Treatment of smooth muscle cell-mediated diseases

Abstract

To provide a pharmaceutical preparation for the diagnosis, treatment and prophylaxis of diseases and conditions associated with smooth muscle cell (SMC) dysfunction.SOLUTION: A pharmaceutical preparation contains as an active ingredient an antibody or antigen-binding fragment thereof capable of binding to IL-11 and inhibiting IL-11-mediated signaling, or an antibody or antigen-binding fragment thereof capable of binding to the IL-11 receptor (IL-11Rα) and inhibiting IL-11-mediated signaling.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the diagnosis, treatment, and prevention of diseases and conditions associated with smooth muscle cell (SMC) dysfunction.

Background Art

[0002] SMC dysfunction is seen in various diseases and conditions, in which normal SMCs stimulated by various upstream disease factors abnormally proliferate, hypertrophy, migrate, dedifferentiate, and produce extracellular matrix.

[0003] SMCs with this disease-related phenotype are called by various names in the art, such as "secretory SMCs" (see, for example, Rainger and Nash Circ Res 88 (6), 615-622 (2001)), "synthetic SMCs" (see, for example, Beamish et al., Tissue Eng Part B Rev. (2010) 16(5): 467-491), and "migratory SMCs" (see, for example, Sandison et al., J Physiol. (2016);594(21):6189-6209). In this specification, SMCs of this phenotype are referred to as "secretory SMCs".

[0004] SMC dysfunction is caused by various upstream factors, and there are several common downstream factors that maintain the harmful disease-related secretory SMC phenotype.

[0005] Diseases characterized by vascular smooth muscle cell (VSMC) dysfunction include atherosclerosis, hypertension, aneurysm, vascular stenosis and restenosis, atherosclerosis, supravalvular stenosis, pulmonary hypertension, plexiform lesions, fibromuscular dysplasia, telangiectasia, etc. SMCs are components of various internal organs and constitute the contraction apparatus of the esophagus, stomach, small intestine, large intestine, rectum, ureter, and bladder. Abnormal functions of SMCs in internal organs may cause achalasia, dysphagia, intestinal stenosis, pyloric stenosis, diarrhea, constipation, diverticular disease, and kidney and bladder diseases. In addition, SMCs also play an important role in lung function. The cell mass of SMCs found in the airways of the bronchus and the contraction of SMCs in the airways of the bronchus are involved in the pathology of asthma, cystic fibrosis, COPD, ARDS, and other respiratory diseases. The dysfunction of SMCs in asthma occurs when the phenotype and behavior of SMCs change in response to environmental and chemical stimuli (such as chemokines, interleukins, and other cytokines).

Summary of the Invention

Means for Solving the Problems

[0006] The present invention relates to treating conditions associated with the activity of smooth muscle cells (SMCs) (such as the activity of secretory SMCs) by suppressing IL-11-mediated signal transduction.

[0007] In one aspect, the present invention provides an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction for use in a method of treating or preventing a disease in which smooth muscle cells (SMCs) are pathologically involved.

[0008] In another aspect, the present invention provides the use of an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction in the manufacture of a pharmaceutical for use in a method of treating or preventing a disease in which smooth muscle cells (SMCs) are pathologically involved.

[0009] In another aspect, the present invention provides a method for treating or preventing a disease pathologically involving smooth muscle cells (SMCs), the method comprising administering to a subject in need of treatment a therapeutically effective amount of an agent capable of suppressing interleukin 11 (IL-11)-mediated signaling.

[0010] According to various aspects of the present invention, in some embodiments, the smooth muscle cells (SMCs) are secretory smooth muscle cells. Thus, in some embodiments, the disease pathologically involving smooth muscle cells (SMCs) is a disease pathologically involving secretory smooth muscle cells. According to various aspects of the present invention, the smooth muscle cells and / or the secretory smooth muscle cells are vascular smooth muscle cells (VSMCs). Thus, in some embodiments, the disease pathologically involving smooth muscle cells or secretory smooth muscle cells is a disease pathologically involving vascular smooth muscle cells (VSMCs).

[0011] According to various aspects of the present invention, in some embodiments, the agent is an agent capable of binding to IL-11 or the IL-11 receptor. In some embodiments, the agent is selected from the group consisting of an antibody or an antigen-binding fragment thereof, a polypeptide, a peptide, an oligonucleotide, an aptamer, and a small molecule. In some embodiments, the agent is an antibody or an antigen-binding fragment thereof. In some embodiments, the agent is a decoy receptor for IL-11. In some embodiments, the agent is an agent capable of decreasing the expression of IL-11 or the IL-11 receptor. In some embodiments, the agent is an oligonucleotide or a small molecule.

[0012] According to various aspects of the present invention, in some embodiments, the disease is a disease of the circulatory system, digestive system, excretory system, respiratory system, renal system or genital system. In some embodiments, the diseases in which smooth muscle cells (SMCs) are pathologically involved include atherosclerosis, hypertension, aneurysm, Marfan syndrome, aortic aneurysm, Furlong syndrome, Spritzen-Goldberg syndrome, Royce-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome, cerebral aneurysm, vascular stenosis and restenosis, atherosclerosis, fibromuscular dysplasia (FMD), supravalvular stenosis, renal artery stenosis, pulmonary arterial hypertension (PAH), plexiform lesion, fibromuscular dysplasia, telangiectasia, achalasia, dysphagia, diarrhea, constipation, inflammatory bowel disease (IBD), celiac disease, irritable bowel syndrome, Crohn's disease, ulcerative colitis, intestinal stenosis, diverticulosis, kidney disease, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, crescentic glomerulonephritis, lupus nephritis, diabetic nephropathy (DN), bladder disease, lung disease, asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), systemic sclerosis, Hutchinson-Gilford progeria syndrome (HGPS), leiomyoma, leiomyosarcoma and Hermansky-Pudlak syndrome (HPS), and is selected from the group consisting of.

[0013] According to various aspects of the present invention, in some embodiments, the treatment method or the prevention method includes the step of administering the drug to a subject in which the expression of IL-11 or the IL-11 receptor is upregulated. In some embodiments, the treatment method or the prevention method includes the step of administering the drug to a subject in which upregulation of the expression of IL-11 or the IL-11 receptor has been confirmed. In some embodiments, the treatment method or the prevention method includes the step of determining whether the expression of IL-11 or the IL-11 receptor is upregulated in a subject, and the step of administering the drug to a subject in which the expression of IL-11 or the IL-11 receptor is upregulated.

[0014] In another aspect, the present invention provides the use of an agent capable of suppressing interleukin 11 (IL-11)-mediated signaling for suppressing the activity of smooth muscle cells (SMCs).

[0015] In another aspect, the present invention provides a method of suppressing the activity of smooth muscle cells (SMCs), the method comprising the step of contacting the smooth muscle cells (SMCs) with an agent capable of suppressing interleukin 11 (IL-11)-mediated signaling.

[0016] In another aspect, the present invention provides a method of suppressing the activity of smooth muscle cells (SMCs) in a subject, the method comprising the step of administering to the subject an agent capable of suppressing interleukin 11 (IL-11)-mediated signaling.

[0017] In another aspect, the present invention provides a method for determining whether a subject is suitable for the treatment or prevention of a disease pathologically involving smooth muscle cells (SMCs) using an agent capable of suppressing the action of interleukin 11 (IL-11), the method comprising the step of determining (optionally in vitro) whether the expression of IL-11 or interleukin 11 receptor (IL-11R) is upregulated in the subject.

[0018] In another aspect, the present invention provides a method for selecting a subject for the treatment or prevention of a disease pathologically involving smooth muscle cells (SMCs) using an agent capable of suppressing interleukin 11 (IL-11)-mediated signaling, the method comprising the step of determining (optionally in vitro) whether the expression of IL-11 or the IL-11 receptor is upregulated in the subject.

[0019] In another aspect, the present invention provides a method for diagnosing a disease in which smooth muscle cells (SMCs) are pathologically involved in a subject or diagnosing the risk of onset of the disease, the method comprising the step of determining (optionally in vitro) whether the expression of interleukin 11 (IL-11) or the IL-11 receptor is upregulated in a sample obtained from the subject. In some embodiments, the method is a method for confirming the diagnosis of the disease in a subject suspected of suffering from a disease in which smooth muscle cells (SMCs) are pathologically involved. In some embodiments, the method further comprises the step of selecting a subject for treatment with an agent capable of suppressing IL-11-mediated signaling.

[0020] In another aspect, the present invention provides a method for providing a prognosis to a subject suffering from a disease in which smooth muscle cells (SMCs) are pathologically involved or a subject suspected of suffering from the disease, the method comprising the step of determining (optionally in vitro) whether the expression of interleukin 11 (IL-11) or the IL-11 receptor is upregulated in a sample obtained from the subject, and the step of providing a prognosis for the treatment of the subject with an agent capable of suppressing IL-11-mediated signaling based on the determination. In some embodiments, the method further comprises the step of selecting a subject in whom upregulation of the expression of IL-11 or the IL-11 receptor has been confirmed for treatment with an agent capable of suppressing IL-11-mediated signaling.

[0021] In another aspect, the present invention provides a method for diagnosing a disease in which smooth muscle cells (SMCs) are pathologically involved in a subject or diagnosing the risk of onset of the disease, the method comprising measuring (optionally in vitro) in the subject one or more genetic factors that predict upregulation of the expression of IL-11 or the IL-11 receptor or upregulation of IL-11-mediated signal transduction. In some embodiments, the method is a method for determining the diagnosis of a disease in a subject suspected of having a disease in which smooth muscle cells (SMCs) are pathologically involved. In some embodiments, the method further comprises a step of selecting a subject for treatment with an agent capable of suppressing IL-11-mediated signal transduction.

[0022] In another aspect, the present invention provides a method for providing a prognosis to a subject suffering from a disease in which smooth muscle cells (SMCs) are pathologically involved or a subject suspected of suffering from the disease, the method comprising measuring (optionally in vitro) in the subject one or more genetic factors that predict upregulation of the expression of IL-11 or the IL-11 receptor or upregulation of IL-11-mediated signal transduction.

Mode for Carrying Out the Invention

[0023] Interleukin 11 and IL-11 receptor Interleukin 11 (IL-11), also known as an adipocyte differentiation inhibitor, is a pleiotropic cytokine and a member of the IL-6 cytokine family, including IL-6, IL-11, IL-27, IL-31, oncostatin, leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), ciliary neurotrophic factor (CNTF) and neuropoetin (NP-1).

[0024] Interleukin 11 (IL-11) is expressed in various mesenchymal cells 1。The genomic sequence of IL-11 is mapped to the centromeric region of chromosome 7 and chromosome 19 1 , and it is transcribed with the addition of a classical signal peptide that efficiently secretes IL-11 from cells. The activator protein complex (cJun / AP-1) within the promoter sequence of the IL-11 gene is important for the basal transcriptional regulation of IL-11 1 . The precursor of human IL-11 is a polypeptide consisting of 199 amino acids, and mature IL-11 is a protein consisting of 178 amino acid residues (Garbers and Scheller., Biol. Chem. 2013; 394(9):1145-1161). The amino acid sequence of human IL-11 is available from the UniProt accession number P20809 (P20809.1 GI:124294; SEQ ID NO: 1). Recombinant human IL-11 (oprelvekin) is also commercially available. IL-11s from several other species, such as mouse, rat, pig, bovine, several species of teleost fish, and primates, have also been cloned and their sequences determined.

[0025] As used herein, IL-11 refers to IL-11 from any species and includes isoforms, fragments, variants or homologs of IL-11 obtained from any species. In a preferred embodiment, the species is human (Homo sapiens). The isoforms, fragments, variants or homologs of IL-11 may be characterized by having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of IL-11 precursor or mature IL-11 derived from a specific species (e.g., human). The isoforms, fragments, variants or homologs of IL-11 may be characterized by the ability to stimulate signal transduction in cells expressing IL-11Rα and gp130 by binding to IL-11Rα (e.g., the ability as described in Curtis et al. Blood, 1997, 90(11) or Karpovich et al. Mol. Hum. Reprod. 2003 9(2): 75-80). The length of the IL-11 fragment may be of any length (amino acid length), but may be at least 25% of the length of mature IL-11, and at most 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of mature IL-11. The length of the IL-11 fragment may be as short as 10 amino acids in length, and as long as 15 amino acids in length, 20 amino acids in length, 25 amino acids in length, 30 amino acids in length, 40 amino acids in length, 50 amino acids in length, 100 amino acids in length, 110 amino acids in length, 120 amino acids in length, 130 amino acids in length, 140 amino acids in length, 150 amino acids in length, 160 amino acids in length, 170 amino acids in length, 180 amino acids in length, 190 amino acids in length or 195 amino acids in length.

[0026] IL-11 signals through homodimers of the ubiquitously expressed glycoprotein 130 (gp130; also known as glycoprotein 130, IL-6ST, IL-6-β, or CD130). gp130 is a transmembrane protein and is a receptor subunit that forms a type I cytokine receptor by associating with the IL-6 receptor family. Specificity is conferred by the individual IL-11α receptor (IL-11Rα). Although the IL-11α receptor is not directly involved in signaling, when a cytokine binds to the α receptor, it associates with gp130 to form the final complex.

[0027] Human gp130 (including a signal peptide consisting of 22 amino acids) is a protein consisting of 918 amino acids, and its mature form consists of 866 amino acids, including an extracellular domain consisting of 597 amino acids, a transmembrane domain consisting of 22 amino acids, and an intracellular domain consisting of 277 amino acids. The extracellular domain of human gp130 contains the cytokine-binding module (CBM) of gp130. The CBM of gp130 contains the Ig-like domain D1 and the fibronectin type III domains D2 and D3 of gp130. The amino acid sequence of human gp130 is available from UniProt accession number P40189-1 (SEQ ID NO: 2).

[0028] Human IL-11Rα is a polypeptide consisting of 422 amino acids (UniProt Q14626; SEQ ID NO: 3), and has approximately 85% nucleotide sequence identity and amino acid sequence identity with mouse IL-11Rα (Du and Williams., Blood Vol, 89, No,11, June 1, 1997). It has been reported that there are two isoforms of IL-11Rα with different intracellular domains (Du and Williams, supra). The IL-11 receptor α-chain (IL-11Rα) has many similarities with the IL-6 receptor α-chain (IL-6Rα) in terms of structure and function. The extracellular domains of IL-11Rα and IL-6Rα have 24% amino acid identity and contain the characteristic Trp-Ser-X-Trp-Ser (WSXWS) conserved motif. The short intracellular domains (34 amino acids in length) of IL-11Rα and IL-6α do not contain the Box1 region and Box2 region required for the activation of the JAK / STAT signaling pathway.

[0029] The receptor binding sites of mouse IL-11 have been mapped and three sites (site I, site II and site III) have been identified. Substitutions in the site II region or the site III region result in a decrease in the binding affinity to gp130. The site III mutation does not show detectable agonist activity and shows antagonist activity against IL-11Rα (Cytokine Inhibitors Chapter 8; edited by Gennaro Ciliberto, Rocco Savino, Marcel Dekker, Inc. 2001).

[0030] As used herein, the IL-11 receptor refers to a polypeptide or polypeptide complex capable of binding to IL-11. In some embodiments, the IL-11 receptor can bind to IL-11 and can induce signal transduction in cells that express itself.

[0031] The IL-11 receptor may be derived from any species and includes isoforms, fragments, variants or homologs of the IL-11 receptor obtained from any species. In a preferred embodiment, the species is human (Homo sapiens).

[0032] In some embodiments, the IL-11 receptor may be IL-11Rα. In some embodiments, the IL-11 receptor may be a polypeptide complex comprising IL-11Rα. In some embodiments, the IL-11 receptor may be a polypeptide complex comprising IL-11Rα and gp130. In some embodiments, the IL-11 receptor may be gp130 or a gp130-containing complex to which IL-11 binds.

[0033] An isoform, fragment, variant or homolog of IL-11Rα may be characterized by having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of IL-11Rα derived from a particular species (e.g., human). An isoform, fragment, variant or homolog of IL-11Rα may be characterized by the ability to stimulate signal transduction in cells expressing IL-11Rα and gp130 by binding to IL-11 (e.g., the ability as described in Curtis et al. Blood, 1997, 90(11) or Karpovich et al. Mol. Hum. Reprod. 2003 9(2): 75-80), preferably derived from the same species. The length of the fragment of the IL-11 receptor may be of any length (amino acid length), but may be at least 25% of the length of mature IL-11Rα, and at most 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of mature IL-11Rα. The length of the fragment of the IL-11 receptor may be as short as 10 amino acids in length, and as long as 15 amino acids in length, 20 amino acids in length, 25 amino acids in length, 30 amino acids in length, 40 amino acids in length, 50 amino acids in length, 100 amino acids in length, 110 amino acids in length, 120 amino acids in length, 130 amino acids in length, 140 amino acids in length, 150 amino acids in length, 160 amino acids in length, 170 amino acids in length, 180 amino acids in length, 190 amino acids in length, 200 amino acids in length, 250 amino acids in length, 300 amino acids in length, 400 amino acids in length or 415 amino acids in length.

[0034] IL-11 signaling IL-11 binds to IL-11Rα with low affinity (Kd = approximately 10 nmol / L) and cannot transmit biological signals by the interaction between these binding partners alone. Co-expression of IL-11Rα and gp130 is required for the formation of a receptor that can bind with high affinity (Kd = approximately 400 - 800 pmol / L) and transmit signals (Curtis et al (Blood 1997 Dec 1; 90 (11):4403-12; Hilton et al., EMBO J 13:4765, 1994; Nandurkar et al., Oncogene 12:585, 1996). When IL-11 binds to IL-11Rα on the cell surface, heterodimerization, tyrosine phosphorylation, activation of gp130, and downstream signal transduction are induced, and this signal transduction is mainly carried out via the mitogen-activated protein kinase (MAPK) cascade and the Janus kinase / signal transducer and activator of transcription (Jak / STAT) pathway (Garbers and Scheller, supra).

[0035] Furthermore, in principle, since soluble IL-11Rα can bind to IL-11 to form a biologically active soluble complex (Pflanz et al., 1999 FEBS Lett, 450, 117-122), similar to IL-6, IL-11 is thought to bind to soluble IL-11Rα before binding to gp130 on the cell surface (Garbers and Scheller, supra). Curtis et al. (Blood 1997 Dec 1; 90 (11):4403-12) reported that they expressed soluble mouse IL-11 receptor α chain (sIL-11R) and examined signal transduction in gp130-expressing cells. In this study, it was reported that in the absence of transmembrane IL-11R but in the presence of gp130, soluble IL-11R induced IL-11-dependent M1 leukemia cell differentiation, Ba / F3 cell proliferation, and early intracellular events (such as phosphorylation of gp130, STAT3, and SHP2) similar to signal transduction through transmembrane IL-11R. Activation of signal transduction through membrane-bound gp130 by IL-11 bound to soluble IL-11Rα has been demonstrated in recent years (Lokau et al., 2016 Cell Reports 14, 1761-1773). This so-called trans-signaling of IL-11 may be important in the pathogenesis of diseases, but its role in human diseases awaits further study.

[0036] As used herein, "trans-signaling of IL-11" refers to the signal transduction induced by IL-11 bound to IL-11Rα further binding to gp130. IL-11 can bind to IL-11Rα by non-covalent bonds to form a complex. gp130 is membrane-bound, expressed by cells, and signal transduction occurs when the IL-11:IL-11Rα complex binds to gp130. In some embodiments, IL-11Rα may be soluble IL-11Rα. In some embodiments, soluble IL-11Rα is a soluble (secreted) isoform of IL-11Rα (e.g., lacking a transmembrane domain). In some embodiments, soluble IL-11Rα is a product released by proteolysis of the extracellular domain of membrane-bound IL-11Rα. In some embodiments, IL-11Rα may be membrane-bound, and signal transduction via gp130 may be induced by IL-11 bound to membrane-bound IL-11Rα further binding to gp130. This is referred to as "cis-signaling of IL-11".

[0037] IL-11-mediated signal transduction has been shown to stimulate hematopoiesis, stimulate osteoclast activity, stimulate neurogenesis, suppress adipogenesis, decrease the expression of pro-inflammatory cytokines, regulate the metabolism of the extracellular matrix (ECM), and mediate normal growth control of gastrointestinal epithelial cells 1 。

[0038] The physiological role of interleukin 11 (IL-11) has not yet been elucidated. IL-11 has been most strongly recognized for its association with the activation of hematopoietic cells and the production of platelets, but it has been found to have not only pro-inflammatory effects, but also anti-inflammatory effects and angiogenesis-promoting effects, suggesting that it is important for tumor formation. It is also known that TGFβ1 and tissue damage may induce the expression of IL-11 (Zhu, M. et al. PLOS ONE 10, (2015); Yashiro, R. et al. J. Clin. Periodontol. 33, 165-71 (2006); Obana, M. et al. Circulation 121, 684-91 (2010); Tang, W et al. J. Biol. Chem. 273, 5506-13 (1998)).

[0039] IL-11 is an important post-transcriptional regulator of TGFβ-mediated signal transduction. TGFβ1 has been shown to stimulate the AP-1 promoter region of IL-11, and it has been shown that when the secretion of IL-11 is induced by TGFβ, this induces the activation of ERK p42 / 44 kinase and p38 MAP kinase in intestinal myofibroblasts (Bamba et al. Am J Physiol Gastrointest Liver Physiol. (2003) 285(3):G529-38). MAP kinase inhibitors can significantly reduce the secretion of TGFβ-induced IL-11, and it has been shown that the stabilization of mRNA via p38 MAP kinase is extremely important for the secretion of TGFβ-induced IL-11.

[0040] As used herein, "IL-11 signal transduction" and "IL-11-mediated signal transduction" refer to signal transduction through the binding of IL-11 to the IL-11 receptor, or signal transduction through the binding of an IL-11 fragment having the function of the mature IL-11 molecule to the IL-11 receptor.

[0041] Smooth muscle cells (SMC) Smooth muscle cells (SMCs) are a type of mesenchymal cells found in many organs in vivo. Vascular smooth muscle cells (VSMCs) form the middle membrane layer in all large arteries and arterioles of the vascular system and are essential for maintaining vascular tone and blood pressure. In addition, SMCs are also found in various internal organs and constitute the contraction apparatus of the esophagus, stomach, small intestine, large intestine, rectum, ureter, and bladder. Furthermore, SMCs are also found in the airways of the respiratory system (e.g., of the lungs).

[0042] In embodiments according to various aspects of the present invention, the smooth muscle cell (SMC) may be any of vascular smooth muscle cells (VSMCs), intestinal smooth muscle cells (iSMCs), airway smooth muscle cells (ASMCs), smooth muscle cells derived from blood vessels, arteries, arterioles, internal organs, digestive system organs, urinary system organs, esophagus, stomach, small intestine, large intestine, rectum, ureter, bladder, kidneys (e.g., mesangial cells), respiratory system organs, airways, trachea, lungs, bronchi, or bronchioles.

[0043] In embodiments according to various aspects of the present invention, the smooth muscle cell (SMC) may be any of vascular smooth muscle cells (VSMCs), intestinal smooth muscle cells (iSMCs), smooth muscle cells derived from blood vessels, arteries, arterioles, internal organs, digestive system organs, urinary system organs, esophagus, stomach, small intestine, large intestine, rectum, ureter, bladder, or kidneys (e.g., mesangial cells). In another embodiment, the SMC is not an airway smooth muscle cell (ASMC) nor a smooth muscle cell derived from respiratory system organs, airways, trachea, lungs, bronchi, or bronchioles. In another embodiment, the SMC is not a vascular smooth muscle cell (VSMC). In another embodiment, the SMC is not an intestinal smooth muscle cell (iSMC). In another embodiment, the SMC is not any of the smooth muscle cells derived from blood vessels, arteries, arterioles, internal organs, digestive system organs, urinary system organs, esophagus, stomach, small intestine, large intestine, rectum, ureter, bladder, kidneys (e.g., mesangial cells).

[0044] Under normal physiological conditions, SMCs have a contractile phenotype characterized by, for example, an elongated spindle shape and a low growth rate when cultured (Beamish et al., Tissue Eng Part B Rev (2010) 16(5):467-491; Rzucidlo (2009) Vascular 17(Suppl 1):S15-S20). Furthermore, the contractile phenotype may be characterized by the expression of, for example, myocardin, myosin 11, smoothelin, smooth muscle myosin heavy chain (SMMHC), α-smooth muscle actin (αSMA), SM22α, h1-caldesmon, h-caldesmon, α1β1 integrin, α7β1 integrin and / or dystrophin glycoprotein complex (DGPC) (Owens et al., Physiol Rev (2004) 84(3):767-801, Xie et al., Arteriosclerosis, Thrombosis, and Vascular Biology (2011) 31:1485-1494; Beamish et al., Tissue Eng Part B Rev (2010) 16(5):467-491; Rzucidlo (2009) Vascular 17(Suppl 1):S15-S20). Contractile vascular smooth muscle cells (VSMCs) can be identified by specific cell markers. αSMA and SM22α are early markers of SMCs at the developmental stage, and caldesmon, caldesmon and SMMHC are late markers. 3 .

[0045] Contractile SMCs may be induced to change to a "secretory" (also called "synthetic" or "migratory") phenotype in response to specific genetic, mechanical, endocrine, inflammatory, lipid and neurohumoral stimuli, and secretory SMCs are characterized by increased growth rate and migratory ability, as well as the expression and / or secretion of pro-inflammatory factors and extracellular matrix components (e.g., type I collagen).

[0046] Secretory SMCs show a decrease in the expression of smooth muscle cell-related genes encoding contractile proteins (e.g., myocardin, SM22α, SMMHC), and an increase in the expression of osteopontin, l-caldesmon, non-muscle myosin heavy chain B (NM-B MHC), vimentin, tropomyosin 4, and intracellular retinol-binding protein-1 (CRBP-1). Secretory SMCs also show a decrease in the number of actin filaments, an increase in the number of secretory vesicles, an increase in cell size, a "hill-and-valley" morphology when cultured, and an increase in the expression of α4β1 integrin.

[0047] SMCs having a phenotype referred to herein as secretory (i.e., secretory SMCs) may be characterized by one or more of the following: expression of one or more pro-inflammatory factors; expression and / or secretion of one or more extracellular matrix components (e.g., type I collagen); expression and / or secretion of IL-11; expression of one or more of osteopontin, l-caldesmon, non-muscle myosin heavy chain B (NM-B MHC), vimentin, tropomyosin 4, and intracellular retinol-binding protein-1 (CRBP-1); secretory vesicles; a "hill-and-valley" morphology when cultured in vitro; and expression of α4β1 integrin. In some embodiments, secretory SMCs are characterized by one or more of the following, as compared to non-secretory equivalent SMCs (e.g., contractile SMCs): an increase in proliferation rate; an increase in migration rate; an increase in the expression of one or more pro-inflammatory factors; an increase in the expression and / or secretion of one or more extracellular matrix components (e.g., type I collagen); an increase in the expression and / or secretion of IL-11; an increase in the expression of one or more of osteopontin, l-caldesmon, non-muscle myosin heavy chain B (NM-B MHC), vimentin, tropomyosin 4, and intracellular retinol-binding protein-1 (CRBP-1); an increase in the number of secretory vesicles; a decrease in the number of actin filaments; an increase in the expression of α4β1 integrin; and a decrease in the expression of one or more contractile proteins (e.g., myocardin, SM22α, SMMHC).

[0048] An SMC having a phenotype called the contractile type (i.e., contractile SMC) in this specification may be characterized by one or more of the expressions of myocardin, myosin 11, smoothelin, smooth muscle myosin heavy chain (SMMHC), α-smooth muscle actin (αSMA), SM22α, h1-caldesmon, h-caldesmon, α1β1 integrin, α7β1 integrin, and dystrophin glycoprotein complex (DGPC); actin filaments; and an elongated spindle shape when cultured in vitro. In some embodiments, the contractile SMC has a decrease in proliferation rate, a decrease in migration rate, a decrease in the expression of one or more pro-inflammatory factors, a decrease in the expression and / or secretion of one or more extracellular matrix components (e.g., type I collagen), a decrease in the expression and / or secretion of IL-11, a decrease in the expression of one or more of osteopontin, l-caldesmon, non-muscle cell myosin heavy chain B (NM-B MHC), vimentin, tropomyosin 4, and intracellular retinol-binding protein-1 (CRBP-1), a decrease in the number of secretory vesicles, an increase in the number of actin filaments, a decrease in the expression of α4β1 integrin, and an increase in the expression of one or more of myocardin, myosin 11, smoothelin, SMMHC, αSMA, SM22α, h1-caldesmon, h-caldesmon, α1β1 integrin, α7β1 integrin, and dystrophin glycoprotein complex (DGPC) when evaluated relative to a non-contractile equivalent SMC (e.g., secretory SMC).

[0049] In some embodiments, the secretory SMCs may exhibit one or more of a decrease in the expression of SM22α when compared to non-secretory equivalent SMCs (e.g., contractile SMCs), a decrease in the expression of myocardin when compared to non-secretory equivalent SMCs (e.g., contractile SMCs), an increase in the expression and / or secretion of collagen when compared to non-secretory equivalent SMCs (e.g., contractile SMCs), or an increase in the expression and / or secretion of IL-11 when compared to non-secretory equivalent SMCs (e.g., contractile SMCs). In some embodiments, the secretory SMCs may exhibit one or more of an increase in proliferation, an increase in migration, or an increase in invasion when compared to non-secretory equivalent SMCs (e.g., contractile SMCs).

[0050] As used herein, an "equivalent SMC" may be, for example, an SMC derived from the same organ or the same tissue as the SMC being compared.

[0051] As used herein, "expression" may refer to gene expression or protein expression. Gene expression can be measured, for example, by quantitative real-time PCR (qRT-PCR), by a method of detecting mRNA encoding a marker, or by a method using a reporter. Protein expression can be measured, for example, by methods known to those skilled in the art using antibodies, such as by detecting a protein, and such methods include Western blot, immunohistochemical methods, immunocytochemical methods, flow cytometry, ELISA, etc. Protein expression can be measured by a method using a reporter, such as an assay for analyzing the function of a protein.

[0052] Cell proliferation can be measured by analyzing cell division over a period of time. Cell division can be analyzed, for example, by analyzing the incorporation of H-thymidine in vitro or by CFSE dilution assay, as described in, for example, Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564 (which is hereby incorporated by reference in its entirety). 3 Proliferating cells can also be identified by analyzing the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) by appropriate assays, as described in, for example, Buck et al., Biotechniques. 2008 Jun; 44(7):927-9 and Sali and Mitchison, PNAS USA 2008 Feb 19; 105(7): 2415-2420 (which are hereby incorporated by reference in their entirety).

[0053] Cell migration can be analyzed, for example, by analyzing wound healing in a scratch assay in vitro, as described in, for example, Liang et al., Nat Protoc. (2007) 2(2):329-33 (which is hereby incorporated by reference in its entirety) and Example 9. Cell migration can also be analyzed using a Boyden chamber assay, as described in Chen, Methods Mol Biol. (2005) 294:15-22 (which is hereby incorporated by reference in its entirety) and Example 9.

[0054] Aspects of the invention include inhibiting the activity of secreted SMCs. That is, aspects of the invention include inhibiting the functionality of secreted SMCs (i.e., reducing their functional level).

[0055] In some embodiments, the activity of secretory SMCs may be one or more of proliferation, migration, invasion, expression and / or secretion of one or more extracellular matrix components (e.g., type I collagen), expression and / or secretion of one or more matrix-modifying enzymes (e.g., TIMP1), expression and / or secretion of one or more pro-inflammatory cytokines (e.g., TNFα), expression and / or secretion of IL-11, and expression of one or more pro-inflammatory factors.

[0056] Inhibition of the activity of secretory SMCs may be achieved, for example, by inhibiting one or more activities of secretory SMCs or by reducing the number of SMCs.

[0057] Inhibition of the activity of secretory SMCs may be performed in vitro or in vivo. In some embodiments, inhibition of one or more activities of secretory SMCs may be performed in a tissue, organ or subject. In some embodiments, reduction of the number of secretory SMCs may be performed in a tissue, organ or subject.

[0058] TGFβ and IL-11 signaling in SMC The role of TGFβ-mediated signaling in the phenotypic conversion of SMCs is not well understood. Also, the role of IL-11-mediated signaling in the phenotypic conversion of SMCs is unclear.

[0059] In some experimental models, TGFβ has been shown to promote the contractile phenotype of vascular smooth muscle cells (VSMCs) and inhibit the migration and proliferation of VSMCs, 4 whereas in another study, TGFβ has been shown to be extremely important for the migration of SMCs. 5。Specific disruption of TGFβ signaling has been reported and elaborated in the literature as a genetic factor in ascending thoracic aortic aneurysm (e.g., Loeys-Dietz syndrome (LDS) due to mutations in TGFBR1, TGFBR2, SMAD3, and TGFB2). In LDS and Marfan syndrome, loss of function has been demonstrated upstream of the TGFβ pathway, but downstream effector activation is paradoxically observed.

[0060] In Taki et al. Atherosclerosis (1999)144(2):375-80, the role of IL-11 signaling in VSMCs was investigated. Taki et al. found that TGFβ, IL-1A, and TNFα stimulate the expression of the IL-11 gene and the production of IL-11 protein in VSMCs, and proposed that this exerts an anti-atherogenic effect. 5,6 。In another study, in cultures of VSMCs derived from the aorta of healthy subjects stimulated with bFGF, it was shown that bFGF-induced VSMC proliferation was decreased in a concentration-dependent manner by IL-11. In this model, attenuation of two NF-κB-dependent cytokines (IL-8 and IL-6) was shown to be due to suppression of NF-κB induced by IL-11. 7 。

[0061] The inventors have identified in the experimental examples of the present disclosure that TGFβ-mediated signaling and IL-11-mediated signaling promote the phenotypic conversion of smooth muscle cells from a contractile type to a secretory type. IL-11-mediated signaling has been shown to be an important effector downstream of TGFβ-mediated signaling, and it has been shown that the effect of TGFβ is inhibited by specifically suppressing IL-11-mediated signaling.

[0062] Agents capable of suppressing the action of IL-11 Aspects of the present invention include suppression of IL-11-mediated signaling.

[0063] As used herein, "suppression" refers to a decrease, reduction or diminution as compared to a control condition. For example, suppression of the action of IL-11 by an agent capable of suppressing IL-11-mediated signal transduction refers to a decrease, reduction or diminution in the intensity / degree of IL-11-mediated signal transduction in the absence of the agent and / or in the presence of an appropriate control agent.

[0064] Also, as used herein, "suppression" may refer to neutralization or antagonism. That is, an agent capable of suppressing IL-11-mediated signal transduction (e.g., an interaction, signal transduction or other activity via IL-11 or an IL-11-containing complex) may be referred to as a "neutralizing" agent or an "antagonistic" agent with respect to the relevant function or process. For example, an agent capable of suppressing IL-11-mediated signal transduction may be referred to as an agent capable of neutralizing IL-11-mediated signal transduction, or as an antagonist of IL-11-mediated signal transduction.

[0065] There are multiple routes by which the signal transduction pathway of IL-11 can be suppressed. An agent capable of suppressing IL-11-mediated signal transduction may suppress the signal transduction of IL-11, for example, by suppressing the action of one or more factors involved in signal transduction via the IL-11 receptor, or the action of one or more factors required for signal transduction via the IL-11 receptor.

[0066] For example, suppression of the signal transduction of IL-11 may be achieved by disrupting the interaction between IL-11 (or an IL-11-containing complex, such as a complex consisting of IL-11 and IL-11Rα) and the IL-11 receptor (e.g., IL-11Rα, a receptor complex containing IL-11Rα, gp130, or a receptor complex containing IL-11Rα and gp130). In some embodiments, suppression of IL-11-mediated signal transduction is achieved, for example, by suppressing the expression of one or more genes or proteins among IL-11, IL-11Rα and gp130.

[0067] In another embodiment, inhibition of IL-11-mediated signaling is achieved by disrupting IL-11-mediated cis-signaling without disrupting IL-11-mediated trans-signaling. For example, inhibition of IL-11-mediated signaling is achieved by inhibiting the gp130-mediated cis-complex containing membrane-bound IL-11Rα. In another embodiment, inhibition of IL-11-mediated signaling is achieved by disrupting IL-11-mediated trans-signaling without disrupting IL-11-mediated cis-signaling, i.e., inhibition of IL-11-mediated signaling is achieved by inhibiting the gp130-mediated trans-signaling complex such as IL-11 bound to soluble IL-11Rα or IL-6 bound to soluble IL-6R. In another embodiment, inhibition of IL-11-mediated signaling is achieved by disrupting both IL-11-mediated cis-signaling and IL-11-mediated trans-signaling. Any of the agents described herein may be used for the inhibition of IL-11-mediated cis-signaling and / or IL-11-mediated trans-signaling.

[0068] In another example, inhibition of IL-11 signaling may be achieved by disrupting the signaling pathway downstream of IL-11 / IL-11Rα / gp130.

[0069] In some embodiments, the method of the present invention uses an agent capable of suppressing JAK / STAT signaling. In some embodiments, an agent capable of suppressing JAK / STAT signaling is capable of suppressing the actions of JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and / or STAT6. For example, an agent capable of suppressing JAK / STAT signaling may be capable of suppressing the activation of JAK / STAT proteins, may be capable of suppressing the interaction between JAK proteins or STAT proteins and cell surface receptors (such as IL-11Rα or gp130), may be capable of suppressing the phosphorylation of JAK proteins, may be capable of suppressing the interaction between JAK proteins and STAT proteins, may be capable of suppressing the phosphorylation of STAT proteins, may be capable of suppressing the dimerization of STAT proteins, may be capable of suppressing the transport of STAT proteins into the nucleus, may be capable of suppressing the binding of STAT proteins to DNA, and / or may be capable of promoting the degradation of JAK proteins and / or STAT proteins. In some embodiments, the JAK / STAT inhibitor is selected from ruxolitinib (Jakafi), tofacitinib (Xeljanz), oclacitinib (Apoquel), baricitinib (Olumiant), filgotinib (G-146034 / GLPG-0634), gandotinib (LY-2784544), lestaurtinib (CEP-701), momelotinib (GS-0387 / CYT-387), pacritinib (SB1518), PF-04965842, upadacitinib (ABT-494), peficitinib (ASP015K / JNJ-54781532), fedratinib (SAR302503), cucurbitacin I (JSI-124), and CHZ868.

[0070] In some embodiments, the method of the present invention uses an agent capable of suppressing MAPK / ERK signaling. In some embodiments, the agent capable of suppressing MAPK / ERK signaling can suppress the action of GRB2, can suppress the action of RAF kinase, can suppress the action of MEK protein, can suppress the activation of MAP3K / MAP2K / MAPK and / or Myc, and / or can suppress the phosphorylation of STAT protein. In some embodiments, the agent capable of suppressing ERK signaling can suppress ERK p42 / 44. In some embodiments, the ERK inhibitor is selected from SCH772984, SC1, VX-11e, and DEL-22379. In an embodiment, the ERK inhibitor is selected from sorafenib (Nexavar; Bayer / Onyx), SB590885, PLX4720, XL281, RAF265 (Novartis), encorafenib (LGX818 / Braftovi; Array BioPharma), dabrafenib (Tafinlar; GSK), vemurafenib (Zelboraf; Roche), cobimetinib (Cotellic; Roche), CI-1040, PD0325901, binimetinib (MEK162 / Mektovi; Array BioPharma), selumetinib (AZD6244; Array / AstraZeneca), and trametinib (GSK1120212 / Mekinist; Novartis).

[0071] Binding agents In some embodiments, an agent capable of suppressing IL-11-mediated signaling may bind to IL-11. In some embodiments, an agent capable of suppressing IL-11-mediated signaling may bind to an IL-11 receptor (e.g., IL-11Rα, gp130, or a complex comprising IL-11Rα and / or gp130). When such an agent binds to IL-11 or an IL-11 receptor, the binding ability of IL-11 to the IL-11 receptor is reduced / inhibited, IL-11-mediated signaling is suppressed, and as a result, downstream signaling may be suppressed. Also, when such an agent binds to IL-11 or an IL-11 receptor, the binding ability of IL-11 to the IL-11 receptor (e.g., IL-11Rα and / or gp130) is reduced / inhibited, IL-11-mediated cis-signaling and / or IL-11-mediated trans-signaling is suppressed, and as a result, downstream signaling may be suppressed. The agent may bind to a trans-signaling complex such as a complex consisting of IL-11 and soluble IL-11Rα to suppress gp130-mediated signaling.

[0072] An agent that can bind to IL-11 or an IL-11-containing complex or an IL-11 receptor may be of any type, but in some embodiments, the agent may be an antibody, an antigen-binding fragment thereof, a polypeptide, a peptide, a nucleic acid, an oligonucleotide, an aptamer, or a small molecule. The agent may be provided in an isolated or purified form, or formulated as a pharmaceutical composition or a drug.

[0073] Antibodies and antigen-binding fragments In some embodiments, the agent capable of binding to IL-11 or an IL-11-containing complex or an IL-11 receptor is an antibody or an antigen-binding fragment thereof. In some embodiments, the agent capable of binding to IL-11 or an IL-11-containing complex or an IL-11 receptor is a polypeptide, such as a decoy receptor molecule. In some embodiments, the agent capable of binding to IL-11 or an IL-11-containing complex or an IL-11 receptor may be an aptamer.

[0074] In some embodiments, the agent capable of binding to IL-11 or an IL-11-containing complex or an IL-11 receptor is an antibody or an antigen-binding fragment thereof. As used herein, the term "antibody" is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit binding to a relevant target molecule.

[0075] According to recent techniques related to monoclonal antibody technology, it is possible to produce antibodies against most antigens. The antigen-binding portion may be part of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single-chain Fv fragment SIt may also be [cFv]. Monoclonal antibodies against the selected antigen may be produced by known techniques, such as those described in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982). Chimeric antibodies have also been reported by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799). Monoclonal antibodies (mAbs) are particularly useful in the method of the present invention. A monoclonal antibody (mAb) is a homogeneous population of antibodies that specifically target a single epitope on an antigen.

[0076] In addition, in the method of the present invention, polyclonal antibodies are also useful. Monospecific polyclonal antibodies are preferred. Suitable polyclonal antibodies can be produced using methods well known in the art.

[0077] Antigen-binding fragments of antibodies such as Fab fragments and Fab2 fragments may be used / provided, and recombinant antibodies and recombinant antibody fragments may also be used / provided. The heavy chain variable (V H ) region and the light chain variable (V L ) region of an antibody are known to be involved in antigen recognition, which was first discovered by initial protease digestion experiments and confirmed in experiments where rodent antibodies were "humanized". Antibodies that retain the antigen specificity of the parental rodent antibody can be produced by fusing the variable region derived from a rodent to the constant region derived from a human (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855).

[0078] The antibodies and antigen-binding fragments according to the present disclosure comprise complementarity-determining regions (CDRs) of antibodies that can bind to related target molecules (i.e., IL-11 / IL-11-containing complexes / IL-11 receptors).

[0079] Examples of antibodies that can bind to IL-11 include, for example, the monoclonal mouse anti-human IL-11 antibody clone #22626 used in Bockhorn et al. Nat. Commun. (2013) 4(0):1393; catalog No. MAB218 (R&D Systems, Minneapolis, MN, USA), clone 6D9A (Abbiotec), clone KT8 (Abbiotec), clone M3103F11 (BioLegend), clone 1F1 (Abnova Corporation), clone 3C6 (Abnova Corporation), clone GF1 (LifeSpan Biosciences), clone 13455 (Source BioScience), and anti-IL-11 antibodies disclosed in US Patent Publication No. 2009 / 0202533 (A1), WO99 / 59608 (A2), and WO2018 / 109174 (A2).

[0080] Examples of antibodies that can bind to IL-11Rα include the monoclonal antibody clone 025 (Sino Biological), clone EPR5446 (Abcam), clone 473143 (R&D Systems), clones 8E2 and 8E4 described in US Patent Publication No. 2014 / 0219919 (A1), monoclonal antibodies described in Blanc et al. (J. Immunol Methods. 2000 Jul 31;241(1-2);43-59), antibodies disclosed in WO2014121325 (A1) and US Patent Publication No. 2013 / 0302277 (A1), and anti-IL-11Rα antibodies disclosed in US Patent Publication No. 2009 / 0202533 (A1), WO99 / 59608 (A2), and WO2018 / 109170 (A2).

[0081] The antibody / fragment may be an antagonist antibody / fragment that suppresses or reduces the biological activity of IL-11. The antibody / fragment may be a neutralizing antibody that neutralizes the biological action of IL-11, for example, a neutralizing antibody that neutralizes the ability of IL-11 to stimulate protein synthesis signal transduction via the IL-11 receptor. The neutralizing activity may be measured by evaluating the neutralizing ability against IL-11-induced proliferation in the T11 mouse myeloma cell line (Nordan, R. P. et al. (1987) J. Immunol. 139:813).

[0082] Antibodies typically contain six CDRs consisting of three CDRs (LC-CDR1, LC-CDR2, and LC-CDR3) in the light chain variable region (VL) and three CDRs (HC-CDR1, HC-CDR2, and HC-CDR3) in the heavy chain variable region (VH). These six CDRs together define the paratope of the antibody, which refers to the part of the antibody that binds to the target molecule. There are several conventional methods for defining the CDRs of an antibody, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), as well as VBASE2 described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671-D674, etc.

[0083] The antibodies and antigen-binding fragments according to the present disclosure may be designed and prepared using the sequences of monoclonal antibodies (mAbs) that can bind to the relevant target molecules. Also, antigen-binding regions of antibodies such as single-chain variable fragments (scFv), Fab fragments, Fab2 fragments, etc. may be used / provided. An "antigen-binding region" is an antibody fragment capable of binding to the target to which the original antibody exhibits specificity.

[0084] In some embodiments, the antibody / fragment comprises the VL and VH regions of an antibody capable of binding to IL-11, an IL-11-containing complex or an IL-11 receptor. The VL and VH regions in the antigen-binding region of the antibody together constitute the Fv region. In some embodiments, the antibody / fragment comprises or consists of the Fv region of an antibody capable of binding to IL-11, an IL-11-containing complex or an IL-11 receptor. The Fv region may be expressed as a single chain comprising the VH and VL regions covalently linked, for example, by a flexible oligopeptide. Thus, the antibody / fragment may comprise or consist of an scFv comprising the VL and VH regions of an antibody capable of binding to IL-11, an IL-11-containing complex or an IL-11 receptor.

[0085] The variable light chain region (VL) and constant light chain region (CL) and the variable heavy chain region (VH) and constant heavy chain region 1 (CH1) in the antigen-binding region of the antibody together constitute the Fab region. In some embodiments, the antibody / fragment comprises or consists of the Fab region of an antibody capable of binding to IL-11, an IL-11-containing complex or an IL-11 receptor.

[0086] In some embodiments, the antibody / fragment comprises, or consists of, a full-length antibody capable of binding to IL-11, an IL-11-containing complex, or an IL-11 receptor. A "full-length antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). For example, Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52 (which is hereby incorporated by reference in its entirety) describes various types of immunoglobulins and their structures. Immunoglobulin G (i.e., IgG) is a glycoprotein of approximately 150 kDa containing two heavy chains and two light chains. The heavy chain contains, in the direction from the N-terminus to the C-terminus, a heavy chain variable region (VH) and a heavy chain constant region containing three subsequent constant regions (CH1, CH2, and CH3). Similarly, the light chain contains a light chain variable region (VL) and a subsequent light chain constant region (CL). Immunoglobulins may be classified into IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM depending on the type of heavy chain. The light chain may be a kappa (κ) chain or a lambda (λ) chain.

[0087] Since Fab antibody fragments, Fv antibody fragments, scFv antibody fragments, and dAb antibody fragments can all be expressed and secreted in E. coli, they can be easily mass-produced.

[0088] Full-length antibodies and F(ab’)2 fragments are "bivalent". "Bivalent" means that full-length antibodies and F(ab’)2 fragments have two antigen-binding sites. In contrast, Fab fragments, Fv fragments, scFv fragments, and dAb fragments are monovalent because they have only one antigen-binding site. Synthetic antibodies capable of binding to IL-11, an IL-11-containing complex, or an IL-11 receptor can also be produced using phage display technology well known in the art.

[0089] The antibody may be prepared by an affinity maturation method for producing a modified antibody having an improved affinity for an antigen as compared to the unmodified parental antibody. The affinity matured antibody may be prepared by techniques known in the art, and techniques for producing the affinity matured antibody are described, for example, in Marks et al., Rio / Technology 10:779-783 (1992); Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-159 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0090] The antibody / fragment includes bispecific antibodies, which are composed of, for example, two fragments each derived from two antibodies, thereby enabling binding to two antigens. The bispecific antibodies include the antibody / fragments described herein that can bind to IL-11, IL-11-containing complexes or IL-11 receptors. The bispecific antibody may include another fragment having an affinity for a second antigen, and this second antigen may be any antigen as desired. Techniques for producing bispecific antibodies are well known in the art. See, for example, Mueller, D et al. (2010 Biodrugs 24 (2): 89-98), Wozniak-Knopp G et al. (2010 Protein Eng Des 23 (4): 289-297.), and Baeuerle, PA et al. (2009 Cancer Res 69 (12): 4941-4944). The bispecific antibodies and bispecific antigen-binding fragments may be provided in any suitable form, for example, in the form described in Kontermann MAbs 2012, 4(2): 182-197 (which is hereby incorporated by reference in its entirety). For example, the bispecific antibody or bispecific antigen-binding fragment may be a bispecific antibody complex (such as IgG2, F(ab’)2 or CovX-Body), a bispecific IgG or IgG-like molecule (such as IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, 2 in 1-IgG, mAb 2 , or a Tandemab with a shared light chain (LC)), an asymmetric bispecific IgG or IgG-like molecule (such as kih IgG, kih IgG with a shared light chain (LC), CrossMab, kih IgG-scFab, mAb-Fv, IgG with a charge pair, or SEED-body), a small bispecific antibody molecule (such as Diabody (Db), dsDb, DART, scDb, tandAb, tandem scFv (taFv), tandem dAb / VHH, triple body, triple head, Fab-scFv or F(ab’)2-scFv2), a bispecific Fc-CH 3 fusion proteins (e.g., taFv-Fc, Di-diabody, scDb-C H 3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc or scFv-kih-C H 3), or any of the bispecific fusion proteins (e.g., scFv2-albumin, scDb-albumin, taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine2). Specifically, see Figure 2 of Kontermann MAbs 2012, 4(2): 182-19.

[0091] As a method for producing bispecific antibodies, for example, as described in Segal and Bast, 2001. Production of Bispecific Antibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16 (this document is hereby incorporated by reference in its entirety), methods for chemically crosslinking antibodies or antibody fragments through, for example, reducible disulfide bonds or non-reducible thioether bonds can be mentioned. For example, by using N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) to chemically crosslink, for example, Fab fragments through the SH-groups in the hinge region, bispecific F(ab)2 heterodimers linked by disulfide bonds can be produced.

[0092] Another method for producing bispecific antibodies is, for example, as described in D. M. and Bast, B. J. 2001. Production of Bispecific Antibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16, a method of fusing hybridomas that produce antibodies using, for example, polyethylene glycol to produce quadroma cells that can secrete bispecific antibodies.

[0093] Bispecific antibodies and bispecific antigen-binding fragments can also be produced by recombinant techniques, for example, as described in Chapter 40: Production of Bispecific Antibodies: Diabodies and Tandem scFv (Hornig and Farber-Schwarz) of Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), or French, How to make bispecific antibodies, Methods Mol. Med. 2000; 40:333-339. For example, bispecific antibodies and bispecific antigen-binding fragments may be expressed from a nucleic acid construct encoding the polypeptide sequences of the antigen-binding molecules.

[0094] For example, a DNA construct can be prepared by molecular cloning techniques that encodes the light and heavy chain variable regions of two antigen-binding domains (i.e., the light and heavy chain variable regions of an antigen-binding domain capable of binding to IL-11, an IL-11-containing complex, or an IL-11 receptor, and the light and heavy chain variable regions of an antigen-binding domain capable of binding to another target protein), and includes a sequence encoding an appropriate linker or dimerization region that links these antigen-binding domains. Subsequently, this DNA construct can be expressed in a suitable host cell (e.g., a mammalian host cell) (e.g., in vitro) to produce a recombinant bispecific antibody, and the expressed recombinant bispecific antibody can be purified as needed.

[0095] Decoy receptors Peptide-based or polypeptide-based agents capable of binding to IL-11 or an IL-11-containing complex may be based on the IL-11 receptor, for example, may be based on the IL-11-binding fragment of the IL-11 receptor.

[0096] In some embodiments, the binding agent may comprise the IL-11 binding fragment of the IL-11Rα chain, may preferably be soluble, and / or may not comprise one or more transmembrane domains and may not comprise any transmembrane domains at all. In some embodiments, the binding agent may comprise the IL-11 binding fragment of gp130, may preferably be soluble, and / or may not comprise one or more transmembrane domains and may not comprise any transmembrane domains at all. Such molecules may be referred to as decoy receptors.

[0097] Curtis et al. (Blood 1997 Dec 1; 90 (11):4403-12) reported that soluble mouse IL-11 receptor α chain (sIL-11R) was able to exert an antagonistic effect on the activity of IL-11 when tested in cells expressing transmembrane IL-11R and gp130. It has been proposed that the antagonistic effect of sIL-11R on IL-11 observed in this study depends on the number of available gp130 molecules on cells already expressing transmembrane IL-11R.

[0098] The use of soluble decoy receptors for the purpose of suppressing signal transduction and therapeutic intervention has also been reported for other signal transduction molecule and receptor pairs, such as VEGF and VEGF receptor (De-Chao Yu et al., Molecular Therapy (2012); 20 5, 938-947; Konner and Dupont Clin Colorectal Cancer 2004 Oct;4 Suppl 2:S81-5).

[0099] Thus, in some embodiments, the binding agent may be a decoy receptor, for example, a soluble receptor for IL-11 and / or an IL-11-containing complex. It has been reported that competition for IL-11 and / or an IL-11-containing complex occurs by the decoy receptor, and antagonistic effects against IL-11 are exerted (Curtis et al., supra). Decoy receptors for IL-11 are also described in WO 2017 / 103108 (A1) and WO 2018 / 109168 (A1) (these documents are incorporated herein by reference in their entirety).

[0100] Preferably, the decoy receptor for IL-11 can inhibit the binding of IL-11 and / or an IL-11-containing complex to gp130, IL-11Rα, and / or the gp130:IL-11Rα receptor by binding to IL-11 and / or an IL-11-containing complex. Thus, the decoy receptor for IL-11 acts as a "decoy" receptor for IL-11 and an IL-11-containing complex in a manner very similar to etanercept, which acts as a decoy receptor for TNFα. IL-11-mediated signaling is reduced compared to signaling in the absence of the decoy receptor.

[0101] The decoy receptor of IL-11 preferably binds to IL-11 via one or more cytokine binding modules (CBMs). The CBM is a CBM of the natural IL-11 receptor molecule, is derived from the CBM of the natural IL-11 receptor molecule, or is homologous to the CBM of the natural IL-11 receptor molecule. For example, the decoy receptor of IL-11 may include one or more CBMs of gp130 and / or IL-11Rα, may consist of one or more CBMs of gp130 and / or IL-11Rα, may include one or more CBMs derived from the CBM of gp130 and / or IL-11Rα, may consist of one or more CBMs derived from the CBM of gp130 and / or IL-11Rα, may include one or more CBMs homologous to the CBM of gp130 and / or IL-11Rα, or may consist of one or more CBMs homologous to the CBM of gp130 and / or IL-11Rα.

[0102] In some embodiments, the decoy receptor of IL-11 may comprise an amino acid sequence corresponding to the cytokine binding module of gp130 or may consist of an amino acid sequence corresponding to the cytokine binding module of gp130. In some embodiments, the decoy receptor of IL-11 may comprise an amino acid sequence corresponding to the cytokine binding module of IL-11Rα. As used herein, an amino acid sequence "corresponding to" a reference region or reference sequence of a particular peptide / polypeptide has at least 60% sequence identity with the amino acid sequence of the reference region / reference sequence, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of the reference region / reference sequence.

[0103] In some embodiments, the decoy receptor may be capable of binding to IL-11 with a binding affinity of at least 100 μM or less, 10 μM or less, 1 μM or less, 100 nM or less, or a binding affinity of about 1 to 100 nM. In some embodiments, the decoy receptor may comprise the whole or a part of the IL-11 binding domain and may lack the whole or a part of the transmembrane domain. The decoy receptor may be fused to the constant region of an immunoglobulin (such as the IgG Fc region).

[0104] Inhibitors The present invention contemplates the use of inhibitor molecules that can bind to one or more of IL-11, IL-11-containing complexes, IL-11Rα, gp130, or complexes containing IL-11Rα and / or gp130 and inhibit IL-11-mediated signal transduction.

[0105] In some embodiments, the agent is a peptide-based or polypeptide-based binding agent based on IL-11, such as a variant, variant or binding fragment of IL-11. Suitable peptide-based or polypeptide-based agents may inhibit the initiation of signal transduction or cause only insufficient signal transduction by binding to the IL-11 receptor (such as a complex containing IL-11Rα, gp130, or IL-11Rα and / or gp130). Such types of IL-11 variants may act as competitive inhibitors of endogenous IL-11.

[0106] For example, W147A is an IL-11 antagonist in which the so-called "site III" of IL-11 is disrupted by mutating the 147th amino acid from tryptophan to alanine. This mutant can bind to IL-11Rα, but does not associate with the gp130 homodimer, and as a result, IL-11 signaling is efficiently blocked (Underhill-Day et al., 2003; Endocrinology 2003 Aug;144(8):3406-14). In addition, Lee et al. (Am J respire Cell Mol Biol. 2008 Dec; 39(6):739-746) reported the generation of an IL-11 antagonist mutant ("mutant") that can specifically inhibit the binding of IL-11 to IL-11Rα. The IL-11 mutant is also described in WO 2009 / 052588 (A1).

[0107] Menkhorst et al. (Biology of Reproduction May 1, 2009 vol.80 no.5 920-927) reported a pegylated IL-11 antagonist PEGIL11A (CSL Limited, Parkville, Victoria, Australia) that can effectively suppress the action of IL-11 in female mice.

[0108] Furthermore, Pasqualini et al. (Cancer (2015) 121(14):2411-2421) reported a ligand-targeting peptide mimetic, bone metastasis-targeting peptidomimetic-11 (BMTP-11), that can bind to IL-11Rα.

[0109] In some embodiments, the binding agent that can bind to the IL-11 receptor may be provided in the form of a small molecule inhibitor of IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130. In some embodiments, the binding agent may be provided in the form of a small molecule inhibitor of IL-11 or an IL-11-containing complex, for example, in the form of the IL-11 inhibitor described in Lay et al., Int. J. Oncol. (2012); 41(2): 759-764 (this document is incorporated herein by reference in its entirety).

[0110] Aptamers In some embodiments, the agent that can bind to IL-11 or an IL-11-containing complex or an IL-11 receptor (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130) is an aptamer. An aptamer, also called a nucleic acid ligand / peptide ligand, is a nucleic acid molecule or peptide molecule characterized by the ability to bind to a target molecule with high specificity and high affinity. Most of the aptamers identified to date are non-natural molecules.

[0111] An aptamer that binds to a specific target (IL-11, IL-11-containing complex or IL-11 receptor) is Systematic Evolution of Ligands by EXponential enrichment (SELEX TM) They may be identified and / or prepared by the SELEX method or alternatively, may be identified and / or prepared by constructing SOMAmer (slow off-rate modified aptamers) (Gold et al. (2010) PLoS ONE 5(12):e15004). The aptamer and the SELEX method have been reported by Tuerk and Gold (Science (1990) 249(4968):505-10) and are also described in WO91 / 19813. In the SELEX method and SOMAmer technology, for example, functional groups mimicking amino acid side chains are added to expand the chemical diversity of aptamers. As a result, aptamers with high affinity for the target can be enriched and identified.

[0112] The aptamer may be a DNA molecule, an RNA molecule, single-stranded or double-stranded. Further, the aptamer may contain a chemically modified nucleic acid, for example, a nucleic acid in which the sugar, phosphate and / or base is chemically modified. Such modifications may improve the stability of the aptamer, confer resistance to degradation on the aptamer, or may include modifications at the 2'-position of ribose.

[0113] The aptamer may be synthesized by methods well known to those skilled in the art. For example, the aptamer may be chemically synthesized, for example, on a solid support. A solid-phase synthesis method using the phosphoramidite method may be used. Specifically, after detritylating the immobilized nucleotide, it is coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester bond. Then capping can be performed, and the phosphite triester can be oxidized with an oxidizing agent (usually iodine). By repeating this cycle, the aptamer can be constructed (see, for example, Sinha, N.D.; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539; and Beaucage, S.L.; Lyer, R.P. (1992). Tetrahedron 48 (12): 2223).

[0114] The lower limit of the length of a suitable nucleic acid aptamer may be any of 10 nucleotides in length, 11 nucleotides in length, 12 nucleotides in length, 13 nucleotides in length, 14 nucleotides in length, 15 nucleotides in length, 16 nucleotides in length, 17 nucleotides in length, 18 nucleotides in length, 19 nucleotides in length, 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, 30 nucleotides in length, 31 nucleotides in length, 32 nucleotides in length, 33 nucleotides in length, 34 nucleotides in length, 35 nucleotides in length, 36 nucleotides in length, 37 nucleotides in length, 38 nucleotides in length, 39 nucleotides in length, 40 nucleotides in length. The upper limit of the length of a suitable nucleic acid aptamer may be any of 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, 30 nucleotides in length, 31 nucleotides in length, 32 nucleotides in length, 33 nucleotides in length, 34 nucleotides in length, 35 nucleotides in length, 36 nucleotides in length, 37 nucleotides in length, 38 nucleotides in length, 39 nucleotides in length, 40 nucleotides in length, 41 nucleotides in length, 42 nucleotides in length, 43 nucleotides in length, 44 nucleotides in length, 45 nucleotides in length, 46 nucleotides in length, 47 nucleotides in length, 48 nucleotides in length, 49 nucleotides in length, 50 nucleotides in length, 51 nucleotides in length, 52 nucleotides in length, 53 nucleotides in length, 54 nucleotides in length, 55 nucleotides in length, 56 nucleotides in length, 57 nucleotides in length, 58 nucleotides in length, 59 nucleotides in length, 60 nucleotides in length, 61 nucleotides in length, 62 nucleotides in length, 63 nucleotides in length, 64 nucleotides in length, 65 nucleotides in length, 66 nucleotides in length, 67 nucleotides in length, 68 nucleotides in length, 69 nucleotides in length, 70 nucleotides in length, 71 nucleotides in length, 72 nucleotides in length, 73 nucleotides in length, 74 nucleotides in length, 75 nucleotides in length, 76 nucleotides in length, 77 nucleotides in length, 78 nucleotides in length, 79 nucleotides in length, 80 nucleotides in length.Suitable lengths of nucleic acid aptamers may be any of 10 nucleotide lengths, 11 nucleotide lengths, 12 nucleotide lengths, 13 nucleotide lengths, 14 nucleotide lengths, 15 nucleotide lengths, 16 nucleotide lengths, 17 nucleotide lengths, 18 nucleotide lengths, 19 nucleotide lengths, 20 nucleotide lengths, 21 nucleotide lengths, 22 nucleotide lengths, 23 nucleotide lengths, 24 nucleotide lengths, 25 nucleotide lengths, 26 nucleotide lengths, 27 nucleotide lengths, 28 nucleotide lengths, 29 nucleotide lengths, 30 nucleotide lengths, 31 nucleotide lengths, 32 nucleotide lengths, 33 nucleotide lengths, 34 nucleotide lengths, 35 nucleotide lengths, 36 nucleotide lengths, 37 nucleotide lengths, 38 nucleotide lengths, 39 nucleotide lengths, 40 nucleotide lengths, 41 nucleotide lengths, 42 nucleotide lengths, 43 nucleotide lengths, 44 nucleotide lengths, 45 nucleotide lengths, 46 nucleotide lengths, 47 nucleotide lengths, 48 nucleotide lengths, 49 nucleotide lengths, 50 nucleotide lengths, 51 nucleotide lengths, 52 nucleotide lengths, 53 nucleotide lengths, 54 nucleotide lengths, 55 nucleotide lengths, 56 nucleotide lengths, 57 nucleotide lengths, 58 nucleotide lengths, 59 nucleotide lengths, 60 nucleotide lengths, 61 nucleotide lengths, 62 nucleotide lengths, 63 nucleotide lengths, 64 nucleotide lengths, 65 nucleotide lengths, 66 nucleotide lengths, 67 nucleotide lengths, 68 nucleotide lengths, 69 nucleotide lengths, 70 nucleotide lengths, 71 nucleotide lengths, 72 nucleotide lengths, 73 nucleotide lengths, 74 nucleotide lengths, 75 nucleotide lengths, 76 nucleotide lengths, 77 nucleotide lengths, 78 nucleotide lengths, 79 nucleotide lengths, 80 nucleotide lengths.

[0115] An aptamer may be a peptide selected or constructed to bind to a specific target molecule. Peptide aptamers, as well as methods for their preparation and identification, are reviewed in Reverdatto et al., Curr Top Med Chem. (2015) 15(12):1082-101 (this document is incorporated herein by reference in its entirety). The lower limit of the length of the peptide aptamer may be any of 2 amino acid lengths, 3 amino acid lengths, 4 amino acid lengths, 5 amino acid lengths, 6 amino acid lengths, 7 amino acid lengths, 8 amino acid lengths, 9 amino acid lengths, 10 amino acid lengths. The upper limit of the length of the peptide aptamer may be any of 15 amino acid lengths, 16 amino acid lengths, 17 amino acid lengths, 18 amino acid lengths, 19 amino acid lengths, 20 amino acid lengths, 21 amino acid lengths, 22 amino acid lengths, 23 amino acid lengths, 24 amino acid lengths, 25 amino acid lengths, 26 amino acid lengths, 27 amino acid lengths, 28 amino acid lengths, 29 amino acid lengths, 30 amino acid lengths, 31 amino acid lengths, 32 amino acid lengths, 33 amino acid lengths, 34 amino acid lengths, 35 amino acid lengths, 36 amino acid lengths, 37 amino acid lengths, 38 amino acid lengths, 39 amino acid lengths, 40 amino acid lengths, 41 amino acid lengths, 42 amino acid lengths, 43 amino acid lengths, 44 amino acid lengths, 45 amino acid lengths, 46 amino acid lengths, 47 amino acid lengths, 48 amino acid lengths, 49 amino acid lengths, 50 amino acid lengths. Suitable lengths of the peptide aptamer may be any of 2-30 amino acid lengths, 2-25 amino acid lengths, 2-20 amino acid lengths, 5-30 amino acid lengths, 5-25 amino acid lengths, 5-20 amino acid lengths.

[0116] The aptamer may have a K in the nM order or pM order, and K d may be, for example, less than 500 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 500 pM, less than 100 pM. d

[0117] Properties of IL-11 binding agents The agent of the present invention that can bind to IL-11 or an IL-11-containing complex or an IL-11 receptor may exhibit any one or more of the following characteristics. ·Specific binding to IL-11 or an IL-11-containing complex or the IL-11 receptor ·K of 10 μM or less D , preferably K of 5 μM or less, 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 100 pM or less D Binding to IL-11 or an IL-11-containing complex or the IL-11 receptor at ·Inhibition of the interaction between IL-11 and IL-11Rα ·Inhibition of the interaction between IL-11 and gp130 ·Inhibition of the interaction between IL-11 and the IL-11Rα:gp130 receptor complex ·Inhibition of the interaction between the IL-11:IL-11Rα complex and gp130

[0118] These properties can be measured by analyzing relevant factors in an appropriate assay, which may include comparing with appropriate controls and performance. One skilled in the art can determine the appropriate control conditions in a specific assay.

[0119] For example, a negative control suitable for analyzing the binding ability of a test antibody / antigen-binding fragment to IL-11 / IL-11-containing complex / IL-11 receptor may be an antibody / antigen-binding fragment against a non-target protein (i.e., an antibody / antigen-binding fragment not specific to IL-11 / IL-11-containing complex / IL-11 receptor). A suitable positive control may be a verified (e.g., commercially available) known IL-11-binding antibody or IL-11 receptor-binding antibody. The control may be of the same isotype as the putative IL-11 / IL-11-containing complex / IL-11 receptor-binding antibody / antigen-binding fragment being analyzed, for example, having the same constant region.

[0120] In some embodiments, the agent may be specifically bindable to IL-11, an IL-11-containing complex, or an IL-11 receptor (e.g., IL-11Rα, gp130, or a complex comprising IL-11Rα and / or gp130). An agent that specifically binds to a particular target molecule preferably binds to the target molecule with a higher affinity and / or longer duration of binding than to other non-target molecules.

[0121] In some embodiments, the agent may bind to IL-11 or an IL-11-containing complex with a higher affinity than to one or more of the other members of the IL-6 cytokine family (e.g., IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine (CLC)). In some embodiments, the agent may bind to an IL-11 receptor (e.g., IL-11Rα, gp130, or a complex comprising IL-11Rα and / or gp130) with a higher affinity than to one or more of the other members of the IL-6 receptor family. In some embodiments, the agent may bind to IL-11Rα with a higher affinity than to one or more of IL-6Rα, leukemia inhibitory factor receptor (LIFR), oncostatin M receptor (OSMR), and ciliary neurotrophic factor receptor α (CNTFRα).

[0122] In some embodiments, when measured, for example, by ELISA, SPR, biolayer interferometry (BLI), microscale thermophoresis (MST), or radioimmunoassay (RIA), the degree of binding of a binding agent to a non-target molecule is less than about 10% of the degree of binding of the binding agent to the target molecule. Alternatively, the binding specificity may be reflected as binding affinity, in which case the binding agent has a K D that is at least 0.1 orders of magnitude (i.e., 0.1×10 n (n represents an integer number of orders of magnitude)) smaller than the K DIt binds to IL-11, an IL-11-containing complex, or an IL-11 receptor. This digit number may be any of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0.

[0123] The binding affinity of a specific binding agent for a target is often represented by its dissociation constant (K D ). The binding affinity can be measured by methods known in the art, such methods include, for example, ELISA, surface plasmon resonance (SPR; see, for example, Hearty et al., Methods Mol Biol (2012) 907:411-442; or Rich et al., Anal Biochem. 2008 Feb 1; 373(1):112-20), biolayer interferometry (see, for example, Lad et al., (2015) J Biomol Screen 20(4): 498-507; or Concepcion et al., Comb Chem High Throughput Screen. 2009 Sep; 12(8):791-800), microscale thermophoresis (MST) analysis (see, for example, Jerabek-Willemsen et al., Assay Drug Dev Technol. 2011 Aug; 9(4): 342-353), or radiolabeled antigen binding assay (RIA), etc.

[0124] In some embodiments, the agent has a K D of 50 μM or less, preferably 10 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, 1 μM or less, 500 nM or less, 100 nM or less, 75 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 15 nM or less, 12.5 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, or 100 pM or less of K Dand can bind to IL-11, the IL-11-containing complex, or the IL-11 receptor.

[0125] In some embodiments, the agent has an EC (when measured by, for example, ELISA) 50 of 10,000 ng / ml or less, preferably an EC 50 of 5,000 ng / ml or less, 1000 ng / ml or less, 900 ng / ml or less, 800 ng / ml or less, 700 ng / ml or less, 600 ng / ml or less, 500 ng / ml or less, 400 ng / ml or less, 300 ng / ml or less, 200 ng / ml or less, 100 ng / ml or less, 90 ng / ml or less, 80 ng / ml or less, 70 ng / ml or less, 60 ng / ml or less, 50 ng / ml or less, 40 ng / ml or less, 30 ng / ml or less, 20 ng / ml or less, 15 ng / ml or less, 10 ng / ml or less, 7.5 ng / ml or less, 5 ng / ml or less, 2.5 ng / ml or less, or 1 ng / ml or less, and binds to IL-11, the IL-11-containing complex, or the IL-11 receptor. ELISA can be performed, for example, according to the description in Antibody Engineering, vol. 1 (2 nd Edn), Springer Protocols, Springer (2010), Part V, pp657-665.

[0126] In some embodiments, the agent binds to IL-11 or the IL-11-containing complex in a region important for binding to the IL-11 receptor or the receptor of the IL-11-containing complex (such as gp130 or IL-11Rα), thereby suppressing the interaction between IL-11 or the IL-11-containing complex and the IL-11 receptor and / or suppressing signal transduction via the IL-11 receptor. In some embodiments, the agent binds to the IL-11 receptor in a region important for binding to IL-11 or the IL-11-containing complex, thereby suppressing the interaction between IL-11 or the IL-11-containing complex and the IL-11 receptor and / or suppressing signal transduction via the IL-11 receptor.

[0127] The inhibitory ability of a specific binding agent (e.g., an agent capable of binding to IL-11 or an IL-11-containing complex or an IL-11 receptor) against the interaction between two proteins can be measured, for example, by analyzing the interaction between these interaction partners in the presence of the binding agent or after incubating the binding agent with one or both of the interaction partners. A preferred assay for determining whether a specific binding agent can inhibit the interaction between two interaction partners is competitive ELISA.

[0128] Binding agents that can inhibit specific interactions (e.g., the interaction between IL-11 and IL-11Rα, the interaction between IL-11 and gp130, the interaction between IL-11 and IL-11Rα:gp130, or the interaction between IL-11:IL-11Rα and gp130) are identified by a decrease in the degree of interaction between these interaction partners in the presence of the binding agent, or after incubating the binding agent with one or both of the interaction partners, compared to the degree of interaction in the absence of the binding agent (or in the presence of a suitable control binding agent). Suitable assays can be performed in vitro, for example, using recombinant interaction partners or cells expressing the interaction partners. Cells expressing the interaction partners may endogenously express the interaction partners or may express the interaction partners from nucleic acids introduced into the cells. For the purpose of performing such assays, one or both of the interaction partners and / or the binding agent may be labeled with a detectable substance or used with such a label to detect and / or measure the degree of interaction. For example, the binding agent may be labeled with a radioactive atom, a dye molecule, a fluorescent molecule, or any other molecule that can be easily detected. Suitable detectable molecules include fluorescent proteins, luciferase, enzyme substrates, and radioactive labels. The binding agent may be labeled directly with a detectable label or indirectly. For example, the binding agent may not be labeled and may be detected using another labeled binding agent. Alternatively, a second binding agent may be conjugated to biotin and a labeled streptavidin may be conjugated to the biotin to indirectly label the first binding agent.

[0129] Moreover, the inhibitory ability of a binding agent against the interaction between two binding partners can also be identified by analyzing the outcome of a function downstream of such an interaction (e.g., IL-11-mediated signaling). For example, the outcomes of functions downstream of the interaction between IL-11 and IL-11Rα:gp130 or between IL-11:IL-11Rα and gp130 may include, for example, IL-11-mediated processes, the generation of myofibroblasts from fibroblasts, the proliferation or migration of secretory SMCs, or gene expression / protein expression of, for example, collagen or IL-11.

[0130] The inhibitory ability of a binding agent against the interaction between IL-11 or an IL-11-containing complex and the IL-11 receptor can be analyzed, for example, by stimulating fibroblasts with TGFβ1, incubating the cells in the presence of the binding agent, and analyzing the proportion of cells having an αSMA-positive phenotype after a predetermined time has elapsed. In such an example, the inhibition of the interaction between IL-11 or an IL-11-containing complex and the IL-11 receptor can be identified from a decrease in the proportion of cells having an αSMA-positive phenotype as compared to a positive control condition in which the cells are treated with TGFβ1 in the absence of the binding agent (or in the presence of an appropriate control binding agent) or in the presence of an appropriate control binding agent. Such an assay is also suitable for analyzing the inhibitory ability of a binding agent against IL-11-mediated signaling. The inhibition of the interaction between IL-11 or an IL-11-containing complex and the IL-11 receptor can be, for example, as described in Curtis et al. Blood, 1997, 90(11) and Karpovich et al. Mol. Hum. Reprod. 2003 9(2): 75-80, 3 an H-thymidine incorporation assay, and / or a Ba / F3 cell proliferation assay can also be used for the analysis. Ba / F3 cells co-express IL-11Rα and gp130.

[0131] In some embodiments, the binding agent may be able to inhibit the interaction between IL-11 and IL-11Rα to less than 100%, for example, 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less, as compared to the degree of interaction between IL-11 and IL-11Rα in the absence of the binding agent (or in the presence of a suitable control binding agent). In some embodiments, the binding agent may be able to inhibit the interaction between IL-11 and IL-11Rα to less than 1-fold, for example, 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less, as compared to the degree of interaction between IL-11 and IL-11Rα in the absence of the binding agent (or in the presence of a suitable control binding agent).

[0132] In some embodiments, the binding agent is capable of suppressing the interaction between IL-11 and gp130 to less than 100%, for example, 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less, compared to the degree of interaction between IL-11 and gp130 in the absence of the binding agent (or in the presence of a suitable control binding agent). In some embodiments, the binding agent is capable of suppressing the interaction between IL-11 and gp130 to less than 1-fold, for example, 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less, compared to the degree of interaction between IL-11 and gp130 in the absence of the binding agent (or in the presence of a suitable control binding agent).

[0133] In some embodiments, the binding agent may be able to inhibit the interaction between IL-11 and IL-11Rα:gp130 to less than 100%, such as 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less, compared to the degree of interaction between IL-11 and IL-11Rα:gp130 in the absence of the binding agent (or in the presence of a suitable control binding agent). In some embodiments, the binding agent may be able to inhibit the interaction between IL-11 and IL-11Rα:gp130 to less than 1-fold, such as 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less, compared to the degree of interaction between IL-11 and IL-11Rα:gp130 in the absence of the binding agent (or in the presence of a suitable control binding agent).

[0134] In some embodiments, the binding agent may be able to inhibit the interaction between the IL-11:IL-11Rα complex and gp130 to less than 100%, such as 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less, compared to the degree of interaction between the IL-11:IL-11Rα complex and gp130 in the absence of the binding agent (or in the presence of a suitable control binding agent). In some embodiments, the binding agent can inhibit the interaction between the IL-11:IL-11Rα complex and gp130 to less than 1-fold, such as 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less, compared to the degree of interaction between the IL-11:IL-11Rα complex and gp130 in the absence of the binding agent.

[0135] Agents capable of reducing the expression of IL-11 or IL-11 receptor In aspects of the present invention, there may be provided an agent capable of inhibiting IL-11-mediated signal transduction by blocking or reducing the expression of one or more of IL-11, IL-11Rα, or gp130.

[0136] The expression may be gene expression, protein expression, or may be measured by the methods described herein. The expression may be expression by cells / tissues / organs / organ systems in a subject. For example, the expression may be blocked / reduced in smooth muscle cells.

[0137] The suitable agent can be of any kind, but in some embodiments, an agent capable of blocking or reducing the expression of one or more of IL-11, IL-11Rα or gp130 may be a small molecule or an oligonucleotide.

[0138] An agent capable of blocking or reducing the expression of one or more of IL-11, IL-11Rα or gp130 may, for example, block or reduce the expression of one or more of IL-11, IL-11Rα or gp130 through suppression of transcription of the gene encoding IL-11, IL-11Rα or gp130, suppression of post-transcriptional processing of the RNA encoding IL-11, IL-11Rα or gp130, reduction of the stability of the RNA encoding IL-11, IL-11Rα or gp130, promotion of degradation of the RNA encoding IL-11, IL-11Rα or gp130, suppression of post-translational processing of the IL-11 polypeptide, IL-11Rα polypeptide or gp130 polypeptide, reduction of the stability of the IL-11 polypeptide, IL-11Rα polypeptide or gp130 polypeptide, or promotion of degradation of the IL-11 polypeptide, IL-11Rα polypeptide or gp130 polypeptide.

[0139] Taki et al. (Clin Exp Immunol (1998) Apr; 112(1): 133-138) reported that the expression of IL-11 was decreased in rheumatoid synovial cells treated with indomethacin, dexamethasone or interferon γ (IFNγ).

[0140] Furthermore, the present invention contemplates the use of antisense nucleic acids for blocking / reducing the expression of IL-11, IL-11Rα or gp130. In some embodiments, an agent capable of blocking or reducing the expression of IL-11, IL-11Rα or gp130 may reduce the expression by RNA interference (RNAi).

[0141] In some embodiments, the agent may be an inhibitory nucleic acid such as antisense RNA or small interfering RNA, including but not limited to shRNA or siRNA.

[0142] In some embodiments, the inhibitory nucleic acid is provided by being incorporated into a vector. For example, in some embodiments, the agent may be a lentiviral vector encoding shRNA against one or more of IL-11, IL-11Rα or gp130.

[0143] Oligonucleotide molecules (especially RNA) may be used to control gene expression. Such oligonucleotide molecules include antisense oligonucleotides; degradation of mRNA targeted by small interfering RNA (siRNA); post-transcriptional gene silencing (PTG); inhibition of mRNA translation specific to sequences regulated during development using microRNA (miRNA); and targeted transcriptional gene silencing.

[0144] An antisense oligonucleotide is an oligonucleotide (preferably a single-stranded oligonucleotide) that targets a target oligonucleotide (such as mRNA) and binds to it through complementary sequence binding. When the target oligonucleotide is mRNA, the binding of the antisense oligonucleotide to the mRNA inhibits the translation of the mRNA and thus inhibits the expression of the gene product. The antisense oligonucleotide may be designed to bind to the sense strand of genomic nucleic acid to suppress the transcription of the target nucleotide sequence.

[0145] Taking into account the known nucleic acid sequences of IL-11, IL-11Rα and gp130 (e.g., accession numbers: BC012506.1 GI:15341754 (human IL-11), BC134354.1 GI:126632002 (mouse IL-11), AF347935.1 GI:13549072 (rat IL-11), NM_001142784.2 GI:391353394 (human IL-11Rα), NM_001163401.1 GI:254281268 (mouse IL-11Rα), NM_139116.1 GI:20806172 (rat IL-11Rα), NM_001190981.1 GI:300244534 (human gp130), NM_010560.3 GI:225007624 (mouse gp130), NM_001008725.3 GI:300244570 (rat gp130), known mRNA sequences available from GenBank), oligonucleotides may be designed to suppress or silence the expression of IL-11, IL-11Rα or gp130.

[0146] Such oligonucleotides may be of any length, but are preferably short, for example less than 100 nucleotides in length, such as 10 to 40 nucleotides in length, or 20 to 50 nucleotides in length, and have a nucleotide sequence of the corresponding length in the target oligonucleotide (e.g., IL-11 mRNA, IL-11Rα mRNA or gp130 mRNA), and may contain a nucleotide sequence having complete complementarity or substantial complementarity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity). The complementary region of the nucleotide sequence may be of any length, but is preferably at least 5 nucleotides in length and may be 50 nucleotides in length or less, for example, 6 nucleotides in length, 7 nucleotides in length, 8 nucleotides in length, 9 nucleotides in length, 10 nucleotides in length, 11 nucleotides in length, 12 nucleotides in length, 13 nucleotides in length, 14 nucleotides in length, 15 nucleotides in length, 16 nucleotides in length, 17 nucleotides in length, 18 nucleotides in length, 19 nucleotides in length, 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, 30 nucleotides in length, 31 nucleotides in length, 32 nucleotides in length, 33 nucleotides in length, 34 nucleotides in length, 35 nucleotides in length, 36 nucleotides in length, 37 nucleotides in length, 38 nucleotides in length, 39 nucleotides in length, 40 nucleotides in length, 41 nucleotides in length, 42 nucleotides in length, 43 nucleotides in length, 44 nucleotides in length, 45 nucleotides in length, 46 nucleotides in length, 47 nucleotides in length, 48 nucleotides in length, 49 nucleotides in length, 50 nucleotides in length.

[0147] By suppressing the expression of IL-11, IL-11Rα or gp130, it is preferable that the amount of IL-11, IL-11Rα or gp130 expressed by cells / tissues / organs / organ systems / subjects is decreased. For example, by suppressing IL-11, IL-11Rα or gp130 in specific cells by administration of appropriate nucleic acids, the amount of IL-11, IL-11Rα or gp130 expressed by the cells is decreased compared to untreated cells. The suppression may be partial. The degree of suppression is preferably at least 50%, more preferably any one of at least 60%, 70%, 80%, 85%, 90%. When the degree of suppression is 90% to 100%, the expression or function is considered to be "silenced".

[0148] In the targeting of heterochromatin complexes and the epigenetic gene silencing of specific chromosomal loci, the roles played by the RNAi mechanism and small RNAs have been demonstrated. Double-stranded RNA (dsRNA)-dependent post-transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which a dsRNA complex can target homologous portions of a specific gene and silence it in a short time. RNAi acts as a signal that promotes the degradation of mRNAs with sequence identity. A 20-nt siRNA is usually long enough to induce gene-specific silencing and short enough to avoid the host response. The reduction in the expression of the target gene product can induce silencing reaching 90% by using several siRNA molecules. Therapeutic agents using RNAi have advanced to Phase I, Phase II and Phase III clinical trials for various indications (Nature 2009 Jan 22; 457(7228):426-433).

[0149] In the art, RNA sequences such as those described above are referred to as "short interfering RNA or small interfering RNA" (siRNA) or "microRNA" (miRNA) depending on their origin. Using these RNA sequences, gene expression may be down-regulated by binding to complementary RNA to induce elimination of mRNA (RNAi), or gene expression may be down-regulated by inhibiting translation from mRNA to protein. siRNA is obtained by processing long double-stranded RNA, and natural siRNA is usually exogenous. Microinterfering RNA (miRNA) is an endogenous small non-coding RNA obtained by processing a short hairpin structure. Both siRNA and miRNA can suppress the translation of mRNA having a partially complementary target sequence without cleaving the RNA, and can degrade mRNA having a complete complementary sequence.

[0150] An siRNA ligand is usually double-stranded, and in order to optimize the effectiveness of down-regulation of the function of a target gene by this RNA, it is preferably long enough for the siRNA to be accurately recognized by the RISC complex that mediates recognition of the target mRNA by the siRNA, and short enough to keep the host response low. The length of the siRNA molecule is selected so that it is short enough to keep the host response low.

[0151] An miRNA ligand is usually single-stranded and has a partially complementary region capable of forming a hairpin structure. miRNA is an RNA gene that is transcribed from DNA but not translated into protein. The DNA sequence encoding the miRNA gene is longer than the miRNA and contains the miRNA sequence and a sequence that is almost complementary to it in the reverse direction. When this DNA sequence is transcribed into a single-stranded RNA molecule, a partially double-stranded RNA segment is formed from the base pairs consisting of the miRNA sequence and its reverse complementary sequence. The design of microRNA sequences has been reported in John et al, PLoS Biology, 11(2), 1862-1879, 2004.

[0152] The RNA ligand that mimics the effect of siRNA or miRNA is usually 10 - 40 ribonucleotides in length (or its synthetic analog), more preferably 17 - 30 ribonucleotides in length, even more preferably 19 - 25 ribonucleotides in length, and most preferably 21 - 23 ribonucleotides in length. In some embodiments of the present invention using double-stranded siRNA, the double-stranded siRNA molecule may have a symmetric 3' end overhang, which may be composed of, for example, 1 or 2 (ribo)nucleotides, and is usually a 3' end UU or dTdT overhang. Those skilled in the art can easily design appropriate siRNA sequences and appropriate miRNA sequences based on the disclosure herein, for example, using a library such as Ambion siRNA finder. The siRNA sequences and miRNA sequences can be synthetically prepared and added extracellularly to induce gene down-regulation, or can also be prepared using an expression system (such as a vector). In a preferred embodiment, the siRNA is synthetically prepared.

[0153] Long double-stranded RNAs may be processed intracellularly to produce siRNAs (see, for example, Myers (2003) Nature Biotechnology 21:324-328). The long dsRNA molecule may have symmetric 3' or 5' terminal overhangs, which may consist of, for example, one or two (ribo) nucleotides, or the long dsRNA molecule may have blunt ends. The long dsRNA molecule may be 25 nucleotides in length or more. The long dsRNA molecule preferably has a length of 25 to 30 nucleotides. The long dsRNA molecule more preferably has a length of 25 to 27 nucleotides. The long dsRNA molecule most preferably has a length of 27 nucleotides. dsRNAs of 30 nucleotides in length or more may be expressed using the pDECAP vector (Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).

[0154] Alternatively, an RNA molecule with a short hairpin structure (shRNA) is expressed in cells. shRNA is more stable than synthetic siRNA. shRNA consists of short inverted repeat sequences linked by a short loop sequence. One of the inverted repeat sequences is complementary to the target gene. Inside the cell, shRNA undergoes processing by DICER to become siRNA, and this siRNA degrades the mRNA of the target gene, suppressing its expression. In a preferred embodiment, shRNA is endogenously (inside the cell) generated by transcription from a vector. shRNA may be generated inside the cell by transfecting the cell with a vector encoding the shRNA sequence under the control of an RNA polymerase III promoter (such as the human H1 promoter or the human 7SK promoter) or an RNA polymerase II promoter. Alternatively, shRNA may be exogenously (in vitro) synthesized by transcription from a vector. Then, the obtained shRNA may be directly introduced into the cell. The shRNA molecule preferably contains a partial sequence of IL-11, IL-11Rα, or gp130. The length of the shRNA sequence is preferably 40 to 100 bases long, more preferably 40 to 70 bases long. The length of the stem portion of the hairpin structure is preferably 19 to 30 base pairs. The stem portion may contain G-U pairs to stabilize the hairpin structure.

[0155] The siRNA molecule, long double-stranded RNA molecule, or miRNA molecule may be prepared by recombinant techniques by transcription of a nucleic acid sequence (preferably incorporated into a vector). The siRNA molecule, long double-stranded RNA molecule, or miRNA molecule preferably contains a partial sequence of IL-11, IL-11Rα, or gp130.

[0156] In one embodiment, siRNA, long dsRNA or miRNA is endogenously (intracellularly) produced by transcription from a vector. The vector may be introduced into cells by any method known in the art. The expression of these RNA sequences can be controlled using a tissue-specific (e.g., heart, liver, kidney or eye-specific) promoter, if necessary. In a further embodiment, siRNA, long dsRNA or miRNA may be exogenously (in vitro) produced by transcription from a vector.

[0157] A suitable vector may be an oligonucleotide vector configured to express an oligonucleotide drug capable of suppressing IL-11, IL-11Rα or gp130. Such a vector may be a viral vector or a plasmid vector. The oligonucleotide therapeutic agent may be incorporated into the genome of the viral vector or may be operably linked to a regulatory sequence (e.g., a promoter) that induces expression. "Operably linked" may include a state in which a selected nucleotide sequence and a regulatory nucleotide sequence are covalently linked such that the nucleotide sequence is expressed under the influence or control of the regulatory sequence. Thus, if a regulatory sequence is capable of inducing transcription of a nucleotide sequence that constitutes all or part of a selected nucleotide sequence, the regulatory sequence is operably linked to the selected nucleotide sequence.

[0158] Viral vectors encoding siRNA sequences whose expression is induced by a promoter are known in the art and have the advantage of being able to express oligonucleotide therapeutics over a long period of time. Examples of viral vectors include lentivirus (Nature 2009 Jan 22; 457(7228):426-433), adenovirus (Shen et al., FEBS Lett 2003 Mar 27;539(1-3)111-4), and retrovirus (Barton and Medzhitov PNAS November 12, 2002 vol.99, no.23 14943-14945).

[0159] In another embodiment, a carrier configured to assist in the delivery of an oligonucleotide therapeutic agent to a site where suppression of the expression of IL-11, IL-11Rα, or gp130 is required may be used. Such carriers generally include carriers with a positive charge complexed with an oligonucleotide (e.g., cell-penetrating cationic peptides, cationic polymers, cationic dendrimers, and cationic lipids); small molecules (e.g., cholesterol, bile acids, and lipids), polymers, antibodies, and RNAs bound to the oligonucleotide; or oligonucleotides encapsulated in nanoparticle formulations (Wang et al., AAPS J. 2010 Dec; 12(4): 492-503).

[0160] In one embodiment, the vector may contain nucleic acid sequences in both the sense strand direction and the antisense strand direction such that when the nucleic acid sequence is expressed as RNA, the sense strand portion and the antisense strand portion associate to form double-stranded RNA.

[0161] Alternatively, the siRNA molecule may be synthesized using standard solid-phase or liquid-phase synthesis methods known in the art. The bond between nucleotides may be a phosphodiester bond or other bonds, for example, P(O)S (thioate); P(S)S (dithioate); P(O)NR’2; P(O)R’; P(O)OR6; CO; or CONR’2 (wherein R is H (or salt) or alkyl (C 1~12 ) and R6 is alkyl (C 1~9 )), and examples include those in which a linking group represented by the formula is linked to adjacent nucleotides via -O- or -S-.

[0162] In addition to natural bases, modified nucleotide bases can be used, and the modified nucleotide bases can impart advantageous properties to siRNA molecules containing them.

[0163] For example, the modified base can improve the stability of the siRNA molecule, thereby reducing the amount of siRNA molecule required for silencing. By adding a modified base, an siRNA molecule with improved or reduced stability compared to unmodified siRNA can be produced.

[0164] "Modified nucleotide base" includes nucleotides to which a modified base and / or a modified sugar are covalently bonded. For example, modified nucleotides include nucleotides having a sugar to which a low molecular weight organic group other than the 3'-hydroxyl group and 5'-phosphate group is covalently bonded. Thus, modified nucleotides may further include 2'-position substituted sugars such as 2'-O-methylribose, 2'-O-alkylribose, 2'-O-allylribose, 2'-S-alkylribose, 2'-S-allylribose, 2'-fluororibose, 2'-haloribose, 2'-azidoribose; carbocyclic sugar analogs; α-anomer sugars; epimer sugars such as arabinose, xylose, lyxose; and may include pyranose sugars, furanose sugars, and sedoheptulose.

[0165] Modified nucleotides are known in the art and include, for example, alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other heterocycles. Such classes of pyrimidines and purines are known in the art and include, for example, pseudoisocytosine, N 4 ,N 4 -ethanocytosine, 8-hydroxy-N 6 -methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N 6 -isopentyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, -D-mannosylqueuosine, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, methyl ester of uracil-5-oxyacetic acid, pseudouracil, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of N-uracil-5-oxyacetic acid, uracil-5-oxyacetic acid, queuosine, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine, 2,6-diaminopurine, methylpseudouracil, 1-methylguanine and 1-methylcytosine.

[0166] Methods for silencing genes in C. elegans, Drosophila, plants and mammals using RNAi are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619 and Elbashir S M, et al., 2001 Nature 411:494-498).

[0167] Accordingly, the present invention provides a nucleic acid capable of suppressing the expression of IL-11, IL-11Rα or gp130 by the RNAi method when appropriately introduced or expressed in mammalian cells (e.g., human cells) expressing IL-11, IL-11Rα or gp130.

[0168] Taking into account the known nucleic acid sequences of IL-11, IL-11Rα and gp130 (for example, accession numbers: BC012506.1 GI:15341754 (human IL-11), BC134354.1 GI:126632002 (mouse IL-11), AF347935.1 GI:13549072 (rat IL-11), NM_001142784.2 GI:391353394 (human IL-11Rα), NM_001163401.1 GI:254281268 (mouse IL-11Rα), NM_139116.1 GI:20806172 (rat IL-11Rα), NM_001190981.1 GI:300244534 (human gp130), NM_010560.3 GI:225007624 (mouse gp130), NM_001008725.3 GI:300244570 (rat gp130), the known mRNA sequences available from GenBank), oligonucleotides that suppress or silence the expression of IL-11, IL-11Rα or gp130 may be designed.

[0169] The nucleic acid may have substantial sequence identity with a part of the mRNA of IL-11, IL-11Rα or gp130, for example, the sequence represented by GenBank accession number NM_000641.3 GI:391353405 (IL-11), NM_001142784.2 GI:391353394 (IL-11Rα) or NM_001190981.1 GI:300244534 (gp130) or a part such as a sequence complementary to these mRNAs may have substantial sequence identity.

[0170] The nucleic acid may be double-stranded siRNA. (As will be fully understood by those skilled in the art, the siRNA molecule may further contain a short DNA sequence at the 3' end, which will be described in detail below.)

[0171] Alternatively, the nucleic acid may be DNA (usually double-stranded DNA), and when this DNA is transcribed in mammalian cells, RNA having two complementary portions linked via a spacer is obtained, and this RNA takes a hairpin structure when the two complementary portions hybridize with each other. In mammalian cells, this hairpin structure portion can be cleaved from the RNA molecule by an enzyme called DICER to obtain a double-stranded RNA in which two different RNA molecules are hybridized.

[0172] In some of the preferred embodiments, the nucleic acid typically targets any one of the sequences shown in SEQ ID NOs: 6-9 (IL-11) or any one of the sequences shown in SEQ ID NOs: 10-13 (IL-11Rα).

[0173] It is expected that only the single-stranded region of the mRNA transcript (i.e., the region not self-hybridized) is suitable as a target for RNAi. Therefore, among the mRNA transcripts of IL-11 or IL-11Rα, other sequences that are very similar to the sequences shown by any of SEQ ID NOs: 6-9 and 10-13 are also considered suitable as targets for RNAi. The length of such target sequences is preferably 17-23 nucleotides in length, and it is preferred that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides or all 19 nucleotides overlap with any of SEQ ID NOs: 6-9 and 10-13 (at one end).

[0174] Therefore, the present invention provides a nucleic acid capable of suppressing the expression of IL-11 or IL-11Rα by the RNAi method when appropriately introduced or expressed in mammalian cells expressing IL-11 or IL-11Rα, and this nucleic acid typically targets the sequence shown by any of SEQ ID NOs: 6-9 and 10-13.

[0175] "Targeting a normal target" means that the nucleic acid may target a sequence overlapping with any of SEQ ID NOs: 6-9 and 10-13. Specifically, the nucleic acid may target the same human IL-11 mRNA sequence or human IL-11Rα mRNA sequence (preferably 17-23 nucleotides in length) as these sequences, except that it is slightly longer or slightly shorter than the sequences represented by any of SEQ ID NOs: 6-9 and 10-13.

[0176] Although it is preferred that there is complete identity / complementarity between the nucleic acid of the present invention and the target sequence, it is expected that this is not essential. Therefore, the nucleic acid of the present invention may contain a single base mismatch compared to IL-11 mRNA or IL-11Rα mRNA. However, even if it is a single base mismatch, since its presence is expected to cause a decrease in efficiency, it is preferred that there is no mismatch. When a 3'-terminal overhang is present, the 3'-terminal overhang may not be considered as the number of mismatches.

[0177] "Complementarity" is not limited to the base pairing between nucleic acids composed of natural ribonucleotides and / or deoxyribonucleotides as commonly seen, but also includes the base pairing between the nucleic acid of the present invention containing unnatural nucleotides and mRNA.

[0178] In one embodiment, the nucleic acid (referred to as double-stranded siRNA herein) contains the double-stranded RNA sequences shown in SEQ ID NOs: 14-17. In another embodiment, the nucleic acid (referred to as double-stranded siRNA herein) contains the double-stranded RNA sequences shown in SEQ ID NOs: 18-21.

[0179] However, even a sequence that is slightly shorter or slightly longer and targets the same IL-11 mRNA region or IL-11Rα mRNA region is expected to be effective. Specifically, a double-stranded sequence with a length of 17-23 bp is expected to be effective.

[0180] Each strand constituting the double-stranded RNA may have a short 3'-terminal overhang of two bases, and this overhang may be DNA or RNA. The 3'-terminal DNA overhang shows no effect on siRNA activity compared to the case where a 3'-terminal RNA overhang is used, but the cost of chemically synthesizing the nucleic acid strand is reduced (Elbashir et al., 2001c). For this reason, DNA of two bases may be preferred in some cases.

[0181] When there are 2-base overhangs at both 3'-ends, these overhangs may be symmetric to each other, but symmetry is not essential. In fact, the 3'-terminal overhang of the sense strand (upper strand) is not involved in the recognition and degradation of mRNA and is thus not related to RNAi activity (Elbashir et al., 2001a, 2001b, 2001c).

[0182] In RNAi experiments in Drosophila, it has been shown that the 3'-terminal overhang of the antisense strand may be involved in the recognition and targeting of mRNA (Elbashir et al., 2001c), but in mammalian cells, the 3'-terminal overhang is not considered necessary for the RNAi activity of siRNA. Therefore, it is considered that even if the 3'-terminal overhang causes incorrect annealing, there is little effect in mammalian cells (Elbashir et al., 2001c; Czauderna et al., 2003).

[0183] Therefore, in the antisense strand of siRNA, any 2-base overhang may be used. However, the 2-base overhang is preferably -UU or -UG (when the overhang is DNA, it is -TT or -TG), and more preferably -UU (or -TT). The 2-base overhang consisting of -UU (or -TT) is the most effective and coincides with the transcription termination signal of RNA polymerase III (the transcription termination signal is TTTTT) (that is, it can constitute a part of the transcription termination signal). Therefore, these 2 bases are the most preferred. Although 2 bases of AA, CC, and GG can also be used, they are not so effective and thus less preferred.

[0184] Furthermore, siRNA may not contain any 3'-end overhang at all.

[0185] Furthermore, the present invention provides a single-stranded nucleic acid having one of the double-stranded nucleic acids described above as a constituent strand (referred to as single-stranded siRNA herein). This single-stranded siRNA preferably has a 3'-end overhang, but may not have a 3'-end overhang. Furthermore, the present invention provides a kit containing a pair of such single-stranded nucleic acids, and these single-stranded nucleic acids can hybridize with each other in vitro to form the double-stranded siRNA described above, and this double-stranded siRNA may then be introduced into cells.

[0186] Furthermore, the present invention provides DNA that is transcribed into RNA (also referred to as shRNA herein) in mammalian cells, in which two complementary parts can self-hybridize to form a double-stranded motif. Examples of the double-stranded motif formed include sequences selected from the group consisting of SEQ ID NOs: 14 to 17 and 18 to 21, or sequences in which a single base pair is substituted in any of these sequences.

[0187] The complementary parts are usually linked by a spacer, which has an appropriate length and sequence such that the two complementary parts can hybridize to each other. The two complementary parts (i.e., the sense strand and the antisense strand) may be linked at the 5' end and the 3' end, and either may be on the 5' end side. The spacer may usually be a short sequence about 4 to 12 nucleotides in length, preferably 4 to 9 nucleotides in length, more preferably 6 to 9 nucleotides in length.

[0188] The 5' end of the spacer (immediately after the 3' end of the upstream complementary part) preferably consists of two bases, -UU- or -UG-, and here, -UU- is more preferred (however, here too, the use of these specific two bases is not essential). A preferred spacer recommended for use in the pSuper system of OligoEngine (Seattle, Washington, USA) is UUCAAGAGA. When using this spacer or other spacers, the two ends of the spacer hybridize to each other, so that, for example, a double-stranded motif that is only a few base pairs (e.g., 1 or 2 base pairs) longer than the sequence itself shown in SEQ ID NOs: 14 to 17 or 18 to 21 can be obtained.

[0189] Similarly, the transcribed RNA preferably contains a 3' end overhang derived from the downstream complementary part. Here too, -UU or -UG is preferred as this overhang, and -UU is more preferred.

[0190] As described above, such shRNA molecules may be cleaved by the DICER enzyme in mammalian cells to form double-stranded siRNAs in which one or both of the single strands constituting the hybridized dsRNA contain a 3' end overhang.

[0191] Needless to say, the techniques for synthesizing the nucleic acids of the present invention are well known in the art.

[0192] A person skilled in the art could easily construct a transcription vector suitable for the DNA of the present invention using well-known techniques and commercially available materials. Specifically, regulatory sequences such as promoters and transcription termination sequences are ligated to the DNA of the present invention.

[0193] The pSuper system and pSuperior system, which are commercially available products from OligoEngine (Seattle, WA, USA), are particularly suitable. In these systems, a polymerase III promoter (H1) and a T5 transcription termination sequence are used, and the T5 transcription termination sequence adds two U residues to the 3' end of the transcript (when this transcript is processed by DICER, siRNA with a 3' end UU overhang added to one RNA strand is obtained).

[0194] Another suitable system is described by Shin et al. (RNA, 2009 May; 15(5): 898-910), and in this system, another polymerase III promoter (U6) is used.

[0195] The double-stranded siRNA of the present invention may suppress the expression of IL-11 or the IL-11 receptor by introducing it into mammalian cells in vitro or in vivo using known techniques as described below.

[0196] Similarly, the transcription vector containing the DNA of the present invention may be introduced into tumor cells in vitro or in vivo using known techniques as described below, and the expression of IL-11 or the IL-11 receptor may be suppressed by transiently or stably expressing the RNA.

[0197] Accordingly, the present invention further provides a method for suppressing the expression of IL-11 or the IL-11 receptor in mammalian (e.g., human) cells, which method comprises administering the double-stranded siRNA of the present invention or the transcription vector of the present invention to said cells.

[0198] Similarly, the present invention further provides a method for treating a disease / condition in which secretory smooth muscle cells are pathologically involved, the method comprising administering the double-stranded siRNA of the present invention or the transcription vector of the present invention to a subject.

[0199] Furthermore, the present invention provides the double-stranded siRNA of the present invention and the transcription vector of the present invention for use in a method of treatment, preferably a method of treating a disease / condition in which secretory smooth muscle cells are pathologically involved.

[0200] Furthermore, the present invention provides the use of the double-stranded siRNA of the present invention and the transcription vector of the present invention in the manufacture of a medicament for treating a disease / condition in which secretory smooth muscle cells are pathologically involved.

[0201] Furthermore, the present invention provides a composition comprising a mixture of the double-stranded siRNA of the present invention or the transcription vector of the present invention and one or more pharmaceutically acceptable carriers. Suitable carriers include lipophilic carriers or vesicles that can improve cell membrane permeability.

[0202] Materials and methods suitable for the administration of the double-stranded siRNA and DNA vectors of the present invention are well known in the art, and since RNAi technology holds various possibilities, improved methods are under development.

[0203] When introducing nucleic acids into mammalian cells, various techniques can usually be utilized. The technique to be used is selected depending on whether the nucleic acids are to be introduced into cultured cells in vitro or into the cells of a patient in vivo. Techniques suitable for introducing nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, the DEAE dextran method, and the calcium phosphate precipitation method. Gene transfer techniques in vivo include transfection using a viral vector (usually a retroviral vector), transfection using a viral coat protein-liposome complex (Dzau et al. (2003) Trends in Biotechnology 11, 205-210).

[0204] Specifically, techniques suitable for administering the nucleic acids of the present invention to cells in vitro or in vivo are described in the following references.

[0205] Review: Borkhardt, A. 2002. Blocking oncogenes in malignant cells by RNA interference--new hope for a highly specific cancer treatment? Cancer Cell. 2:167-8. Hannon, G.J. 2002. RNA interference. Nature. 418:244-51. McManus, M.T., and P.A. Sharp. 2002. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet. 3:737-47. Scherr, M., M.A. Morgan, and M. Eder. 2003b. Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem. 10:245-56. Shuey, D.J., D.E. McCallus, and T. Giordano. 2002. RNAi: gene-silencing in therapeutic intervention. Drug Discov Today. 7:1040-6.

[0206] Editor-in-Chief: Lewis, DL, JE Hagstrom, AG Loomis, JA Wolff, and H. Herweijer. 2002. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Night Genet. 32:107-8 Paul, CP, Good PD, Winer I, and English DR. 2002. Effective expression of small interfering RNA in human cells. Nat Biotechnol. 20:505-8 Song , E. , SK Lee , J. Wang , N. Ince , N. Ouyang , J. Min , J. Chen , P. Shankar , and J. Lieberman . 2003. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 9:347-5 Sorensen , DR , Leirdal M , and M Sioud . 2003. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol. 327:761-6

[0207] Viral-mediated introduction: Abbas-Terki, T., W. Blanco-Bose, N. Deglon, W. Pralong, and P. Aebischer. 2002. Lentiviral-mediated RNA interference. Hum Gene Ther. 13:2197-201. Barton, G.M., and R. Medzhitov. 2002. Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A. 99:14943-5. Devroe, E., and P.A. Silver. 2002. Retrovirus-delivered siRNA. BMC Biotechnol. 2:15. Lori, F., P. Guallini, L. Galluzzi, and J. Lisziewicz. 2002. Gene therapy approaches to HIV infection. Am J Pharmacogenomics. 2:245-52. Matta, H., B. Hozayev, R. Tomar, P. Chugh, and P.M. Chaudhary. 2003. Use of lentiviral vectors for delivery of small interfering RNA. Cancer Biol Ther. 2:206-10. Qin, X.F., D.S. An, I.S. Chen, and D. Baltimore. 2003. Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A. 100:183-8. Scherr, M., K. Battmer, A. Ganser, and M. Eder. 2003a.Modulation of gene expression by lentiviral-mediated delivery of small interfering RNA. Cell Cycle. 2:251-7. Shen, C., A.K. Buck, X. Liu, M. Winkler, and S.N. Reske. 2003. Gene silencing by adenovirus-delivered siRNA. FEBS Lett. 539:111-4.

[0208] Peptide delivery: Morris, M.C., L. Chaloin, F. Heitz, and G. Divita. 2000. Translocating peptides and proteins and their use for gene delivery. Curr Opin Biotechnol. 11:461-6. Simeoni, F., M.C. Morris, F. Heitz, and G. Divita. 2003. Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. Nucleic Acids Res. 31:2717-24. Other techniques considered suitable for delivery of siRNA to target cells include methods using nanoparticles or nanocapsules, as described in U.S. Patent No. 6,649,192(B) and U.S. Patent No. 5,843,509(B).

[0209] Suppression of IL-11-mediated signaling In embodiments of the present invention, the agent capable of suppressing the action of IL-11 may have one or more of the following functional characteristics. · Suppression of IL-11-mediated signal transduction ·Suppression of signal transduction via the binding of IL-11 to the IL-11Rα:gp130 receptor complex ·Suppression of signal transduction (i.e., trans-signaling of IL-11) via the binding of the IL-11:IL-11Rα complex to gp130 ·Suppression of processes mediated by IL-11 ·Suppression of the generation of myofibroblasts ·Suppression of the proliferation / migration of smooth muscle cells ·Suppression of gene expression / protein expression of collagen or IL-11

[0210] These properties can be measured by analyzing relevant factors in an appropriate assay, which may include comparing the performance of the agent with an appropriate control. One skilled in the art can determine appropriate control conditions in a specific assay.

[0211] IL-11-mediated signal transduction and / or IL-11-mediated processes include signal transduction via an IL-11 fragment and signal transduction via a polypeptide complex containing IL-11 or a fragment thereof. IL-11-mediated signal transduction may be signal transduction via human IL-11 and / or mouse IL-11. IL-11-mediated signal transduction may be signal transduction that occurs when an IL-11 or IL-11-containing complex binds to a receptor to which the IL-11 or IL-11-containing complex binds.

[0212] In some embodiments, the agent of the present invention may be capable of suppressing the biological activity of IL-11 or an IL-11-containing complex.

[0213] In some embodiments, the agent of the present invention is an antagonist to one or more signaling pathways activated by signal transduction through a receptor comprising IL-11Rα and / or gp130 (e.g., IL-11Rα:gp130). In some embodiments, the agent of the present invention can suppress signal transduction through one or more immunoreceptor complexes comprising IL-11Rα and / or gp130 (e.g., IL-11Rα:gp130).

[0214] In some embodiments, the agent of the present invention can suppress IL-11-mediated signal transduction to less than 100%, such as 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less, compared to the amount of IL-11-mediated signal transduction in the absence of the agent (or in the presence of an appropriate control agent). In some embodiments, the agent of the present invention can suppress IL-11-mediated signal transduction to less than 1-fold, such as 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less, compared to the amount of IL-11-mediated signal transduction in the absence of the agent (or in the presence of an appropriate control agent).

[0215] In some embodiments, IL-11-mediated signal transduction may be signal transduction through the binding of IL-11 to the IL-11Rα:gp130 receptor. Such signal transduction can be analyzed, for example, by treating cells expressing IL-11Rα and gp130 with IL-11 or by stimulating the production of IL-11 in cells expressing IL-11Rα and gp130.

[0216] IC of the suppression of IL-11-mediated signal transduction by the agent of the present invention 50 For example, Ba / F3 cells expressing IL-11Rα and gp130 are cultured in the presence of human IL-11 and the agent of the present invention, and the incorporation of 3 H-thymidine into DNA may be determined by measurement. In some embodiments, the IC of the agent of the present invention in such an assay 50 value may be 10 μg / ml or less, preferably 5 μg / ml or less, 4 μg / ml or less, 3.5 μg / ml or less, 3 μg / ml or less, 2 μg / ml or less, 1 μg / ml or less, 0.9 μg / ml or less, 0.8 μg / ml or less, 0.7 μg / ml or less, 0.6 μg / ml or less, or 0.5 μg / ml or less.

[0217] In some embodiments, IL-11-mediated signal transduction may be signal transduction via the binding of the IL-11:IL-11Rα complex to gp130. In some embodiments, the IL-11:IL-11Rα complex may be soluble, for example, a complex of the extracellular domain of IL-11Rα and IL-11, or a complex of a soluble IL-11Rα isoform / fragment and IL-11. In some embodiments, the soluble IL-11Rα is a soluble (secreted) isoform of IL-11Rα or a product released by proteolysis of the extracellular domain of membrane-bound IL-11Rα.

[0218] In some embodiments, the IL-11:IL-11Rα complex may be membrane-bound, for example, a complex consisting of membrane-bound IL-11Rα and IL-11. Signal transduction via the binding of the IL-11:IL-11Rα complex to gp130 can be analyzed by treating cells expressing gp130 with the IL-11:IL-11Rα complex, for example, with a recombinant fusion protein (such as hyper IL-11 described herein) containing IL-11 linked to the extracellular domain of IL-11Rα by a peptide linker.

[0219] In some embodiments, the agent of the present invention may be capable of suppressing signal transduction through the binding of the IL-11:IL-11Rα complex to gp130, and may also be able to suppress signal transduction through the binding of IL-11 to the IL-11Rα:gp130 receptor.

[0220] In some embodiments, the agent of the present invention may be capable of suppressing processes mediated by IL-11, such as after stimulation with TGFβ1. Processes mediated by IL-11 include, for example, the generation of myofibroblasts from fibroblasts, the proliferation / migration of smooth muscle cells (SMCs), and gene / protein expression of, for example, collagen and IL-11, which can be evaluated in vitro or in vivo.

[0221] In some embodiments, the agent of the present invention may be capable of suppressing the generation of myofibroblasts from fibroblasts, such as after exposing fibroblasts to a fibrosis-promoting factor (such as TGFβ1). The generation of myofibroblasts from fibroblasts can be examined by analyzing myofibroblast markers.

[0222] Fibroblasts may be obtained from any tissue, including fibroblasts obtained from the liver, lung, kidney, heart, blood vessels, eye, skin, pancreas, spleen, intestinal tract (such as the large intestine or small intestine), brain, and bone marrow. In certain embodiments, the fibroblasts may be any of cardiac fibroblasts (such as atrial fibroblasts), skin fibroblasts, lung fibroblasts, kidney fibroblasts, or liver fibroblasts. Fibroblasts may be characterized by the expression of one or more of the genes or proteins COL1A, ACTA2, prolyl-4-hydroxylase, MAS516, FSP1. Myofibroblast markers include an increase in αSMA, an increase in vimentin, an increase in palladin, an increase in cofilin, and an increase in desmin (when compared to the expression level by equivalent fibroblasts (such as fibroblasts derived from the same tissue)).

[0223] The generation of myofibroblasts from fibroblasts can be analyzed by measuring the expression level of αSMA protein using an Operetta high-content imaging system after stimulating the fibroblasts with TGFβ1. See, for example, WO 2017 / 103108 (A1), which is hereby incorporated by reference in its entirety.

[0224] In some embodiments, the agent of the present invention can suppress the generation of myofibroblasts from fibroblasts to less than 100%, for example, 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less, compared to the number of myofibroblasts generated from fibroblasts in the absence of the agent (or in the presence of a suitable control agent). In some embodiments, the agent of the present invention can suppress the generation of myofibroblasts from fibroblasts to less than 1-fold, for example, 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less, compared to the number of myofibroblasts generated from fibroblasts in the absence of the agent (or in the presence of a suitable control agent).

[0225] In some embodiments, the agent of the present invention may be capable of suppressing the proliferation of smooth muscle cells (such as secretory smooth muscle cells), for example, after stimulation with TGFβ1. The proliferation of smooth muscle cells can be measured, for example, using 3 an H-thymidine incorporation assay, a CFSE dilution assay, or an EdU incorporation assay as described herein.

[0226] In some embodiments, the agent of the present invention can suppress the proliferation of smooth muscle cells to less than 100%, for example, 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less, compared to the amount of smooth muscle cell proliferation in the absence of the agent (or in the presence of an appropriate control agent). In some embodiments, the agent of the present invention can suppress the proliferation of smooth muscle cells to less than 1-fold, for example, 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less, compared to the amount of smooth muscle cell proliferation in the absence of the agent (or in the presence of an appropriate control agent).

[0227] In some embodiments, the agent of the present invention may be capable of suppressing the migration of smooth muscle cells (such as secretory smooth muscle cells), for example, after stimulation with TGFβ1. The migration of smooth muscle cells can be measured using, for example, the scratch assay described in Example 9 and Liang et al., Nat Protoc. (2007) 2(2):329-33, or using the Boyden chamber assay described in Example 9 and Chen, Methods Mol Biol. (2005) 294:15-22.

[0228] In some embodiments, the agent of the present invention can suppress the migration of smooth muscle cells to less than 100%, for example, 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less, compared to the degree of smooth muscle cell migration in the absence of the agent (or in the presence of an appropriate control agent). In some embodiments, the agent of the present invention can suppress the migration of smooth muscle cells to less than 1-fold, for example, 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less, compared to the degree of smooth muscle cell migration in the absence of the agent (or in the presence of an appropriate control agent).

[0229] In some embodiments, the agent of the present invention may be capable of suppressing the gene expression / protein expression of collagen or IL-11. Gene expression and / or protein expression can be measured according to the description herein.

[0230] In some embodiments, the agent of the present invention can suppress the gene expression / protein expression of collagen or IL-11 to less than 100%, for example, 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less, as compared to the gene expression level / protein expression level of collagen or IL-11 in the absence of the agent (or in the presence of an appropriate control agent). In some embodiments, the agent of the present invention can suppress the gene expression / protein expression of collagen or IL-11 to less than 1-fold, for example, 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, or 0.1-fold or less, as compared to the gene expression level / protein expression level of collagen or IL-11 in the absence of the agent (or in the presence of an appropriate control agent).

[0231] Smooth muscle cell dysfunction and smooth muscle cell diseases Smooth muscle cell (SMC) dysfunction is observed in various diseases / conditions, in which SMCs abnormally proliferate, hypertrophy, migrate, infiltrate, produce and / or modify the extracellular matrix, and lead to cell death.

[0232] Secreted SMCs are pathogenic effectors of diseases / conditions involving SMC dysfunction. The onset or progression of such diseases / conditions and / or their symptoms may be positively correlated with one or more activities of secreted SMCs. That is, the activity of secreted SMCs may cause the onset / progression of the said diseases / conditions and / or their symptoms, or contribute thereto (for example, it may worsen or enhance them).

[0233] In some cases, the disease / condition may be caused by abnormal phenotypic conversion of SMC from the contractile type to the secretory type, and may be exacerbated thereby. In another case, the disease / condition may be caused by an increase in the number / ratio of secretory SMC in a particular tissue / organ / organ system / patient (e.g., as compared to the number / ratio of secretory SMC in the absence of the disease / condition), and may be exacerbated thereby.

[0234] Diseases characterized by vascular smooth muscle cell (VSMC) dysfunction include atherosclerosis, hypertension, aneurysm, vascular stenosis and restenosis, atherosclerosis, supravalvular stenosis, pulmonary hypertension, plexiform lesion, fibromuscular dysplasia, telangiectasia, etc. SMC dysfunction in internal organs is involved in, for example, dysphagia, diarrhea, constipation, kidney and bladder diseases, and SMC dysfunction is also involved in respiratory diseases such as asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD) and acute respiratory distress syndrome (ARDS).

[0235] Diseases / conditions in which SMC is pathologically involved will be further described in the following section.

[0236] Systemic sclerosis / scleroderma Systemic sclerosis (SSc) is a connective tissue disease characterized by vascular remodeling, vasospasm and vascular occlusion due to the contribution of complex interactions among endothelial cells, vascular smooth muscle cells (VSMCs), extracellular matrix and circulating mediators. 8 VSMCs are involved in the formation of fibrotic intimal lesions in systemic sclerosis. 9 Patients with systemic sclerosis show excessive reactivity to TGFβ signaling, which plays an important role in the pathogenesis of the disease. 2 .

[0237] Pulmonary hypertension Pulmonary arterial hypertension (PAH) is a rare disease but a common complication in connective tissue diseases (most commonly systemic sclerosis (SSc)). The mechanism in which, following endothelial injury, SMC migration, proliferation, and extracellular matrix deposition are activated, and at the same time endothelial cell proliferation occurs, is extremely important as the fundamental pathology of PAH. 10 Vascular smooth muscle cells (VSMCs) transform their phenotype from contractile to secretory in the presence of pro-inflammatory stimuli, hypoxia, and mitogenic stimuli, causing the lesions seen in PAH. 11 .

[0238] The major genetic factor in PAH is loss-of-function mutations in BMPR2, a negative regulator of smooth muscle function and TGFβ. 12 In familial cases, BMPR2 mutations associated with VSMC proliferation and PAH development are found in up to 70% of patients.

[0239] Plexiform lesions are usually present at the branch points of muscular arteries, are characteristic of PAH, and consist of a vascular network partitioned by endothelial cells with myofibroblasts at the core. 10 Loss of antiproliferative signaling from TGFβ is thought to cause the abnormal proliferation of plexiform lesions. 10 .

[0240] Marfan syndrome, aortic aneurysm and other related diseases Marfan syndrome (MFS) is an autosomal dominant connective tissue disease affecting multiple organ systems. 13 In aortic samples from MFS patients, increased protein levels of pSMAD2 / 3 and RhoA are seen, indicating upregulated TGFβ signaling. 14 The upregulation of TGFβ signaling in MFS has been accepted by researchers for over a decade as a central principle in the pathogenesis of MFS, based on a series of experiments by the Dietz laboratory. 14~17 TGFβ neutralizing antibodies can reduce the rate of aortic root dilation, improve the maintenance of aortic wall structure and elastic fibers, reduce aortic wall thickness, and decrease collagen deposition in MFS models. 18。

[0241] Furlong syndrome and Spritzen-Goldberg syndrome are MFS-like diseases that exhibit premature cranial suture closure, aortic dissection, premature cranial suture closure, and mental retardation. 13 。Mutations in TβRI and TGFβR2 have been identified in these diseases, and it is thought that they explain the overlapping phenotypes among these diseases. 13 。Similarly, in Loeys-Dietz syndrome (somatic mutations in TGFβR1 and TGFβR2), familial thoracic aortic aneurysm syndrome (germline mutations in TGFβR2 and missense mutations in TGFβR1), and arterial tortuosity syndrome, vascular symptoms similar to those of MFS are observed, highlighting the importance of TGFβ signaling. 19 。

[0242] Brain aneurysm Brain aneurysms occur in cerebral arteries, mainly at branch points where hemodynamic shear stress is high. 20 。Changes in the phenotype of VSMCs (changes to a pro-inflammatory secretory phenotype) increase the expression of MMPs and cause the disappearance of the internal elastic lamina. Most brain aneurysms are formed in this way. 20 。Furthermore, studies have shown that morphological changes in SMCs (changes from spindle-shaped cells to spider-like cells) and decreases in the expression and staining of contractile SMC markers (smooth muscle myosin heavy chain and smooth muscle α-actin) are observed in response to changes in the phenotype of VSMCs. 21 。SMCs are associated with TGFβ 20 、and are also associated with other factors that are thought to have an indirect relationship with the IL-11 signaling pathway. Therefore, suppression of IL-11-mediated signaling is considered useful for the prevention or treatment of brain aneurysms.

[0243] Restenosis Restenosis is characterized by fibrosis, proliferation of vascular smooth muscle cells (VSMCs), and remodeling resulting from iatrogenic vascular injury (e.g., angioplasty). 13Apoptosis of VSMCs induces aggregation of platelets and fibrin. Thrombin is also a potent factor that induces production of growth factors, proliferation of VSMCs, and deposition of ECM. Furthermore, activated platelets release mitogens such as PDGF (a potent inducer), EGF, TGFβ, and vasoconstrictors (thromboxane, serotonin), worsening the proliferation of VSMCs. In addition, in human restenosis samples, the mRNA level of TGFβ is significantly upregulated. 22 Furthermore, overexpression of TGFβ in intact porcine arteries increases ECM deposition and cell proliferation in the arterial wall. 23 Similarly, it has been shown that inhibition of TGFβ by an antibody suppresses restenosis in balloon catheter-injured rabbit carotid arteries. 24 In addition, in human stenotic lesions, SMAD3 is upregulated compared to early plaques of atherosclerosis. 25 Overexpression of SMAD3 causes an increase in the intima / media ratio and an increase in the proliferation of cells (PCNA-positive cells) in the intima and subintima. 26 Furthermore, overexpression of SMAD7 (which has an inhibitory effect) reduces restenosis after angioplasty. 27 These studies suggest a strong association between restenosis and TGFβ signaling.

[0244] Atherosclerosis Atherosclerosis is a chronic inflammatory reaction of the arterial wall induced by, for example, damage caused by chemical aggression (hyperglycemia), modified low-density lipoprotein (LDL), or physical force (hypertension). 28 Proliferation and migration of vascular smooth muscle cells (VSMCs) are extremely important for the stability of atherosclerotic plaques. 29 In addition, in the late stage of atherosclerosis, it is known that plaque disruption occurs due to apoptosis of VSMCs by the activity of inflammatory cells. Furthermore, TGFβ signaling has been shown to suppress the proliferation and migration of VSMCs and endothelial cells and stimulate apoptosis of VSMCs and endothelial cells. 13VSMCs isolated from human atherosclerotic lesions have been shown to be resistant to the antiproliferative and apoptotic effects of TGFβ, have mutations in TβRII, and / or have reduced expression of TβRII. 30 Furthermore, atherosclerotic disease is exacerbated by systemic inhibition of TGFβ signaling using neutralizing antibodies 31 , expression of dominant negative type II receptors 32 , and targeted deletion of one allele. 33 In contrast, administration of tamoxifen increases TGFβ and improves atherosclerotic disease. 34 .

[0245] Fibromuscular dysplasia Fibromuscular dysplasia (FMD) is a rare non-atherosclerotic disease that affects medium-sized arteries and is known to cause arterial stenosis, beading, dissection, and aneurysm. 35 FMD is most commonly seen in the renal arteries (60 - 75%), followed by the carotid and intracranial arteries (25 - 30%), visceral arteries (9%), and limb arteries (5%). 36 Histopathologically, FMD lesions are classified based on the arterial layer in which the lesion is predominantly found (tunica media, intima, or adventitia) and the composition of the arterial lesion (deposition of collagen known as fibroplasia, or, less frequently, hyperplasia of smooth muscle cells). 35 In patients with FMD, increased secretion of TGFβ1 and TGFβ2, as well as increased blood concentrations of TGFβ1 and TGFβ2, have been shown compared to corresponding controls. 37 .

[0246] Renal artery stenosis Renal artery stenosis (RAS) is a disease that includes three major clinical syndromes consisting of ischemic nephropathy, hypertension, and cardiac disorder syndrome. 38 The most common causes of RAS are atherosclerosis (90%) and fibromuscular dysplasia (10%), and pathological functional changes occur after these diseases develop. 38 .

[0247] Hypertension Angiotensin II (AGII) controls vascular smooth muscle cell (VSMC) proliferation signaling via MAPK (ERK1 / 2, JNK, and p38 kinases), Janus kinase (JAK) / signal transducer and activator of transcription (STAT), NF-κB, and phosphatidylinositol 3-kinase (PI3K). 39 The deposition of ECM by VSMCs and the proliferation of VSMCs play important roles in vascular remodeling in hypertension, especially in age-related hypertension where vascular compliance decreases and systolic blood pressure increases. 39 In hypertensive rats, the tendency of VSMC proliferation and migration is higher compared to wild-type controls. 40 Since the decrease in contractile markers, namely smooth muscle actin (SMA) and SM22α, in rats occurs along with the decrease in PPAR-γ, it is possible that in hypertension, the phenotypic transformation of VSMCs is regulated through the suppression of PPAR-γ-induced PI3K / Akt signaling. 40 In a recent very large-scale genome-wide association study (GWAS), the function of VSMCs has been strongly associated as an important determinant of blood pressure. 41

[0248] Kidney diseases Focal segmental glomerulosclerosis (FSGS), IgA nephropathy, crescentic glomerulonephritis, lupus nephritis, and diabetic nephropathy (DN) are important kidney diseases. 42 The proliferation of mesangial cells plays an important role in the progression of these kidney diseases and causes glomerulosclerosis through a process very similar to atherosclerosis. Mesangial cells are very similar to vascular smooth muscle cells (VSMCs) in terms of their origin, microscopic anatomical features, histochemical features, and contractility. 43 ​It is considered to be a subtype of VSMC. Mesangial cells secrete mesangial matrix and surround themselves with this matrix, playing a central role in supporting the structure of the glomerulus. Mesangial cells under pathological conditions dedifferentiate into a myofibroblast-like phenotype (mesangioblast), similar to the phenotypic conversion of VSMC in response to vascular injury, and overproduce matrix components. 44 Mesangial cells also upregulate the expression of markers such as αSMA during dedifferentiation. An increase in the size of the mesangial region due to the deposition of mesangial matrix, and the proliferation and hypertrophy of mesangial cells are characteristics of glomerulosclerosis. 45 Also, similar to VSMC, PDGF has been identified to act as a potent inducer of mesangial cell proliferation. 42 Other important transcription factors that promote mesangial cell proliferation include c-fos, c-myc, and c-jun. c-fos dimerizes with c-jun to form the AP-1 complex, and this AP-1 complex transactivates various target genes. 42 Dysfunction of the glomerulus and hypertension can occur due to the proliferation of mesangial cells and / or vascular smooth muscle cells (VSMC) or the impaired relaxation of the efferent arterioles of the glomerulus.

[0249] Lung diseases Airway smooth muscle cells (ASMC) are deeply involved in lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). 46 Airway smooth muscle cells are characterized by relatively low expression of contractile proteins such as smooth muscle myosin heavy chain, calponin, and smooth muscle α-actin, and maintain a proliferative mode. 47 TGFβ increases the expression of smooth muscle contractile proteins such as smooth muscle α-actin and calponin in airway smooth muscle and airway fibroblasts, and increases the size and number of airway smooth muscle cells. 48 。

[0250] Asthma Asthma is a chronic disease affecting over 300 million patients worldwide, with 250,000 deaths annually attributed to asthma. Asthma is characterized by airway inflammation, hyperresponsiveness, and remodeling. 49 . Structural remodeling occurs when airway smooth muscle cells (ASMCs) are frequently stimulated by contractile agonists, inflammatory mediators, and growth factors, resulting in irreversible airway obstruction in the late stage of asthma. Among various mediators, TGFβ has been identified. 50 . McMillan et al. showed that treating mice with anti-TGFβ antibodies significantly reduced peribronchial extracellular matrix deposition, ASMC proliferation, and mucus production in the lung without affecting established airway inflammation and Th2 cytokine production. 51 . The central role of ASMCs in asthma is emphasized in the initial treatment of acute exacerbations of asthma using β-blockers that relax ASMC contraction. 52 . Furthermore, ASMCs contribute to the disease pathogenesis by producing collagen and secreting pro-inflammatory cytokines. 53 .

[0251] Chronic obstructive pulmonary disease (COPD) COPD is a chronic lung disease estimated to cause 3 million deaths worldwide annually. COPD is characterized by airway wall thickening and tissue repair and epithelial metaplasia that cause airway obstruction. 48 . Previous studies have shown that the amount of airway smooth muscle is inversely correlated with lung function and that relaxation induced by bronchodilators such as β-agonists and anticholinergics can be inhibited by peribronchial adventitial fibrosis. 48 . TGFβ has been shown to be overexpressed in the airway epithelium and airway smooth muscle cells (ASMCs) of smokers with COPD compared to smokers without COPD. 54 .

[0252] Intestinal pathology Intestinal smooth muscle cells (iSMCs) play an important role in the formation of strictures in the intestinal wall (e.g., ileal strictures). This process is commonly seen in inflammatory bowel diseases (e.g., celiac disease, irritable bowel syndrome, Crohn's disease, and ulcerative colitis) and other diseases that cause inflammation and thickening of the intestinal wall. Intestinal smooth muscle cells (iSMCs) are contractile and non-proliferative under physiological conditions and are required for normal intestinal function 55 . However, in response to various pathological conditions, intestinal smooth muscle cells (iSMCs) dedifferentiate, re-enter the cell cycle, hypertrophy, and undergo a phenotypic conversion to secretory SMCs 55 . Interstitial cells of Cajal of the intestinal muscle are a special type of SMC that controls intestinal peristalsis. Interstitial cells of Cajal are particularly sensitive to the transformation into secretory SMCs that adversely affect intestinal contraction (Vetuschi et al., Eur J Clin Invest. (2006) 36(1):41-8). Intestinal SMCs have the ability to rapidly proliferate and synthesize and secrete extracellular matrix (ECM) such as collagen. In in vitro experiments, it has been shown that in intestinal smooth muscle cells (iSMCs), TGFβ increased the absolute amount of collagen synthesis per cell by 100% 56 .

[0253] Hutchinson-Gilford progeria syndrome (HGPS) Hutchinson-Gilford progeria syndrome (HGPS) is a severe human premature aging disease caused by a mutant form of lamin A called progerin. Generally, death due to cardiovascular disease in progeria patients is due to a severe deficiency of vascular smooth muscle cells (VSMCs) 57 . The expression of progerin downregulates PARP1, causing mitotic cell death and the death of SMCs 57 . In progeria, TGFβ and SMAD are upregulated, and the MAPK pathway is one of the things that change in this disease 58 .

[0254] Leiomyoma and leiomyosarcoma Leiomyomas, known as fibroid tumors, are benign smooth muscle tumors that can occur in any organ. Leiomyomas generally occur in the uterus (i.e., uterine leiomyomas / fibroid tumors), esophagus, stomach, and intestine. Most leiomyomas arise from the proliferation of a single smooth muscle cell and contain vascular smooth muscle cells. It has also been found that fibroid tumors contain differentiated cell populations such as fibroblasts and fibroid-related fibroblasts. 65 。

[0255] Leiomyomas can also occur in the skin, for example, solitary cutaneous leiomyoma, multiple cutaneous (or pilosebaceous) leiomyomas arising from arrector pili muscles, angioleiomyomas (vascular leiomyomas) arising from vascular smooth muscle, leiomyomas of the genital mesothelium, mesothelium (or genital) leiomyomas occurring in the areola and nipple, and angiolipoleiomyomas.

[0256] Changes in the signaling of 17β-estradiol (E2) in leiomyomas have been reported to increase the level of phosphorylated ERK1 / 2, thereby causing MAPK activation and pathological cell proliferation. 66 。

[0257] On the other hand, leiomyosarcoma (LMS) is a malignant smooth muscle tumor that can occur in any organ. Leiomyomas are not usually considered to develop into malignant leiomyosarcoma (LMS), but leiomyosarcoma (LMS) is sometimes seen in association with fibroid tumors (e.g., uterine fibroid tumors). 67 。Leiomyosarcoma (LMS) usually expresses smooth muscle actin (SMA), desmin, and caldesmon, and may also show the phenotype of secretory SMC.

[0258] Hermansky-Pudlak syndrome (HPS) Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism and platelet function abnormalities. HPS patients may develop fatal pulmonary fibrosis, inflammation of the gastrointestinal tract and / or colon (colitis), and / or renal failure. 68The mouse HPS model develops fibrosis in the lungs and exhibits high TGFβ1 levels. 69 。

[0259] Treatment / prevention of smooth muscle cell-related diseases / conditions The present invention provides methods and compositions for the treatment / prevention of diseases and conditions associated with smooth muscle cell (SMC) dysfunction. The diseases / conditions treated / prevented by the present invention may also be referred to as smooth muscle cell-related diseases / conditions or smooth muscle cell-mediated diseases / conditions.

[0260] More specifically, the present invention provides methods and compositions for the treatment / prevention of diseases and conditions in which secreted smooth muscle cells are pathologically involved.

[0261] The methods of the present invention generally involve suppressing the activity of secreted smooth muscle cells, i.e., suppressing the functional properties of secreted smooth muscle cells (reducing the level of functional properties). This is achieved by suppressing IL-11-mediated signal transduction.

[0262] That is, the present invention provides the treatment / prevention of diseases / conditions caused / worsened by secreted SMCs by suppressing IL-11-mediated signal transduction, for example, in cells or tissues / organs / organ systems / subjects.

[0263] One of ordinary skill in the art will readily understand that the therapeutic and prophylactic utilities of the present invention are applicable to substantially any disease / condition that can benefit from a reduction in the number or activity of secreted SMCs.

[0264] Diseases / conditions in which secretory SMCs are "pathologically involved" may be, for example, diseases / conditions in which their occurrence, onset, progression and / or the severity of one or more symptoms are positively correlated with an increase in secretory SMCs or their number / percentage, or diseases / conditions in which an increase in secretory SMCs or their number / percentage is a risk factor for their occurrence, onset or progression. Secretory SMCs may be present in the organs / tissues affected by the disease (for example, the organs / tissues in which the symptoms of the disease / condition are observed). The percentage of secretory SMCs may be determined as the percentage relative to the total number of secretory SMCs and non-secretory SMCs (for example, contractile SMCs) in the relevant organs / tissues.

[0265] In some embodiments, the disease / condition to be treated / prevented according to the present invention is a disease characterized in that, for example, in the organs / tissues affected by the disease / condition (for example, the organs / tissues in which the symptoms of the disease / condition are observed), the number / percentage / activity of secretory SMCs is increased.

[0266] In some embodiments, the disease / condition to be treated / prevented according to the present invention is such that in the organs / tissues / subjects affected by the disease, for example, compared to normal (i.e., disease-free) organs / tissues / subjects, one or more of the expressions of extracellular matrix components (such as type I collagen), IL-11, osteopontin, l-caldesmon, NM-B MHC, vimentin, tropomyosin 4, CRBP-1, secretory vesicles and α4β1 integrin and one or more of the number / percentage / activity of secretory SMCs are increased. In some embodiments, the disease / condition to be treated / prevented according to the present invention is such that in the organs / tissues / subjects affected by the disease, for example, compared to normal (i.e., disease-free) organs / tissues / subjects, one or more of the expressions of extracellular matrix components, collagen and IL-11 and one or more of the number / percentage / activity of secretory SMCs are increased.

[0267] In some embodiments, the disease / condition being treated / prevented according to the present invention is such that in the organ / tissue / subject affected by the disease, for example, compared to a normal (i.e., disease-free) organ / tissue / subject, one or more of the expressions of myosin 11, smoothelin, SMMHC, αSMA, SM22α, h1-caldesmon, h-caldesmon, α1β1 integrin, α7β1 integrin, actin filaments, and dystrophin glycoprotein complex (DGPC) and one or more of the number / percentage of contractile SMCs are decreased. In some embodiments, the disease / condition being treated / prevented according to the present invention is such that in the organ / tissue / subject affected by the disease, for example, compared to a normal (i.e., disease-free) organ / tissue / subject, one or more of the expression of myocardin, the expression of SM22α, and the number / percentage of contractile SMCs are decreased.

[0268] The disease / condition may affect any tissue, organ, or organ system. In some embodiments, the disease / condition may affect several tissue / organs / organ systems.

[0269] In some embodiments, the disease / condition is a disease / condition that affects one or more of the cardiovascular system, digestive system, excretory system, respiratory system, renal system, and genital system.

[0270] In some embodiments, the disease / condition for which treatment / prevention is carried out according to the present invention is a disease / condition that affects one or more organs of the cardiovascular system, for example, a disease / condition that affects blood vessels (i.e., a vascular disease / condition). In some embodiments, the disease / condition is one or more of atherosclerosis, hypertension, aneurysm, Marfan syndrome, aortic aneurysm, Furlong syndrome, Spritzen-Goldberg syndrome, Royce-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome, cerebral aneurysm, vascular stenosis and restenosis, atherosclerosis, fibromuscular dysplasia (FMD), supravalvular stenosis, renal artery stenosis, pulmonary arterial hypertension (PAH), plexiform lesion, fibromuscular dysplasia, telangiectasia, systemic sclerosis, Hutchinson-Gilford progeria syndrome (HGPS), leiomyoma and leiomyosarcoma.

[0271] In some embodiments, the disease / condition for which treatment / prevention is carried out according to the present invention is a disease / condition that affects one or more organs of the digestive system or excretory system. In some embodiments, the disease / condition is one or more of achalasia, dysphagia, diarrhea, constipation, inflammatory bowel disease (IBD), intestinal stenosis, pyloric stenosis, celiac disease, irritable bowel syndrome, diverticulitis, Crohn's disease, ulcerative colitis and Hermansky-Pudlak syndrome (HPS).

[0272] In some embodiments, the disease / condition for which treatment / prevention is carried out according to the present invention is a disease / condition that affects one or more organs of the respiratory system, for example, a disease / condition that affects the airways (i.e., a respiratory disease / condition). In some embodiments, the disease / condition is one or more of lung disease, asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS) and Hermansky-Pudlak syndrome (HPS).

[0273] In some embodiments, the disease / condition being treated / prevented according to the present invention is a disease / condition that affects one or more organs of the renal system, such as a disease / condition that affects the kidney or bladder (i.e., a renal disease / condition). In some embodiments, the disease / condition is one or more of kidney disease, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, crescentic glomerulonephritis, lupus nephritis, diabetic nephropathy (DN), bladder disease, and Hermansky-Pudlak syndrome (HPS).

[0274] In some embodiments, the disease / condition being treated / prevented according to the present invention is atherosclerosis, hypertension, aneurysm, Marfan syndrome, aortic aneurysm, Furlong syndrome, Spritzen-Goldberg syndrome, Royce Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome, cerebral aneurysm, vascular stenosis and restenosis, atherosclerosis, fibromuscular dysplasia (FMD), supravalvular stenosis, renal artery stenosis, pulmonary arterial hypertension (PAH), plexiform lesion, fibromuscular dysplasia, telangiectasia, achalasia, dysphagia, diarrhea, constipation, inflammatory bowel disease (IBD), intestinal stenosis, pyloric stenosis, celiac disease, irritable bowel syndrome, diverticulitis, Crohn's disease, ulcerative colitis, kidney disease, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, crescentic glomerulonephritis, lupus nephritis, diabetic nephropathy (DN), bladder disease, lung disease, asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), systemic sclerosis, Hutchinson-Gilford progeria syndrome (HGPS), leiomyoma, leiomyosarcoma, and Hermansky-Pudlak syndrome (HPS).

[0275] In some embodiments, the disease / condition being treated / prevented according to the present invention is not a disease / condition that affects one or more organs of the respiratory system, e.g., not a disease / condition that affects the airways (i.e., not a respiratory disease / condition). In some embodiments, the disease / condition being treated / prevented according to the present invention is atherosclerosis, hypertension, aneurysm, Marfan syndrome, aortic aneurysm, Furlong syndrome, Spritzen-Goldberg syndrome, Royce-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome, cerebral aneurysm, vascular stenosis and restenosis, atherosclerosis, fibromuscular dysplasia (FMD), supravalvular stenosis, renal artery stenosis, pulmonary arterial hypertension (PAH), plexiform lesion, fibromuscular dysplasia, telangiectasia, achalasia, dysphagia, diarrhea, constipation, inflammatory bowel disease (IBD), intestinal stenosis, pyloric stenosis, celiac disease, irritable bowel syndrome, diverticulitis, Crohn's disease, ulcerative colitis, kidney disease, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, crescentic glomerulonephritis, lupus nephritis, diabetic nephropathy (DN), bladder disease, systemic sclerosis, Hutchinson-Gilford progeria syndrome (HGPS), leiomyoma, leiomyosarcoma, and one or more of the airway / lung-unrelated lesions of Hermansky-Pudlak syndrome (HPS). In some embodiments, the disease / condition being treated / prevented according to the present invention is not any of the lung diseases, asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), airway / lung-related lesions of Hermansky-Pudlak syndrome (HPS).

[0276] The treatment / prevention of diseases and conditions by the present invention may be the treatment / prevention of diseases / conditions associated with upregulation of IL-11, e.g., the treatment / prevention of diseases / conditions associated with upregulation of IL-11 in cells or tissues where symptoms are observed or in cells or tissues that may develop symptoms, or the treatment / prevention of diseases / conditions associated with upregulation of extracellular IL-11 or IL-11Rα.

[0277] The treatment may be effective in preventing the progression of the disease / condition, for example, may be effective in reducing / delaying / preventing the worsening of the disease / condition, and may be effective in reducing / delaying / preventing the onset of the disease / condition. In some embodiments, the disease / condition may be improved by the treatment, for example, the severity of the symptoms of the disease / disorder may be reduced, and / or the symptoms of the disease / disorder may improve. In some embodiments, survival may be prolonged by the treatment.

[0278] "Prevention" may be the prevention of the onset of the disease / condition and / or the prevention of the worsening of the disease / condition, for example, may be the prevention of the progression to the late or chronic stage of the disease / condition.

[0279] Administration Administration of an agent capable of suppressing IL-11-mediated signaling is preferably carried out in a "therapeutically effective" amount or "preventively effective" amount sufficient for the subject to benefit.

[0280] The actual dosage, dosing rate and time course after administration depend on the characteristics and severity of the disease / condition and the characteristics of the agent. Prescription of treatment (such as determination of dosage, etc.) is carried out under the responsibility of a general practitioner and other medical professionals, usually taking into account the disease / condition to be treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors well known to the physician. Examples of such techniques and protocols are described in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0281] The agent of the present invention may be provided in multiple doses. Another therapeutic agent may be administered simultaneously or sequentially with one or more doses or each dose.

[0282] The multiple doses may be administered at predetermined intervals, which may be selected from 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or 31 days, or 1 month, 2 months, 3 months, 4 months, 5 months or 6 months. As an example, it may be administered once every 7 days, once every 14 days, once every 21 days, or once every 28 days (±3 days, 2 days or 1 day).

[0283] In therapeutic use, an agent capable of suppressing IL-11-mediated signal transduction is preferably formulated as a pharmaceutical or a pharmaceutical preparation together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art. Other pharmaceutically acceptable ingredients include, but are not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (such as wetting agents), masking agents, colorants, flavoring agents and sweetening agents.

[0284] As used herein, "pharmaceutically acceptable" refers to a compound, ingredient, material, composition, dosage form, etc. that is suitable for use in contact with the tissues of a subject (e.g., a human) within the scope of sound medical judgment, without causing excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit / risk ratio. Further, carriers, adjuvants, excipients, etc. must each be "acceptable" in terms of compatibility with the other ingredients in the formulation.

[0285] Suitable carriers, adjuvants, excipients, etc. are described in standard textbooks on pharmaceuticals such as Remington’s Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0286] The preparation may be prepared by any method as long as it is a method well known in the pharmaceutical field. Such methods include the step of mixing a carrier constituting one or more accessory components with the active compound. Generally, the preparation is prepared by uniformly and intimately mixing a carrier (such as a liquid carrier, a finely divided solid carrier, etc.) and the active compound, and shaping the resulting mixture as needed.

[0287] The preparation may be prepared as a preparation to be administered by a topical administration route, a parenteral administration route, a systemic administration route, an intravenous administration route, an intra-arterial administration route, an intramuscular administration route, an intrathecal administration route, an intraocular administration route, an intracorneal administration route, a subcutaneous administration route, an oral administration route, a transdermal administration route (which may include injection). The injection preparation may contain a drug selected in a sterile solvent or an isotonic solvent. The preparation and the administration method may be selected according to the drug of the present invention and the disease / condition to be treated.

[0288] A drug capable of suppressing IL-11-mediated signal transduction may be administered in the treatment described herein in combination with another treatment for diseases and conditions associated with smooth muscle cell dysfunction. Appropriate other treatments are known to those skilled in the art. The drug of the present invention may be administered alone or in combination with another treatment, simultaneously or sequentially, according to the disease / condition to be treated. For example, the drug of the present invention may be administered before, simultaneously with or after another treatment. The drug of the present invention and another treatment may be formulated together or separately in the form of a preparation as described above, for example.

[0289] Detection of IL-11 and IL-11 receptor Some aspects and embodiments of the present invention relate to the detection of the expression of IL-11 or the IL-11 receptor (e.g., IL-11Rα, gp130, or a complex comprising IL-11Rα and / or gp130) in a sample obtained from a subject.

[0290] In some aspects and embodiments, the present invention relates to the detection of upregulation (overexpression) of the expression of IL-11 or the IL-11 receptor (as a protein or as an oligonucleotide encoding IL-11 or the IL-11 receptor) as an indicator of suitability for treatment with an agent that can suppress the action of IL-11 or an agent that can inhibit or reduce the expression of IL-11 or the IL-11 receptor.

[0291] Upregulation of expression includes expression that exceeds the amount of expression normally expected in a particular type of cell or tissue. Upregulation may be measured by measuring the amount of expression of a relevant factor in a cell or tissue. The amount of expression of a relevant factor in a cell sample or tissue sample obtained from a subject may be compared with a reference amount of the relevant factor (e.g., a numerical value or range of numerical values indicating the normal amount of expression of the relevant factor in the same type of cell or tissue or corresponding cell or tissue). In some embodiments, the reference amount may be determined by detecting the expression of IL-11 or the IL-11 receptor in a control sample (e.g., corresponding cells or tissues obtained from a healthy subject, or corresponding cells or tissues obtained from healthy tissue of the same subject). In some embodiments, the reference amount may be obtained from a standard curve or a standard dataset.

[0292] The amount of expression may be quantified by absolute comparison or by relative comparison.

[0293] In some embodiments, when the expression level in the measurement sample is at least 1.1 times the reference level, it may be considered that IL-11 or the IL-11 receptor (for example, IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130) is upregulated. More preferably, the expression level when upregulated may be selected from at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, and at least 10.0 times the reference level.

[0294] The expression level may be measured by any of various known in vitro analysis techniques such as an assay using PCR, an in situ hybridization assay, a flow cytometry assay, an immunological assay, an immunohistochemical assay, and the like.

[0295] As an example, a suitable technique involves contacting a sample with an agent capable of binding to IL-11 or the IL-11 receptor and detecting the formation of a complex consisting of IL-11 or the IL-11 receptor and the agent, thereby detecting the amount of IL-11 or the IL-11 receptor in the sample. The agent may be any suitable binding molecule, for example, an antibody, polypeptide, peptide, oligonucleotide, aptamer, small molecule, etc., and may be labeled so that the formed complex can be detected (e.g., visualized). Labels and methods suitable for detecting such complexes are well known in the art, for example, fluorescent labels (e.g., fluorescein, rhodamine, eosin, NDB, green fluorescent protein (GFP); rare earth element chelates such as europium (Eu), terbium (Tb), samarium (Sm); tetramethylrhodamine, Texas red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, Cy5), isotope markers, radioisotopes (e.g., 32 P, 33 P, 35 S), chemiluminescent labels (e.g., acridinium ester, luminol, isoluminol), enzymes (e.g., peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, luciferase), antibodies, ligands, and receptors. Detection techniques are well known to those skilled in the art and can be selected according to the labeled agent. Suitable techniques include PCR amplification of oligonucleotide tags, mass spectrometry, detection of fluorescence or color (e.g., generated by enzymatic conversion of a substrate by a reporter protein), or detection of radioactivity.

[0296] The assay may be configured to be able to quantify the amount of IL-11 or the IL-11 receptor in the sample. The amount of IL-11 or the IL-11 receptor quantified from the measurement sample may be compared with a reference amount, and such a comparison may be used to determine whether the amount of IL-11 or the IL-11 receptor contained in the measurement sample is higher or lower than the reference value by a selected degree of statistical significance.

[0297] By quantifying the detected IL-11 or IL-11 receptor, upregulation, downregulation or amplification of the gene encoding IL-11 or IL-11 receptor may also be measured. When the measurement sample contains fibrotic cells, the presence or absence of a statistically significant difference may be determined by comparing such upregulation, downregulation or amplification with a reference amount.

[0298] The sample obtained from the subject may be of any kind. The biological sample may be obtained from any tissue or body fluid, for example, it may be any of a blood sample, a blood-derived sample, a serum sample, a lymph fluid sample, a semen sample, a saliva sample, a synovial fluid sample. The blood-derived sample may be a selected fraction derived from the patient's blood, for example, it may be any of a selected cell-containing fraction, a plasma fraction, a serum fraction. The sample may contain a tissue sample or a biopsy sample, or cells isolated from the subject. Also, the sample may be collected by known techniques such as biopsy or aspiration biopsy. Further, the sample may be stored until the expression level of IL-11 is measured and / or processed before measuring the expression level of IL-11.

[0299] The sample obtained from the subject may be used to measure the upregulation of IL-11 or IL-11 receptor in the subject.

[0300] In some preferred embodiments, the sample may be a tissue sample (for example, a biopsy sample) obtained from vascular tissue or heart tissue, visceral organ tissue or respiratory system organ tissue. The sample may contain cells, preferably, it may contain smooth muscle cells (SMC).

[0301] Based on the confirmation of upregulation of the expression of IL-11 or the IL-11 receptor (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130) in a subject, a subject to be treated / prevented according to the present invention may be selected. Also, the upregulation of the expression of IL-11 or the IL-11 receptor may be used as a marker for a disease / condition pathologically involved by SMCs that is suitable for treatment with an agent capable of suppressing IL-11-mediated signal transduction.

[0302] The upregulation may be an upregulation in a specific tissue or an upregulation in selected cells derived from a specific tissue. Preferred tissues may be any of vascular tissue, cardiac tissue, visceral tissue, and respiratory organ tissue. Preferred cells may be smooth muscle cells (SMCs). The upregulation of the expression of IL-11 or the IL-11 receptor may be measured in a circulating body fluid (e.g., blood) or in a blood-derived sample. The upregulation may be an upregulation of extracellular IL-11 or IL-11Rα. In some embodiments, the expression may be upregulated locally or systemically.

[0303] In the description given in the following section, an agent capable of suppressing IL-11-mediated signal transduction may be administered to a subject.

[0304] Diagnosis and prognosis The detection of upregulation of the expression of IL-11 or the IL-11 receptor (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130) may be used in a method for diagnosing a disease / condition pathologically involved by secretory SMCs and identifying a subject at risk of developing such a disease / condition, or in a method for diagnosing or predicting the responsiveness of a subject to treatment with an agent capable of suppressing IL-11-mediated signal transduction.

[0305] In some embodiments, for example, based on the presence or absence of other symptoms indicating a disease / condition in which secreted SMCs are pathologically involved in a subject's living body or selected cells / tissues derived therefrom, it may be determined that the subject is suspected of having the disease, or, for example, there is a genetic predisposition known to be a risk factor for a disease / condition in which secreted SMCs are pathologically involved, or there is exposure to environmental conditions known to be risk factors for a disease / condition in which secreted SMCs are pathologically involved, it may be considered that there is a risk of developing the disease / condition. By confirming the upregulation of the expression of IL-11 or the IL-11 receptor, the diagnosis or a diagnosis of suspicion may be confirmed, or it may be confirmed that the subject is at risk of developing the disease. Also, by confirming the upregulation of the expression of IL-11 or the IL-11 receptor, it may be diagnosed that the condition or predisposition is suitable for treatment with an agent capable of suppressing IL-11-mediated signal transduction.

[0306] Accordingly, provided is a method for providing a prognosis for a subject suffering from or suspected of suffering from a disease / condition in which secreted smooth muscle cells are pathologically involved, the method comprising: determining, in a sample obtained from the subject, whether the expression of IL-11 or the IL-11 receptor is upregulated; and providing a prognosis for the treatment of the subject with an agent capable of suppressing IL-11-mediated signal transduction based on the determination.

[0307] In some embodiments, a method of diagnosing a subject's responsiveness to treatment with an agent capable of suppressing IL-11-mediated signaling, or diagnosing the prognosis of or predicting a subject's responsiveness to such treatment, may not require measurement of the expression level of IL-11 or the IL-11 receptor, but may be based on measuring, in the subject, a genetic factor that predicts upregulation of the expression or activity of IL-11 or the IL-11 receptor. Such genetic factors include measurement of gene mutations, single nucleotide polymorphisms (SNPs), or gene amplifications of IL-11, IL-11Rα, and / or gp130 that correlate with and / or are predictive of the expression or activity of IL-11 or the IL-11 receptor or upregulation of IL-11-mediated signaling. The use of genetic factors to predict disease predisposition or responsiveness to treatment is known in the art; see, for example, Peter Starkel Gut 2008;57:440-442; Wright et al., Mol. Cell. Biol. March 2010 vol. 30 no. 6 1411-1420.

[0308] The genetic factors may be analyzed by methods known to those of skill in the art, such as assays using PCR, for example quantitative PCR or competitive PCR. For example, in a sample obtained from a subject, etc., the diagnosis may be confirmed by determining the presence or absence of the genetic factor, the subject may be classified as being at risk of developing the disease / condition, and / or the subject may be identified as being suitable for treatment with an agent capable of suppressing IL-11-mediated signaling.

[0309] Some methods may involve identifying the presence or absence of one or more SNPs associated with susceptibility to the development of diseases / conditions pathologically involving secreted SMCs or with the secretion of IL-11. SNPs are typically biallelic and can thus be readily identified using any of a variety of conventional assays known to those of skill in the art (see, e.g., Anthony J. Brookes. The essence of SNPs. Gene Volume 234, Issue 2, 8 July 1999, 177-186; Fan et al., Highly Parallel SNP Genotyping. Cold Spring Harb Symp Quant Biol 2003. 68: 69-78; Matsuzaki et al., Parallel Genotyping of Over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array. Genome Res. 2004. 14: 414-425).

[0310] The method may involve identifying which SNP alleles are present in a sample obtained from the subject. In some embodiments, by identifying the presence or absence of the minor allele, an increased susceptibility to the development of diseases / conditions pathologically involving secreted SMCs or an increased secretion of IL-11 may be identified.

[0311] Accordingly, in one aspect of the invention, a method of screening a subject, comprising: obtaining a nucleic acid sample from the subject; and identifying which allele is present at the polymorphic nucleotide positions of one or more SNPs listed in FIG. 33, FIG. 34, or FIG. 35 of WO 2017 / 103108 (A1) (this document is incorporated herein by reference) in the sample, or identifying an SNP that shows a linkage disequilibrium of r 2 ≧0.8 with any one of the SNPs listed in these figures is provided.

[0312] The step of identifying an allele or SNP may include identifying the presence or absence of a minor allele at the position of the selected polymorphic nucleotide in the sample. Further, the step may include identifying the presence or absence of 0, 1, or 2 minor alleles.

[0313] The screening method may be a method for identifying the susceptibility of a subject to the onset of a disease / condition pathologically involved by secreted SMC, or may be the aforementioned diagnostic method or prognostic diagnostic method, and may constitute a part of these methods.

[0314] For example, when the subject is identified as having a minor allele at the polymorphic nucleotide position, the method may further include a step of identifying the subject as having susceptibility to the onset of a disease / condition pathologically involved by secreted SMC, or as having a high risk of developing the disease / condition. The method may further include a step of selecting a subject for treatment with an agent capable of suppressing IL-11-mediated signal transduction, and / or administering an agent capable of suppressing IL-11-mediated signal transduction to the subject to treat the disease / condition of the subject, or to prevent the onset or progression of the disease / condition of the subject.

[0315] Examples of SNPs that can be specified include one or more of the SNPs listed in FIGS. 33, 34, or 35 of WO 2017 / 103108 (A1) (this document is incorporated herein by reference). In some embodiments, the method may include a step of specifying one or more of the SNPs listed in FIG. 33 of WO 2017 / 103108 (A1). In some embodiments, the method may include a step of specifying one or more of the SNPs listed in FIG. 34 of WO 2017 / 103108 (A1). In some embodiments, the method may include a step of specifying one or more of the SNPs listed in FIG. 35 of WO 2017 / 103108 (A1). The SNPs may be selected based on being specified as having a low P-value or FDR (false discovery rate).

[0316] In some embodiments, the SNPs are selected as factors that can preferably predict responsiveness to anti-IL-11 treatment based on the control of VST by SNPs located on different chromosomes (trans) (FIG. 33 of WO 2017 / 103108 (A1)). In some embodiments, the method of the present invention may include a step of specifying which allele is present in one or more SNPs selected from rs10831850, rs4756936, rs6485827, rs7120273, and rs895468. In some embodiments, the SNPs are selected as factors that can preferably predict responsiveness to anti-IL-11 treatment based on the control of VST by SNPs located on the same chromosome (cis) stim -VST stim -VST unstim (FIG. 34 of WO 2017 / 103108 (A1)).

[0317] In some embodiments, the SNPs are selected as factors that can preferably predict responsiveness to anti-IL-11 treatment based on the control of VST by SNPs located on different chromosomes (trans) stim -VST unstimBased on the control (Figure 35 of WO 2017 / 103108(A1)), it is selected as a factor that can well predict the responsiveness to anti-IL-11 treatment. In some embodiments, the method of the present invention may include a step of identifying which allele exists in one or more SNPs selected from rs7120273, rs10831850, rs4756936, and rs6485827 (Figure 35 of WO 2017 / 103108(A1)).

[0318] The rs7120273 SNP, rs10831850 SNP, rs4756936 SNP, and rs6485827 SNP are in strong linkage disequilibrium (LD) with each other on chromosome 11 (so-called linkage disequilibrium block), and thus are very likely to be inherited together.

[0319] The square of the correlation coefficient of gene frequencies (r 2 ) reflects the degree of linkage disequilibrium (LD) between two SNPs. When neighboring SNPs are in linkage disequilibrium (LD), since these genomic regions are inherited together, the genotype of a specific SNP can be inferred by identifying the genotype of the tag / proxy SNP. The LD threshold used in the art to identify tag SNP / proxy SNP pairs is r 2 = 0.8 (Wang et al. 2005, Nat. Rev. Genet. 6(2): 109-18; Barrett et al. 2006, Nat Genet., 38 (6): 659-662). Therefore, the genotype of a specific SNP can be inferred by identifying the genotype of the tag / proxy SNP with a linkage disequilibrium of r 2 ≧0.8.

[0320] The nucleotide sequence of the SNP is indicated using the "rs" number. The full-length sequence of the SNP is available from the Single Nucleotide Polymorphism (dbSNP) database of the National Center for Biotechnology Information (NCBI) accessible at https: / / www.ncbi.nlm.nih.gov / snp.

[0321] The diagnostic method or prognostic diagnostic method may be performed in vitro using a sample obtained from a subject, or may be performed in vitro after processing a sample obtained from a subject. The patient from whom the sample is collected does not need to wait until the in vitro diagnostic method or prognostic diagnostic method is performed, and thus these methods do not need to be performed on a human or animal body.

[0322] The method of the present invention may be used in combination with other diagnostic tests or prognostic tests, thereby enhancing the accuracy of diagnosis or prognostic diagnosis, or confirming the results obtained using the test methods described herein.

[0323] Subject The subject may be an animal or a human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but is more preferably a human. The subject may be male or female. The subject may be a patient. The patient may have the disease / condition described herein. The subject may be a subject diagnosed with a disease / condition that requires treatment, a subject suspected of having such a disease / condition, or a subject at risk of developing such a disease / condition.

[0324] In an embodiment according to the present invention, the subject is preferably a human subject. In some embodiments, the subject to be treated by the treatment method or prevention method of the present invention is a subject having cancer or a subject at risk of developing cancer. In an embodiment according to the present invention, a subject to be treated by the method of the present invention may be selected based on the characteristics of a specific marker of such a disease / disorder / condition. The subject may be diagnosed with a disease or disorder that requires treatment, or may be suspected of having such a disease / disorder / condition.

[0325] Sequence identity Pairwise sequence alignment and multiple sequence alignment for determining the identity (%) between two or more amino acid sequences or nucleic acid sequences can be performed using various methods known to those skilled in the art. For example, ClustalOmega software (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee software (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign software (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)), MAFFT software (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780), and other publicly available computer software can be used. When using such software, it is preferable to use the default parameters, for example, in the case of gap penalty and extension penalty.

[0326] [Table 1] TIFF0007704806000002.tif85170

[0327] A series of statements In the following numbered sections, specific aspects and embodiments of the present invention are described.

[0328] Item 1. An agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction for use in a method for treating or preventing a disease in which smooth muscle cells (SMCs) are pathologically involved.

[0329] Item 2. Use of an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction in the manufacture of a pharmaceutical for use in a method for treating or preventing a disease in which smooth muscle cells (SMCs) are pathologically involved.

[0330] Item 3. A method for treating or preventing a disease pathologically involving smooth muscle cells (SMC), the method comprising administering to a subject in need of treatment a therapeutically effective amount of an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction.

[0331] Item 4. The agent, use or method according to item 1, wherein the disease is a disease pathologically involving secretory smooth muscle cells.

[0332] Item 5. The agent, use or method according to any one of items 1 to 4, wherein the agent is an agent capable of binding to IL-11 or an IL-11 receptor.

[0333] Item 6. The agent, use or method according to item 5, wherein the agent is selected from the group consisting of an antibody or an antigen-binding fragment thereof, a polypeptide, a peptide, an oligonucleotide, an aptamer and a small molecule.

[0334] Item 7. The agent, use or method according to item 6, wherein the agent is an antibody or an antigen-binding fragment thereof.

[0335] Item 8. The agent, use or method according to item 6, wherein the agent is a decoy receptor for IL-11.

[0336] Item 9. The agent, use or method according to any one of items 1 to 4, wherein the agent is an agent capable of reducing the expression of IL-11 or an IL-11 receptor.

[0337] Item 10. The agent, use or method according to item 9, wherein the agent is an oligonucleotide or a small molecule.

[0338] Item 11. The agent, use or method according to any one of items 1 to 10, wherein the disease is a disease of the circulatory system, digestive system, excretory system, respiratory system, renal system or genital system.

[0339] Item 12. The drug, use or method according to any one of Items 1 to 11, wherein the disease is selected from the group consisting of atherosclerosis, hypertension, aneurysm, Marfan syndrome, aortic aneurysm, Furlong syndrome, Spritzen-Goldberg syndrome, Royce-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome, cerebral aneurysm, vascular stenosis and restenosis, atherosclerosis, fibromuscular dysplasia (FMD), supravalvular stenosis, renal artery stenosis, pulmonary arterial hypertension (PAH), plexiform lesion, fibromuscular dysplasia, telangiectasia, achalasia, dysphagia, diarrhea, constipation, inflammatory bowel disease (IBD), intestinal stenosis, pyloric stenosis, celiac disease, irritable bowel syndrome, diverticulitis, Crohn's disease, ulcerative colitis, kidney disease, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, crescentic glomerulonephritis, lupus nephritis, diabetic nephropathy (DN), bladder disease, lung disease, asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), systemic sclerosis, and Hutchinson-Gilford progeria syndrome (HGPS).

[0340] Item 13. The drug, use or method according to any one of Items 1 to 12, wherein the method comprises administering the drug to a subject in which the expression of IL-11 or the IL-11 receptor is upregulated.

[0341] Item 14. The drug, use or method according to any one of Items 1 to 13, wherein the method comprises administering the drug to a subject in which upregulation of the expression of IL-11 or the IL-11 receptor has been confirmed.

[0342] Item 15. The drug, use or method according to any one of Items 1 to 14, wherein the method comprises a step of determining whether the expression of IL-11 or the IL-11 receptor is upregulated in a subject, and a step of administering the drug to a subject in which the expression of IL-11 or the IL-11 receptor is upregulated.

[0343] Use of an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction for suppressing the activity of smooth muscle cells (SMC).

[0344] A method for suppressing the activity of smooth muscle cells (SMC), the method comprising the step of contacting the smooth muscle cells (SMC) with an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction.

[0345] A method for suppressing the activity of smooth muscle cells (SMC) in a subject, the method comprising the step of administering to the subject an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction.

[0346] The use or method according to any one of items 16 to 18, wherein the smooth muscle cells (SMC) are secretory smooth muscle cells.

[0347] The use or method according to any one of items 16 to 19, wherein the agent is an agent capable of binding to IL-11 or the IL-11 receptor.

[0348] The use or method according to any one of items 16 to 20, wherein the agent is selected from the group consisting of an antibody or an antigen-binding fragment thereof, a polypeptide, a peptide, an oligonucleotide, an aptamer, and a small molecule.

[0349] The use or method according to any one of items 16 to 21, wherein the agent is an antibody or an antigen-binding fragment thereof.

[0350] The use or method according to any one of items 16 to 21, wherein the agent is a decoy receptor for IL-11.

[0351] The use or method according to any one of items 16 to 21, wherein the agent is an agent capable of reducing the expression of IL-11 or the IL-11 receptor.

[0352] Use or method according to any one of items 16 to 21, wherein the agent is an oligonucleotide or a small molecule.

[0353] Item 26. A method for determining whether a subject is suitable for the treatment or prevention of a disease pathologically involving smooth muscle cells (SMC) using an agent capable of suppressing the action of interleukin 11 (IL-11), the method comprising the step of determining (optionally in vitro) whether the expression of IL-11 or interleukin 11 receptor (IL-11R) is upregulated in the subject.

[0354] Item 27. A method for selecting a subject for the treatment or prevention of a disease pathologically involving smooth muscle cells (SMC) using an agent capable of suppressing IL-11-mediated signal transduction, the method comprising the step of determining (optionally in vitro) whether the expression of IL-11 or the IL-11 receptor is upregulated in the subject.

[0355] Item 28. A method for diagnosing a disease pathologically involving smooth muscle cells (SMC) in a subject or diagnosing the risk of onset of the disease, the method comprising the step of determining (optionally in vitro) whether the expression of interleukin 11 (IL-11) or the IL-11 receptor is upregulated in a sample obtained from the subject.

[0356] Item 29. The method according to item 28, which is a method for confirming the diagnosis of the disease in a subject suspected of suffering from a disease pathologically involving smooth muscle cells (SMC).

[0357] Item 30. The method according to item 28 or 29, further comprising the step of selecting a subject for treatment using an agent capable of suppressing IL-11-mediated signal transduction.

[0358] Item 31. The method according to any one of items 26 to 30, wherein the disease is a disease pathologically involving secretory smooth muscle cells.

[0359] Item 32. A method for providing a prognosis to a subject suffering from a disease pathologically involving smooth muscle cells (SMCs) or suspected of suffering from such a disease, comprising: determining (optionally in vitro) whether the expression of interleukin 11 (IL-11) or the IL-11 receptor is upregulated in a sample obtained from the subject, and providing a prognosis for the treatment of the subject with an agent capable of suppressing IL-11-mediated signal transduction based on the determination. A method comprising the above.

[0360] Item 33. The method according to Item 32, further comprising selecting a subject in whom upregulation of the expression of IL-11 or the IL-11 receptor has been confirmed for treatment with an agent capable of suppressing IL-11-mediated signal transduction.

[0361] Item 34. A method for diagnosing a disease pathologically involving smooth muscle cells (SMCs) or diagnosing the risk of onset of such a disease in a subject, comprising measuring (optionally in vitro) in the subject one or more genetic factors that predict upregulation of the expression of IL-11 or the IL-11 receptor or upregulation of IL-11-mediated signal transduction.

[0362] Item 35. The method according to Item 34, which is a method for confirming the diagnosis of the disease in a subject suspected of suffering from a disease pathologically involving smooth muscle cells (SMCs).

[0363] Item 36. The method according to Item 34 or 35, further comprising selecting a subject for treatment with an agent capable of suppressing IL-11-mediated signal transduction.

[0364] Item 37. A method for providing a prognosis to a subject suffering from a disease pathologically involving smooth muscle cells (SMCs) or suspected of suffering from such a disease, the method comprising the step of measuring (optionally in vitro) in said subject one or more genetic factors that predict upregulation of the expression of IL-11 or the IL-11 receptor or upregulation of IL-11-mediated signaling.

[0365] Item 38. The method according to any one of Items 32 to 37, wherein the disease is a disease pathologically involving secretory smooth muscle cells.

[0366] The present invention encompasses the aspects and preferred feature combinations described herein, except when such combinations are clearly unacceptable or clearly to be avoided.

[0367] The section headings used herein are provided solely for the purpose of presenting the invention in an organized manner and should not be construed as limiting the subject matter described herein.

[0368] As an example, aspects and embodiments of the present invention will be described below with reference to the accompanying drawings. Further aspects and embodiments will be readily understood by those skilled in the art. All documents cited herein are incorporated by reference as part of this specification.

[0369] Throughout this specification, including the claims that follow, unless otherwise specified, the term "comprise", and variations thereof such as "comprises" and "comprising", are to be understood to mean the inclusion of the stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0370] As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Numerical ranges are herein expressed as ranges from about a particular value, and / or to about another particular value. When such a range is recited, another embodiment includes the particular value(s) that are not approximations, and / or the range from the particular value(s) that are not approximations to the other particular value. Similarly, when a particular value is described as about a value by use of the antecedent "about", it is understood that another embodiment can be constituted by the particular value that is not an approximation.

[0371] The methods described herein may be performed in vitro, ex vivo, or in vivo, and the products of the invention may be in vitro products, ex vivo products, or in vivo products. "In vitro" encompasses experiments using materials, biological substances, cells, and / or tissues under laboratory conditions or in culture. In contrast, "in vivo" encompasses experiments and manipulations using live multicellular organisms. "Ex vivo" refers to something that exists outside the body, such as outside a human or animal body, or is performed outside the body, and may be present in or performed on a tissue (such as an entire organ) or cells taken from an organism.

[0372] When a nucleic acid sequence is disclosed herein, its reverse complementary strand is also clearly contemplated.

[0373] Embodiments and experiments demonstrating the principles of the invention will now be described with reference to the accompanying drawings.

Brief Description of the Drawings

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Example

[0410] In the following examples, the inventors demonstrate that when smooth muscle cells (SMCs) are treated with TGFβ1, the expression of the IL-11 gene and IL-11 protein in SMCs is upregulated in response; when SMCs are stimulated with IL-11, IL-11 is produced by an autocrine loop; when SMCs are stimulated with TGFβ1 or IL-11, the expression of the contractile phenotype, which is the normal phenotype of SMCs, is decreased and the expression of the secretory markers, which are the pathological phenotypes, is upregulated; and when IL-11-mediated signaling is inhibited with an anti-IL-11 neutralizing antibody, the effect of TGFβ1 stimulation on the SMC phenotype / activity is inhibited.

[0411] The phenotype of SMCs can switch between a phenotype with physiological contractile / relaxation ability and a pathological proliferative / hyperplastic / extracellular matrix synthetic phenotype. 3 The latter pathological phenotype is involved in several diseases often associated with increased TGFβ1 signaling and activation of other pathways.

[0412] Although TGFβ1 and its receptor have been suggested to be potential therapeutic targets for SMC-related diseases, suppressing them causes severe side effects. 59,60The present inventors attempted to identify factors that are required for the action of TGFβ1 signaling in SMCs and that can be targeted downstream of TGFβ1. Using a systematic and integrated target discovery platform and primary human vascular smooth muscle cells (VSMCs) obtained from several individuals, a robust signature indicating the effect of TGFβ1 action in SMCs was identified.

[0413] Example 1: Patient cohort and preparation of vascular smooth muscle cells (VSMC) Patients aged 21 - 81 years who underwent coronary artery bypass grafting (CABG) at the National Heart Centre Singapore were recruited for the study. Patients with valvular heart disease and those who had previously received atrial treatment were excluded. Aortic button (AB) and left internal mammary artery (LIMA) tissues were harvested by punching out the aorta in a button shape, and explant culture was performed using these samples to grow primary vascular smooth muscle cells (VSMCs). Additionally, biopsy samples of the aortic button and / or left internal mammary artery were taken from 15 patients who underwent coronary artery bypass grafting (CABG) (AB: n = 6; LIMA: n = 11). Vascular smooth muscle cells (VSMCs) were prepared from these samples as follows.

[0414] During open - chest surgery, biopsy samples of the aortic button (AB) and left internal mammary artery (LIMA) were taken from coronary artery bypass grafting (CABG) patients. After removing the adventitial layer and carefully scraping off the endothelium with forceps, the media layer was cut into 1 - 2 mm 3The tissue pieces were minced into small pieces and placed in 6-cm dishes. The distance between adjacent tissue pieces was set at approximately 5 mm. Human vascular smooth muscle cells (VSMCs) were cultured in vitro at 37 °C in a humidified atmosphere of 95% air / 5% CO2 in M231 medium (M-231-500, Life Technologies) supplemented with smooth muscle growth supplement (SMGS; S-007-25, Life Technologies) and 1% antibiotic-antimycotic (15240062, Life Technologies). The cell culture medium was replaced with fresh medium every 2 - 3 days to remove cell debris and maintain physiological pH. When the confluence reached 80 - 90%, the cells were detached and passaged using trypsin (A6964, Sigma-Aldrich) with standard cell dissociation techniques. After 1 - 2 passages, magnetic separation was performed using LD columns (130-042-901, Miltenyi Biotec) with microbeads conjugated with CD90 for fibroblast removal (Thy-1, 130-096-253, Miltenyi Biotec) and microbeads conjugated with CD144 for endothelial cell removal (VE-cadherin, 130-097-857, Miltenyi Biotec) to remove fibroblasts and endothelial cells from the cell culture. The VSMCs remaining in the culture after negative selection were used for further passage. All experiments were performed using cells with a low passage number (≤P4). The cells were synchronized by culturing them in serum-starved state for 16 hours in M231 basal medium supplemented with 0.2% fetal bovine serum (10500064, Life Technologies), and then treated in serum-free M231 medium.

[0415] To evaluate the characteristics of TGFβ1-induced phenotypic transition of VSMCs, molecular phenotypic analysis and cellular phenotypic analysis were performed, integrating the results obtained from the large database of gene expression in human tissues (GTEx 61 ) and the large database of gene expression in various cell types (FANTOM 62 ).

[0416] Example 2: RNA-seq analysis RNA-seq analysis of various types of cells was performed as follows.

[0417] Total RNA was isolated using the Trizol Plus RNA mini kit (12183555, Life Technologies). RNA was quantified using the Qubit RNA high sensitivity assay kit (Life Technologies), and RNA degradation was evaluated based on the RNA integrity number (RIN value) determined using the LabChip GX RNA Assay Reagent Kit (PerkinElmer). The transcript amount was evaluated using the TruSeq Stranded mRNA Library Prep kit (Illumina) according to the manufacturer's standard instructions. Briefly, poly(A)+ RNA was purified from 0.8 - 1 μg of total RNA with an RIN value exceeding 7, fragmented, and cDNA was synthesized using the resulting RNA fragments. Subsequently, 3'-end adenylation, adapter ligation, and PCR amplification were performed. According to the manufacturer's instructions, the finally obtained library was quantified using the KAPA library quantification kit (KAPA Biosystems) in the StepOnePlus real-time PCR system (Applied Biosystems). The quality and average fragment size of the final library were measured using the LabChip GX DNA High Sensitivity Reagent Kit (PerkinElmer). The libraries were pooled and sequenced on a NextSeq 500 benchtop sequencer using 75bp paired-end sequencing chemistry.

[0418] Using bcl2fastq v2.16.0.10 from Illumina, raw sequence data (.bcl files) were demultiplexed based on unique index pairs and separated into separate FastQ read files. Trimmomatic v0.36 6We used it to trim adapter sequences and low-quality reads / base sequences and evaluated the read quality with FastQC v0.11.5. High-quality reads were mapped to the Ensembl human GRCh38 v86 reference genome or the mouse GRCm38 v86 reference genome by Spliced Transcripts Alignment to a Reference (STAR) v2.5.2b 7 . The options of the STAR alignment tool were selected based on the parameters used in the ENCODE project. featureCounts 8 was used to summarize the strand-specific raw count numbers of reads mapped to only one location (uniquely mapped reads) (paired-end) and quantify gene features at the gene level (featureCounts -t exon -g gene_id -s 2 -p). The differential expression (DE) analysis was performed with DESeq2 v1.14.1 using the raw read count numbers obtained by featureCounts. Minimal pre-filtering was performed to exclude genes with no reads or only one read from all samples, reducing the data size and improving the speed of the analysis process. To remove the batch effect by samples and improve the sensitivity to detect differences between states, the sample ID was included as a covariate in the design formula of DESeq2. In pairwise comparisons, the basal state was always used as the reference quantity. A shrunk estimated MA plot showing the log2 fold change relative to the mean value of the normalized count numbers was created, and each point was shown in red when the adjusted p-value was less than 0.1.

[0419] Primary human VSMCs were sequenced at a read depth of approximately 20M per sample. Most of the reads were mapped to only one location on the genome. Reads aligned to only one location were counted, and the expression levels of all annotated genes were evaluated (Figure 1).

[0420] Example 3: Verification of the purity of vascular smooth muscle cell (VSMC) cultures To confirm that the VSMC cultures were pure, principal component analysis (PCA) was performed by comparing RNA-seq data obtained from VSMC cultures (not stimulated with TGFβ1) with RNA-seq data obtained from primary cardiac fibroblasts (FIB) and human umbilical vein endothelial cells (EC) respectively.

[0421] Primary human fibroblasts were obtained by performing explantation using atrial biopsy samples taken from the right atrium of patients (n = 84) undergoing coronary artery bypass grafting (CABG). Human cardiac fibroblasts (FIB) were prepared as follows. The weight of the biopsy sample derived from the right atrium was measured and minced into small pieces of 1 - 2 mm 3 and placed in a 6 cm dish. Human cardiac fibroblasts (FIB) were grown and maintained at 37 °C in 5% CO2 in a humidified atmosphere in DMEM (Life Technologies) supplemented with 20% fetal bovine serum (FBS, HyClone) and 1% penicillin / streptomycin (Gibco). The medium was changed with fresh medium every 2 - 3 days. When the cells reached 80 - 90% confluence, the cells were passaged using a standard trypsinization method. All experiments were performed using cells with a low passage number (<P4), and the cells were cultured in serum-free DMEM medium for 16 hours before being treated.

[0422] Human umbilical vein endothelial cells (EC) (CC - 2519) were obtained from Lonza. Human umbilical vein endothelial cells (EC) were grown and maintained at 37 °C in 5% CO2 in a humidified atmosphere in a 10 cm dish containing EGM-2 Bullet Kit medium (Lonza, CC - 3162). The medium was changed with fresh medium every 2 - 3 days. When the cells reached 80 - 90% confluence, the cells were passaged using a standard trypsinization method. All experiments were performed using cells with a low passage number (<P4), and the cells were cultured in serum-free EBM-2 basal medium for 16 hours before being treated.

[0423] The results of the principal component analysis are shown in Figure 2. All of these cells were found to be classified into different groups, and it was confirmed that VSMC cultures derived from the aortic button (AB) and VSMC cultures derived from the left internal mammary artery (LIMA) did not correspond to umbilical vein endothelial cells (EC) or cardiac fibroblasts (FIB). Also, from this analysis, it was shown that VSMCs derived from the aortic button (AB) and VSMCs derived from the left internal mammary artery (LIMA) are different from each other.

[0424] Furthermore, when the RNA expression levels of marker genes for EC, FIB, and VSMC were analyzed, the results of the principal component analysis were reconfirmed. CD31 (endothelial cell marker gene) was highly expressed in EC, but no expression was seen in VSMC cultures or FIB cultures. From this result too, it was confirmed that EC was not present in the VSMC cultures. Also, VSMC had lower expression of THY-1 (fibroblast marker) compared to other types of cells, and had high expression levels of elastin (ELN) and fibulin (FBLN), which are vascular smooth muscle markers (Figures 3A - 3D).

[0425] Furthermore, the morphological differences between EC, fibroblasts, and VSMC confirmed by microscopic observation are shown in Figure 4. Combining the results shown in Figures 2, 3, and 4, it was demonstrated that the research results described below were obtained using pure cultures of primary human VSMCs.

[0426] Example 4: RNA-seq analysis of changes in RNA expression levels related to TGFβ1 signaling RNA-seq analysis was performed on VSMCs collected from the aortic button (AB) and the left internal mammary artery (LIMA) and passaged a small number of times (3 - 4 times) to evaluate genome-wide changes in RNA expression in response to TGFβ1 signaling. VSMCs were stimulated with TGFβ1 (5 ng / ml; 24 hours), and RNA-seq analysis was performed in the same manner as in Example 2 (Figure 5).

[0427] Next, the RNA transcription levels were compared between TGFβ1-stimulated VSMCs and unstimulated VSMCs to identify genes whose expression was upregulated by TGFβ1 stimulation. At each locus, reads aligned to only one location were counted, and differential expression was detected using the DESeq2 63 package.

[0428] The analysis results are shown in Figure 6. It was found that IL-11 was significantly upregulated in VSMCs derived from the aortic button (AB) and the left internal mammary artery (LIMA) in response to TGFβ1 stimulation (fold change = 4.39 and fold change = 3.16, respectively; adjusted P-value = 1.69 e-11 and adjusted P-value = 4.55 e-07 ), respectively). Since IL-11 was highly significantly upregulated in both VSMCs derived from the aortic button (AB) and the left internal mammary artery (LIMA), it was confirmed that IL-11 was upregulated at the RNA level by TGFβ1 in various types of VSMCs and multiple subjects.

[0429] Next, the inventors performed ELISA analysis in triplicate on cell culture supernatants obtained from unstimulated VSMCs and VSMCs stimulated with TGFβ1 (5 ng / ml, 24 hours) and confirmed that a robust expression signature indicating upregulation of IL-11 was also observed at the protein level. A 39-fold increase in IL-11 was detected in the cell culture supernatant of VSMCs stimulated with TGFβ1 (Figure 7). The increase in IL-11 secretion induced by TGFβ1 stimulation was greater than the increase in IL-11 at the RNA level (comparison of Figure 6C and Figure 6D with Figure 7). This suggests that TGFβ1 may affect IL-11 concentration through post-transcriptional regulation.

[0430] Example 5: Analysis of the targets of IL-11 To investigate whether IL-11 secreted from VMSCs in response to TGFβ1 stimulation acts on VMSCs themselves or only signals to other types of neighboring cells, PHANTOM 62 analyzed the expression of interleukin-11 receptor α (IL-11RA) in more than 500 cell lines listed in the catalog.

[0431] The expression levels of all genes and their replicate data for various primary cells were downloaded from the FANTOM5 62 web resource (119 cell types). Since the FANTOM5 data is the expression level of transcription start sites (TSSs) measured by CAGE sequencing, the gene expression level was determined by summing all counts attributed to a specific gene. Next, the obtained gene expression levels were normalized by the library size to obtain the TPM for each gene. To compare the expression profiles of the IL-11RA gene and the IL-6R gene, the TPMs of these two genes were extracted from various primary cell samples covering cell types from all cell lineages. In both cases, cell types with an expression level of the IL-11RA gene or the IL-6R gene higher than the noise level were highlighted and each cell was classified according to the FANTOM5 cell ontology.

[0432] The results are shown in Fig. 8. Each cell tended to express either the IL-11 receptor or the IL-6 receptor, and almost no cells were found to express both of these receptors simultaneously. Most of the expression of the IL-6 receptor was seen in immune cells, and the expression of the IL-11 receptor was detected in mesenchymal cells and smooth muscle cells (highlighted in Fig. 8).

[0433] Example 6: Production of IL-11 by VSMC in response to IL-11 stimulation Since some smooth muscle cell lines express the IL-11 receptor, it was suggested that IL-11 not only is secreted but also acts directly on VMSCs. From this, if IL-11 induces its own expression on VMSCs, it was suggested that an autocrine loop of IL-11 might exist. To test this hypothesis, hyper IL-1164 The IL-11:IL-11RA fusion protein called hyper IL-11 was prepared by recombinant DNA technology and protein expression technology. Using a fragment of IL-11RA (amino acid residues 1 to 317 constituting domains 1 to 3; UniProtKB: Q14626), IL-11 (amino acid residues 22 to 199 of UniProtKB: P20809), and a 20-amino acid-long linker (SEQ ID NO: 5), hyper IL-11 was constructed. The amino acid sequence of hyper IL-11 is shown in SEQ ID NO: 4.

[0434] Similar to the IL-6:IL-6R fusion protein reported in Lokau et al., Cell Reports (2016) 14, 1761-1773, hyper IL-11 is a potent stimulator of IL-11 signal transduction. The inventors confirmed that hyper IL-11 is not recognized in the ELISA used for the detection of secreted soluble IL-11 (Figure 9). Briefly, according to the manufacturer's protocol, various concentrations of IL-11 in an equal volume of cell culture medium were added to each well of an ELISA plate, and human IL-11 Quantikine ELISA kit (D1100, R&D Systems) was used to quantify IL-11.

[0435] Next, the inventors analyzed the secretion of IL-11 into the cell culture medium by VSMCs stimulated with hyper IL-11 using the same ELISA kit. Briefly, after culturing VSMCs for 24 hours in the presence of 0.2 ng / ml, 0.5 ng / ml, or 1 ng / ml of hyper IL-11, the human IL-11 Quantikine ELISA kit was used to analyze IL-11 in the cell culture supernatant. In this way, the inventors were able to confirm whether VSMCs produce IL-11 autocrinely through IL-11-mediated signal transduction (induced by hyper IL-11) in VSMCs.

[0436] The results are shown in Fig. 10. It was found that the secretion of IL-11 from VSMCs was dose-dependently induced by hyper IL-11.

[0437] Example 7: Effect of IL-11 stimulation on gene expression in VSMC Next, the present inventors analyzed the effect of IL-11 stimulation on RNA expression in VSMCs. VSMCs derived from human aortic buttons (AB) and VSMCs derived from the human left internal mammary artery (LIMA) were cultured for 24 hours in the presence of 5 ng / ml of recombinant human interleukin 11 (IL-11; PHC0115, Life Technologies), and RNA-seq analysis was performed in the same manner as in Example 2.

[0438] The results are shown in Fig. 11. It was found that IL-11 did not induce a strong transcriptional response in VSMCs. Furthermore, since stimulation with IL-11 did not strongly upregulate RNA expression or IL-11 RNA, it was suggested that the increase in the expression of IL-11 protein in response to treatment with IL-11 (Fig. 10) was achieved through post-transcriptional regulation.

[0439] Example 8: Effect of IL-11 treatment on the phenotype of VSMC Next, the present inventors further investigated the effect of IL-11 on the phenotype and activity of VSMCs by analyzing various phenotypic markers of SMCs using the Operetta platform.

[0440] VSMCs were seeded at 1×10 in a 96-well black CellCarrier plate (PerkinElmer) 4Cells were seeded at a density of cells per well and incubated in the medium for 24 hours. Next, the cells were cultured without stimulation or stimulated with TGFβ1 (5 ng / ml) or IL-11 (5 ng / ml) and cultured for 24 hours. Then, the cells were rinsed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (28908, Life Technologies) for 15 minutes. A PBS solution containing 0.1% Triton X-100 (Sigma Aldrich) was added and incubated for 10 minutes to permeabilize the cells, and the cells were rinsed with PBS and a wash buffer (a PBS solution containing 0.25% BSA and 0.1% Tween-20). Nonspecific sites were blocked using a wash buffer containing 0.25% BSA (blocking solution; 30 minutes). Anti-transgelin (SM22α) antibody (1:200; AB14106, Abcam), anti-type I collagen (Col1) antibody (1:500; AB292, Abcam), and anti-myocardin (MYOCD) antibody (1:200; AB203614, Abcam) were added to the cells and incubated overnight at 4°C. All of these primary antibodies were diluted with the blocking solution. After rinsing with the wash buffer, goat anti-mouse AF488 (AB150113, Abcam) or anti-rabbit AF488 (AB150077, Abcam) was added to the cells and incubated for 1 hour at room temperature (RT) in the dark. These secondary antibodies were diluted 1:1000 with the blocking solution. The cells were counterstained (for 1 hour) with a blocking solution containing rhodamine phalloidin (1:1000, R415, Life Technologies) and DAPI (1 μg / ml, D1306, Life Technologies). The plate was scanned using an Operetta high-content imaging system 1438 (PerkinElmer) with a 10x objective lens, and images were acquired. For each condition, at least two wells were used, and at least 7 fields of view were analyzed per well. Harmony software version 3.5.2 (PerkinElmer) was used to quantify SM22α-positive cells. Columbus 2.7.1 (PerkinElmer) was used to measure the fluorescence intensity of type I collagen and the fluorescence intensity of MYOCD per region.

[0441] Furthermore, the deposition of collagen was analyzed using a colorimetric assay. The total amount of collagen secreted into the cell culture supernatant was measured using a Sirius Red Collagen Detection Kit (9062, Chondrex) according to the manufacturer's instructions.

[0442] The experimental results are shown in Figures 12A - 12E. It was found that both TGFβ1 and IL-11 decreased the expression of phenotypic markers of contractile VSMCs (i.e., SM22α, myocardin) and increased the expression of type I collagen, a phenotypic marker of secretory VSMCs.

[0443] These results suggest that IL-11 is a driver of the pathological transition of the VSMC phenotype from contractile to secretory and is not a protective response to TGFβ1 stimulation.

[0444] Example 9: Effect of IL-11 treatment on the migration of VSMC In vitro scratch assays and Boyden chamber assays were performed to analyze the effect of IL-11 stimulation on VSMC migration.

[0445] In vitro scratch wound healing assays and Boyden chamber assays were performed in duplicate for each patient sample. The scratch wound healing assay was performed using confluent monolayers of VSMCs. After synchronizing the cells by culturing them in low-serum medium (M231 medium containing 0.2% FBS) for 24 hours, a linear scratch was created using a sterile pipette tip, and the cells were treated with IL-11 (5 ng / ml) or TGFβ1 (5 ng / ml) for 24 hours. The wound sites were photographed at 0 hours and 24 hours, and the migration ability was calculated using ImageJ software. Briefly, the migration ability of VSMCs was calculated using the formula "migration = (A0 - A1) / A0 × 100", where A0 is the area of the wound at 0 hours and A1 is the area not covered by VSMCs after 24 hours. Six to ten regions were randomly selected and analyzed for each treatment to obtain the average value.

[0446] The Boyden chamber assay was performed using a Cell Migration Assay kit (CBA-100, Cell Biolabs) according to the manufacturer's protocol. VSMCs (5×10 4 cells / well) were seeded inside the Transwell insert, and the lower well of the Boyden chamber was filled with cell culture medium or cell culture medium supplemented with TGFβ1 (5 ng / ml) or IL-11 (5 ng / ml). After 24 hours, the OD at 560 nm was measured by colorimetry to determine the migratory ability of VSMCs into the lower well.

[0447] The experimental results are shown in Figures 13 and 14. Treatment with IL-11 or TGFβ1 significantly increased the wound healing area (Figures 13A and 13B). Also, in terms of the migratory ability of VSMCs, a tendency for increased migration into the compartment containing TGFβ1 or IL-11 was observed (Figures 14A and 14B; P = 0.15).

[0448] To inhibit IL-11 signaling, cells were treated with an IL-11 neutralizing antibody (2 μg / ml, MAB218, R&D Systems) or mouse IgG2a (2 μg / ml, MAB003, R&D Systems) for 24 hours in the presence of TGFβ1.

[0449] Example 10: Analysis of the neutralizing effect of IL-11 on TGFβ1-mediated effects in VSMC The inventors then investigated whether IL-11 is required for TGFβ1-mediated effects on VSMC phenotype and activity.

[0450] In the same manner as in Example 8, VSMCs were seeded on a 96-well black CellCarrier plate and incubated in the medium for 24 hours. Next, the cells were cultured without stimulation in the presence of EdU (10 μM / ml), or in the presence or absence of TGFβ1 (5 ng / ml) or IL-11 (5 ng / ml) in the presence of EdU (10 μM / ml) and in the presence or absence of an IgG control antibody or an anti-IL-11 neutralizing antibody (2 μg / ml) for 24 hours. Thereafter, the cells were rinsed, fixed, and stained in the same manner as in Example 8, and analyzed. Using the Click-iT EdU labeling kit (C10350, Life Technologies), EdU incorporated into the cells was labeled with AlexaFluor (AF) 488. 100 μl per well of a Click-iT reaction cocktail consisting of 85 μl of Click-iT reaction buffer, 4 μl of copper sulfate, 0.25 μl of AF488 azide, and 10 μl of reaction buffer additive was used. After adding this reaction cocktail and incubating at room temperature for 30 minutes, the cells were washed once with 100 μl of Click-iT reaction rinse buffer. Further, the cells were rinsed with a wash buffer (PBS solution containing 0.25% BSA and 0.1% Tween-20). In the same manner as in Example 8, the plate was scanned to obtain an image. The Harmony software version 3.5.2 (PerkinElmer) was used to quantify EdU-positive cells.

[0451] The results are shown in FIGS. 15A to 15C. It was found that suppressing IL-11-mediated signal transduction using an anti-IL-11 neutralizing antibody suppressed the growth stimulation of VSMCs (FIG. 15A) and the production stimulation of type I collagen (FIGS. 15B and 15C) via TGFβ1.

[0452] Furthermore, in the same manner as in Example 9, an in vitro scratch wound healing assay was performed using cells treated with IL-11 (5 ng / ml) or TGFβ1 (5 ng / ml) for 24 hours in the presence of an anti-IL-11 neutralizing antibody (2 μg / ml, MAB218, R&D Systems) or mouse IgG2a (2 μg / ml, MAB003, R&D Systems). In the same manner as in Example 9, images of the wound sites were acquired and analyzed.

[0453] The results are shown in FIGS. 16A and 16B. It was found that suppression of IL-11-mediated signaling using an anti-IL-11 neutralizing antibody inhibited the increase in the wound healing area by VSMCs via TGFβ1.

[0454] Cell proliferation and collagen production induced by TGFβ1 (FIG. 13) were decreased by the use of an IL-11 neutralizing antibody. The migration of VSMCs in wound healing was also decreased by the use of an IL-11 neutralizing antibody (FIG. 14).

[0455] Example 11: Statistical analysis High-content imaging and statistical analysis of protein data were performed using GraphPad Prism 6 software. Fluorescence intensity (type I collagen, MYOCD) was normalized by the number of cells detected in the field of view and recorded for 7 fields per well. EdU-expressing cells and SM22α-expressing cells were quantified using the aforementioned software, and the percentage of EdU-positive VSMCs or SM22α-positive VSMCs was determined for each field of view. Outliers (ROUT 2%, Prism software) were excluded before analysis. When comparing several experimental groups to one condition (i.e., unstimulated cells), the P value was corrected by the Dunnett method. Also, when comparing several conditions in one experiment, multiple test correction was performed by the Holm-Sidak method. The criterion for statistical significance was set at P < 0.05. * indicates a P value < 0.05, ** indicates a P value < 0.01, *** indicates a P value < 0.001, and **** indicates a P value < 0.0001.

[0456] Example 12: Conclusion From the above data, it was suggested that IL-11 acts downstream of TGFβ1 signaling in VSMCs, induces the pathological conversion of VSMCs from the contractile type to the secretory type, and is required for TGFβ1-mediated actions in VSMCs.

[0457] Therefore, inhibition of IL-11-mediated signaling was identified as a therapeutic option for diseases and conditions involving the phenotypic transition of VSMCs from the contractile type to the secretory type and / or diseases and conditions involving the action of TGFβ1 signaling in VSMCs.

[0458] Example 13: IL-11 increases the amount of intestinal smooth muscle cell aggregates and collagen Ten-week-old Col1a1-GFP reporter male mice were subcutaneously injected daily for 20 days with a dose of 100 μg / kg of recombinant mouse IL-11 (rmIL11) or the same dose of PBS (PBS: n = 3, IL-11: n = 4). After sacrificing the mice, the colon was fixed according to a standard cryosection preparation protocol. The frozen blocks were sectioned at a thickness of 10 μm. Serial sections were fixed, blocked with 5% bovine serum albumin, and incubated overnight at 4°C with rabbit anti-αSMA antibody (diluted 1:200, Ab5694, Abcam) as the primary antibody. After washing the sections with PBS, they were incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 647) antibody (diluted 1:500, Ab150079, Abcam) and counterstained with DAPI nuclear staining. After mounting the sections, images were acquired under an Olympus BX51 microscope by fluorescence microscopy using ImagePro software.

[0459] The results are shown in Figure 17. It was found that IL-11 induced the hypertrophy of the muscularis mucosa, circular muscle layer, and longitudinal muscle layer of the mouse colon and increased the number of collagen-secreting smooth muscle cells in these layers.

[0460] Therefore, in various tissues, it was shown that IL-11-mediated signaling increases the number and activity of secretory smooth muscle cells.

[0461] Example 14: Overexpression of IL-11 contributes to the pathology of smooth muscle cells in the heart / aorta Mice with conditional expression of IL-11 in smooth muscle cells induced by tamoxifen were used to investigate the effect of increased expression of IL-11 on cardiac fibrosis.

[0462] Smooth muscle cell-specific Cre male mice (B6.FVB-Tg(Myh11-cre / ERT2)1Soff / J) were purchased from The Jackson Laboratory (01979; Bar Harbor, Maine), and mated with female mice (C57BL / 6N-Gt(ROSA)26Sor tm1(CAG-Il11)Cook / J)(031928) having the ROSA-IL11 gene available from The Jackson Laboratory to generate mice with conditional expression of murine IL-11 only in smooth muscle cells (SMRS). Induction with tamoxifen was started at 6 weeks of age, and tamoxifen at a dose of 1 mg / kg was intraperitoneally injected 3 times a week for the next 1 week as a washout period. Littermates expressing only smooth muscle-specific Cre (SMWT) were used as a control mouse line, and corn oil was administered as a solvent control for tamoxifen.

[0463] Figure 18A shows that the expression of IL-11 protein in the hearts of 8-week-old SMRS mice was increased compared to the SMWT control when IL-11 protein was detected by immunoblotting after induction with tamoxifen (n = 6-7 per group) for 2 weeks. Figure 18B shows that the heart weight / body weight (HW / BW) ratio in 8-week-old SMRS mice was increased compared to the SMWT control (n = 8 per group).

[0464] Heart sections prepared from SMRS mice and SMWT mice were stained with Masson's trichrome to evaluate collagen. An increase in the expression / secretion of extracellular matrix (ECM) components such as collagen indicates that the cells are secretory smooth muscle cells. Heart tissues were fixed in 10% neutral buffered formalin for 24 - 48 hours, dehydrated, and embedded in formalin. Sections (5 μm) were cut thinly and stained with Masson's trichrome. Furthermore, hydroxyproline was detected by colorimetric quantification using a Quickzyme total collagen quantification assay kit (Quickzyme Biosciences) to quantify the amount of collagen in ventricular tissues.

[0465] Figure 18C shows representative heart sections stained with Masson's trichrome (n = 3 per group). Perivascular fibrosis was observed in heart tissues obtained from SMRS mice compared to the SMWT control. Figure 18D shows that higher expression of collagen was observed in the ventricles of SMRS mice compared to the SMWT control from the evaluation results by quantification of hydroxyproline (HPA) (n = 5 - 6 per group). Statistical analysis was performed using an unpaired two-sided t-test. * indicates P < 0.01, and **** indicates P < 0.0001.

[0466] Therefore, overexpression of IL-11 in smooth muscle cells contributes to perivascular fibrosis of the heart.

[0467] Expression of extracellular matrix (ECM) genes and inflammatory genes The expression of genes for various ECM components and various inflammatory genes in heart tissues was quantified by RT-PCR. Heart tissue samples were collected from mice in which IL-11 was overexpressed in smooth muscle cells by tamoxifen-inducible Cre.

[0468] After treating the snap-frozen tissue with Trizol reagent (Invitrogen), total RNA was extracted by purification with the Purelink RNA mini kit (Invitrogen). The iScript cDNA synthesis kit was used according to the manufacturer's instructions, and cDNA was prepared using 1 μg of total RNA for each reaction. Quantitative RT-PCR gene expression analysis was performed on duplicate samples by the fast SYBR green method (Qiagen) using QuantStudio (Applied Biosystems). The expression data were normalized to the expression level of GAPDH mRNA, and the fold change was calculated using the 2 -ΔΔCt method. Specific primer-probes were obtained from Integrated DNA Technologies and are shown in Table 1.

[0469]

Table 2

[0470] The results are shown in Fig. 18E. Overexpression of IL-11 increases the expression of extracellular matrix components and inflammatory genes in cardiac smooth muscle cells. Each bar represents the mean gene expression level (normalized to the expression level of GAPDH), the bar on the left represents the SMWT control, and the bar on the right represents the SMRS overexpression group (n = 5 per group). As extracellular matrix genes, collagen (Col1a1, Col1a2, Col3a1), fibronectin (FN1), matrix metalloproteinase (MMP2), and tissue matrix metalloproteinase inhibitor (TIMP-1) were analyzed. As inflammatory genes, interleukin 6 (IL-6), tumor necrosis factor α (TNFα), C-C motif chemokine ligand 2 (CCL2), and C-C motif chemokine ligand 5 (CCL5) were analyzed. Statistical analysis was performed using an unpaired two-sided t-test. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0471] Heart size and function Mice overexpressing IL-11 under the induction of tamoxifen-inducible Cre were used to analyze the effects of IL-11 overexpression on heart size and function.

[0472] IL-11 expression was induced as described above. Using a Vevo 2100 (VisualSonics) equipped with an MS400 linear array ultrasound probe in the frequency range of 18 - 38 MHz, with the genotypes and treatment groups blinded, transthoracic echocardiography was performed on all mice by a single trained echocardiographer. Mice were anesthetized with 2% isoflurane and maintained under anesthesia with 0.6 - 1.0% isoflurane, and body temperature was maintained at 37 °C on a heating mat. The chest and neck hair were removed with depilatory cream, and an acoustic coupling gel was applied to the chest. According to a previously reported method (Gao S, et al. Curr. Protoc Mouse Biol 2011, 1, 71 - 83), at the level of the papillary muscle in the center of the ventricle, standard two-dimensional short-axis tomographic images in the M-mode were taken and saved for an average of 10 cardiac cycles, and the size and wall thickness of the left ventricle were analyzed offline. The left ventricular ejection fraction was calculated using a modified method of Quinone's method (Tortoledo FA, et al. Circulation 1983, 67, 579 - 584). The diameter of the left atrium (LA) was measured in the parasternal long-axis tomographic image, and the average value of three measurements was obtained. The left ventricular weight was estimated according to past literature (Fard CY, et al. J Am Soc Echocardiogr 2000;13: 582 - 7).

[0473] The results are shown in Figures 19 - 21. Figure 19A shows that the body weight of the SMRS mice measured before echocardiography was less than that of the SMWT control. From the left ventricular weight estimated by echocardiography, it was shown that the heart weight of the SMRS mice was less than that of the SMWT control, but the left ventricular weight ratio corrected for body weight was shown to be increased (Figures 19B and 19C). Figure 19D shows the diameter of the left atrium (LA) measured in the parasternal long-axis tomographic image, indicating that the size of the left atrium (LA) of the SMRS mice was increased compared to the SMWT control.

[0474] Figures 20A - 20C show the thickness of the anterior wall, the inner diameter of the left ventricle, and the thickness of the left ventricular posterior wall at the end of diastole corrected for body weight, respectively. All three of these measurements in SMRS mice were increased compared to the SMWT control.

[0475] Figures 21A - 21C show the thickness of the anterior wall, the inner diameter of the left ventricle, and the thickness of the left ventricular posterior wall at the end of systole corrected for body weight, respectively. All three of these measurements in SMRS mice were increased compared to the SMWT control.

[0476] Figure 21D shows that the ejection fraction in SMRS mice is maintained compared to the SMWT control.

[0477] In Figures 19 - 21, each point represents an individual mouse. Statistical analysis was performed using an unpaired two - tailed t - test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates p < 0.001, and **** indicates P < 0.0001.

[0478] Thus, as evaluated by echocardiography, it was found that overexpression of IL - 11 in smooth muscle cells by tamoxifen - inducible Cre leads to left ventricular (LV) hypertrophy and ventricular stiffening, while systolic function is maintained.

[0479] Aortic remodeling Mice overexpressing IL - 11 by tamoxifen - inducible Cre were used to analyze the effect of IL - 11 overexpression on aortic smooth muscle cells.

[0480] Examination was performed in 8 - week - old SMRS mice induced with tamoxifen for 2 weeks in the same manner as described above (n = 6 - 7 per group).

[0481] The Vevo 2100 (VisualSonics) equipped with an MS400 linear array ultrasonic probe with a frequency range of 18 - 38 MHz was used. The genotypes and treatment groups were blinded, and transthoracic echocardiography of all mice was performed by one trained echocardiographer. The mice were anesthetized with 2% isoflurane and maintained under anesthesia with 0.6 - 1.0% isoflurane, and the body temperature was maintained at 37 °C on a heating mat. The body hair on the chest and neck was removed with depilatory cream, and an acoustic coupling gel was applied to the chest. The sizes of the aortic root and ascending aorta were evaluated from parasternal long-axis tomograms in B-mode and M-mode using the inner interspace measurement method widely accepted in US and European guidelines (Lang RM, et al. Recommendations for chamber quantification. Eur J Echocardiogr 7, 79 - 108 (2006)). The peak value of the blood flow velocity in the aorta was determined by taking a suprasternal notch cross-sectional image from the aortic arch to the aortic valve using pulsed Doppler method. All measured values were averaged every three cardiac cycles.

[0482] The results are shown in Fig. 22. Each point represents an individual mouse. Statistical analysis was performed using an unpaired two-sided t-test. * indicates P < 0.05, and **** indicates P < 0.0001.

[0483] Fig. 22A shows that the expression of IL-11 protein in the proximal part of the thoracic aorta of 8-week-old SMRS mice is increased compared to the SMWT control (detected by immunoblotting). Figs. 22B and 22C show that the inner diameter of the aortic root of SMRS mice measured at the end of diastole and end of systole and corrected for body weight is larger than that of the SMWT control. Fig. 22D shows that the inner diameter of the ascending aorta of SMRS mice measured at the end of systole and corrected for body weight is larger than that of the SMWT control. Fig. 22E shows that the peak value of the blood flow velocity in the aorta of SMRS mice is maintained compared to the control.

[0484] Thus, when IL-11 is overexpressed in smooth muscle cells by tamoxifen-inducible Cre, although the blood flow velocity of the aorta is maintained, aortic remodeling occurs.

[0485] Example 15: Overexpression of IL-11 contributes to the pathology of smooth muscle cells in the lung Using a mouse model in which IL-11 is overexpressed in smooth muscle cells by tamoxifen-inducible Cre, the effect of increased expression of IL-11 on pulmonary fibrosis was investigated.

[0486] Examination was conducted in 8-week-old SMRS mice in which induction by tamoxifen was carried out for 2 weeks in the same manner as described above (n = 3 per group). In the same manner as in Example 14, the expression of collagen was measured by quantification of hydroxyproline (n = 6 per group). Furthermore, in the same manner as in Example 14, representative lung sections were stained with Masson's trichrome (n = 3 per group).

[0487] The results are shown in Fig. 23. Fig. 23A shows that the expression of IL-11 protein in the lungs of 8-week-old SMRS mice is increased compared to the SMWT control (detected by immunoblotting). Fig. 23B shows that the lung weight / body weight ratio of SMRS mice is increased compared to the SMWT control (n = 8 per group). Fig. 23C shows that the amount of collagen expression in the lungs of SMRS mice measured by quantification of hydroxyproline and corrected by the lung weight / body weight ratio is higher than that of the control. Fig. 23D shows a representative lung section stained with Masson's trichrome, and it is recognized that pulmonary fibrosis is increased and infiltrating cells are infiltrated in the lungs of SMRS mice compared to the SMWT control.

[0488] Thus, when IL-11 is overexpressed in smooth muscle cells by tamoxifen-inducible Cre, pulmonary fibrosis increases.

[0489] Expression of extracellular matrix (ECM) genes and inflammatory genes RT-PCR was carried out in the same manner as in Example 14.

[0490] Figure 24 shows that overexpression of IL-11 in smooth muscle cells by tamoxifen-inducible Cre increases the expression of extracellular matrix genes and inflammatory genes in the lung. Each bar represents the average gene expression level (normalized to the expression level of GAPDH). The left bar represents the SMWT group, and the right bar represents the SMRS group (n = 5 per group). As extracellular matrix genes, collagen (Col1a1, Col1a2, Col3a1), fibronectin (FN1), matrix metalloproteinase (MMP2), and tissue matrix metalloproteinase inhibitor (TIMP-1) were analyzed. As inflammatory genes, interleukin 6 (IL-6), tumor necrosis factor α (TNFα), C-C motif chemokine ligand 2 (CCL2), and C-C motif chemokine ligand 5 (CCL5) were analyzed. Statistical analysis was performed using an unpaired two-sided t-test. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0491] Example 16: Overexpression of IL-11 contributes to the pathology of smooth muscle cells in the liver A mouse model in which IL-11 is overexpressed in smooth muscle cells by tamoxifen-inducible Cre was used to investigate the effect of increased expression of IL-11 on liver fibrosis.

[0492] Similar to Example 14, induction with tamoxifen and evaluation by quantification of hydroxyproline were performed.

[0493] The results are shown in FIGS. 25A to 25C. FIG. 25A shows that when induction with tamoxifen was performed for 2 weeks, the expression of IL-11 protein in the liver of 8-week-old SMRS mice increased compared to the SMWT control (n = 6 to 7 per group; detected by immunoblotting). FIG. 25B shows that the liver weight / body weight ratio of SMRS mice did not change compared to the control (n = 8 per group). FIG. 25C shows that from the evaluation results by quantification of hydroxyproline, higher expression of collagen was observed in the liver of SMRS mice compared to the control (n = 5 to 6 per group). Statistical analysis was performed using an unpaired two-sided t-test. * indicates p < 0.05.

[0494] Thus, when IL-11 is overexpressed in smooth muscle cells by tamoxifen-inducible Cre, hepatic fibrosis increases.

[0495] Expression of extracellular matrix (ECM) genes and inflammatory genes RT-PCR was performed in the same manner as in Example 14.

[0496] Figure 26 shows that when IL-11 is overexpressed in smooth muscle cells by tamoxifen-inducible Cre, the expression of extracellular matrix proteins in the liver increases. Each bar represents the average gene expression level (normalized to the expression level of GAPDH), the bar on the left represents the SMWT group, and the bar on the right represents the SMRS group (n = 5 per group). As extracellular matrix genes, collagen (Col1a1, Col1a2, Col3a1), fibronectin (FN1), matrix metalloproteinase (MMP2), and tissue matrix metalloproteinase inhibitor (TIMP-1) were analyzed. As inflammatory genes, interleukin 6 (IL-6), tumor necrosis factor α (TNFα), C-C motif chemokine ligand 2 (CCL2), and C-C motif chemokine ligand 5 (CCL5) were analyzed. Statistical analysis was performed using an unpaired two-tailed t-test. * indicates P < 0.05, and *** indicates P < 0.001.

[0497] Example 17: Overexpression of IL-11 contributes to the pathology of smooth muscle cells in the kidney Using a mouse model in which IL-11 is overexpressed in smooth muscle cells by tamoxifen-inducible Cre, the effect of increased IL-11 expression on renal fibrosis was investigated.

[0498] In the same manner as in Example 14, induction by tamoxifen and evaluation by hydroxyproline quantification were performed.

[0499] The results are shown in FIGS. 27A to 27C. FIG. 27A shows that when induction with tamoxifen was performed for 2 weeks, the expression of IL-11 protein in the kidneys of 8-week-old SMRS mice increased compared to the SMWT control (n = 6 - 7 per group; detected by immunoblotting). FIG. 27B shows that the kidney weight / body weight ratio of SMRS mice increased compared to the SMWT control (n = 8 per group). FIG. 27C shows that from the evaluation results by hydroxyproline quantification, a tendency for higher expression of collagen in the kidneys of SMRS mice was observed compared to the control (P = 0.12, n = 5 per group). Statistical analysis was performed using an unpaired two-sided t-test. * indicates p < 0.05.

[0500] Thus, overexpression of IL-11 in smooth muscle cells by tamoxifen-inducible Cre increases renal fibrosis.

[0501] Expression of extracellular matrix (ECM) genes and inflammatory genes RT-PCR was performed in the same manner as in Example 14.

[0502] FIG. 28 shows that overexpression of IL-11 in smooth muscle cells by tamoxifen-inducible Cre increases the expression of extracellular matrix proteins in the kidneys. Each bar represents the average gene expression level (normalized by the expression level of GAPDH). The left bar represents the SMWT group, and the right bar represents the SMRS group (n = 5 per group). As extracellular matrix genes, collagen (Col1a1, Col1a2, Col3a1), fibronectin (FN1), matrix metalloproteinase (MMP2), and tissue matrix metalloproteinase inhibitor (TIMP-1) were analyzed. As inflammatory genes, interleukin 6 (IL-6), tumor necrosis factor α (TNFα), C-C motif chemokine ligand 2 (CCL2), and C-C motif chemokine ligand 5 (CCL5) were analyzed. Statistical analysis was performed using an unpaired two-sided t-test. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0503] Example 18: Overexpression of IL-11 contributes to the pathology of smooth muscle cells in inflammatory bowel disease Using a mouse model that overexpresses IL-11 in smooth muscle cells by tamoxifen-inducible Cre, the effect of increased expression of IL-11 on inflammatory bowel disease was investigated.

[0504] In the same manner as in Example 14, induction was performed with tamoxifen. The fecal calprotectin (S100A8 / A9) concentration was quantified by using Mouse S100A8 / S100A9 Heterodimer Duoset ELISA (DY8596-05) according to the manufacturer's instructions. Fecal calprotectin was extracted using a fecal extraction buffer (0.1 M Tris, 0.15 M NaCl, 1.0 M urea, 10 mM CaCl2, 0.1 M citric acid monohydrate, 5 g / L BSA).

[0505] Figure 29A shows the rectums of SMRS mice and SMWT control mice after administration of the solvent (corn oil) or tamoxifen (1 mg / kg / day, three times). The SMRS mice administered tamoxifen showed a swollen red rectum (arrow) compared to the other mouse groups, indicating that the intestine was in an inflammatory state.

[0506] Figure 29B shows representative photographs of fecal samples obtained from SMRS mice and SMWT mice after treatment with tamoxifen. The feces of SMRS mice were softer and lighter in color compared to those of the SMWT control.

[0507] Figure 29C shows that the concentration of calprotectin (S100A8 / A9), which reflects the activity of intestinal inflammatory cells, was increased in fecal samples of SMRS mice compared to the SMWT control (n = 8 per group).

[0508] Thus, when IL-11 is overexpressed in smooth muscle cells by tamoxifen-inducible Cre, an inflammatory phenotype is observed in the intestine of SMRS mice.

[0509] Example 19: Overexpression of IL-11 contributes to the pathology of smooth muscle cells in the gastrointestinal tract Using a mouse model that overexpresses IL-11 in smooth muscle cells by tamoxifen-inducible Cre, the effect of increased expression of IL-11 on the gastrointestinal tract was investigated.

[0510] In the same manner as in Example 14, induction with tamoxifen and Masson's trichrome staining were performed.

[0511] Figure 30A shows that the gastrointestinal tract collected from SMRS mice is reddish and swollen compared to the SMWT control. Figure 30B shows that when induction with tamoxifen was performed over a 2-week period, the expression of IL-11 in the colon of 8-week-old SMRS mice increased compared to the SMWT control (n = 3 per group; detected by immunoblotting). Figure 30C shows representative sections of the small intestine and colon collected from SMWT mice and SMRS mice that were stained with Masson's trichrome (n = 3 per group). The intestinal wall of SMRS mice was thicker in wall thickness and showed intestinal fibrosis compared to the control.

[0512] Thus, when IL-11 is overexpressed in smooth muscle cells by tamoxifen-inducible Cre, inflammation occurs in the gastrointestinal tract and intestinal fibrosis develops.

[0513] Expression of extracellular matrix (ECM) genes and inflammatory genes RT-PCR was performed in the same manner as in Example 14.

[0514] Figure 31 shows that overexpression of IL-11 in smooth muscle cells by tamoxifen-inducible Cre increases the expression of extracellular matrix proteins in the colon. Each bar represents the average gene expression level (normalized to the expression level of GAPDH). The left bar represents the SMWT group, and the right bar represents the SMRS group (n = 5 per group). As extracellular matrix genes, collagen (Col1a1, Col1a2, Col3a1), fibronectin (FN1), matrix metalloproteinase (MMP2), and tissue matrix metalloproteinase inhibitor (TIMP-1) were analyzed. As inflammatory genes, interleukin 6 (IL-6), tumor necrosis factor α (TNFα), C-C motif chemokine ligand 2 (CCL2), and C-C motif chemokine ligand 5 (CCL5) were analyzed. Statistical analysis was performed using an unpaired two-tailed t-test. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0515] Example 20: Expression of IL-11 in Marfan syndrome Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder associated with increased TGFβ signaling. The expression of IL-11 was investigated using MFS mice.

[0516] All mice used in this experiment had a C57BL / 6 genetic background, were housed and bred in the same room, and were allowed free access to food and water. Marfan syndrome (MFS) (B6.129-Fbn1 tm1Hcd / J) mice were purchased from The Jackson Laboratory (012885; Bar Harbor, ME). Heterozygous mice showing classical features of human disease (including aortic aneurysm and lung abnormalities) were used in the experiment.

[0517] Total proteins were extracted from the hearts, lungs, and thoracic aortas of mice and subjected to Western blot analysis. Each frozen tissue was homogenized gently with shaking in lysis buffer (RIPA buffer containing protease inhibitor and phosphatase inhibitor (Roche)), and then centrifuged to clarify the lysate. Equal amounts of each protein lysate were separated by SDS-PAGE, transferred to a PVDF membrane, and incubated overnight with anti-IL11 antibody (MAB218, R&D Systems) and anti-GAPDH antibody (2118, Cell Signaling). Anti-rabbit HRP (7074, Cell Signaling) or anti-mouse HRP (7076, Cell Signaling) was added as the appropriate secondary antibody, and proteins were visualized using an ECL detection system (Pierce).

[0518] Figure 32 shows that IL-11 is upregulated in the hearts, lungs, and aortas of Marfan syndrome (MFS) mice. Figure 32A shows, by evaluation in Western blot analysis, an increase in the expression of IL-11 in the heart, lung, and aorta tissues of MFS mice compared with wild-type (WT) controls. Figures 32B to 32D show the evaluation results of comparing the expression of IL-11 and the expression of GAPDH in the hearts, lungs, and aortas of MFS mice using a densitometer.

[0519] Example 21: Effect of IL-11 inhibition on aortic remodeling Transverse aortic constriction (TAC) was performed on mice, and the effect of suppressing IL-11-mediated signaling on TAC-induced aortic remodeling by smooth muscle cells was analyzed.

[0520] All the mice used in this experiment had a C57BL / 6 genetic background. They were all housed in the same room, fed and given free access to water. Thoracotomy was performed on the mice while keeping them alive to create ascending aortic stenosis. The final test was conducted 2 weeks after aortic constriction (TAC). Sham controls with the same age were given thoracotomy without TAC. Using transthoracic Doppler echocardiography, an increase in the pressure gradient (exceeding 40 mmHg) indicating the success of TAC was confirmed. To perform histological and molecular evaluations, the mice were euthanized 2 weeks after TAC. As postoperative drug therapy, anti-IL-11 antibody, anti-IL-11Rα antibody or IgG control antibody was intraperitoneally administered at a dose of 20 mg / kg twice a week for 2 consecutive weeks.

[0521] The results are shown in Figures 33A - 33D. It is observed that by suppressing IL-11-mediated signaling using anti-IL-11RA antibody, TAC-induced aortic remodeling is reduced despite the maintenance of pressure load in the mice.

[0522] Figures 33A and 33B show the inner diameters of the aortic base at the end of systole and the end of diastole. Figure 33C shows the peak value of blood flow velocity in the aortic arch, and Figure 33D shows the pressure gradient. Statistical analysis was performed by one-way analysis of variance and Sidak's multiple comparison as a post hoc test. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0523] Representative sections of the proximal part of the thoracic aorta were fixed in 10% neutral buffered formalin for 24 - 48 hours, dehydrated, and embedded in formalin. To evaluate collagen, sections (5 μm) were stained with Masson's trichrome staining in the same manner as in Example 14.

[0524] Figure 34 shows that TAC-induced aortic remodeling is alleviated by IL-11 neutralizing antibody and IL-11Rα neutralizing antibody (see arrows). Representative sections of the proximal part of the thoracic aorta were stained with Masson's trichrome staining (n = 5 per group). The scale bar indicates 100 μm.

[0525] Example 22: Effect of inhibition of IL-11-mediated signaling on the migration of VSMCs in the aorta Mouse VSMCs were isolated and cultured using a modified protocol adopted from published literature (Metz, Richard P., et al. Cardiovascular Development. Humana Press, Totowa, NJ, 2012. 169 - 176; Weber, Sven C., et al. Pediatric research 70.3 (2011): 236). Thoracic aortas were harvested from mice treated with recombinant mouse IL - 11 (5 ng / ml), mice treated with recombinant mouse TGFβ1 (5 ng / ml) and anti - IL - 11 antibody (2 μg / ml) or IgG isotype control at the same concentration, and mice treated with recombinant mouse TGFβ1 (5 ng / ml) without antibody treatment. The harvested aortic tissues were minced and digested at 37°C for 45 minutes with gentle shaking in M231 medium containing 1% antibiotic - antifungal agent mixture solution and 0.25 mg / mL Liberase TM (Roche), and then explant - cultured at 37°C in complete M231 medium supplemented with SMGS and 1% antibiotic - antifungal agent mixture solution. The mixed cells obtained from the digested aortic tissues were cultured, and when they reached 80 - 90% confluence at the first passage, magnetic beads against CD45 (leukocytes; 130 - 052 - 301, Miltenyi Biotec), magnetic beads against CD90.2 (fibroblasts; 130 - 049 - 101, Miltenyi Biotec), and magnetic beads against CD31 (endothelial cells; 130 - 097 - 418, Miltenyi Biotec) were used for negative selection to enrich VSMCs according to the manufacturer's instructions. Mouse aortic VSMCs with a small number of passages (3 - 5) were used in subsequent experiments. Using confluent monolayer mouse VSMCs, an in vitro scratch wound healing assay was performed over 24 hours to evaluate the migration of VSMCs.

[0526] Figures 35A and 35B show that neutralization of TGFβ1-mediated migration of mouse aortic-derived VSMCs is achieved by suppressing IL-11-mediated signaling with an antibody. Representative photographs (Figure 35A) and cumulative plots (Figure 35B) of VSMCs obtained from mice treated with recombinant mouse IL-11 (5 ng / ml), mice treated with recombinant mouse TGFβ1 (5 ng / ml) and anti-IL-11 antibody (2 μg / ml) or IgG isotype control at the same concentration, and mice treated with recombinant mouse TGFβ1 (5 ng / ml) without antibody treatment, when allowed to migrate over 24 hours. Wound sites were photographed at 0 hours (upper panel) and 24 hours (lower panel), and migration ability was calculated using the MRI wound healing tool in ImageJ software as described below. The scale bar indicates 200 μm. All data are shown as mean ± SD. Two-way ANOVA and Sidak's multiple comparisons were used to test for statistically significant differences. * indicates P < 0.05 and ** indicates P < 0.01.

[0527] Furthermore, in another study, the effects of various known VSMC migration stimulants on mouse aortic-derived VSMCs with IL-11Rα removed were evaluated.

[0528] Mouse VSMCs were isolated and cultured using a modified protocol adopted from published literature (Metz, Richard P., et al. Cardiovascular Development. Humana Press, Totowa, NJ, 2012. 169 - 176; Weber, Sven C., et al. Pediatric research 70.3 (2011): 236). Briefly, 4 - 6-week-old mice lacking the functional allele of IL11ra1 (Il11ra1− / −, KO) and their wild-type littermates (Il11ra1+ / +, WT) were euthanized, and the thoracic aorta was harvested for culturing VSMCs. The thoracic aorta collected from WT and KO mice was minced and incubated in a 1% antibiotic - antifungal mixture solution and 0.25 mg / mL Liberase TMDigested at 37°C for 45 minutes with gentle shaking in M231 medium containing (Roche), and the explants were cultured at 37°C in complete M231 medium supplemented with SMGS and 1% antibiotic - antifungal agent mixture solution. The mixed cells obtained from the digested aortic tissue were cultured, and when they reached 80 - 90% confluence at the first passage, according to the manufacturer's instructions, a MidiMACS separator was used for negative selection with magnetic beads against CD45 (leukocytes; 130 - 052 - 301, Miltenyi Biotec), magnetic beads against CD90.2 (fibroblasts; 130 - 049 - 101, Miltenyi Biotec), and magnetic beads against CD31 (endothelial cells; 130 - 097 - 418, Miltenyi Biotec) to enrich VSMCs. VSMCs derived from mouse aorta with a small number of passages (3 - 5) were used in subsequent experiments.

[0529] An in vitro scratch wound healing assay was performed using confluent monolayer mouse VSMCs to evaluate VSMC migration. After culturing in low - serum medium (M231 containing 0.2% FBS) for 24 hours to induce serum starvation, a linear scratch was made using a sterile pipette tip, and the cells were treated with M231 alone (unstimulated), angiotensin II (ANGII, 100 μM) (Sigma - Aldrich), mouse IL - 11 (5 ng / ml) (Genscript), or mouse TGFβ1 (5 ng / ml) (R&D Systems) for 48 hours. The wound sites were analyzed using ImageJ with the "MRI wound healing tool" plugin (available from http: / / dev.mri.cnrs.fr / projects / imagej - macros / wiki / Wound_Healing_Tool). Photographs of the wound sites were taken at 0 hours and 48 hours, and the migration ability was calculated using the formula "migration=(A0 - A1) / A0×100", where A0 is the area of the wound at 0 hours and A1 is the area not covered by VSMCs after 24 hours or 48 hours. Six to ten regions were randomly selected and analyzed for each treatment to obtain the average value. In the mouse stimulation tests using VSMCs derived from WT mice and KO mice, the treatment time was 48 hours.

[0530] The results are shown in FIGS. 36A and 36B. Representative photographs (FIG. 36A) and cumulative plots (FIG. 36B) showing wild-type (WT) mice and IL11ra1 knockout (KO) mice treated for 48 hours with no stimulant, angiotensin II (ANGII, 100 μM), recombinant mouse TGFβ1 (5 ng / ml), or recombinant mouse IL-11 (5 ng / ml). Wound sites were photographed at 0 hours (upper panel) and 48 hours (lower panel), and migration ability was calculated using the MRI wound healing tool in ImageJ software. Scale bars indicate 200 μm. Data are shown as mean ± SD for all. Statistical significance was tested by two-way ANOVA and Dunnett's multiple comparisons. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0531] Thus, by removing IL-11Rα in VSMCs of the mouse aorta, a protective effect against various known VSMC migration stimulants (including IL-11) is obtained.

[0532] The present invention includes the following inventions. [1] An agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction for use in a method of treating or preventing a disease in which smooth muscle cells (SMCs) are pathologically involved. [2] Use of an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction in the manufacture of a pharmaceutical for use in a method of treating or preventing a disease in which smooth muscle cells (SMCs) are pathologically involved. [3] A method of treating or preventing a disease in which smooth muscle cells (SMCs) are pathologically involved, the method comprising administering a therapeutically effective amount of an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction to a subject in need of treatment. ​[4] The agent, use or method according to [1], wherein the disease is a disease pathologically involving secretory smooth muscle cells. [5] The agent, use or method according to any one of [1] to [4], wherein the disease is a disease pathologically involving vascular smooth muscle cells (VSMCs). [6] The agent, use or method according to any one of [1] to [5], wherein the agent is an agent capable of binding to IL-11 or an IL-11 receptor. [7] The agent, use or method according to [6], wherein the agent is selected from the group consisting of an antibody or an antigen-binding fragment thereof, a polypeptide, a peptide, an oligonucleotide, an aptamer and a small molecule. [8] The agent, use or method according to [6] or [7], wherein the agent is an antibody or an antigen-binding fragment thereof. [9] The agent, use or method according to [6] or [7], wherein the agent is a decoy receptor for IL-11.

[10] The agent, use or method according to any one of [1] to [5], wherein the agent is an agent capable of decreasing the expression of IL-11 or an IL-11 receptor.

[11] The agent, use or method according to

[10] , wherein the agent is an oligonucleotide or a small molecule.

[12] The agent, use or method according to any one of [1] to

[11] , wherein the disease is a disease of the circulatory system, digestive system, excretory system, respiratory system, renal system or genital system.

[13] The disease is selected from the group consisting of atherosclerosis, hypertension, aneurysm, Marfan syndrome, aortic aneurysm, Furlong syndrome, Spritzen-Goldberg syndrome, Royce-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome, cerebral aneurysm, vascular stenosis and restenosis, atherosclerosis, fibromuscular dysplasia (FMD), supravalvular stenosis, renal artery stenosis, pulmonary arterial hypertension (PAH), plexiform lesion, fibromuscular dysplasia, telangiectasia, achalasia, dysphagia, diarrhea, constipation, inflammatory bowel disease (IBD), intestinal stenosis, pyloric stenosis, celiac disease, irritable bowel syndrome, diverticulitis, Crohn's disease, ulcerative colitis, kidney disease, focal segmental glomerulosclerosis (FSGS), IgA nephropathy, crescentic glomerulonephritis, lupus nephritis, diabetic nephropathy (DN), bladder disease, lung disease, asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), systemic sclerosis, Hutchinson-Gilford progeria syndrome (HGPS), leiomyoma, leiomyosarcoma, and Hermansky-Pudlak syndrome (HPS), the agent, use or method according to any one of [1] to

[12] .

[14] The method includes the step of administering the agent to a subject in which the expression of IL-11 or the IL-11 receptor is upregulated, the agent, use or method according to any one of [1] to

[13] .

[15] The method includes the step of administering the agent to a subject in which upregulation of the expression of IL-11 or the IL-11 receptor has been confirmed, the agent, use or method according to any one of [1] to

[14] .

[16] The method includes the step of determining whether the expression of IL-11 or the IL-11 receptor is upregulated in a subject, and the step of administering the agent to a subject in which the expression of IL-11 or the IL-11 receptor is upregulated, the agent, use or method according to any one of [1] to

[15] .

[17] Use of an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction for suppressing the activity of smooth muscle cells (SMC).

[18] A method for suppressing the activity of smooth muscle cells (SMCs), comprising the step of contacting smooth muscle cells (SMCs) with an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction.

[19] A method for suppressing the activity of smooth muscle cells (SMCs) in a subject, comprising the step of administering to the subject an agent capable of suppressing interleukin 11 (IL-11)-mediated signal transduction.

[20] The use or method according to any one of

[17] to

[19] , wherein the smooth muscle cells (SMCs) are secretory smooth muscle cells.

[21] The use or method according to any one of

[17] to

[20] , wherein the smooth muscle cells (SMCs) are vascular smooth muscle cells (VSMCs).

[22] The use or method according to any one of

[17] to

[21] , wherein the agent is an agent capable of binding to IL-11 or the IL-11 receptor.

[23] The use or method according to any one of

[17] to

[22] , wherein the agent is selected from the group consisting of an antibody or an antigen-binding fragment thereof, a polypeptide, a peptide, an oligonucleotide, an aptamer, and a small molecule.

[24] The use or method according to any one of

[17] to

[23] , wherein the agent is an antibody or an antigen-binding fragment thereof.

[25] The use or method according to any one of

[17] to

[23] , wherein the agent is a decoy receptor for IL-11.

[26] The use or method according to any one of

[17] to

[21] , wherein the agent is an agent capable of reducing the expression of IL-11 or the IL-11 receptor.

[27] The use or method according to

[26] , wherein the agent is an oligonucleotide or a small molecule.

[28] A method for determining whether a subject is suitable for the treatment or prevention of a disease pathologically involving smooth muscle cells (SMC) using an agent capable of suppressing the action of interleukin 11 (IL-11), the method comprising the step of determining (optionally in vitro) whether the expression of IL-11 or interleukin 11 receptor (IL-11R) is upregulated in the subject.

[29] A method for selecting a subject for the treatment or prevention of a disease pathologically involving smooth muscle cells (SMC) using an...

Claims

1. A pharmaceutical for inhibiting the activity of secretory smooth muscle cells (SMCs), comprising, as an active ingredient, an agent that inhibits interleukin 11 (IL-11)-mediated signal transduction, wherein the agent that inhibits IL-11-mediated signal transduction is an antibody or an antigen-binding fragment thereof that can bind to IL-11 and inhibit IL-11-mediated signal transduction, or an antibody or an antigen-binding fragment thereof that can bind to the IL-11 receptor (IL-11Rα) and inhibit IL-11-mediated signal transduction. A pharmaceutical characterized by this.

2. The pharmaceutical according to claim 1, wherein the secretory smooth muscle cells (SMCs) are vascular smooth muscle cells (VSMCs).

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