Treatment and prevention of metabolic diseases

Abstract

A method for treating and preventing metabolic diseases by inhibiting interleukin 11 (IL-11)-mediated signaling, as well as a preparation for use in this method, is disclosed.

Description

Technology Field

[0001] This application claims priority from GB 1906291.8 filed May 3, 2019, GB 1906597.8 filed May 10, 2019, GB 2001013.8 filed January 24, 2020, GB 2001896.6 filed February 12, 2020, and GB 2002030.1 filed February 14, 2020, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] Technology field

[0003] The present invention relates to the diagnosis, treatment, and prevention of metabolic diseases. Background Technology

[0004] Obesity, diabetes, and related conditions

[0005] Obesity is defined by the WHO as excessive fat accumulation that is harmful to health, and a BMI of ≥30 kg / m² 2Obesity is diagnosed in [location]. Obesity substantially increases the risk of metabolic diseases (including type 2 diabetes mellitus (T2D) and fatty liver disease), cardiovascular diseases (including hypertension, myocardial infarction and stroke), musculoskeletal diseases (e.g., osteoarthritis), Alzheimer's disease, depression and some types of cancer (breast, ovarian, prostate, liver, kidney and colon) - see, for example, the literature [Bluher, et al., Nat Rev Endocrinol. (2019) 15:288-298] and the literature [Prospective Studies Collaboration, Lancet. (2009) 373(9669):1083-96]. Furthermore, obesity causes reduced quality of life, unemployment, lower productivity, and social vulnerability (Reference [Berrington de Gonzalez et al., N Engl J Med (2010) 363:2211-2219]). Obesity is also associated with reduced life expectancy, with an estimated loss of 5 to 20 years depending on the severity of pathology and comorbid disorders (Reference [Fontaine et al., JAMA (2003) 289: 187-193]).

[0006] Over the past 50 years, the prevalence of obesity has increased to pandemic levels. The global prevalence of obesity also increased rapidly among children and adolescents between 1975 and 2016, rising from 0.7% to 5.6% for boys and from 0.9% to 7.8% for girls (Reference [NCD-RisC, Lancet (2017) 390(10113):26227-2642]). The 'Westernization' of lifestyle and high-calorie foods appeared to be the main causes of obesity.

[0007] Another major threat to global human health is the increasing incidence of type 2 diabetes, along with obesity. The literature [Menke et al., JAMA (2015) 314: 1021-1029] examined trends by BMI categories and found that diabetes increased only among obese subjects (18.0% to 20.1%), suggesting that most of the increase in diabetes prevalence is due to the rising prevalence of obesity. In particular, Asians are 30% to 50% more likely to develop diabetes despite having lower BMIs than Caucasians, and the high prevalence of diabetes is a concern (Lee et al., Diabetes Care (2011) 34, 353-357).

[0008] Metformin has demonstrated therapeutic potential and has been used as a first-line treatment for diabetes, along with adopting a healthy lifestyle that includes regular exercise to reap its anti-obesity benefits (Reference [Yerevanian et al., Curr Obes Rep (2019)]). Recently, interleukin-1 has also been shown to be a therapeutic target for diabetes to regulate low-grade inflammation in T2D (Reference [Kataria et al., Semin Immunopathol. (2010)]).

[0009] Non-alcoholic steatohepatitis (NASH)

[0010] The global prevalence of non-alcoholic fatty liver disease (NAFLD) is estimated at 25% (Friedman et al., Nat Med. (2018) 24(7): 908-922), and while NAFLD is reversible, it can progress to non-alcoholic steatohepatitis (NASH). NASH is characterized by steatosis-driven inflammation, hepatocyte death, and liver fibrosis that eventually lead to liver failure. Hepatic stellate cells (HSCs) are pivotal in the pathogenesis of NASH and generate up to 95% of liver myofibroblasts (Mederacke et al., Nat Commun (2013) 4:2823), which drive many of the major pathogenesiss of NASH, namely liver fibrosis, inflammation, and parenchymal dysfunction (Friedman, Physiol Rev (2008) 88:125-172; Friedman, J Biol Chem (2000) 275:2247-2250; Higashi et al., Adv Drug Deliv Rev (2017) 121:27-42).

[0011] Canonical pro-fibrotic factors, transforming growth factor-β1 (TGFβ1) and platelet-derived growth factor (PDGF; [Hellerbrand J Hepatol (1999) 30:77-87]; [Tsuchida and Friedman Nat Rev Gastroenterol Hepatol (2017) 14:397-411]) and also pro-inflammatory factors, such as CCL2, TNFα, and CCL5 ([Friedman, J Biol Chem (2000) 275:2247-2250]; [Tsuchida and Friedman Nat Rev Gastroenterol Hepatol (2017) 14:397-411]; [Kim et al., Sci Rep (2018) Many factors, including [8:7499]), are involved in HSC activation and transformation. Perhaps reflecting this complexity and inherent redundancy, no single upstream initiator has been successfully targeted in NASH, and no approved NASH drug exists. Currently, while many drugs for NASH are in clinical trials, many of them target metabolism, and it is unclear whether this will improve liver fibrosis, which predicts clinical outcomes (References [Friedman et al., Nat Med. (2018) 24(7): 908-922]; References [Banini et al., Curr Opin Gastroenterol (2017) 33:134-141]).

[0012] Quiescent HSCs are vitamin A storage cells and are very distinct from fibroblasts. However, a common factor activates both cell types and stimulates the transition to myofibroblasts, which possess shared traits (Mederacke et al., Nat Commun (2013) 4:2823; Iwaisako et al., Proc Natl Acad Sci USA 2014;111:E3297-305). IL-11 has recently been identified as a critical factor for the transformation of cardiovascular and pulmonary fibroblasts into myofibroblasts (Schafer et al., Nature 2017;552:110-115; Cook et al., (2018) https: / / doi.org / 10.1101 / 336537). Although there is very limited insight into IL-11 in the liver, recombinant human IL-11 has been reported to have a protective effect when injected into rodents at very high doses (references [Zhu et al., PLoS One (2015) 10:e0126296]; references [Yu et al., Clin Res Hepatol Gastroenterol (2016) 40:562-570]), and has been tested in humans in an attempt to reduce inflammation in advanced hepatitis (references [Lawitz et al., Am J Gastroenterol 2004;99:2359-2364]).

[0013] wasting disease

[0014] Wasting can be defined as the loss of muscle mass, either with or without the loss of fat mass, which may manifest as weight loss. Various acute and chronic diseases, as well as aging, are often associated with wasting. Wasting can lead to deterioration of nutritional status, loss of muscle mass and function, impaired quality of life, and an increased risk of morbidity and mortality. While muscle wasting is the most common denominator of wasting, adipose tissue wasting can also occur isolated or in combination with muscle wasting. Examples of wasting disorders include cachexia, sarcopenia (e.g., age-related or use-lack loss of skeletal muscle mass and strength), anorexia (lack or loss of appetite for food), myopenia (a term generally proposed to describe muscle wasting), lipodystrophy (specific wasting of fat accumulation), and lipoatrophy. The currently accepted definition of cachexia is: "a multifactorial syndrome characterized by an ongoing loss of skeletal muscle mass (with or without loss of fat mass) that cannot be fully reversed by conventional nutritional support and causes progressive functional impairment" (Reference [Evans et al. Clin Nutr. 2008 (6):793-9]).

[0015] Wasting is highly prevalent in patients with late-stage chronic diseases. According to the Sarcopenia, Cachexia and Wasting Disorders Society (SCWS), approximately 5% to 15% of patients with chronic heart failure or chronic obstructive pulmonary disease exhibit wasting disorders, while wasting disorders are experienced by 60% to 80% of patients with advanced cancer. Cachexia is directly responsible for 20% of cancer deaths (Skipworth et al., Clin Nutr. 2007; 26:667-76). Wasting has been noted in patients with infectious diseases, such as HIV / AIDS, malaria, and tuberculosis, as well as in chronic conditions, such as cystic fibrosis, liver cirrhosis, renal failure, Crohn's disease, rheumatoid arthritis, stroke, and neurological degenerative disease. Traumatic injury, post-surgery, weightlessness, chronic alcoholism, and sepsis are also associated with the onset of wasting (Farkas et al. J Cachexia Sarcopenia Muscle (2013) 4:173-178).

[0016] Wasting and wasting disorders often have an adverse effect on the treatment of underlying diseases: they impair the patient's response to treatment, compromise the immune system, and cause exacerbated symptoms of the underlying pathology. Therefore, treatments that alleviate wasting can increase the efficacy of treatment for the underlying disease / pathology and improve the patient's prognosis.

[0017] For example, advanced cancer-related cachexia often leads to a poor prognosis for anticancer treatment. Cancer patients who experience weight loss up to and during chemotherapy receive lower initial doses and experience more frequent and severe dose-limiting toxicities compared to weight-stable patients; consequently, they may receive significantly less treatment or actually be excluded from the treatment regimen at the start of treatment (Vaughan et al. J Cachexia Sarcopenia Muscle (2013) 4:95-109). Effective treatment for wasting will lead to more positive outcomes for these patients.

[0018] Currently, treatment for wasting includes appetite stimulants and exercise to build muscle mass. However, nutritional support or pharmacological manipulation of appetite alone often fails to reverse the wasting process, particularly in severe or terminal stages. Eicosapentaenoic acid (EPA; an omega-3 fatty acid from fish oil) has been tested for its effects in improving cachexia in the context of cancer, but the results have been contradictory (Jatoi et al., J Clin Oncol. 2004; 22:2469-76). Antioxidants and non-steroidal anti-inflammatory drugs have been tested, and combinations of these therapies, for example with EPA, oxidative stress inhibitors, and / or appetite stimulants, are thought to have potential in treating wasting. Inhibition of the ubiquitin proteolytic pathway (UPP) is of particular interest. Inhibition of the muscle inhibitor myostatin has never been successful in clinical trials.

[0019] The multifactorial nature of wasting combined with underlying disease means that there are no globally effective or accepted treatments for wasting, or even any approved drug therapies. In fact, it is generally accepted that the only way to treat disease-related wasting is to cure the underlying disease (Vaughan et al. J Cachexia Sarcopenia Muscle (2013) 4:95-109). Therefore, new therapies for wasting are needed.

[0020] The present invention provides a preparation capable of inhibiting interleukin 11 (IL-11)-mediated signaling for use in a method of treating or preventing metabolic diseases.

[0021] In addition, in the manufacture of a drug to be used in a method for treating or preventing metabolic diseases, the use of a drug capable of inhibiting interleukin 11 (IL-11)-mediated signaling is provided.

[0022] Additionally, a method for treating or preventing metabolic diseases is provided, and the method includes the step of administering to a subject a therapeutically or prophylactically effective amount of an agent capable of inhibiting interleukin 11 (IL-11)-mediated signaling.

[0023] In some embodiments, metabolic diseases include obesity, type 2 diabetes (T2D), type 1 diabetes (T1D), pre-diabetes, being overweight, metabolic syndrome, pregnancy-associated hyperglycemia, cholestatic liver disease, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, wasting, cachexia, chemotherapy-associated weight loss, pancreatic insufficiency, pancreatitis, acute pancreatitis, chronic pancreatitis, steatosis, lipotoxicity, Non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), lipodystrophy, lipohypertrophy, lipoatrophy, insulin resistance, or hyperglucagonemia, or includes these.

[0024] In some embodiments, the formulation is capable of preventing or reducing the binding of interleukin 11 (IL-11) to the receptor for interleukin 11 (IL-11R).

[0025] In some embodiments, the formulation may bind to interleukin 11 (IL-11) or a receptor for interleukin 11 (IL-11R).

[0026] In some embodiments, the formulation is selected from the group consisting of an antibody or its antigen-binding fragment, polypeptide, peptide, nucleic acid, oligonucleotide, aptamer, or small molecule.

[0027] In some embodiments, the formulation is an antibody or its antigen-binding fragment.

[0028] In some embodiments, the agent is a decoy receptor.

[0029] In some embodiments, the formulation is an anti-IL-11 antibody antagonist of IL-11-mediated signaling, or an antigen-binding fragment thereof. In some embodiments, the formulation is an anti-IL-11Rα antibody antagonist of IL-11-mediated signaling, or an antigen-binding fragment thereof.

[0030] In some embodiments, the formulation is an attractant receptor for IL-11. In some embodiments, the attractant receptor for IL-11 comprises: (i) an amino acid sequence corresponding to the cytokine binding module of gp130 and (ii) an amino acid sequence corresponding to the cytokine binding module of IL-11Rα.

[0031] In some embodiments, the formulation is IL-11 mutain. In some embodiments, the IL-11 mutain is W147A.

[0032] In some embodiments, the agent may prevent or reduce the expression of interleukin 11 (IL-11) or the receptor for interleukin 11 (IL-11R).

[0033] In some embodiments, the formulation is an oligonucleotide or a small molecule.

[0034] In some embodiments, the formulation is an antisense oligonucleotide capable of preventing or reducing the expression of IL-11. In some embodiments, the antisense oligonucleotide capable of preventing or reducing the expression of IL-11 comprises the sequence of SEQ ID NO: 12, 13, 14, or 15. IL11 It is siRNA targeted to. In some embodiments, the formulation is an antisense oligonucleotide capable of preventing or reducing the expression of IL-11Rα. In some embodiments, the antisense oligonucleotide capable of preventing or reducing the expression of IL-11Rα comprises the sequence of SEQ ID NO: 16, 17, 18, or 19. IL11RA It is siRNA targeted by...

[0035] In some embodiments, the interleukin 11 receptor is IL-11Rα or includes it.

[0036] In some embodiments, the method includes the step of administering a preparation to a subject whose expression of interleukin 11 (IL-11) or a receptor for IL-11 (IL-11R) is upregulated.

[0037] In some embodiments, the method may include the step of administering a preparation to a subject in whom the expression of interleukin 11 (IL-11) or a receptor for interleukin 11 (IL-11R) was determined to be upregulated.

[0038] In some embodiments, the method comprises the steps of determining whether the expression of interleukin 11 (IL-11) or a receptor for IL-11 (IL-11R) is upregulated in a subject, and administering a preparation to a subject in which the expression of interleukin 11 (IL-11) or a receptor for IL-11 (IL-11R) is upregulated.

[0039] explanation

[0040] Receptors for Interleukin 11 and IL-11

[0041] Interleukin 11 (IL-11), also known as an adipogenesis inhibitor, is a pleiotropic cytokine and is a member of the IL-6 family of cytokines, which includes IL-6, IL-11, IL-27, IL-31, oncostatin, leukemia inhibitor (LIF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), ciliary neurotrophic factor (CNTF), and neuropoetin-1 (NP-1).

[0042] Interleukin 11 (IL-11) is expressed in various mesenchymal cell types. The IL-11 genome sequence is mapped to the centromeric regions of chromosomes 19 and 7 and is transcribed along with canonical signaling peptides that ensure efficient secretion from cells. The IL-11 activator protein complex, cJun / AP-1, is located within its promoter sequence and is important for the basal transcriptional regulation of IL-11 (Du and Williams., Blood 1997, Vol 89: 3897-3908). The immature form of human IL-11 is a 199-amino acid polypeptide, whereas the mature form of IL-11 encodes a protein of 178 amino acid residues (Garbers and Scheller., Biol. Chem. 2013; 394(9):1145-1161). The human IL-11 amino acid sequence is available under UniProt accession number P20809 (P20809.1 GI:124294; SEQ ID NO:1). Recombinant human IL-11 (oprelvekin) is also commercially available. IL-11 from other species, including mice, rats, pigs, cattle, several species of bony fish, and primates, has also been cloned and sequenced.

[0043] In this specification, "IL-11" refers to IL-11 from any species and includes isoforms, fragments, variants, or homologues of IL-11 from any species. In a preferred embodiment, the species is human (Homo sapiens). The isoforms, fragments, variants, or homologues of IL-11 may optionally 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 respect to the amino acid sequence of immature or mature IL-11 from a given species, e.g., human. Isoforms, fragments, variants, or homologs of IL-11 may be characterized by the ability to selectively bind to IL-11Rα (preferably from the same species) and stimulate signal transduction in cells expressing IL-11Rα and gp130 (e.g., Curtis et al. Blood, 1997, 90(11)]; or as described in the literature [Karpovich et al. Mol. Hum. Reprod. 2003 9(2): 75-80]). The IL-11 fragment can be of any length (by the number of amino acids), but optionally can be at least 25% of the length of mature IL-11 and can have a maximum length of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of mature IL-11. The IL-11 fragment may have a minimum length of 10 amino acids, and a maximum length of one of 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 195 amino acids.

[0044] IL-11 signals through a homodimer of the ubiquitously expressed glycoprotein 130 (gp130; also known as glycoprotein 130, IL-6ST, IL-6-beta, or CD130). Gp130 is a transmembrane protein that forms one subunit of type I cytokine receptors together with the IL-6 receptor family. Specificity is achieved through the individual interleukin 11 receptor subunit alpha (IL-11Rα), which does not directly participate in signal transduction but triggers the formation of a final complex with gp130 through an initial cytokine binding event to the α-receptor.

[0045] Human gp130 (including a 22-amino acid signal peptide) is a 918-amino acid protein, and the mature form is 866 amino acids, comprising a 597-amino acid extracellular domain, a 22-amino acid transmembrane domain, and a 277-amino acid intracellular domain. The extracellular domain of the protein 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 under UniProt accession number P40189-1 (SEQ ID NO:2).

[0046] Human IL-11Rα is a 422-amino acid polypeptide (UniProt Q14626; SEQ ID NO:3) and shares approximately 85% nucleotide and amino acid sequence identity with murine IL-11Rα. Two isoforms of IL-11Rα have been reported, which differ in their cytoplasmic domains (Du and Williams cited above). IL-11 receptor α-chain (IL-11Rα) shares many structural and functional similarities with IL-6 receptor α-chain (IL-6Rα). The extracellular domain exhibits 24% amino acid identity, including a characteristic conserved Trp-Ser-X-Trp-Ser (WSXWS) motif. The short cytoplasmic domain (34 amino acids) lacks the box 1 and 2 regions required for the activation of the JAK / STAT signaling pathway.

[0047] Receptor binding sites on murine IL-11 were mapped, and three sites—Sites I, II, and III—were identified. Binding to gp130 is reduced by substitutions in the Site II region and by substitutions in the Site III region. Site III mutants do not exhibit detectable agonist activity but possess IL-11Rα antagonist activity (Cytokine Inhibitors Chapter 8; edited by Gennaro Ciliberto and Rocco Savino, Marcel Dekker, Inc. 2001).

[0048] In this specification, a receptor for IL-11 (IL-11R) 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 induce signal transduction in a cell expressing the receptor.

[0049] The IL-11 receptor may be from any species and includes isoforms, fragments, variants, or homologs of the IL-11 receptor from any species. In a preferred embodiment, the species is human (Homo sapiens).

[0050] In some embodiments, the IL-11 receptor may be IL-11Rα. In some embodiments, the receptor for IL-11 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 a complex comprising gp130, or gp130 to which IL-11 binds.

[0051] An isoform, fragment, variant, or homolog of IL-11Rα may optionally 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 from a given species, e.g., human. Isoforms, fragments, variants, or homologs of IL-11Rα may be characterized by the ability to selectively bind to IL-11 (preferably from the same species) and stimulate signal transduction in cells expressing IL-11Rα and gp130 (e.g., as described in the literature [Curtis et al. Blood, 1997, 90(11)] or [Karpovich et al. Mol. Hum. Reprod. 2003 9(2): 75-80]). The fragment of the IL-11 receptor can be of any length (by the number of amino acids), but optionally can be at least 25% of the length of mature IL-11Rα and can have a maximum length of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of mature IL-11Rα. The IL-11 receptor fragment may have a minimum length of 10 amino acids and a maximum length of one of 15, 20, 25, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, or 415 amino acids.

[0052] IL-11 signaling

[0053] IL-11 binds to IL-11Rα with low affinity (Kd ~10 nmol / L), and the interaction between these binding partners alone is insufficient to transmit biological signals. The development of high-affinity receptors capable of signal transduction (Kd ~400 to 800 pmol / L) requires the co-expression of IL-11Rα and gp130 (Curtis et al., Blood 1997; 90 (11):4403-12; Hilton et al., EMBO J 13:4765, 1994; Nandukar et al., Oncogene 12:585, 1996). Binding of IL-11 to cell-surface IL-11Rα induces heterodimerization, tyrosine phosphorylation, activation of gp130, and downstream signaling primarily through the mitogen-activated protein kinase (MAPK) cascade and Janus kinase / transcriptional signaler and activator (Jak / STAT) pathways (Garbers and Scheller cit.).

[0054] In principle, soluble IL-11Rα also forms a biologically active soluble complex with IL-11 (reference [Pflanz et al., 1999 FEBS Lett, 450, 117-122]), and, similar to IL-6, can increase the likelihood that IL-11 may bind to soluble IL-11Rα before binding to cell-surface gp130 in some cases (Garbers and Scheller cit.). Curtis et al. (reference [Blood 1997 Dec 1;90 (11):4403-12]) described the expression of soluble murine IL-11 receptor alpha chain (sIL-11R) and investigated signaling in cells expressing gp130. In the presence of gp130, sIL-11R, rather than transmembrane IL-11R, mediated early intracellular events, including IL-11-dependent differentiation of M1 leukemia cells, proliferation in Ba / F3 cells, and phosphorylation of gp130, STAT3, and SHP2, similar to signaling via transmembrane IL-11R. The activation of signaling via cell-membrane bound gp130 by IL-11 bound to soluble IL-11Rα has recently been demonstrated (Lokau et al., 2016 Cell Reports 14, 1761-1773). These so-called IL-11 trans Although signaling can be important for disease onset, its role in human diseases has not yet been studied.

[0055] As used herein, 'IL-11 trans"Signaling" is used to refer to signaling triggered by IL-11 bound to IL-11Rα binding to gp130. IL-11 may bind to IL-11Rα as a non-covalent complex. gp130 is membrane-bound and expressed by the cell, so signaling occurs after 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 liberated product of proteolytic cleavage of the extracellular domain of membrane-bound IL-11Rα. In some embodiments, IL-11Rα may be membrane-bound, and signaling via gp130 is "IL-11 cis The binding of IL-11 to cell-membrane-bound IL-11Rα, referred to as "signaling," can be triggered. In a preferred embodiment, inhibition of IL-11-mediated signaling is IL-11-mediated cis It is achieved by disrupting signaling.

[0056] IL-11-mediated signaling has been shown to stimulate hematopoiesis and thrombopoiesis, stimulate osteoclasts, stimulate neurogenesis, inhibit adipogenesis, reduce pro-inflammatory cytokine expression, regulate extracellular matrix (ECM) metabolism, and mediate the control of normal growth of gastrointestinal epithelial cells (Du and Williams cited above).

[0057] The physiological role of interleukin 11 (IL-11) remains unclear. IL-11 has been most strongly associated with the activation of hematopoietic cells and platelet production. IL-11 has also been shown to induce protection against graft-versus-host disease, inflammatory arthritis, and inflammatory bowel disease, and is considered to be an anti-inflammatory cytokine (Putoczki and Ernst, J Leukoc Biol 2010, 88(6):1109-1117). However, IL-11 is suggested to be not only pro-inflammatory but also anti-inflammatory, pro-angiogenic, and important for neoplasmia. Recent studies have shown that IL-11 is easily detectable during virus-induced inflammation in mouse arthritis models and cancer, suggesting that IL-11 expression may be induced by pathological stimuli. IL-11 is also associated with the Stat3-dependent activation of tumor-promoting target genes in neoplastic gastrointestinal epithelium (Putoczki and Ernst, cit.).

[0058] As used herein, "IL-11 signaling" and "IL-11-mediated signaling" refer to signaling mediated by IL-11, or a fragment thereof having the function of a mature IL-11 molecule, binding to a receptor for IL-11. "IL-11 signaling" and "IL-11-mediated signaling" will be understood to refer to signaling initiated by IL-11 / its functional fragment through binding to a receptor for IL-11, for example. In other words, "signaling" refers to signal transduction and other cellular processes governing cellular activity.

[0059] metabolic diseases

[0060] The present invention relates to the treatment and / or prevention of metabolic diseases.

[0061] As used herein, "metabolic disease" refers to any disease or pathological condition caused by or characterized by abnormal metabolism. In this context, "metabolism" refers to the conversion / processing within the body of components consumed to provide nutrition, for example, as a source of energy for energy and / or storage.

[0062] "Normal metabolism" may be the metabolism of a healthy subject who does not have a disease, for example, who does not have a metabolic disease or possesses symptoms / correlations of a metabolic disease.

[0063] Subjects with metabolic diseases may exhibit abnormal metabolism. Subjects with metabolic diseases may have symptoms or correlations of abnormal metabolism. Subjects with metabolic diseases may have been diagnosed with a metabolic disease. Subjects may meet the diagnostic criteria for the diagnosis of a metabolic disease.

[0064] In some embodiments, metabolic diseases are or include obesity, type 2 diabetes (T2D), type 1 diabetes (T1D), prediabetes, overweight, metabolic syndrome, pregnancy-related hyperglycemia (i.e., gestational diabetes), cholestatic liver disease, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, wasting, cachexia, chemotherapy-related weight loss, pancreatic insufficiency, pancreatitis, acute pancreatitis, chronic pancreatitis, steatosis, lipotoxicity, non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), lipodystrophy, lipohypertrophy, lipoatrophy, insulin resistance, and hyperglucagonemia (e.g., characterized therein).

[0065] An aspect of the present invention relates to the treatment and / or prevention of aberrant and / or insufficient function of cells / tissue(s) / organ(s) / organ systems having a role in metabolism. In particular, the treatment and / or prevention of aberrant and / or insufficient function of pancreatic cells / pancreatic tissue / pancreas is considered herein, as are the treatment and / or prevention of aberrant and / or insufficient function of liver cells / liver tissue / liver.

[0066] In some embodiments, the metabolic disease is or includes obesity. Obesity is characterized by excessive body fat. The diagnosis of obesity is reviewed, for example, in the literature [Orzano and Scott, J Am Board Fam Pract (2004) 17(5): 359-369], the full contents of which are incorporated herein by reference. Obese subjects have 30 kg / m² 2 Has an excess body mass index (BMI; calculated by dividing a person's weight by the square of their height). In some embodiments, the metabolic disease is or includes being overweight. Overweight is 25 kg / m² 2 Exceeding 30 kg / m² 2 It is characterized by having a BMI of less than (Data Table N°311", WHO (2015)). Obesity and overweight are generally caused by a combination of excessive food intake, lack of physical activity, and genetic susceptibility.

[0067] In some embodiments, the metabolic disease is or includes diabetes mellitus (often simply referred to as 'diabetes'). Diabetes mellitus refers to a group of metabolic disorders characterized by high blood sugar levels over a prolonged period (Diabetes Reference Table N°312". WHO, (2013)). According to the American Diabetes Association (ADA), a diagnosis of diabetes mellitus requires a hemoglobin A1c level of ≥6.5%, a fasting plasma glucose (FPG) level of ≥126 mg / dl (7.0 mmol / l) (defined as no caloric intake for at least 8 hours), the detection of plasma glucose 2 hours after the intake of a 75 g oral glucose load of ≥200 mg / dl (11.1 mmol / l), or the detection of a random plasma glucose level of ≥200 mg / dl (11.1 mmol / l) in patients with typical symptoms of hyperglycemia or a hyperglycemic crisis (American Diabetes Association, literature [Diabetes Care]). (2010) 33(Suppl 1): S62-S69]). Symptoms of diabetes include frequent urination, increased thirst, and increased hunger. The underlying cause of diabetes is usually insufficient insulin production by the pancreas, or cells in the body that do not respond properly to the insulin produced.

[0068] There are three major types of diabetes, which are listed, for example, in Diabetes Reference Table No. 312". WHO, (2013). Type 1 diabetes (T1D) results from the pancreas's failure to produce sufficient insulin due to an insufficient number of insulin-producing β cells from the pancreatic islets. T1D and its diagnosis have been reviewed, for example, by the literature [Kahanovitz et al., Point Care. (2017) 16(1): 37-40], the full contents of which are incorporated herein by reference. Type 2 diabetes (T2D) results from the subject's cells' failure to respond adequately to insulin and may also progress to include insufficient insulin production. T2D is most commonly caused by excessive body weight and insufficient exercise. T2D has been reviewed by the literature [DeFronzo, Nature Reviews Disease Primers (2015) 1:15019], the full contents of which The full text is incorporated herein by reference. Gestational diabetes (also referred to as pregnancy-related hyperglycemia) occurs when a pregnant woman develops high blood glucose levels. Gestational diabetes is caused by the insufficient production of excess insulin required during pregnancy in the context of pregnancy-related insulin resistance. Gestational diabetes is reviewed, for example, in the literature [Kampmann et al., World J Diabetes. (2015) 6(8):1065-1072], the full text of which is incorporated herein by reference.

[0069] In some embodiments, the metabolic disease is or includes insulin deficiency. In some embodiments, the metabolic disease is or includes insulin resistance. In some embodiments, the metabolic disease is or includes hyperglycemia. In some embodiments, the metabolic disease is or includes type 2 diabetes mellitus (T2D). In some embodiments, the metabolic disease is or includes type 1 diabetes mellitus (T1D). In some embodiments, the metabolic disease is or includes pregnancy-related hyperglycemia.

[0070] In some embodiments, the metabolic disease is or includes prediabetes. Prediabetes refers to a state of hyperglycemia in which blood glucose levels rise for an extended period but to a level less than that required for the diagnosis of diabetes. Prediabetes and the diagnosis thereof are reviewed, for example, in the literature [Bansal World J Diabetes. (2015) 6(2):296-303], the full contents of which are incorporated herein by reference. The WHO defines prediabetes as an intermediate hyperglycemic state diagnosed by determining FPG levels of 6.1 to 6.9 mmol / L (110 to 125 mg / dL) and 2 h plasma glucose levels of 7.8 to 11.0 mmol / L (140 to 200 mg / dL) after the intake of a 75 g oral glucose load. Diagnosis of prediabetes according to the ADA requires a 2 h plasma glucose level of 7.8 to 11.0 mmol / L (140 to 200 mg / dL) after intake of 75 g of oral glucose, an FPG level of 100 to 125 mg / dL, and a hemoglobin A1c level of 5.7% to 6.4%.

[0071] In some embodiments, the metabolic disease is or includes metabolic syndrome. Metabolic syndrome has been reviewed, for example, by the literature [Rochlani et al., Cardiovascular Disease (2017) 215-225], the full contents of which are incorporated herein by reference. The WHO defines metabolic syndrome as the presence of insulin resistance (impaired fasting glucose, impaired glucose tolerance, or T2D) in addition to two of obesity, hyperlipidemia (hypertriglyceridemia or low high-density lipoprotein (HDL) cholesterol), hypertension, or microalbuminuria. Several other definitions of metabolic syndrome exist, which are summarized in Table 1 of Rochlani et al.

[0072] In some embodiments, the metabolic disease is or includes cholestasis. Cholestasis refers to reduced flow of bile from the liver to the duodenum. In some embodiments, the metabolic disease is or includes cholestatic liver disease. Cholestatic liver disease results from insufficient bile synthesis, secretion, and / or flow through the bile ducts, as described, for example, in the literature [Jansen et al., Hepatology (2017) 65(2):722-738] and the literature [Pollock and Minuk, J Gastroenterol Hepatol (2017) 32(7):1303-1309], the full contents of both of which are incorporated herein by reference. Cholestatic liver disease includes primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).

[0073] In some embodiments, the metabolic disease is or includes hyperlipidemia. Hyperlipidemia refers to elevated levels of lipids or lipoproteins in the blood. Hyperlipidemia includes hypertriglyceridemia, hypercholesterolemia, and combined hyperlipidemia (a combination of hypertriglyceridemia and hypercholesterolemia). Hyperlipidemia is associated with, for example, atherosclerosis and cardiovascular disease.

[0074] In some embodiments, the metabolic disease is or includes hypertriglyceridemia. Hypertriglyceridemia is described, for example, in the literature [Berglund et al., J. Clin. Endocrinol. Metab. (2012) 97(9):2969-89] and is defined by blood triglyceride levels of ≥150 mg / dL (≥1.7 mmol / L).

[0075] In some embodiments, the metabolic disease is or includes hypercholesterolemia. Hypercholesterolemia is described, for example, in the literature [Bhatnagar et al., BMJ (2008) 337:a993]. The UK NHS defines it as a blood total cholesterol level ≥5 mmol / L or a blood low-density lipoprotein (LDL) level ≥3 mmol / L. The US NIH defines hypercholesterolemia as a blood total cholesterol level ≥240 mg / dL.

[0076] In some embodiments, the metabolic disease is or includes pancreatic insufficiency. Pancreatic insufficiency may be endocrine or exocrine. Endocrine pancreatic insufficiency may be characterized by insufficient production of one or more of insulin, amylin, glucagon, somatostatin, ghrelin, and pancreatic polypeptide (PP). Exocrine pancreatic insufficiency may be characterized by insufficient production of one or more of pancreatic juice, digestive enzymes, trypsinogen, chymotrypsinogen, elastase, carboxypeptidase, pancreatic lipase, nuclease, and amylase, and consequently, an inability to properly digest food. Pancreatic insufficiency is generally caused by the loss of pancreatic cells that produce the relevant factors, e.g., islet cells in the case of endocrine function, and acinar cells in the case of exocrine function. Exocrine pancreatic insufficiency is described, for example, in the literature [Struyvenberg et al., BMC Med (2017) 15:29], the full contents of which are incorporated herein by reference. The most common cause of pancreatic insufficiency is pancreatitis, but it can also be caused by cystic fibrosis, surgery, celiac disease, and diabetes mellitus.

[0077] In some embodiments, the metabolic disease is or includes pancreatic injury. As used herein, "injury" refers to injury to the relevant organ and / or tissues or cells of the organ. Injury to cells / tissues / organs may result from an attack on cells / tissues / organs, e.g., chemical or physical treatment / experience. In some embodiments, the injury may result from a chemical attack, e.g., in the case of drug-induced injury. In some embodiments, the injury may result from a physical attack, e.g., injury as a result of a surgical injury that may occur during surgery to treat a disease and / or for transplantation (e.g., the injury may have an iatrogenic cause). In some embodiments, the injury may result from hypoxia, e.g., as a result of ischemia, or due to reperfusion. In some embodiments, the injury may result from an infection, an immune response to an infection, cancer, and / or autoimmunity. The injury may be reversible or irreversible. Injury to a cell / tissue / organ may be characterized by changes in the structure and / or function of the cell / tissue / organ. For example, injury to a cell / tissue / organ may be characterized by a decrease in the correlation level of normal function of the cell / tissue / organ and / or an increase in the correlation of weakened function of the cell / tissue / organ. Injury to a cell / tissue / organ may be characterized by apoptosis, for example, the death of cells in the injured organ / tissue. The apoptosis may be due to apoptosis (i.e., programmed cell death) or necrosis (premature cell death as a result of injury).

[0078] In some embodiments, the metabolic disease is or includes pancreatitis. Pancreatitis is characterized by inflammation of the pancreas. Pancreatitis may be acute or chronic. Acute pancreatitis is reviewed, for example, in the literature [Shah et al., J Inflamm Res (2018) 11:77-85], the full contents of which are incorporated herein by reference. Acute pancreatitis is most commonly caused by gallstones, but may also be caused by alcohol and metabolic diseases, among other things. Chronic pancreatitis is reviewed, for example, in the literature [Pham et al. Version 1. F1000Res (2018) 7: F1000 Faculty Rev-607], the full contents of which are incorporated herein by reference. Chronic pancreatitis is a syndrome involving the loss of acinar cells and pancreatic islet cells, which may occur in chronic inflammation, fibrosis, and exocrine and endocrine insufficiency.

[0079] In some embodiments, the metabolic disease is or includes steatosis. Steatosis refers to the abnormal retention of lipids within a cell / tissue / organ. Steatosis may be macrovesicular or microvesicular.

[0080] In some embodiments, the metabolic disease is characterized by the accumulation of molecules (or derivatives thereof) containing lipid moiety in non-fat tissue. In some embodiments, the metabolic disease is lipotoxic, includes, is characterized by, or is associated with lipotoxicity.

[0081] As used herein, 'lipotoxicity' refers to damage, dysfunction, and / or death of cells / tissues resulting from the accumulation of molecules (or derivatives thereof) containing lipid moiety in non-fatty tissues. In some embodiments, according to the present disclosure, lipotoxicity is lipotoxicity of liver cells, kidneys, hearts, and / or skeletal muscles. In some embodiments, lipotoxicity is lipotoxicity of liver cells (e.g., hepatocytes).

[0082] Metabolic diseases characterized by and associated with lipotoxicity include, for example, non-alcoholic fatty liver disease (NAFLD) and NASH. The accumulation of lipids in hepatocytes and their association with NAFLD, particularly NASH, are described in the literature [Friedman et al., Nat. Med. (2018) 24(7):908-922] and [Farrell et al., Adv. Exp. Med. Biol. (2018) 1061: 19-44] (the full contents of both are incorporated herein by reference).

[0083] NAFLD, such as NASH, is thought to occur as a result of lipotoxicity to hepatocytes. Lipotoxic factors are thought to include free (non-esterified) cholesterol, saturated free fatty acids (e.g., palmitic acid), diacylglycerol, lysophosphatidyl-choline, sphingolipids, and ceramides. Hepatocytes are unable to sequester these chemically reactive lipid molecules, leading to mitochondrial damage, endoplasmic reticulum (ER) stress, and autophagy. Lipotoxicity causes hepatocyte apoptosis, as well as necrosis, necroptosis, and pyroptosis, which activate the innate immune system and trigger the expression of pro-inflammatory factors. Pro-inflammatory cytokines and chemokines recruit inflammatory cells, such as macrophages and neutrophils.

[0084] In the present embodiment (particularly Example 5), the inventors demonstrate that autocrine IL-11-mediated signaling is an important component of lipid-toxic signaling (e.g., in hepatocytes) and that lipid toxicity (and its downstream consequences) can be inhibited by antagonizing IL-11-mediated signaling. Accordingly, aspects of the present disclosure provide for the treatment or prevention of lipotoxicity or diseases characterized by lipotoxicity or related diseases through the antagonization of IL-11-mediated signaling.

[0085] Importantly, in Example 5.3.5 of the present invention, the inventors [discuss] IL-11-mediated signaling is associated with NAFL and NASH of HSCs into myofibroblasts. Upstream and separately It demonstrates that IL-11-mediated activation is a significant component of lipotoxicity in hepatocytes. Accordingly, aspects of the present disclosure provide the treatment / prevention of NAFLD (e.g., NASH), including the inhibition of lipotoxicity in hepatocytes through the antagonization of IL-11-mediated signaling.

[0086] In some embodiments, the metabolic disease is or includes non-alcoholic fatty liver disease (NAFLD). NAFLD is described, for example, in the literature [Benedict and Zhang, World J Hepatol. (2017) 9(16): 715-732] and in the literature [Albhaisi et al., Version 1. F1000Res. (2018) 7: F1000 Faculty Rev-720], the full contents of both of which are incorporated herein by reference. NAFLD is characterized by steatosis of the liver, particularly of hepatocytes. NAFLD includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NAFL is characterized by steatosis of the liver involving more than 5% of the tissue parenchyma without evidence of hepatocyte damage. NAFL can progress to NASH, which is steatosis combined with inflammation and / or fibrosis (steatohepatitis).

[0087] In some embodiments, the metabolic disease is or includes lipodystrophy. Lipodystrophy is reviewed, for example, in the literature [Fiorenza et al., Nature Reviews Endocrinology (2011) 7: 137-150], the full contents of which are incorporated herein by reference. Lipodystrophy refers to the inability to produce and / or maintain healthy adipose tissue and encompasses the complete or partial loss of adipose tissue (lipoatrophy), which may occur together with the pathological accumulation of adipose tissue (lipohypertrophy). Lipodystrophy may be hereditary or acquired, but hereditary lipodystrophy syndromes are rare. In some embodiments, the metabolic disease is or includes lipoatrophy. In some embodiments, the metabolic disease is or includes lipohypertrophy.

[0088] In some embodiments, the metabolic disease is or includes hyperglucagonemia. Hyperglucagonemia is described, for example, in the literature [Wewer Albrechtsen et al., Biomark Med. (2016) (11):1141-1151], the full contents of which are incorporated herein by reference.

[0089] In some embodiments, metabolic disease is wasting or includes it. As used herein, the term “wasting” refers to involuntary weight loss, which may be progressive and / or generative. Wasting may be defined as the loss of muscle with or without the loss of fat mass, typically accompanied by a significant and usually involuntary loss of body mass (including skeletal muscle), and may or may not include the loss of adipose tissue. In some cases, adipose tissue wasting may occur in isolation, as seen in lipodystrophic disease. Wasting may be characterized by a negative protein and energy balance driven by a variable combination of reduced food intake and abnormal metabolism (Fearon et al. Lancet Oncol. (2011) 12(5):489-95). Wasting may cause progressive functional impairment, reduced quality of life, and increased risk of morbidity and mortality. In some cases, wasting causes asthenia (abnormal physical weakness or lack of energy) and / or anemia (deficiency of red blood cells or hemoglobin in the blood). In some cases, wasting cannot be completely reversed by conventional nutritional support or by the therapeutic interventions tested to date. Death typically occurs once weight loss reaches 30% of the patient's historic stable body weight (Reference [Tisdale, Nature Reviews Cancer, 2, 862-871 (2002)]).

[0090] Diseases / conditions characterized by wasting include cachexia (non-age-related loss of muscle mass), sarcopenia (loss of muscle mass: e.g., age-related, disuse, spatial displacement, or denervation), anorexic disorder (protein-energy malnutrition), muscular atrophy, lipodystrophy (e.g., abnormal or degenerative condition of adipose tissue), lipoatrophy (age-related loss of subcutaneous fat in the face and other tissues), and sarcopenia (muscle wasting in any chronic disease; as suggested by the literature [Fearon et al. J Cachexia Sarcopenia Muscle. 2011; 2:1-3]). In this document, diseases / conditions characterized by wasting are also referred to as "wasting disorders." In some embodiments, the wasting disorder according to the present disclosure is cachexia, pre-cachexia, refractory cachexia, sarcopenia, anorexia, lipodystrophy, lipoatrophy, and / or sarcopenia. In some embodiments, according to various aspects described herein, the wasting disorder is cachexia, pre-cachexia, and / or refractory cachexia.

[0091] Wasting disorders resulting from chronic disease may include "mild muscular wasting disease" (with or without frailty), "moderate muscular wasting disease" (with or without frailty; sometimes also known as "total cachexia"), or "severe muscular wasting disease" (sometimes called "cachexia," often with frailty present).

[0092] Cachexia is a complex inflammatory / metabolic syndrome characterized by wasting and associated with an underlying disease (which may be acute or chronic). The primary clinical features of cachexia are weight loss in adults (corrected for fluid retention) or growth failure in children (excluding endocrine disorders). Anorexia, inflammation, insulin resistance, increased muscle protein breakdown, and increased basal metabolic rate are frequently associated with cachexia. Low lipid levels and fatty liver in cachexic patients suggest a role for hepatic metabolism in the development of cachexia. Therefore, therapies that target the liver and prevent fatty liver, liver injury, or hepatic metabolism may have a direct link to cachexia. Cachexia is distinct from starvation, age-related loss of muscle mass, primary depression, malabsorption, and hyperthyroidism, and is associated with an increased morbidity (Evans et al. Clin Nutr. 2008 (6):793-9]).

[0093] Cachexia is <20 kg / m² with involuntary weight loss of >5% from historical stable weight and >2% random weight loss. 2 (Persons under 65 years of age) or <22 kg / m² 2 It can be defined as a body mass index (BMI) (in individuals aged 65 years or older) or a skeletal muscle index consistent with sarcopenia accompanied by any degree of weight loss of >2%. Subjects may also exhibit <10% body fat and / or low blood albumin levels of <35 g / l. These criteria can also help identify groups 'at risk' of developing wasting disorders (Fearon et al. Lancet Oncol. 2011; 12(5):489-95).

[0094] A three-stage classification of cachexia has been proposed, and severity is classified according to the degree of depletion of energy storage and body protein (BMI) combined with the degree of progressive weight loss.

[0095] 1. Total cachexia - when the patient has <5% body weight loss but has not yet developed severe complications.

[0096] 2. Cachexia - When the syndrome is progressing and weight loss exceeds the aforementioned parameters but is still potentially treatable.

[0097] 3. Refractory cachexia - This is when the disease is no longer responsive to treatment or when the benefits of treatment do not outweigh the burden and risks (Fearon et al. above). Often, the refractory stage is determined by the full stage of the underlying disease described below and the patient's condition.

[0098] Metabolic diseases may exist in acute or chronic settings. An aspect of the present invention provides treatment or prevention of diseases / pathological conditions associated with metabolic diseases. Diseases / pathological conditions associated with metabolic diseases include diseases / pathological conditions that are positively related to the occurrence of metabolic diseases. In some embodiments, diseases / pathological conditions associated with metabolic diseases are those that can cause, cause, or have caused (i.e., can cause, or have caused) metabolic diseases.

[0099] Diseases / pathologies related to metabolic diseases also include diseases / pathologies that are caused by or exacerbated (become worse, progress, or develop complications) by metabolic diseases. In some embodiments, diseases / pathologies related to metabolic diseases may be positively related to the occurrence of metabolic diseases and may also be exacerbated by metabolic diseases. "Related" diseases / pathologies may include metabolic disease-related pathologies.

[0100] In an embodiment of the present invention, a metabolic disease, or a disease / pathological condition related to a metabolic disease, may be present in any organ / tissue, e.g., heart, liver, kidney, brain, skin, muscular system, stomach, small intestine, large intestine, pancreas, mouth, salivary gland, pharynx, esophagus, gallbladder, trachea, larynx, bladder, ovary, uterus, testis, glands of the endocrine system, e.g., pituitary gland or thyroid gland, lymphatic system, e.g., spleen.

[0101] In an embodiment of the present invention, a disease / condition associated with a metabolic disease may be one or more of cancer, heart disease, kidney disease, lung disease, liver disease, chronic infection, neurodegenerative disease, acute injury, traumatic injury / trauma, post-operative condition, or aging / senescence.

[0102] In some embodiments, metabolic diseases may be recognized / identified / diagnosed using one or more biomarkers or correlations of metabolic diseases.

[0103] Agents capable of inhibiting the action of IL-11

[0104] The present invention involves inhibition of IL-11-mediated signaling.

[0105] In this document, 'inhibition' refers to a reduction, decrease, or attenuation compared to control conditions. For example, inhibition of the action of IL-11 by an agent capable of inhibiting IL-11-mediated signaling refers to a reduction, decrease, or attenuation of the magnitude / degree of IL-11-mediated signaling in the absence of the agent and / or in the presence of an appropriate control agent.

[0106] In this document, inhibition may also be referred to as neutralization or antagonism. In other words, an agent capable of inhibiting IL-11-mediated signaling (e.g., interactions, signaling, or other activities mediated by IL-11 or IL-11-containing complexes) may be referred to as a 'neutralizing' or 'antagonistic' agent with respect to the relevant function or process. For example, an agent capable of inhibiting IL-11-mediated signaling may be referred to as an agent capable of neutralizing IL-11-mediated signaling or as an antagonist of IL-11-mediated signaling.

[0107] The IL-11 signaling pathway provides multiple pathways for the inhibition of IL-11 signaling. Agents capable of inhibiting IL-11-mediated signaling can do so, for example, by inhibiting the action of one or more factors involved in or necessary for signaling via receptors for IL-11.

[0108] For example, inhibition of IL-11 signaling can be achieved by interfering with the interaction between IL-11 (or an IL-11-containing complex, e.g., a complex of IL-11 and IL-11Rα) and a receptor for IL-11 (e.g., IL-11Rα, a receptor complex containing IL-11Rα, gp130, or a receptor complex containing IL-11Rα and gp130). In some embodiments, inhibition of IL-11-mediated signaling is achieved, for example, by inhibiting the expression of one or more genes or proteins of IL-11, IL-11Rα, and gp130.

[0109] In an embodiment, inhibition of IL-11-mediated signaling is IL-11-mediated trans It does not interfere with signaling, but IL-11-mediated cis This is achieved by interfering with signaling, that is, inhibition of IL-11-mediated signaling is gp130-mediated involving newly bound IL-11Rα cisThis is achieved by inhibiting the complex. In an embodiment, the inhibition of IL-11-mediated signaling is IL-11-mediated cis It does not interfere with signaling, but IL-11-mediated trans This is achieved by interfering with signaling, that is, inhibition of IL-11-mediated signaling is gp130-mediated trans This is achieved by inhibiting a signaling complex, e.g., IL-11 bound to soluble IL-11Rα or IL-6 bound to soluble IL-6R. In an embodiment, inhibition of IL-11-mediated signaling is achieved by IL-11-mediated cis Signaling and IL-11-mediated trans This is achieved by interfering with signaling. Any preparation as described herein is IL-11-mediated cis and / or trans It can be used to inhibit signaling.

[0110] In another example, inhibition of IL-11 signaling can be achieved by interfering with the downstream signaling pathway of IL-11 / IL-11Rα / gp130. In other words, in some embodiments, inhibition / antagonism of IL-11-mediated signaling involves inhibition of downstream signaling pathways / processes / factors of signaling through the IL-11 / IL-11 receptor complex.

[0111] In some embodiments, inhibition / antagonism of IL-11-mediated signaling comprises inhibition of signaling through an intracellular signaling pathway activated by the IL-11 / IL-11 receptor complex. In some embodiments, inhibition / antagonism of IL-11-mediated signaling comprises inhibition of one or more factors whose expression / activity is upregulated as a result of signaling through the IL-11 / IL-11 receptor complex.

[0112] In some embodiments, the method of the present invention uses a preparation capable of inhibiting JAK / STAT signaling. In some embodiments, the preparation capable of inhibiting JAK / STAT signaling may inhibit the action of JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and / or STAT6. For example, the agent may inhibit the activation of JAK / STAT proteins and / or inhibit the interaction between JAK or STAT proteins and cell surface receptors, e.g., IL-11Rα or gp130, and / or inhibit the phosphorylation of JAK proteins and / or inhibit the interaction between JAK proteins and STAT proteins, and / or inhibit the phosphorylation of STAT proteins, and / or inhibit the dimerization of STAT proteins, and / or inhibit the translocation of STAT proteins into the cell nucleus, and / or inhibit the binding of STAT proteins to DNA, and / or promote the degradation of JAK and / or STAT proteins. In some embodiments, the JAK / STAT inhibitors are ruxolitinib (Jakafi / Jakavi; Incyte), tofacitinib (Xeljanz / Jakvinus; NIH / Pfizer), oclacitinib (Apoquel), and baricitinib (Olumiant; Incyte / Eli Lilly). Filgotinib (G-146034 / GLPG-0634; Galapagos NV), Gandotinib (LY-2784544; Eli Lilly), Lestaurtinib (CEP-701; Teva), Momelotinib (GS-0387 / CYT-387; Gilead Sciences), Pacritinib (SB1518; CTI), PF-04965842 (Pfizer), UpadacitinibIt is selected from (ABT-494; AbbVie), peficitinib (ASP015K / JNJ-54781532; Astellas), fedratinib (SAR302503; Celgene), cucurbitacin I (JSI-124), and CHZ868.

[0113] In some embodiments, the method of the present invention utilizes an agent capable of inhibiting MAPK / ERK signaling. In some embodiments, the agent capable of inhibiting MAPK / ERK signaling may inhibit the action of GRB2 and / or inhibit the action of RAF kinase and / or inhibit the action of MEK protein and / or inhibit the activation of MAP3K / MAP2K / MAPK and / or Myc, and / or inhibit the phosphorylation of STAT protein. In some embodiments, the agent capable of inhibiting ERK signaling may inhibit ERK p42 / 44. In some embodiments, the ERK inhibitors are SCH772984, SC1, VX-11e, DEL-22379, 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), It is selected from selumetinib (AZD6244; Array / AstraZeneca) and trametinib (GSK1120212 / Mekinist; Novartis). In some embodiments, the method of the present invention utilizes an agent capable of inhibiting c-Jun N-terminal kinase (JNK) signaling / activation. In some embodiments, the agent capable of inhibiting JNK signaling / activation may inhibit the action and / or phosphorylation of JNK (e.g., JNK1, JNK2).In some embodiments, the JNK inhibitor is selected from SP600125, CEP 1347, TCS JNK 6o, c-JUN peptide, SU3327, AEG 3482, TCS JNK 5a, BI78D3, IQ3, SR3576, IQ1S, JIP-1(153-163) and CC401 dihydrochloride.

[0114] In this embodiment, the inventors demonstrate that NOX4 expression and activity are upregulated by signaling via IL-11 / IL-11Rα / gp130. NOX4 is a source of NADPH oxidase and reactive oxygen species (ROS). Nox4 expression is upregulated in transgenic mice with hepatocyte-specific Il11 expression, and primary human hepatocytes stimulated with IL11 upregulate NOX4 expression.

[0115] In some embodiments, the present invention utilizes a agent capable of inhibiting NOX4 expression (gene or protein expression) or function. In some embodiments, the present invention utilizes a agent capable of inhibiting IL-11-mediated upregulation of NOX4 expression / function. An agent capable of inhibiting NOX4 expression or function may be referred to herein as a NOX4 inhibitor. For example, a NOX4 inhibitor may reduce the expression of NOX4 (e.g., gene and / or protein expression), reduce the level of RNA encoding NOX4, reduce the level of NOX4 protein, and / or reduce the level of NOX4 activity (e.g., reduce NOX4-mediated NADPH oxidase activity and / or NOX4-mediated ROS production).

[0116] NOX4 inhibitors include NOX4-binding molecules and molecules capable of reducing NOX4 expression. NOX4-binding inhibitors include peptide / nucleic acid aptamers, antibodies (and antibody fragments) and fragments of interacting partners for NOX4, which act as antagonists of NOX4 function and small molecule inhibitors of NOX4. Molecules capable of reducing NOX4 expression include antisense RNAs for NOX4 (e.g., siRNA, shRNA). In some embodiments, NOX4 inhibitors are selected from NOX4 inhibitors described in the literature [Altenhofer et al., Antioxid Redox Signal. (2015) 23(5): 406-427] or the literature [Augsburder et al., Redox Biol. (2019) 26: 101272], such as GKT137831.

[0117] binder

[0118] In some embodiments, an agent capable of inhibiting IL-11-mediated signaling may bind to IL-11. In some embodiments, an agent capable of inhibiting IL-11-mediated signaling may bind to a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130). Binding of such an agent may inhibit IL-11-mediated signaling by inhibiting downstream signaling by reducing / preventing the ability of IL-11 to bind to a receptor for IL-11. Binding of such an agent may inhibit IL-11-mediated signaling by inhibiting downstream signaling by reducing / preventing the ability of IL-11 to bind to a receptor for IL-11, e.g., IL-11Rα and / or gp130. cis and / or trans - It can inhibit signaling. The preparation is trans - It can bind to signaling complexes, such as IL-11 and soluble IL-11Rα, and inhibit gp130-mediated signaling.

[0119] The IL-11 / IL-11 containing complex, or the formulation capable of binding to a receptor for IL-11, may be of any type, but in some embodiments, the formulation may be an antibody, its antigen-binding fragment, polypeptide, peptide, nucleic acid, oligonucleotide, aptamer, or small molecule. The formulation may be provided in an isolated or purified form, or may be formulated as a pharmaceutical composition or drug.

[0120] Antibody and antigen-binding fragment

[0121] In some embodiments, the IL-11 / IL-11 containing complex, or the agent capable of binding to a receptor for IL-11, is an antibody or an antigen-binding fragment thereof. In some embodiments, the IL-11 / IL-11 containing complex, or the agent capable of binding to a receptor for IL-11, is a polypeptide, e.g., an attractant receptor molecule. In some embodiments, the IL-11 / IL-11 containing complex, or the agent capable of binding to a receptor for IL-11, may be an aptamer.

[0122] In some embodiments, the IL-11 / IL-11-containing complex, or the preparation capable of binding to a receptor for IL-11, is an antibody or an antigen-binding fragment thereof. The term "antibody" is used herein in the broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they indicate binding to a target molecule.

[0123] In terms of current techniques related to monoclonal antibody technology, antibodies can be produced for most antigens. The antigen-binding portion can be a part of the antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single-stranded Fv fragment [ScFv]). Monoclonal antibodies against selected antigens can be produced by known techniques, for example, those disclosed in the literature ["Monoclonal Antibodies: A manual of techniques", H. Zola (CRC Press, 1988)] and the literature ["Monoclonal Hybridoma Antibodies: Techniques and Applications", J.GR. Hurrell (CRC Press, 1982)]. Chimeric antibodies have been discussed in the literature [Neuberger et al. (1988, 8th International Biotechnology Symposium Part 2, 792-799)]. Monoclonal antibodies (mAbs) are particularly useful for the method of the present invention and are a homogeneous population of antibodies that specifically target a single epitope on an antigen.

[0124] Polyclonal antibodies are also useful for the method of the present invention. Monospecific polyclonal antibodies are preferred. Suitable polyclonal antibodies can be prepared using methods well known in the art.

[0125] Antigen-binding fragments of antibodies, such as Fab and Fab2 fragments, may also be used or provided, as may genetically engineered antibodies and antibody fragments. The variable heavy chain (VH) and variable light chain (VL) domains of antibodies are involved in antigen recognition, a fact first recognized by early protease degradation experiments. Further confirmation was revealed by the "humanization" of rodent antibodies. A variable domain of rodent origin can be fused to a constant domain of human origin so that the resulting antibody retains the antigen specificity of the rodent parented antibody (Reference [Morrison et al. (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855]).

[0126] The antibody and antigen-binding fragment according to the present disclosure comprise a complementarity-determining region (CDR) of the antibody capable of binding to a relevant target molecule (i.e., IL-11 / IL-11-containing complex / receptor for IL-11).

[0127] Antibodies capable of binding to IL-11 include, for example, monoclonal mouse anti-human IL-11 antibody clone #22626, such as the one used in the literature [Bockhorn et al. Nat. Commun. (2013) 4(0):1393]; Catalog number MAB218 (R&D Systems, 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), 11h3 / 19.6.1 (also published as [Hermann et al., Arthritis Rheum. (1998) 41(8):1388-97]), AB-218-NA (R&D Systems), X203 (also published as [Ng et al., Sci Transl Med. (2019) 11(511) pii: eaaw1237]; doi: https: / / doi.org / 10.1101 / 336537) and US Includes anti-IL-11 antibodies disclosed in 2009 / 0202533 A1, WO 99 / 59608 A2, WO 2018 / 109174 A2 and WO 2019 / 238882 A1.

[0128] In particular, the anti-IL-11 antibody clone 22626 (also known as MAB218) has been shown to be an antagonist of IL-11-mediated signaling, for example, in the literature [Schaefer et al., Nature (2017) 552(7683):110-115]. The monoclonal antibody 11h3 / 19.6.1 is disclosed in the literature [Hermann et al., Arthritis Rheum. (1998) 41(8):1388-97] as a neutralizing anti-IL-11 IgG1. For example, AB-218-NA from R&D Systems, used in the literature [McCoy et al., BMC Cancer (2013) 13:16], is another example of a neutralizing anti-IL-11 antibody. WO 2018 / 109174 A2 and WO 2019 / 238882 A1 disclose exemplary anti-IL-11 antibody antagonists for further addition to IL-11-mediated signaling. Literature [Ng, et al., "IL-11 is a therapeutic target in idiopathic pulmonary fibrosis." bioRxiv 336537]; X203 (also referred to as Enx203) disclosed in doi: https: / / doi.org / 10.1101 / 336537 and WO 2019 / 238882 A1 is an anti-IL-11 antibody antagonist of IL-11-mediated signaling and comprises a VH region according to SEQ ID NO:92 of WO 2019 / 238882 A1 (SEQ ID NO:22 of the present disclosure) and a VL region according to SEQ ID NO:94 of WO 2019 / 238882 A1 (SEQ ID NO:23 of the present disclosure). A humanized version of X203, including hEnx203 comprising a VH area according to SEQ ID NO:117 of WO 2019 / 238882 A1 (SEQ ID NO:30 of the present disclosure) and a VL area according to SEQ ID NO:122 of WO 2019 / 238882 A1 (SEQ ID NO:31 of the present disclosure), is described in WO 2019 / 238882 A1.Enx108A is an additional example of an anti-IL-11 antibody antagonist of IL-11-mediated signaling and comprises a VH region according to SEQ ID NO:8 of WO 2019 / 238882 A1 (SEQ ID NO:26 of the present disclosure) and a VL region according to SEQ ID NO:20 of WO 2019 / 238882 A1 (SEQ ID NO:27 of the present disclosure).

[0129] Antibodies capable of binding to IL-11Rα include, for example, the monoclonal antibody clone 025 (Sino Biological), clone EPR5446 (Abcam), clone 473143 (R & D Systems), clones 8E2, 8D10, and 8E4 and affinity-maturity variants of 8E2 described in US 2014 / 0219919 A1, Blanc et al. (Literature [ J. Immunol Methods. The monoclonal antibody X209 described in [2000 Jul 31;241(1-2);43-59]), [Widjaja et al., Gastroenterology (2019) 157(3):777-792], [Widjaja, et al., "IL-11 neutralizing therapies target hepatic stellate cell-induced liver inflammation and fibrosis in NASH." bioRxiv 470062]; also disclosed as doi: https: / / doi.org / 10.1101 / 470062), the antibody disclosed in WO 2014121325 A1 and US 2013 / 0302277 A1, and US 2009 / 0202533 A1, WO 99 / 59608 A2, WO 2018 / 109170 A2 and WO It includes the anti-IL-11Rα antibody disclosed in 2019 / 238884 A1.

[0130] In particular, the anti-IL-11Rα antibody clone 473143 (also known as MAB1977) has been shown to be an antagonist of IL-11-mediated signaling, for example, in the literature [Schaefer et al., Nature (2017) 552(7683):110-115]. US 2014 / 0219919 A1 provides sequences for anti-human IL-11Rα antibody clones 8E2, 8D10, and 8E4 and discloses their ability to antagonize IL-11-mediated signaling—see, for example,

[0489] to

[0490] of US 2014 / 0219919 A1. Furthermore, US 2014 / 0219919 A1 provides sequence information for an additional 62 affinity-maturity variants of clone 8E2, 61 of which are disclosed as antagonizing IL-11-mediated signaling—see Table 3 of US 2014 / 0219919 A1. WO 2018 / 109170 A2 and WO 2019 / 238884 A1 disclose further exemplary anti-IL-11Rα antibody antagonists of IL-11-mediated signaling. Reference [Widjaja, et al., "IL-11 neutralizing therapies target hepatic stellate cell-induced liver inflammation and fibrosis in NASH." bioRxiv 470062]; X209 (also referred to as Enx209) disclosed in doi: https: / / doi.org / 10.1101 / 470062 and WO 2019 / 238884 A1 is an anti-IL-11Rα antibody antagonist of IL-11-mediated signaling and comprises a VH region according to SEQ ID NO:7 of WO 2019 / 238884 A1 (SEQ ID NO:24 of the present disclosure) and a VL region according to SEQ ID NO:14 of WO 2019 / 238884 A1 (SEQ ID NO:25 of the present disclosure).A humanized version of X209, including hEnx209 comprising a VH area according to SEQ ID NO:11 of WO 2019 / 238884 A1 (SEQ ID NO:32 of the present disclosure) and a VL area according to SEQ ID NO:17 of WO 2019 / 238884 A1 (SEQ ID NO:33 of the present disclosure), is described in WO 2019 / 238884 A1.

[0131] Those skilled in the art are well aware of techniques for generating antibodies suitable for therapeutic use in a given species / subject. For example, the procedure for generating antibodies suitable for therapeutic use in humans is described in the literature [Park and Smolen Advances in Protein Chemistry (2001) 56: 369-421 (the whole of which is incorporated herein by reference).

[0132] Antibodies against a given target protein (e.g., IL-11 or IL-11Rα) are generated in model species (e.g., rodents, lagomorphs) and can subsequently be engineered to improve their suitability for therapeutic use in a given species / subject. For example, one or more amino acids of a monoclonal antibody generated by immunization of a model species can be substituted to arrive at an antibody sequence more similar to the human germline immunoglobulin sequence (thus reducing the potential for an anti-xenogenic antibody immune response in human subjects treated with the antibody). Modifications in the antibody variable domain can focus on the framework region to preserve the antibody paratope. Antibody humanization is a matter of routine practice in the field of antibody technology and has been reviewed, for example, in the literature [Almagro and Fransson, Frontiers in Bioscience (2008) 13:1619-1633], the literature [Safdari et al., Biotechnology and Genetic Engineering Reviews (2013) 29(2): 175-186], and the literature [Lo et al., Microbiology Spectrum (2014) 2(1)], all of which are incorporated herein by reference. The requirement for humanization can be avoided by generating antibodies against a given target protein (e.g., IL-11 or IL-11Rα) in a genetically modified model species expressing a human immunoglobulin gene, and thus, antibodies generated in such animals are fully human (e.g., described in the literature [Bruggemann et al., Arch Immunol Ther Exp (Warsz) (2015) 63(2):101-108], the whole of which is incorporated herein by reference).

[0133] Phage display techniques can also be used to identify antibodies against a given target protein (e.g., IL-11 or IL-11Rα), as is well known to those skilled in the art. The use of phage display for the identification of fully human antibodies against human target proteins has been reviewed, for example, in the literature [Hoogenboom, Nat. Biotechnol. (2005) 23, 1105-1116] and in the literature [Chan et al., International Immunology (2014) 26(12): 649-657], the entirety of which is incorporated herein by reference.

[0134] The antibody / fragment may be an antagonist antibody / fragment that inhibits or reduces the biological activity of IL-11. The antibody / fragment may be a neutralizing antibody that neutralizes the biological effects of IL-11, e.g., its ability to stimulate productivity signaling through IL-11 receptors. Neutralizing activity can be measured by the ability to neutralize IL-11-induced proliferation in T11 mouse plasmacytoma cell lines (reference [Nordan, RP et al. (1987) J. Immunol. 139:813]).

[0135] IL-11-binding or IL-11Rα-binding antibodies for their ability to antagonize IL-11-mediated signaling, for example, US 2014 / 0219919 A1 or Blanc et al. (Literature [ J. Immunol Methods It can be evaluated using the assay described in . 2000 Jul 31;241(1-2);43-59]). Briefly, IL-11-binding and IL-11Rα-binding antibodies for the ability to inhibit the proliferation of Ba / F3 cells expressing IL-11Rα and gp130 from appropriate species in response to stimulation by IL-11 from appropriate species In a test tubeAlternatively, IL-11-binding and IL-11Rα-binding antibodies can be evaluated for their ability to inhibit the transition from fibroblasts to myofibroblasts after TGFβ1 stimulation by assessing αSMA expression. In a test tube It may be analyzed (e.g., as described in WO 2018 / 109174 A2 (Example 6) and WO 2018 / 109170 A2 (Example 6), the literature [Ng et al., Sci Transl Med. (2019) 11(511) pii: eaaw1237] and the literature [Widjaja et al., Gastroenterology (2019) 157(3):777-792]).

[0136] The antibody generally comprises six CDRs; three within the light chain variable region (VL): LC-CDR1, LC-CDR2, and LC-CDR3, and three within the heavy chain variable region (VH): HC-CDR1, HC-CDR2, and HC-CDR3. Together, the six CDRs define the paratopes of the antibody, and these paratopes are the parts of the antibody that bind to the target molecule. The VH and VL regions each contain a framework region (FR) on the side of the respective CDR, which provides a scaffold for the CDR. From the N-terminus to the C-terminus, the VH region comprises the following structure: N-terminus–[HC-FR1]–[HC-CDR1]–[HC-FR2]–[HC-CDR2]–[HC-FR3]–[HC-CDR3]–[HC-FR4]–C-terminus; The VL region includes the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0137] Several different conventions for defining antibody CDR and FR, e.g., literature [Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)], literature [Chothia et al., J. Mol. Biol. VBASE2 as described in [196:901-917 (1987)] and in the literature [Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671-D674] is present. The CDR and FR of the VH and VL regions of the antibodies described herein are defined according to the Kabat system.

[0138] In some embodiments, the antibody according to the present disclosure, or its antigen-binding fragment, is derived from an antibody that specifically binds to IL-11 (e.g., Enx108A, Enx203, or hEnx203). In some embodiments, the antibody according to the present disclosure, or its antigen-binding fragment, is derived from an antibody that specifically binds to IL-11Rα (e.g., Enx209 or hEnx209).

[0139] The antibody and antigen-binding fragment according to the present disclosure preferably inhibit IL-11-mediated signaling. Such antibody / antigen-binding fragment may be described as an antagonist of IL-11-mediated signaling and / or may be described as having the ability to neutralize IL-11-mediated signaling.

[0140] In some embodiments, the antibody / antigen-binding fragment comprises a CDR of an antibody that binds to IL-11. In some embodiments, the antibody / antigen-binding fragment comprises a CDR of a CDR of an IL-11-binding antibody described herein (e.g., Enx108A, Enx203, or hEnx203), or a CDR derived therefrom.

[0141] In some embodiments, the antibody / antigen-binding fragment incorporates the following CDR into the VH region:

[0142] (1)

[0143] HC-CDR1 having the amino acid sequence of SEQ ID NO:34

[0144] HC-CDR2 having the amino acid sequence of SEQ ID NO:35

[0145] HC-CDR3 having the amino acid sequence of SEQ ID NO:36,

[0146] or includes a variant thereof in which 1, 2, or 3 amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0147] In some embodiments, the antibody / antigen-binding fragment is a VL region incorporating the following CDR:

[0148] (2)

[0149] LC-CDR1 having the amino acid sequence of SEQ ID NO:37

[0150] LC-CDR2 having the amino acid sequence of SEQ ID NO:38

[0151] LC-CDR3 having the amino acid sequence of SEQ ID NO:39,

[0152] or includes a variant thereof in which 1, 2, or 3 amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0153] In some embodiments, the antibody / antigen-binding fragment incorporates the following CDR into the VH region:

[0154] (3)

[0155] HC-CDR1 having the amino acid sequence SEQ ID NO:40

[0156] HC-CDR2 having the amino acid sequence of SEQ ID NO:41

[0157] HC-CDR3 having the amino acid sequence of SEQ ID NO:42,

[0158] or includes a variant thereof in which 1, 2, or 3 amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0159] In some embodiments, the antibody / antigen-binding fragment is a VL region incorporating the following CDR:

[0160] (4)

[0161] LC-CDR1 having the amino acid sequence of SEQ ID NO:43

[0162] LC-CDR2 having the amino acid sequence of SEQ ID NO:44

[0163] LC-CDR3 having the amino acid sequence of SEQ ID NO:45,

[0164] or includes a variant thereof in which 1, 2, or 3 amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0165] In some embodiments, the antibody / antigen-binding fragment comprises a VH region incorporating a CDR according to (1) and a VL region incorporating a CDR according to (2). In some embodiments, the antibody / antigen-binding fragment comprises a VH region incorporating a CDR according to (3) and a VL region incorporating a CDR according to (4).

[0166] In some embodiments, the antibody / antigen-binding fragment comprises the VH region and VL region of an antibody that binds to IL-11. In some embodiments, the antibody / antigen-binding fragment comprises the VH region and VL region of the IL-11-binding antibody described herein (e.g., Enx108A, Enx203, or hEnx203), or the VH region and VL region derived therefrom.

[0167] In some embodiments, the antibody / antigen-binding fragment comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:26, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen-binding fragment comprises a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:27, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen-binding fragment comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:26, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of the amino acid sequences of SEQ ID NO:26 and at least 70% sequence identity with the amino acid sequence of SEQ ID NO:27, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, It includes a VL region containing an amino acid sequence having one of 99% or 100% sequence identity.

[0168] In some embodiments, the antibody / antigen-binding fragment comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:22, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen-binding fragment comprises a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:23, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen-binding fragment comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:22, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of the amino acid sequences of SEQ ID NO:22, and at least 70% sequence identity with the amino acid sequence of SEQ ID NO:23, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, It includes a VL region containing an amino acid sequence having one of 99% or 100% sequence identity.

[0169] In some embodiments, the antibody / antigen-binding fragment comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:30, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen-binding fragment comprises a VL region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:31, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen-binding fragment comprises a VH region having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:30, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NO:30 and at least 70% sequence identity with the amino acid sequence of SEQ ID NO:31, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, It includes a VL region containing an amino acid sequence having one of 99% or 100% sequence identity.

[0170] In some embodiments, the antibody / antigen-binding fragment comprises a CDR of an antibody that binds to IL-11Rα. In some embodiments, the antibody / antigen-binding fragment comprises a CDR of the CDR of the IL-11Rα-binding antibody described herein (e.g., Enx209 or hEnx209), or a CDR derived therefrom.

[0171] In some embodiments, the antibody / antigen-binding fragment incorporates the following CDR into the VH region:

[0172] (5)

[0173] HC-CDR1 having the amino acid sequence of SEQ ID NO:46

[0174] HC-CDR2 having the amino acid sequence of SEQ ID NO:47

[0175] HC-CDR3 having the amino acid sequence of SEQ ID NO:48,

[0176] or includes a variant thereof in which 1, 2, or 3 amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino acid.

[0177] In some embodiments, the antibody / antigen-binding fragment is a VL region incorporating the following CDR:

[0178] (6)

[0179] LC-CDR1 having the amino acid sequence of SEQ ID NO:49

[0180] LC-CDR2 having the amino acid sequence of SEQ ID NO:50

[0181] LC-CDR3 having the amino acid sequence of SEQ ID NO:51,

[0182] or includes a variant thereof in which 1, 2, or 3 amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are substituted with another amino acid.

[0183] In some embodiments, the antibody / antigen-binding fragment comprises a VH region incorporating a CDR according to (5) and a VL region incorporating a CDR according to (6).

[0184] In some embodiments, the antibody / antigen-binding fragment comprises the VH region and VL region of an antibody that binds to IL-11Rα. In some embodiments, the antibody / antigen-binding fragment comprises the VH region and VL region of the IL-11Rα-binding antibody (e.g., Enx209 or hEnx209) described herein, or the VH region and VL region derived therefrom.

[0185] In an embodiment according to the present invention in which one or more amino acids of a reference amino acid sequence (e.g., the CDR sequence, VH region sequence, or VL region sequence described herein) are substituted with another amino acid, the substitution may be, for example, a conservative substitution according to the table below. In some embodiments, amino acids within the same block of the middle column are substituted. In some embodiments, amino acids on the same line of the far right column are substituted:

[0186]

[0187] In some embodiments, the substitution(s) may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of the antibody / fragment containing the substitution compared to an equivalent unsubstituted molecule.

[0188] In some embodiments, the substitution(s) may be focused on specific regions or areas of the VH region or VL region sequence relative to the reference VH region or VL region sequence. For example, variations from the reference VH region or VL region sequence may be focused on one or more framework regions (FR1, FR2, FR3 and / or FR4).

[0189] The antibody and antigen-binding fragment according to the present disclosure may be designed and manufactured using the sequence of a monoclonal antibody (mAb) capable of binding to a relevant target molecule. The antigen-binding region of the antibody, e.g., a single-chain variable fragment (scFv), Fab, and Fab2 fragments may also be used or provided. An 'antigen-binding region' or 'antigen-binding fragment' is any fragment of an antibody capable of binding to a specific target.

[0190] In some embodiments, the antibody / fragment comprises VL and VH regions of an antibody capable of binding to IL-11, an IL-11-containing complex, or a receptor for IL-11. The VL and VH regions of the antigen-binding region of the antibody together form an Fv region. In some embodiments, the antibody / fragment comprises or is composed of an Fv region of an antibody capable of binding to IL-11, an IL-11-containing complex, or a receptor for IL-11. The Fv region may be represented as a single chain, wherein the VH and VL regions are covalently linked, for example, by a flexible oligopeptide. Accordingly, the antibody / fragment may comprise or be composed of a scFv comprising VL and VH regions of an antibody capable of binding to IL-11, an IL-11-containing complex, or a receptor for IL-11.

[0191] The VL and light chain constant (CL) regions of the antigen-binding region of the antibody, and the VH region and heavy chain constant 1 (CH1) region together form a Fab region. In some embodiments, the antibody / fragment comprises or is composed of a Fab region of an antibody capable of binding to IL-11, an IL-11-containing complex, or a receptor for IL-11.

[0192] In some embodiments, the antibody / fragment comprises or consists of IL-11, an IL-11-containing complex, or a whole antibody capable of binding to a receptor for IL-11. "Whole antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Different types of immunoglobulins and their structures are described, for example, in the literature [Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52], the entirety of which is incorporated herein by reference. Type G immunoglobulin (i.e., IgG) is a glycoprotein of about 150 kDa comprising two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain comprises VH followed by a heavy chain constant region containing three constant domains (CH1, CH2, and CH3), and similarly, the light chain comprises VL followed by CL. Depending on the heavy chain, the immunoglobulin may be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain may be kappa (κ) or lambda (λ).

[0193] In some embodiments, the antibody / antigen-binding fragment of the present disclosure comprises an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence may be a human immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is a heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, e.g., human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM, or is derived therefrom. In some immunoglobulins, the heavy chain constant sequence is the heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17) or is derived therefrom.

[0194] In some embodiments, the immunoglobulin heavy chain constant sequence is the constant region sequence of human immunoglobulin G1 constant (IGHG1; UniProt: P01857-1, v1) or is derived therefrom. In some embodiments, the immunoglobulin heavy chain constant sequence is the constant region sequence of human immunoglobulin G1 constant (IGHG1; UniProt: P01857-1, v1) containing the substitutions K214R, D356E, and L358M (i.e., G1m3 allotype) or is derived therefrom. In some embodiments, the antibody / antigen-binding fragment comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:52, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0195] In some embodiments, the immunoglobulin heavy chain invariant sequence is the invariant region sequence of human immunoglobulin G4 invariant (IGHG4; UniProt: P01861, v1) or is derived therefrom. In some embodiments, the immunoglobulin heavy chain invariant sequence is the invariant region sequence of human immunoglobulin G4 invariant (IGHG4; UniProt: P01861, v1) containing the substitutions S241P and / or L248E or is derived therefrom. While the S241P mutation causes hinge stabilizing, the L248E mutation further reduces the already low ADCC effector function of IgG4 (Davies and Sutton, Immunol Rev. 2015 Nov; 268(1):139-159; Angal et al. Mol Immunol. 1993 Jan;30(1):105-8). In some embodiments, the antibody / antigen-binding fragment comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:53, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0196] In some embodiments, the antibody / antigen-binding fragment of the present disclosure comprises an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence may be a human immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is a kappa (κ) or lambda (λ) light chain, e.g., human immunoglobulin kappa constant (IGKC; Cκ; UniProt: P01834-1, v2), or human immunoglobulin lambda constant (IGLC; Cλ), e.g., IGLC1 (UniProt: P0CG04-1, v1), IGLC2 (UniProt: P0DOY2-1, v1), IGLC3 (UniProt: P0DOY3-1, v1), IGLC6 (UniProt: P0CF74-1, v1) or IGLC7 (UniProt: A0M8Q6-1, v3) or derived therefrom.

[0197] In some embodiments, the antibody / antigen-binding fragment comprises an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; Cκ; UniProt: P01834-1, v2; SEQ ID NO:90) or is derived therefrom. In some embodiments, the immunoglobulin light chain constant sequence is human immunoglobulin lambda constant (IGLC; Cλ), e.g., IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In some embodiments, the antibody / antigen-binding fragment comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:54, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antibody / antigen-binding fragment comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:55, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0198] In some embodiments, the antibody / antigen-binding fragment comprises: (i) a polypeptide comprising or composed of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:28; and (ii) a polypeptide comprising or composed of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:29.

[0199] In some embodiments, the antibody / antigen-binding fragment comprises (i) a polypeptide comprising or composed of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:56, and (ii) a polypeptide comprising or composed of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:57.

[0200] In some embodiments, the antibody / antigen-binding fragment comprises (i) a polypeptide comprising or composed of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:58, and (ii) a polypeptide comprising or composed of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:59.

[0201] Fab, Fv, ScFv, and dAb antibody fragments are all expressed in E. coli and can be secreted from it, which enables the easy production of large amounts of the said fragments.

[0202] The whole antibody and the F(ab')2 fragment are "bivalent." "Bivalent" means that the antibody and the F(ab')2 fragment have two antigenic combination sites. In contrast, the Fab, Fv, ScFv, and dAb fragments are monovalent, having only one antigenic combination site. Synthetic antibodies capable of binding to IL-11, IL-11-containing complexes, or receptors for IL-11 can also be prepared using phage display technology, as is well known in the art.

[0203] Antibodies can be generated by an affinity maturation process, in which a modified antibody is produced that has an improved affinity for the antigen compared to an unmodified parent antibody. Affinity-matured antibodies are produced by procedures known in the art, for example, in the literature [Marks et al., Rio / Technology 10:779-783 (1992)]; literature[Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994)]; literature[Schier et al., Gene 169:147-155 (1995)]; literature[Yelton et al., J. Immunol. 155:1994-2004 (1995)]; literature[Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995)]; and literature[Hawkins et al., J. Mol. Biol. It can be generated by 226:889-896 (1992)].

[0204] The antibody / fragment comprises, for example, a bispecific antibody composed of two different fragments of two different antibodies, wherein the bispecific antibody binds to two types of antigens. The bispecific antibody comprises an antibody / fragment as described herein capable of binding to IL-11, an IL-11-containing complex, or a receptor for IL-11. The antibody may contain different fragments having an affinity for a second antigen, which may be any desired antigen. Techniques for preparing bispecific antibodies are well known in the art, for example, in the literature [Mueller, D et al., (2010 Biodrugs 24 (2): 89-98)], literature[Wozniak-Knopp G et al., (2010 Protein Eng Des 23 (4): 289-297)], and literature [Baeuerle, PA et al., (2009 Cancer ResRefer to 69 (12): 4941-4944). Bispecific antibodies and bispecific antigen-binding fragments may be provided in any suitable format, e.g., in the format described in the literature [Kontermann MAbs 2012, 4(2): 182-197], the whole of which is incorporated herein by reference. For example, bispecific antibodies or bispecific antigen-binding fragments include bispecific antibody conjugates (e.g., IgG2, F(ab')2, or CovX-Body), bispecific IgG or IgG-like molecules (e.g., IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, 2-in 1-IgG, mAb2, or tandem ab common LC), asymmetric bispecific IgG or IgG-like molecules (e.g., kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair, or SEED-body), and small bispecific antibody molecules (e.g., Diabody (Db), dsDb, DART, scDb, tandAbs, tandem It may be scFv(taFv), tandem dAb / VHH, triple body, triple head, Fab-scFv, or F(ab')2-scFv2), bispecific Fc and CH3 fusion proteins (e.g., taFv-Fc, Di-diabody, scDb-CH3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-CH3), or bispecific fusion proteins (e.g., scFv2-albumin, scDb-albumin, taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine2). In particular, refer to Fig. 2 of the literature [Kontermann MAbs 2012, 4(2): 182-19].

[0205] A method for producing a bispecific antibody comprises the step of chemically crosslinking the antibody or antibody fragment with, for example, a reducing disulfide or non-reducing thioether bond, as described in the literature [Segal and Bast, 2001. Production of Bispecific Antibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16], the entirety of which is incorporated herein by reference. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) may be used to chemically crosslink the Fab fragment, for example, through a hinge region SH- group, to produce a disulfide-linked bispecific F(ab)2 heterodimer.

[0206] Another method for generating bispecific antibodies includes the step of generating quadroma cells capable of secreting bispecific antibodies by fusing antibody-producing hybridomas with polyethylene glycol, for example, as described in the literature [DM and Bast, BJ 2001. Production of Bispecific Antibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16].

[0207] Bispecific antibodies and bispecific antigen-binding fragments can also be recombinantly produced by expression from a nucleic acid construct encoding a polypeptide for an antigen-binding molecule, for example, as described in the literature [Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antibodies: Diabodies and Tandem scFv (Hornig and Farber-Schwarz)], or the literature [French, How to make bispecific antibodies, Methods Mol. Med. 2000; 40:333-339].

[0208] For example, a DNA construct comprising a sequence encoding light and heavy chain variable domains for two antigen-binding domains (i.e., a light and heavy chain variable domain for an antigen-binding domain capable of binding to IL-11, an IL-11-containing complex, or a receptor for IL-11, and a light and heavy chain variable domain for an antigen-binding domain capable of binding to another target protein) and encoding a suitable linker or dimerization domain between the antigen-binding domains can be prepared by molecular cloning techniques. Subsequently, a recombinant bispecific antibody can be generated by the expression of the construct in a suitable host cell (e.g., a mammalian host cell) (e.g., in vitro), and the expressed recombinant bispecific antibody can then be selectively purified.

[0209] Attracting receptors

[0210] A peptide or polypeptide-based formulation capable of binding to IL-11 or an IL-11-containing complex may be based on an IL-11 receptor, for example, an IL-11 binding fragment of an IL-11 receptor.

[0211] In some embodiments, the binder may comprise an IL-11-binding fragment of an IL-11Rα chain, preferably excluding one or more of soluble and / or transmembrane domain(s). In some embodiments, the binder may comprise an IL-11-binding fragment of gp130, preferably excluding one or more of soluble and / or transmembrane domain(s). Such molecules may be described as attractant receptors. Binding of such agents may inhibit IL-11-mediated cis and / or trans-signaling by inhibiting downstream signaling by reducing / preventing the ability of IL-11 to bind to a receptor for IL-11, e.g., IL-11Rα or gp130.

[0212] Curtis et al. (Blood 1997 Dec 1;90 (11):4403-12) report that soluble murine IL-11 receptor alpha chain (sIL-11R) was able to antagonize the activity of IL-11 when tested on cells expressing transmembrane IL-11R and gp130. They suggested that the observed IL-11 antagonism by sIL-11R depended on a limited number of gp130 molecules on cells already expressing transmembrane IL-11R.

[0213] The use of soluble attractant receptors as a basis for the inhibition of signal transduction and therapeutic intervention has also been reported for other signaling molecule:receptor pairs, e.g., VEGF and VEGF receptors (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).

[0214] As such, in some embodiments, the binder may be an attractant receptor, e.g., a receptor for soluble IL-11 and / or an IL-11-containing complex. Competition for IL-11 and / or an IL-11-containing complex provided by the attractant receptor has been reported to induce IL-11 antagonistic action (Curtis et al. cited above). Attractant IL-11 receptors are also described in WO 2017 / 103108 A1 and WO 2018 / 109168 A1, the entirety of which is incorporated herein by reference.

[0215] The attracting IL-11 receptor preferably binds to IL-11 and / or IL-11-containing complexes, making these species unavailable for binding to gp130, IL-11Rα, and / or gp130:IL-11Rα receptors. In this way, it acts as an 'attracting' receptor for IL-11 and IL-11-containing complexes, in much the same manner that etanercept acts as an attracting receptor for TNFα. IL-11-mediated signaling is reduced compared to signaling levels in the absence of the attracting receptor.

[0216] The attracting IL-11 receptor preferably binds to IL-11 through one or more cytokine binding modules (CBMs). The CBM is the CBM of a naturally occurring receptor molecule for IL-11, is derived therefrom, or is homologous thereto. For example, the attracting IL-11 receptor may comprise or be composed of one or more CBMs that are derived from, are derived from, or are homologous thereto the CBM of gp130 and / or IL-11Rα.

[0217] In some embodiments, the attracting IL-11 receptor may comprise or be composed of an amino acid sequence corresponding to the cytokine binding module of gp130. In some embodiments, the attracting IL-11 receptor may comprise an amino acid sequence corresponding to the cytokine binding module of IL-11Rα. Wherever the amino acid sequence 'corresponding' to a reference region or sequence of a given peptide / polypeptide has at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of the amino acid sequence of the reference region / sequence.

[0218] In some embodiments, the attractant receptor may bind to IL-11 with a binding affinity of, for example, at least 100 μM or less, optionally 10 μM or less, 1 μM or less, 100 nM or less, or about 1 to 100 nM. In some embodiments, the attractant receptor may include all or part of the IL-11 binding domain and optionally lack all or part of the transmembrane domain. The attractant receptor may optionally be fused to an immunoglobulin constant region, for example, an IgG Fc region.

[0219] inhibitor

[0220] The present invention considers the use of an inhibitor molecule capable of inhibiting IL-11-mediated signaling by binding to one or more of IL-11, IL-11-containing complexes, IL-11Rα, gp130, or complexes containing IL-11Rα and / or gp130.

[0221] In some embodiments, the formulation is a peptide-based or polypeptide-based binder based on IL-11, e.g., a mutant, variant, or binding fragment of IL-11. A suitable peptide or polypeptide-based formulation may bind to a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130) in a manner that does not induce the initiation of signal transduction or produces suboptimal signaling. This type of IL-11 mutant may act as a competitive inhibitor of endogenous IL-11.

[0222] For example, W147A is an IL-11 antagonist in which amino acid 147 is mutated from tryptophan to alanine, and it disrupts the so-called 'site III' of IL-11. This mutant can bind to IL-11Rα, but engagement with the gp130 homodimer fails, resulting in the efficient blockade of IL-11 signaling (Underhill-Day et al., 2003; Endocrinology 2003 Aug;144(8):3406-14]). Lee et al. (Literature[ Am J respire Cell Mol Biol [. 2008 Dec; 39(6):739-746]) also reports the occurrence of an IL-11 antagonist mutant ("mutane") that can specifically inhibit the binding of IL-11 to IL-11Rα. IL-11 mutain is also described in WO 2009 / 052588 A1.

[0223] Menkhorst et al. (Biology of Reproduction May 1, 2009 vol.80 no.5 920-927) describe a PEGylated IL-11 antagonist, PEGIL11A (CSL Limited, Parkville, Victoria, Australia), which is effective in inhibiting IL-11 action in female mice.

[0224] Literature [Pasqualini et al. Cancer

[2015] 121(14):2411-2421] describes a ligand-directed, peptidomimetic drug, bone metastasis-targeted peptidomimetic-11 (BMTP-11), which can bind to IL-11Rα.

[0225] In some embodiments, a binder capable of binding to a receptor for IL-11 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 binder may be provided in the form of a small molecule inhibitor of IL-11 or an IL-11-containing complex, e.g., an IL-11 inhibitor described in the literature [Lay et al., Int. J. Oncol. (2012); 41(2): 759-764], the entirety of which is incorporated herein by reference.

[0226] Aptamer

[0227] In some embodiments, the agent capable of binding to an IL-11 / IL-11-containing complex, or to a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130), is an aptamer. Aptamers, also known as nucleic acid / peptide ligands, are nucleic acid or peptide molecules characterized by the ability to bind to a target molecule with high specificity and high affinity. Most aptamers identified to date are non-naturally occurring molecules.

[0228] Aptamers for a given target (e.g., IL-11, IL-11-containing complexes, or receptors for IL-11) can be identified and / or generated by the systematic evolution of ligands by exponential enrichment (SELEX™) method, or by developing SOMAmers (slow off-rate modified aptamers) (Reference [Gold L et al. (2010) PLoS ONE 5(12):e15004]). Aptamers and SELEX are described in [Tuerk and Gold, Science (1990) 249(4968):505-10] and WO 91 / 19813. Applying SELEX and SOMAmer techniques involves, for example, the step of adding functional groups that mimic amino acid side chains to expand the chemical diversity of the aptamer. As a result, aptamers with high affinity for the target can be enriched and identified.

[0229] Aptamers can be DNA or RNA molecules and can be single-stranded or double-stranded. Aptamers may comprise chemically modified nucleic acids, for example, in which sugars and / or phosphates and / or bases have been chemically modified. Such modifications may improve the stability of the aptamer or make the aptamer more resistant to degradation and may include modifications at the 2' position of ribose.

[0230] Aptamers can be synthesized by methods well known to those skilled in the art. For example, aptamers can be chemically synthesized on a solid support. Solid-phase synthesis may utilize phosphoramidite chemistry. Briefly, a solid-supported nucleotide is detritylated and then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester linkage. Subsequently, capping occurs, followed by oxidation of the phosphite triester by an oxidizing agent, typically iodine. Subsequently, the cycle is repeated to assemble aptamers (see, e.g., [Sinha, ND; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539]; and [Beaucage, SL; Lyer, RP (1992). Tetrahedron 48 (12): 2223]).

[0231] Suitable nucleic acid aptamers may optionally have a minimum length of one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. Suitable nucleic acid aptamers are selectively, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, It can have a maximum length of one of 79 or 80 nucleotides. Suitable nucleic acid aptamers are selectively, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, It can have a length of one of 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.

[0232] Aptamers may be peptides selected or engineered to bind to specific target molecules. Peptide aptamers and methods of their generation and identification are reviewed in the literature [Reverdatto et al., Curr Top Med Chem. (2015) 15(12):1082-101], the entirety of which is incorporated herein by reference. Peptide aptamers may optionally have a minimum length of one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The peptide aptamer may optionally have a maximum length of one of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. The suitable peptide aptamer may optionally have a length of one of 2-30, 2-25, 2-20, 5-30, 5-25, or 5-20 amino acids.

[0233] Aptamers are in the nM or pM range, for example, K less than one of 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, and 100 pM. D Can have.

[0234] Characteristics of IL-11 binder

[0235] The IL-11 / IL-11 containing complex according to the present invention, or the formulation capable of binding to a receptor for IL-11, may exhibit one or more of the following characteristics:

[0236] · IL-11 / IL-11-containing complex, or specific binding to a receptor for IL-11;

[0237] · Binding to an IL-11 / IL-11-containing complex or a receptor for IL-11 with a KD of 10 μM or less, preferably ≤ 5 μM, ≤ 1 μM, ≤ 500 nM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 100 pM;

[0238] · Inhibition of the interaction between IL-11 and IL-11Rα;

[0239] · Inhibition of the interaction between IL-11 and gp130;

[0240] · Inhibition of the interaction between IL-11 and the IL-11Rα:gp130 receptor complex;

[0241] · Inhibition of the interaction between the IL-11:IL-11Rα complex and gp130.

[0242] These characteristics can be determined by the analysis of the relevant formulation in an appropriate test, and such a test may involve a comparison of the performance of the formulation with that of a suitable control formulation. A person skilled in the art can identify appropriate control conditions for a given test.

[0243] For example, a suitable negative control for the analysis of the ability of a test antibody / antigen-binding fragment to bind to the IL-11 / IL-11-containing complex / IL-11 receptor may be an antibody / antigen-binding fragment to a non-target protein (i.e., an antibody / antigen-binding fragment not specific to the IL-11 / IL-11-containing complex / IL-11 receptor). A suitable positive control may be a known, proven (e.g., commercially available) IL-11-binding or IL-11 receptor-binding antibody. The control may be the same isotype as the putative IL-11 / IL-11-containing complex / IL-11 receptor-binding antibody / antigen-binding fragment being analyzed, and may, for example, have the same invariant region.

[0244] In some embodiments, the formulation may specifically bind to IL-11 or an IL-11-containing complex, or to a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130). The formulation that specifically binds to a given target molecule preferably binds to the target with greater affinity and / or for a longer period than it binds to other non-target molecules.

[0245] In some embodiments, the formulation may bind to IL-11 or an IL-11-containing complex with a greater affinity than its binding affinity to one or more other members of the IL-6 cytokine family (e.g., IL-6, leukemia inhibitor factor (LIF), oncostatin M (OSM), cardiotropin-1 (CT-1), ciliary neuroaffinity factor (CNTF), and cardiotropin-like cytokine (CLC)). In some embodiments, the formulation may bind to a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130) with a greater affinity than its binding affinity to one or more other members of the IL-6 receptor family. In some embodiments, the formulation may bind to IL-11Rα with a greater affinity than its binding affinity to one or more of IL-6Rα, leukemia inhibitor factor receptor (LIFR), oncostatin M receptor (OSMR), ciliary neuroaffinity factor receptor alpha (CNTFRα) and cytokine receptor-like factor 1 (CRLF1).

[0246] In some embodiments, the binding scale of the binder to a non-target is less than about 10% of the binding of the agent to the target, as measured, for example, by ELISA, SPR, Bio-Layer Interferometry (BLI), Microscale Thermophoresis (MST), or radioimmunoassay (RIA). Alternatively, binding specificity may be reflected in terms of binding affinity, where the binder is K for another non-target molecule D K that is at least 0.1 order of magnitude (i.e., 0.1 x 10n, where n is an integer representing the number of digits) larger than D It binds to IL-11, an IL-11-containing complex, or a receptor for IL-11. This may optionally be at least one of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

[0247] The binding affinity of such a binder for a given target is often its dissociation constant (K D ; dissociation constant) is described in terms of binding affinity. Binding affinity is determined by methods known in the art, e.g., ELISA, surface plasmon resonance (SPR; e.g., in the literature [Hearty et al., Methods Mol Biol (2012) 907:411-442]; or literature[Rich et al., Anal Biochem. [See 2008 Feb 1; 373(1):112-20]), biolayer interferometer (e.g., literature [Lad et al., (2015) J Biomol Screen 20(4): 498-507]; or literature [Concepcion et al., Comb Chem High Throughput Screen [See . 2009 Sep; 12(8):791-800]), microscale thermophoresis (MST) analysis (e.g., literature [Jerabek-Willemsen et al., Assay Drug Dev Technol.It can be measured by [see 2011 Aug; 9(4): 342-353] or by radiolabeled antigen binding assay (RIA).

[0248] In some embodiments, the formulation is 50 μM or less, preferably ≤ 10 μM, ≤5 μM, ≤4 μM, ≤3 μM, ≤2 μM, ≤1 μM, ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, or ≤100 pM, of one of K D It can bind to IL-11 or an IL-11-containing complex, or a receptor for IL-11.

[0249] In some embodiments, the formulation has an EC50 = 10,000 ng / ml or less, preferably ≤5,000 ng / ml, ≤1000 ng / ml, ≤900 ng / ml, ≤800 ng / ml, ≤700 ng / ml, ≤600 ng / ml, ≤500 ng / ml, ≤400 ng / ml, ≤300 ng / ml. ng / ml, ≤200 ng / ml, ≤100 ng / ml, ≤90 ng / ml, ≤80 ng / ml, ≤70 ng / ml, ≤60 ng / ml, ≤50 ng / ml, ≤40 ng / ml, ≤30 ng / ml, ≤20 ng / ml, ≤15 ng / ml, ≤10 ng / ml, ≤7.5 ng / ml, ≤5 ng / ml, It binds to IL-11, an IL-11-containing complex, or a receptor for IL-11 with a binding affinity of ≤2.5 ng / ml or ≤1 ng / ml (e.g., as determined by ELISA). Such ELISA can be performed, for example, as described in the literature [Antibody Engineering, vol. 1 (2nd Edn), Springer Protocols, Springer (2010), Part V, pp657-665].

[0250] In some embodiments, the formulation binds to an IL-11 or IL-11-containing complex within a region critical for binding to a receptor for IL-11 or an IL-11-containing complex, e.g., gp130 or IL-11Rα, thereby inhibiting the interaction between the IL-11 or IL-11-containing complex and the receptor for IL-11, and / or signaling through the receptor. In some embodiments, the formulation binds to a receptor for IL-11 within a region critical for binding to an IL-11 or an IL-11-containing complex, thereby inhibiting the interaction between the IL-11 or IL-11-containing complex and the receptor for IL-11, and / or signaling through the receptor.

[0251] The ability of a given binder (e.g., an IL-11 / IL-11-containing complex, or a agent capable of binding to a receptor for IL-11) to inhibit the interaction between two proteins can be determined, for example, by analyzing the interaction in the presence of the binder, or after incubation of the binder with one or both of the interaction partners. An example of a suitable assay for determining whether a given binder can inhibit the interaction between two interaction partners is a competitive ELISA.

[0252] A binder capable of inhibiting a given interaction (e.g., between IL-11 and IL-11Rα, or between IL-11 and gp130, or between IL-11 and IL-11Rα:gp130, or between IL-11:IL-11Rα and gp130) is identified by observing a decrease / decrease in the level of the interaction between the interaction partners in the presence of the binder—or after incubation of one or both of the interaction partners with the binder—compared to the level of the interaction in the absence of the binder (or in the presence of an appropriate control binder). A suitable assay may be performed in vitro, for example, using a recombinant interaction partner or using cells expressing the interaction partner. Cells expressing the interaction partner may do so endogenously or from nucleic acids introduced into the cell. For the purpose of this assay, one or both of the interaction partners and / or the binder may be labeled with or used with a detectable entity for the purpose of detecting and / or measuring the level of the interaction. For example, the formulation may be labeled with a radioactive atom, a colored molecule, a fluorescent molecule, or any other molecule that can be easily detected. Suitable detectable molecules include fluorescent proteins, luciferases, enzyme substrates, and radiolabels. The binder may be directly labeled with a detectable label, or the binder may be indirectly labeled. For example, the binder may be unlabeled and detected by another binder that is labeled itself. Alternatively, the second binder may have biotin bound thereto, and the binding of labeled streptavidin to biotin may be used to indirectly label the second binder.

[0253] The ability of a binder to inhibit the interaction between two binding partners can also be determined by analyzing the downstream functional consequences of such interactions, e.g., IL-11-mediated signaling. For example, the downstream functional consequences of the interaction between IL-11 and IL-11Rα:gp130 or between IL-11:IL-11Rα and gp130 may include, e.g., processes mediated by IL-11, or, e.g., gene / protein expression of collagen or IL-11.

[0254] Inhibition of the interaction between IL-11 or IL-11-containing complexes and receptors for IL-11 is 3H-thymidine incorporation and / or Ba / F3 cell proliferation assays, e.g., e.g., literature [Curtis et al.]. Blood , 1997, 90(11)] and literature [Karpovich et al. Mol. Hum. Reprod It can be analyzed using the one described in [2003 9(2): 75-80]. Ba / F3 cells co-express IL-11Rα and gp130.

[0255] In some embodiments, the binder may inhibit the interaction between IL-11 and IL-11Rα to less than 100% of the level of interaction between IL-11 and IL-11Rα in the absence of the binder (or in the presence of a suitable control binder), e.g., 99% or less, 95% or less, 90% or less, 85% 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. In some embodiments, the binder may inhibit the interaction between IL-11 and IL-11Rα to less than 1 time the level of the interaction between IL-11 and IL-11Rα in the absence of the binder (or in the presence of a suitable control binder), e.g., ≤0.99 time, ≤0.95 time, ≤0.9 time, ≤0.85 time, ≤0.8 time, ≤0.75 time, ≤0.7 time, ≤0.65 time, ≤0.6 time, ≤0.55 time, ≤0.5 time, ≤0.45 time, ≤0.4 time, ≤0.35 time, ≤0.3 time, ≤0.25 time, ≤0.2 time, ≤0.15 time, ≤0.1 time.

[0256] In some embodiments, the binder may inhibit the interaction between IL-11 and gp130 to less than 100% of the level of interaction between IL-11 and gp130 in the absence of the binder (or in the presence of a suitable control binder), e.g., 99% or less, 95% or less, 90% or less, 85% 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. In some embodiments, the binder may inhibit the interaction between IL-11 and gp130 to less than 1 time the level of the interaction between IL-11 and gp130 in the absence of the binder (or in the presence of a suitable control binder), e.g., ≤0.99 time, ≤0.95 time, ≤0.9 time, ≤0.85 time, ≤0.8 time, ≤0.75 time, ≤0.7 time, ≤0.65 time, ≤0.6 time, ≤0.55 time, ≤0.5 time, ≤0.45 time, ≤0.4 time, ≤0.35 time, ≤0.3 time, ≤0.25 time, ≤0.2 time, ≤0.15 time, ≤0.1 time.

[0257] In some embodiments, the binder may inhibit the interaction between IL-11 and IL-11Rα:gp130 to less than 100% of the level of interaction between IL-11 and IL-11Rα:gp130 in the absence of the binder (or in the presence of a suitable control binder), e.g., 99% or less, 95% or less, 90% or less, 85% 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. In some embodiments, the binder may inhibit the interaction between IL-11 and IL-11Rα:gp130 to less than 1 time the level of the interaction between IL-11 and IL-11Rα:gp130 in the absence of the binder (or in the presence of a suitable control binder), e.g., ≤0.99 time, ≤0.95 time, ≤0.9 time, ≤0.85 time, ≤0.8 time, ≤0.75 time, ≤0.7 time, ≤0.65 time, ≤0.6 time, ≤0.55 time, ≤0.5 time, ≤0.45 time, ≤0.4 time, ≤0.35 time, ≤0.3 time, ≤0.25 time, ≤0.2 time, ≤0.15 time, ≤0.1 time.

[0258] In some embodiments, the binder may inhibit the interaction between the IL-11:IL-11Rα complex and gp130 to less than 100% of the level of interaction between the IL-11:IL-11Rα complex and gp130 in the absence of the binder (or in the presence of a suitable control binder), e.g., 99% or less, 95% or less, 90% or less, 85% 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. In some embodiments, the binder may inhibit the interaction between the IL-11:IL-11Rα complex and gp130 to less than 1 time the level of the interaction between the IL-11:IL-11Rα complex and gp130 in the absence of the binder, for example, up to one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times.

[0259] Agents capable of reducing the expression of IL-11 or IL-11 receptors

[0260] In an embodiment of the present invention, an agent capable of inhibiting IL-11-mediated signaling can prevent or reduce the expression of one or more of IL-11, IL-11Rα, or gp130.

[0261] Expression may be gene or protein expression and may be determined as described herein or by methods of the art well known to those skilled in the art. Expression may be by the cells / tissues / organs / organ systems of the subject.

[0262] Suitable agents may be of any type, but in some embodiments, agents capable of preventing or reducing the expression of one or more of IL-11, IL-11Rα or gp130 may be small molecules or oligonucleotides.

[0263] Agents capable of preventing or reducing the expression of one or more of IL-11, IL-11Rα, or gp130 may do so, for example, by inhibiting the transcription of a gene encoding IL-11, IL-11Rα, or gp130; inhibiting post-transcriptional processing of RNA encoding IL-11, IL-11Rα, or gp130; reducing the stability of RNA encoding IL-11, IL-11Rα, or gp130; promoting the degradation of RNA encoding IL-11, IL-11Rα, or gp130; inhibiting post-translational processing of IL-11, IL-11Rα, or gp130 polypeptides; reducing the stability of IL-11, IL-11Rα, or gp130 polypeptides; or promoting the degradation of IL-11, IL-11Rα, or gp130 polypeptides.

[0264] Literature [Taki et al. Clin Exp Immunol

[1998] Apr; 112(1): 133-138] reported a decrease in the expression of IL in rheumatoid synovial cells upon treatment with indomethacin, dexamethasone, or interferon-gamma (IFNγ).

[0265] The present invention considers the use of antisense nucleic acids for preventing / reducing the expression of IL-11, IL-11Rα, or gp130. In some embodiments, the agent capable of preventing or reducing the expression of IL-11, IL-11Rα, or gp130 may cause reduced expression by RNA interference (RNAi).

[0266] In some embodiments, the preparation may be an antisense or small interfering RNA comprising, but not limited to, an inhibitory nucleic acid, such as shRNA or siRNA.

[0267] In some embodiments, the inhibitory nucleic acid is provided in the vector. For example, in some embodiments, the preparation may be a lentiviral vector encoding shRNA for one or more of IL-11, IL-11Rα or gp130.

[0268] Oligonucleotide molecules, particularly RNA, can be utilized to regulate gene expression. This includes antisense oligonucleotides, targeted degradation of mRNA by small interfering RNA (siRNA), post-transcriptional gene silencing (PTG), developmentally regulated sequence-specific translation repression of mRNA by microRNA (miRNA), and targeted transcriptional gene silencing.

[0269] An antisense oligonucleotide is an oligonucleotide, preferably single-stranded, that targets and binds to a target oligonucleotide, e.g., mRNA, by binding to a complementary sequence. When the target oligonucleotide is mRNA, binding of the antisense to the mRNA blocks the translation of the mRNA and the expression of the gene product. An antisense oligonucleotide can be designed to bind to a sense genomic nucleic acid and inhibit the transcription of the target nucleotide sequence.

[0270] Known nucleic acid sequences for IL-11, IL-11Rα, and gp130 (e.g., Accession No.: 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 In terms of known mRNA sequences available from GenBank under gp130), NM_010560.3 GI:225007624 (mouse gp130), NM_001008725.3 GI:300244570 (rat gp130), oligonucleotides can be designed to suppress or silence the expression of IL-11, IL-11Rα, or gp130.

[0271] These oligonucleotides may be of any length, but preferably short, and may be, for example, fewer than 100 nucleotides, for example, 10 to 40 nucleotides, or 20 to 50 nucleotides, and may include a nucleotide sequence having full-complementary or near-complementary (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) with a nucleotide sequence of corresponding length in a target oligonucleotide, for example, IL-11, IL-11Rα, or gp130 mRNA. The complementary region of the nucleotide sequence may be of any length, but preferably at least 5, optionally 50 or fewer nucleotides, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

[0272] Inhibition of the expression of IL-11, IL-11Rα, or gp130 will preferably result in a decrease in the amount of IL-11, IL-11Rα, or gp130 expressed by a cell / tissue / organ / organ system / subject. For example, in a given cell, inhibition of IL-11, IL-11Rα, or gp130 by administration of a suitable nucleic acid will result in a decrease in the amount of IL-11, IL-11Rα, or gp130 expressed by that cell compared to an untreated cell. A preferred degree of inhibition is at least 50%, more preferably at least 60%, 70%, 80%, 85%, or 90%. A level of inhibition of 90% to 100% is considered to be 'silencing' of expression or function.

[0273] The roles of RNAi machinery and small RNAs in the targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci have been demonstrated. Double-stranded RNA (dsRNA)-dependent post-transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific homologous genes for silencing within a short period. This acts as a signal to promote the degradation of sequence-identical mRNAs. 20-nt siRNA is generally long enough to induce gene-specific silencing but short enough to avoid host reactions. The reduction in expression of targeted gene products can be extensive, ranging from 90% silencing induced by a few molecules of siRNA. RNAi-based therapeutics have progressed to Phase I, II, and III clinical trials for many indications (Literature [ Nature 2009 Jan 22; 457(7228):426-433]).

[0274] In the art, these RNA sequences are referred to as "short or small interfering RNA" (siRNA) or "microRNA" (miRNA) depending on their origin. Both types of sequences can be used to downregulate gene expression by binding to complementary RNA and triggering mRNA removal (RNAi) or inhibiting mRNA translation into protein. siRNA is derived by the processing of long double-stranded RNA and is typically of exogenous origin when found in nature. Micro-interfering RNA (miRNA) is an endogenously encoded small non-coding RNA derived by the processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNA containing partially complementary target sequences without RNA cleavage and can degrade mRNA containing fully complementary sequences.

[0275] siRNA ligands are typically double-stranded, and to optimize the effectiveness of RNA-mediated downregulation of the function of a target gene, the length of the siRNA is selected to ensure proper recognition of the siRNA by the RISC complex mediating the recognition of the mRNA target by the siRNA, and thus the siRNA is desirable to be short enough to reduce the host response.

[0276] miRNA ligands are typically single-stranded and possess a region that is partially complementary, enabling the ligand to form a hairpin. miRNA is an RNA gene that is transcribed from DNA but is not translated into protein. The DNA sequence encoding the miRNA gene is longer than the miRNA. This DNA sequence contains the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded DNA molecule, the miRNA sequence and its reverse complement form base pairs to form a partially double-stranded RNA segment. The design of microRNA sequences is [John et al., PLoS Biology This is discussed in , 11(2), 1862-1879, 2004.

[0277] Typically, an RNA ligand intended to mimic the effect of siRNA or miRNA has 10 to 40 ribonucleotides (or synthetic analogs thereof), more preferably 17 to 30 ribonucleotides, more preferably 19 to 25 ribonucleotides, and most preferably 21 to 23 ribonucleotides. In some embodiments of the present invention utilizing double-stranded siRNA, the molecule may have, for example, a symmetric 3' overhang of 1 or 2 (ribonucleotides), typically a dTdT 3' overhang. Based on the disclosures provided herein, those skilled in the art can easily design suitable siRNA and miRNA sequences using resources such as, for example, an Ambion siRNA finder. SiRNA and miRNA sequences may be synthetically generated and exogenously added to cause gene downregulation, or may be generated using an expression system (e.g., a vector). In a preferred embodiment, siRNA is synthesized synthetically.

[0278] Longer double-stranded RNA can be processed in cells to generate siRNA (see, for example, [Myers (2003) Nature Biotechnology 21:324-328]). Longer dsRNA molecules may have, for example, symmetric 3' or 5' overhangs of one or two (ribonucleotides) or blunt ends. Longer dsRNA molecules may be 25 nucleotides or longer. Preferably, longer dsRNA molecules are 25 to 30 nucleotides long. More preferably, longer dsRNA molecules are 25 to 27 nucleotides long. Most preferably, longer dsRNA molecules are 27 nucleotides long. dsRNAs with a length of 30 nucleotides or more can be expressed using the vector pDECAP (Reference [Shinagawa et al., Genes and Dev., 17, 1340-5, 2003]).

[0279] Another alternative is the expression of short hairpin RNA molecules (shRNA) in cells. shRNA is more stable than synthetic siRNA. shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complementary to the gene target. In cells, shRNA is processed into siRNA by Dicer, which degrades the target gene mRNA and inhibits expression. In a preferred embodiment, shRNA is generated endogenously (in the cell) by transcription from a vector. In the cell, shRNA can be generated by transfecting cells with a vector encoding an shRNA sequence under the control of an RNA polymerase III promoter, such as a human H1 or 7SK promoter, or an RNA polymerase II promoter. Alternatively, shRNA can be synthesized exogenously (in vitro) by transcription from a vector. Subsequently, shRNA can be directly introduced into the cell. Preferably, the shRNA molecule comprises a partial sequence of IL-11, IL-11Rα, or gp130. Preferably, the shRNA sequence is 40 to 100 base pairs long, more preferably 40 to 70 base pairs long. The stem of the hairpin is preferably 19 to 30 base pairs long. The stem contains GU pairings, which can stabilize the hairpin structure.

[0280] siRNA molecules, longer dsRNA molecules, or miRNA molecules can preferably be recombinantly produced by transcription of a nucleic acid sequence contained within a vector. Preferably, the siRNA molecules, longer dsRNA molecules, or miRNA molecules contain a partial sequence of IL-11, IL-11Rα, or gp130.

[0281] In one embodiment, siRNA, longer dsRNA, or miRNA is generated endogenously (in a cell) by transcription from the vector. The vector may be introduced into the cell in any manner known in the art. Optionally, the expression of the RNA sequence may be regulated using a tissue-specific (e.g., heart, liver, or kidney-specific) promoter. In a further embodiment, siRNA, longer dsRNA, or miRNA is generated exogenously (in vitro) by transcription from the vector.

[0282] A suitable vector may be an oligonucleotide vector positioned to express an oligonucleotide preparation capable of inhibiting IL-11, IL-11Rα, or gp130. Such a vector may be a viral vector or a plasmid vector. The therapeutic oligonucleotide may be incorporated into the genome of a viral vector and operably linked to a regulatory sequence, e.g., a promoter, that drives its expression. The term "operably linked" may include a situation in which a selected nucleotide sequence and a regulatory nucleotide sequence are covalently linked so that the expression of the nucleotide sequence occurs under the influence or control of the regulatory sequence. Thus, the regulatory sequence is operably linked to the selected nucleotide sequence if the regulatory sequence can perform the transcription of the nucleotide sequence that forms part or all of the selected nucleotide sequence.

[0283] Viral vectors encoding promoter-expressed siRNA sequences are known in the art and offer the benefit of long-term expression of therapeutic oligonucleotides. Examples include lentiviruses (literature [ Nature 2009 Jan 22; 457(7228):426-433]), adenovirus (literature [Shen et al., FEBS Lett 2003 Mar 27;539(1-3)111-4]) and retroviruses (Literature [Barton and Medzhitov PNASIncludes [November 12, 2002 vol.99, no.23 14943-14945]).

[0284] In other embodiments, the vector may be positioned to facilitate the delivery of therapeutic oligonucleotides to a site requiring inhibition of IL-11, IL-11Rα, or gp130 expression. Such vectors typically involve complexing the oligonucleotides with a positively charged vector (e.g., cationic cell-penetrating peptides, cationic polymers and dendrimers, and cationic lipids); conjugating the oligonucleotides with small molecules (e.g., cholesterol, bile acids, and lipids), polymers, antibodies, and RNA; or encapsulating the oligonucleotides in a nanoparticle formulation (reference [Wang et al., AAPS It involves J. 2010 Dec; 12(4): 492-503).

[0285] In one embodiment, the vector may contain nucleic acid sequences in both sense orientation and antisense orientation, so that when expressed as RNA, the sense compartment and the antisense compartment will associate to form double-stranded RNA.

[0286] Alternatively, siRNA molecules can be synthesized using standard solid-phase or solution-phase synthesis techniques known in the art. The linkage between nucleotides may be a phosphodiester bond, or alternatively, for example, the linker of the formula P(O)S, (thioate); P(S)S, (dithioate); P(O)NR'2; P(O)R'; P(O)OR6; CO; or CONR'2 is conjugated to adjacent nucleotides through -O- or -S-, where R is H (or salt) or alkyl (1-12C) and R6 is alkyl (1-9C).

[0287] Modified nucleotide bases other than naturally occurring bases can be used, and advantageous properties can be imparted to siRNA molecules containing them.

[0288] For example, modified bases can increase the stability of siRNA molecules, thereby reducing the amount required for silencing. Providing modified bases can also provide siRNA molecules that are more or less stable than unmodified siRNA.

[0289] The term 'modified nucleotide base' encompasses nucleotides having covalently modified bases and / or sugars. For example, modified nucleotides include nucleotides having sugars covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Therefore, modified nucleotides may also include 2'-substituted sugars, e.g., 2'-O-methyl-; 2'-O-alkyl; 2'-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro-; 2'-halo or azido-ribose, carboxylic sugar analogs, α-anomeric sugars; epimeric sugars, e.g., arabinose, xylose or lyxose, pyranose sugars, furanose sugars, and sedoheptulose.

[0290] Modified nucleotides are known in the art and include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles. These classes of pyrimidines and purines are known in the art, including pseudoisocytosine, N4,N4-ethanocytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, -D-mannosylqueosine, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-methyl oxyacetate, pseudouracil, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-methyl oxyacetate, uracil 5-oxyacetate, queosine, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine, and It includes 2,6-diaminopurine, methylpseudouracil, 1-methylguanine, and 1-methylcytosine.

[0291] Methods for the use of RNAi to silence genes in C. elegans, fruit flies, plants, and mammals are known in the art (Reference [Fire A, et al., 1998 Nature 391:806-811]; Literature[Fire, A. Trends Genet. 15, 358-363 (1999)]; Literature[Sharp, PA RNA interference 2001. Genes Dev. 15, 485-490 (2001)]; Literature[Hammond, SM, et al., Nature Rev . Genet . 2, 110-1119 (2001)]; literature[Tuschl, T. Chem. Biochem . 2, 239-245 (2001)]; literature[Hamilton, A. et al., Science 286, 950-952 (1999)]; literature[Hammond, SM, et al., Nature 404, 293-296 (2000)]; literature[Zamore, PD, et al., Cell 101, 25-33 (2000)]; Literature[Bernstein, E., et al., Nature 409, 363-366 (2001)]; literature[Elbashir, SM, et al., Genes Dev . 15, 188-200 (2001)]; WO0129058; WO9932619, and literature[Elbashir SM, et al., 2001 Nature 411:494-498]).

[0292] Accordingly, the present invention provides a nucleic acid capable of inhibiting the expression of IL-11, IL-11Rα, or gp130 by RNAi when introduced into or expressed within mammalian, e.g., human, cells expressing IL-11, IL-11Rα, or gp130, suitably.

[0293] Nucleic acid sequences for IL-11, IL-11Rα, and gp130 (e.g., Accession No.: 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 Known mRNA sequences available from GenBank under gp130), NM_010560.3 GI:225007624 (mouse gp130), NM_001008725.3 GI:300244570 (rat gp130) oligonucleotides can be designed to inhibit or silence the expression of IL-11, IL-11Rα, or gp130.

[0294] The nucleic acid may have substantial sequence identity with IL-11, IL-11Rα, or gp130 mRNA, or a portion of the sequence complementary to said mRNA, as defined under, for example, GenBank accession numbers NM_000641.3 GI:391353405(IL-11), NM_001142784.2 GI:391353394(IL-11Rα), NM_001190981.1 GI:300244534(gp130).

[0295] The nucleic acid may be double-stranded siRNA (as will be understood by those skilled in the art and as further explained below, siRNA molecules may also contain a short 3' DNA sequence).

[0296] Alternatively, the nucleic acid can be DNA (typically double-stranded DNA), which, when transcribed in mammalian cells, yields RNA with two complementary parts joined by spacers, and the RNA takes on a hairpin shape when the complementary parts hybridize with each other. In mammalian cells, the hairpin structure can be cleaved from the molecule by the enzyme Dicer, yielding two distinct but hybridized RNA molecules.

[0297] In some preferred embodiments, the nucleic acid is generally targeted to one of the sequences of SEQ ID NO 4 to 7 (IL-11) or one of SEQ ID NO 8 to 11 (IL-11Rα).

[0298] Only single-stranded (i.e., non-self-hybridized) regions of mRNA transcripts are expected to be suitable targets for RNAi. Accordingly, it is suggested that other sequences in the IL-11 or IL-11Rα mRNA transcript that are very close to the sequence indicated by SEQ ID NO 4 to 7 or 8 to 11 may also be suitable targets for RNAi. These target sequences are preferably 17 to 23 nucleotide long and preferably overlap with one of SEQ ID NO 4 to 7 or 8 to 11 by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or all 19 nucleotides (at either end of SEQ ID NO 4 to 7 or 8 to 11).

[0299] Accordingly, the present invention provides a nucleic acid capable of inhibiting the expression of IL-11 or IL-11Rα by RNAi when introduced into or expressed within a mammalian cell expressing IL-11 or IL-11Rα, suitably, the nucleic acid is generally targeted to one of the sequences of SEQ ID NO 4 to 7 or 8 to 11.

[0300] "Generally targeted" means that the nucleic acid may target sequences that overlap with SEQ ID NO 4 to 7 or 8 to 11. In particular, the nucleic acid may target sequences within the mRNA of human IL-11 or IL-11Rα that are slightly longer or shorter than one of SEQ ID NO 4 to 7 or 8 to 11 (preferably 17 to 23 nucleotides long), but identical to one of SEQ ID NO 4 to 7 or 8 to 11.

[0301] Perfect identity / complementarity between the nucleic acid of the present invention and the target sequence is expected to be desirable but not essential. Accordingly, the nucleic acid of the present invention may contain a single mismatch compared to the mRNA of IL-11 or IL-11Rα. However, since the presence of even a single mismatch tends to cause reduced efficiency, the absence of a mismatch is expected to be desirable. When present, a 3' overhang may be excluded in consideration of the number of mismatches.

[0302] The term “complementarity” is not limited to conventional base pairing between nucleic acids composed of naturally occurring ribo- and / or deoxyribonucleotides, but also includes base pairing between the mRNA and nucleic acid of the present invention comprising non-natural nucleotides.

[0303] In one embodiment, the nucleic acid (referred herein as double-stranded siRNA) comprises double-stranded RNA sequences indicated by SEQ ID NOs 12 to 15. In another embodiment, the nucleic acid (referred herein as double-stranded siRNA) comprises double-stranded RNA sequences indicated by SEQ ID NOs 16 to 19.

[0304] However, it is also expected that slightly shorter or longer sequences regarding the same region of IL-11 or IL-11Rα mRNA will be effective. In particular, double-stranded sequences of 17 to 23 bp in length are also expected to be effective.

[0305] The strands forming double-stranded RNA may have a short 3' dinucleotide overhang, which can be DNA or RNA. The use of a 3' DNA overhang has no effect on siRNA activity compared to a 3' RNA overhang, but it reduces the cost of chemical synthesis of the nucleic acid strand (Elbashir et al., 2001c). For this reason, a DNA dinucleotide may be preferred.

[0306] When present, dinucleotide overhangs may be symmetrical to each other, but are not essential. In fact, the 3' overhang of the sense (top) strand is irrelevant to RNAi activity because it does not participate in mRNA recognition and degradation (Reference [Elbashir et al., 2001a, 2001b, 2001c]).

[0307] Although RNAi experiments in Drosophila indicate that the antisense 3' overhang may participate in mRNA recognition and targeting (Elbashir et al. 2001c), the 3' overhang does not appear to be required for the RNAi activity of siRNA in mammalian cells. Therefore, improper annealing of the 3' overhang is thought to have little effect in mammalian cells (references [Elbashir et al. 2001c; Czauderna et al. 2003]).

[0308] Therefore, any dinucleotide overhang can be used on the antisense strand of siRNA. However, the dinucleotide is preferably -UU or -UG (or -TT or -TG if the overhang is DNA), more preferably -UU (or -TT). The -UU (or -TT) dinucleotide overhang is most effective and is consistent with the RNA polymerase III end of the transcription signal (the terminator signal is TTTTT) (i.e., it can form a part of it). Accordingly, such dinucleotides are most preferred. Dinucleotides AA, CC, and GG may also be used, but are less effective and consequently less preferred.

[0309] Furthermore, the 3' overhang can be entirely omitted from the siRNA.

[0310] The present invention also provides a single-stranded nucleic acid (referred herein as single-stranded siRNA) composed of one component strand of each of the aforementioned double-stranded nucleic acids, preferably having a 3'-overhang but optionally not having one. The present invention also provides a kit containing a pair of such single-stranded nucleic acids capable of hybridizing with each other in vitro to form the aforementioned double-stranded siRNA, which can then be introduced into a cell.

[0311] The present invention also provides DNA that yields RNA (also referred to herein as shRNA) having two complementary parts capable of self-hybridizing to produce a double-stranded motif when transcribed in a mammalian cell, for example, by a sequence selected from the group consisting of SEQ ID NO: 12 to 15 or 16 to 19 or a different sequence by single base pair substitution from any one of the aforementioned sequences.

[0312] The complementary parts will generally be joined by a spacer, and this spacer has a length and sequence suitable for the two complementary parts to hybridize with each other. The two complementary (i.e., sense and antisense) parts can be joined 5'-3' in any order. The spacer will typically be a short sequence of approximately 4 to 12 nucleotides, preferably 4 to 9 nucleotides, more preferably 6 to 9 nucleotides.

[0313] Preferably, the 5' end of the spacer (immediately 3' of the upstream complementary part) consists of the nucleotide -UU- or -UG-, also preferably -UU- (although the use of these specific dinucleotides is not essential). A suitable spacer recommended for use in the pSuper system of OligoEngine (Seattle, Washington, USA) is UUCAAGAGA. In this case and in other cases, the ends of the spacer are hybridized to extend the double-strand motif by a small number (e.g., 1 or 2) of base pairs, for example, the exact sequence of SEQ ID NO 12 to 15 or 16 to 19.

[0314] Similarly, the transcribed RNA preferably contains a 3' overhang from the downstream complementary portion. Again, this is preferably -UU or -UG, more preferably -UU.

[0315] After that, these shRNA molecules can be cleaved by the enzyme Dicer in mammalian cells to yield double-stranded siRNA as described above, wherein one or each strand of the hybridized dsRNA contains a 3' overhang.

[0316] The technique for synthesizing nucleic acids according to the present invention is, of course, well known in the art.

[0317] Those skilled in the art can construct transcription vectors suitable for the DNA of the present invention using well-known techniques and commercially available materials. In particular, the DNA will be associated with control sequences including a promoter and a transcription termination signal.

[0318] Particularly suitable are the commercially available pSuper and pSuperior systems from OligoEngine (Seattle, Washington, USA). These utilize a polymerase-III promoter (H1) and a T5 transcription terminator sequence consisting of two U residues at the 3' end of the transcript (which provides a 3' UU overhang of a single strand of siRNA after Dicer processing).

[0319] Another suitable system is described in the literature [Shin et al. (RNA, 2009 May; 15(5): 898-910)] and uses another polymerase-III promoter (U6).

[0320] The double-stranded siRNA of the present invention is produced using known techniques as described below. In a test tube or In vivo It can be introduced into mammalian cells and inhibit the expression of IL-11 or receptors for IL-11.

[0321] Similarly, the DNA-containing transcription vector of the present invention is used for the transient or stable expression of RNA using known techniques as described below. In a test tube or In vivo It is introduced into tumor cells and can also inhibit the expression of IL-11 or receptors for IL-11.

[0322] Accordingly, the present invention also provides a method for inhibiting the expression of IL-11 or a receptor for IL-11 in mammalian, e.g., human, cells, and the method comprises the step of administering the double-stranded siRNA of the present invention or the transcription vector of the present invention to the cells.

[0323] Similarly, furthermore, the present invention provides a method for treating metabolic diseases, the method comprising the step of administering the double-stranded siRNA of the present invention or the transcription vector of the present invention to a subject.

[0324] Furthermore, the present invention provides a double-stranded siRNA of the present invention or a transcription vector of the present invention for use in a treatment method, preferably a method for treating metabolic diseases.

[0325] Furthermore, the present invention provides the use of the double-stranded siRNA of the present invention or the transcription vector of the present invention in the manufacture of a drug for the treatment of metabolic diseases.

[0326] Furthermore, the present invention provides a composition comprising the double-stranded siRNA of the present invention or the transcription vector of the present invention together with one or more pharmaceutically acceptable carriers. Suitable carriers include lipophilic carriers or vehicles, which can facilitate permeation of the cell membrane.

[0327] Materials and methods suitable for administering the siRNA duplex and DNA vector of the present invention are well known in the art, and improved methods are being developed considering the potential of RNAi technology.

[0328] Generally, many techniques are available to introduce nucleic acids into mammalian cells. The choice of technique depends on the nucleic acid In a test tube into cultured cells or In vivo It will depend on whether it is transferred to the patient's cells. In a test tube Suitable techniques for transferring nucleic acids into mammalian cells include the use of liposomes, electroporation, microinjection, cell fusion, DEAE, dextran, and calcium phosphate precipitation. In vivo Gene transfer techniques include transfection by viral (typically retrovirus) vectors and viral envelope protein-liposome-mediated transfection (reference [Dzau et al. (2003) Trends in Biotechnology Includes 11, 205-210]).

[0329] In particular, the nucleic acid of the present invention In vitro and In vivo Techniques suitable for intracellular administration in both are disclosed in the following paper:

[0330] General review: Literature [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, GJ 2002. RNA interference. Nature. 418:244-51]. McManus, MT, and PA Sharp. 2002. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet. 3:737-47]. Scherr, M., MA Morgan, and M. Eder. 2003b. Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem. 10:245-56]. Shuey, DJ, DE McCallus, and T. Giordano. 2002. RNAi: gene-silencing in therapeutic intervention. Drug Disco Today. 7:1040-6].

[0331] Systemic delivery using liposomes: Literature [Lewis, DL, JE Hagstrom, AG Loomis, JA Wolff, and H. Herweijer. 2002. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat Genet. 32:107-8]. Literature [Paul, CP, PD Good, I. Winer, and DR Engelke. 2002. Effective expression of small interfering RNA in human cells. Nat Biotechnol. 20:505-8]. Literature [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. 9:347-51]. Literature [Sorensen, DR, M. Leirdal, and M. Sioud. 2003. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol. 327:761-6].

[0332] virus mediated transfer: [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, GM, and R. Medzhitov. 2002. Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci US A. 99:14943-5]. [Devroe, E., and PA 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 PM Chaudhary. 2003. Use of lentiviral vectors for delivery of small interfering RNA. Cancer Biol Ther. 2:206-10]. [Qin, XF, DS An, IS Chen, and D. Baltimore. 2003. Inhibition of HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci US 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., AK Buck, X. Liu, M. Winkler, and SN Reske. 2003. Gene silencing by adenovirus-delivered siRNA. FEBS Lett. 539:111-4].

[0333] Peptide delivery: Literature [Morris, MC, L. Chaloin, F. Heitz, and G. Divita. 2000. Translocating peptides and proteins and their use for gene delivery. Curr Opin Biotechnol. 11:461-6]. Literature [Simeoni, F., MC 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 that may be suitable for delivering siRNA to target cells are based on nanoparticles or nanocapsules, e.g., those described in U.S. Patents No. 6,649,192B and 5,843,509B.

[0334] Inhibition of IL-11-mediated signaling

[0335] In an embodiment of the present invention, a formulation capable of inhibiting the action of IL-11 may possess one or more of the following functional characteristics:

[0336] · Inhibition of IL-11-mediated signaling;

[0337] · Inhibition of signaling mediated by the binding of IL-11 to the IL-11Rα:gp130 receptor complex;

[0338] · Signaling mediated by the binding of the IL-11:IL-11Rα complex to gp130 (i.e., IL-11 trans Inhibition of signaling;

[0339] · Inhibition of IL-11-mediated processes;

[0340] · Inhibition of gene / protein expression of IL-11 and / or IL-11Rα.

[0341] These characteristics can be determined by the analysis of the relevant formulation in an appropriate test, and such a test may involve a comparison of the performance of the formulation with that of a suitable control formulation. A person skilled in the art can identify appropriate control conditions for a given test.

[0342] IL-11-mediated signaling and / or processes mediated by IL-11 include signaling mediated by fragments of IL-11 and polypeptide complexes containing IL-11 or fragments thereof. IL-11-mediated signaling may be signaling mediated by human IL-11 and / or mouse IL-11. Signaling mediated by IL-11 may occur after IL-11 or said complex binds to a receptor to which IL-11 or said complex binds.

[0343] In some embodiments, the formulation may inhibit the biological activity of IL-11 or an IL-11-containing complex.

[0344] In some embodiments, the formulation is an antagonist of one or more signaling pathways activated by signaling through receptors comprising IL-11Rα and / or gp130, e.g., IL-11Rα:gp130. In some embodiments, the formulation may inhibit signaling through one or more immune receptor complexes comprising IL-11Rα and / or gp130, e.g., IL-11Rα:gp130. In various aspects of the invention, the formulation provided herein is an IL-11-mediated cis and / or trans It may inhibit signaling. In some embodiments, according to various aspects of the present invention, the formulation provided herein is IL-11-mediated cis It can inhibit signaling.

[0345] In some embodiments, the agent may inhibit IL-11-mediated signaling to less than 100% of the level of signaling in the absence of the agent (or in the presence of a suitable control agent), e.g., 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. In some embodiments, the agent may reduce IL-11-mediated signaling to less than 1 time the level of signaling in the absence of the agent (or in the presence of an appropriate control agent), e.g., ≤0.99 time, ≤0.95 time, ≤0.9 time, ≤0.85 time, ≤0.8 time, ≤0.75 time, ≤0.7 time, ≤0.65 time, ≤0.6 time, ≤0.55 time, ≤0.5 time, ≤0.45 time, ≤0.4 time, ≤0.35 time, ≤0.3 time, ≤0.25 time, ≤0.2 time, ≤0.15 time, ≤0.1 time.

[0346] In some embodiments, IL-11-mediated signaling may be signaling mediated by the binding of IL-11 to the IL-11Rα:gp130 receptor. Such signaling may be analyzed, for example, by treating cells expressing IL-11Rα and gp130 with IL-11, or by stimulating IL-11 production in cells expressing IL-11Rα and gp130.

[0347] IC of agents for inhibition of IL-11-mediated signaling 50 This can be determined by culturing Ba / F3 cells expressing IL-11Rα and gp130 in the presence of human IL-11 and the agent, and measuring the incorporation of 3H-thymidine into the DNA. In some embodiments, the agent has an IC of 10 μg / ml or less in this assay, preferably ≤ 5 μg / ml, ≤ 4 μg / ml, ≤ 3.5 μg / ml, ≤ 3 μg / ml, ≤ 2 μg / ml, ≤ 1 μg / ml, ≤ 0.9 μg / ml, ≤ 0.8 μg / ml, ≤ 0.7 μg / ml, ≤ 0.6 μg / ml, or ≤ 0.5 μg / ml. 50 It can represent.

[0348] In some embodiments, IL-11-mediated signaling may be signaling mediated by the binding of an IL-11:IL-11Rα complex to gp130. In some embodiments, the IL-11:IL-11Rα complex, e.g., 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, may be soluble. In some embodiments, the soluble IL-11Rα is a soluble (secreted) isoform of IL-11Rα or a free product of proteolytic cleavage of the extracellular domain of IL-11Rα bound to a cell membrane.

[0349] In some embodiments, the IL-11:IL-11Rα complex may be cell-bound, for example, a complex of IL-11Rα and IL-11 bound to a cell membrane. Signaling mediated by the binding of the IL-11:IL-11Rα complex to gp130 can be analyzed by treating cells expressing gp130 with a recombinant fusion protein comprising the IL-11:IL-11Rα complex, for example, IL-11 bound to the extracellular domain of IL-11Rα, for example, hyper IL-11, by a peptide linker. Hyper IL-11 was constructed using a fragment of IL-11Rα (amino acid residues 1 to 317 consisting of domains 1 to 3; UniProtKB: Q14626) and IL-11 (amino acid residues 22 to 199 of UniProtKB: P20809) with a 20-amino acid length linker (SEQ ID NO: 20). The amino acid sequence for Hyper IL-11 is indicated by SEQ ID NO: 21.

[0350] In some embodiments, the formulation may inhibit signaling mediated by the binding of the IL-11:IL-11Rα complex to gp130, and may also inhibit signaling mediated by the binding of IL-11 to the IL-11Rα:gp130 receptor.

[0351] In some embodiments, the formulation may inhibit the process mediated by IL-11.

[0352] In some embodiments, the formulation may inhibit gene / protein expression of IL-11 and / or IL-11Rα. Gene and / or protein expression may be measured as described herein or by methods of the art well known to those skilled in the art.

[0353] In some embodiments, the agent may inhibit the gene / protein expression of IL-11 and / or IL-11Rα to less than 100% of the expression level in the absence of the agent (or in the presence of a suitable control agent), e.g., 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. In some embodiments, the agent may inhibit the gene / protein expression of IL-11 and / or IL-11Rα to less than 1 of the expression level in the absence of the agent (or in the presence of an appropriate control agent), e.g., ≤0.99, ≤0.95, ≤0.9, ≤0.85, ≤0.8, ≤0.75, ≤0.7, ≤0.65, ≤0.6, ≤0.55, ≤0.5, ≤0.45, ≤0.4, ≤0.35, ≤0.3, ≤0.25, ≤0.2, ≤0.15, ≤0.1.

[0354] Treatment / Prevention of metabolic diseases

[0355] The present invention provides a method and articles (preparations and compositions) for the treatment and / or prevention of metabolic diseases, for example, metabolic diseases as described herein.

[0356] Treatment is achieved by inhibition of IL-11-mediated signaling (i.e., antagonism of IL-11-mediated signaling). In other words, the present invention provides treatment / prevention of metabolic diseases through inhibition of IL-11-mediated signaling in, for example, cells, tissues / organs / organ systems / subjects. In some embodiments, inhibition of IL-11-mediated signaling according to the present disclosure includes inhibition of IL-11-mediated signaling in liver cells (e.g., hepatocytes).

[0357] Accordingly, the present invention provides a preparation capable of inhibiting interleukin 11 (IL-11)-mediated signaling for use in a method of treating or preventing metabolic diseases.

[0358] In addition, the use of a preparation capable of inhibiting interleukin 11 (IL-11)-mediated signaling is provided for use in the manufacture of a drug to be used in a method for treating or preventing metabolic diseases.

[0359] Furthermore, a method for treating or preventing metabolic diseases is provided, and the method includes the step of administering a therapeutically effective amount of an agent capable of inhibiting interleukin 11 (IL-11)-mediated signaling to a subject requiring treatment.

[0360] The applicability of the present invention extends to the treatment / prevention of any metabolic disease. The present invention also provides for the treatment / prevention of diseases / pathological conditions caused or exacerbated by metabolic diseases. In some embodiments, the present invention provides for the treatment / prevention of diseases / pathological conditions in subjects in whom metabolic diseases provide a poor prognosis.

[0361] In some embodiments, the metabolic disease to be treated / prevented may be characterized by an increase in the expression of IL-11 and / or IL-11Rα (i.e., gene and / or protein expression) in an organ / tissue / subject affected by the metabolic disease compared to a normal organ / tissue / subject (i.e., absence of the metabolic disease), for example.

[0362] The treatment / prevention of a metabolic disease according to the present invention may be a metabolic disease associated with the upregulation of IL-11, for example, the upregulation of IL-11 in cells or tissues where symptoms of the disease may be manifested or occur, or the upregulation of extracellular IL-11 or IL-11Rα.

[0363] Metabolic diseases may affect any tissue, organ, or organ system. In some embodiments, metabolic diseases may affect several tissues / organs / organ systems.

[0364] In some embodiments, metabolic disease affects one or more of the liver, pancreas, cardiovascular system, digestive system, excretory system, respiratory system, renal system, reproductive system, circulatory system, muscular system, endocrine system, exocrine system, lymphatic system, immune system, nervous system, and / or skeletal system.

[0365] According to various embodiments disclosed herein, in some embodiments, the metabolic disease is characterized by reduced function of the liver or reduced function of liver cells (e.g., hepatocytes) compared to function in the absence of the metabolic disease. In some embodiments, the metabolic disease is characterized by increased levels of ALT and / or AST and / or decreased levels of GSH (e.g., in serum) compared to levels in the absence of the metabolic disease.

[0366] In some embodiments, the metabolic disease is characterized by inflammation and / or fibrosis of the liver. In some embodiments, the metabolic disease is characterized by increased gene / protein expression of pro-inflammatory and / or fibrosis-promoting factors (e.g., IL-11, IL-6, CCL2 and / or CCL5) by liver cells (e.g., hepatocytes) compared to levels in the absence of the metabolic disease. In some embodiments, the metabolic disease is characterized by increased gene / protein expression of collagen by liver cells and / or increased collagen content of the liver compared to levels in the absence of the metabolic disease.

[0367] In some embodiments, the metabolic disease is characterized by an increased number / ratio of myofibroblasts in the liver compared to the number / ratio of myofibroblasts in the liver in the absence of the metabolic disease.

[0368] In some embodiments, the metabolic disease is characterized by increased apoptosis and / or necrosis of liver cells (e.g., hepatocytes) compared to the level in the absence of the metabolic disease. In some embodiments, the metabolic disease is characterized by an increase in the number / ratio of apoptotic and / or necrotic liver cells compared to the number / ratio in the absence of the metabolic disease.

[0369] In some embodiments, the metabolic disease is characterized by increased gene / protein expression of fatty acid synthase (FASN) by liver cells (e.g., hepatocytes) compared to levels in the absence of the metabolic disease. In some embodiments, the metabolic disease is characterized by increased levels of reactive oxygen species (ROS) in liver cells (e.g., hepatocytes) compared to levels in the absence of the metabolic disease. In some embodiments, the metabolic disease is characterized by increased gene / protein expression of NOX4 by liver cells (e.g., hepatocytes) compared to levels in the absence of the metabolic disease. In some embodiments, the metabolic disease is characterized by increased levels of ERK and / or JNK activation in liver cells (e.g., hepatocytes) compared to levels in the absence of the metabolic disease.

[0370] In some embodiments, the metabolic disease is characterized by increased triglyceride levels in the liver or in liver cells (e.g., hepatocytes) compared to levels in the absence of the metabolic disease. In some embodiments, the metabolic disease is characterized by hyperglycemia. In some embodiments, the metabolic disease is characterized by hypertriglyceridemia. In some embodiments, the metabolic disease is characterized by hypercholesterolemia. In some embodiments, the metabolic disease is characterized by increased body weight compared to body weight in the absence of the metabolic disease. In some embodiments, the metabolic disease is characterized by increased liver weight compared to liver weight in the absence of the metabolic disease.

[0371] Treatment may be effective in reducing, delaying, or preventing the onset or progression of metabolic disease. Treatment may be effective in reducing, delaying, or preventing the worsening of one or more symptoms of metabolic disease. Treatment may be effective in improving one or more symptoms of metabolic disease. Treatment may be effective in reducing the severity of one or more symptoms of metabolic disease and / or reversing them. Treatment may be effective in reversing the effects of metabolic disease.

[0372] Prevention may refer to the prevention of the onset of metabolic disease and / or the prevention of the exacerbation of metabolic disease, for example, the prevention of progression to the terminal / chronic stage of metabolic disease.

[0373] According to various embodiments of the present invention, a method for treating and / or preventing metabolic diseases according to the present invention may include one or more of the following:

[0374] Decrease in blood lipid levels;

[0375] Decrease in blood glucose levels;

[0376] Increase in glucose tolerance (e.g., of glucose-intolerance subjects);

[0377] Increase in insulin tolerance (e.g., of insulin-resistant subjects);

[0378] Increase in pancreatic function;

[0379] Reduction in body weight (e.g., of overweight / obese subjects);

[0380] Reduction in body fat mass;

[0381] Increase in lean mass;

[0382] Decrease in fasting blood glucose levels;

[0383] Decrease in serum triglyceride levels;

[0384] Decrease in serum cholesterol levels;

[0385] Increased glucose tolerance;

[0386] Increase in pancreatic function (e.g., exocrine and / or endocrine function);

[0387] Increased growth of pancreatic tissue;

[0388] Regeneration of pancreatic tissue;

[0389] Increase in pancreatic weight;

[0390] Inhibition of PSC-to-myofibroblast transition by PSCs;

[0391] Decrease in the number / ratio of pancreatic myofibroblasts;

[0392] Decrease in pancreatic hydroxyproline levels;

[0393] Decrease in pancreatic collagen levels;

[0394] Reduction of pancreatic injury;

[0395] Reduction of pancreatic islet cell hyperplasia;

[0396] Decrease in glucagon expression;

[0397] Increase in insulin expression;

[0398] Increase in body weight (e.g., of a subject with wasting disease, e.g., cachexia);

[0399] Decrease in the expression of IL-11 protein in the liver;

[0400] Decrease in hepatic Erk activation;

[0401] Decrease in intrahepatic JNK activation;

[0402] Reduction in intrahepatic caspase-3 cleavage;

[0403] Reduction in liver ROS levels;

[0404] Decrease in hepatic NOX4 expression;

[0405] For example, reduction of liver steatosis;

[0406] Decrease in liver triglyceride levels;

[0407] Decrease in fatty acid synthase expression;

[0408] Decrease in serum ALT and / or AST levels;

[0409] Reduction in the expression of pro-inflammatory factors (e.g., TNFα, CCL2, CCL5, IL-6, CXCL5, and / or CXCL1);

[0410] Decrease in the expression of fibrosis-promoting factors (e.g., IL-11, TIMP1, ACTA2, TGFβ1, MMP2, TIMP2, MMP9, COL1A2, COL1A1 and / or COL3A1);

[0411] Decrease in serum TGFβ1 levels;

[0412] Reduction in the expression / production of IL-11, ACTA2, MMP2, TGFβ1, PDGF, ANG II, bFGF, CCL2, and / or H2O2 by HSC;

[0413] Inhibition of HSC-to-myofibroblast transition by HSCs;

[0414] Decrease in the number / ratio of myofibroblasts in the liver;

[0415] Decrease in liver hydroxyproline levels;

[0416] Decrease in liver collagen levels;

[0417] Increase in liver function;

[0418] Increase in serum GSH levels;

[0419] Increased function of organs / tissues affected by metabolic diseases;

[0420] Reduction of liver damage;

[0421] Reduction in hepatocyte apoptosis;

[0422] Reduction in apoptosis as a result of lipotoxicity;

[0423] Reduction of IL-11-mediated signaling within hepatocytes; and

[0424] Decrease in the number / ratio of CD45+ cells in the liver.

[0425] According to the various aspects and embodiments described herein, treatment / prevention of metabolic disease specifically includes, for example, inhibition, reduction, or prevention of lipotoxicity in a given organ system / organ / tissue / cell type. In some embodiments, treatment / prevention of metabolic disease includes inhibition / reduction / prevention of lipotoxicity in the liver, for example, in hepatocytes.

[0426] administration

[0427] The administration of an agent capable of inhibiting IL-11-mediated signaling is preferably present in a "therapeutic effective" or "prophylactic effective" amount, which is sufficient to provide benefit to the subject.

[0428] The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease and the nature of the formulation. Prescription of treatment, e.g., decisions regarding dosage, etc., falls under the responsibility of general practitioners and other medical doctors, who typically consider the disease / pathology to be treated, the condition of the individual subject, the delivery site, the method of administration, and other factors known to the physician. Examples of the aforementioned techniques and protocols can be found in the literature [Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins].

[0429] Multiple dosage formulations may be provided. One or more of the dosages, or each of them, may be accompanied by the simultaneous or sequential administration of another therapeutic agent.

[0430] Multiple doses may be separated by scheduled time intervals, and these time intervals may be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. For example, doses may be given once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0431] In therapeutic applications, a formulation capable of inhibiting IL-11-mediated signaling may preferably be formulated as a pharmaceutical or pharmaceutical preparation with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, coloring agents, flavoring agents, and sweetening agents.

[0432] As used herein, the term “pharmaceuticalally acceptable” refers to a compound, component, substance, composition, dosage form, etc., suitable for use in contact with tissues in the subject (e.g., human) without excessive toxicity, irritation, allergic reaction, or other problems or complications, in proportion to a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Each carrier, adjuvant, excipient, etc., must also be “acceptable” in the sense of compatibility with other components of the formulation.

[0433] Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical literature, for example, [Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990]; and [Handbook of Pharmaceutical Excipients, 2nd edition, 1994].

[0434] The formulation may be prepared by any method well known in the pharmaceutical field. Such methods include the step of associating an active compound with a carrier comprising one or more accessory components. Generally, the formulation is prepared by uniformly and closely associating the active compound with a carrier (e.g., a liquid carrier, a finely divided solid carrier, etc.) and then shaping the product if necessary.

[0435] The formulation may be prepared for local, parenteral, systemic, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, subcutaneous, oral, or transdermal routes of administration, including injection. The injectable formulation may contain the selected preparation in a sterile or isotonic medium. The formulation and method of administration may be selected depending on the preparation and the disease to be treated.

[0436] Detection of IL-11 and receptors for IL-11

[0437] Some aspects and embodiments of the present invention relate to the detection of expression of IL-11 or a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130) in a sample obtained from a subject.

[0438] In some aspects and embodiments, the present invention relates to the upregulation (overexpression) of the expression of IL-11 or a receptor for IL-11 (as a protein or oligonucleotide encoding each IL-11 or a receptor for IL-11), and the detection of such upregulation as an indicator of suitability for treatment by a agent capable of inhibiting the action of IL-11 or by a agent capable of preventing or reducing the expression of IL-1 or a receptor for IL-11.

[0439] Upregulated expression involves expression at a level greater than would typically be expected for a given type of cell or tissue. Upregulation can be determined by measuring the expression level of a relevant factor in a cell or tissue. Comparison may be made between the expression level of the relevant factor in a cell or tissue sample from a subject and a reference expression level, for example, a value or range of values ​​representing the normal expression level of the relevant factor for the same or corresponding cell or tissue type. In some embodiments, the reference level may be determined by detecting the expression of IL-11 or a receptor for IL-11 in a control sample, for example, in a corresponding cell or tissue from a healthy subject or from a healthy tissue of the same subject. In some embodiments, the reference level may be obtained from a standard curve or a data set.

[0440] Expression levels can be quantified for absolute comparison or relative comparison can be performed.

[0441] In some embodiments, upregulation of IL-11 or a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130) may be considered to exist when the expression level in the test sample is at least 1.1 times the reference level. More preferably, the expression level may be selected from one of at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.5, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, or at least 10.0 times the reference level.

[0442] Expression levels are many bases In vitro It can be determined by one of the assay techniques, such as a PCR basic assay, an in situ hybridization assay, a flow cytometry assay, or an immunological or immunohistochemical assay.

[0443] For example, a suitable technique involves a method for detecting the level of IL-11 or the receptor for IL-11 in a sample by contacting a sample with a preparation capable of binding to IL-11 or the receptor for IL-11 and detecting the formation of a complex between the preparation and IL-11 or the receptor for IL-11. The preparation may be any suitable binding molecule, e.g., an antibody, polypeptide, peptide, oligonucleotide, aptamer, or small molecule, and may be optionally labeled to allow detection, e.g., visualization of the formed complex. Suitable labels and means of detecting the same are well known to those skilled in the art, and fluorescent labels (e.g., fluorescein, rhodamine, eosin and NDB, green fluorescent protein (GFP), chelates of rare earths, e.g., europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, Cy5), isotope markers, radioisotopes (e.g., 32P, 33P, 35S), chemiluminescent labels (e.g., acridinium ester, luminol, isoluminol), enzymes (e.g., peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, luciferase), antibodies, It includes ligands and receptors. Detection techniques are well known to those skilled in the art and can be selected to correspond to labeling agents. Suitable techniques include PCR amplification of oligonucleotide tags, mass spectrometry, for example, detection of fluorescence or color upon enzymatic conversion of a substrate by a reporter protein, or radioactive detection.

[0444] The test may be configured to quantify the amount of IL-11 or IL-11 receptors in a sample. The quantified amount of IL-11 or IL-11 receptors from the test sample may be compared to a reference value, and this comparison may be used to determine, to a selected degree of statistical significance, whether the test sample contains a higher or lower amount of IL-11 or IL-11 receptors than the reference value.

[0445] Quantification of detected IL-11 or IL-11 receptors can be used to determine upregulation, downregulation, or amplification of the gene encoding IL-11 or IL-11 receptors. If the test sample contains fibrotic cells, such upregulation, downregulation, or amplification can be compared to a reference value to determine whether any statistically significant difference exists.

[0446] Samples obtained from the subject may be of any type. Biological samples may be obtained from any tissue or body fluid, e.g., blood samples, blood-derived samples, serum samples, lymph samples, semen samples, saliva samples, synovial fluid samples. Blood-derived samples may be selected fractions of the patient's blood, e.g., selected cell-containing fractions or plasma or serum fractions. Samples may comprise tissue samples or biopsies; or cells isolated from the subject. Samples may be collected by known techniques, e.g., biopsy or needle aspiration. Samples may be stored and / or processed for subsequent determination of IL-11 expression levels.

[0447] The sample can be used to determine the upregulation of IL-11 or receptors for IL-11 in the subject from which the sample was obtained.

[0448] In some preferred embodiments, the sample may be a tissue sample obtained from a tissue / organ affected by a metabolic disease, e.g., a biopsy. The sample may contain cells.

[0449] Subjects may be selected for therapeutic / prevention according to the present invention based on the determination that such subjects have an upregulated level of expression of IL-11 or a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130). Upregulated expression of IL-11 or a receptor for IL-11 may serve as a marker of a metabolic disease suitable for treatment with an agent capable of inhibiting IL-11-mediated signaling.

[0450] Upregulation may be present in a given tissue or in a selected sample from a given tissue. A preferred tissue may be liver tissue or pancreatic tissue. Upregulation of the expression of IL-11 or its receptor may also be determined in circulating fluids, e.g., blood, or in blood-derived samples. Upregulation may be of extracellular IL-11 or IL-11Rα. In some embodiments, expression may be upregulated locally or systemically.

[0451] After selection, the subject may be administered an agent capable of inhibiting IL-11-mediated signaling.

[0452] Diagnosis and prognosis

[0453] Detection of the upregulation of expression of IL-11 or receptors for IL-11 (e.g., IL-11Rα, gp130, or complexes containing IL-11Rα and / or gp130) can also be used in methods for diagnosing metabolic diseases and identifying subjects at risk of developing metabolic diseases, and in methods for prognosing or predicting a subject's response to treatment with agents capable of inhibiting IL-11-mediated signaling.

[0454] Other forms of "causing to cause," "onset," and "to cause to cause" may refer to the onset of a disorder / disease, or the continuation or progression of a disorder / disease.

[0455] In some embodiments, a subject may have or be suspected of having a metabolic disease based, for example, on the presence of other symptoms indicating a metabolic disease in the subject's body or in selected cells / tissues of the subject's body, or may be considered at risk of developing a metabolic disease due, for example, to exposure to a genetic predisposition or environmental conditions known as risk factors for metabolic disease. Determination of the upregulation of expression of IL-11 or receptors for IL-11 may confirm a diagnosis or suspected diagnosis, or confirm that the subject is at risk of developing a metabolic disease. The determination may also diagnose the metabolic disease or predisposition as suitable for treatment with an agent capable of inhibiting IL-11-mediated signaling.

[0456] Thus, a method for providing a prognosis for a subject having or suspected of having a metabolic disease may be provided, and the method comprises the steps of determining whether the expression of IL-11 or a receptor for IL-11 is upregulated in a sample obtained from the subject, and, based on the determination, providing a prognosis for the treatment of the subject with an agent capable of inhibiting IL-11-mediated signaling.

[0457] In some embodiments, diagnostic methods or methods for prognosing or predicting a subject's response to treatment with agents capable of inhibiting IL-11-mediated signaling may not require the determination of expression of IL-11 or its receptor, but may be based on determining genetic factors in the subject that predict the upregulation of expression or activity. Such genetic factors may include the determination of genetic mutations, single nucleotide polymorphisms (SNPs), or gene amplifications in IL-11, IL-11Rα, and / or gp130, which correlate with and / or predict expression or activity and / or the upregulation of IL-11-mediated signaling. The use of genetic factors to predict predisposition to a disease state or therapeutic response is known in the art, for example, in the literature [Peter Starkel Gut 2008;57:440-442]; in the literature [Wright et al., Mol. Cell. Biol. March 2010 vol. 30 no. Refer to 6 1411-1420.

[0458] Genetic factors may be tested by methods known to those skilled in the art, including PCR-based assays, e.g., quantitative PCR, competitive PCR. For example, by determining the presence of genetic factors in a sample obtained from a subject, a diagnosis may be confirmed and / or the subject may be classified as being at risk of developing a metabolic disease and / or the subject may be identified as suitable for treatment with an agent capable of inhibiting IL-11-mediated signaling.

[0459] Some methods may include determining the presence of one or more SNPs associated with IL-11 secretion or susceptibility to the onset of metabolic diseases. SNPs are typically bi-allelic and can therefore be easily determined using one of many conventional assays known to those skilled in the art (see, for example, [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]).

[0460] The method may include a step of determining whether an SNP allele is present in a sample obtained from a subject. In some embodiments, determining the presence of a minor allele may be related to increased IL-11 secretion or susceptibility to the onset of metabolic disease.

[0461] Accordingly, in one aspect of the present invention, a method for screening an object is provided, and the method

[0462] A step of obtaining a nucleic acid sample from a target;

[0463] r whether a specific allele is present in the sample at a polymorphic nucleotide position of one or more SNPs listed in FIG. 33, 34, or 35 of WO 2017 / 103108 A1 (incorporated herein by reference), or an SNP that is in linkage disequilibrium with one of the listed SNPs 2 Step to determine ≥ 0.8

[0464] Includes

[0465] The determination step may include a step of determining whether a minority allele is present at a selected polymorphic nucleotide position within the sample. This may include a step of determining whether 0, 1, or 2 minority alleles are present.

[0466] A screening method may be a method for determining a subject's susceptibility to the onset of a metabolic disease, or a diagnostic or prognostic method as described herein, or may form a part thereof.

[0467] The method may further include the step of identifying a subject as having susceptibility to or an increased risk of a metabolic disease, for example, if it is determined that the subject has a minority allele at a polymorphic nucleotide position. The method may further include the step of selecting a subject for treatment with an agent capable of inhibiting IL-11-mediated signaling and / or administering an agent capable of inhibiting IL-11-mediated signaling to the subject, in order to provide treatment for the metabolic disease in the subject or to prevent the onset or progression of the metabolic disease in the subject.

[0468] In some embodiments, a method for diagnosing a metabolic disease and identifying a subject at risk of developing a metabolic disease, and a method for prognosing or predicting the subject's response to treatment with an agent capable of inhibiting IL-11-mediated signaling, utilizes an indicator other than the detection of upregulation of expression of IL-11 or a receptor for IL-11, or a genetic factor.

[0469] In some embodiments, a method for diagnosing a metabolic disease and identifying a subject at risk of developing a metabolic disease, and a method for prognosing or predicting the subject's response to treatment with an agent capable of inhibiting IL-11-mediated signaling, is based on detecting, measuring, and / or identifying one or more indicators of metabolic function.

[0470] Diagnostic or prognostic methods are based on a sample obtained from the subject, or in vitro after processing of a sample obtained from the subject It can be performed. Once the sample is collected, the patient will be performed In vitro It is not necessary for a diagnostic or prognostic method to exist, and therefore, the method may not be performed on a human or animal body. The sample obtained from the subject may be of any type as described above in this invention.

[0471] Other diagnostic or prognostic tests may be used in conjunction with those described herein to enhance the accuracy of the diagnosis or prognosis or to confirm the results obtained by using the tests described herein.

[0472] object

[0473] The subject may be an animal or a human. The subject may preferably be a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. The subject may be a patient.

[0474] The patient may have metabolic diseases as described herein. The subject may have been diagnosed with a metabolic disease requiring treatment, may be suspected of having such a metabolic disease, or may be at risk of developing a metabolic disease.

[0475] In an embodiment, according to the present invention, the subject is preferably a human subject. In an embodiment, according to the present invention, the subject may be selected for treatment according to a method based on characterization of a predetermined marker of a metabolic disease.

[0476] Additional methods and uses provided

[0477] The present invention also provides a agent capable of inhibiting IL-11-mediated signaling for use in the following methods, or the use of a agent capable of inhibiting IL-11-mediated signaling: a decrease in blood lipid levels, a decrease in blood glucose levels, an increase in glucose tolerance (e.g., in subjects with glucose intolerance), an increase in insulin tolerance (e.g., in subjects with insulin resistance), an increase in pancreatic function, a decrease in body weight (e.g., in overweight / obese subjects), a decrease in body fat mass, an increase in lean body mass, a decrease in fasting blood glucose levels, a decrease in serum triglyceride levels, a decrease in serum cholesterol levels, an increase in glucose tolerance, an increase in pancreatic function (e.g., exocrine and / or endocrine function), an increase in pancreatic tissue growth, pancreatic tissue regeneration, an increase in pancreatic weight, inhibition of PSC-to-myofibroblast migration by PSCs, a decrease in the number / ratio of myofibroblasts in the pancreas, a decrease in pancreatic hydroxyproline levels, and pancreatic collagen levels Decrease, decrease in pancreatic injury, decrease in pancreatic islet cell hyperplasia, decrease in glucagon expression, increase in insulin expression, increase in body weight (e.g., in subjects with wasting diseases, e.g., cachexia), decrease in hepatic IL-11 protein expression, decrease in hepatic Erk activation, decrease in hepatic JNK activation; decrease in hepatic caspase-3 cleavage; decrease in hepatic ROS levels;Decrease in hepatic NOX4 expression, e.g., decrease in hepatic steatosis, decrease in hepatic triglyceride levels, decrease in fatty acid synthase expression, decrease in serum ALT and / or AST levels, decrease in the expression of pro-inflammatory factors (e.g., TNFα, CCL2, CCL5, IL-6, CXCL5, and / or CXCL1), decrease in the expression of fibrosis-promoting factors (e.g., IL-11, TIMP1, ACTA2, TGFβ1, MMP2, TIMP2, MMP9, COL1A2, COL1A1 and / or COL3A1), decrease in serum TGFβ1 levels, decrease in the expression / production of IL-11, ACTA2, MMP2, TGFβ1, PDGF, ANG2 II, bFGF, CCL2 and / or H2O2 by HSCs, inhibition of HSC-to-myofibroblast transition by HSCs, decrease in the number / ratio of hepatic myofibroblasts, Decrease in hepatic hydroxyproline levels, decrease in hepatic collagen levels, increase in liver function, increase in serum GSH levels, increase in the function of organs / tissues affected by metabolic diseases, decrease in liver injury, decrease in hepatocyte apoptosis; decrease in cell (e.g., hepatocyte) apoptosis as a result of lipotoxicity; decrease in IL-11-mediated signaling within hepatocytes or decrease in the number / ratio of CD45+ cells within the liver.;

[0478] The present invention also provides for the use of agents capable of inhibiting IL-11-mediated signaling for use in the preparation of compositions for use in the following methods: reduction of blood lipid levels, reduction of blood glucose levels, increase in glucose tolerance (e.g., in subjects with glucose intolerance), increase in insulin tolerance (e.g., in subjects with insulin resistance), increase in pancreatic function, reduction in body weight (e.g., in overweight / obese subjects), reduction in body fat mass, increase in lean body mass, reduction in fasting blood glucose levels, reduction in serum triglyceride levels, reduction in serum cholesterol levels, increase in glucose tolerance, increase in pancreatic function (e.g., exocrine and / or endocrine function), increase in pancreatic tissue growth, regeneration of pancreatic tissue, increase in pancreatic weight, reduction in pancreatic islet cell hyperplasia, reduction in glucagon expression, increase in insulin expression, increase in body weight (e.g., in subjects with wasting disease, e.g., cachexia), reduction in the expression of IL-11 protein in the liver, Decrease in hepatic Erk activation, decrease in hepatic JNK activation; decrease in hepatic caspase-3 cleavage; decrease in hepatic ROS levels;Decrease in hepatic NOX4 expression, e.g., decrease in hepatic steatosis, decrease in hepatic triglyceride levels, decrease in fatty acid synthase expression, decrease in serum ALT and / or AST levels, decrease in the expression of pro-inflammatory factors (e.g., TNFα, CCL2, CCL5, IL-6, CXCL5, and / or CXCL1), decrease in the expression of fibrosis-promoting factors (e.g., IL-11, TIMP1, ACTA2, TGFβ1, MMP2, TIMP2, MMP9, COL1A2, COL1A1 and / or COL3A1), decrease in serum TGFβ1 levels, decrease in the expression / production of IL-11, ACTA2, MMP2, TGFβ1, PDGF, ANG2 II, bFGF, CCL2 and / or H2O2 by HSCs, inhibition of HSC-to-myofibroblast transition by HSCs, decrease in the number / ratio of hepatic myofibroblasts, Decrease in hepatic hydroxyproline levels, decrease in hepatic collagen levels, increase in liver function, increase in serum GSH levels, increase in the function of organs / tissues affected by metabolic diseases, decrease in liver injury, decrease in hepatocyte apoptosis; decrease in cell (e.g., hepatocyte) apoptosis as a result of lipotoxicity; decrease in IL-11-mediated signaling within hepatocytes or decrease in the number / ratio of CD45+ cells within the liver.;

[0479] The present invention also provides the following method: a decrease in blood lipid levels, a decrease in blood glucose levels, an increase in glucose tolerance (e.g., in subjects with glucose intolerance), an increase in insulin tolerance (e.g., in subjects with insulin resistance), an increase in pancreatic function, a decrease in body weight (e.g., in overweight / obese subjects), a decrease in body fat mass, an increase in lean body mass, a decrease in fasting blood glucose levels, a decrease in serum triglyceride levels, a decrease in serum cholesterol levels, an increase in glucose tolerance, an increase in pancreatic function (e.g., exocrine and / or endocrine function), an increase in pancreatic tissue growth, pancreatic tissue regeneration, an increase in pancreatic weight, a decrease in pancreatic islet cell hyperplasia, a decrease in glucagon expression, an increase in insulin expression, an increase in body weight (e.g., in subjects with wasting disease, e.g., cachexia), a decrease in the expression of IL-11 protein in the liver, a decrease in Erk activation in the liver, a decrease in JNK activation in the liver; Decrease in intrahepatic caspase-3 cleavage; decrease in intrahepatic ROS levels;Decrease in hepatic NOX4 expression, e.g., decrease in hepatic steatosis, decrease in hepatic triglyceride levels, decrease in fatty acid synthase expression, decrease in serum ALT and / or AST levels, decrease in the expression of pro-inflammatory factors (e.g., TNFα, CCL2, CCL5, IL-6, CXCL5, and / or CXCL1), decrease in the expression of fibrosis-promoting factors (e.g., IL-11, TIMP1, ACTA2, TGFβ1, MMP2, TIMP2, MMP9, COL1A2, COL1A1 and / or COL3A1), decrease in serum TGFβ1 levels, decrease in the expression / production of IL-11, ACTA2, MMP2, TGFβ1, PDGF, ANG2 II, bFGF, CCL2 and / or H2O2 by HSCs, inhibition of HSC-to-myofibroblast transition by HSCs, decrease in the number / ratio of hepatic myofibroblasts, Decrease in hepatic hydroxyproline levels, decrease in hepatic collagen levels, increase in liver function, increase in serum GSH levels, increase in the function of organs / tissues affected by metabolic diseases, decrease in liver injury, decrease in hepatocyte apoptosis; decrease in cell (e.g., hepatocyte) apoptosis as a result of lipotoxicity; decrease in IL-11-mediated signaling within hepatocytes or decrease in the number / ratio of CD45+ cells within the liver.;

[0480] Sequence identity

[0481] Pairwise and multiple sequence alignments for determining the percentage of identity between two or more amino acid or nucleic acid sequences are known to those skilled in the art in various ways, for example, publicly available computer software, such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960]), T-coffee (Literature [Notredame et al. 2000, J. Mol. Biol . (2000) 302, 205-217]), Kalign(literature[Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)]) and MAFFT(literature[Katoh and Standley 2013, Molecular Biology and Evolution This can be achieved using software. When using such software, default parameters for gap penalty and extension penalty are preferably used, for example.

[0482]

[0483]

[0484]

[0485]

[0486] The present invention comprises combinations of the described embodiments and preferred features, except where such combinations are clearly unacceptable or clearly avoided.

[0487] The features disclosed in the foregoing description, in the following claims, or in the attached drawings in terms of specific forms thereof, means for performing the disclosed function, or methods or processes for obtaining the disclosed result, may be utilized to realize the present invention in various forms thereof, separately or in combination of such features, if appropriate.

[0488] To avoid any doubt, any theoretical descriptions provided herein are intended to enhance the reader's understanding. The inventors do not intend to associate any of these theoretical descriptions with the reader.

[0489] Any section headings used herein are for structural purposes only and are not intended to limit the subject matter described.

[0490] Throughout this specification, including subsequent claims, unless otherwise required by the context, the words “comprise” and “include” and their variations, such as “comprises,” “comprising,” and “including,” will be understood to imply the inclusion of the mentioned integer or step or group of integers or steps, rather than the exclusion of any other integer or step or group of integers or steps.

[0491] It should be noted that, as used in this specification and the appended claims, the singular forms (“a,” “an,” and “the”) include the plural forms unless the context clearly indicates otherwise. Ranges may be expressed herein as “about” from one specific value and / or “about” another specific value. When such ranges are expressed, another embodiment includes from one specific value and / or to another specific value. Similarly, when values ​​are expressed as approximations, the use of the antecedent “about” will be understood to mean that the specific value forms another embodiment. With respect to numerical values, the term “about” is optional and means, for example, + / - 10%.

[0492] In vitro, in vitro or In vivo , the method disclosed herein may be performed or a product may exist. The term " In a test tube " is intended to encompass experiments using materials, biological components, cells, and / or tissues under laboratory conditions or in cultures, whereas the term " In vivo " is intended to encompass experiments and procedures using multicellular organisms. In some embodiments, In vivo The method being performed can be performed on non-human animals. In vitro" refers to something that exists or occurs outside of an organism, for example, outside of the human or animal body, and may be on tissues (e.g., whole organs) or cells obtained from the organism.

[0493] Where a nucleic acid sequence is disclosed herein, its reverse complement is also clearly taken into account.

[0494] For standard molecular biology techniques, refer to the literature [Sambrook, J., Russell, DW Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press].

[0495] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All references in this document are incorporated herein by reference in their entirety. Although the present invention has been described with the exemplary embodiments described below, many equivalent variations and changes will be apparent to those skilled in the art when given such disclosure. Accordingly, the exemplary embodiments of the present invention presented above are considered exemplary and are not intended to be limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the present invention. Brief explanation of the drawing

[0496] Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the attached drawings. FIGS. 1a and FIGS. 1b . ( 1a ) Normal feed diet (NCD), or ( 1b This is a graph showing the percentage change in body weight over time for IL-11RA knockout (Il11ra1- / -) or wild-type, IL-11RA expressing (Il11ra1+ / +) mice fed a Western diet (WDF) with fructose. FIGS. 2a and FIGS. 2b . ( 2a ) Normal feed diet (NCD), or ( 2b This is a bar chart showing the percentage change in total body fat mass for IL-11RA knockout (Il11ra1KO) or wild-type, IL-11RA expressing (Il11raWT) mice raised on a Western diet (WDF) with fructose. Fig. 3 This is a graph showing fasting blood glucose levels (mM) in IL-11RA knockout (KO) or wild-type, IL-11RA expressing (WT) mice fed a normal diet (NC) or a Western diet with fructose (WDF). Fig. 4 This is a graph showing serum triglyceride levels (mg / g) for IL-11RA knockout (KO) or wild-type, IL-11RA expressing (WT) mice fed a normal diet (NC) or a Western diet with fructose (WDF). Figs. 5a and 5b . ( 5a ) Normal feed diet (NC), or ( 5b This is a graph showing serum cholesterol levels (mg / dl) for IL-11RA knockout (KO) or wild-type, IL-11RA expressing (WT) mice raised on a Western diet (WDF) with fructose. Figures 6a and 6b These are graphs and box plots illustrating changes in body weight for mice fed on a normal diet (NC) or a Western diet (WDF) with fructose and treated with anti-IL-11RA antibodies or IgG controls. 6a ) Plots the percentage change in weight over time (week). 6b ) Plots the percentage difference between total body fat mass and lean body mass. *P<0.05. Figures 7a and 7bGraphs, schematics, and bar charts illustrating glucose tolerance in mice fed a Western diet (WDF) with fructose and treated with anti-IL-11RA antibodies or IgG controls, as determined by the intraperitoneal glucose tolerance test (ipGTT). 7a Plot the change in glucose level (mM) from the 1-minute mark. 7b ) Plot the area under the curve. *P<0.05, ** P<0.01. Fig. 8 This is a box plot showing pancreatic weight for mice fed a normal diet (NCD) or a Western diet (WDF) with fructose and treated with anti-IL-11RA antibodies or IgG controls at different time points. ****P<0.0001. FIGS. 9a to 9c . For mice fed a normal diet (NCD) or a Western diet (WDF) with fructose and treated with anti-IL-11RA antibodies or IgG controls ( 9a ) Serum cholesterol level (mg / dl), ( 9b ) Serum triglyceride levels (mg / g) and ( 9c This is a box plot showing fasting blood glucose levels (mM). FIGS. 10a and FIGS. 10b . of pancreatic tissue sections obtained at 24 weeks from mice fed a normal diet (NCD), or mice fed a Western diet (WDF) with fructose and treated with anti-IL-11RA antibodies or IgG controls from 16 weeks ( 10a ) Glucagon content and ( 10b This is an image illustrating the results of the immunohistochemical analysis of insulin content. FIGS. 11a and FIGS. 11b This is a graph and image illustrating the effect of anti-IL-11 / anti-IL-11Rα antibody treatment on cachexia-related weight loss. 11aMice fed a cachexia-induced high-fat methionine-choline deficiency (HFMCD) diet returned to normal or near-normal body weight when treated with anti-IL-11 or anti-IL-11Rα antibodies at 2x / week. Control mice were fed a normal diet (NC) or fed an HFMCD diet and treated with an IgG isotype control. 11b This is a comparison of the body size of mice raised on an HFMCD diet and treated with IgG or anti-IL-11 antibody or anti-IL-11Rα antibody. FIGS. 12a to 12c This is a graph illustrating the effect of anti-IL-11 / anti-IL-11Rα antibody treatment on body weight in a model of cachexia-related body weight loss. Mice fed the HFMCD diet were treated with 0.5, 1, 5, or 10 mg / kg anti-IL-11Rα antibody ( 12a ) or one of two anti-IL-11 antibodies ( 12b and 12c ) were treated 2x / week. Control mice were fed normal feed (NC) or fed on the HFMCD diet and treated with IgG isotype controls. FIGS. 13a to 13c This is a graph illustrating the effect of anti-IL-11 / anti-IL-11Rα antibody treatment on food consumption in a cachexia-related body weight loss model. Mice fed the HFMCD diet were treated with 0.5, 1, 5, or 10 mg / kg anti-IL-11Rα antibody ( 13a ) or one of two anti-IL-11 antibodies ( 13b and 13c ) were treated 2x / week. Control mice were fed normal feed (NC) or fed on the HFMCD diet and treated with IgG isotype controls. FIGS. 14a and FIGS. 14b This is a graph illustrating the effect of anti-IL-11 / anti-IL-11Rα antibody treatment on body weight in cachexia-related weight loss following folate-induced acute renal injury. 14aMice with folate-induced renal injury were treated with anti-IL-11Rα antibodies, anti-IL-11 antibodies, or IgG controls from 1 hour prior to injury until 28 days after injury. 'Control' mice were administered the vehicle alone. 14b Mice with folate-induced renal injury were treated with anti-IL-11 antibodies or IgG controls starting on day 21 after injury. FA = folic acid. Fig. 15. This graph illustrates the effect of anti-IL-11 antibody treatment on body weight in cachexia-related weight loss following unilateral ureter obstruction (UUO)-induced acute renal injury. Mice with UUO-induced renal injury were treated with anti-IL-11 antibodies or IgG controls for 10 days after injury. FIGS. 16a and FIGS. 16b This is a graph illustrating the effect of IL-11 overexpression on body weight gain. 16a Administration of recombinant mouse IL-11 (rmIL11) demonstrated progress in normal mouse body weight gain. 16b Induction of the IL-11 transplant gene (IL-11 Tg) in mice resulted in body weight loss over time. FIGS. 17a to 17n. IL-11 induces HSC activation and liver fibrosis. a ) IL-11 RNA is upregulated in HSCs stimulated by TGFβ1. b IL-11 protein is secreted from HSCs stimulated by TGFβ1. c Human precision-cut liver slices were stimulated with TGFβ1, and IL-11 protein was measured in the supernatant. d Immunofluorescence images of IL6R and IL11RA expression in HSCs and activated THP-1 cells (scale bar, 100 μm). e ) Immunofluorescence images (scale bar, 100 μm) and ( fThis is a Western blot of ACTA2 in HSCs after incubation with TGFβ1, PDGF, or IL-11 without stimulation (-). g ) HSC and ( for collagen I staining h This is an immunofluorescence image (scale bar, 100 μm) of collagen secretion in the supernatant of HSCs stimulated with TGFβ1, PDGF, or IL-11. i ) It is dose-dependent Matrigel invasion of HSCs induced by IL-11. j Hyper-IL-11 induces IL-11 protein secretion from HSCs (ELISA). ac, eh, j ) TGFβ1 (5 ng / ml), hyper IL-11 (0.2 ng / ml), PDGF (20 ng / ml), IL-11 (5 ng / ml); 24h; ( k ) 48 h. ( k This is a schematic diagram of mice receiving daily injections of saline (control) or rmIl-11 (100 μg / kg). l-n ) 1 K This is data (n≥7 / group) for the rmIl-11 injection experiment as described in [Figure]. l ) Relative hydroxyproline content, ( m ) Hepatic mRNA expression of fibrosis-promoting and pro-inflammatory markers, and ( n ) It is the serum ALT level. ab, hj, l, n ) data is displayed as mean ± sd; ( c, m ) The box-and-whisker plot displays the median (median line), 25th–75th percentiles (box), and minimum–maximum percentiles (whisker). ac, j, ln ) Two-tailed Student's t-test t -test); ( hi ) Two-tailed Dunnett's test. FC: Change in drainage; I / A: Intensity / Area. FIGS. 18a to 18n. Il11ra1 Mice deficient in [it] are protected from NASH liver pathology, hyperlipidemia, and hyperglycemia. a Western blots of liver Il-11, Gapdh, p-Erk, and Erk in mice on the HFMCD diet for 1, 4, 6, and 10 weeks. After 10 weeks of the HFMCD diet Il11ra + / + (WT) and Il11ra - / - (KO) In the liver of a mouse ( b Representative Masson's Trichrome image (scale bar, 100 μm), ( c ) liver triglycerides, ( d ) serum ALT, and ( e ) is the level of pro-inflammatory mRNA expression (n≥5 / group). fn This is data on WT and KO mice for WDF over 16 weeks. f This is a Western blot of liver Il-11 and Gapdh. g ) Relative mRNA expression levels of liver pro-inflammatory markers, ( h ) Serum ALT levels, ( i ) Relative hydroxyproline content (n≥4 / group). j This is a representative Mason tricolor image (scale bar, 100 μm) between ). k Western blot of the ) Erk active state, ( l ) Fasting blood glucose, ( m ) serum triglyceride and (n) serum cholesterol levels (n≥3 / group). ce, gi The ) data is plotted as box-and-whiskers with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers); ( LN) Data are displayed as mean ± sd, and the dotted line represents the mean value of WT for NC; Sidoc-Calibration Students t -Check(Sidak-corrected Student's t -test) FC: Digestive change; NC: Normal feed; HFMCD: High fat methionine-deficient and choline-deficient; WDF: Western diet + 15% (w / v) fructose. FIGS. 19a to 19j. Anti-IL-11 therapies inhibit the transformation of HSCs into myofibroblasts in an ERK-dependent manner and have a favorable metabolic safety profile. a Dose-response curves and IC50 of X203 and X209 (61 pg / ml to 4 μg / ml; 4-fold dilution) in inhibiting MMP2 secretion induced by TGFβ1-stimulated HSCs 50 It is the value. b ) ELISA of IL-11 secretion from HSCs stimulated with various NASH factors (n≥5 / group). (c) ACTA2 from HSCs treated with TGFβ1 and other NASH factors in the presence of IgG, X203, or X209. +ve Representative fluorescence image and quantification of the cells (scale bar, 100 μm, and the dotted line represents the median value of the baseline). d ) This is the effect of X203 and X209 on PDGF-induced or CCL2-induced HSC invasion. e Western blot of p-ERK and ERK in HSC lysate-stimulated IL-11 (top panel) or with various NASH factors in the presence of IgG or X209 (bottom panel). f ) ACTA2 in HSCs treated with IL-11 and important NASH factors in the presence of the ERK / MEK inhibitors U0126 or PD98059 +ve Representative fluorescence image and quantification of the cells (scale bar, 100 μm, and the dotted line represents the median value of the baseline). af) TGFβ1 (5 ng / ml), IL-11 (5 ng / ml), PDGF (20 ng / ml), AngII (100 nM), bFGF (10 ng / ml), CCL2 (5 ng / ml), H202 (0.2 mM), IgG, μM); ( a,c,ef ) 24 h; ( b,d ) 48 h. From mice injected every other week with 10 mg / kg of X203 and X209 for 5 months ( g ) Peripheral platelet count, ( h ) Serum ALT levels, ( i ), serum triglyceride levels, and ( j ) Serum cholesterol level (n≥5 / group). b, d ) Data are plotted as mean ± sd; ( c, f, gj ) The data is plotted as box-and-whiskers, along with the median (median line), 25th–75th percentiles (box), and min-maximum percentiles (whiskers). b, d, gj ) Two-sided Dunnett test; ( c, f ) Both sides, Türkiye-corrected students t -black . FC: Multiplier change. FIGS. 20a to 20n Therapeutic targeting of Il-11 inhibits and reverses NASH pathology in preclinical models. a ) ( be This is a schematic diagram illustrating the therapeutic use of X203 and X209 (10 mg / kg, every other week) in HFMCD-bred mice for the experiment described in ). X203, X209, or IgG isotype controls were administered from week 6 to week 10 of the HFMCD diet. b ) Representative liver histological image (Mason tricolor staining; scale bar, 100 μm), ( c ) Relative hydroxyproline content, ( d ) Relative levels of pro-inflammatory mRNA expression (n≥6 / group) and ( e) Serum ALT level. f ) Western blot with liver Erk activated. g ) hn MCD-breeding for the experiment described in db / db This is a schematic diagram of X203 or IgG administration to mice. Liver ( h ) Il-11 and Gapdh, ( i ) This is a Western blot of p-Erk and Erk. j ) X203 or IgG-treated MCD-retaining db / db This is a representative Macone tricolor image of the liver from a mouse (scale bar, 100 μm). ( k ) liver triglycerides, ( l ) Relational hydroxyproline, ( m ) serum ALT, and ( n ) is the level of mRNA expression of liver pro-inflammatory markers (n≥5 / group). ( ce, kn ) Data are plotted as box-and-whisker with the median (median line), 25th–75th percentiles (box), and min–maximum percentiles (whisker); two-sided, turkey-corrected Students' t-test. FC: Changes in drainage; NC: Normal feed; HFMCD: High fat methionine-deficient and choline-deficient; MCD: Methionine-deficient and choline-deficient. FIGS. 21a to 21l. Inhibition of Il-11 signaling reverses NASH pathology in preclinical models and HSC-to-myofibroblast transformation. a ) ( bg This is a schematic diagram illustrating the therapeutic dosing regimens in the NASH reversal experiment for the data presented in ). Mice were fed WDF for 16 weeks to induce NASH, then treated with X209 (10 mg / kg) or IgG for 8 weeks, while these mice remained in continuous WDF feeding. In mice in NC and IgG-treated and X209-treated WDF ( b ) Total liver hydroxyproline content, ( c) liver triglycerides, ( d ) Serum ALT, ( e ), fasting blood glucose, (f) serum triglycerides, and ( g ) is the level of serum cholesterol (n≥5 / group). H This is a schematic diagram illustrating a reversal experiment in which fibrosis was established by rearing mice on HFMCD for 10 weeks, then replacing them with NC and initiating antibody (X203 and X209) therapy. Mice were euthanized at the indicated time points. i ) Total liver hydroxyproline content (the dotted line indicates the average value of NC=0.93) and ( j ) at weeks 1, 3, and 6 after concurrent metabolic intervention (diet switch) and X203, X209, or IgG treatment Mmp2 / Timp1 It is the relative mRNA expression (n≥3 / group). Before or after the addition of X203, X209, or IgG ( k ) together with TGFβ1 or ( l ) ACTA2 after incubation with PDGF +ve This is the quantification of immunohistochemistry (scale bar, 200 μm) (n=5 / group). k-l ) TGFβ1 (5 ng / ml), PDGF (20 ng / ml), IgG, X203, and X209 (2 μg / ml). ( bg, kl The data is plotted as box-and-whiskers with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers); ( i ) data are plotted as mean ± sd; ( j The data is displayed as a line chart (average), and transparency is represented by the standard deviation. b ) Two-sided Student's t-test ( cg, kl ) Two-sided, Türkiye-corrected Students' t-test; ( ij) Two-sided ANOVA. FC: Change in drainage; NC: Normal diet; WDF: Western diet + 15% (w / v) fructose; HFMCD: High fat, methionine-deficient, and choline-deficient. FIGS. 22a to 22k. Neutralization of Il-11 signaling reverses liver damage in early NASH. a Relative hepatic mRNA expression of fibrosis and inflammation markers from mice fed the NC or HFMCD diet at the indicated time points. b This is a schematic diagram of the initial anti-IL-11 therapy experiment in the HFMCD diet NASH model. Antibody therapy was initiated on week 1 after the start of the NASH diet when X209, X203, or IgG (10 mg / kg, every other week) was administered intraperitoneally for 5 weeks. cg ) This is data for the experiment as illustrated in Fig. 21b. After 5 weeks of IgG or X209 treatment ( c ) Representative overall liver image and ( d This is a tricolor-stained image of the liver (scale bar, 100 μm). e ) Liver triglyceride levels (n≥5 / group), ( f ) Hepatic hydroxyproline content in X209-treated and IgG-treated mice (n≥5 / group), ( g ) Serum ALT level (n≥5 / group). h Immunofluorescence image of IL6R and IL11RA expression in hepatocytes (scale bar, 100 μm). i IL-11 and ( j ) This is the dose-dependent effect on stress fiber formation (rhodamine-phaloidine staining) in hepatocytes (scale bar, 200 μm). k IL-11 protein is secreted from primary human hepatocytes stimulated with TGFβ1 (5 ng / ml); 24 h. ( bThe data is plotted as box-and-whiskers with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers); ( fg, i, k The data is plotted as mean ± sd. e ) Turkish-corrected students t -black; ( f, g ) Two-sided ANOVA; ( i, k ) Two-sided Dunnett test. FC: Changes in drainage; NC: Normal feed; HFMCD: High fat, methionine-deficient and choline-deficient. FIGS. 23a to 23g. Anti-IL11RA therapy inhibits immune cell activation while reversing the molecular signature of NASH toward a normal liver profile. ag ) This is data for the experiment as illustrated in Fig. 22b. a ) 6B This is the principal component analysis (PCA) of liver gene expression in mice in NC or HFMCD in the presence of IgG, X203, or X209 antibodies for the time shown in Figure. The arrows illustrate the transition from normal gene expression (NC) to perturbed gene expression in NASH (HFMCD+IgG), to moderately recovered gene expression (HFMCD+Abs (3w)), and to normalized gene expression (HFMCD+Abs (6w)). b ) Fibrosis-promoting and pro-inflammatory gene expression heatmap (scaled transcripts per million, TPM (Transcripts Per Million)). c ) by qPCR Tnf , Ccl2, and Ccl5 mRNA expression (n≥5 / group). d ) liver CD45 +ve Number of immune cells, ( e Total 45 CDs +ve Ly6C within the group +ve TGFβ1+ve Cell, ( f) Ly6C +ve TGFβ1+ve Representative pseudocolor plot (n≥4 / group) illustrating a gating strategy used to detect cells. g ) Serum TGFβ levels (n≥5 / group). ( ce, g The data is plotted as box-and-whiskers, along with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers). c, g ) Two-sided, Türkiye-corrected Students' t-test; ( de ) Two-sided Students' t-test. FC: Digestive change; NC: Normal feed; HFMCD: High fat, methionine-deficient, and choline-deficient. FIGS. 24a to FIGS. 24k. HSCs secrete IL-11 and respond to it, and IL-11 injection into mice causes liver fibrosis. a ) Genome-wide changes in RNA expression in HSCs after TGFβ1 stimulation (n=3, RNAseq). ( b ) Human HSC (RNA-seq 14 It is stiffness-induced RNA upregulation in ), and genes are ranked according to fragments per kilobase million (FPKM), and IL-11 Upregulation is the most highly upregulated gene genome-wide. c ) in human cardiac fibroblasts (HCF), human pulmonary fibroblasts (HLF), and human HSCs IL11RA It is a transcriptome. IL-11 and GAPDH in human liver samples from patients with alcoholic liver disease (ALD), primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), and non-alcoholic steatohepatitis (NASH) ( d ) Western blot and ( e ) It is densitometry. After incubation without stimulation (-), with TGFβ1, PDGF, or IL-11 ( f) ACTA2 +ve Cells and ( g ) It is automated fluorescence quantification for collagen I immunostaining. h ) This is the concentration of MMP-2 in the HSC supernatant with TGFβ1 or IL-11 by ELISA, without stimulation (-). a, fh ) TGFβ1 and IL-11 (5 ng / ml), PDGF (20 ng / ml); 24 h stimulation. ( i ) Representative (scale bar, 100 μm) and ( j ) Quantification of Masone tricolor staining images of liver sections from mice injected with saline or rmIl-11. k ) injected daily with rmIl-11 or saline Col1a1-GFP Schematic diagram of the mouse and representative fluorescence image GFP +ve These are cells. Sections were immunostained for Acta2 and counterstained with DAPI (scale bar, 200 μm). ( c, fg The data is plotted as box-and-whiskers with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers); ( e, h, j ) Data is displayed as mean ± sd. fh ) Two-sided Dunnett test; ( j ) Two-sided Student's t-test. FC: Variation in multiples; TPM: Transcriptome per million. FIGS. 25a to 25i. Genetic inhibition of Il-11 signaling protects mice from HFMCD-induced NASH disease. Liver ( a ) Il-11 mRNA and ( b This is the effect of the 16-week HFMCD diet compared to the NC diet on Il-11 protein levels. ab RNA and proteins were extracted from the same mice (n=5 / group). c ) Relative hydroxyproline content and ( d) Serum ALT levels from mice fed the NC or HFMCD diet for 1, 4, 6, or 10 weeks (n≥5 / group). ei ) After 10 weeks of the HFMCD diet Il11ra + / + (WT) and Il11ra - / - (KO) This is data regarding mice. Between ( e ) Relative hydroxyproline content, ( f ) Representative (scale bar, 100 μm) and ( g ) It is the quantification of Mason tricolor images. h ) Acta2 , Col1a1 , Col1a2 , and Col3a1 It is the relative mRNA expression level between groups (n≥5). i This is a Western blot of phosphorylated Erk and total Erk after 10 weeks of NC and HFMCD diets. a, e, h The data is plotted as box-and-whiskers with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers); ( cd, g ) Data is displayed as mean ± sd. a, g ) Two-sided Student's t-test; ( CD ) Two-sided ANOVA; ( e, h ), Sidoc-corrected Students' t-test. ( c The values ​​for NC and HFMCD 6 weeks are the same as those used in FIG. 20c; and the values ​​for NC and HFMCD 1 week are the same as those used in FIG. 22f. d The values ​​for HFMCD week 6 are the same as those used in Fig. 20d; the values ​​for NC and HFMCD week 1 are the same as those used in Fig. 22g. FC: Digestive change; NC: Normal feed; HFMCD: High fat, methionine-deficient, and choline-deficient. FIGS. 26a to 26e. Genetic inhibition of Il-11 signaling protects mice from WDF-induced NASH disease. a ) Il11ra + / + (WT) and Il11ra - / - (KO) This is the effect of 16 weeks of WDF on mouse body weight (n≥6 / group). b ) liver triglyceride levels, ( c ) Representative (scale bar, 100 μm) and ( d Quantification of the Mason tricolor staining image of the liver, ( e These are the relative mRNA expression levels (n≥5 / group) for pro-fibrosis genes in WT and KO mice after 16 weeks of NC and WDF. a, d ) Data are plotted as mean ± sd, two-sided Student's t-test; ( b, e Data are plotted as box-and-whisker with the median (midline), 25th–75th percentiles (box), and min-max percentiles (whisker), Sidac-corrected Students' t-test. FC: Dilution variation; NC: Normal feed; WDF: Western diet + 15% (w / v) fructose. FIGS. 27a to 27f. It is the development of neutralizing anti-IL-11RA monoclonal antibodies. a ) Purified mouse monoclonal anti-IL11RA candidate (6 μg ml -1 ACTA2 of human atrial fibroblasts stimulated with TGFβ1- (top) and HyperIL-11- (middle) and mouse atrial fibroblasts stimulated with TGFβ1- (bottom) +ve It is the inhibition of cell transformation. b It is an X209 interaction with IL11RA as determined by SPR(1:1 Langmuir). c ) In mouse (n=5) 125 This is the hemodynamics of the I-X209. The results were fitted to a two-phase exponential decay model (R 2 =0.92). ( d ) by the liver (n=5) at the indicated time after retro-orbital injection125 I-X209 is the percentage of absorption. ef ) ( e ) in the presence of an IgG control, X203, or X209, or ( f Representative fluorescence images (scale bar, 100 μm) and quantification of Collagen 1 immunostaining of HSCs treated with various NASH factors in the presence of MEK / ERK inhibitors (U0126 or PD98059). a, ef ) TGFβ1 (5 ng / ml), IL-11 (5 ng / ml), PDGF (20 ng / ml), AngII (100 nM), bFGF (10 ng / ml), CCL2 (5 ng / ml), H202 (0.2 mM), IgG, μM); 24 h stimulation. ( CD ) data is displayed as mean + sd; ( ef The data is plotted as Box-and-Whisker with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers); the dashed line represents the mean of the baseline values, and it is a Turkey-corrected Student's t-test. FC: Variation in fold; I / A: Intensity / Area. FIGS. 28a to 28f. Neutralizing anti-IL-11 and anti-IL11RA antibodies inhibit HFMCD-induced and WDF-induced NASH pathogenesis. ad ) Data on the therapeutic use of X203 and X209 in HFMCD-bred mice as shown in Fig. 20a. ( a ) Quantification of Mason tricolor staining of liver sections (dotted line indicates mean NC value). b ) Relative mRNA expression levels of fibrosis genes and ( c ) Liver triglyceride content (n≥5 / group). d This is a Western blot of hepatic ERK activation from NC, IgG-treated, and X203-treated mice (10 mg / kg, bi-weekly) on an HFMCD diet. e) Quantification of Mason tricolor staining of liver sections, and the dotted line is 12 weeks of age db / db Represents the average value of the steatosis from (see Fig. 20g), ( f steatosis and MCD-reproduction injected with IgG or X203 as illustrated in the schematic diagram db / db Relative fibrosis-promoting mRNA expression levels in mouse liver (Fig. 20g, n≥5 / group). a, e ) Data is displayed as mean ± sd; ( bc, f The data is plotted as box-and-whiskers, along with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers). ac, ef ) Two-sided, turkey-corrected Students' t-test. FC: Digestive change; NC: Normal feed; HFMCD: High fat methionine-deficient and choline-deficient; MCD: Methionine-deficient and choline-deficient. FIGS. 29a to 29e. Neutralizing anti-IL11RA therapy reverses WDF-induced NASH symptoms. ab Data on anti-IL-11RA therapeutic intervention in mice on the WDF diet as illustrated in the schematic diagram (Fig. 21a). Mice on the WDF were treated with IgG or X209 (10 mg / kg) every other week for 8 weeks, starting at week 16 and ending at sacrifice (week 24). a Western blots of p-Erk and Erk from livers of mice in NC or WDF for 24 weeks. b ) Bimonthly body weight measurements (n≥4 / group). From mice treated with WDF for 16 weeks (left), IgG injections at weeks 16–24 for 24 weeks (middle), and X209 treatment at weeks 16–24 for 24 weeks (right) ( c ) Representative (scale bar, 100 μm) and ( d This is the quantification of the Mason tricolor staining image between ) and the dotted line represents the average value of NC. ( e ) Relative mRNA expression levels of pro-inflammatory genes (n≥5 / group). b, d ) Data are plotted as mean ± sd; ( e The data is plotted as box-and-whiskers, along with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers). d, e ) Two-sided, Türkiye-corrected Students' t-test. FIGS. 30a to FIGS. 30g. Neutralizing anti-IL-11 or anti-IL11RA antibodies reverse HFMCD-induced liver fibrosis and the transformation of HSCs into myofibroblasts. ad ) 5G This is data from mice treated with IgG, X203, or X209 for 1, 3, or 6 weeks as shown in the (HFMCD reversal experiment). a ) This is a Western blot of the hepatic ERK activation state. From mice treated with IgG, X203, or X209 for 6 weeks ( b ) Representative (scale bar, 100 μm) and ( c ) is the quantification of the liver using Mason tricolor staining. dg ) Data from HSC transformation experiments as illustrated in FIGS. 21k to 21l; TGFβ1(5 ng / m l), PDGF (20 ng / ml), IgG, X203, and X209 (2 μg / ml) . ( d ) ACTA2 after incubation with TGFβ1 or PDGF before or after the addition of X203, X209, or IgG +ve This is a representative fluorescence image of immunohistochemistry (scale bar, 200 μm). Before or after the addition of IgG, X203, or X209 ( e ) TGFβ1 or ( f ) This is the amount of collagen secreted by PDGF-stimulated HSCs (n=5 / group). g This is a Western blot of ERK activation status after X203 and X209 treatment in TGFβ1-treated HSCs. c ) data are plotted as mean ± sd; ( ef The data is plotted as box-and-whiskers, along with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers). c, ef ) Two-sided, Türkiye-corrected Students' t-test. FC: Changes in drainage; NC: Normal feed; HFMCD: High fat, methionine deficiency, and choline deficiency. FIGS. 31a to 31h. Neutralizing anti-IL-11 and anti-IL11RA antibodies protect HFMCD-bred mice from liver fibrosis and inflammation. a ) CCL2 in the supernatant of HSC (n=4 / group) by ELISA, without stimulation (-) in the presence of IgG, X203, or X209, with IL-11, or with TGFβ1; IL-11 (5 ng ml -1 ), TGFβ1(5 ng ml -1 ), IgG, X203, and X209 (2 μg ml -1 ). ( bi ) This is data for the therapeutic drug trial as shown in Fig. 22b. After 5 weeks of initial X203 and X209 treatment ( b ) Representative overall liver image, ( c Western blot with ERK enabled between ) d ) Representative (scale bar, 100 μm) and ( e It is the quantification of the tricolor-stained images of the liver. f ) Liver hydroxyproline content (values ​​for the NC and HFMCD 1-week diets are the same as those used in Fig. 25c, and IgG 3-week and 6-week diets are the same as those used in Fig. 22f, n≥5 / group), ( g ) Relative RNA expression levels of fibrosis markers in the liver after 5 weeks of treatment with X203 and X209 by qPCR, which confirms the data from RNA-seq and ( Col1a1 , Col1a2 , Col3a1, and Acta2The NC 6-week values ​​for are the same as those shown in FIG. 28d, n≥5 / group), and ( h ) Serum ALT levels (values ​​for NC and HFMCD at week 1 are the same as those used in Fig. 25d, and values ​​for IgG at weeks 3 and 6 (weeks 2 and 5 treatments, respectively) are the same as those used in Fig. 22g, n≥5 / group). a, ef, h ) data is displayed as mean ± sd; ( g The data is plotted as box-and-whiskers, along with the median (median line), 25th–75th percentiles (box), and min-max percentiles (whiskers). a, e, g ) Two-sided, Türkiye-corrected Students' t-test; ( f, h ) Two-sided ANOVA. FC: Changes in drainage; NC: Normal feed; HFMCD: High fat, methionine-deficient, and choline-deficient. FIGS. 32a to 32d. Neutralizing anti-IL-11 or anti-IL11RA antibodies reverse the molecular signature of NASH toward a normal liver profile. ad ) Data for RNA-seq and gene setting enrichment analysis for the initial therapeutic dosing experiment as shown in Fig. 22a (n=3 / group). ab ) IgG and ( a ) X209 or ( b This is a heatmap plotting gene expression levels (scaled transcriptome-mapped reads per million, TPM) across samples for all genes that were statistically differentially expressed between X203 treatments. As expression profiles for the anti-IL-11 treatment cluster along with profiles in NC, this suggests a nearly complete reversal of the transcriptional effects of the HFMCD diet. c As a heatmap of lipogenesis and β-oxidation gene expression, it shows that X209 improved liver lipid metabolism more than X203 compared to IgG. dThis is a bubblemap illustrating the results of Gene Set Enrichment Analysis (GSEA) for differentially expressed genes after 6 weeks of NC or HFMCD diet and antibody therapy. Each dot represents the Normalized Enrichment Score (NES) and its FDR-corrected significance level for the gene set, summarized by color and size, respectively. The gene sets for the enrichment test were selected from the "H-Hallmark" collection in MSigDB. FC: Digestive change; NC: Normal diet; HFMCD: High fat, methionine-deficient, and choline-deficient. FIGS. 33a to 33k. Scatter plots, box plot histograms, and images regarding the expression of receptors for IL-11 and IL-6 and the effects of IL-11 and IL-6 signaling in primary human hepatocytes. (a) Representative flow cytometry forward scatter (FSC) and fluorescence intensity plots of IL11RA, IL6R, and gp130 staining on hepatocytes. (b) In hepatocytes at the basal level based on RNA-seq (left) and ribo-seq (right). IL11RA1 and IL6R Abundance of reads (transcripts per million, TPM: transcripts per million). (c and d) (c) based on RNA-seq and ribo-seq of human hepatocytes (n=3) IL11RA1 and (d) IL6RTranscriptome readout coverage. (e and f) (e) Western blot showing the activation status of ERK, JNK, and STAT3, and (f) ALT secretion by hepatocytes after stimulation with HyperIL11 or HyperIL6 across dose ranges. (g) ALT levels in the supernatant of hepatocytes stimulated with HyperIL11 alone or in the presence of increasing amounts of soluble gp130 (sgp130). (h and i) (h) Phosphorylated ERK and JNK and their respective total expression in response to HyperIL11 stimulation alone or with sgp130, and (i) Western blot of hepatocytes showing phosphorylated STAT3 and total STAT3 in response to HyperIL6 stimulation with and without sgp130. (j) Representative FSC plot of propidium iodide (PI) staining of IL11-stimulated hepatocytes in the presence of sgp130 or soluble IL11RA (sIL11RA). (k) Western blot showing p-ERK, p-JNK, and their respective total expression in hepatocytes in response to IL11 stimulation alone or in the presence of sgp130 or sIL11RA. (a to k) Primary human hepatocytes; (e to k) 24-hour stimulation; (e to k) HyperIL11, HyperIL6, IL11 (20 ng / ml), sgp130, sIL11RA (1 μg / ml). (b, f to g) Data are plotted as box-and-whiskers with median values ​​(centerline), 25-75 percentiles (box), and min-max values ​​(whisker). FIGS. 34a to FIGS. 34k. Graphs, scatter plots, and images showing lipid-laden hepatocytes secreting IL11, which is autocrine IL11 cis- Induces multiple lipid-toxicity phenotypes through signaling. (ak) Data for palmitate loading experiments in primary human hepatocytes in the presence of IgG (2 μg / ml), anti-IL11RA (X209, 2 μg / ml), or sgp130 (1 μg / ml). (a) IL11, (b) IL6, (c) CCL2, and (d) CCL5 protein secretion levels as measured by ELISA of the supernatant. (e and f) Representative FSC plot and (f) quantification of palmitate-stimulated PI+ve hepatocytes. (g) ALT levels in the supernatant. (h) Hepatocyte glutathione (GSH) levels. (i) Representative fluorescence image of DCFDA staining (ROS detection; scale bar, 100 μm). (j) Western blot of pERK, ERk, pJNK, JNK, truncated Caspase 3, Caspase 3, NOX4, FASN, and GAPDH, and (k) representative image of Oil Red O staining (scale bar, 100 μm). (ad, fh) mean ± SD; Turkey-corrected Students' t-test. FIGS. 35a to FIGS. 35p. IL6 family cytokines trans- Schematic, image, and box plot showing that inhibition of signaling has no effect on NASH or metabolic phenotypes in mice on a fructose-supplemented Western diet. (a) Schematic of WDF rearing in mice with hepatocyte-specific expression of sgp130 for the data shown in (bp). At 3 weeks after injection of AAV8-Alb-Null or AAV8-Alb-sgp130 viruses, mice were reared on WDF for 16 weeks. (b) Western blot showing liver levels of sgp130, IL11, IL6, and GAPDH as an internal control. (c) Serum IL11 levels. (d) Serum IL6 levels. (e) Representative whole anatomy and H&E-stained images of the liver. (f) Liver weight. (g) Liver triglyceride content. (h) Serum ALT levels. (i) Serum AST levels. (j) Liver collagen levels. (k) Fasting blood glucose level. (l) Serum triglyceride level. (m) Serum cholesterol level. (n) Liver GSH content. (o) Heatmap of liver pro-inflammatory and fibrotic gene expression (values ​​shown in Figures 41d and 41e). (p) Western blot of liver p-ERK, ERK, p-JNK, JNK, p-STAT3, and STAT3. (cn) Data are the median (midline), 25 th -75 th Plotted as Box-and-Whisker with percentiles (boxes) and min-max values ​​(whiskers), it is a Turkey-corrected Student's t-test; the conditions shown from left to right are as follows: NC Invalid, WDF Invalid, WDF sgp130 . FIGS. 36a to 36k. IL11 cis - Schematic diagram, images, graphs, and box plots illustrating that hepatocyte-specific inhibition of signaling protects against cachexia and NASH in mice on the HFMCD diet. (a) AAV8-Alb-Cre injected for the experiment shown in (bk) Il11ra1 loxP / loxP This is a schematic diagram of the HFMCD rearing regimen for (conditional knockout; CKO) mice. Il11ra1 loxP / loxP Mice were intravenously injected with AAV8-Alb-invalid or AAV8-Alb-Cre to specifically delete Il11ra1 in hepatocytes at week 3 prior to the start of the HFMCD diet. (b) Western blot of hepatic IL11RA and GAPDH. (c) Body weight (shown as a percentage of initial body weight). (d) Representative whole anatomy and H&E-stained images of the liver. (e) Hepatic triglyceride content. (f) Serum ALT levels. (g) Serum AST levels. (h) Hepatic GSH content. (i) Hepatic collagen levels. (j) Pro-inflammatory markers ( Tnf , Ccl2 , Ccl5 ) and fibrosis markers( Col1a1 , Col1a2 , Col3a1 , Acta2 (k) This is a heatmap plotting liver mRNA expression. The values ​​are shown in Figures 43a and 43b. (k) This is a Western blot showing the liver ERK and JNK activation status. (c) Data are plotted as a two-sided ANOVA with mean ± SEM and Turkey multiple comparison test, and statistical significance is indicated by the P-value between HFMCD WT and CKO; (ei) Data are the median (midline), 25 th -75 th Presented as a Box-and-Whisker, Sidoc-corrected Student's t-test with percentiles (boxes) and min-max values ​​(whiskers); the conditions from left to right are as follows: NC WT, NC CKO, HFMCD WT, HFMCD CKO. FIGS. 37a to FIGS. 37m. IL11 cisSchematics, images, graphs, and box plots illustrating that mice with hepatocyte-specific inhibition of signaling are protected against WDF-induced obesity and NASH. (a) Schematic of WDF-fed control and CKO mice for the data shown in (bm). At 3 weeks after AAV8-Alb-inactivated or AAV8-Alb-Cre virus injection, CKO mice were fed WDF for 16 weeks. (b) Western blot showing liver levels of IL11RA and GAPDH. (c) Body weight (expressed as a percentage of initial body weight). (d) Fat mass. (e) Representative whole anatomy and H&E-stained images of the liver. (f) Liver triglyceride content. (g) Liver weight. (h) Serum ALT levels. (i) Serum AST levels. (j) Liver GSH content. (k) Liver collagen levels. (l) Hepatic pro-inflammatory and fibrotic gene expression in the heatmap (values ​​are shown in Figs. 44a and 44b). (m) Western blot showing the activation status of liver ERK and JNK. (c and d) Data are plotted as two-sided ANOVA with mean ± SEM and Turkey multiple comparison test, and statistical significance is indicated by P-values ​​between WDF WT and CKO; (fk) Data are the median (midline), 25 th -75 th Presented as a Box-and-Whisker, Sidoc-corrected Student's t-test with percentiles (boxes) and min-max values ​​(whiskers); the conditions from left to right are as follows: NC WT, NC CKO, WDF WT, WDF CKO. FIGS. 38a to 38n. IL11 trans - Not signaling, but hepatocyte-specific IL11 cis - Schematic diagram, image, and box plot illustrating that signaling induces fatty liver hepatitis in mice in WDF. (a) For the experiment illustrated in (bn) Il11ra1+ / + (WT) and Il11ra1- / -(b) A schematic diagram illustrating the WDF rearing regimen in (KO) mice. KO mice injected with AAV8-Alb-inactivated, AAV8-Alb-mbIl11ra1 (full-membrane-conjugated Il11ra1), and AAV8-Alb-sIl11ra1 (soluble form of Il11ra1) received 16 weeks of WDF rearing starting at week 3 after virus administration. (b) Western blot showing liver levels of IL11RA and GAPDH. (c) Representative whole anatomy and H&E stained images of the liver. (d) Liver weight. (e) Liver triglyceride content. (f) Serum ALT levels. (g) Serum AST levels. (h) Liver GSH content. (i) Liver collagen content. (j) Heatmap of liver pro-inflammatory and fibrotic gene expression (values ​​shown in Figs. 45c and 45d). (k) Western blot showing the activation status of hepatic ERK and JNK. (l) Fasting blood glucose level. (m) Serum triglyceride level. (n) Serum cholesterol level. (di, ln) Data are the median (midline), 25 th -75 th Presented as a Box-and-Whisker, Turkey-corrected Student's t-test with percentiles (boxes) and min-max values ​​(whiskers); the conditions shown from left to right are as follows: NC, Invalid WT, WDF; Invalid WT, WDF; Invalid KO, WDF mbIl11ra1 KO, WDF sIl11ra1 KO. Fig. 39.This is a schematic diagram of the proposed mechanism of IL11 signaling in NASH. Excessive lipid accumulation in hepatocytes leads to lipotoxicity, inducing the generation of reactive oxygen species that trigger IL11 protein translation and secretion. IL11 binds to IL11RA and gp130 on hepatocytes, initiating the activation of autocrine ERK, JNK, and Caspase 3 to induce adipocyte apoptosis. IL11 also acts paracrinely to induce the transformation of resting hepatic astrocytes (HSCs) into activated myofibroblasts. Cytokines and chemokines released from lipotoxic hepatocytes and HSCs activate and mobilize immune cells, thereby inducing inflammation. Therefore, autocrine IL11 in hepatocytes cis Signaling is a critical initiation event for all NASH beds. FIGS. 40a to 40i.Scatter plots, box plot histograms, images, and graphs regarding the expression of receptors for IL-11 and IL-6 and the effects of IL-11 and IL-6 signaling in primary human hepatocytes. (a) Representative FSC plot of IL11RA, IL6R, and gp130 staining on activated THP-1 cells. (b) gp130 transcripts in primary human hepatocytes based on RNA-seq and ribo-seq (transcripts per million, TPM). (c) Read coverage of gp130 transcripts based on RNA-seq and ribo-seq in primary human hepatocytes (n=3). (d) Immunofluorescence images (scale bar, 100 μm) of IL11RA, IL6R, gp130, and albumin expression in primary human hepatocytes and activated THP-1 cells. (e) Basal level of soluble IL6R in hepatocyte medium. (f) Quantification of PI staining on IL11-stimulated primary human hepatocytes (PI+ve cells) in the presence of sgp130 or sIL11RA. (g) Dose-dependent effect of increasing concentrations of IL11 in the presence of 1 μg / ml of sgp130 or sIL11RA on ALT levels secreted by primary human hepatocytes. (h) Dose-dependent effect of increasing concentrations of sgp130 or sIL11RA on IL11-induced ALT secretion. (i) Hepatocyte triglyceride levels after palmitate stimulation in the presence of IgG (2 μg / ml), anti-IL11RA (X209, 2 μg / ml), or sgp130 (1 μg / ml). (b, g to h) Data are plotted as box-and-whiskers with median (centerline), 25–75 percentiles (box), and min–maximum values ​​(whisker); (e to f, i) Data are plotted as mean ± SEM; (f to i) Turkey-corrected Students' t-test. (g) From left to right for each concentration of IL-11, the conditions shown are as follows: BL, sgp130, sIL11RA.(h) For each concentration of IL11 + sgp130 / IL11RA, the conditions shown from left to right are as follows: sgp130, sIL11RA. FIGS. 41a to 41e. This is a schematic diagram, box plot, and graph illustrating that sgp130 expression does not protect mice from WDF-induced liver and obesity phenotypes. (a) Schematic diagram of the gp130 protein domain structure and its amino acid positions (left) and the domain used to construct sgp130 in this study (right). (be) WDF-sgp130 as shown in Fig. 35a In vivo Data for the experiment. (b) Serum gp130 levels in NC-bred control mice and WDF-bred AAV8-Alb-inactivated-injected mice and AAV8-Alb-sgp130-injected mice. (c) Effect of 16 weeks of WDF on body weight in AAV8-Alb-inactivated-injected mice and AAV8-Alb-sgp130-injected mice. Data are expressed as mean ± SEM. (d and e) As shown in Fig. 35o, (d) pro-inflammatory markers ( Tnf , Ccl2 , Ccl5 ) and (e) fibrosis markers ( Col1a1 , Col1a2 , Col3a1 , Acta2 This is the liver mRNA expression of ). (b, de) Data are the median (midline), 25 th -75 th Plotted as a box-and-whisker, turkey-corrected Student's t-test with percentiles (box) and min-max values ​​(whiskers); the conditions shown from left to right are as follows: NC invalid, WDF invalid, WDF sgp130 . FIGS. 42a to 42n. Estimated members of the IL6 family trans- Schematic, image, and box plot illustrating that inhibition of signaling has no effect on the NASH phenotype in mice on the HFMCD diet. (a) Schematic of mice with hepatocyte-specific expression in mice on the HFMCD diet for the data shown in (bn). Mice were intravenously injected with AAV8-Alb-invalid or AAV8-Alb-sgp130 for 4 weeks and reared on HFMCD. (b) Western blot showing liver levels of sgp130, IL11, and IL6 along with GAPDH shown as an internal control. (c) Serum gp130 levels. (d) Serum IL11 levels. (e) Serum IL6 levels. (f) Representative whole anatomy and H&E-stained image of the liver. (g) Liver triglyceride content. (h) Serum ALT levels. (i) Serum AST levels. (j) Liver GSH content. (k) Liver collagen levels. (l and m) (l) Pro-inflammatory markers ( Tnf , Ccl2 , Ccl5 ) and (m) fibrosis markers( Col1a1 , Col1a2 , Col3a1 , Acta2 This is the liver mRNA expression of ). (n) Western blot of liver p-ERK, ERK, p-JNK, JNK, p-STAT3, and STAT3. (ce, gm) Data are the median (midline), 25 th -75 th Presented as a Box-and-Whisker, Turkey-corrected Student's t-test with percentiles (box) and min-max values ​​(whiskers); the conditions shown from left to right are as follows: NC invalid, HFMCD invalid, HFMCD sgp130 . Figs. 43a and 43b. Il11ra1This is a box plot illustrating that mice with hepatocyte-specific deletions are protected from HFMCD-induced gene dysregulation. (a and b) (a) pro-inflammatory markers (from control and CKO mice on NC and HFMCD diets as shown in Fig. 36j) Tnf , Ccl2 , Ccl5 ) and (b) fibrosis markers ( Col1a1 , Col1a2 , Col3a1 , Acta2 This is the liver mRNA expression of ). (ab) Data is the median (midline), 25 th -75 th Box-and-whisker, Sidac-corrected Students' t-tests are presented with percentiles (boxes) and min-max values ​​(whiskers); for each gene, the conditions shown from left to right are as follows: NC WT, NC CKO, HFMCD WT, HFMCD CKO. FIGS. 44a to 44e. Hepatocyte-specific Il11ra1 This is a box plot illustrating that the deleted mice are protected from the WDF-induced NASH phenotype. (ae) Data for control and CKO mice on NC and WDF diets as shown in Fig. 37a. (a and b) As shown in Fig. 37l, (a) pro-inflammatory markers ( Tnf , Ccl2 , Ccl5 ) and (b) fibrosis markers ( Col1a1 , Col1a2 , Col3a1 , Acta2 (a) Liver mRNA expression. (c) Fasting blood glucose level. (d) Serum triglyceride level. (e) Serum cholesterol level. (ae) Data are the median (midline), 25 th -75 thBox-and-whisker, Sidac-corrected Students' t-tests are presented along with percentiles (boxes) and min-max values ​​(whiskers). (a and b) For each gene, the conditions shown from left to right are as follows: NC WT, NC CKO, WDF WT, WDF CKO. (CE) The conditions shown from left to right are as follows: NC WT, NC CKO, WDF WT, WDF CKO. FIGS. 45a to 45d. IL11 trans - Not signaling, but hepatocyte-specific IL11 cis - A schematic diagram and box plot illustrating that signaling induces WDF-induced steatohepatitis in WDF-infected mice. (a) A schematic diagram of the full-length membrane-bound IL11RA protein domain and its structural amino acid positions (left) and the domain used to construct soluble IL11RA (right). (bd) When AAV8-Alb-inactivated, AAV8-Alb-mbIl11ra1 (full-length membrane-bound Il11ra1), or AAV8-Alb-sIl11ra1 (soluble form of Il11ra1) were injected as exemplified in Fig. 38a. Il11ra1+ / + (WT) mice and mice with total deletion for Il11ra ( Il11ra1- / - (b) Data on WDF rearing regimen in ;KO mice. (c) Serum IL11RA levels in AAV8-Alb-inactivated and AAV8-Alb-sIl11ra1-injected KO mice in WDF. (c and d) (c) Pro-inflammatory markers ( Tnf , Ccl2 , Ccl5 ) and (d) fibrosis markers ( Col1a1 , Col1a2 , Col3a1 , Acta2 This is the liver mRNA expression of ). (bd) Data are the median (midline), 25 th -75 thPresented as a box-and-whisker, turkey-corrected Student's t-test with percentiles (box) and min-max values ​​(whisker). (c and d) For each gene, the conditions shown from left to right are as follows: NC invalid WT, WDF invalid WT, WDF invalid KO, WDF mbIl11ra1 KO, WDF sIl11ra1 KO. FIGS. 46a to 46l. IL11 trans - Not signaling, but hepatocyte-specific IL11 cis - Schematic diagram, images, and box plots illustrating that signaling induces steatohepatitis in mice on HFMCD. (a) Schematic diagram of HFMCD-bred WT and KO mice for the experiment illustrated in (bl). KO mice were intravenously injected with AAV8-Alb-inactivated, AAV8-Alb-mbIl11ra1, or AAV8-ALB-sIl11ra1; WT mice received AAV8-Alb-inactivated as a control. At 3 weeks after viral administration, mice were started on HFMCD for 4 weeks. (b) Western blot showing liver levels of IL11RA and GAPDH. (c) Serum IL11RA levels. (d) Representative whole anatomy and H&E-stained images of the liver. (e) Liver triglyceride content. (f) Serum ALT levels. (g) Serum AST levels. (h) Liver GSH levels. (i) Liver collagen content. (j and k) (j) Pro-inflammatory markers ( Tnf , Ccl2 , Ccl5 ) and (k) fibrosis markers( Col1a1 , Col1a2 , Col3a1 , Acta2 (l) This is the liver mRNA expression of ). (l) This is a Western blot showing the activation status of liver ERK and JNK. (c, ek) Data are the median (midline), 25 th -75 thPresented as a Box-and-Whisker, Turkey-corrected Student's t-test with percentiles (boxes) and min-max values ​​(whiskers). (ei) The conditions shown from left to right are as follows: NC Invalid WT, HFMCD Invalid WT, HFMCD Invalid KO, HFMCD mbIl11ra1 KO, HFMCD sIl11ra1 KO. (j and k) For each gene, the conditions shown from left to right are as follows: NC Invalid WT, HFMCD Invalid WT, HFMCD Invalid KO, HFMCD mbIl11ra1 KO, HFMCD sIl11ra1 KO. Figs. 47a and 47b. Graphs and images showing that pancreatic stellate cells (PSCs) express IL-11Rα and gp130, rather than IL-6Rα. (a) In mouse PSCs and ductal cells Il6st (encoded in gp130), Il11ra1 and Il6ra (b) Single-cell RNA sequencing analysis of expression. (b) Immunofluorescence analysis of expression of gp130, IL11RA, and IL6RA proteins by human PSCs. Figs. 48a and 48b. Box plot and images showing the activation of pancreatic astrocytocytes (PSCs) into an αSMA-positive, collagen-expressing fibrogenic phenotype. (a) PSCs for 24 hours with the indicated factors in the presence or absence of a neutralizing anti-IL-11RA antibody or an IgG isotype control antibody. In vitro (b) Quantification of the high-content imaging assay for the percentage of ACTA2-positive cells and collagen I intensity / area after stimulation. In vitro This is a representative image of the high-content imaging analysis of collagen I intensity / area after stimulation. FIGS. 49a to 49c. In genetically modified mice with inducible, fibroblast-specific expression of IL-11 In vivoSchematic diagram, box plot, and image regarding the induction of pancreatic fibrosis. (a) Gene transplantation Col1a2-CreER Rosa26Il11 / + (b) A schematic representation of an experiment in which IL11 Tg mice are induced to express IL-11 in fibroblasts by treatment with tamoxifen. (b) Hydroxyproline content in pancreatic tissues of control mice and IL11 Tg mice after 24 days. (c) Representative image of Mason tricolor staining of pancreatic tissues from control mice and IL11 Tg mice after 24 days. FIGS. 50a to 50c. Schematic diagram, box plot, and images regarding the effects of antagonism on IL-11-mediated signaling in a pancreatic duct ligation (PDL) model of pancreatic injury. (a) Schematic representation of an experiment in which pancreatic injury is induced by PDL and mice are subsequently treated with a neutralizing anti-IL-11RA antibody or an IgG isotype control antibody. (b) Ligated lobe weights for mice treated with a neutralizing anti-IL-11RA antibody or an IgG isotype control antibody at day 14. (c) Representative images of Mason tristaining of pancreatic tissue from mice treated with a neutralizing anti-IL-11RA antibody or an IgG isotype control antibody at day 14. Specific details for implementing the invention

[0497] Examples

[0498] In the following examples, the inventors demonstrate that inhibition of IL-11-mediated signaling reduces and reverses the severity of symptoms of a wide range of metabolic diseases.

[0499] Example 1: General method for Examples 1 to 4

[0500] IL-11-RA-Knockout Mouse

[0501] Mice lacking the functional allele for Il11rα (Il11rα- / -) were in the C57Bl / 6J genetic background (B6.129S1-Il11rαtm1Wehi / J, Jackson's Laboratory).

[0502] Treatment using anti-IL-11 or anti-IL-11Rα antibodies

[0503] Mice were injected intraperitoneally with 10 mg / kg of an antagonist anti-IL-11 antibody, an antagonist anti-IL-11Rα antibody, or an equal amount of an isotype-matched IgG control antibody. The anti-IL-11 and anti-IL-11Rα antibodies bind to mouse IL-11 and mouse IL-11Rα, respectively, and inhibit IL-11-mediated signaling.

[0504] Specifically, the anti-IL-11 antibody used in this embodiment is mouse anti-mouse IL-11 IgG X203, which is described, for example, in the literature [Ng et al., Sci Transl Med. (2019) 11(511) pii: eaaw1237] (also disclosed in the literature [Ng, et al., "IL-11 is a therapeutic target in idiopathic pulmonary fibrosis." bioRxiv 336537]; doi: https: / / doi.org / 10.1101 / 336537). X203 is also referred to as "Enx203" and includes a VH region according to SEQ ID NO:92 of WO 2019 / 238882 A1 (SEQ ID NO:22 of the present disclosure) and a VL region according to SEQ ID NO:94 of WO 2019 / 238882 A1 (SEQ ID NO:23 of the present disclosure).

[0505] The anti-IL-11Rα antibody used in this embodiment is mouse anti-mouse IL-11Rα IgG X209, which is described, for example, in the literature [Widjaja et al., Gastroenterology (2019) 157(3):777-792] (also disclosed in the literature [Widjaja, et al., "IL-11 neutralizing therapies target hepatic stellate cell-induced liver inflammation and fibrosis in NASH." bioRxiv 470062]; doi: https: / / doi.org / 10.1101 / 470062). X209 is also referred to as "Enx209" and includes a VH region according to SEQ ID NO:7 of WO 2019 / 238884 A1 (SEQ ID NO:24 of the present disclosure) and a VL region according to SEQ ID NO:14 of WO 2019 / 238884 A1 (SEQ ID NO:25 of the present disclosure).

[0506] diet

[0507] Using a Western diet (WDF) with fructose, metabolic disorders closely resembling those in humans during obesity, T2D, and NAFLD were established (Baena et al., Sci Rep (2016) 6: 26149; Macado et al., PLoS One (2015) 10:e0127991).

[0508] To establish metabolic diseases, such as obesity and T2D, mice were fed a Western diet (D12079B, Research Diets) (WDF) supplemented with 15% by weight / volume fructose in their water for 16 weeks starting from 12 weeks of age.

[0509] Cachexia-like metabolic disorders were established using a high-fat methionine and choline-deficient diet (HFMCD). To establish cachexia body weight loss and lean mass loss, C57BL / 6N mice were fed a methionine and choline-deficient (HFMCD) diet supplemented with 60 kcal% fat (A06071301B, Research Diets).

[0510] Control subjects were raised on normal feed (NC, Specialty Feeds) and drinking water.

[0511] Echo MRI analysis of body composition

[0512] Total body fat and lean mass measurements were performed every two weeks by EchoMRI analysis using a 4-in-1 body composition analyzer on living small animals.

[0513] Fasting blood glucose measurement

[0514] To measure fasting blood glucose, mice were fasted for 6 hours prior to blood collection (via tail snip), and fasting glucose was measured using an Accu-Chek blood glucose meter.

[0515] Intraperitoneal glucose tolerance test (ipGTT)

[0516] For the intraperitoneal glucose tolerance test, mice were fasted for 6 hours prior to receiving the ipGTT. Basal fasting glucose was measured using an Accu-Chek blood glucose meter via tail snip. 2 g / kg lean mass glucose was injected intraperitoneally, and glucose measurements were performed every 15 minutes for 2 hours. The area under the curve (AUC) was calculated and plotted as a bar graph.

[0517] Histology of the pancreas for the islets of Langerhans, glucagon, and insulin

[0518] For histological analysis, pancreatic samples were resected, fixed in 4% neutral-buffered formalin (NBF) at RT for 24 hours, and stored in 30% sucrose. 5 μm frozen sections were stained overnight with glucagon or insulin antibodies, visualized according to standard protocol using the ImmPRESS HRP IgG polymer detection kit (Vector Laboratories) with ImmPACT DAB peroxidase substrate (Vector Laboratories), and examined by light microscopy.

[0519] Example 2: Antagonism of IL-11-mediated signaling in obesity-related disorders

[0520] To investigate the effects of antagonism on IL-11-mediated signaling on obesity and related disorders, e.g., T2D, using diet-induced mouse models of these metabolic diseases, either by using IL-11 receptor alpha knockout (IL11-RA- / -) mice or by treating mice with antagonist anti-mouse IL-11 antibodies or antagonist anti-mouse IL11-RA antibodies. In vivo The experiment was performed.

[0521] IL11RA knockout mice fed on a normal diet (NCD) or WDF showed an improved metabolic phenotype compared to wild-type IL11RA-expressing littermates.

[0522] Figures 1a and 1b show that body weight increased more in wild-type mice than in IL11RA knockout mice. Figures 2a and 2b show that IL11RA knockout mice had significantly lower total body fat mass compared to wild-type mice. Figure 3 shows that IL11RA knockout mice had significantly lower fasting blood glucose levels compared to wild-type mice. Figure 4 shows that IL11RA knockout mice had significantly lower serum triglyceride levels compared to wild-type mice. Figures 5a and 5b show that IL11RA knockout mice had significantly lower serum cholesterol levels compared to wild-type mice.

[0523] The results suggested that a reduction in IL-11-mediated signaling has beneficial effects on metabolism.

[0524] Next, the inventors investigated the effects of an antagonist anti-IL-11RA antibody or a control IgG antibody on mice reared with WDF.

[0525] Surprisingly, anti-IL-11RA antibody-treated mice fed with WDF showed a significant decrease in body weight compared to control IgG antibody-treated mice fed with WDF (Fig. 6a). Similar to IL11RA KO mice (Fig. 3), these anti-IL-11RA antibody-treated mice also showed significantly reduced fat mass (Fig. 6b).

[0526] Interestingly, an increase in lean body mass was also observed in mice treated with the anti-IL-11RA antibody compared to IgG control-treated mice, suggesting that inhibition of IL-11 signaling during WDF-induced metabolic onset restored muscle mass. Furthermore, the results of the intraperitoneal glucose tolerance test (ipGTT) showed a significant improvement in glucose tolerance in mice treated with the anti-IL-11RA antibody, along with fasting glucose (Figs. 7a and 7b).

[0527] The analysis was extended to include effects on the pancreas. Unexpectedly, anti-IL-11RA antibody-treated mice reared with WDF were found to exhibit remarkable protection against WDF-induced loss of the pancreas compared to IgG control-treated mice, whether treated for 8 to 16 weeks (to protect against metabolic disease-related effects) or 16 to 24 weeks (to reverse metabolic disease-related effects) (Fig. 8).

[0528] Figure 9a shows that anti-IL-11RA antibody-treated mice fed with WDF had significantly lower serum cholesterol levels compared to control IgG antibody-treated mice fed with WDF, and Figure 9b shows that anti-IL-11RA antibody-treated mice fed with WDF had significantly lower serum triglyceride levels compared to control IgG antibody-treated mice fed with WDF. Figure 9c shows that anti-IL-11RA antibody-treated mice fed with WDF had significantly lower fasting blood glucose levels compared to control IgG antibody-treated mice fed with WDF.

[0529] Furthermore, immunohistology of the pancreas also revealed increased glucagon and insulin staining in the pancreatic islets, along with pancreatic islet hyperplasia, in IgG-treated WDF-bred mice (Figs. 10a and 10b), which is a typical characteristic of T2D (Bonner-Weir and O'Brien Diabetes (2008) 57:2899-2904). Treatment with anti-IL-11RA antibodies in WDF-bred mice from 16 to 24 weeks not only significantly reduced pancreatic islet hyperplasia and glucagon staining but also enhanced insulin expression in the pancreatic islets (Figs. 10a and 10b), suggesting that antagonism of IL-11-mediated signaling is useful for improving and reversing metabolic diseases caused by a Western-type diet.

[0530] Example 3: IL-11-mediated signaling and antagonism of cachexia

[0531] The effects of the anti-IL-11 treatment were evaluated in a mouse model of cachexia.

[0532] Rearing mice on a methionine-choline-deficient (MCD) diet induces severe non-alcoholic steatohepatitis (NASH), liver inflammation, and fibrosis, resulting in severe and sustained body weight loss (up to 30% of body weight after 3 weeks of the MCD diet). Mice on the MCD diet have a 36% higher metabolic rate than mice on a normal feed diet (NCD) and possess a strong appetite-stimulating environment (low leptin, low glucose, low TGs / cholesterol, low insulin), while this does not increase their food consumption (Rizki et al. J. Lipid Res. (2006) 47:2280-2290). As such, the MCD diet is a well-recognized model of cachexia and shares many traits with cancer-related cachexia. Steatohepatitis is frequently documented in the literature in experimental and human cancer cachexia and represents an important but poorly understood role in wasting syndromes.

[0533] Five-week-old male mice were fed a methionine-deficient and choline-deficient (MCD) diet along with 60 kcal% fat (A06071301B, Research Diets), designated as a high-fat MCD (HFMCD) diet, which causes NASH more severe than the MCD diet alone. Control mice were fed a normal diet (NC; Specialty Feeds). One week after receiving HFMCD for the same treatment sequence, these mice were intraperitoneally injected with 10 mg / kg of anti-IL-11 antibody or anti-IL-11RA antibody, or the same concentration of IgG isotype control, twice per week. Body weight was measured weekly.

[0534] The results are shown in Figures 11a and 11b. Anti-IL-11 therapy was found to have a distinct positive effect on body weight, indicating that inhibition of IL-11-mediated signaling can improve cachexia-related body weight loss. While all HFMCD treatment groups (n≥5 mice / group) lost approximately 15% of their body weight after 1 week on the steatohepatitis-induced HFMCD diet, the groups receiving anti-IL-11 or anti-IL-11RA therapy rapidly regained body weight and subsequently returned to normal or near-normal weight by 5 weeks (Figure 11a). Mice fed the NC diet gained steady body weight, whereas mice fed the HFMCD diet and treated with an IgG control lost >30% of their body weight over the course of treatment. A comparison of mouse sizes in examples is shown in Figure 11b. Therefore, inhibition of IL-11-mediated signaling in mouse models of anorexia / cachexia In vivo It was found to reverse cachexia.

[0535] To further investigate the effects of inhibiting IL-11-mediated signaling on cachexia, dose ranges of anti-IL-11 therapies were studied in an MCD model. Five-week-old male mice were fed the HFMCD or NC diet for one week as before, after which cachexia was induced, resulting in a loss of approximately 15% of body weight in MCD mice. After the initial week, mice were intraperitoneally injected with 0.5, 1, 5, or 10 mg / kg of anti-IL-11 or anti-IL-11RA antibodies twice per week. Three antibodies were studied: two anti-IL-11 antibodies ((1) and (2)), and one anti-IL-11RA antibody. 10 mg / kg of an IgG isotype antibody was used as a control.

[0536] Body weight and food consumption were measured weekly. For food consumption, average food intake was measured in a food hopper from the cage (n=3 mice per cage) (g / mouse / week).

[0537] Body weight results are shown in Figures 12a through 12c. All three anti-IL-11 therapies were found to provide dose-dependent gains in body weight, indicating a reversal of cachexia. The highest dose showed the greatest cachexia-reversal effect. Mice fed on the NC diet gained steady body weight, while mice fed on the HFMCD diet and treated with IgG controls lost about 30% of their body weight over the course of treatment.

[0538] The results of food consumption are illustrated in Figures 13a through 13c. All three anti-IL-11 therapies were found to provide a dose-dependent increase in food consumption. The highest dose had the greatest effect on food consumption, whereas mice treated with the IgG control showed a slight decrease in food consumption.

[0539] Anti-IL-11RA antibody treatment was found to be the most effective in reversing weight loss and was associated with the greatest increase in food intake.

[0540] Acute diseases, such as trauma or sepsis, may also be associated with anorexia and cachexia; therefore, the inventors next investigated the effects of IL-11-mediated signaling antagonism on anorexia and cachexia in a mouse model of acute kidney injury.

[0541] Renal injury was induced in 10-week-old male mice by IP injection of folic acid (180 mg / kg) in vehicle (0.3 M NaHCO3); vehicle alone was administered to control mice. Animals were sacrificed on day 28 after injection. Starting 1 hour before folic acid administration, mice were injected intraperitoneally with 20 mg / kg of anti-IL-11 antibody, anti-IL-11RA antibody, or the same concentration of IgG isotype control every 3 days until the mice were sacrificed.

[0542] The results are shown in Fig. 14a. Folate-induced renal injury resulted in rapid anorexia / cachexia-related weight loss associated with the acute phase of severe bilateral renal injury. Mice (n=7 / group) receiving anti-IL-11Rα or anti-IL-11 treatment at the time of injury and throughout the duration of injury regained weight more rapidly compared to IgG controls and subsequently returned to normal or near-normal weight by 3 weeks.

[0543] In the second experiment, renal injury was induced by IP injection of folic acid as in the previous experiment. Starting 21 days after renal injury, mice were treated with either anti-IL-11 antibodies or IgG controls. Animal body weight was evaluated before and after antibody treatment. Healthy mice that did not receive folic acid were used as controls.

[0544] The results are shown in Fig. 14b. Animals treated with the anti-IL-11 antibody began to regain body weight at the start of treatment, which demonstrates that wasting-related weight loss can be improved in terminal disease.

[0545] In additional experiments, mice were subjected to unilateral urinary tract obstruction (UUO)-induced acute renal injury. UUO surgery was performed on 12-week-old mice. Briefly, mice were anesthetized by IP injection of ketamine (100 mg / kg) / xylazine (10 mg / kg), and the total depth of anesthesia was assessed by the pedal reflex. Subsequently, the left side of the mouse's abdomen was shaved. A vertical incision was made through the skin using a scalpel, followed by a second incision through the peritoneum to expose the kidney. Using forceps, the kidney was brought to the surface, and the ureter was ligated twice beneath the kidney with surgical silk. The ligated kidney was gently returned to its correct anatomical position, and sterile saline was added to replenish fluid loss. Afterward, the incisions were sutured. Animals were treated postoperatively with the antibiotic enrofloxacin (15 mg / kg, SC) and the analgesic buprenorphine (0.1 mg / kg, SC) for 3 consecutive days. Mice were sacrificed on the 10th day after ligation. From the 4th day after surgery until the mice were sacrificed, mice were injected intraperitoneally with 20 mg / kg (2x / week) of anti-IL-11 antibody or the same concentration of IgG isotype control.

[0546] The results are shown in Fig. 15. Animals from both groups initially lost a similar amount of body weight (about 6%) due to surgical trauma-related anorexia. Animals receiving anti-IL-11 treatment (20 mg / kg 2x / week from day 4 after UUO until the mice were sacrificed) regained their body weight more rapidly than mice receiving IgG controls and returned to normal body weight within 4 days.

[0547] Therefore, the antagonism of IL-11-mediated signaling is associated with the therapeutic recovery of body weight in models of acute disease.

[0548] Next, the inventors investigated the effect of IL-11 overexpression on mouse body weight through the injection of recombinant mouse IL-11 or the induction of IL-11 transplant gene expression.

[0549] 50 μg ml of recombinant mouse IL-11 (rmIL11) in saline -1 It was reconstituted to the concentration of . 10-week-old male wild-type C57BL / 6J mice were fed 100 μg kg in saline for 21 days. -1 They received daily subcutaneous injections of rmIL11 (n=19) or an equal volume of saline (n=15).

[0550] The results are shown in Figure 16a. Administration of rmIL11 was found to slow the progression of normal body weight gain. Mice that received daily injections of rmIL11 for 21 days gained less body weight compared to mice that received saline alone during the course of treatment.

[0551] IL-11 transgenic (IL-11-Tg) mice were generated. Heterozygous Rosa26-IL11 mice were crossed with Col1a2-CreER mice to generate double heterozygous Col1a2-CreER:Rosa26-IL11 offspring (IL-11-Tg mice) with IL-11 transgenic expression in fibroblasts. For Cre-mediated IL-11 transgenic induction, IL-11-Tg mice were administered 50 mg / kg for 10 consecutive days at 6 weeks of age. -1 Tamoxifen (Sigma-Aldrich) was injected intraperitoneally, and the animals were sacrificed on day 21 (n=14). Rosa26:Il11 mice (lacking the Col1a2-CreER allele) were injected with the same dose of tamoxifen as the control group (n=10) for 10 consecutive days.

[0552] The results are shown in Fig. 16b. IL-11-Tg mice showed early signs of cachexia, ceased gaining weight, and experienced weight loss over time. Therefore, IL-11-mediated signaling was found to contribute to wasting-related weight loss.

[0553] Example 4: Antagonism of IL-11-mediated signaling in a mouse model of non-alcoholic steatohepatitis (NASH)

[0554] The inventors investigated the role of IL-11 signaling in the development of non-alcoholic fatty liver disease (NASH).

[0555] 4.1 Method

[0556] Hepatic stellate cells (HSCs) or hepatocytes were stimulated with IL-11, and their effects were evaluated using cellular and high-content imaging, immunoblotting, ELISA, and invasion assays. Genetic and pharmacological IL-11 function-gain or function-loss experiments were conducted. In a test tube and In vivo IL-11 signaling was investigated using ERK inhibitors. The efficacy of anti-IL-11 or anti-IL11RA therapies was evaluated in three preclinical NASH models using a methionine / choline-deficient diet or a Western diet with liquid fructose. Phenotyping was performed using the hydroxyproline assay, qPCR, RNA-seq, Western blotting, histology, CyTOF, and lipid and metabolic biomarkers.

[0557] animal testing

[0558] All animal procedures were approved and performed in accordance with the SingHealth Institutional Animal Care and Use Committee (IACUC). All mice were provided with food and water at will.

[0559] NASH mouse models

[0560] Wild-type mice fed a high-fat methionine and choline-deficient (HFMCD) diet

[0561] 5-week-old male C57BL / 6N mice were fed a methionine and choline-deficient diet supplemented with 60 kcal% fat (A06071301B16, Research Diets); control mice were fed a normal diet (NC, Specialty Feeds). The duration of diet and antibody treatment is described.

[0562] db / db mice fed a methionine and choline-deficient (MCD) diet

[0563] Male BKS.Cg-Dock7m+ / +LeprdbJ(db / db) mice with a C57BL / 6J genetic background were 12 weeks old and in the stage of hepatic steatosis when fed a methionine-deficient and choline-deficient diet (MCD, A02082002BRi, Research Diets) for 8 weeks; control mice were of the same genotype. The duration of diet and antibody therapy is described.

[0564] Wild-type mice fed a fructose-supplemented Western diet (WDF).

[0565] Ten-week-old male C57Bl / 6J mice were fed a Western diet (D12079B, Research Diets) supplemented with 15% by weight / volume fructose in their drinking water to mimic NAFLD / NASH-like human17,18, while control mice were fed normal feed and tap water. The duration of the diet and antibody treatment is described.

[0566] Il11ra-deleted mouse

[0567] Mice lacking the functional allele for Il11rα (Il11rα- / -) were in the C57Bl / 6J genetic background (B6.129S1-Il11rαtm1Wehi / J, Jackson's Laboratory). Both Il11rα- / - mice and their wild-type littermates (Il11rα+ / +) were fed (1) HFMCD for 10 weeks from 5 weeks of age and (2) WDF for 16 weeks from 12 weeks of age to induce NASH; control mice were fed NC for the same consecutive periods.

[0568] In vivo administration of Il-11

[0569] 10-week-old male Col1a1-GFP reporter mice19 and wild-type C57BL / 6J mice received daily subcutaneous injections of 100 μg / kg of recombinant mouse IL-11 (rmIL-11) or an equal volume of saline for 21 days.

[0570] In vivo administration of anti-IL-11 or anti-IL11RA monoclonal antibodies

[0571] Mice were injected intraperitoneally with an antagonist anti-IL-11 antibody, an antagonist anti-IL11RA antibody, or an equal amount of an IgG isotype control during the treatment period described in Figure 5.

[0572] Fasting blood glucose measurement

[0573] Mice were fasted for 6 hours prior to blood collection (via tail snip), and fasting glucose measurements were obtained using an Accu-Chek blood glucose meter.

[0574] cell culture

[0575] Cells (atrial fibroblasts, HSCs, and hepatocytes) were grown and maintained at 37°C and 5% CO2. The growth medium was refreshed every 2 to 3 days, and cells were subcultured at 80% to 90% confluence using standard trypsin treatment techniques. All experiments were performed at low cell subculture levels (P1 to P2). Cells were serum-depleted (starve) for 16 hours prior to stimulation. Stimulated cells were compared to unstimulated cells that had been grown for the same continuous period under the same conditions (serum-free medium) but without stimulation.

[0576] Primary human atrial fibroblasts

[0577] Human atrial fibroblasts were prepared and cultured as previously described11.

[0578] Primary human liver astrocytes (HSCs)

[0579] HSC (5300, ScienCell) was cultured on a poly-L-lysine-coated plate (2 μg / cm2, 0403, ScienCell) in astrocyte complete medium (5301, ScienCell).

[0580] Primary human hepatocytes

[0581] Human hepatocytes (5200, ScienCell) were grown and maintained in hepatocyte medium (5201, ScienCell) supplemented with 2% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0582] THP-1

[0583] THP-1 (ATCC) was cultured in RPMI 1640 (A1049101, Thermo Fisher) supplemented with 10% FBS and 0.05 mM β-mercaptoethanol. THP-1 cells were differentiated for 48 hours with 10 ng / ml of Fovol 12-myristate 13-acetate (PMA, P1585, Sigma) in RPMI 1640.

[0584] Operetta high throughput phenotyping assay

[0585] The operetta assay was performed as previously described, with minimal modifications to the present invention11: HSCs or hepatocytes were placed in 96-well Black cell carrier plates (PerkinElmer) at a rate of 5 x 10⁶ per well. 3 Canine cells were seeded at a density. Depending on the experimental conditions, cells were fixed in 4% paraformaldehyde (PFA, 28908, Thermo Fisher Scientific), permeated with 0.1% Triton X-100 (Sigma) in PBS, and non-specific sites were blocked with 0.5% BSA and 0.1% Tween-20 in PBS. Cells were incubated overnight (4°C) with the primary antibody (1:500) and subsequently incubated with the appropriate AlexaFluor488 secondary antibody (1:1000). Rhodamine phaloidin staining (1:1000, R415, Thermo Fisher) was performed by overnight incubation (4°C). Cells were counterstained with 1 μg / ml DAPI (D1306, Thermo Fisher) in the blocking solution. Each condition was imaged using the Operetta high-content imaging system 1483 (PerkinElmer) with 2 wells and at least 7 fields per well. ACTA2 was quantified using Harmony v3.5.2 (PerkinElmer), and the percentage of activated fibroblasts to total cells (ACTA2+ve) was determined for each field. Measurement of the fluorescence intensity per area of ​​Collagen I (normalized by the number of cells) was performed using Columbus 2.7.1 (PerkinElmer).

[0586] Immunofluorescence

[0587] Human HSCs and hepatocytes at 24 hours prior to staining were placed on 8-well chamber slides (1.5 x 10⁶ per well). 4Cells were seeded onto a substrate. Cells were fixed in 4% PFA for 20 minutes, washed with PBS, and non-specific sites were blocked with 5% BSA in PBS for 2 hours. Cells were incubated overnight (4°C) with anti-IL11RA or anti-IL6R antibodies, followed by incubation for 1 hour with the appropriate Alexa Fluor 488 secondary antibody. The chamber slides were dried in the dark, and after adding 5 drops of mounting medium with DAPI to the slides for 15 minutes, they were imaged using a fluorescence microscope (Leica).

[0588] Mass cytometry by Time of Flight (CyTOF)

[0589] Immune cells were isolated from the liver as previously described20. Liver tissue was finely ground and lysed with 100 μg / ml collagenase IV and 20 U / ml DNase I at 37°C for 1 hour. After lysation, the cells were passed through a strainer to obtain a single-cell suspension and subjected to percoll gradient centrifugation for the isolation of immune cells. CyTOF staining was performed as previously described21. The cells were thawed and stained with cisplatin (Fluidigm) to identify viable cells, followed by staining with a metal-conjugated CD45 antibody for barcoding purposes. After barcoding, the cells were stained with a metal-conjugated cell surface antibody (Ly6C). Subsequently, the cells were fixed with 1.6% PFA, permeated with 100% methanol, and subjected to intracellular antibody staining (TGFβ1). Cells were labeled with a DNA intercalator and acquired on a Helios mass flow cytometer (Fluidigm). For analysis, the initial living single cell was identified, followed by debarcoding to identify individual samples. Manual gating was performed using Flowjo software.

[0590] Statistical analysis

[0591] Statistical analysis was performed using GraphPad Prism software (version 6.07). P-values ​​were corrected for multiplexed assays based on the d'Net (when several experimental groups were compared to a single condition), turcica (when several conditions were compared within a single experiment), and sidac (when several conditions from two different genotypes were compared). For the comparison of two different groups, an analysis of two parameters (antibody efficacy over time) was performed using binary ANOVA. The criterion for statistical significance was P < 0.05.

[0592] Data availability

[0593] The high-throughput sequencing data generated for this study can be downloaded from GSE128940. All other data are presented in the copy or in the supplementary methods.

[0594] 4.2 Results

[0595] outline

[0596] When stimulated by NASH, factor HSCs secrete IL-11, which induces an autocrine, ERK-dependent signaling loop necessary for the transformation of HSCs into myofibroblasts. IL-11 is upregulated in human and murine NASH; IL-11 injection induces hepatic injury, inflammation, and fibrosis in mice, while Il11ra1-deficient mice are protected from NASH in two preclinical models. Therapeutic antibodies against IL11RA or IL-11 consistently inhibit and reverse fibrosis and steatosis in three murine NASH models. Unexpectedly, IL-11 causes hepatocellular injury and promotes stromal-mediated inflammation, and anti-IL-11 therapies reverse NASH-related hepatotoxicity and hepatitis. Genetic or pharmacological inhibition of IL-11 signaling in NASH is associated with lower serum triglycerides, cholesterol, and glucose.

[0597] IL-11 activates HSCs and induces liver fibrosis.

[0598] Genome-wide RNA-seq analysis showed that TGFβ1 in HSCs IL-11 Strongly upregulated (14.9-fold, P = 3.40x10 -145 It revealed that ) was confirmed by qPCR, verified at the protein level, and replicated in experiments using precision-cut human liver slices (Figs. 17a to 17c, Fig. 24a). Independently generated RNA-seq data 22 Is, IL-11 This shows that it is the gene most upregulated in HSCs when grown on a stiff substrate to model sclerotic liver (Fig. 24b). HSCs have higher levels of IL-11 receptor subunit alpha than cardiac or lung fibroblasts. IL11RAIt expresses ) which is reactive to IL-11 (Fig. 24b). Immunohistochemical analysis confirmed high IL11RA expression and undetectable IL6R expression in HSCs (Fig. 17d). Western blots of human liver samples showed increased IL-11 in patients with fibrotic liver disease, including NASH (Figs. 24d to 24e). These data demonstrate that HSCs are both a source and a target of IL-11 in human livers, and that IL-11 is elevated in human liver disease.

[0599] To investigate the effects of IL-11 on HSCs, cells were stimulated with IL-11, TGFβ1, or PDGF. IL-11 activated HSCs to an extent similar to that of TGFβ1 or PDGF, causing resting HSCs to ACTA2 +ve They were transformed into myofibroblasts, which secrete collagen and matrix-modifying enzymes (Figs. 17e to 17h, Figs. 24f to 24h). IL-11 also promoted dose-dependent matrix invasion by HSCs, which is NASH 23 This is an important aspect of the HSC lesion (Fig. 17i). HyperIL-11 11 HSCs stimulated by also secreted IL-11, confirming the autocrine supply-forward loop of IL-11 signaling (Fig. 17j).

[0600] Recombinant mouse IL-11 (rmIL-11) treated with Col1a1-GFP Reporter Mouse 19 GFP expression in the liver Col1a1 +veIt caused the accumulation of myofibroblasts, which further confirmed the effect of Il-11 on the transformation of HSCs to myofibroblasts in vivo. Subcutaneous administration of rmIl-11 to mice for 21 days also increased liver collagen content, the expression of major fibrosis-promoting and pro-inflammatory genes, and serum alanine aminotransferase (ALT) (Figs. 17k to 17n, Figs. 24i to 24k). This implies that rmIl-11 causes hepatocellular injury and inflammation in addition to fibrosis.

[0601] Deletion of Il11ra1 protects mice from NASH-related inflammation, hepatotoxicity, and fibrosis, and lowers serum lipids and glucose.

[0602] High-fat methionine-deficient and choline-deficient (HFMCD) diet 16 A study was conducted in a preclinical model of severe NASH using [the substance]. In this model, Il-11 mRNA levels were slightly elevated, whereas protein levels were highly upregulated, suggesting post-transcriptional regulation of Il-11 expression in the liver (Figs. 25a to 25b). The gradual induction of Il-11 protein during NASH was reflected by Erk activation, which is related to NASH onset. 24 , increased collagen and elevated serum ALT levels may be important (Fig. 18a, Figs. 25c to 25d).

[0603] To evaluate the pathophysiological relevance of increased Il-levels in NASH, the inventors used a genetic loss-of-function model: Il-11 receptor subunit alpha-deleted mice ( Il11ra1 - / - ) 25 . In the HFMCD diet Il11ra1 - / - The mice were strongly protected from fibrosis and had less steatosis and liver damage compared to the control group (Figs. 18b to 18d, Figs. 25e to 25h). Furthermore, significantly less liver inflammation Il11ra1 - / - It was observed in mice (Fig. 18e), suggesting that Il-11 plays an important role across multiple NASH lesions.

[0604] The HFMCD model features early-onset steatosis followed by fibrosis. However, this model is not obese or insulin resistant. Western diet supplemented with liquid fructose (WDF) 26 Using this, we established another NASH model that is obese, insulin-resistant, and hyperglycemic, which is human NASH 18 This reflected... After 16 weeks of WDF rearing, NASH was established, and Il-11 protein was upregulated in the liver (Fig. 18f). In WDF... Il11ra1 - / - Mice had similar body weight compared to control mice but were protected from hepatic steatosis, inflammation, hepatocellular damage, and fibrosis (Figs. 18g to 18j, Fig. 26). Il11ra1 - / - Erk activation in mice was attenuated in both the HFMCD and WDF models, implying that Il-11-led Erk activation is important for NASH (Fig. 18k, Fig. 25i).

[0605] The primary cause of mortality in NASH is cardiovascular: myocardial infarction, renal failure, and stroke. 27,28 . Biomarkers of cardiovascular risk after 16 weeks on the WDF diet Il11ra1 - / - It was measured in mice. Compared to a litter control, in the WDF Il11ra1 - / - Mice had lower levels of fasting blood glucose, serum cholesterol, and triglycerides (Figs. 18l to 18n).

[0606] Neutralizing anti-IL-11 antibodies or anti-IL11RA antibodies block HSC activation.

[0607] Mice were genetically immunized with IL11RA to generate neutralizing anti-IL11RA antibodies. Clones with blocked fibroblast transformation11 This was identified, clone X209(IgG1κ, K D X209 prioritized MMP2 secretion from HSCs at an IC5.8 pM. 50 It was blocked by and has an in vivo half-life of approximately 18 days with good hepatic absorption (Fig. 19a, Figs. 27a to 27d). To ensure therapeutic specificity for IL-11 signaling, the neutralizing anti-IL-11 antibody X203 12 (IC for IgG1κ, HSC activation) 50 = 40.1 pM) was developed and used in experiments.

[0608] The inventors discovered that, in addition to TGFβ1 (Figs. 17a to 17c), other major NASH stimuli, such as PDGF, CCL2, angiotensin II, bFGF, or oxidative stress, induce IL-11 secretion from HSCs (Fig. 19b). This suggested that IL-11 plays a role downstream of HSC activation by multiple factors. To test this, HSCs were stimulated with various NASH factors, and it was found that all stimuli relied on intact IL-11 signaling to induce ACTA2 or collagen expression (Figs. 19c, 27e). In a separate assay, the pro-invasive effects of PDGF or CCL2 on HSCs were also found to be IL-11-dependent (Fig. 19d).

[0609] Il11ra1 - / - In mice, hepatoprotection in the HFMCD or WDF diets was associated with reduced ERK activation. The inventors discovered that IL-11 directly activates ERK in HSCs, and that any stimulus inducing IL-11 secretion from HSCs also induces ERK activation. X209 neutralized the ERK phosphorylation of all factors, including IL-11 itself, and the downstream of HSC transformation. ERK inhibitors blocked the IL-11 effects of all NASH triggers and the downstream of HSC activation, suggesting that IL-11-induced ERK phosphorylation is of central importance for HSC transformation (Figs. 19e to 19f, Fig. 27f).

[0610] Although published literature on IL-11 in the liver is limited, injection of high doses of recombinant human IL-11 into rodents has a protective effect. 13,14 It has been related to, and platelet biology 25 There is confusion regarding the role of IL-11. To rule out safety issues, the inventors conducted a long-term (5-month) high-dose (10 mg / kg x 2 / week) preclinical toxicology study of X209 and X209 and observed no effects on serum ALT levels or platelets (Figs. 19g to 19h). Il11ra - / - Consistent with the data from mice, anti-IL-11 therapy tended to lower serum lipid levels or lower them during this treatment period (Figs. 19i to 19j).

[0611] Therapeutic targeting of IL-11 or IL11RA is effective in three preclinical NASH models.

[0612] Subsequently, the inventors tested the X209 and X203 therapies in vivo and initiated antibody administration after a 6-week HFMCD diet when IL-11 was strongly upregulated, collagen was accumulated, and steatohepatitis was established (Figs. 18a, 20a, and 25c to 25d). After 4 weeks of treatment, both antibodies inhibited or reversed liver fibrosis, inflammation, and injury, while steatosis remained unchanged (Figs. 20b to 20e, 28a to 28c). Furthermore, both antibodies neutralized Erk activation, which indicates target engagement and coverage (Figs. 20f, 28d).

[0613] The inventors also [described] a 20-week-old patient who was obese, had diabetes, and had steatosis liver when on a NASH-induced methionine-choline-deficient (MCD) diet for 8 weeks db / db Anti-IL-11 therapy was tested in mice (Fig. 20g) 29-31 Consistent with other models of the inventors, IL-11 expression and Erk activation are MCD-fed db / db Erk activation was increased in the livers of mice, and was inhibited by the treatment (Figs. 20h to 20i). In this model, the anti-IL-11 treatment lowered ALT levels while reducing hepatic steatosis, fibrosis, and inflammation (Figs. 20j to 20n, Figs. 28e to 28f).

[0614] Using a third model of WDF-induced NASH, the effects of anti-IL-11 therapy were tested in the context of obesity, insulin resistance, and diabetes. 18Mice were fed WDF for 16 weeks, at which point they were obese and insulin-resistant, along with hepatic steatosis, inflammation, and fibrosis. Subsequently, treatment with an anti-IL11RA (X209) regimen was initiated (Fig. 21a). When IL-11 signaling was targeted, hepatic Erk activation was inhibited in NASH livers (Fig. 29a). Despite similar body weight gain, reversal of hepatic fibrosis, steatosis, and inflammation, as well as a decrease in serum ALT levels, were observed in mice treated with the anti-IL11RA regimen. This was accompanied by a decrease in serum glucose, triglyceride, and cholesterol levels (Figs. 21b to 21g and Figs. 29b to 29e).

[0615] Effects of Combined Metabolic Intervention and Anti-IL-11 Intervention on Liver Fibrosis

[0616] The inventors' data showed that anti-IL-11 therapy reversed fibrosis, but did not evaluate whether this effect was sustained or gradual. Furthermore, combination therapy may be beneficial in reversing fibrosis in NASH. 1 To address this, severe liver fibrosis was established using HFMCD for 10 weeks, then the mice were switched to a normal diet to mimic a potent metabolic intervention, and anti-IL-11 treatment was initiated in combination (Fig. 21h).

[0617] Upon removal of metabolic stimulation, Erk activation gradually regressed, which was accelerated by X203 or X209 treatment (Fig. 30a). Fibrosis remained unchanged in IgG-treated animals throughout the duration of the experiment, suggesting that complete metabolic correction alone does not reverse fibrosis or reverses it very slowly. In contrast, liver collagen content was significantly reversed after 3 weeks of antibody treatment (reversal: 18%, X203; 24%, X209) and further reversed at 6 weeks (reversal: 37%, X203; 46%, X209), demonstrating a gradual and sustained effect (Fig. 21i, Figs. 30b to 30c).

[0618] Regression of fibrosis is associated with lower TIMP and higher MMP levels, which favors matrix reconstruction 3,32 It promotes. Consistent with this, X203 or X209 treated mice with severe fibrosis rapidly Mmp2 Adjust upward and Timp1 It downregulated (Fig. 21j). Transformed HSCs apoptosis 33 , senility 34,35 and / or inactive ACTA2 -ve situation 36 Reversal of liver fibrosis is preferred when undergoing regression. To check whether IL-11 is required to maintain HSCs in a transformed state, HSCs were stimulated with TGFβ1 or PDGF, followed by inhibition of IL-11 signaling. Within 24 hours of IL-11 inhibition, ACTA2 +ve The percentage of cells and the amount of secreted collagen were reversed to near baseline levels, and ERK activity was largely attenuated despite ongoing TGFβ1 / PDGF stimulation (Figs. 21k to 21l, Figs. 30d to 30g).

[0619] Effects of Anti-IL-11 Therapy on Liver Health During Acute Necrotitis in Early NASH

[0620] The transition from NAFLD to NASH is characterized by the onset of steatotic hepatitis, inflammation, and apoptosis (necrotic inflammation). HSCs are pro-inflammatory factors 3,8,37,38 It plays a central role in this process through the secretion of. The inventors investigated whether IL-11 affects the HSC-led inflammatory pathway and found that IL-11 stimulated the HSC population of CCL2, whereas IL-11 inhibition blocked CCL2 secretion (Fig. 31a). This suggests that across a NASH diet Il11ra1 - / - It demonstrates an unrecognized pro-inflammatory role for IL-11 in hepatic immunity in maintaining consistently low levels of inflammation observed in the liver of X203-treated or X209-treated mice.

[0621] In the HFMCD model of NASH, a fibrotic phase follows early inflammation (Fig. 22a). Therapeutic targeting of IL-11 during early steatohepatitis remarkably reduced hepatic steatosis, accompanied by less Erk activation (Figs. 22b–22e, Figs. 31b–31c). Lipid droplets were not observed in the livers of mice receiving X203- and X209, and these mice did not develop fibrosis (Figs. 22d, 22f, and Figs. 31d–31g). The HFMCD diet also induces acute and severe necrotizing inflammation (a >20-fold increase in ALT by 1 week), and a substantial reversal of liver injury using anti-IL-11 therapy was unexpectedly observed over a 3-week period (Figs. 22g, Fig. 31h). This rapid therapeutic benefit advances the fibrotic stage of the disease and, in previous preclinical models Il11ra1 - / - X203 or X209-treated mice consistently showed lower ALT levels, suggesting a direct damaging effect of IL-11 on hepatocytes.

[0622] Primary human hepatocytes were found to express IL11RA rather than IL6R (Fig. 22h). When hepatocytes were stimulated with physiological levels of IL-11, dose-dependent release of ALT was observed. This corresponds to a gradual increase in the expression of stress fibers in hepatocytes (Figs. 22i to 22j). Interestingly, he...

Claims

Claim 1 A pharmaceutical composition for treating or preventing metabolic diseases, comprising an agent capable of inhibiting interleukin 11 (IL-11)-mediated signaling, wherein the metabolic disease is or includes obesity, type 2 diabetes (T2D), pre-diabetes, being overweight, metabolic syndrome, pregnancy-associated hyperglycemia, hyperglycemia, hyperlipidaemia, hypertriglyceridemia, hypercholesterolemia, pancreatic insufficiency, pancreatitis, acute pancreatitis, chronic pancreatitis, lipotoxicity, insulin resistance, or hyperglucagonemia; A pharmaceutical composition capable of inhibiting interleukin 11 (IL-11)-mediated signaling is (i) an anti-IL-11 antibody antagonist of IL-11-mediated signaling or an antigen-binding fragment thereof, or (ii) an anti-IL-11Rα antibody antagonist of IL-11-mediated signaling or an antigen-binding fragment thereof. Claim 2 A pharmaceutical composition according to claim 1, wherein the preparation is a preparation capable of preventing or reducing the binding of interleukin 11 (IL-11) to the receptor for interleukin 11 (IL-11R). Claim 3 A pharmaceutical composition according to claim 1 or 2, wherein the formulation is an anti-IL-11 antibody antagonist of IL-11-mediated signaling or an antigen-binding fragment thereof. Claim 4 A pharmaceutical composition according to claim 1 or 2, wherein the formulation is an anti-IL-11Rα antibody antagonist of IL-11-mediated signaling or an antigen-binding fragment thereof. Claim 5 A pharmaceutical composition according to paragraph 2, wherein the receptor for interleukin 11 is IL-11Rα or comprises the same. Claim 6 A pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is administered to a subject in whom the expression of interleukin 11 (IL-11) or a receptor for IL-11 (IL-11R) is upregulated. Claim 7 A pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is administered to a subject in whom the expression of interleukin 11 (IL-11) or a receptor for IL-11 (IL-11R) was determined to be upregulated. Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete

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