including the signal peptide. The split site 3 of IL-6 is defined as amino acids R68-N88 at site 3-1, L129-R141 at site 3-2, and L179-R196 at site 3-3. LIF consists of 202 amino acids, with site 3 located at 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 located at amino acids L56-P78 for site 3-1, L113-Q137 for site 3-2, and P176-H196 for site 3-3. The new cytochimeras are larger than the native cytokines, with GIL-6 at 240 amino acids and GIO-6 at 229 amino acids.

[0161] In silico modeling using structure-based alignment suggested that transposition of the entire site 3 does not interfere with the overall architecture. IL-6 forms a hexameric receptor complex containing 2 × IL-6:2 × IL-6R:2 × gp130, whereas the α-receptor-independent signaling complexes for LIF and OSM are trimers containing LIF:gp130:LIFR, OSM:gp130:OSMR, and OSM:gp130:LIFR. However, the cytochimeras GIL-6 and GIO-6, using IL-6R as the α-receptor, specifically form tetrameric complexes consisting of GIL-6:IL-6R:gp130:LIFR, GIO-6:IL-6R:gp130:LIFR, and GIO-6:IL-6R:gp130:LIFR.

[0162] Example 9 - Cytochimeric GIL-6 and GIL-6 promote signaling and cell proliferation through a non-native cytokine receptor complex As is known in the art, IL-3 is required to induce proliferation of the murine pre-B cell line Ba / F3. After introduction of cDNAs encoding human gp130 and additional receptors of the family including IL-6R, IL-11R, OSMR, LIFR, and CNTFR (FIG. 13), proliferation of Ba / F3 cells became responsive to each IL-6-type cytokine receptor combination.

[0163] To determine the signaling properties of the cytochimeric GIL-6 and GIO-6, we used a Ba / F3 cell collection expressing the human IL-6 receptor gp130, IL-6R:gp130, gp130:OSMR, gp130:LIFR, IL-6R:gp130:OSMR, IL-6R:gp130:LIFR, CNTFR:gp130:LIFR, and CNTFR:gp130:OSMR. GIO-6 induced proliferation of both IL-6R:gp130:LIFR-expressing cells and IL-6R:gp130:OSMR-expressing cells, whereas GIL-6 exclusively induced proliferation of Ba / F3-IL-6R:gp130:LIFR cells (Figure 9A). Hyper-IL-6 (a soluble IL-6R:IL-6 fusion protein), IL-6, LIF, OSM, and the cytochimera IC7 were used as controls to demonstrate the receptor specificity of the Ba / F3 cell repertoire.

[0164] As expected, Hyper-IL-6 induced proliferation of all cell lines due to the general expression of gp130. After the addition of IL-6R, the derivative cell lines proliferated with IL-6. The LIFR:gp130-expressing cell lines proliferated with LIF and OSM, and OSM additionally induced proliferation of OSMR:gp130 cells. The IL-6 / CNTF hybrid cytokine IC7 induced proliferation of Ba / F3-IL-6R:gp130:LIFR cells and, unexpectedly, Ba / F3-IL-6R:gp130:OSMR cells, suggesting a novel receptor complex for IC7 (Figure 9A). Next, we analyzed STAT3 phosphorylation induced by the cytochimeras GIL-6, GIO-6, and IC7, as well as the cytokines HIL-6, IL-6, LIF, and OSM in the Ba / F3 cell collection. Importantly, STAT3 phosphorylation in Ba / F3 cells after cytokine stimulation (Fig. 9B) closely resembled the data obtained in the proliferation assay (Fig. 9A). STAT3 phosphorylation was observed in Ba / F3 cells expressing gp130 after stimulation with IL-6; in Ba / F3 cells expressing IL-6R:gp130 after stimulation with IL-6; in Ba / F3 cells expressing gp130:LIFR after stimulation with LIF; and in Ba / F3 cells expressing gp130:LIFR or gp130:OSMR after stimulation with OSM. IC7 and GIO-6 induced STAT3 phosphorylation in Ba / F3 cells expressing IL-6R:gp130:LIFR and IL-6R:gp130:OSMR, whereas GIL-6 induced STAT3 phosphorylation only in Ba / F3-IL-6R:gp130:LIFR cells (Fig. 9B). Because all cytokines and cytochimeras tested had an effect on Ba / F3-IL-6R:gp130:LIFR cells, this cell line was used to characterize key signaling pathways in more detail. Phosphorylation of STATs 1, 3, and 5, as well as ERK and Akt, was assessed after stimulation with HIL-6, IL-6, LIF, OSM, GIL-6, GI0-6, and IC7 (Fig. 9C).As expected, all cytokines and cytochimeras induced phosphorylation of STAT3, ERK, and Akt. STAT1 phosphorylation was most pronounced for IL-6, LIF, GIO-6, and IC7, and to a lesser extent for HIL-6, OSM, and GIL-6. Weak STAT5 phosphorylation was observed for LIF, GIO-6, GIL-6, and IC7, indicating a significant LIFR-mediated STAT1 and STAT5 phosphorylation compared with signaling through gp130 alone (Figure 9C). Human IL-6, LIF, CNTF, OSM, and IC7 are cross-species cross-reactive in mice. Therefore, 10 μg of GIL-6 or GIO-6 was injected intraperitoneally into wild-type C57BL / 6N mice. Heart, spleen, and liver were analyzed for STAT3 phosphorylation. Both GIL-6 and GIO-6 induced STAT3 phosphorylation in the liver, but only weak STAT3 phosphorylation was observed in the spleen, and no signal was observed in the heart (FIG. 9D).

[0165] Taken together, our data demonstrate that GIL-6 and GIO-6 have biological activity against both mouse and human receptors and induce signaling in cells expressing IL-6R:gp130:LIFR and IL-6R:gp130:OSMR, respectively. Furthermore, the previously described cytochimera IC7 also induced signaling through the IL-6R:gp130:OSMR complex. Because of these novel findings, GIL-6 is the first IL-6R:LIFR-selective cytochimera.

[0166] Example 10 - GIL-6 and GIO-6 are cytochimeras with biological activity similar to the natural cytokines To elucidate the biological properties of GIL-6 and GIO-6, dose-dependent proliferation was determined. Consistent with previous activity assays (Fig. 9A, B), GIL-6 did not induce proliferation of Ba / F3 cells expressing receptor combinations other than IL-6R:gp130:LIFR, even at the highest applied concentration of 1000 ng / ml. For Ba / F3-IL-6R:gp130:LIFR cells, an EC of 1.21 ng / ml was observed. 50 was determined for IL-6, which is comparable to the commercially available cytokine IL-6 (EC 50 :0.11ng / ml) and LIF (EC 50 :0.08ng / ml), but IC7 (EC 50 : 1.11 ng / ml) (Fig. 10A, B).

[0167] GIO-6 inhibited the proliferation of Ba / F3 cells expressing IL-6R:gp130:OSMR with an EC 50 The proliferation of Ba / F3 cells expressing Ba / F3-IL-6R:gp130:LIFR was suppressed by an EC 50 This was due to the efficient induction of IL-6, OSM, and LIF (EC 50 Although the efficacy was lower than that of the control (0.11, 0.12 ng / ml, and 0.08 ng / ml), the IC7 (EC 50 : 1.21 ng / ml for IL-6R:gp130:LIFR and 4.8 ng / ml for IL-6R:gp130:OSMR) (Fig. 10A, C, D).

[0168] Next, we analyzed the phosphorylation of STAT3 and ERK 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 of GIL-6 and IL-6 was sufficient to induce phosphorylation of STAT3 and ERK, whereas 2 ng / ml of LIF and 200 ng / ml of GIO-6 were required for sustained signaling (Figure 10E).

[0169] To determine the time response of signaling, Ba / F3-IL-6R:gp130:OSMR and Ba / F3-IL-6R:gp130:LIFR cells were stimulated with the indicated cytokines, including LIF, OSM, IL-6, GI0-6, and GIL-6, at intervals up to 240 min. As expected, STAT3 phosphorylation was typically induced as early as 5–10 min after cytokine addition and decreased between 120 and 240 min (Figure 10F), likely due to negative feedback by SOCS3.

[0170] Taken together, our data showed that the activity of GIL-6 and GIO-6 was comparable to that of IC7 but weaker than that of the natural cytokines IL-6, LIF, and OSM. Both GIO-6 and IC7 showed cross-reactivity with LIFR and OSMR.

[0171] Example 11 - Cytochimeric GIL-6 and GIO-6 are ineffective inducers of trans-signaling IL-6 requires a non-signaling membrane-anchored IL-6R before signaling via gp130 homodimerization, a process termed classical signaling. IL-6 can also form a complex with gp130 after binding to soluble IL-6R, a process termed trans-signaling. It is noteworthy that IL-6 trans-signaling is a major response in chronic inflammatory diseases. Therefore, the ability of cytochimeras 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 (sIL-6R). For comparison, increasing concentrations of IL-6 were also used. The IL-6:sIL-6R complex exhibited an EC of 5.3 ng / ml for IL-6. 50At 100 ng / ml sIL-6R, IC7 induced proliferation of Ba / F3-gp130:LIFR cells in a dose-dependent manner, whereas GIL-6 and GIO-6 failed to induce proliferation of Ba / F3-gp130:LIFR cells in the presence of 100 ng / ml sIL-6R at any of the concentrations tested (Fig. 11A, B, C). Interestingly, GIO-6 and IC7 induced weak proliferation of Ba / F3-gp130:OSMR and Ba / F3-gp130:LIFR cells, respectively, in the presence of sIL-6R at high concentrations of GIO-6 exceeding 500 ng / ml (Fig. 11D, E). Our data indicated that GIL-6, GIO-6, and IC7 are ineffective inducers of trans-signaling.

[0172] Example 12 – CNTF signals through an alternative CNTFR:gp130:OSMR complex IC7 has been described to signal through a receptor complex consisting of IL-6R:gp130:LIFR. Here, we identified that IC7 also signals through a receptor complex consisting of IL-6R:gp130:OSMR (Figure 9D). Since site 3 of IC7 is transferred from CNTF to IL-6, we investigated whether CNTF additionally interacts with OSMR through site 3. To analyze the ability of CNTF to induce cell proliferation in a dose-dependent manner (0.0002–100 ng / ml), we used Ba / F3 cells expressing CNTFR:gp130:LIFR or CNTFR:gp130:OSMR. Notably, CNTF induced cell proliferation in Ba / F3-CNTFR:gp130:LIFR and Ba / F3-CNTFR:gp130:OSMR cells (Figure 12A). Furthermore, Ba / F3 cells expressing CNTFR:gp130:OSMR (EC 50 EC for CNTF, which induces proliferation of 9.09 pg / ml 50 Values ​​are from Ba / F3 cells expressing CNTFR:gp130:LIFR (EC 50:50.15 pg / ml) (Fig. 12A). Western blotting revealed STAT3 phosphorylation in Ba / F3-CNTFR:gp130:OSMR cells stimulated with 10 ng / ml CNTF and OSM, but not LIF (Fig. 12B). As shown in Fig. 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 through OSMR and LIFR is relatively efficient. CNTF can also recruit IL-6R, as an alternative low-affinity α receptor, to the CNTF:IL-6R:gp130:LIFR complex. Therefore, we tested whether CNTF also signals through 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, or IL-6, or were left untreated. As expected, proliferation of Ba / F3-gp130 cells was not induced by these cytokines (Figure 12E). However, proliferation of Ba / F3-CNTFR:gp130:LIFR and Ba / F3-IL-6R:gp130:LIFR cells was observed after stimulation with CNTF, demonstrating low-affinity crosstalk of CNTF with IL-6R. Although Ba / F3-IL-6R:gp130:OSMR proliferated after stimulation with OSM via the gp130:OSMR complex and after stimulation with IL-6 via the IL-6R:gp130 receptor complex, proliferation was not induced by CNTF, even at the highest applied concentration (50 ng / ml) (Fig. 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. While 10 ng / ml CNTF was sufficient to induce STAT3 phosphorylation in Ba / F3-IL-6R:gp130:LIFR cells, 100 ng / ml CNTF failed to induce STAT3 phosphorylation in Ba / F3-IL-6R:gp130:OSMR cells (Fig. 12G, H). Finally, we investigated whether CNTF directly binds to recombinant OSMR in a cell-free environment via coimmunoprecipitation using purified soluble recombinant protein. HyperCNTF-Fc (CNTF fused to soluble CNTFR and the IgG1 Fc portion) was incubated with soluble OSMR in the presence and absence of soluble gp130, followed by precipitation with protein A beads. Unlike LIF, OSM must bind to gp130 via site 2 before recruitment of OSMR via site 3, suggesting that CNTF binding to OSMR may also be gp130-dependent. As shown in Figure 12H, OSMR was precipitated by HyperCNTF-Fc only in the presence of soluble gp130. This indicates that recruitment of CNTF to OSMR via site 3 depends on prior binding of CNTF to site 2 of gp130. Collectively, our findings indicate that CNTF can utilize OSMR as a distinct receptor after binding to CNTFR, but not after binding to IL-6R.

[0173] Example 13 - Discussion of Experimental Results IL-6, IL-11, and CNTF cannot bind to the β-receptors gp130 and / or LIFR in the absence of α-receptors, suggesting that cytokine interaction with α-receptors shifts binding sites 2 and / or 3 to the correct conformation to enable β-receptor binding. Because we introduced the α-receptor-independent binding site 3 from LIF / OSM into the α-receptor-dependent IL-6 and IL-11 scaffolds, it was entirely unexpected that transferring site 3 from α-receptor-independent cytokines, for example, from LIF to GIL-11 and GIL-6, or from OSM to G1O-6, resulted in cytochimeras that were still α-receptor-dependent. As shown here, GIL-6 and G1O-6 activate the gp130:LIFR and gp130:OSMR receptor complexes only after binding to non-signaling IL-6R. This provides additional evidence that, although site 3 of the original LIF / OSM relative to LIFR / OSMR is α-receptor independent, rearrangement of site 3 of LIF / OSM into the IL-6 scaffold renders site 3 of LIF and OSM α-receptor dependent. This suggests that the interconnection with site 3 mediated by the α-helix shift of site 1 determines whether a cytokine is α-receptor dependent.

[0174] Regarding biological activity, GIL-6 (EC 50 : IL-6R:gp130:LIFR, 1.21 ng / ml) and GIO-6 (IL-6R:gp130:LIFR, EC 50 :6.16ng / ml, IL-6R:gp130:OSMR, EC 50 :1.6ng / ml) was IC7 (for IL-6R:gp130:LIFR, EC 50 IL-6R:gp130:OSMR (EC 4.8 ng / ml) compared to the native cytokines IL-6, LIF, and OSM (EC 4.8 ng / ml for IL-6R:gp130, respectively). 50 :0.11ng / ml, gp130:LIFR, EC 50:0.08ng / ml, gp130:OSMR, EC 50 However, the biological activity of GIL-6 and GIO-6 may be increased by introducing affinity-enhancing mutations into site 1 to increase their affinity for IL-6R.

[0175] To directly compare the activities of IC7 and GIL-6, we reexamined the biological properties of IC7. IC7 had not previously been tested in cells expressing IL-6R, gp130, and OSMR. GIL-6 signaled via IL-6R:gp130:OSMR, and thus IC7, initially considered a negative control, was also found to bind and activate cells via IL-6R:gp130:OSMR, defining a second high-affinity non-natural receptor complex for IC7.

[0176] Therefore, we also tested and demonstrated that CNTF uses the CNTFR:gp130:OSMR as a second high-affinity natural receptor complex. According to the Human Protein Atlas, CNTFR is restricted to a subset of cells and is largely absent in immune cells and cardiomyocytes, whereas OSMR and gp130 are more commonly expressed. Notably, co-expression of all three receptors has been found in mammary gland cells, cholangiocytes, neurons, astrocytes, skeletal muscle cells, smooth muscle cells, hepatocytes, and fibroblasts. Regarding cancer cell lines, LIFR and OSMR expression overlap and is found in nearly all cells, whereas CNTFR expression is restricted to only a subset of cell lines. Therefore, it is not surprising that OSMR was not previously discovered as an alternative CNTFR, since only Ba / F3 cells lack expression of all IL-6-type cytokine receptors except for endogenous IL-27RA. Therefore, heterologous expression of receptor chains in Ba / F3 cells is a reasonable approach to identify and validate receptor complexes of IL-6-type cytokines and cytochimeras.

[0177] Treatment with the human CNTF derivative Axokine induced persistent weight loss in clinical trials but subsequently failed therapeutically due to the rapid development of anti-Axokine / CNTF antibodies for unknown reasons. Recently, IC7 was shown to have CNTF-like effects on weight control in mice. However, it is unknown whether IC7 also induces anti-IC7 antibodies in humans. CNTF also possesses inflammatory properties, and its administration induces inflammatory responses, including the induction of fever and an acute-phase response, although no pro-inflammatory effects have been described for IC7 to date.

[0178] The question then arose as to whether the beneficial effects of CNTF and IC7 on weight maintenance are mediated through LIFR signaling, OSMR signaling, or both. Cytochimeras GIL-6 and GIL-11 distinguish between LIFR and OSMR signaling and are therefore promising therapeutic candidates. If OSMR, but not LIFR, is responsible for the beneficial effects of IC7 / CNTF, we could generate OSMR-selective cytochimeras by transferring the site 3 AB loop from mouse OSM to CNTF / IL-6, which likely prevents LIFR crosstalk.

[0179] In conclusion, our studies demonstrate the development of two cytochimeric IL-6 and GI0-6 receptors that specifically activate the non-native IL-6R:gp130:LIFR and IL-6R:gp130:OSMR complexes with high affinity. Furthermore, we demonstrate that the previously described cytokine IC7 uses OSMR as an alternative β receptor, as does the native cytokine CNTF.

Claims

1. (a) two binding sites derived from the same cytokine of the IL-6 cytokine family, the cytokine requires a cytokine-specific non-signaling alpha receptor subunit as part of a receptor complex to trigger signal transduction; the first binding site is capable of binding to the cytokine-specific non-signaling alpha receptor subunit; the second binding site is capable of binding to the signal transduction receptor gp130; the two binding sites; and (b) a third binding site derived from a further cytokine of the IL-6 cytokine family, the additional cytokine does not require a cytokine-specific non-signaling alpha receptor subunit as part of a receptor complex to cause signaling; the third binding site is capable of binding to a cytokine-specific signaling receptor for the additional cytokine; the third binding site; and A polypeptide comprising:

2. 2. The polypeptide of claim 1, wherein the cytokine that requires a cytokine-specific non-signaling alpha receptor subunit as part of a receptor complex is selected from the group including IL-6 and IL-11.

3. 3. The polypeptide of claim 1 or 2, wherein the cytokine that does not require a cytokine-specific non-signaling alpha receptor subunit as part of the receptor complex is selected from the group including LIF, OSM, and CT-1.

4. 4. The polypeptide of any one of claims 1 to 3, wherein the cytokine that requires a cytokine-specific, non-signaling alpha receptor subunit as part of its receptor complex is IL-11, and the cytokine that does not require a cytokine-specific, non-signaling receptor subunit as part of its receptor complex is LIF.

5. 4. The polypeptide of any one of claims 1 to 3, wherein the cytokine that requires a cytokine-specific, non-signaling alpha receptor subunit as part of its receptor complex is IL-6, and the cytokine that does not require a cytokine-specific, non-signaling receptor subunit as part of its receptor complex is LIF or OSM.

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

7. 7. The polypeptide of any one of claims 1 to 6, comprising an amino acid sequence having 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 having 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 having 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 site is derived from a human cytokine.

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

10. A polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide according to any one of claims 1 to 9, optionally an expression cassette, preferably an expression vector.

11. 11. An expression system comprising the polynucleotide of claim 10, which may be of eukaryotic, prokaryotic or archaeal origin or synthetic in nature, e.g. a cell-free expression system.

12. 12. A host cell comprising the polynucleotide of claim 10 or the expression system of claim 11, optionally 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, such as 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. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

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

15. 14. The polypeptide of any one of claims 1 to 9 or the pharmaceutical composition of claim 13 for use in a method for the prevention and / or treatment of a condition in a patient, wherein the condition is selected from the group consisting of lymphopenia, muscle atrophy, osteoporosis, thrombocytopenia, obesity-related metabolic disorders such as type II diabetes, obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, hypertension, stroke, or cardiovascular disease, neurological disorders such as paraplegia, Alzheimer's disease, and Parkinson's disease.

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