Treatment of hepatotoxicity

Abstract

Disclosed are methods for treating and preventing hepatotoxicity by inhibiting interleukin 11 (IL-11)-mediated signaling, and agents for use in such methods.

Description

[0001] This application claims priority to GB 1900811.9 filed on January 21, 2019, GB 1907839.3 filed on June 3, 2019, and GB 1915003.6 filed on October 17, 2019, the contents and elements of which are incorporated herein by reference for all purposes. Field of the Invention

[0002] The present invention relates to the diagnosis, treatment and prevention of diseases and disorders associated with hepatotoxicity. Background of the Invention

[0004] Hepatotoxicity refers to toxic damage to the liver, specifically the dysfunction and death of hepatocytes within the liver. It is often driven by chemicals, such as damage or injury to the liver caused by drugs, chemicals (such as alcohol), herbs, or dietary supplements. Hepatotoxicity can also be caused by infection (such as by hepatitis viruses), poor nutrition, or genetic disorders.

[0005] The analgesic acetaminophen (APAP, N-acetyl-para-aminophenol, or paracetamol) is commonly used to reduce fever and mild to moderate pain. Acetaminophen overdose is a common cause of liver damage, causing up to 80,000 emergency room visits, 2,500 hospitalizations, and 500 fatal poisonings in the United States each year (Lee WM., Hepatology (2004) 40(1): 6-9; Budnitz DS et al., Am J Prev Med (2011) 40(6): 585-92). In addition, chronic use of low-dose acetaminophen can lead to liver damage, especially in the presence of other predisposing factors, such as chronic alcohol consumption (alcoholism), fasting, malnutrition, HIV infection, hepatitis virus infection, cancer, and drug interactions (McClain et al., Curr. Gastroenterol. Rep., 1999; 1: 42–49).

[0006] The pathophysiology of acetaminophen-induced hepatotoxicity has been extensively studied over the years. Damage to the liver following acetaminophen ingestion is not due to the drug itself, but rather to a toxic metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which is produced by the cytochrome P450 group of enzymes in the liver. NAPQI is normally harmless and is removed from the body after conjugation to endogenous glutathione. However, overdose of acetaminophen results in the accumulation of excess NAPQI through biotransformation by cytochrome P450 (CYP450), primarily the CYP2E1 isoform, due to glutathione depletion. This in turn leads to hepatocellular damage and increased cell death (Jollow et al., J Pharmacol Exp Ther, Oct 1973; 187(1): 195-202; Dahlin et al., Proc Natl Acad Sci USA, Mar 1984; 81(5): 1327-31; Moore et al., Acta Biochimica Sinica, Vol. 260, No. 24, Oct 25, pp. 13035-13040, 1985; Kyle et al., Biochemical Pharmacology, Vol. 40, No. 6, Sept 15, 1990). Liver damage can be limited by administration of N-acetylcysteine, which replenishes liver glutathione levels. Further treatment and prevention methods are needed.

[0007] The cytokine interleukin 11 (IL-11) has been reported to have a protective effect against acetaminophen-induced liver damage and toxicity (Trepicchio WL et al., Toxicol Pathol. 2001; 29(2): 242-9; Nishina T et al., J Biol Chem. 2017; 292(1): 205–216). Activation of the STAT3 pathway by cytokines such as IL-11 has been shown to drive compensatory hepatocyte proliferation, a key principle of liver regeneration, and administration of IL-11 and other STAT3-activating cytokines is considered a treatment for APAP toxicity (Mühl H, Front Immunol, 2016 2; 7: 163). Nishina T et al., Sci Signal., 2012; 5(207): ra5 reported that IL-11 provides a functional link between oxidative stress and compensatory hepatocyte proliferation, and found that IL-11 receptor agonists can promote hepatocyte proliferation and improve oxidative stress in acetaminophen-induced liver injury.

[0008] IL-11 treatment has also been reported to prevent and / or treat other liver diseases, such as hepatic ischemia / reperfusion injury, immune thrombocytopenic purpura (ITP) associated with chronic hepatitis C (HpC-ITP), acute endotoxemia, and T cell-mediated liver injury (Yu J et al., Clin Res Hepatol Gastroenterol., 2016; 40(5): 562-570; Zhu M et al., PLoS ONE 10(5): e0126296; Fontana V et al., Acta Haematol., 2008; 119(2): 126-32; Maeshima et al., Shock., (2004) 21(2): 134-8; Bozza et al., Hepatology (1999) 30(6): 1441-7). SUMMARY OF THE INVENTION

[0010] In contrast to the reported protective role of IL-11 in liver injury, the present invention is directed to treating and / or preventing hepatotoxicity and disorders, diseases or conditions associated with hepatotoxicity by inhibiting IL-11 signaling.

[0011] In one aspect of the present invention, there is provided a use of an agent capable of inhibiting interleukin 11 (IL-11) mediated signaling in a method for treating or preventing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity.

[0012] In another aspect of the invention, there is provided the use of an agent capable of inhibiting interleukin 11 (IL-11) mediated signaling for the manufacture of a medicament for use in a method for treating or preventing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity.

[0013] In another aspect of the present invention, a method for treating or preventing hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity is provided, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an agent capable of inhibiting interleukin 11 (IL-11) mediated signaling.

[0014] In some embodiments, the agent is an agent capable of preventing or reducing the binding of interleukin 11 (IL-11) to interleukin 11 receptor (IL-11R).

[0015] In some embodiments, the agent is capable of binding to interleukin 11 (IL-11) or a receptor for interleukin 11 (IL-11R). In some embodiments, the agent is selected from the group consisting of an antibody or an antigen-binding fragment thereof, a polypeptide, a peptide, a nucleic acid, an oligonucleotide, an aptamer, or a small molecule. The agent may be an antibody or an antigen-binding fragment thereof. The agent may be a decoy receptor.

[0016] In some embodiments, the agent is an anti-IL-11 antibody antagonist of IL-11-mediated signaling, or an antigen-binding fragment thereof. In some embodiments, the agent is an anti-IL-11Rα antibody antagonist of IL-11-mediated signaling, or an antigen-binding fragment thereof.

[0017] In some embodiments, the agent is a decoy receptor for IL-11. In some embodiments, the decoy receptor for IL-11 comprises: (i) an amino acid sequence corresponding to a cytokine binding module of gp130 and (ii) an amino acid sequence corresponding to a cytokine binding module of IL-11Rα.

[0018] In some embodiments, the agent is an IL-11 mutant protein. In some embodiments, the IL-11 mutant protein is W147A.

[0019] In some embodiments, the agent is capable of preventing or reducing the expression of interleukin 11 (IL-11) or interleukin 11 receptor (IL-11R). The agent can be an oligonucleotide or a small molecule.

[0020] In some embodiments, the agent 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 is an siRNA targeting IL11, including the sequence of SEQ ID NO: 12, 13, 14 or 15. In some embodiments, the agent 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 is an siRNA targeting IL11RA, including the sequence of SEQ ID NO: 16, 17, 18 or 19.

[0021] In any of the embodiments provided herein, the interleukin 11 receptor is or comprises IL-11Rα.

[0022] In any of the embodiments provided herein, the agent can be administered prior to, in conjunction with, or after a cause of hepatotoxicity (eg, administration or ingestion of a hepatotoxic drug or exposure to an environmental source of hepatotoxicity).

[0023] In any of the embodiments provided herein, a disorder, disease or condition associated with hepatotoxicity is a disease in which hepatotoxicity is pathologically implicated.

[0024] In some embodiments, the disease pathologically involving liver toxicity is selected from the group consisting of drug-induced liver injury (DILI), acute liver injury (ALI), acute liver failure, acute liver disease, chronic liver disease, liver damage, hepatitis, viral hepatitis, alcoholic hepatitis, hepatic ischemia-reperfusion injury (IRI), warm ischemia-reperfusion (WIR), radiation-induced liver disease (RILD), idiosyncratic drug-induced liver injury (IDILI), autoimmune liver injury, cholestatic liver disease, HIV, and cancer.

[0025] In any embodiment, provided herein are agents, uses and methods for treating and / or preventing drug-induced liver injury (DILI). The DILI can be endogenous and / or specific hepatotoxicity. In any embodiment, provided herein are agents, uses and methods for treating and / or preventing acetaminophen (APAP)-induced hepatotoxicity. In some embodiments, the method further comprises treatment with N-acetylcysteine.

[0026] In any embodiment, the method of treatment or prevention comprises administering the agent to a subject having upregulated expression of interleukin 11 (IL-11) or IL-11 receptor (IL-11R).

[0027] In any embodiment, the method of treating or preventing comprises administering the agent to a subject whose interleukin 11 (IL-11) or interleukin 11 (IL-11R) receptor expression is determined to be upregulated.

[0028] In some embodiments, the method of treatment or prevention comprises determining whether the expression of interleukin 11 (IL-11) or IL-11 receptor (IL-11R) is upregulated in a subject, and administering the agent to a subject in which the expression of interleukin 11 (IL-11) or IL-11 receptor (IL-11R) is upregulated.

[0029] Also provided is a method for determining whether a subject is suitable for treatment or prevention of hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity with an agent capable of inhibiting interleukin 11 (IL-11)-mediated signaling, the method comprising optionally determining in vitro whether expression of interleukin 11 (IL-11) or IL-11 receptor (IL-11R) is upregulated in the subject.

[0030] Also provided is a method for selecting a subject for treatment or prevention of hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity using an agent capable of inhibiting interleukin 11 (IL-11)-mediated signaling, the method comprising optionally determining in vitro whether expression of interleukin 11 (IL-11) or IL-11 receptor (IL-11R) is upregulated in the subject.

[0031] In one aspect, a method for diagnosing hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity in a subject, or the risk of developing hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity is provided, the method comprising optionally determining in vitro the upregulation of interleukin 11 (IL-11) or IL-11 receptor (IL-11R) in a sample obtained from the subject. In some embodiments, the diagnostic method is a method for confirming the diagnosis of hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity in a subject suspected of having hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity. In some embodiments, the method for diagnosing and / or confirming the diagnosis comprises selecting a subject to be treated with an agent capable of inhibiting interleukin 11 (IL-11)-mediated signal transduction.

[0032] Also provided is a method for providing a prognosis for a subject having or suspected of having hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, the method comprising optionally determining in vitro whether expression of interleukin 11 (IL-11) or interleukin 11 receptor (IL-11R) is upregulated in a sample obtained from the subject, and based on the determination, providing a prognosis for the subject treated with an agent capable of inhibiting interleukin 11 (IL-11)-mediated signaling. In some embodiments, the method of providing a prognosis comprises selecting a subject determined to have upregulated expression of interleukin 11 (IL-11) or interleukin 11 receptor (IL-11R) for treatment with an agent capable of inhibiting interleukin 11 (IL-11)-mediated signaling.

[0033] In another aspect, a method for diagnosing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity or a risk of developing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity is provided, the method comprising optionally determining in vitro one or more genetic factors predicted to upregulate expression of IL-11 or IL-11 receptor or upregulate IL-11-mediated signaling in the subject. In some embodiments, the method comprises selecting a subject for treatment with a drug capable of inhibiting interleukin 11 (IL-11)-mediated signaling.

[0034] Also provided is a method for providing a prognosis for a subject having or suspected of having hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, the method comprising optionally determining in vitro one or more genetic factors predicted to upregulate IL-11 or IL-11 receptor expression or upregulation of IL-11-mediated signaling in the subject.

[0035] Details

[0036] There is a long-standing need to effectively prevent and treat hepatotoxicity, such as hepatotoxicity caused by drug-induced liver injury (DILI). Liver damage caused by acetaminophen (APAP) overdose is a common cause of hepatotoxicity.

[0037] Generally speaking, cytokine IL-11 has been repeatedly reported to have a protective effect on APAP-induced liver damage and hepatotoxicity. However, on the contrary, the present inventors have found that inhibiting IL-11-mediated signal transduction can effectively improve APAP-induced hepatotoxicity, thereby proposing a new method for treating and / or preventing hepatotoxicity.

[0038] Interleukin-11 and IL-11 receptor

[0039] Interleukin 11 (IL-11), also known as lipogenesis inhibitory factor, is a pleiotropic cytokine and a member of the IL-6 family of cytokines, including IL-6, IL-11, IL-27, IL-31, oncostatin, leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), ciliary neurotrophic factor (CNTF) and neurogenin (NP-1).

[0040] Interleukin 11 (IL-11) is expressed in many types of mesenchymal cells. The IL-11 genomic sequence has been mapped to the centromeric region of chromosome 19 and chromosome 7 and is transcribed by a classical signal peptide that ensures efficient cell secretion. The IL-11 activation protein complex cJun / AP-1 is located in its promoter sequence and is essential for the basic 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, while 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 available on the market. IL-11 from other species (including mouse, rat, pig, cow, several teleost fish and primates) has also been cloned and sequenced.

[0041] 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). Isoforms, fragments, variants or homologues of IL-11 can optionally be characterized as 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 immature or mature IL-11 from a given species (e.g., human). Isoforms, fragments, variants or homologs of IL-11 may optionally be characterized by their ability to bind to IL-11Rα (preferably from the same species) and stimulate signal transduction in cells expressing IL-11Rα and gp130 (e.g., as described in Curtis et al., Blood, 1997, 90(11); or Karpovich et al., Mol. Hum. Reprod., 2003 9(2):75-80). Fragments of IL-11 may be of any length (in terms of amino acids), although may optionally be at least 25% of the length of mature IL-11, and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of mature IL-11. The minimum length of an IL-11 fragment is 10 amino acids and the maximum length is 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.

[0042] IL-11 signals through homodimers of the ubiquitously expressed glycoprotein 130 (GP130; also known as glycoprotein 130, IL-6ST, IL-6-β, or CD130). Gp130 is a transmembrane protein that forms a subunit of the type I cytokine receptor with the IL-6 receptor family. Specificity is acquired through a separate interleukin 11 receptor subunit, alpha (IL-11Rα), which is not directly involved in signal transduction, although the initial cytokine binding event to the α-receptor leads to the eventual formation of a complex with gp130.

[0043] Human gp130 (including a 22 amino acid signal peptide) is a 918 amino acid protein with a mature form of 866 amino acids, including an extracellular domain of 597 amino acids, a transmembrane domain of 22 amino acids, and an intracellular domain of 277 amino acids. The extracellular domain of the protein includes the cytokine binding module (CBM) of gp130. The CBM of gp130 includes the Ig-like domain D1 of gp130 and the fibronectin type III domains D2 and D3. The amino acid sequence of human gp130 is available under UniProt accession number P40189-1 (SEQ ID NO: 2).

[0044] Human IL-11Rα is a 422 amino acid polypeptide (UniProt Q14626; SEQ ID NO:3) with 85% nucleotide and amino acid sequence identity to mouse IL-11Rα. Two isoforms of IL-11Rα have been reported, which differ in the cytoplasmic domain (Du and Williams, supra). The IL-11 receptor alpha chain (IL-11Rα) and the IL-6 receptor alpha chain (IL-6Rα) have many structural and functional similarities. The extracellular domain shows 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 (Box 1) and box 2 (Box 2) regions required for activation of the JAK / STAT signaling pathway.

[0045] The mouse IL-11 receptor binding site was located and three sites, I, II and III, were identified. Binding to gp130 was reduced by substitutions in the site II region and the site III region. The site III mutant showed no detectable agonist activity and had IL-11Rα antagonist activity (Chapter 8 of Cytokine Inhibitors; edited by Gennaro Ciliberto and RoccoSavino, Marcel Dekker Ltd., 2001).

[0046] In the present specification, IL-11 (IL-11R) receptor refers to a polypeptide or polypeptide complex capable of binding to IL-11. In some embodiments, IL-11 receptor is capable of binding to IL-11 and inducing signal transduction in cells expressing the receptor.

[0047] 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).

[0048] In some embodiments, the IL-11 receptor may be or may comprise IL-11Rα. In some embodiments, the IL-11 receptor may be a polypeptide complex comprising IL-11Rα. In some embodiments, the IL-11 receptor may be or may comprise gp130. In some embodiments, the IL-11 receptor may be a polypeptide complex comprising gp130. In some embodiments, the IL-11 receptor may be a polypeptide complex comprising IL-11Rα and gp130. In some embodiments, the IL-11 receptor may be gp130, or a complex comprising gp130 bound to IL-11.

[0049] An isoform, fragment, variant or homologue of IL-11Rα may optionally be characterized as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence of IL-11Rα from a given species, such as human. An isoform, fragment, variant or homologue of IL-11Rα may optionally be characterized by the ability to bind IL-11 (preferably from the same species) and stimulate signal transduction in cells expressing IL-11Rα and gp130 (e.g., as in Curtis et al., Blood, 1997, 90(11); or Karpovich et al., Mol. Hum. Reprod., 2003 9(2):75-80). Fragments of the IL-11 receptor may be of any length (in terms of number of amino acids), although may optionally be at least 25% of the length of mature IL-11Rα and have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of mature IL-11Rα. The minimum length of a fragment of the IL-11 receptor fragment is 10 amino acids and the maximum length is 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.

[0050] IL-11 signaling

[0051] IL-11 binds to IL-11Rα with low affinity (Kd ~ 10 nmol / L), and the interaction between these binding partners alone is not sufficient to transmit biological signals. The production of high-affinity receptors (Kd ~ 400 to 800 pmol / L) capable of signal transduction 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; Nandurkar et al., Oncogene, 12: 585, 1996). Binding of IL-11 to cell surface IL-11Rα induces heterodimerization, tyrosine phosphorylation, gp130 activation, and downstream signaling primarily through the mitogen-activated protein kinase (MAPK) cascade and the Janus kinase / signal transducer and activator of transcription (JAK / STAT) pathway (Garbers and Scheller, supra).

[0052] In principle, soluble IL-11Rα can also form a biologically active soluble complex with IL-11 (Pflanz et al., 1999 FEBS Lett, 450, 117-122), raising the possibility that, similar to IL-6, IL-11 may bind to soluble IL-11Rα before binding to cell surface gp130 under certain circumstances (Garbers and Scheller, supra). Curtis et al. (Blood, 1997 Dec 1;90(11):4403-12) described the expression of a soluble mouse IL-11 receptor α chain (sIL-11R) and examined signaling in cells expressing gp130. In the presence of gp130 but not transmembrane IL-11R, sIL-11R mediates IL-11-dependent differentiation of M1 leukemia cells and proliferation of BA / F3 cells, as well as early intracellular events similar to those transduced by transmembrane IL-11R, including phosphorylation of gp130, STAT3, and SHP2. Recently, it has been demonstrated that gp130 bound to the cell membrane activates signal transduction by IL-11 binding to soluble IL-11Rα (Lokau et al., 2016 Cell Reports 14, 1761-1773). This so-called IL-11 trans-signaling may play an important role in disease pathogenesis, but its role in human disease has not yet been studied.

[0053] As used herein, "IL-11 trans-signaling" refers to signaling triggered by IL-11 bound to IL-11Rα binding to gp130. IL-11 may bind to IL-11Rα in the form of a non-covalent complex. gp130 is membrane-bound and is expressed by cells in which 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, the soluble IL-11Rα is a soluble (secreted) isoform of IL-11Rα (e.g., lacking a transmembrane domain). In some embodiments, the soluble IL-11Rα is a release product of proteolytic cleavage of the extracellular domain of cell membrane-bound IL-11Rα. In some embodiments, IL-11Rα can be cell membrane bound, and signaling through gp130 can be triggered by binding of IL-11 bound to cell membrane bound IL-11Rα (referred to as "IL-11"), referred to as "cis signaling."

[0054] IL-11-mediated signaling has been shown to stimulate hematopoiesis and platelet production, stimulate osteoclast activity, stimulate neurogenesis, inhibit adipogenesis, reduce proinflammatory cytokine expression, regulate extracellular matrix (ECM) metabolism, and mediate normal growth control of gastrointestinal epithelial cells (Du and Williams, supra).

[0055] The physiological role of interleukin 11 (IL-11) is still unclear. IL-11 is closely associated with the activation of hematopoietic cells and the production of platelets. IL-11 has also been shown to have a protective effect against graft-versus-host disease, inflammatory arthritis, and inflammatory bowel disease, which has led to IL-11 being considered an anti-inflammatory cytokine (Putoczki and Ernst, Jleukoc Biol 2010, 88(6): 1109-1117). However, IL-11 not only has anti-inflammatory effects, but also has pro-inflammatory and pro-angiogenic effects and is important for tumorigenesis. Recent studies have shown that IL-11 is readily detected during virus-induced inflammation in mouse arthritis models and cancer, suggesting that pathological stimuli can induce IL-11 expression. IL-11 is also associated with Stat3-dependent activation of tumor-promoting target genes in tumor gastrointestinal epithelium (Putoczki and Ernst, supra).

[0056] As used herein, "IL-11 signaling" and "IL-11-mediated signaling" refer to signaling mediated by binding of IL-11 or a fragment thereof having the function of a mature IL-11 molecule to an IL-11 receptor. It should be understood that "IL-11 signaling" and "IL-11-mediated signaling" refer to signaling initiated by IL-11 / its functional fragment, for example, by binding to an IL-11 receptor. "Signaling" also refers to signal transduction and other cellular processes that control cell activity.

[0057] Hepatotoxicity

[0058] Aspects of the invention relate to the diagnosis, treatment and prevention of hepatotoxicity and disorders, diseases and conditions characterized by hepatotoxicity.

[0059] As used herein, "hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity" refers to hepatotoxicity, a disorder associated with hepatotoxicity, a disease associated with hepatotoxicity, or a condition associated with hepatotoxicity.

[0060] As used herein, hepatotoxicity refers to damage and / or death of liver cells / tissues. Hepatotoxicity may refer to toxic damage to the liver, particularly the death of hepatocellular cells within the liver. Hepatotoxicity may be determined / diagnosed by detecting one or more hepatotoxicity correlates as described below.

[0061] Hepatotoxicity may be the result of hepatotoxic injury. As used herein, "hepatotoxicity" refers to any treatment, event, or condition that causes hepatotoxicity. For example, hepatotoxic injury may be caused by chemical / physical treatment / experience or gas conditions. In some embodiments, the hepatotoxic injury is chemical, such as in the case of drug-induced liver injury, such as APAP-induced hepatotoxicity. In some embodiments, the hepatotoxic injury is physical, such as in the case of hepatotoxicity caused by surgical injury to liver tissue, which may occur, for example, in the treatment of disease and / or surgery for liver transplantation (e.g., the hepatotoxicity may have iatrogenic causes). In some embodiments, the hepatotoxic injury results from lack of oxygen, such as as a result of ischemia, or may result from reperfusion (e.g., the hepatotoxic injury may result from IRI).

[0062] Hepatotoxicity may be chemically induced liver damage, such as damage or injury caused by drugs, chemicals, ischemia, reperfusion, sepsis, or herbs or dietary supplements. In some embodiments, hepatotoxicity refers to drug-induced liver injury (DILI). In some embodiments, hepatotoxicity refers to liver damage caused by a hepatotoxin. The hepatotoxin may be alcohol. Hepatotoxicity may also be referred to as toxic hepatitis. Hepatotoxicity may refer to acute and / or chronic hepatotoxicity.

[0063] Hepatotoxicity can be caused directly or indirectly by alcohol intake, such as chronic drinking. Hepatotoxicity as described herein can be caused directly or indirectly by fasting, malnutrition, infection with infectious agents (e.g., hepatitis viruses (e.g., hepatitis A, B, C, D, or E), HIV), cancer, or drug interactions.

[0064] Hepatotoxicity may be associated with other disorders, diseases and conditions. Disorders, diseases or conditions associated with hepatotoxicity include acute liver injury (ALI), acute liver failure, acute liver disease, chronic liver disease, liver damage, hepatitis (e.g., viral hepatitis, alcoholic hepatitis), hepatic ischemia-reperfusion injury (IRI) (e.g., "warm" ischemia-reperfusion (WIR)), radiation-induced liver disease (RILD), drug-induced liver injury (DILI), autoimmune liver injury, cholestatic liver disease, HIV and cancer.

[0065] Drug-induced liver injury includes intrinsic and idiosyncratic hepatotoxicity, with idiosyncratic DILI further including allergic and non-allergic reactions. Its intrinsic mechanism is related to dose-dependent hepatotoxicity, while idiosyncratic hepatotoxicity is not dose-dependent and may occur in an unpredictable manner. Allergic idiosyncratic hepatotoxicity is further characterized by the presence of symptoms and signs typical of an adaptive immune system response, including fever, skin reactions, eosinophilia, formation of autoantibodies, and a short latency period, especially after re-exposure (Khoury et al. J Clin Transl Hepatol. 2015 Jun 28;3(2):99–108).

[0066] Aspects of the present invention relate to the diagnosis, treatment and prevention of acetaminophen (APAP)-induced hepatotoxicity. Acetaminophen is also known as N-acetyl-para-aminophenol or paracetamol, or by the brand names Tylenol and Panadol. Acetaminophen poisoning leads to hepatotoxicity, which is associated with increased serum concentrations of hepatocyte leaky enzymes such as aspartate aminotransferase, lactate dehydrogenase and alanine aminotransferase, centrilobular degeneration and necrosis, and activation of Kupffer cells (Trepicchio WL et al., Toxicol Pathol. 2001; 29(2):242-9).

[0067] Drugs that inhibit the action of IL-11

[0068] Aspects of the invention relate to inhibiting IL-11 mediated signaling.

[0069] Here, "inhibit" means to reduce, decrease or mitigate relative to a control condition. For example, inhibition of IL-11 action by a drug capable of inhibiting IL-11-mediated signaling means to reduce, decrease or mitigate the extent / degree of IL-11-mediated signaling in the absence of the agent and / or in the presence of an appropriate control agent.

[0070] Inhibition may also be referred to herein as neutralization or antagonism. That is, an agent capable of inhibiting IL-11-mediated signal transduction (e.g., interactions, signal transduction, or other activities mediated by IL-11 or a complex containing IL-11) may be referred to as a "neutralizing" or "antagonist" agent with respect to a related function or process. For example, an agent capable of inhibiting IL-11-mediated signal transduction may be referred to as an agent capable of neutralizing IL-11-mediated signal transduction, or may be referred to as an antagonist of IL-11-mediated signal transduction.

[0071] The IL-11 signaling pathway provides multiple ways to inhibit IL-11 signaling. Agents capable of inhibiting IL-11 mediated signaling may do so, for example, by inhibiting the action of one or more factors involved in or required for signaling through the IL-11 receptor.

[0072] For example, the inhibition of IL-11 signaling can be achieved by disrupting the interaction between IL-11 (or a complex containing IL-11, such as a complex of IL-11 and IL-11Rα) and an IL-11 receptor (e.g., IL-11Rα, a receptor complex containing IL-11Rα or gp130, or a receptor complex containing IL-11R and gp130). In some embodiments, the inhibition of IL-11-mediated signaling is achieved by inhibiting the gene or protein expression of one or more of IL-11, IL-11Rα, and gp130.

[0073] In an embodiment, the inhibition of IL-11-mediated signaling is achieved by interfering with IL-11-mediated cis signaling but not interfering with IL-11-mediated trans signaling, for example, the inhibition of IL-11-mediated signaling is achieved by inhibiting the cis complex involving membrane-bound IL-11Rα mediated by gp130. In an embodiment, the inhibition of IL-11-mediated signaling is achieved by interfering with IL-11-mediated trans signaling without interfering with IL-11-mediated cis signaling, that is, the inhibition of IL-11-mediated signaling is achieved by inhibiting the gp130-mediated trans signaling complex, such as IL-11 binding to soluble IL-11Rα or IL-6 binding to soluble IL-6R. In an embodiment, the inhibition of IL-11-mediated signaling is achieved by interfering with IL-11-mediated cis signaling and IL-11-mediated trans signaling. Any agent described herein can be used to inhibit IL-11-mediated cis and / or trans signaling. In a preferred embodiment, inhibition of IL-11 mediated signaling is achieved by interfering with IL-11 mediated cis signaling.

[0074] In other embodiments, inhibition of IL-11 signaling can be achieved by interfering with the signaling pathway downstream of IL-11 / IL-11Rα / gp130. That is, in some embodiments, inhibition / antagonism of IL-11-mediated signaling includes inhibiting the signaling pathway / process / factor downstream of signaling through the IL-11 / IL-11 receptor complex.

[0075] In some embodiments, inhibiting / antagonizing IL-11-mediated signal transduction comprises inhibiting signal transduction through an intracellular signal transduction pathway activated by the IL-11 / IL-11 receptor complex. In some embodiments, inhibiting / antagonizing IL-11-mediated signal transduction comprises inhibiting one or more factors whose expression / activity is increased due to signal transduction through the IL-11 / IL-11 receptor complex.

[0076] In some embodiments, the method of the present invention uses an agent capable of inhibiting JAK / STAT signaling. In some embodiments, the agent capable of inhibiting JAK / STAT signals can inhibit the effects of JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and / or STAT6. For example, the agent can inhibit the activation of JAK / STAT proteins, inhibit the interaction of JAK or STAT proteins with cell surface receptors such as IL-11Rα or gp130, inhibit the phosphorylation of JAK proteins, inhibit the interaction between JAK and STAT proteins, inhibit the phosphorylation of STAT proteins, inhibit the dimerization of STAT proteins, inhibit the translocation of STAT proteins to the nucleus, 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 inhibitor is selected from Ruxolitinib (Jakafi / Jakavi; Incyte), Tofacitinib (Xeljanz / Jakvinus; NIH / Pfizer), Oclacitinib (Apoquel), Baricitinib (Olumiant; Incyte / Eli Lilly), Filgotinib (G-146034 / GLPG-0634; Galapagos NV), Gandotinib (LY-2784544; Eli Lilly and Company), Lestaurtinib (CEP-701; Teva Pharmaceutical Industries), Momelotinib (GS-0387 / CYT-387; Gilead Sciences), Pacritinib (SB1518; CTI), PF-04965842 (Pfizer), Upadacitinib (ABT-494; AbbVie), Peficitinib (ASP015K / JNJ-54781532; Astellas Pharma), Fedratinib (SAR302503; Celgene), Cucurbitacin I (JSI-124), and CHZ868.

[0077] In some embodiments, the methods of the present invention use agents that can inhibit MAPK / ERK signaling. In some embodiments, the drug that can inhibit MAPK / ERK signaling can inhibit the action of GRB2, inhibit the action of RAF kinase, inhibit the action of MEK protein, inhibit the activation of MAP3K / MAP2K / MAPK and / or Myc, and / or inhibit the phosphorylation of STAT protein. In some embodiments, the agent that can inhibit ERK signaling can inhibit ERK P42 / 44. In some embodiments, the ERK inhibitor is selected from 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), BioPharma), selumetinib (AZD6244; Array / AstraZeneca) and trametinib (GSK1120212 / Mekinist; Novartis). In some embodiments, the methods of the present invention use agents that can inhibit c-Jun N-terminal kinase (JNK) signaling / activity. In some embodiments, agents that can inhibit JNK signaling / activity can 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 JNK6o, c-JUN peptide, SU3327, AEG 3482, TCS JNK5a, BI78D3, IQ3, SR3576, IQ1S, JIP-1 (153-163) and CC401 dihydrochloride.

[0078] In this example, the inventors demonstrated that NOX4 expression and activity are upregulated by IL-11 / IL-11Rα / gp130 signaling. NOX4 is a NADPH oxidase and a source of reactive oxygen species (ROS). Nox4 expression is upregulated in transgenic mice expressing hepatocyte-specific IL11, and IL11-stimulated primary human hepatocytes upregulate NOX4 expression.

[0079] In some embodiments, the present invention uses an agent capable of inhibiting NOX4 expression (gene or protein expression) or function. In some embodiments, the present invention uses a drug capable of inhibiting IL-11-mediated NOX4 expression / function upregulation. Agents capable of inhibiting NOX4 expression or function may be referred to herein as NOX4 inhibitors. For example, NOX4 inhibitors can reduce NOX4 expression (e.g., gene and / or protein expression), reduce RNA levels encoding NOX4, reduce NOX4 protein levels, and / or reduce NOX4 activity levels (e.g., reduce NOX4-mediated NADPH oxidase activity and / or NOX4-mediated ROS production).

[0080] 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 NOX4 interaction partners as NOX4 functional antagonists and small molecule inhibitors of NOX4. Molecules capable of reducing NOX4 expression include antisense RNA (such as siRNA, shRNA) of NOX4. In some embodiments, the NOX4 inhibitor is selected from Altenhofer et al., Antioxid Redox Signal. (2015) 23 (5): 406-427 or Augsburder et al., Redox Biol. (2019) 26: NOX4 inhibitors described in 101272, such as GKT137831.

[0081] Binder

[0082] In some embodiments, agents capable of inhibiting IL-11-mediated signal transduction can be combined with IL-11. In some embodiments, agents capable of inhibiting IL-11-mediated signal transduction can bind to IL-11 receptors (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130). The binding of these agents can inhibit IL-11-mediated signal transduction by reducing / preventing the ability of IL-11 to bind to the IL-11 receptor, thereby inhibiting downstream signal transduction. The binding of these agents may inhibit IL-11-mediated signal transduction by reducing / preventing the ability of IL-11 to bind to IL-11 receptors (such as IL-11Rα and / or gp130), thereby inhibiting cis and / or trans signal transduction, thereby inhibiting downstream signal transmission. The agent can bind to a trans-signaling complex, such as IL-11 and soluble IL-11Rα, and inhibit gp130-mediated signal transduction.

[0083] The agent capable of binding to IL-11 / IL-11 complex or IL-11 receptor may be of any type, but in some embodiments, the agent may be an antibody, an antigen binding fragment thereof, a polypeptide, a peptide, a nucleic acid, an oligonucleotide, an aptamer or a small molecule. The agent may be provided in an isolated or purified form, or may be formulated as a pharmaceutical composition or a drug.

[0084] Antibodies and antigen-binding fragments

[0085] In some embodiments, the agent capable of binding to IL-11 / complex containing IL-11 or IL-11 receptor is an antibody or an antigen binding fragment thereof. In some embodiments, the agent capable of binding to IL-11 / complex containing IL-11 or IL-11 receptor is a polypeptide, such as a decoy receptor molecule. In some embodiments, the agent capable of binding to IL-11 / complex containing IL-11 or IL-11 receptor may be an aptamer.

[0086] In some embodiments, the agent capable of binding to IL-11 / IL-11-containing complexes or IL-11 receptors is an antibody or an antigen-binding fragment thereof. "Antibody" is used in the broadest sense herein and includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they show binding to the relevant target molecule.

[0087] In view of the current technology related to monoclonal antibody technology, antibodies against most antigens can be prepared. The antigen binding portion can be a part of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single-chain Fv fragment [ScFv]). Monoclonal antibodies against selected antigens can be prepared by known techniques, such as those described in Monoclonal Antibodies: A Technical Manual, HZola (CRC Press, 1988) and Monoclonal Hybridoma Antibodies: Technology and Applications, JG RHurrell (CRC Press, 1982). Chimeric antibodies are discussed by Neuberger et al. (1988, Part 2 of the 8th International Biotechnology Symposium, 792-799). Monoclonal antibodies (mAbs) are particularly useful in the method of the present invention and are homogeneous antibody groups that specifically target a single epitope on an antigen.

[0088] Polyclonal antibodies are also useful in the methods of the invention. Monospecific polyclonal antibodies are preferred. Suitable polyclonal antibodies can be prepared using methods well known in the art.

[0089] Antigen-binding fragments of antibodies, such as Fab and Fab2 fragments, can also be used / provided as genetically engineered antibodies and antibody fragments. The variable heavy (VH) and variable light (VL) domains of antibodies are involved in antigen recognition, a fact first recognized in early protease digestion experiments. Further confirmation was carried out by the "humanization" of rodent antibodies. The variable domains of rodent origin can be fused with the constant domains of human origin so that the resulting antibodies retain the antigenic specificity of the rodent parent antibody (Morrison et al. (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855).

[0090] The antibodies and antigen-binding fragments according to the present invention comprise the complementarity determining regions (CDRs) of the antibody capable of binding to the relevant target molecule (ie IL-11 / IL-11 containing complex / IL-11 receptor).

[0091] Antibodies capable of binding to IL-11 include, for example, monoclonal mouse anti-human IL-11 antibody clone #22626; catalog number MAB218 (R&D Systems, Minnesota, USA), such as used in Bockhorn et al., Nat. Commun. (2013) 4(0): 1393, clone 6D9A (Abbiotec), clone KT8 (Abbiotec), clone M3103F11 (BioLegend), clone 1F1 (Abnova Biotech, Inc.), clone 3C6 (Abnova Biotech, Inc.), clone GF1 (LifeSpan Biosciences), clone 13455 (Source BioScience), 11H3 / 19.6.1 (Hermann et al., Arthritis Rheum. (1998) 41(8): 1388-97), AB-218-NA (R&D Systems), X203 (Ng et al., Sci Transl. Med.(2019)11(511)pii:eaaw1237) and anti-IL-11 antibodies disclosed in US 2009 / 0202533 A1, WO 99 / 59608 A2 and WO 2018 / 109174 A2.

[0092] In particular, 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 Schaefer et al., Nature (2017) 552(7683):110-115. Monoclonal antibody 11H3 / 19.6.1 was disclosed as a neutralizing anti-IL-11 IgG1 in Hermann et al., Arthritis & Rheumatology (1998) 41(8):1388-97. AB-218-NA from R&D Systems, used for example in McCoy et al., BMC Cancer (2013) 13:16, is another example of a neutralizing anti-IL-11 antibody. WO 2018 / 109174A2 discloses yet another exemplary anti-IL-11 antibody antagonist of IL-11-mediated signaling.

[0093] Antibodies capable of binding to IL-11Rα include, for example, monoclonal antibody clone 025 (Sino Biologic), clone EPR5446 (Abcam), clone 473143 (R&D Systems), clones 8E2, 8D10 and 8E4, and variants of affinity-matured 8E2 described in US 2014 / 0219919A1, monoclonal antibodies described by Blanc et al. (J. Immunol Methods. 2000 Jul 31; 241(1-2); 43-59), X209 (Widjaja et al., Gastroenterology (2019) 157(3):777-792), antibodies disclosed in WO 2014121325 A1 and US 2013 / 0302277 A1, and antibodies disclosed in US 2009 / 0202533 A1, WO 99 / 59608 A2 and WO The anti-IL-11Rα antibody disclosed in 2018 / 109170 A2.

[0094] In particular, 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 Schaefer et al., Nature (2017) 552 (7683): ​​110-115. US 2014 / 0219919 A1 provides sequences of 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. In addition, US 2014 / 0219919 A1 also provides sequence information for another 62 affinity mature variants of clone 8E2, 61 of which are disclosed to antagonize IL-11-mediated signaling - see Table 3 of US 2014 / 0219919 A1. WO 2018 / 109170 A2 discloses yet another exemplary anti-IL-11Rα antibody antagonist of IL-11-mediated signaling.

[0095] The skilled person is well aware of techniques for producing antibodies suitable for use in treatment of a given species / subject. For example, methods for producing antibodies suitable for human therapeutic use are described in Park and Smolen, Advances in Protein Chemication (2001) 56:369-421 (which is incorporated herein by reference in its entirety).

[0096] Antibodies against a given target protein (e.g., IL-11 or IL-11Rα) can be produced in model species (e.g., rodents, lagomorphs), and subsequently engineered to improve their suitability for treatment in a given species / subject. For example, one or more amino acids of a monoclonal antibody produced by immunizing a model species can be replaced to obtain an antibody sequence more similar to a human germline immunoglobulin sequence (thereby reducing the possibility of an anti-xenogeneic antibody immune response in a human subject treated with the antibody). The modification of the antibody variable domain can be concentrated in the framework region to retain the antibody binding site. Antibody humanization is a routine practice problem in the field of antibody technology, as reviewed in Almagro and Fransson, Frontiers in Bioscience (2008) 13: 1619-1633, Safdari et al., Review of Biotechnology and General Engineering (2013) 29 (2): 175-186, and Lo et al., Microbial Spectrum (2014) 2 (1), all of which are incorporated herein by reference in their entirety. The requirement for humanization can be circumvented by producing antibodies against a given target protein (e.g., IL-11 or IL-11Rα) in a transgenic model species expressing human immunoglobulin genes, such that the antibodies produced in such animals are fully human (e.g., as described in Brüggemann et al., Arch Immunol Ther Exp (Warsz) (2015) 63(2):101-108, which is incorporated herein by reference in its entirety).

[0097] Phage display technology can also be used to identify antibodies against a given target protein (e.g., IL-11 or IL-11Rα), which is well known to those skilled in the art. For example, Hoogenboom, Nat. Biotechnol. (2005) 23, 1105-1116 and Chan et al., International Immunology (2014) 26 (12): 649-657 review the application of phage display in identifying fully human antibodies against human target proteins, which are incorporated herein by reference in their entirety.

[0098] The antibody / fragment can be an antagonist antibody / fragment that inhibits or reduces the biological activity of IL-11. The antibody / fragment can be a neutralizing antibody that neutralizes the biological effects of IL-11, such as its ability to stimulate productive signaling through the IL-11 receptor. Neutralizing activity can be determined by the ability to neutralize IL-11-induced proliferation in the T11 mouse plasmacytoma cell line (Nordan, RP et al. (1987) J. Immunol. 139: 813).

[0099] IL-11 or IL-11Rα binding antibodies can be evaluated for their ability to antagonize IL-11-mediated signaling, for example using the methods described in US 2014 / 0219919 A1 or Blanc et al., J. Immunol Methods. 2000 Jul 31; 241(1-2); 43-59. Briefly, the ability of IL-11 and IL-11Rα binding antibodies to inhibit the proliferation of Ba / F3 cells expressing IL-11Rα and gp130 from an appropriate species in response to stimulation with IL-11 from an appropriate species can be evaluated in vitro. Alternatively, the ability of IL-11 and IL-11Rα binding antibodies to inhibit the transition of fibroblasts to myofibroblasts after stimulation of fibroblasts with TGFβ1 can be analyzed in vitro by assessing αSMA expression (as described in WO 2018 / 109174 A2 (Example 6) and WO 2018 / 109170 A2 (Example 6), Ng et al., Sci Transl Med. (2019) 11(511) pii:eaaw1237 and Widjaja et al., Gastroenterology (2019) 157(3):777-792).

[0100] Antibodies typically contain six CDRs; three in the light chain variable region (VL): LC-CDR1, LC-CDR2, LC-CDR3, and three in the heavy chain variable region (VH): HC-CDR1, HC-CDR2, and HC-CDR3. The six CDRs together define the paratope of the antibody, which is the portion of the antibody that binds to the target molecule. There are several different conventions for defining antibody CDRs, such as the convention described by Kabat et al., Protein Sequences of Immunological Significance, 5th ed., National Institutes of Health, Public Health Service, Bethesda, Maryland (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and VBASE2, as described in Retter et al., Nucl. Natl. Acad. Sci. (2005) 33(Suppl 1):D671-D674.

[0101] Antibodies and antigen binding fragments of the present invention can be designed and prepared using monoclonal antibody (mAb) sequences that can bind to relevant target molecules. Antigen binding regions of antibodies, such as single-chain variable fragments (scFv), Fab and Fab2 fragments, can also be used / provided. An "antigen binding region" or "antibody binding fragment" is any fragment of an antibody that can bind to a target specific to a given antibody.

[0102] In some embodiments, the antibody / fragment comprises the VL and VH regions of an antibody, which can bind to IL-11, a complex containing IL-11, or a receptor for IL-11. The VL and VH regions of the antigen binding region of the antibody together constitute the Fv region. In some embodiments, the antibody / fragment comprises or consists of the Fv region of an antibody that can bind to IL-11, a complex containing IL-11, or an IL-11 receptor. The Fv region can be expressed as a single chain, in which the VH and VL regions are covalently linked, for example, by a flexible oligopeptide. Therefore, the antibody / fragment can comprise or consist of a scFv comprising the VL and VH regions of an antibody, which can bind to IL-11, a complex containing IL-11, or an IL-11 receptor.

[0103] The VL and light chain constant (CL) regions of the antigen-binding region of the antibody, as well as the VH region and heavy chain constant 1 (CH1) region together constitute the Fab region. In some embodiments, the antibody / fragment comprises or consists of the Fab region of an antibody capable of binding to IL-11, an IL-11 complex or an IL-11 receptor.

[0104] In some embodiments, the antibody / fragment includes or consists of a full antibody capable of binding to IL-11, a IL-11 complex or an IL-11 receptor. A "full antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). For example, different types of immunoglobulins and their structures are described in Schroeder and Cavacini's J Allergy Clin Immunol. (2010) 125 (202): S41-S52, which is incorporated herein by reference in its entirety. G-type immunoglobulins (i.e., IgG) are glycoproteins 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 comprising three constant domains (CH1, CH2 and CH3), and similarly, the light chain comprises VL, followed by CL. According to the heavy chain, immunoglobulins can be classified into IgG (eg, IgG1, IgG2, IgG3, IgG4), IgA (eg, IgA1, IgA2), IgD, IgE, or IgM. The light chain can be κ (kappa) or λ (lambda).

[0105] Fab, Fv, ScFv, and dAb antibody fragments can all be expressed in or secreted from E. coli, allowing large amounts of the fragments to be readily produced.

[0106] Whole antibodies and F(ab')2 fragments are "bivalent". "Bivalent" means that the antibodies and F(ab')2 fragments have two antigen binding sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent and have only one antigen binding site. Synthetic antibodies that can bind to IL-11, IL-11-containing complexes or IL-11 receptors can also be prepared using phage display technology well known in the art.

[0107] Antibodies can be produced through the process of affinity maturation, wherein the modified antibody produced has an improved affinity for the antigen compared to the unmodified parent antibody. Affinity matured antibodies can be produced by methods known in the art, such as Marks et al., Rio / Technology 10:779-783 (1992); Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154 (7):331 0-15 9 (1995) and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0108] Antibodies / fragments include bispecific antibodies, for example, composed of two different fragments of two different antibodies, so that the bispecific antibody binds to two types of antigens. The bispecific antibodies include antibodies / fragments described herein that can bind to IL-11, IL-11 complexes or IL-11 receptors. The antibody may contain different fragments with affinity for a second antigen, which may be any desired antigen. The technology for preparing bispecific antibodies is well known in the art, for example, see Mueller, D et al., (2010 Biodrugs 24 (2): 89-98), Wozniak-Knopp G et al., (2010 Protein Eng Des 23 (4): 289-297) and Baeuerle, PA et al., (2009 Cancer Res 69 (12): 4941–4944). Bispecific antibodies and bispecific antigen-binding fragments can be provided in any suitable form, such as those described in Kontermann Mabs 2012, 4 (2): 182-197, which is incorporated herein by reference in its entirety. For example, the bispecific antibody or bispecific antigen-binding fragment can be a bispecific antibody conjugate (e.g., IgG2, F(ab')2, or CovX-body), a bispecific IgG or IgG-like molecule (e.g., IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, 1-IgG, mAb2, or Tandemab common LC), an asymmetric bispecific IgG or IgG-like molecule (e.g., kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair or SEED body), small bispecific antibody molecules (e.g., diabody (Db), dsDb, DART, scDb, tandAbs, tandem scFv (taFv), tandem dAb / VHH, tribody, tri-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-cytokine 2). See Kontermann MAbs 2012, 4(2): Figure 2 of 182-19 for details.

[0109] Methods for producing bispecific antibodies include chemical cross-linking of antibodies or antibody fragments, such as through reducible disulfide bonds or non-reducible thioether bonds, as described in Segal and Bast, 2001, "Production of Bispecific Antibodies," Current Protocols in Immunology 14: Iv: 2.13: 2.13.1-2.13.16, which is incorporated herein by reference in its entirety. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used for chemical cross-linking, such as Fab fragments via hinge region SH-groups to produce disulfide-linked bispecific F(ab)2 heterodimers.

[0110] Other methods for producing bispecific antibodies include fusing antibody-producing hybridomas, for example, with polyethylene glycol to produce a quadrivalent cell capable of secreting the bispecific antibody, as described in DM and Bast, BJ, 2001, "Production of Bispecific Antibodies," Current Protocols in Immunology 14: IV: 2.13: 2.13.1–2.13.16.

[0111] Bispecific antibodies and bispecific antigen-binding molecules can also be produced recombinantly, for example by expression from a nucleic acid construct encoding a polypeptide of the antigen-binding molecule, for example, Antibody Engineering: Methods and Protocols, 2nd Edition (Humana Press, 2012), Chapter 40: Generation of Bispecific Antibodies: Diabodies and Tandem scFvs (Hornig et al., 2012). -Schwarz, or in French, How to make bispecific antibodies, Methods in Mol Medicine, 2000;40:333-339.

[0112] For example, a DNA construct encoding a light chain and a heavy chain variable domain of two antigen binding domains (i.e., a light chain and a heavy chain variable domain of an antigen binding domain capable of binding to IL-11, a complex containing IL-11, or a receptor of IL-11, and a light chain and a heavy chain variable domain of an antigen binding domain capable of binding to another target protein), and including a sequence encoding a suitable linker or dimerization domain between the antigen binding domains, which can be prepared by molecular cloning techniques. Recombinant bispecific antibodies can be produced by expressing (e.g., in vitro) constructs in suitable host cells (e.g., mammalian host cells), and the expressed recombinant bispecific antibodies can then be optionally purified.

[0113] Decoy receptor

[0114] The peptide or polypeptide-based agent capable of binding IL-11 or a complex containing IL-11 may be based on the IL-11 receptor, such as an IL-11 binding fragment of the IL-11 receptor.

[0115] In some embodiments, the binding agent may include an IL-11 binding fragment of the IL-11Rα chain, and may preferably be soluble and / or exclude one or more or all transmembrane domains. In some embodiments, the binding agent may include an IL-11 binding fragment of gp130, and may preferably be soluble and / or exclude one or more or all transmembrane domains. These molecules may be described as decoy receptors. The binding of these agents may inhibit cis and / or trans signaling by reducing / preventing the ability of IL-11 to bind to IL-11 receptors (such as IL-11Rα or gp130), thereby inhibiting downstream signaling.

[0116] Curtis et al. (Blood 1997 Dec 1;90(11):4403-12) reported that soluble mouse 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 proposed that the observed antagonism of IL-11 by sIL-11R was dependent on the number of gp130 molecules restricted to cells that already expressed transmembrane IL-11R.

[0117] The use of soluble decoy receptors as a basis for inhibiting signaling and therapeutic intervention has also been reported for other signaling molecule: receptor pairs, such as VEGF and VEGF receptor (De-Chao Yu et al., Molecular Therapy (2012); 20 5, 938-947; Konner and Dupont Clin Colorectal Cancer, 2004 Oct; 4 Supplement 2: S81-5).

[0118] Thus, in some embodiments, the binding agent may be a decoy receptor, such as a soluble receptor for IL-11 and / or a complex containing IL-11. Competition for IL-11 and / or IL-11-containing complexes provided by decoy receptors has been reported to result in IL-11 antagonist effects (Curtis et al., supra). Decoy IL-11 receptors are also described in WO 2017 / 103108 A1 and WO2018 / 109168 A1, which are incorporated herein by reference in their entirety.

[0119] Decoy IL-11 receptors preferentially bind IL-11 and / or IL-11-containing complexes, thereby rendering these species unable to bind to gp130, IL-11Rα, and / or gp130:IL-11Rα receptors. Thus, they act as "decoy" receptors for IL-11 and IL-11-containing complexes, just as etanercept acts as a decoy receptor for TNFα. IL-11-mediated signaling is reduced compared to the level of signaling in the absence of the decoy receptor.

[0120] The decoy IL-11 receptor preferably binds to IL-11 through one or more cytokine binding modules (CBMs). These CBMs are or are derived from or are homologous to CBMs of naturally occurring IL-11 receptor molecules. For example, the decoy IL-11 receptor may include or consist of one or more CBMs derived from or homologous to CBMs of gp130 and / or IL-11Rα.

[0121] In some embodiments, the decoy IL-11 receptor may include or consist of an amino acid sequence corresponding to a cytokine binding module of gp130. In some embodiments, the decoy IL-11 receptor may comprise an amino acid sequence corresponding to a cytokine binding module of IL-11Rα. Here, an amino acid sequence "corresponding" to a reference region or sequence of a given peptide / polypeptide has at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of the reference region / sequence.

[0122] In some embodiments, the decoy receptor may be capable of binding to IL-11, for example, with a binding affinity of at least 100 μm or less, optionally 10 μm or less, 1 μm or less, 100 nm or less, or one of about 1 to 100 nm. In some embodiments, the decoy receptor may include all or part of the IL-11 binding domain, and may optionally lack all or part of the transmembrane domain. The decoy receptor may optionally be fused to an immunoglobulin constant region, such as an IgG Fc region.

[0123] Inhibitors

[0124] The present invention contemplates the use of inhibitor molecules that are capable of binding to one or more of IL-11, IL-11-containing complexes, IL-11Rα, gp130, or complexes containing IL-11Rα and / or gp130 and inhibiting IL-11-mediated signaling.

[0125] In some embodiments, the agent is a peptide or polypeptide binding agent based on IL-11, such as a mutant, variant or binding fragment of IL-11. Suitable peptide or polypeptide-based drugs can bind to IL-11 receptors (e.g., IL-11Rα, gp130, or a complex comprising IL-11Rα and / or gp130) in a manner that does not cause signal transduction initiation or produces suboptimal signal transduction. Such IL-11 mutants can act as competitive inhibitors of endogenous IL-11.

[0126] For example, W147A is an IL-11 antagonist in which amino acid 147 is mutated from tryptophan to alanine, disrupting the so-called "site III" of IL-11. This mutant can bind to IL-11Rα, but fails to bind to the gp130 homodimer, resulting in effective blockade of IL-11 signaling (Underhill-Day et al., 2003; Endocrinology 2003 August; 144(8): 3406-14). Lee et al. (Am J respire Cell Mol Biol. 2008 December; 39(6): 739-746) also reported the generation of an IL-11 antagonist mutant (a "mutant") that can specifically inhibit the binding of IL-11 to IL-11Rα. IL-11 mutants are also described in WO 2009 / 052588 A1.

[0127] Menkhorst et al. (Biology of Reduction 2009 May 1, Vol. 80 No. 5 920-927) describe a pegylated IL-11 antagonist, PEGIL11A (CSL Ltd., Parkville, Victoria, Australia) that effectively inhibits the action of IL-11 in female mice.

[0128] Pasqualini et al., Cancer (2015) 121(14):2411-2421, described a ligand-directed peptidomimetic drug, bone metastasis targeting peptide multimer-11 (BMTP-11), which is able to bind to IL-11Rα.

[0129] In some embodiments, the binding agent capable of binding to the IL-11 receptor can be provided in the form of a small molecule inhibitor of one of IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130. In some embodiments, the binding agent can be provided in the form of a small molecule inhibitor of IL-11 or a complex containing IL-11, such as the IL-11 inhibitor described in Lay et al., Int. J. Oncol. (2012); 41(2):759-764, which is incorporated herein by reference in its entirety.

[0130] Aptamer

[0131] In some embodiments, the agent capable of binding to IL-11 / complex containing IL-11 or IL-11 receptor (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 high specificity and high affinity binding to target molecules. To date, almost all aptamers are non-naturally occurring molecules.

[0132] Aptamers for a given target (e.g., IL-11, IL-11-containing complexes, or IL-11 receptors) can be identified and / or produced by a method of systematic evolution of ligands by exponential enrichment (SELEX™), or by developing SOMAmers (slowly modified aptamers) (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. Application of SELEX and SOMAmer technology includes the addition of functional groups that mimic amino acid side chains to expand the chemical diversity of aptamers. Thus, high affinity aptamers for a target can be enriched and identified.

[0133] Aptamers can be DNA or RNA molecules, and can be single-stranded or double-stranded. Aptamers can include chemically modified nucleic acids, for example, where sugars and / or phosphates and / or bases are chemically modified. Such modifications can increase the stability of the aptamer or make the aptamer more resistant to degradation, and can include modifications at the 2' position of the ribose.

[0134] Aptamers can be synthesized by methods known to those skilled in the art. For example, aptamers can be chemically synthesized, such as on a solid support. Solid phase synthesis can employ phosphoramidite chemistry. Briefly, the nucleotides of the solid support are denitrogenated and then coupled with appropriately activated nucleoside phosphoramidites to form phosphite triester bonds. Capping can then be performed, followed by oxidation of the phosphite triester with an oxidizing agent, typically iodine. This cycle can then be repeated to assemble the aptamer (e.g., see Sinha, ND; Biernat, J.; McManus, J.; , H., Nuclear Acids Res., 1984, 12, 4539; and Beaucage, SL; Lyer, RP (1992). Tetrahedron 48(12):2223).

[0135] Suitable nucleic acid aptamers 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. 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length. 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, or 80 nucleotides in length.

[0136] An aptamer can be a peptide selected or engineered to bind to a specific target molecule. Peptide aptamers and methods for their production and identification are reviewed in Reverdatto et al., Curr Top Med Chem. (2015) 15 (12): 1082-101, which is incorporated herein by reference in its entirety. Peptide aptamers may optionally have a minimum length of one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Peptide aptamers 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. Suitable peptide aptamers may optionally have a length of one of 2-30, 2-25, 2-20, 5-30, 5-25, or 5-20 amino acids.

[0137] K D It may be in the nM or pM range, for example less than one of 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM.

[0138] Properties of IL-11 binders

[0139] The agent of the present invention capable of binding to IL-11 / IL-11-containing complex or IL-11 receptor may exhibit one or more of the following properties:

[0140] ·Specific binding to IL-11 / IL-11-containing complexes or IL-11 receptors;

[0141] Binds to IL-11 / complex containing IL-11 or IL-11 receptor with a KD of 10 μm or less, preferably one of ≤5 μm, ≤1 μm, ≤500 nm, ≤100 nm, ≤10 nm, ≤1 nm or ≤100 pm;

[0142] Inhibits the interaction between IL-11 and IL-11Rα;

[0143] Inhibits the interaction between IL-11 and gp130;

[0144] Inhibits the interaction of IL-11 with the IL-11Rα:gp130 receptor complex;

[0145] Inhibits the interaction of IL-11:IL-11Rα complex with gp130.

[0146] These properties can be determined by analyzing the relevant agent in appropriate experiments, which may involve comparing the performance of the agent with an appropriate control agent. Those skilled in the art will be able to determine appropriate control conditions for a given experiment.

[0147] For example, a suitable negative control for analyzing the ability of the test antibody / antigen binding fragment to bind to IL-11 / complex containing IL-11 / IL-11 receptor can be an antibody / antigen binding fragment directed against a non-target protein (i.e., an antibody / antigen binding fragment that is non-specific to IL-11 / complex containing IL-11 / IL-11 receptor). A suitable positive control can be a known, validated (e.g., commercially available) IL-11 or IL-11 receptor binding antibody. The control can be the same isotype of the hypothetical IL-11 / complex containing IL-11 / IL-11 receptor binding antibody / antigen binding fragment being analyzed, and can, for example, have the same constant region.

[0148] In some embodiments, the agent may be able to specifically bind to IL-11 or a complex containing IL-11, or a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130). An agent that specifically binds to a given target molecule preferably binds to the target with greater affinity and / or longer duration than it binds to other non-target molecules.

[0149] In some embodiments, the agent can bind to IL-11 or a complex containing IL-11 with an affinity greater than the affinity of binding to one or more other members of the IL-6 cytokine family (e.g., IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine (CLC)). In some embodiments, the agent can bind to an IL-11 receptor (e.g., IL-11Rα, gp130, or containing IL-11Rα and / or gp130) with an affinity greater than the affinity of binding to one or more other members of the IL-6 receptor family. In some embodiments, the agent binds to IL-11Rα with an affinity greater than the affinity of binding to one or more of IL-6Rα, leukemia inhibitory factor receptor (LIFR), oncostatin M receptor (OSMR), ciliary neurotrophic factor receptor α (CNTFRα), and cytokine receptor-like factor 1 (CRLF1).

[0150] In some embodiments, the extent of binding of the binding agent 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, biolayer interferometry (BLI), microscale thermophoresis (MST), or by radioimmunoassay (RIA). Alternatively, binding specificity can be reflected in terms of binding affinity, wherein the K of the binding agent to IL-11, IL-11-containing complex, or IL-11 receptor is less than about 10%. D It is at least comparable to the K of other non-target molecules. D 0.1 order of magnitude greater (ie 0.1 x 10n, where n is an integer representing an order of magnitude). 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.

[0151] The binding affinity of a given binding agent for its target is often expressed in terms of its dissociation constant (K D). Binding affinity can be measured by methods known in the art, such as by ELISA, surface plasmon resonance (SPR; see Hearty et al., Methods Mol Biol (2012) 907:411-442; or Rich et al., Anal Biochem. 2008 Feb 1; 373(1): 112-20), biolayer interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4): 498-507; or Concepcion et al., Comb Chem High Throughput Screen. 2009 Sep; 12(8): 791-800), microscale thermophoresis (MST) analysis (see Jerabek-Willemsen et al., Assay Drug Dev Technol. 2011 Aug; 9(4): 342-353), or by radiolabeled antigen binding assay (RIA).

[0152] In some embodiments, the agent is capable of binding to IL-11 or a complex containing IL-11, or a K of a receptor for IL-11. D Less than or equal to 50μm, preferably one of ≤10μm, ≤5μm, ≤4μm, ≤3μm, ≤2μm, ≤1μm, ≤500nM, ≤100nM, ≤75nM, ≤50nM, ≤40nM, ≤30nM, ≤20nM, ≤15nM, ≤12.5nM, ≤10nM, ≤9nM, ≤8nM, ≤7nM, ≤6nM, ≤4nM, ≤3nM, ≤300pM, ≤200pM or ≤100pM.

[0153] In some embodiments, the binding affinity of the agent to IL-11, a complex containing IL-11 or an IL-11 receptor (e.g., as determined by ELISA) EC50 = 10,000 ng / ml or less, preferably one of ≤5,000 ng / ml, ≤1000 ng / ml, ≤900 ng / ml, ≤800 ng / ml, ≤600 ng / ml, ≤500 ng / ml, ≤400 ng / ml, ≤200 ng / ml, ≤100 ng / ml, ≤80 ng / ml, ≤40 ng / ml, ≤30 ng / ml, ≤15 ng / ml, ≤10 ng / ml, ≤7.5 ng / ml, ≤5 ng / ml, ≤2.5 ng / ml, or ≤1 ng / ml. Such ELISAs can be performed, for example, as described in Antibody Engineering, Volume 1 (2nd Edition), Springer Protocol, Springer (2010), Part V, pages 657-665.

[0154] In some embodiments, the agent binds to IL-11 or a complex containing IL-11 in an area important for binding to a receptor (e.g., gp130 or IL-11Rα) to thereby inhibit the interaction between IL-11 or a complex containing IL-11 and the IL-11 receptor, and / or signaling through the receptor. In some embodiments, the agent binds to the IL-11 receptor in an area important for binding to IL-11 or a complex containing IL-11 to thereby inhibit the interaction between IL-11 or a complex containing IL-11 and the IL-11 receptor, and / or signaling through the receptor.

[0155] The ability of a given binding agent (e.g., an agent capable of binding to an IL-11 / IL-11 complex or an IL-11 receptor) to inhibit the interaction between two proteins can be determined by, for example, an interaction assay in the presence of the binding agent or after incubation of the binding agent with one or both interaction partners. An example of a suitable assay for determining whether a given binding agent is capable of inhibiting the interaction between two interaction partners is a competitive ELISA.

[0156] A binding agent 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 and IL-11Rα:gp130, or between IL-11:IL-11Rα and gp130) is determined by observing a decrease / reduction in the level of interaction between the interaction partners in the presence of the binding agent or after incubation of one or both interaction partners with the binding agent compared to the level of interaction in the absence of the binding agent (or in the presence of an appropriate control binding agent). Suitable analyses can be performed in vitro, for example using recombinant interaction partners or using cells expressing interaction partners. Cells expressing interaction partners may be endogenous or may be expressed by nucleic acids introduced into the cells. For the purpose of such an assay, one or both interaction partners and / or binding agents may be labeled or used in conjunction with a detectable entity to detect and / or measure the level of interaction. For example, the agent may be labeled with a radioactive atom or a colored molecule or a fluorescent molecule or a molecule that is easily detected in any other manner. Suitable detectable molecules include fluorescent proteins, luciferases, enzyme substrates and radioactive labels. The binding agent can be directly labeled with a detectable marker, or can be indirectly labeled. For example, the binding agent can be unlabeled and detected by another binding agent that is itself labeled. Alternatively, the second binding agent can be combined with biotin, and the combination of the labeled streptavidin and biotin is used to indirectly label the first binding agent.

[0157] The ability of a binding agent to inhibit the interaction between two binding partners can also be determined by analyzing the downstream functional consequences of such interaction, such as 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 can include, for example, processes mediated by IL-11, or gene / protein expression of, for example, collagen or IL-11.

[0158] Inhibition of the interaction between IL-11 or IL-11-containing complexes and IL-11 receptors can be analyzed using 3H-thymidine incorporation and / or BA / F3 cell proliferation assays, such as those described in Curtis et al., Blood, 1997, 90(11) and Karpovich et al., Mol. Hum. Reprod., 2003 9(2):75-80. BA / F3 cells co-express IL-11Rα and gp130.

[0159] In some embodiments, the binding agent can inhibit the interaction of 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 binding agent (or in the presence of an appropriate control binding agent), for example, one of 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, a binding agent may be capable of inhibiting the interaction between IL-11 and IL-11Rα to less than 1-fold, e.g., one of ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, above the level of interaction between IL-11 and IL-11Rα in the absence of the binding agent (or in the presence of an appropriate control binding agent).

[0160] In some embodiments, the binding agent can inhibit the interaction of IL-11 and gp130 to less than 100% of the level of interaction between IL-11 and gp130 in the absence of the binding agent (or in the presence of an appropriate control binding agent), for example, one of 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, a binding agent may be capable of inhibiting the interaction between IL-11 and gp130 to less than 1 fold, e.g., one of ≤0.99 fold, ≤0.95 fold, ≤0.9 fold, ≤0.85 fold, ≤0.8 fold, ≤0.75 fold, ≤0.7 fold, ≤0.65 fold, ≤0.6 fold, ≤0.55 fold, ≤0.5 fold, ≤0.45 fold, ≤0.4 fold, ≤0.35 fold, ≤0.3 fold, ≤0.25 fold, ≤0.2 fold, ≤0.15 fold, ≤0.1 fold, above the level of interaction between IL-11 and gp130 in the absence of the binding agent (or in the presence of an appropriate control binding agent).

[0161] In some embodiments, the binding agent can inhibit the interaction of 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 binding agent (or in the presence of an appropriate control binding agent), for example, one of 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, a binding agent may be capable of inhibiting the interaction between IL-11 and IL-11Rα:gp130 to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, above the level of interaction between IL-11 and IL-11Rα:gp130 in the absence of the binding agent (or in the presence of an appropriate control binding agent).

[0162] In some embodiments, the binding agent can inhibit the interaction of the IL-11:IL-11Rα complex with gp130 to less than 100% of the level of interaction between IL-11:IL-11Rα and gp130 in the absence of the binding agent (or in the presence of an appropriate control binding agent), for example, one of 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 binding agent is capable of inhibiting the interaction between the IL-11:IL-11Rα complex and gp130 to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, compared to the level of interaction between the IL-11:IL-11Rα complex and gp130 in the absence of the binding agent (or in the presence of an appropriate control binding agent).

[0163] Agents capable of reducing the expression of IL-11 or IL-11 receptor

[0164] In various aspects of the invention, the agent capable of inhibiting IL-11 mediated signaling is capable of preventing or reducing the expression of one or more of IL-11, IL-11Rα, or gp130.

[0165] Expression can be gene or protein expression and can be determined as described herein or by methods well known to those skilled in the art. Expression can be by a cell / tissue / organ / organ system of a subject.

[0166] Suitable agents may be of any kind, but in some embodiments, the agent capable of preventing or reducing the expression of one or more of IL-11, IL-11Rα, or gp130 may be a small molecule or an oligonucleotide.

[0167] An agent capable of preventing or reducing the expression of one or more of IL-11, IL-11Rα or gp130 can exert its effect by inhibiting the transcription of genes encoding IL-11, IL-11Rα or gp130, inhibiting the 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 the 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.

[0168] Taki et al., Clin Exp Immunol (1998) April; 112(1): 133-138 reported that the expression of IL-11 in rheumatoid synoviocytes was reduced after treatment with indomethacin, dexamethasone or interferon gamma (IFNγ).

[0169] The present invention contemplates the use of antisense nucleic acids to prevent / reduce the expression of IL-11, IL-11Rα or gp130. In some embodiments, the drug capable of preventing or reducing the expression of IL-11, IL-11Rα or gp130 may cause reduced expression by RNA interference (RNAi).

[0170] In some embodiments, the agent can be an inhibitory nucleic acid, such as an antisense or small interfering RNA, including but not limited to shRNA or siRNA.

[0171] In some embodiments, the inhibitory nucleic acid is provided in a vector. For example, in some embodiments, the agent can be a lentiviral vector encoding a shRNA for one or more of IL-11, IL-11Rα, or gp130.

[0172] Oligonucleotide molecules, especially RNA, can be used to modulate gene expression. These include antisense oligonucleotides, targeted degradation of mRNA by small interfering RNA (siRNAs), posttranscriptional gene silencing (PTGs), developmental regulatory sequence-specific translational inhibition of mRNA by microRNA (miRNAs), and targeted transcriptional gene silencing.

[0173] An antisense oligonucleotide is an oligonucleotide, preferably single-stranded, that binds, targets and binds to a target oligonucleotide, such as mRNA, through complementary sequence binding. When the target oligonucleotide is mRNA, the binding of the antisense to the mRNA blocks the translation of the mRNA and the expression of the gene product. Antisense oligonucleotides can be designed to bind to sense genomic nucleic acids and inhibit the transcription of the target nucleotide sequence.

[0174] In view of the known nucleic acid sequences of IL-11, IL-11Rα and gp130 (e.g., known mRNA sequences obtained from GenBank, accession numbers: BC012506.1GI:15341754 (human IL-11), BC134354.1GI:126632002 (mouse IL-11), AF347935.1GI:13549072 (rat IL-11), NM_001142784.2GI:391353394 (human IL-11Rα), NM_00116340 1.1GI:254281268 (mouse IL-11Rα), NM_139116.1GI:20806172 (rat IL-11Rα), NM_001190981.1GI:300244534 (human gp130), NM_010560.3GI:225007624 (mouse gp130), NM_001008725.3GI:300244570 (rat gp130)) oligonucleotides can inhibit or silence the expression of IL-11, IL-11Rα or gp130.

[0175] Such oligonucleotides can be of any length, but are preferably short, e.g., less than 100 nucleotides, e.g., 10-40 nucleotides, or 20-50 nucleotides, and can include a nucleotide sequence that is completely or nearly complementary (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary) to a nucleotide sequence of a corresponding length in a target oligonucleotide, e.g., IL-11, IL-11Rα or gp130 mRNA. The region of complementarity to the nucleotide sequence may be of any length, but is preferably at least 5, and optionally no more than 50 nucleotides in length, for example one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 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.

[0176] Inhibition of the expression of IL-11, IL-11Rα or gp130 will preferably result in a reduction in the amount of IL-11, IL-11Rα or gp130 expressed by cells / tissues / organs / organ systems / subjects. For example, in a given cell, inhibition of the expression of IL-11, IL-11Rα or gp130 by administering appropriate nucleic acids will result in a reduction in the amount of IL-11, IL-11Rα or gp130 expressed by the cell relative to untreated cells. Inhibition may be partial. The preferred degree of inhibition is at least 50%, more preferably at least one of 60%, 70%, 80%, 85% or 90%. Inhibition levels between 90% and 100% are considered to be "silencing" of expression or function.

[0177] The role of RNAi mechanisms and small RNAs in targeting specific chromatin complexes and epigenetic gene silencing at specific chromosomal loci has been demonstrated. Posttranscriptional silencing dependent on double-stranded RNA (dsRNA), also known as RNA interference (RNAi), refers to the phenomenon that dsRNA complexes target homologous specific genes for silencing in a short period of time. It acts as a signal to promote the degradation of mRNAs with sequence identity. A 20nt siRNA is generally long enough to induce gene-specific silencing, but short enough to escape host responses. The reduction in expression of the targeted gene product can be extensive, with silencing induced by a few siRNA molecules reaching up to 90%. RNAi-based therapies have entered Phase I, II, and III clinical trials for many indications (Nature, 2009 Jan 22;457(7228):426-433).

[0178] In the art, these RNA sequences are referred to as "short or small interfering RNA" (siRNA) or "microRNAs" (miRNA), depending on their source. Both types of sequences can downregulate gene expression by binding to complementary RNA and triggering mRNA elimination (RNAi), or by preventing the translation of mRNA into protein. siRNAs are processed from long double-stranded RNAs and are usually exogenous when found in nature. Microinterfering RNAs (miRNAs) are endogenously encoded small non-coding RNAs processed from short hairpins. Both siRNAs and miRNAs can inhibit the translation of mRNAs containing partially complementary target sequences without cleaving the RNA, and degrade mRNAs containing fully complementary sequences.

[0179] siRNA ligands are typically double-stranded, and to optimize the effectiveness of RNA-mediated downregulation of target gene function, the length of the siRNA molecule is preferably selected to ensure proper recognition of the siRNA by the RISC complex that mediates recognition of the siRNA to the mRNA target, and to make the siRNA short enough to reduce host response.

[0180] miRNA ligands are usually single-stranded with partially complementary regions that form hairpins. miRNAs are RNA genes that are transcribed from DNA but are not translated into proteins. The DNA sequence encoding the miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence forms a partial double-stranded RNA fragment with its reverse complementary base pair. The design of microRNA sequences is discussed in John et al., PLoS Biology, 11 (2), 1862-1879, 2004.

[0181] Typically, the RNA ligand intended to simulate the siRNA or miRNA effect has 10-40 ribonucleotides (or its synthetic analogs), more preferably 17-30 ribonucleotides, more preferably 19-25 ribonucleotides, most preferably 21-23 ribonucleotides. In some embodiments of the present invention using double-stranded siRNA, the molecule can have a symmetrical 3' protrusion, such as one or two (ribose) nucleotides, typically dTdT 3' protruding UU. Based on the disclosure provided herein, those skilled in the art can easily design suitable siRNA and miRNA sequences, for example, using resources such as Ambion siRNA finder. siRNA and miRNA sequences can be synthesized and exogenously added to cause gene downregulation or produced using an expression system (such as a vector). In a preferred embodiment, the siRNA is synthetically synthesized.

[0182] Longer double-stranded RNA can be processed in cells to produce siRNA (e.g., see Myers (2003) Nature Biotechnology 21: 324-328). Longer dsRNA molecules may have symmetrical 3' or 5' overhangs, such as one or two (ribo) nucleotides, or may have blunt ends. Longer dsRNA molecules may be 25 nucleotides or longer. Preferably, the longer dsRNA molecule length is between 25 and 30 nucleotides. More preferably, the longer dsRNA molecule length is between 25 and 27 nucleotides. Most preferably, the longer dsRNA molecule length is 27 nucleotides. Lengths of 30 or longer dsRNAs can be expressed using the vector pDECAP (Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).

[0183] Another option is to express a short hairpin RNA molecule (shRNA) in a cell. shRNA is more stable than synthetic siRNA. An shRNA consists of a short inverted repeat sequence, separated by a small loop sequence. An inverted repeat gene target is complementary. In the cell, shRNA is processed into siRNA by DICER, which degrades the target gene mRNA and inhibits expression. In a preferred embodiment, shRNA is produced endogenously (in the cell) by transcription from a vector. Under the control of an RNA polymerase III promoter such as a human H1 or 7SK promoter or an RNA polymerase II promoter, shRNA can be produced in the cell by transfecting cells with a vector encoding the shRNA sequence. Alternatively, shRNA can be synthesized exogenously (in vitro) by transcription from a vector. Then 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 length of the shRNA sequence is between 40 and 100 bases, more preferably between 40 and 70 bases. The length of the stem of the hairpin is preferably between 19 and 30 base pairs. The stem may contain a GU pairing to stabilize the hairpin structure.

[0184] The siRNA molecule, the longer dsRNA molecule or the miRNA molecule can be recombined by transcribing a nucleic acid sequence, which is preferably contained in a vector. Preferably, the siRNA molecule, the longer dsRNA molecule or the miRNA molecule comprises a partial sequence of IL-11, IL-11Rα or gp130.

[0185] In one embodiment, siRNA, longer dsRNA or miRNA are produced endogenously (in cells) by transcribing from a vector. The vector can be introduced into the cell in any manner known in the art. Optionally, tissue-specific (e.g., heart, liver or kidney-specific) promoters can be used to regulate the expression of the RNA sequence. In another embodiment, siRNA, longer dsRNA or miRNA are produced exogenously (in vitro) by transcribing from a vector.

[0186] Suitable vectors can be oligonucleotide vectors configured to express oligonucleotide agents capable of inhibiting IL-11, IL-11Rα or gp130. Such vectors can be viral vectors or plasmid vectors. Therapeutic oligonucleotides can be integrated into the genome of viral vectors and operably connected to regulatory sequences that drive their expression, such as promoters. The term "operably connected" can include situations in which a selected nucleotide sequence and a regulatory nucleotide sequence are covalently linked so that the expression of the nucleotide sequence is affected or controlled by the regulatory sequence. Therefore, if the regulatory sequence can affect the transcription of a nucleotide sequence that forms part or all of a selected nucleotide sequence, the regulatory sequence is operably connected to the selected nucleotide sequence.

[0187] Viral vectors encoding siRNA sequences expressed by promoters are known in the art and have the benefit of long-term expression of therapeutic oligonucleotides. Examples include slow viruses (Nature January 22, 2009; 457(7228): 426-433), adenoviruses (Shen et al., FEBS Lett March 27, 2003; 539(1-3)111-4) and retroviruses (Barton and Medzhitov PNAS November 12, 2002, Vol. 99, No. 23, 14943-14945).

[0188] In other embodiments, the carrier can be configured to facilitate delivery of the therapeutic oligonucleotide to the site where inhibition of IL-11, IL-11Rα or gp130 expression is desired. Such carriers generally include complexing the oligonucleotide with a positively charged carrier (e.g., cationic cell penetrating peptides, cationic polymers and dendrimers, and cationic lipids); conjugating the oligonucleotide with small molecules (e.g., cholesterol, bile acids and lipids), polymers, antibodies and RNA; or encapsulating the oligonucleotide in a nanoparticle formulation (Wang et al., AAPS J. 2010 Dec; 12(4): 492-503).

[0189] In one embodiment, the vector can include nucleic acid sequences in sense and antisense orientations such that when expressed as RNA, the sense and antisense segments will combine to form double-stranded RNA.

[0190] Alternatively, siRNA molecules can be synthesized using standard solid phase or liquid phase synthesis techniques known in the art. The linkage between nucleotides can be a phosphodiester bond or a surrogate bond, for example, a linker of the formula P(O)S, (sulfate); P(S)S, (disulfide); P(O)NR′2; P(O)R′; P(O)OR6; CO; or CONR′2, wherein R is H (or salt) or alkyl (1-12C), and R6 is alkyl (1-9C) connected to adjacent nucleotides via -O- or -S-.

[0191] Modified nucleotide bases can be used in addition to naturally occurring bases and can impart advantageous properties to siRNA molecules containing them.

[0192] For example, modified bases can increase the stability of siRNA molecules, thereby reducing the number required for silencing. The provision of modified bases can also provide siRNA molecules that are more stable or less stable than unmodified siRNA.

[0193] The term "modified nucleotide base" includes nucleotides with covalently modified bases and / or sugars. For example, modified nucleotides include nucleotides with sugars covalently linked to low molecular weight organic groups that are not hydroxyl groups at the 3' position and not phosphate groups at the 5' position. Thus, modified nucleotides may also include 2' substituted sugars such as 2'-O-methyl-; 2'-O-alkyl; 2'-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro-; 2'-halogenated or azido-ribose, carbocyclic sugar analogs, α-isomeric sugars; isomeric sugars such as arabinose, xylose or lyxose, pyranose, furanose and sedoheptulose.

[0194] Modified nucleotides are known in the art, including alkylated purines and pyrimidines, acylated purines and pyrimidines and other heterocycles. These types of pyrimidines and purines are known in the art, including pseudoisocytosine, N4, N4-ethycytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromopyrimidine, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl -2-thiouracil, -D-mannosylquinoside, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine uracil-5-oxoacetate methyl ester, pseudouracil, 2-thiopyrimidine, 5-methyl-2-thiopyrimidine, 2-thiopyrimidine, 4-thiopyrimidine, 5-methyluracil, N-uracil-5-oxoacetate methyl ester, uracil-5-oxoacetate, queosine, 2-thiopyrimidine, 5-propyluracil, 5-propylcytosine, 5-ethylcytosine, 5-butyluracil, 5-pentylcytosine, 2,6-diaminopurine, methylthiopyrimidine, 1-methylguanine, 1-methylcytosine.

[0195] Methods for using RNAi to silence genes in C. elegans, Drosophila, plants and mammals are known in the art (Fire A et al., 1998 Nature 391:806-811; Fire, A., Trends Genet. 15, 358-363 (1999); Sharp, PA, RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, SM et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, SM et al., Nature 404, 293-296 (2000); Zamore, PD et al., Cell 101, 25-33 (2000); Bernstein, E. et al., Nature 409, 363-366 (2001); Elbashir, SM et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619 and Elbashir SM et al., 2001 Nature 411: 494-498).

[0196] Thus, the present invention provides nucleic acids which, when suitably introduced into or expressed in mammalian (eg human) cells which, among other things, express IL-11, IL-11Rα or gp130, are capable of inhibiting the expression of IL-11, IL-11Rα or gp130 by RNAi.

[0197] Nucleic acid sequences of IL-11, IL-11Rα and gp130 oligonucleotides (e.g., known mRNA sequences obtained from GenBank, accession numbers: BC012506.1GI:15341754 (human IL-11), BC134354.1GI:126632002 (mouse IL-11), AF347935.1GI:13549072 (rat IL-11), NM_001142784.2GI:391353394 (human IL-11Rα), NM_001163 401.1GI:254281268 (mouse IL-11Rα), NM_139116.1GI:20806172 (rat IL-11Rα), NM_001190981.1GI:300244534 (human gp130), NM_010560.3GI:225007624 (mouse gp130), NM_001008725.3GI:300244570 (rat gp130)) can inhibit or silence the expression of IL-11, IL-11Rα or gp130.

[0198] The nucleic acid may have substantial sequence identity to a portion of IL-11, IL-11Rα or gp130 mRNA, for example, as defined in GenBank Accession Nos.: NM_000641.3GI:391353405 (IL-11), NM_001142784.2GI:391353394 (IL-11Rα), NM_001190981.1GI:300244534 (gp130), or a complementary sequence thereof.

[0199] The nucleic acid may be a double-stranded siRNA. (As will be appreciated by those skilled in the art, and as further explained below, siRNA molecules may also include a short 3' DNA sequence.)

[0200] Alternatively, the nucleic acid may be DNA (usually double-stranded DNA) which, when transcribed in a mammalian cell, produces an RNA having two complementary portions connected by a spacer, such that when the complementary portions hybridize to each other, the RNA takes the form of a hairpin. In mammalian cells, the hairpin structure can be cleaved by the DICER enzyme to split the molecule, producing two different but hybridized RNA molecules.

[0201] In some preferred embodiments, the nucleic acid generally targets a sequence of one of SEQ ID NOs: 4 to 7 (IL-11) or a sequence of one of SEQ ID NOs: 8 to 11 (IL-11Rα).

[0202] Only single-stranded (i.e., non-self-hybridized) regions of mRNA transcripts are expected to be suitable targets for RNAi. Therefore, other sequences in IL-11 or IL-11Rα mRNA transcripts that are very close to the sequences represented by SEQ ID NOs: 4 to 7 or 8 to 11 may also be suitable RNAi targets. The length of these target sequences is preferably 17-23 nucleotides, and preferably overlaps with one of SEQ ID NOs: 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 one of SEQ ID NOs: 4 to 7 or 8 to 11).

[0203] Thus, the present invention provides nucleic acids that are capable of inhibiting the expression of IL-11 or IL-11Rα by RNAi when appropriately introduced or expressed in a mammalian cell that otherwise expresses IL-11 or IL-11Rα, wherein the nucleic acid generally targets one of SEQ ID NOs: 4 to 7 or 8 to 11.

[0204] By "general targeting", the nucleic acid may target a sequence overlapping with SEQ ID NOs: 4 to 7 or 8 to 11. In particular, the nucleic acid may target a sequence in the mRNA of human IL-11 or IL-11Rα that is slightly longer or shorter than one of SEQ ID NOs: 4 to 7 or 8 to 11 (preferably 17 to 23 nucleotides in length), but is otherwise identical to one of SEQ ID NOs: 4 to 7 or 8 to 11.

[0205] Complete identity / complementarity between the nucleic acid of the invention and the target sequence is desired, although preferred, but not required. Thus, the nucleic acid of the invention may include a single mismatch compared to the mRNA of IL-11 or IL-11Rα. However, even the presence of a single mismatch may result in reduced efficiency, so the absence of mismatches is preferred. When present, 3' overhangs may be excluded from consideration of the number of mismatches.

[0206] The term "complementarity" is not limited to conventional base pairing between nucleic acids composed of natural ribonucleotides and / or deoxyribonucleotides, but also includes base pairing between mRNA and the nucleic acid of the present invention including non-natural nucleotides.

[0207] In one embodiment, the nucleic acid (referred to herein as double-stranded siRNA) comprises the double-stranded RNA sequence shown in SEQ ID NOs: 12 to 15. In another embodiment, the nucleic acid (referred to herein as double-stranded siRNA) comprises the double-stranded RNA sequence shown in SEQ ID NOs: 16 to 19.

[0208] However, it is also expected that slightly shorter or longer sequences directed against the same region of IL-11 or IL-11Rα mRNA will also be effective. In particular, it is expected that double-stranded sequences between 17 and 23 bp in length will also be effective.

[0209] The strands forming the double-stranded RNA may have short 3' dinucleotide overhangs, which may be DNA or RNA. The use of 3' DNA overhangs has no effect on siRNA activity compared to 3' RNA overhangs, but reduces the cost of chemical synthesis of the nucleic acid strand (Elbashir et al., 2001c). Therefore, DNA dinucleotides may be preferred.

[0210] When present, the dinucleotide overhangs may be symmetrical to each other, although this is not necessary. In fact, the 3' overhang of the sense (upstream) strand is not associated with RNAi activity as it is not involved in mRNA recognition and degradation (Elbashir et al., 2001a, 2001b, 2001c).

[0211] While RNAi experiments in Drosophila have shown that antisense 3' overhangs may be involved in mRNA recognition and targeting (Elbashir et al., 2001c), 3' overhangs do not appear to be required for RNAi activity of siRNAs in mammalian cells. Therefore, incorrect annealing of 3' overhangs is thought to have little effect in mammalian cells (Elbashir et al., 2001c; Czauderna et al., 2003).

[0212] Therefore, any overhanging dinucleotide can be used for the antisense strand of siRNA. However, the dinucleotide is preferably -UU or -UG (-TT or -TG if the overhang is DNA), more preferably -UU (or -TT). The -UU (or -TT) dinucleotide overhang is the most effective and is consistent with (i.e., can form part of) the end of the RNA polymerase III transcription signal (the termination signal is TTTTT). Therefore, the dinucleotide is most preferred. The dinucleotides AA, CC and GG can also be used, but the effect is poor and therefore less preferred.

[0213] Furthermore, 3' overhangs can be completely removed from siRNAs.

[0214] The present invention also provides single-stranded nucleic acids (referred to herein as single-stranded siRNAs), each consisting of a constituent strand of one of the above double-stranded nucleic acids, preferably with a 3'-overhang, but optionally without. The present invention also provides a kit comprising such a pair of single-stranded nucleic acids, which can hybridize with each other in vitro to form the above double-stranded siRNA, which is then introduced into cells.

[0215] The present invention also provides DNA, which, when transcribed in mammalian cells, produces RNA (also referred to herein as shRNA) having two complementary parts that are capable of self-hybridization to produce a double-stranded motif, for example, comprising a sequence selected from the group consisting of SEQ ID NO: 12 to 15 or 16 to 19 or a sequence that is a single base pair substituted sequence different from any of the above sequences.

[0216] The complementary parts are usually connected by a spacer, which has an appropriate length and sequence to allow the two complementary parts to hybridize with each other. The two complementary parts (i.e., sense and antisense) can be connected together in a 5'-3' order. The spacer is usually a short sequence, about 4-12 nucleotides, preferably 4-9 nucleotides, and more preferably 6-9 nucleotides.

[0217] Preferably, the 5' end of the spacer (immediately adjacent to the 3' end of the upstream complementary portion) consists of the nucleotides -UU- or -UG-, preferably -UU- (however, again, the use of these specific dinucleotides is not required). A suitable spacer recommended for use in the pSuper system of OligoEngine (Seattle, Washington, USA) is UUCAAGAGA. In this and other cases, the ends of the spacer can hybridize to each other, for example, extending the double-stranded motif a small number (e.g., 1 or 2) base pairs beyond the exact sequence of SEQ ID NO: 12 to 15 or 16 to 19.

[0218] Similarly, the transcribed RNA preferably includes a 3' overhang from the downstream complementary portion. Again, this is preferably -UU or -UG, more preferably -UU.

[0219] This shRNA molecule can then be cleaved in mammalian cells by the enzyme DICER to generate a double-stranded siRNA as described above, wherein one or each strand of the hybridized dsRNA includes a 3' overhang.

[0220] Techniques for synthesizing the nucleic acids of the present invention are of course well known in the art.

[0221] Those skilled in the art can use well-known techniques and commercially available materials to construct suitable transcription vectors for the DNA of the present invention. In particular, the DNA will be associated with control sequences, including promoter and transcription termination sequences.

[0222] Particularly suitable are the commercially available pSuper and pSuperior systems from OligoEngine (Seattle, WA, USA). These use a polymerase III promoter (H1) and a T5 transcription terminator sequence that contributes two U residues at the 3' end of the transcript (providing a 3' UU overhang on one strand of the siRNA following DICER treatment).

[0223] Another suitable system is described in Shin et al. (RNA 2009 May; 15(5): 898-910), which uses another polymerase-III promoter (U6).

[0224] The double-stranded siRNA of the present invention can be introduced into mammalian cells in vitro or in vivo using known techniques, as described below, to inhibit the expression of IL-11 or IL-11 receptor.

[0225] Similarly, transcription vectors containing the DNA of the invention can be introduced into tumor cells in vitro or in vivo using known techniques, as described below, for transient or stable expression of RNA, again to inhibit expression of IL-11 or IL-11 receptor.

[0226] Therefore, the present invention also provides a method for inhibiting the expression of IL-11 or IL-11 receptor in mammalian (eg, human) cells, the method comprising administering the double-stranded siRNA of the present invention or the transcription vector of the present invention to the cells.

[0227] Similarly, the present invention also provides a method for treating hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity, the method comprising administering the double-stranded siRNA of the present invention or the transcription vector of the present invention to a subject.

[0228] The present invention also provides the double-stranded siRNA of the present invention and the transcription vector of the present invention for use in a method of treatment, preferably a method of treating hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity.

[0229] The present invention also provides the use of the double-stranded siRNA of the present invention and the transcription vector of the present invention in preparing a drug for treating hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity.

[0230] The present invention also provides a composition comprising the double-stranded siRNA of the present invention or the transcription vector of the present invention mixed with one or more pharmaceutically acceptable carriers. Suitable carriers include lipophilic carriers or vesicles, which may help penetrate the cell membrane.

[0231] Materials and methods suitable for administration of the siRNA duplexes and DNA vectors of the invention are well known in the art, and given the potential of RNAi technology, improved methods are under development.

[0232] In general, many techniques can be used to introduce nucleic acids into mammalian cells. The choice of technique will depend on whether the nucleic acid is transferred into cells cultured in vitro or into cells in the patient. Techniques suitable for transferring nucleic acids into mammalian cells in vitro include liposomes, electroporation, microinjection, cell fusion, DEAE, dextran, and calcium phosphate precipitation. In vivo gene transfer techniques include transfection with viral (usually retroviral) vectors and transfection mediated by viral coat protein liposomes (Dzau et al., (2003) Trends in Biotechnology 11, 205-210).

[0233] In particular, suitable techniques for cellular administration of the nucleic acids of the invention in vitro and in vivo are disclosed in the following articles:

[0234] General review: Borkhardt, A., 2002. RNA interference blocks oncogenes in malignant tumor cells-a new hope for highly specific cancer therapy? 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 RNA, Nat Rev Genet. 3: 737-47; Scherr, M., M.A. Morgan, and M. Eder., 2003b. Small interfering RNA-mediated gene silencing in mammalian cells, Curr Med Chem. 10: 245-56. Shuey, DJ, DE M.Callus and T. Giordano, 2002. RNAi: gene silencing in therapeutic intervention, Drug Discov Today. 7: 1040-6.

[0235] Systemic administration using liposomes: Lewis, DL, JE Hagstrom, A G Loomis, JA Wolff, and H. Herweijer., 2002. Efficient delivery of siRNA to inhibit gene expression in postnatal mice, Nat Genet. 32: 107-8. Paul, CP, PD Good, I. Winer, and DR Engelke., 2002. Efficient expression of small interfering RNA in human cells, Nat Biotechnol. 20: 505-8. Song, E., SK Lee, J. Wang, N. Ince, N. Ouyang, J. Min, J. Chen, P. Shankar, and J. Lieberman., 2003. RNA interference targeting Fas protects against fulminant hepatitis in mice, Nat Med. 9: 347-51. Sorensen, DR, M. Leirdal, and M. Sioud., 2003. Gene silencing in adult mice by systemic delivery of synthetic siRNA. J Mol Biol. 327: 761-6.

[0236] Virus-mediated transfer: Abbas-Terki, T., W. Blanco-Bose, N. Deglon, W. Pralong, and P. Aebischer., 2002. Lentivirus-mediated RNA interference, Hum Gene Ther. 13: 2197-201. Barton, G. M. and R. Medzhitov., 2002. Retroviral delivery of small interfering RNA into primary cells, Proc Natl Acad Sci U SA. 99: 14943-5. Devroe, E. and P. A. Silver., 2002. Retroviral delivery of siRNA, BMC Biotechnol. 2: 15. Lori, F., P. Guallini, L. Galluzzi and J. Lisziewicz., 2002. Gene therapy for HIV infection, Am J Pharmacogenomics. 2: 245-52. Matta, H., B. Hozayev, R. Tomar, P. Chugh, and PM Chaudhary., 2003. Delivery of small interfering RNA using lentiviral vectors, Cancer Biol Ther. 2: 206-10. Qin, X. F., D. S. An, I. S. Chen, and D. Baltimore., 2003. Lentiviral-mediated anti-CCR5 small interfering RNA inhibits HIV-1 infection in human T cells, Proc Natl Acad Sci U S A. 100: 183-8. Scherr, M., K. Battmer, A. Ganser, and M. Eder., 2003a. Regulation of gene expression by lentiviral-mediated delivery of small interfering RNA, Cell Cycle. 2: 251-7. Shen, C., A. B. Puck, X. Liu, M. Winkler, and S. N. Reske., 2003. Gene silencing by adenoviral-delivered siRNA, FEBS Lett. 539: 111-4.

[0237] Peptide delivery: Morris, MC, L. Chaloin, F. Heitz, and G. Divita., 2000. Translocation peptides and proteins and their use in gene delivery, Curr Opin Biotechnol. 11: 461-6. Simeoni, F., MC Morris, F. Heitz, and G. Divita., 2003. Insights into the mechanism of MPG, a peptide-based gene delivery system: implications for siRNA delivery to mammalian cells, Nucleic Acids Res. 31: 2717-24. Other technologies that may be suitable for delivering siRNA to target cells are based on nanoparticles or nanocapsules, such as those described in U.S. Pat. Nos. 6,649,192B and 5,843,509B.

[0238] Inhibits IL-11-mediated signaling

[0239] In an embodiment of the present invention, the agent capable of inhibiting the action of IL-11 may have one or more of the following functional properties:

[0240] Inhibits IL-11-mediated signaling;

[0241] Inhibits signal transduction mediated by IL-11 binding to the IL-11Rα:gp130 receptor complex;

[0242] Inhibits signal transduction mediated by the binding of IL-11:IL-11Rα complex to gp130 (i.e., IL-11 trans-signaling);

[0243] Inhibits a process mediated by IL-11;

[0244] Inhibit the expression of IL-11, IL-11Rα and / or gp130 gene / protein.

[0245] These properties can be determined by analyzing the relevant agent in appropriate experiments, which may involve comparing the performance of the agent with an appropriate control agent. Those skilled in the art will be able to determine appropriate control conditions for a given experiment.

[0246] IL-11-mediated signal transduction and / or processes mediated by IL-11 include signal transduction mediated by IL-11 fragments and polypeptide complexes containing IL-11 or its fragments. IL-11-mediated signal transduction may be signal transduction mediated by human IL-11 and / or mouse IL-11. Signal transduction mediated by IL-11 may occur after IL-11 or a complex containing IL-11 binds to a receptor to which IL-11 or the complex binds.

[0247] In some embodiments, the agent may be capable of inhibiting the biological activity of IL-11 or a complex containing IL-11.

[0248] In some embodiments, the agent is an antagonist of one or more signaling pathways, which are activated by signaling of receptors including IL-11Rα and / or gp130, such as IL-11Rα:gp130. In some embodiments, the agent is capable of inhibiting signaling by one or more immune receptor complexes comprising IL-11Rα and / or gp130, such as IL-11Rα:gp130. In various aspects of the invention, the agents provided herein are capable of inhibiting IL-11-mediated cis and / or trans signaling. In some embodiments according to various aspects of the invention, the agents provided herein are capable of inhibiting IL-11-mediated cis signaling.

[0249] In some embodiments, the agent can 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 an appropriate control agent), for example, one of 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 is capable of reducing IL-11 mediated signaling to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, compared to the level of signaling in the absence of the agent (or in the presence of an appropriate control agent).

[0250] In some embodiments, the IL-11-mediated signal transduction can be signal transduction mediated by the binding of IL-11 to the IL-11Rα:gp130 receptor. For example, such signal transduction can be analyzed by treating cells expressing IL-11Rα and gp130 with IL-11, or by stimulating cells expressing IL-11Rα and gp130 to produce IL-11.

[0251] The IC50 of a drug that inhibits IL-11-mediated signaling can be determined, for example, by culturing BA / F3 cells expressing IL-11Rα and gp130 in the presence of human IL-11 and the agent, and measuring 3H-thymidine incorporation into DNA. In some embodiments, the IC50 of the agent can be 10 μg / ml or less, preferably ≤5 μg / ml, ≤4 μg / ml, ≤3.5 μg / ml, ≤3 μg / ml, ≤2 μg / ml, ≤1 μg / ml, ≤0.9 μg / ml, ≤0.7 μg / ml, ≤0.6 μg / ml, or ≤0.5 μg / ml.

[0252] In some embodiments, the IL-11-mediated signal transduction can be signal transduction mediated by binding of the IL-11:IL-11Rα complex to gp130. In some embodiments, the IL-11:IL-11Rα complex can be soluble, such as a complex of the extracellular domain of IL-11Rα and IL-11, or a complex of a soluble IL-11Rα isoform / fragment and IL-11. In some embodiments, the soluble IL-11Rα is a soluble (secreted) isoform of IL-11Rα, or a release product of proteolytic cleavage of the extracellular region of cell membrane-bound IL-11Rα.

[0253] In some embodiments, the IL-11:IL-11Rα complex can be cell-bound, such as a complex of cell membrane-bound IL-11Rα and IL-11. Signal transduction mediated by the binding of the IL-11:IL-11Rα complex to gp130 can be analyzed by treating cells expressing gp130 with the IL-11:IL-11Rα complex, for example, a recombinant fusion protein comprising IL-11, which is linked to the extracellular region of IL-11Rα via a peptide linker, such as hyper-IL-11. Hyper IL-11 was constructed using an IL-11Rα fragment (amino acid residues 1-317 consisting of domains 1-3; UniprotKB: Q14626) and IL-11 (UniprotKB: P20809 amino acid residues 22-199), with a 20 amino acid long linker (SEQ ID NO: 20). The amino acid sequence of Hyper IL-11 is shown in SEQ ID NO: 21.

[0254] In some embodiments, the agent may be capable of inhibiting signaling mediated by binding of the IL-11:IL-11Rα complex to gp130, and also capable of inhibiting signaling mediated by binding of IL-11 to the IL-11Rα:gp130 receptor.

[0255] In some embodiments, the agent is capable of inhibiting a process mediated by IL-11.

[0256] In some embodiments, the agent is capable of inhibiting gene / protein expression of IL-11 and / or IL-11Rα.Gene and / or protein expression can be measured as described herein or by methods well known to those skilled in the art.

[0257] In some embodiments, the agent can inhibit 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 an appropriate control agent), for example, 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less or 1% or less. In some embodiments, the agent is capable of inhibiting the gene / protein expression of IL-11 and / or IL-11Rα to less than 1 times the expression level in the absence of the agent (or in the presence of an appropriate control agent), for example, ≤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.

[0258] Treatment / prevention of hepatotoxicity

[0259] The present invention provides methods and articles (agents and compositions) for treating / preventing hepatotoxicity and disorders, diseases and conditions associated with hepatotoxicity, for example, as described herein. Methods for treating / preventing hepatotoxicity and disorders, diseases and conditions associated with hepatotoxicity, for example, as described herein are also provided.

[0260] Treatment is achieved by inhibiting IL-11-mediated signaling (i.e., antagonizing IL-11-mediated signaling). That is, the present invention provides treatment / prevention of hepatotoxicity and disorders, diseases, and conditions associated with hepatotoxicity by inhibiting IL-11-mediated signaling, for example, in cells, tissues / organs / organ systems / subjects. In some embodiments, inhibition of IL-11-mediated signaling according to the present disclosure includes inhibiting IL-11-mediated signaling in cells of the liver (e.g., hepatocytes).

[0261] The present invention provides a use of an agent capable of inhibiting interleukin 11 (IL-11)-mediated signaling in a method for treating or preventing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity.

[0262] The present invention also provides a use of an agent capable of inhibiting interleukin 11 (IL-11)-mediated signal transduction in the preparation of a medicament for a method of treating or preventing hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity.

[0263] Also provided is a method of treating or preventing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an agent capable of inhibiting interleukin 11 (IL-11) mediated signaling.

[0264] In some embodiments, the present invention provides treatment / prevention of hepatotoxicity-related pathologies in diseases / disorders. That is, the present invention provides treatment / prevention of diseases / disorders in which hepatotoxicity is pathologically implicated. Hepatotoxicity-related pathologies are described herein.

[0265] It will be clear to those skilled in the art that the therapeutic and preventive uses of the present invention extend substantially to diseases / disorders that would benefit from a reduction in hepatotoxicity and / or hepatotoxicity-related pathology. The therapeutic and preventive uses of the present invention extend to any subject suffering from hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity. The therapeutic and preventive uses of the present invention also extend to any subject suffering from a disease in which a hepatotoxicity-related pathology is present.

[0266] In some embodiments, the present invention provides treatment / prevention of a disease / disorder caused / exacerbated by hepatotoxicity. In some embodiments, treatment / prevention of a disease / disorder in a subject where hepatotoxicity provides a poor prognosis is provided.

[0267] In some embodiments, the hepatotoxicity and / or disorder, disease or condition associated with hepatotoxicity to be treated / prevented can be characterized by one or more of the following features of the affected organ / tissue / subject, e.g., compared to a normal, unaffected organ / tissue / subject (i.e., not affected by hepatotoxicity and / or disorder, disease or condition associated with hepatotoxicity):

[0268] Decreased liver function

[0269] elevated serum concentrations of liver enzymes, such as alanine aminotransferase (ALT / SGPT), lactate dehydrogenase (LDH), and / or aspartate aminotransferase (AST / SGOT);

[0270] AST / ALT ratio greater than 0.5, greater than 1, or greater than 2;

[0271] Elevated blood alkaline phosphatase (ALP) levels;

[0272] Increased levels of gamma-glutamyl transpeptidase (GGT);

[0273] Serum concentrations of cytokines such as TNFα, IL-1β, and interferon γ were elevated;

[0274] Decreased serum albumin levels;

[0275] An increase in total bilirubin (unconjugated (indirect) and conjugated (direct)), such as the reference range described in Van Wagner LB. JAMA. 313(5):516-517, which is incorporated herein by reference in its entirety;

[0276] loss of liver mass;

[0277] Increased actin stress fiber formation in hepatocytes;

[0278] Increased centrilobular necrosis (i.e., necrosis of the central tissue of the liver lobule).

[0279] The characteristics described in the preceding paragraph may also be referred to herein as symptoms / associates of hepatotoxicity.

[0280] Reference levels for laboratory liver tests can be found, for example, in Gowda S et al., Pan Afr Med J. 2009;3:17, which is incorporated herein by reference in its entirety.

[0281] In some embodiments, the hepatotoxicity and / or hepatotoxicity-related disorder, disease or condition to be treated / prevented can be characterized by an increase in one or more of the following in an organ / tissue / subject affected by the hepatotoxicity and / or hepatotoxicity-related disorder, disease or condition, e.g., compared to a normal organ / tissue / subject (i.e., without hepatotoxicity or a hepatotoxicity-related disorder, disease or condition): expression of one or more of IL-11 and IL-11Rα.

[0282] In some embodiments, the present invention provides treatment / prevention of hepatotoxicity in the context of a disease / disorder / condition associated with hepatotoxicity, e.g., as described herein. In some embodiments, the present invention provides treatment / prevention of hepatotoxicity and underlying diseases / disorders / conditions associated with hepatotoxicity. For example, inhibition of IL-11-mediated signaling can antagonize the role of IL-11 in chemotherapy-related hepatotoxicity, as well as the role of IL-11 in cancer itself.

[0283] According to the present invention, the treatment / prevention of hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity can be hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity associated with upregulation of IL-11, for example, upregulation of IL-11 in cells or tissues in which symptoms of the disease / disorder / condition are displayed or may occur, or upregulation of extracellular IL-11 or IL-11Rα.

[0284] A disorder, disease or condition associated with hepatotoxicity may affect any tissue, organ or organ system. In some embodiments, the disease / disorder / condition may affect several tissues / organs / organ systems. In some embodiments, the disease / disorder / condition affects the liver.

[0285] In some embodiments, the disorder, disease, or condition associated with hepatotoxicity affects one or more of the 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.

[0286] In some embodiments, the present invention provides treatment / prevention of hepatotoxicity-related pathologies in acute liver injury (ALI), acute liver failure, acute liver disease, chronic liver disease, liver damage, hepatitis (e.g., viral hepatitis, alcoholic hepatitis), hepatic ischemia-reperfusion injury (IRI) (e.g., "warm" ischemia-reperfusion (WIR)), radiation-induced liver disease (RILD)), drug-induced liver injury (DILI), idiosyncratic drug-induced liver injury (IDILI), autoimmune liver injury, cholestatic liver disease, HIV, and cancer.

[0287] Treatment can effectively prevent the progression of hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity, such as reducing / delaying / preventing the worsening of hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity, or reducing / delaying / preventing the development of hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity. In some embodiments, treatment can result in improvement, such as a reduction in the severity of hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity, and / or an improvement in the symptoms of hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity. In some embodiments, treatment can increase survival. In some embodiments, treatment effectively reverses the effects and / or symptoms of hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity.

[0288] Specifically, the inventors have demonstrated that by antagonizing IL-11 mediated signaling, it is possible to reduce (ie, reverse) the symptoms of hepatotoxicity, acute liver failure (ALF), and ALF-related mortality.

[0289] Prevention can refer to preventing the development of hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, and / or preventing the worsening of hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, e.g., preventing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity from developing to an advanced or chronic stage.

[0290] In some embodiments, the present invention provides treatment / prevention of hepatotoxicity in the context of liver transplantation. Antagonists of IL-11 mediated signaling can be administered to donor subjects prior to transplantation harvest to minimize damage to the graft. Antagonists of IL-11 mediated signaling can be administered to donor subjects prior to and / or after transplantation harvest to treat / prevent hepatotoxicity in donor subjects. Antagonists of IL-11 mediated signaling can be administered to recipient subjects prior to and / or after transplantation to treat / prevent hepatotoxicity in recipient subjects. In some embodiments, treatment can be effective in improving transplant acceptance / reducing transplant rejection.

[0291] According to various aspects of the present invention, the method for treating and / or preventing hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity according to the present invention may include one or more of the following:

[0292] Reduce damage to liver tissue;

[0293] Reduce liver cell death;

[0294] Reduce IL-11-mediated signaling in hepatocytes;

[0295] Reduced activation of CASP3 in the liver;

[0296] Improve liver function;

[0297] Reduce serum ALT levels;

[0298] Reduce serum AST levels;

[0299] Increase liver GSH levels;

[0300] Reduce acute liver failure;

[0301] Reduce fulminant liver failure;

[0302] Reduce mortality associated with acute liver failure;

[0303] Increased liver weight;

[0304] Regenerate liver tissue;

[0305] Reduced activation (i.e., phosphorylation) of ERK and / or JNK in the liver;

[0306] Reduce the expression of pro-inflammatory genes / proteins in the liver;

[0307] Reduce NOX4 gene / protein expression;

[0308] Reduces the production of reactive oxygen species in the liver;

[0309] Increased gene / expression of PCNA, cyclin D1, cyclin D3 and / or cyclin E1 genes in the liver;

[0310] Increases activation (phosphorylation) of Rb in the liver.

[0311] The present invention also provides a drug capable of inhibiting IL-11-mediated signal transduction, which is used to: reduce damage to liver tissue, reduce liver cell death, reduce IL-11-mediated signal transduction in liver cells, reduce CASP3 activation in the liver, and increase liver function; reduce serum ALT levels; reduce serum AST levels; increase liver GSH levels; reduce acute liver failure; reduce fulminant liver failure; reduce acute liver failure-related mortality; increase liver weight; regenerate liver tissue; reduce liver ERK and / or JNK activation (i.e., phosphorylation); reduce pro-inflammatory gene / protein expression in the liver; reduce NOX4 gene / protein expression; reduce the generation of reactive oxygen species in the liver; increase PCNA, cyclin D1, cyclin D3 and / or cyclin E1 gene / protein expression in the liver; and / or increase Rb activation (phosphorylation) in the liver.

[0312] In some embodiments, the present invention provides treatment / prevention of acute liver injury (ALI), acute liver failure, acute liver disease, chronic liver disease, liver damage, hepatitis (e.g., viral hepatitis, alcoholic hepatitis), hepatic ischemia-reperfusion injury (IRI) (e.g., "warm" ischemia-reperfusion (WIR)), radiation-induced liver disease (RILD)), drug-induced liver injury (DILI), idiosyncratic drug-induced liver injury (IDILI), autoimmune liver injury, cholestatic liver disease, HIV, and cancer.

[0313] As used herein, "cancer" may be any unwanted cell proliferation (or any disease manifested by unwanted cell proliferation), neoplasm or tumor, or an increased risk or susceptibility to unwanted cell proliferation, neoplasm or tumor. The cancer may be benign or malignant, and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells, and may be located in any tissue. Examples of tissues include adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph nodes (including abdominal lymph nodes, axillary lymph nodes, cervical lymph nodes, inguinal lymph nodes, mediastinal lymph nodes, pelvic lymph nodes, periaortic lymph nodes), lymphoblasts, maxilla, mediastinum, mesentery, muscularis, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, leukocytes.

[0314] The cancer can be of a specific type. Examples of cancer types include astrocytomas, carcinomas (e.g., adenocarcinomas, hepatocellular carcinomas, medullary carcinomas, papillary carcinomas, squamous cell carcinomas), gliomas, lymphomas, medulloblastomas, melanomas, myeloma, meningiomas, neuroblastomas, sarcomas (e.g., angiosarcomas, chronic sarcomas, osteosarcomas).

[0315] As used herein, "cancer" may include any one or more of the following: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, female reproductive system cancer, male reproductive system cancer, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colorectal cancer, colon cancer, endometrial cancer, endometrial sarcoma, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, Cancer, leukemia, leukemia, liver cancer, lung cancer, malignant fibrous histiocytoma, malignant thymoma, melanoma, mesothelioma, multiple myeloma, myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nervous system cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, plasma cell tumor, primary central nervous system lymphoma, prostate cancer, rectal cancer, respiratory system, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, gastric cancer, gastric cancer, testicular cancer, thyroid cancer, urinary system cancer, uterine sarcoma, vaginal cancer, vascular system, Waldenstrom's macroglobulinemia, Wilms' tumor.

[0316] In certain embodiments, the present invention provides prevention of hepatotoxicity. In some embodiments, inhibition of IL-11 mediated signaling is provided to protect against hepatotoxic injury before hepatotoxic injury (e.g., which may be physical and / or chemical). Inhibition of IL-11 mediated signaling before hepatotoxic injury can reduce one or more hepatotoxic symptoms after hepatotoxic injury.

[0317] In certain embodiments, the present invention provides treatments for hepatotoxicity. In some embodiments, inhibition of IL-11-mediated signaling is provided to reduce hepatotoxicity following a hepatotoxic insult (which may be, for example, physical, perfusion-related, and / or chemical). Inhibition of IL-11-mediated signaling following a hepatotoxic insult may reduce one or more symptoms of hepatotoxicity.

[0318] In a further aspect, the present invention provides an agent capable of inhibiting IL-11-mediated signaling for use in combination with other therapeutic / preventive interventions to treat / prevent hepatotoxicity (e.g., drug-induced liver injury, such as APAP-induced hepatotoxicity). As reviewed in Park et al., BMJ Clin Evid. (2015) 2015: 2101, therapeutic / preventive interventions for APAP overdose (and thereby APAP-induced hepatotoxicity) are incorporated herein by reference in their entirety.

[0319] Such interventions include treatments / procedures designed to minimize gastrointestinal absorption of APAP after ingestion. These treatments / procedures include gastric lavage, administration of emetics, or administration of activated charcoal.

[0320] Further interventions include treatments / procedures aimed at maintaining / increasing glutathione levels. Such treatments / procedures include administration of acetylcysteine ​​(such as N-acetylcysteine; NAC), methionine, cysteamine, or calciferol.

[0321] Further interventions include treatments / procedures aimed at providing the subject with functional liver tissue, for example following liver failure or in anticipation of liver failure. Such treatments / procedures include liver transplantation.

[0322] On the other hand, the present invention provides an agent capable of inhibiting IL-11-mediated signaling for use in a method for treating / preventing APAP-induced hepatotoxicity in a subject, the method comprising administering to the subject (i) an agent capable of inhibiting IL-11-mediated signaling and (ii) another (different) agent for treating / preventing APAP-induced hepatotoxicity. The present invention also provides a combination (e.g., a composition) comprising: an agent capable of inhibiting IL-11-mediated signaling and another (different) agent for treating / preventing APAP-induced hepatotoxicity, the method comprising administering the composition to the subject.

[0323] Also provided is a use of an agent capable of inhibiting IL-11-mediated signal transduction in the preparation of a medicament for treating / preventing APAP-induced hepatotoxicity in a subject, the method comprising administering to the subject (i) an agent capable of inhibiting IL-11-mediated signal transduction and (ii) another (different) agent for treating / preventing APAP-induced hepatotoxicity. Also provided is a use of a combination (e.g., a composition) comprising: an agent capable of inhibiting IL-11-mediated signal transduction and another (different) agent for the preparation of a medicament for treating / preventing APAP-induced hepatotoxicity, the medicament being used for treating / preventing APAP-induced hepatotoxicity in a subject, the method comprising administering the composition to the subject.

[0324] Also provided is a method for treating / preventing APAP-induced hepatotoxicity in a subject, the method comprising administering to the subject (i) an agent capable of inhibiting IL-11-mediated signaling and (ii) another (different) agent for treating / preventing APAP-induced hepatotoxicity. Also provided is a method for treating / preventing APAP-induced hepatotoxicity in a subject, the method comprising administering a combination (e.g., a composition) comprising: (i) an agent capable of inhibiting IL-11-mediated signaling, and (ii) another (different) agent useful for treating / preventing APAP-induced hepatotoxicity.

[0325] In some embodiments according to aspects described herein, the agent useful for treating / preventing APAP-induced hepatotoxicity (other than an agent capable of inhibiting IL-11 mediated signaling) is selected from: acetylcysteine ​​(e.g., NAC), methionine, cysteamine, calcitriol, an emetic, or activated charcoal. In a specific embodiment, the agent useful for treating / preventing APAP-induced hepatotoxicity (other than an agent capable of inhibiting IL-11 mediated signaling) is acetylcysteine ​​(e.g., NAC).

[0326] In some embodiments, the aspect of using an agent capable of inhibiting IL-11 mediated signaling and another (different) agent for treating / preventing APAP-induced hepatotoxicity provides an improved therapeutic effect compared to the effect observed when either agent is used alone (i.e., as a monotherapy). In some embodiments, the aspect of using an agent capable of inhibiting IL-11 mediated signaling and another (different) agent useful for treating / preventing APAP-induced hepatotoxicity achieves a synergistic (i.e., super-additive) therapeutic effect compared to the effect observed when either agent is used alone.

[0327] Drug administration

[0328] Administration of an agent capable of inhibiting IL-11 mediated signaling is preferably in a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to show benefit to the subject.

[0329] In some embodiments, the drug can be administered before, in conjunction with, or after a hepatotoxic insult (i.e., the cause of hepatotoxicity). In some embodiments, the hepatotoxic insult is chemical, e.g., administration or taking of a hepatotoxic agent (e.g., a hepatotoxic drug, such as APAP). In some embodiments, the cause of hepatotoxicity is physical, e.g., physical damage to liver cells / tissue, such as due to surgery, ischemia / reperfusion, or physical injury. In some embodiments, the hepatotoxic insult is an environmental source of hepatotoxicity.

[0330] In some embodiments, the agent capable of inhibiting IL-11-mediated signaling is administered before hepatotoxic injury. The agent may be administered when hepatotoxic injury is expected. The agent may be administered to prevent / reduce the hepatotoxicity caused by subsequent hepatotoxic injury. In some embodiments, the agent is administered within a specified time before hepatotoxic injury. In some embodiments, the agent is administered within 1 week before hepatotoxic injury, for example, within 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 16 hours, 12 hours, 10 hours, 8 hours, 4 hours, 2 hours, 1 hour or 30 minutes before hepatotoxic injury.

[0331] In some embodiments, the agent capable of inhibiting IL-11-mediated signaling is administered at the same time as the hepatotoxic injury (i.e., administered simultaneously). The agent can be used to prevent / reduce hepatotoxicity caused by hepatotoxic injury. In some embodiments, the agent is administered simultaneously with the hepatotoxic injury, for example, within 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes before / after the hepatotoxic injury.

[0332] In some embodiments, the medicament capable of inhibiting IL-11-mediated signal transduction is administered after hepatotoxic injury. The medicament may be administered to prevent / reduce the hepatotoxicity caused by previous hepatotoxic injury. The medicament may be administered after the onset of hepatotoxicity. The medicament may be administered after the detection of hepatotoxicity. In some embodiments, the medicament is administered within a specified time after hepatotoxic injury. In some embodiments, the medicament is administered within 1 month after hepatotoxic injury, for example, within 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour or 30 minutes after hepatotoxic injury. In some embodiments, the agent is administered 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or more than 1 month after the hepatotoxic insult.

[0333] In certain embodiments, the hepatotoxic injury is the administration / intake of a hepatotoxic agent. In some embodiments, the hepatotoxic agent is a chemical substance that directly or indirectly causes hepatotoxicity. In some embodiments, the hepatotoxic agent is acetaminophen.

[0334] In some embodiments, the agent is administered within 48 hours, 36 hours, 24 hours, 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes after an acetaminophen overdose. In some embodiments, the agent is administered more than 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, or 48 hours after an acetaminophen overdose.

[0335] The actual dosage, administration rate and time course will depend on the nature and severity of hepatotoxicity and the nature of the medicament. The treatment prescription (e.g., the decision of dosage, etc.) is responsible for by general practitioners and other doctors, and usually considers the disease to be treated / illness, the condition of individual subjects, administration site, administration method and other factors known to practitioners. The example of the above-mentioned technology and scheme can be found in " Remington's Pharmaceutical Sciences " (Remington's Pharmaceutical Sciences) (the 20th edition) published by Lippincott, William & Wilkins Press in 2000.

[0336] Multiple doses of the agent may be provided. One or more or each dose may be used to administer another therapeutic agent simultaneously or sequentially.

[0337] Multiple doses may be separated by a predetermined time interval, which may be selected as one of 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, a dose may be given once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0338] In therapeutic applications, the agent capable of inhibiting IL-11-mediated signal transduction is preferably formulated into a medicament or drug together 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, colorants, flavoring agents and sweeteners.

[0339] The term "pharmaceutically acceptable" as used herein relates to compounds, ingredients, materials, compositions, dosage forms, etc., which are suitable for use in contact with the tissues of the subject (e.g., human) within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio. Each carrier, adjuvant, excipient, etc. must also be "acceptable", i.e. compatible with the other ingredients of the formulation.

[0340] Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Formula (18th Edition), 18th Edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 2nd Edition, 1994.

[0341] The preparation can be prepared by any method known in the field of pharmaceutics. This method includes the step of combining the active compound with a carrier constituting one or more auxiliary components. Usually, the preparation is prepared by uniformly and closely combining the active compound with a carrier (e.g., a liquid carrier, a fine solid carrier, etc.), and then molding the product when necessary.

[0342] The formulations can be prepared for topical, parenteral, systemic, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subcutaneous, oral or transdermal administration, including injection. Injectable formulations can contain the selected formulation in a sterile or isotonic medium. The formulation and mode of administration can be selected according to the medicament to be treated and the disease / disorder / condition.

[0343] In some cases, the articles described herein (e.g., medicaments / compositions) are combined with treatments for diseases / disorders / conditions associated with hepatotoxicity to perform the treatments described herein. Suitable treatments for diseases / disorders / conditions associated with hepatotoxicity are known in the art. Depending on the disease / disorder / condition to be treated, the composition can be administered alone, in combination with other treatment methods, simultaneously, or sequentially. For example, the article can be administered before, simultaneously with, or after treatment. The article and the treatment can be formulated together, for example, as described above, or separately.

[0344] In some embodiments according to the aspect of using a combination of agents, the agents can be administered simultaneously or sequentially. Simultaneous administration refers to administration as a composition containing two drugs (combination formulations), or immediately after each other and optionally administered by the same route of administration, such as administration to the same artery, vein or other blood vessel. Sequential administration refers to administration of one agent followed by administration of another agent after a given time interval. It is not necessary to administer two agents by the same route, although this is the case in some embodiments. The time interval can be any time interval.

[0345] Detection of IL-11 and its receptor

[0346] Some aspects and embodiments of the invention relate to detecting the expression of IL-11 or an IL-11 receptor (eg, IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130) in a sample obtained from a subject.

[0347] In some aspects and embodiments, the present invention relates to upregulated expression (overexpression) of IL-11 or IL-11 receptor (as a protein or oligonucleotide encoding the corresponding IL-11 or IL-11 receptor), and detecting such upregulation as an indicator of the suitability of treatment with an agent capable of inhibiting the action of IL-11 or with an agent capable of preventing or reducing the expression of IL-11 or IL-11 receptor.

[0348] Up-regulated expression includes expression higher than the usual expected level of cells or tissues of a given type. Up-regulated expression can be determined by measuring the expression level of the related factor in the cell or tissue. Comparison can be made between the expression level in the cell or tissue sample from the subject and the reference expression level of the related factor, for example, representing the value or value range of the normal expression level of the related factor of the same or corresponding cell or tissue type. In some embodiments, the reference level can be determined by detecting the expression of IL-11 or IL-11 receptor in the corresponding cells or tissues from healthy subjects or from the healthy tissues of the same subject in the control sample. In some embodiments, the reference level can be obtained from a standard curve or a data set.

[0349] Expression levels can be quantified for absolute or relative comparisons.

[0350] In some embodiments, when the expression level in the test sample is at least 1.1 times the reference level, it can be considered that there is an upregulation of IL-11 or IL-11 receptor (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130). More preferably, the expression level can be selected from 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 7.0, at least 8.0, at least 9.0 or at least 10.0 times the reference level.

[0351] Expression levels can be determined by one of a number of known in vitro assays, such as PCR-based assays, in situ hybridization assays, flow cytometry assays, immunological or immunohistochemical assays.

[0352] For example, suitable techniques involve methods for detecting the level of IL-11 or IL-11 receptor in a sample by contacting the sample with an agent capable of binding to IL-11 or IL-11 receptor and detecting the formation of a complex between the agent and IL-11 or IL-11 receptor. The agent may be any suitable binding molecule, such as an antibody, polypeptide, peptide, oligonucleotide, aptamer or small molecule, and may optionally be labeled to allow detection of the formed complex, such as visual detection. Suitable labels and methods for their detection are well known to those skilled in the art, including fluorescent labels (e.g., fluorescein, rhodamine, eosin and NDB, green fluorescent protein (GFP), rare earth chelates such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, Cy5), isotope labels, radioisotopes (e.g., 32P, 33P, 35S), chemiluminescent labels (e.g., acridinium esters, luminol, isoluminol), enzymes (e.g., peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, luciferase), antibodies, ligands and receptors. Detection techniques are well known to those skilled in the art, and detection techniques corresponding to the labeling reagents can be selected. Suitable techniques include PCR amplification of oligonucleotide tags, mass spectrometry, fluorescence or color detection, such as enzymatic conversion of substrates or radioactive detection by reporter proteins.

[0353] The assay can be configured to quantify the amount of IL-11 or IL-11 receptor in a sample. The quantified amount of IL-11 or IL-11 receptor from the test sample can be compared to a reference value, and the comparison is used to determine whether the amount of IL-11 or IL-11 receptor in the test sample is higher or lower than the reference value to a selected degree of statistical significance.

[0354] Quantification of the detected IL-11 or IL-11 receptor can be used to determine the upregulation or downregulation or amplification of the gene encoding IL-11 or IL-11 receptor. In the case where the test sample contains fibrotic cells, such upregulation, downregulation or amplification can be compared with a reference value to determine if there are any statistically significant differences.

[0355] The sample obtained from the subject can be of any kind. The biological sample can be extracted from any tissue or body fluid, such as a blood sample, a blood-derived sample, a serum sample, a lymph sample, a semen sample, a saliva sample, a synovial fluid sample. The blood-derived sample can be a selected portion of the patient's blood, such as a selected cell-containing portion or a plasma or serum portion. The sample can include a tissue sample or a biopsy; or a cell isolated from the subject. The sample can be collected by known techniques, such as a biopsy or aspiration. The sample can be stored and / or processed for subsequent determination of IL-11 expression levels.

[0356] The sample may be used to determine upregulation of IL-11 or an IL-11 receptor in the subject from which the sample was taken.

[0357] In some preferred embodiments, the sample may be a tissue sample, such as a biopsy, taken from liver tissue, heart tissue, visceral organ tissue, respiratory system organ tissue, or urinary / renal system tissue. The sample may contain cells.

[0358] According to the present invention, a subject can be selected for treatment / prevention based on a determination that the subject has upregulated expression levels of IL-11 or IL-11 receptor (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130). Upregulated expression of IL-11 or IL-11 receptor can be used as a marker of hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity that is suitable for treatment with an agent capable of inhibiting IL-11-mediated signaling.

[0359] The upregulation may be in a given tissue or in selected cells from a given tissue. A preferred tissue may be liver tissue. The upregulation of IL-11 or IL-11 receptor expression may also be measured in a circulating fluid such as blood or in a blood-derived sample. The upregulation may be extracellular IL-11 or IL-11Rα. In some embodiments, expression may be locally or systemically upregulated.

[0360] Following selection, an agent capable of inhibiting IL-11 mediated signaling may be administered to the subject.

[0361] Diagnosis and Prognosis

[0362] Detection of upregulated expression of IL-11 or an IL-11 receptor (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and / or gp130) can also be used in methods for diagnosing hepatotoxicity and / or disorders, diseases, or conditions associated with hepatotoxicity, identifying subjects at risk for developing hepatotoxicity and / or disorders, diseases, or conditions associated with hepatotoxicity, and methods for prognosing or predicting a subject's response to treatment with an agent capable of inhibiting IL-11-mediated signaling.

[0363] "Occurrence," "progression," and other forms of "development" may refer to the appearance of a disease / condition, or the continuation or progression of a disease / condition.

[0364] In some embodiments, for example, based on the presence of other symptoms indicative of hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity in selected cells / tissues in the subject or in the subject, the subject may be suspected of having or suffering from hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity, or may be considered to be at risk of developing hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity, for example, due to genetic susceptibility or exposure to environmental conditions, which are known to be risk factors for hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity. The determination of upregulated expression of IL-11 or IL-11 receptors may confirm a diagnosis or a suspected diagnosis, or may confirm that the subject is at risk of developing hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity. The determination may also diagnose hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity or susceptibility as diseases suitable for treatment with agents capable of inhibiting IL-11-mediated signaling.

[0365] Thus, a method of providing a prognosis for a subject having or suspected of having hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity can be provided, the method comprising determining whether expression of IL-11 or IL-11 receptor is upregulated in a sample obtained from the subject, and based on the determination, providing a prognosis for treating the subject with an agent capable of inhibiting IL-11-mediated signaling.

[0366] In some aspects, a method of diagnosis or a method of prognosticating or predicting a subject's response to treatment with an agent capable of inhibiting IL-11 mediated signaling may not require determination of IL-11 or IL-11 receptor expression, but may be based on determination of genetic factors in a subject that predict upregulated expression or activity. These genetic factors may include determination of genetic mutations, single nucleotide polymorphisms (SNPs), or gene amplifications of IL-11, IL-11Rα, and / or gp130 that are associated with and / or predictive of upregulated expression or activity and / or IL-11 mediated signaling. The use of genetic factors to predict susceptibility to a disease state or response to treatment is known in the art, e.g., see Peter et al. Gut 2008; 57: 440-442; Wright et al., Mol. Cell. Biol., March 2010, Vol. 20, No. 6, 1411-1420.

[0367] The genetic factor can be analyzed by methods known to those of ordinary skill in the art, including PCR-based analysis, such as quantitative PCR, competitive PCR. By determining the presence of the genetic factor, for example in a sample obtained from a subject, a diagnosis can be confirmed, and / or the subject can be classified as being at risk for developing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, and / or the subject can be identified as suitable for treatment with a drug capable of inhibiting IL-11-mediated signaling.

[0368] Some methods may include determining the presence of one or more SNPs associated with IL-11 secretion or hepatotoxicity and / or a susceptibility to the development of an obstacle, disease or condition associated with hepatotoxicity. SNPs are typically biallelic and can therefore be readily determined using one of many conventional analyses known to those skilled in the art (e.g., see Anthony J. Brookes, The Nature of SNPS, Gene, Vol. 234, No. 2, July 8, 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 More Than 10,000 SNPs Using Single Primer Analysis on High-Density Oligonucleotide Arrays, Genome Res., 2004, 14: 414-425).

[0369] The method may include determining which SNP allele is present in a sample obtained from a subject. In some embodiments, determining the presence of a minor allele may be associated with increased IL-11 secretion or susceptibility to development of hepatotoxicity and / or a disorder, disease, or condition associated with hepatotoxicity.

[0370] Therefore, in one aspect of the present invention, there is provided a method for screening an object, the method comprising:

[0371] obtaining a nucleic acid sample from a subject;

[0372] Determine which allele is present at the polymorphic nucleotide position of one or more SNPs listed in Figure 33, Figure 34 or Figure 35 of WO 2017 / 103108 A1 (which are incorporated herein by reference), or a SNP in linkage disequilibrium with one of the listed SNPs, wherein r 2 ≥0.8.

[0373] The determining step may include determining whether the minor allele is present at a selected polymorphic nucleotide position in the sample. It may include determining whether there are 0, 1 or 2 minor alleles.

[0374] The screening method can be a method for determining a subject's susceptibility to development of hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, or be part of a diagnostic or prognostic method described herein.

[0375] The method may also include a step, for example, if the subject is determined to have a minor allele at the polymorphic nucleotide position, the subject is determined to have an increased susceptibility or risk of developing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity. The method may also include the step of selecting a subject for treatment, treating the subject with an agent capable of inhibiting IL-11-mediated signaling and / or administering to the subject an agent capable of inhibiting IL-11-mediated signaling, so as to provide treatment for hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity in the subject, or to prevent the development or progression of hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity in the subject.

[0376] The SNPs that can be determined include one or more SNPs listed in Figure 33, Figure 34 or Figure 35 of WO 2017 / 103108 A1 (incorporated herein by reference). SNPs can be selected to be determined to have a low P value or FDR (false discovery rate).

[0377] In some embodiments, the methods described herein may include determining the presence or absence of a genetic factor associated with hepatotoxicity in a subject, such as those described in Njoku DB, Int J Mol Sci. 2014; 15(4): 6990-7003; Khoury T et al., J Clin Transl Hepatol. 2015; 3(2): 99-108; Ahmad J and Odin JA, Clin Liver Dis. 2017; 21(1): 5-72; Clare et al., Curr Hepatol Rep. 2017; 16(3): 258-264 and Urban TJ et al., Pharmacogenomics. 2012 May; 13(7): 735-738, which are incorporated herein by reference in their entireties. In some embodiments, the methods described herein may include determining whether the subject to be treated is heterozygous or homozygous for the cytochrome P450 (CYP450) isoform CYP2E1. The methods and uses according to the present invention may comprise the steps of determining the CYP2E1 genotype of a subject and / or selecting a subject for treatment who is heterozygous or homozygous for CYP2E1.

[0378] In some embodiments, a method for diagnosing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, identifying a subject at risk for developing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, and a method for prognosticating or predicting a subject's response to treatment with an agent capable of inhibiting IL-11-mediated signaling uses an indicator other than detecting upregulated expression of IL-11 or IL-11 receptor or genetic factors.

[0379] In some embodiments, methods of diagnosing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, methods of identifying a subject at risk for developing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, and methods of prognosing or predicting a subject's response to treatment with an agent capable of inhibiting IL-11 mediated signaling are based on detecting, measuring and / or identifying one or more of the following:

[0380] Increased serum concentrations of liver enzymes, such as alanine aminotransferase (ALT / SGPT), lactate dehydrogenase (LDH), and / or aspartate aminotransferase (AST / SGOT);

[0381] AST / ALT ratio greater than 0.5, greater than 1, or greater than 2;

[0382] Elevated blood alkaline phosphatase (ALP) levels;

[0383] Increased levels of gamma-glutamyl transpeptidase (GGT);

[0384] Serum concentrations of cytokines such as TNFα, IL-1β, and interferon γ were elevated;

[0385] Decreased serum albumin levels;

[0386] An increase in total bilirubin (unconjugated (indirect) and conjugated (direct)), such as the reference range described in Van Wagner LB. JAMA. 313(5):516-517, which is incorporated herein by reference in its entirety;

[0387] loss of liver mass;

[0388] Increased actin stress fiber formation in hepatocytes;

[0389] Increased centrilobular necrosis (i.e., necrosis of the central tissue of the liver lobule).

[0390] Yellowing of the skin and whites of the eyes (jaundice);

[0391] Itching

[0392] Abdominal pain in the upper right part of the abdomen;

[0393] fatigue;

[0394] Loss of appetite

[0395] Nausea and vomiting

[0396] rash;

[0397] Weight loss

[0398] Dark or tea-colored urine.

[0399] Reference levels for laboratory liver tests can be found, for example, in Gowda S et al., Pan Afr Med J. 2009;3:17, which is incorporated herein by reference in its entirety.

[0400] The diagnostic or predictive method can be performed in vitro on a sample obtained from a subject, or after processing the sample obtained from the subject. Once the sample is collected, the patient does not need to be present for the diagnostic or predictive in vitro method to be performed, and thus the method may be a method that is not practiced on the human or animal body. As described above, the sample obtained from the subject may be of any kind.

[0401] Other diagnostic or prognostic tests may be used in conjunction with the tests described herein to enhance the accuracy of the diagnosis or prognosis or to confirm the results obtained by using the tests described herein.

[0402] Subjects

[0403] The subject may be an animal or a human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. The subject may be a patient. The patient may have hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity as described herein. The subject may have been diagnosed with hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity that requires treatment, may be suspected of having such hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity, or may be at risk of developing hepatotoxicity and / or a disorder, disease or condition associated with hepatotoxicity.

[0404] 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 the characteristics of one or more markers (correlations / symptoms) of hepatotoxicity and / or disorders, diseases or conditions associated with hepatotoxicity.

[0405] In some embodiments, the subject can be selected for treatment according to the present invention based on determining that the subject has experienced, will experience, or is experiencing hepatotoxic injury. In some embodiments, before an expected intervention (e.g., surgery, such as treatment with an agent associated with hepatotoxicity) that will or may result in hepatotoxicity, the subject can be selected for treatment according to the present invention. In some embodiments, after determining that the subject has experienced or is experiencing hepatotoxicity or hepatotoxic injury, the subject can be selected for treatment according to the present invention.

[0406] Further methods and uses provided

[0407] The present invention also provides a preparation capable of inhibiting IL-11-mediated signal transduction for use, or the use of a preparation capable of inhibiting IL-11-mediated signal transduction in the following methods: reducing damage to liver tissue; reducing liver cell death; increasing liver function; reducing serum ALT levels; increasing liver weight; regenerating liver tissue or reducing ERK and / or JNK activation (i.e., phosphorylation) in the liver.

[0408] The present invention also provides the use of an agent capable of inhibiting IL-11-mediated signal transduction, which is used to prepare a composition, wherein the composition is used in the following methods: reducing damage to liver tissue; reducing liver cell death; increasing liver function; reducing serum ALT levels; increasing liver weight; regenerating liver tissue or reducing ERK and / or JNK activation (i.e., phosphorylation) in the liver.

[0409] The present invention also provides the following methods: reducing damage to liver tissue; reducing liver cell death; increasing liver function; reducing serum ALT levels; increasing liver weight; regenerating liver tissue or reducing ERK and / or JNK activation (i.e., phosphorylation) in the liver, the method comprising administering to a subject an effective amount of an agent capable of inhibiting IL-11-mediated signaling.

[0410] Sequence identity

[0411] For the purpose of determining the percent sequence identity between two or more amino acid or nucleic acid sequences, pairwise and multiple sequence alignments can be achieved in a variety of ways known to those skilled in the art, for example, using software such as ClustalOmega ( , J. 2005, Bioinformatics 21, 951-960), T-Coffee (Notredame et al., 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772-780) and other publicly available computer software. When using such software, it is preferred to use the default parameters (e.g., for gap penalties and extension penalties).

[0412] sequence

[0413]

[0414]

[0415] ***

[0416] The present invention includes any combination of the described aspects and preferred features unless such a combination is expressly impermissible or explicitly avoided.

[0417] The features disclosed in the above description, or in the following claims or drawings, in their specific form or as means for performing the disclosed functions, or as methods or processes for obtaining the disclosed results (as the case may be), may be expressed alone or in any combination of these features for implementing the invention in various forms.

[0418] For the avoidance of any doubt, any theoretical explanations provided herein are intended to enhance the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0419] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0420] Throughout the specification, including the appended claims, unless the context requires otherwise, the words "comprise" and "comprising" and variations such as "including", "having" and "including", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0421] It must be noted that the singular forms "a", "an", and "the" used in the specification and the appended claims include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it will be understood that the particular value forms another embodiment. The term "about" relative to a value is optional and refers to, for example, + / - 10%.

[0422] The methods disclosed herein may be performed, or the products may be present, in vitro, ex vivo, or in vivo. The term "in vitro" is intended to include experiments performed with materials, biological substances, cells, and / or tissues under laboratory conditions or under culture conditions, while the term "in vivo" is intended to include experiments and procedures performed with intact multicellular organisms. "Ex vivo" refers to something that exists or occurs outside an organism, such as outside the human body or animal body, possibly on a tissue (e.g., a whole organ) or a cell obtained from an organism.

[0423] Where a nucleic acid sequence is disclosed herein, its reverse complement is also expressly contemplated.

[0424] For standard molecular biology techniques, see Sambrook, J., Russell, DW, Molecular Cloning, A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0425] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. For those skilled in the art, other aspects and embodiments will be apparent. All documents mentioned herein are incorporated herein by reference. Although the present invention has been described in conjunction with the following exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when this disclosure is given. Therefore, the exemplary embodiments of the present invention set forth above are considered to be illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the present invention. Sequence Listing <110> Singapore Health Services Pte Ltd National University of Singapore R. Clegg I (Lesotho only) <120> Treatment of hepatotoxicity <130> 007608896 <150> GB 1900811.9 <151> 2019-01-21 <150> GB 1907839.3 <151> 2019-06-03 <150> GB 1915003.6 <151> 2019-10-17 <160> 29 <170> PatentIn version 3.5 <210> 1 <211> 199 <212> PRT <213> Homo sapiens IL-11 11 (UniProt P20809) <400> 1 Met Asn Cys Val Cys Arg Leu Val Leu Val Val Leu Ser Leu Trp Pro 1 5 10 15 Asp Thr Ala Val Ala Pro Gly Pro Pro Pro Gly Pro Pro Arg Val Ser 20 25 30 Pro Asp Pro Arg Ala Glu Leu Asp Ser Thr Val Leu Leu Thr Arg Ser 35 40 45 Leu Leu Ala Asp Thr Arg Gln Leu Ala Ala Gln Leu Arg Asp Lys Phe 50 55 60 Pro Ala Asp Gly Asp His Asn Leu Asp Ser Leu Pro Thr Leu Ala Met 65 70 75 80 Ser Ala Gly Ala Leu Gly Ala Leu Gln Leu Pro Gly Val Leu Thr Arg 85 90 95 Leu Arg Ala Asp Leu Leu Ser Tyr Leu Arg His Val Gln Trp Leu Arg 100 105 110 Arg Ala Gly Gly Ser Ser Leu Lys Thr Leu Glu Pro Glu Leu Gly Thr 115 120 125 Leu Gln Ala Arg Leu Asp Arg Leu Leu Arg Arg Leu Gln Leu Leu Met 130 135 140 Ser Arg Leu Ala Leu Pro Gln Pro Pro Pro Asp Pro Pro Ala Pro Pro 145 150 155 160 Leu Ala Pro Pro Ser Ser Ala Trp Gly Gly Ile Arg Ala Ala His Ala 165 170 175 Ile Leu Gly Gly Leu His Leu Thr Leu Asp Trp Ala Val Arg Gly Leu 180 185 190 Leu Leu Leu Lys Thr Arg Leu 195 <210> 2 <211> 918 <212> PRT <213> Homo sapiens gp130 (UniProt P40189-1) <400> 2 Met Leu Thr Leu Gln Thr Trp Leu Val Gln Ala Leu Phe Ile Phe Leu 1 5 10 15 Thr Thr Glu Ser Thr Gly Glu Leu Leu Asp Pro Cys Gly Tyr Ile Ser 20 25 30 Pro Glu Ser Pro Val Val Gln Leu His Ser Asn Phe Thr Ala Val Cys 35 40 45 Val Leu Lys Glu Lys Cys Met Asp Tyr Phe His Val Asn Ala Asn Tyr 50 55 60 Ile Val Trp Lys Thr Asn His Phe Thr Ile Pro Lys Glu Gln Tyr Thr 65 70 75 80 Ile Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Thr Asp Ile Ala Ser 85 90 95 Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu Thr Phe Gly Gln Leu Glu 100 105 110 Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser Gly Leu Pro Pro Glu Lys 115 120 125 Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly Lys Lys Met Arg Cys 130 135 140 Glu Trp Asp Gly Gly Arg Glu Thr His Leu Glu Thr Asn Phe Thr Leu 145 150 155 160 Lys Ser Glu Trp Ala Thr His Lys Phe Ala Asp Cys Lys Ala Lys Arg 165 170 175 Asp Thr Pro Thr Ser Cys Thr Val Asp Tyr Ser Thr Val Tyr Phe Val 180 185 190 Asn Ile Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val Thr 195 200 205 Ser Asp His Ile Asn Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro 210 215 220 Pro His Asn Leu Ser Val Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu 225 230 235 240 Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val Ile Ile Leu Lys 245 250 255 Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile 260 265 270 Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser Phe Thr Val Gln Asp 275 280 285 Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg Ile Arg Cys Met Lys Glu 290 295 300 Asp Gly Lys Gly Tyr Trp Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile 305 310 315 320 Thr Tyr Glu Asp Arg Pro Ser Lys Ala Pro Ser Phe Trp Tyr Lys Ile 325 330 335 Asp Pro Ser His Thr Gln Gly Tyr Arg Thr Val Gln Leu Val Trp Lys 340 345 350 Thr Leu Pro Pro Phe Glu Ala Asn Gly Lys Ile Leu Asp Tyr Glu Val 355 360 365 Thr Leu Thr Arg Trp Lys Ser His Leu Gln Asn Tyr Thr Val Asn Ala 370 375 380 Thr Lys Leu Thr Val Asn Leu Thr Asn Asp Arg Tyr Leu Ala Thr Leu 385 390 395 400 Thr Val Arg Asn Leu Val Gly Lys Ser Asp Ala Ala Val Leu Thr Ile 405 410 415 Pro Ala Cys Asp Phe Gln Ala Thr His Pro Val Met Asp Leu Lys Ala 420 425 430 Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp Thr Thr Pro Arg Glu 435 440 445 Ser Val Lys Lys Tyr Ile Leu Glu Trp Cys Val Leu Ser Asp Lys Ala 450 455 460 Pro Cys Ile Thr Asp Trp Gln Gln Glu Asp Gly Thr Val His Arg Thr 465 470 475 480 Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val 485 490 495 Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser Ile Lys Ala 500 505 510 Tyr Leu Lys Gln Ala Pro Pro Ser Lys Gly Pro Thr Val Arg Thr Lys 515 520 525 Lys Val Gly Lys Asn Glu Ala Val Leu Glu Trp Asp Gln Leu Pro Val 530 535 540 Asp Val Gln Asn Gly Phe Ile Arg Asn Tyr Thr Ile Phe Tyr Arg Thr 545 550 555 560 Ile Ile Gly Asn Glu Thr Ala Val Asn Val Asp Ser Ser His Thr Glu 565 570 575 Tyr Thr Leu Ser Ser Leu Thr Ser Asp Thr Leu Tyr Met Val Arg Met 580 585 590 Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe 595 600 605 Thr Thr Pro Lys Phe Ala Gln Gly Glu Ile Glu Ala Ile Val Val Pro 610 615 620 Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys 625 630 635 640 Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro 645 650 655 Asp Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro 660 665 670 Arg His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe 675 680 685 Thr Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe 690 695 700 Pro Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn 705 710 715 720 Thr Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser 725 730 735 Ser Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn 740 745 750 Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg 755 760 765 His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln 770 775 780 Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln Leu Val Asp 785 790 795 800 His Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys 805 810 815 Gln Asn Cys Ser Gln His Glu Ser Ser Pro Asp Ile Ser His Phe Glu 820 825 830 Arg Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu 835 840 845 Lys Gln Gln Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln 850 855 860 Met Lys Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly 865 870 875 880 Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala 885 890 895 Thr Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln 900 905 910 Gly Gly Tyr Met Pro Gln 915 <210> 3 <211> 422 <212> PRT <213> Homo sapiens IL11RA (UniProt Q14626) <400> 3 Met Ser Ser Ser Cys Ser Gly Leu Ser Arg Val Leu Val Ala Val Ala 1 5 10 15 Thr Ala Leu Val Ser Ala Ser Ser Pro Cys Pro Gln Ala Trp Gly Pro 20 25 30 Pro Gly Val Gln Tyr Gly Gln Pro Gly Arg Ser Val Lys Leu Cys Cys 35 40 45 Pro Gly Val Thr Ala Gly Asp Pro Val Ser Trp Phe Arg Asp Gly Glu 50 55 60 Pro Lys Leu Leu Gln Gly Pro Asp Ser Gly Leu Gly His Glu Leu Val 65 70 75 80 Leu Ala Gln Ala Asp Ser Thr Asp Glu Gly Thr Tyr Ile Cys Gln Thr 85 90 95 Leu Asp Gly Ala Leu Gly Gly Thr Val Thr Leu Gln Leu Gly Tyr Pro 100 105 110 Pro Ala Arg Pro Val Val Ser Cys Gln Ala Ala Asp Tyr Glu Asn Phe 115 120 125 Ser Cys Thr Trp Ser Pro Ser Gln Ile Ser Gly Leu Pro Thr Arg Tyr 130 135 140 Leu Thr Ser Tyr Arg Lys Lys Thr Val Leu Gly Ala Asp Ser Gln Arg 145 150 155 160 Arg Ser Pro Ser Thr Gly Pro Trp Pro Cys Pro Gln Asp Pro Leu Gly 165 170 175 Ala Ala Arg Cys Val Val His Gly Ala Glu Phe Trp Ser Gln Tyr Arg 180 185 190 Ile Asn Val Thr Glu Val Asn Pro Leu Gly Ala Ser Thr Arg Leu Leu 195 200 205 Asp Val Ser Leu Gln Ser Ile Leu Arg Pro Asp Pro Pro Gln Gly Leu 210 215 220 Arg Val Glu Ser Val Pro Gly Tyr Pro Arg Arg Leu Arg Ala Ser Trp 225 230 235 240 Thr Tyr Pro Ala Ser Trp Pro Cys Gln Pro His Phe Leu Leu Lys Phe 245 250 255 Arg Leu Gln Tyr Arg Pro Ala Gln His Pro Ala Trp Ser Thr Val Glu 260 265 270 Pro Ala Gly Leu Glu Glu Val Ile Thr Asp Ala Val Ala Gly Leu Pro 275 280 285 His Ala Val Arg Val Ser Ala Arg Asp Phe Leu Asp Ala Gly Thr Trp 290 295 300 Ser Thr Trp Ser Pro Glu Ala Trp Gly Thr Pro Ser Thr Gly Thr Ile 305 310 315 320 Pro Lys Glu Ile Pro Ala Trp Gly Gln Leu His Thr Gln Pro Glu Val 325 330 335 Glu Pro Gln Val Asp Ser Pro Ala Pro Pro Arg Pro Ser Leu Gln Pro 340 345 350 His Pro Arg Leu Leu Asp His Arg Asp Ser Val Glu Gln Val Ala Val 355 360 365 Leu Ala Ser Leu Gly Ile Leu Ser Phe Leu Gly Leu Val Ala Gly Ala 370 375 380 Leu Ala Leu Gly Leu Trp Leu Arg Leu Arg Arg Gly Gly Lys Asp Gly 385 390 395 400 Ser Pro Lys Pro Gly Phe Leu Ala Ser Val Ile Pro Val Asp Arg Arg 405 410 415 Pro Gly Ala Pro Asn Leu 420 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> siRNA targeting IL-11 <400> 4 ccttccaaag ccagatctt 19 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> siRNA targeting IL-11 <400> 5 gcctgggcag gaacatata 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> siRNA targeting IL-11 <400> 6 cctgggcagg aacatatat 19 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> siRNA targeting IL-11 <400> 7 ggttcattat ggctgtgtt 19 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> siRNA targeting IL-11Rα <400> 8 ggaccatacc aaaggagat 19 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> siRNA targeting IL-11Rα <400> 9 gcgtctttgg gaatccttt 19 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> siRNA targeting IL-11Rα <400> 10 gcaggacagt agatccct 18 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> siRNA targeting IL-11Rα <400> 11 gctcaaggaa cgtgtgtaa 19 <210> 12 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> siRNA for IL-11 (NM_000641.3) <220> <221> misc_feature <222> (20)..(21) <223> n = deoxythymidine <220> <221> misc_feature <222> (41)..(42) <223> n = deoxythymidine <400> 12 ccuuccaaag ccagaucuun naagaucugg cuuuggaagg nn 42 <210> 13 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> siRNA for IL-11 (NM_000641.3) <220> <221> misc_feature <222> (20)..(21) <223> n = deoxythymidine <220> <221> misc_feature <222> (41)..(42) <223> n = deoxythymidine <400> 13 gccugggcag gaacauauan nuauauguuc cugcccaggc nn 42 <210> 14 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> siRNA for IL-11 (NM_000641.3) <220> <221> misc_feature <222> (20)..(21) <223> n = deoxythymidine <220> <221> misc_feature <222> (41)..(42) <223> n = deoxythymidine <400> 14 ccugggcagg aacauauaun nauauauguu ccugcccagg nn 42 <210> 15 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> siRNA for IL-11 (NM_000641.3) <220> <221> misc_feature <222> (20)..(21) <223> n = deoxythymidine <220> <221> misc_feature <222> (41)..(42) <223> n = deoxythymidine <400> 15 gguucauuau ggcuguguun naacacagcc auaaugaacc nn 42 <210> 16 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> siRNA for IL-11Rα (U32324.1) <220> <221> misc_feature <222> (20)..(21) <223> n = deoxythymidine <220> <221> misc_feature <222> (41)..(42) <223> n = deoxythymidine <400> 16 ggaccauacc aaaggagaun naucuccuuu gguauggucc nn 42 <210> 17 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> siRNA for IL-11Rα (U32324.1) <220> <221> misc_feature <222> (20)..(21) <223> n = deoxythymidine <220> <221> misc_feature <222> (41)..(42) <223> n = deoxythymidine <400> 17 gcgucuuugg gaauccuuun naaaggauuc ccaaagacgc nn 42 <210> 18 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> siRNA for IL-11Rα (U32324.1) <220> <221> misc_feature <222> (20)..(21) <223> n = deoxythymidine <220> <221> misc_feature <222> (41)..(42) <223> n = deoxythymidine <400> 18 gcaggacagu agaucccuan nuagggaucu acuguccugc nn 42 <210> 19 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> siRNA for IL-11Rα (U32324.1) <220> <221> misc_feature <222> (20)..(21) <223> n = deoxythymidine <220> <221> misc_feature <222> (41)..(42) <223> n = deoxythymidine <400> 19 gcucaaggaa cguguguaan nuuacacacg uuccuugagc nn 42 <210> 20 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> 20 amino acid linker <400> 20 Gly Pro Ala Gly Gln Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Val 20 <210> 21 <211> 516 <212> PRT <213> Artificial Sequence <220> <223> Hyper IL-11 (IL-11RA:IL-11 fusion) <400> 21 Met Ser Ser Ser Cys Ser Gly Leu Ser Arg Val Leu Val Ala Val Ala 1 5 10 15 Thr Ala Leu Val Ser Ala Ser Ser Pro Cys Pro Gln Ala Trp Gly Pro 20 25 30 Pro Gly Val Gln Tyr Gly Gln Pro Gly Arg Ser Val Lys Leu Cys Cys 35 40 45 Pro Gly Val Thr Ala Gly Asp Pro Val Ser Trp Phe Arg Asp Gly Glu 50 55 60 Pro Lys Leu Leu Gln Gly Pro Asp Ser Gly Leu Gly His Glu Leu Val 65 70 75 80 Leu Ala Gln Ala Asp Ser Thr Asp Glu Gly Thr Tyr Ile Cys Gln Thr 85 90 95 Leu Asp Gly Ala Leu Gly Gly Thr Val Thr Leu Gln Leu Gly Tyr Pro 100 105 110 Pro Ala Arg Pro Val Val Ser Cys Gln Ala Ala Asp Tyr Glu Asn Phe 115 120 125 Ser Cys Thr Trp Ser Pro Ser Gln Ile Ser Gly Leu Pro Thr Arg Tyr 130 135 140 Leu Thr Ser Tyr Arg Lys Lys Thr Val Leu Gly Ala Asp Ser Gln Arg 145 150 155 160 Arg Ser Pro Ser Thr Gly Pro Trp Pro Cys Pro Gln Asp Pro Leu Gly 165 170 175 Ala Ala Arg Cys Val Val His Gly Ala Glu Phe Trp Ser Gln Tyr Arg 180 185 190 Ile Asn Val Thr Glu Val Asn Pro Leu Gly Ala Ser Thr Arg Leu Leu 195 200 205 Asp Val Ser Leu Gln Ser Ile Leu Arg Pro Asp Pro Pro Gln Gly Leu 210 215 220 Arg Val Glu Ser Val Pro Gly Tyr Pro Arg Arg Leu Arg Ala Ser Trp 225 230 235 240 Thr Tyr Pro Ala Ser Trp Pro Cys Gln Pro His Phe Leu Leu Lys Phe 245 250 255 Arg Leu Gln Tyr Arg Pro Ala Gln His Pro Ala Trp Ser Thr Val Glu 260 265 270 Pro Ala Gly Leu Glu Glu Val Ile Thr Asp Ala Val Ala Gly Leu Pro 275 280 285 His Ala Val Arg Val Ser Ala Arg Asp Phe Leu Asp Ala Gly Thr Trp 290 295 300 Ser Thr Trp Ser Pro Glu Ala Trp Gly Thr Pro Ser Thr Gly Pro Ala 305 310 315 320 Gly Gln Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 325 330 335 Ser Val Pro Gly Pro Pro Pro Gly Pro Pro Arg Val Ser Pro Asp Pro 340 345 350 Arg Ala Glu Leu Asp Ser Thr Val Leu Leu Thr Arg Ser Leu Leu Ala 355 360 365 Asp Thr Arg Gln Leu Ala Ala Gln Leu Arg Asp Lys Phe Pro Ala Asp 370 375 380 Gly Asp His Asn Leu Asp Ser Leu Pro Thr Leu Ala Met Ser Ala Gly 385 390 395 400 Ala Leu Gly Ala Leu Gln Leu Pro Gly Val Leu Thr Arg Leu Arg Ala 405 410 415 Asp Leu Leu Ser Tyr Leu Arg His Val Gln Trp Leu Arg Arg Ala Gly 420 425 430 Gly Ser Ser Leu Lys Thr Leu Glu Pro Glu Leu Gly Thr Leu Gln Ala 435 440 445 Arg Leu Asp Arg Leu Leu Arg Arg Leu Gln Leu Leu Met Ser Arg Leu 450 455 460 Ala Leu Pro Gln Pro Pro Pro Asp Pro Pro Ala Pro Pro Leu Ala Pro 465 470 475 480 Pro Ser Ser Ala Trp Gly Gly Ile Arg Ala Ala His Ala Ile Leu Gly 485 490 495 Gly Leu His Leu Thr Leu Asp Trp Ala Val Arg Gly Leu Leu Leu Leu 500 505 510 Lys Thr Arg Leu 515 <210> twenty two <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> Il11 genotyping primer F <400> twenty two ggagggaggg gacgccaatg acc 23 <210> twenty three <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> l11 Genotyping Primer R <400> twenty three tctgcctccc ctgcctgttt ctcg 24 <210> twenty four <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Il11-luciferase genotyping primer F <400> twenty four aattccgtgg tgttgtcg 18 <210> 25 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Il11-luciferase genotyping primer R <400> 25 tctgcctccc ctgcctgttt ctcg 24 <210> 26 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Il11-EGFP genotyping primer F <400> 26 gaaatgagag cctagagtcc agag 24 <210> 27 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Il11-EGFP genotyping primer R <400> 27 gaggcttgga agaatgcaca atta 24 <210> 28 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Conserved motif <220> <221> misc_feature <222> (3) <223> Xaa can be any naturally occurring amino acid <400> 28 Trp Ser Xaa Trp Ser 1 5 <210> 29 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Knock-in site <400> 29 aacccctggc ctgtggggac atgaactgta agttggttcc agttcatggg tgag 54

[0426] BRIEF DESCRIPTION OF THE DRAWINGS

[0427] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying drawings.

[0428] Figures 1A to 1C Effects of IL-11 on hepatocytes. (1A) Primary human hepatocytes express IL-11Rα receptor. (1B) After IL-11 treatment, ALT levels and the number of actin stress fibers in the supernatant of hepatocytes increased in a dose-dependent manner (0.019-10 ng / ml). (1C) IL-11 expression in H2O2-induced hepatocytes.

[0429] Figures 2A to 2E Effects of anti-IL-11 therapy on APAP-induced liver toxicity in mice. IgG antibody was used as control. (2A) Schematic diagram showing the treatment schedule. (2B) Serum IL-11 levels after APAP-induced toxicity. (2C) ALT levels showing the extent of liver damage. (2D) Extent of APAP-induced liver weight loss. (2E) Hematoxylin and eosin (H&E) staining showing the extent of centrilobular necrosis in liver tissue of mice treated with anti-IL-11Rα antibody or IgG control.

[0430] Figure 3 .Dot plot showing that anti-IL11Ra antibody can prevent APAP-mediated hepatocyte death. Human hepatocytes were treated with APAP (20 mM) in the presence or absence of anti-IL11RA (X209, 2 μg / ml) or IgG control antibody. Cells were then stained with Annexin V and PI, and cell death was analyzed by flow cytometry. BL = baseline.

[0431] Figure 4 Western blot images show that anti-IL11Ra antibody blocks APAP-mediated Erk and Jnk activation. Human hepatocytes were treated with APAP (10 mM) in the presence or absence of anti-IL11RA (X209, 2 μg / ml) or IgG control antibody. Cell extracts were prepared and Western blots were performed to assess the activation (phosphorylation) status of Erk and Jnk kinases. BL = baseline.

[0432] Figure 5A and 5B .Box plots and images show that anti-IL11RA treatment given 16 hours before APAP overdose protects against acute liver injury. Mice were intraperitoneally administered 20 mg / kg of anti-IL11Ra antibody (ENx209) or IgG control antibody, and then given a severe overdose of APAP (400 mg / kg) 16 hours later. Mice were euthanized 24 hours later. (5A) Serum alanine aminotransferase (ALT) was measured as a marker of acute liver injury and hepatocellular death. (5B) Livers were harvested, fixed with 10% neutral buffered formalin, dehydrated, embedded in paraffin blocks, sectioned, and stained with hematoxylin and eosin to observe the characteristic centrilobular hepatocellular necrosis seen in APAP overdose.

[0433] Fig. 6A and 6B Images and boxplots show acute liver injury in mice treated with anti-IL11RA 10 h after APAP overdose. Mice were given a severe overdose of APAP (400 mg / kg) and 10 h later were intraperitoneally administered with 20 mg / kg of anti-IL11RA antibody (ENx209) or IgG control antibody. (6A) Livers were harvested at the indicated time points and fixed in 10% neutral buffered formalin, and gross morphology and appearance were recorded. (6B) Serum alanine aminotransferase (ALT) was measured as a marker of acute liver damage and hepatocyte death.

[0434] Figure 7Western blot images show that anti-IL11RA treatment 10 hours after APAP overdose inhibits Jnk and ERK activation. Mice were given a severe overdose of APAP (400 mg / kg) and 10 hours later, 20 mg / kg of anti-IL11RA antibody (ENx209) or IgG control antibody was intraperitoneally administered. Livers were harvested at the indicated time points and Western blots were performed to assess the activation (phosphorylation) status of Erk and Jnk kinases.

[0435] Figures 8A to 8C .Graphs, images, and boxplots show that anti-IL11RA treatment 10 h after APAP overdose prevents mortality from acute liver injury and restores liver function. Mice were given a lethal overdose of APAP (550 mg / kg) and 10 h later were intraperitoneally administered 20 mg / kg of anti-IL11RA antibody (ENx209) or IgG control antibody. (8A) Graphs show mortality within 8 days after overdose in both treatment groups. (8B) Livers were harvested at the indicated time points and fixed in 10% neutral buffered formalin, and gross morphology and appearance were recorded. (8C) Serum alanine aminotransferase (ALT) was measured in ENx209-treated mice as a marker of liver damage and hepatocellular death 8 days after overdose and compared with normal controls.

[0436] Figures 9A to 9J .Graphs and images show that acetaminophen-induced IL11 secretion from injured hepatocytes leads to cell death. (A) Serum IL11 levels in APAP-treated mice. (B) Hepatic IL11 mRNA after APAP injury. (C) Representative images of luciferase activity in the livers of control and APAP-challenged IL11-luciferase mice. (D) Western blotting showing IL11 expression in the livers of APAP-treated mice. (E) Representative immunofluorescence images of EGFP and cleaved Caspase3 (Cl.CASP3) expression in liver tissues of IL11-EGFP mice after APAP treatment (scale bar, 100 μm). (AE) APAP, 400 mg kg -1 (F) ELISA detection of IL11 secretion from APAP-stimulated hepatocytes. (G) Western blotting of phosphorylated ERK, JNK and CI. CASP3 protein and its corresponding total expression in hepatocytes in response to rhIL11 stimulation. (H) Propidium iodide-positive (PI) +VE ) Cell quantification. (I) Western blotting showing ERK, JNK and CASP3 activation status and (J) in the presence of IgG or anti-IL11RA (x209; 2 μg ml -1 ) in the presence of APAP-treated hepatocytes (20 mM). +VEQuantification of cells. (FJ) Primary human hepatocytes (F, HJ) 24h. (A, B, F, H I) Data are shown as whisker plots of median (center line), 25-75 percentiles (boxes), and minimum-maximum values ​​(whiskers). (A, B) Two-tailed Student's t-test; (F, H) Two-tailed Dunnett's test; (J) Two-tailed, Tukey-corrected Student's t-test.

[0437] Figures 10A to 10L Images, graphs, and diagrams show that recombinant human IL11 inhibits the effects of mouse IL11 in mouse hepatocytes. (A) Recombinant human IL11 (rhIL11, 10 ng ml -1 or recombinant mouse IL11 (rmIL11, 10 ng ml -1 ) on the activation status of ERK, JNK and CASP3 in mouse hepatocytes. (B) rmIL11 (10 ng ml -1 ) or by increasing the dose of rhIL11 (1, 5, 10, 15 and 20 ng ml -1 ) stimulation. (C) Mice received a single subcutaneous injection of saline, rhIL11, or rmIl11 (500 μg kg -1 ) Schematic diagram of (D) Western blot analysis of liver p-ERK, p-JNK and Cl.CASP3 and (E) Fig. 10CSerum ALT levels for the experiments shown; for each time period (6h, 24h), the boxes from left to right correspond to saline, rmIL11, and rhIL11 treatment, respectively. (F) Schematic representation of mice injected subcutaneously with saline, rhIL11, or rmIL11 2h before APAP OD. Effects of rhIL11 or rmIL11 injection before APAP OD on (G) 6 and 24h serum ALT measurements (for each time period (6h, 24h), the boxes from left to right correspond to saline, rmIL11, and rhIL11 treatment, respectively) and (H) ERK and JNK activities in the liver 24h after APAP administration. (I) Sensorgrams showing the binding of mIL11RA1 to immobilized rhIL11 (left) and rmIL11 (center), and the binding of hIL11RA to rhIL11 (right). Experimental data and theoretical fitting curves (1:1 Langmuir) are shown. (J) Binding of biotinylated rmIL11 to mIL11RA1 in the presence of two-fold dilutions of rmIL11 (dark gray dots, upper line) and rhIL11 (light gray dots, lower line) by competition ELISA. Dose-dependent inhibitory effect of rhIL11 on rmIL11-induced (K) ALT secretion and (L) CASP3 activation by mouse hepatocytes. (A, B, K, L) 24 h. (B, K) Data are shown as mean ± SD; (E, G) Data are shown as whisker plots of median (center line), 25th-75th percentiles (boxes), and minimum-maximum values ​​(whiskers). (B, K) Two-tailed, Tukey-corrected Student's t-test; (E) Two-tailed Student's t-test; (G) Two-tailed Dunnett's test. FC: Fold change

[0438] Figures 11A to 11Q Schematic diagrams, images, and figures show that IL11 induces liver failure through NOX4-dependent glutathione depletion. (A) ROSA26 IL11 / +Schematic diagram of mice injected with a single intravenous injection of AAV8-ALB-Null (control) or AAV8-ALB-Cre (IL11-Tg) to specifically induce IL11 overexpression in albumin-expressing cells (hepatocytes); ALB: albumin. (B) Representative gross anatomy of the liver, (C) liver weight, (D) serum ALT level, (E) representative H&E-stained liver images (scale bar, 100 μm), (F) Western blot for p-ERK, p-JNK, and CL.CASP3, (G) liver GSH levels, and (H) NOX4 mRNA expression levels 3 weeks after injection in control and IL11-Tg mice. (I) Time course of GSH levels, (J) dose-dependent decrease in GSH levels, and (K) Western blot showing increased NOX4 protein expression in rhIL11-treated primary human hepatocytes. (L) Western blot of NOX4 in mouse hepatocytes stimulated with rhIL11 or rmIL11. (M) Western blot of NOX4 expression and (N) GSH levels in APAP-stimulated human hepatocytes (20 mM) treated with IgG and X209. (O) Dose-dependent inhibitory effects of GKT-13781 on GSH levels and CASP3 activation in rhIL11-stimulated human hepatocytes. Effects of siNOX4 on rhIL11-induced (P) ERK, JNK, and CASP3 activation and (Q) GSH depletion levels in human hepatocytes. (IQ) rhIL11 / rmIL11 (10 ngml -1 Unless otherwise stated), APAP (20 mM), IgG / x209 (2 μg ml -1 ), siNT (non-targeting siRNA control) / siNOX4 (50nM). (IK, MQ) Primary human hepatocytes (L) Primary mouse hepatocytes. (J, LQ) 24h. (CD, GJ, N, O, Q) Data are shown as whisker plots of median (center line), 25-75 percentiles (boxes) and minimum-maximum values ​​(whiskers). (CD, GH) Two-tailed Student's t-test; (IJ) Two-tailed Dunnett's test; (N, O, Q) Two-tailed, Tukeyx-corrected Student's t-test.

[0439] Figures 12A to 12H Schematic diagrams, images, and figures show that hepatocyte-specific loss of IL11RA1 protects mice from APAP-induced liver damage. (A) IL11RA1 loxP / loxP Schematic diagram of APAP injury induction in mice. loxP / loxPMice were intravenously injected with AAV8-ALB-Null (control group) or AAV8-ALB-Cre (CKO) to specifically delete IL11RA1 in hepatocytes. Three weeks after viral administration, control and CKO mice were fasted overnight and injected with APAP (400 mg kg -1 ) or saline. ALB: albumin. (B) Representative liver gross anatomy and (C) H&E images from saline- and APAP-injected control and CKO mice (scale bar, 500 μm). (D) Serum ALT levels, (E) serum AST levels, (F) liver GSH levels, (G) protein immunoblots of IL11RA, p-ERK, ERK, p-JNK, JNK, Cl.CASP3, CASP3, and GAPDH, and (H) relative liver mRNA expression levels of proinflammatory genes. (DF, H) Data are presented as whisker plots of median (midline), 25-75 percentiles (boxes), and minimum-maximum values ​​(whiskers); Student's t-test with Sidak correction.

[0440] Figures 13A to 13L Schematic diagrams, images, and figures show the treatment of APAP-induced liver damage with anti-IL11Ra antibody and / or NAC. (A) Schematic diagram of the preventive administration of anti-IL11RA (X209) to APAP OD mice; X209 or IgG (10 mg kg -1 ) at the beginning of the fasting period, i.e. APAP (400 mg kg -1 ) were administered 16 h before injection; control mice received saline injection. (B) Serum ALT levels, (C) Fig.13A Representative H&E images (bar, 500 μm) and liver GSH levels of the experiments shown. (E) Schematic diagram of the anti-IL11RA (X209) dose-finding experiment; X209 (2.5-10 mg kg -1 ) or IgG (10 mg kg-1) was administered to mice 3 h after APAP injection. (F) Serum ALT levels (the values ​​for saline were the same as those used in 5B), (G) liver GSH levels, and (H) Western blots for liver ERK and JNK activation, as Fig.13E Experiment shown. (I) Schematic diagram showing the reaction of X209 and N-acetylcysteine ​​(NAC, 500 mg kg -1 ) alone or in combination with X209 (5 mg kg -1). Overnight fasted mice were treated with IgG, NAC, or NAC+X209 3 h after APAP injection, and the data are shown in (JL). Effects of NAC, NAC+X209 treatment on (H) serum ALT levels, (I) liver GSH levels, and (J) p-ERK, p-JNK, and Cl.CASP3 expression levels. (B, C, F, G, J, K) Data are presented as whisker plots of median (center line), 25-75 percentiles (boxes), and minimum-maximum values ​​(whiskers); two-tailed, Tukey-corrected Student's t-test.

[0441] Figures 14A to 14K Schematic diagrams, images and figures showing liver regeneration and reversal of liver failure with late anti-IL11RA treatment. (A) Schematic diagram of late treatment doses in APAP-injured mice. IgG / X209 (20 mg / kg) was administered to mice fasted overnight 10 h after APAP. -1 ). (B) Representative gross anatomy of the liver, (C) Representative H&E-stained liver images (bar, 500 μm), (D) Serum Il11 levels, (E) Serum ALT levels, (F) Western immunoblotting of p-ERK, p-JNK, Cl.CASP3, PCNA, Cyclin D1 / D3 / E1, and p-RB, (G) Representative EdU-stained liver images (bar, 100 μm) from APAP mice that received a late X209 dose (10 h after APAP), as shown. Fig.14A (H) Western blotting shows the expression levels of PCNA, Cyclin D1 / D3 / E1, and p-RB proteins in the liver of APAP mice treated with NAC or NAC+X209 ( Figure 13G (I) At lethal APAP OD (550 mg / kg -1 ) 10 h after mice were treated with X209 (20 mg kg -1 ) Schematic diagram of treatment, and data are shown in (JK). (J) Survival curves of mice treated with IgG or X209 10 h after lethal APAP OD. (K) Gross liver dissection of control (D8), IgG (24 h), and X209-treated mice (D8). (D,E) Data are mean ± SD; 2-way ANOVA; (J) Gehan-Breslow-Wilcoxon test.

[0442] Fig.15 . Schematic diagram related to the generation of Il11-luciferase knock-in mice. Strategy for knocking in Kozak-Luciferase-WPRE-polyA located in exon 1 of IL11 using CRISPR / Cas9. Woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE).

[0443] Fig.16 . Schematic diagram related to the generation of IL11-EGFP knock-in mice. Strategy for knocking in the A-EGFP cassette into exon 5 of the IL11 gene, replacing the TGA stop codon resulting in the translation of the Il11-2A-EGFP protein. The 2A linker is cleaved, resulting in the retention of EGFP in cells that express and secrete IL11.

[0444] Fig.17A and 17B Scatter plots showing the hepatotoxic effects of IL11. (A) Increased rhIL11 and (B) the presence of IgG or X209 (2 μg ml -1 Representative flow cytometry forward scatter plots of propidium iodide (PI) staining of APAP-stimulated human primary hepatocytes at 4 ℃ and 8 ℃.

[0445] Figures 18A to 18F Images, graphs, and tables show species-specific effects of human or mouse IL11 on human or mouse hepatocytes. (A) Recombinant human IL11 (rhIL11, 10 ng ml -1 or recombinant mouse IL11 (rmIL11, 10 ng ml -1 ) on the activation status of ERK, JNK and CASP3 in human hepatocytes. (B) rhIL11 (10 ng ml -1 ) or increasing doses of rmIL11 (1, 5, 10, 15, and 20 ng ml -1 ) ALT levels in the supernatant of human hepatocytes stimulated with rhIL11 and rmIL11 alone ( Fig. 10C ) or (D) APAP administration ( Fig.10F ) on serum AST levels in mice; for each time period (6 h, 24 h), the boxes from left to right correspond to saline, rmIL11, and rhIL11 treatment, respectively. (E) Binding affinity and kinetic constants of mouse IL11RA interaction with mouse IL11 or human IL11 and human IL11 interaction with human IL11. (F) Western blotting shows the effect of rhIL11 on rmIL11 (10 ng ml -1 , 24h) stimulated mouse hepatocytes p-ERK, ERK, p-JNK, JNK inhibition in a dose-dependent manner, (B) Data are shown as mean ± SD; (C, D) Data are shown as whisker plots of median (center line), 25-75 percentiles (boxes) and minimum-maximum values ​​(whiskers). (B) Two-tailed, Tukey-corrected Student's t-test; (C) Two-tailed Student's t-test; (D) Two-tailed Dunnett's test. FC: fold change.

[0446] Figures 19A to 19E.Graphs and images show that hepatocyte-specific overexpression of IL11 leads to liver necroinflammation. Control and IL11-Tg mice ( Fig.11A ) of (A) heart, lung, and kidney weights (B) serum AST levels, (C) quantification of portal vein diameter, (D) total ERK, total JNK, and CASP3 protein immunoblots, and (E) relative liver mRNA expression levels of proinflammatory markers. (AC, E) Data are presented as whisker plots of median (midline), 25-75 percentiles (boxes), and minimum-maximum values ​​(whiskers); two-tailed Student's t-test.

[0447] Figures 20A to 20D Images and graphs show that only species-specific IL11 induces NOX4 and glutathione depletion in hepatocytes. rhIL11 and rmIL11 (10 ng ml -1 ) on (A) NOX4 protein expression, (B) GSH level in human hepatocytes, and (C) GSH level in mouse hepatocytes. (D) Fig. 10C ) Hepatic GSH levels after rhIL11 or rmIL11; for each time period (6h, 24h), the boxes from left to right correspond to saline, rmIL11 and rhIL11 treatment, respectively. (BD) Data are presented as whisker plots of median (center line), 25-75 percentiles (boxes) and minimum-maximum values ​​(whiskers); two-tailed Dunnett's test.

[0448] Fig.21A and 21B .Graphs show that recombinant human IL11 (rhIL11) restores GSH levels in damaged mouse liver. (A) Dose-dependent inhibitory effect of rhIL11 on GSH levels in mouse primary hepatocytes stimulated by rmIL11; two-tailed, Tukey-corrected Student's t-test. (B) Effects of rhIL11 or rmIL11 on GSH levels in mouse liver after APAP injury, as shown in Fig.10F Two-tailed Dunnett's test. For each time period (6h, 24h), the boxes from left to right correspond to saline, rmIL11, and rhIL11 treatment, respectively. (A, B) Data are shown as whisker plots of median (center line), 25-75 percentiles (boxes), and minimum-maximum values ​​(whiskers).

[0449] Fig.22A and 22B Images and figures show that the NOX4 inhibitor GKT-137831 prevents the hepatotoxic effects of IL11. NOX4 inhibitor GKT-137831 has an effect on (A) ERK and JNK activation and (B) rhIL11 (10 ng ml -1Dose-dependent inhibition of ALT secretion in human hepatocytes stimulated with 5% paraformaldehyde (PNA) for 24 h. (B) Data are shown as whisker plots of median (centerline), 25th-75th percentiles (boxes), and minimum-maximum values ​​(whiskers); two-tailed, Tukey-corrected Student's t-test. FC: fold change.

[0450] Fig.23A and 23B Images and figures show that NOX4 is essential for the hepatotoxic effect of IL11. (A) Western blotting shows the knockdown efficiency of siNOX4. (B) Effects of siNOX4 on rhIL11-induced primary human hepatocyte cell death and ALT release. (AB) rhIL11 (10 ng ml -1 ), siNT (non-targeting siRNA control) / siNOX4 (50 nM); 24 h; data are shown as whisker plots of median (center line), 25-75 percentiles (boxes) and min-max values ​​(whiskers); two-tailed, Tukey-corrected Student's t-test. FC: fold change.

[0451] Fig.24A and 24B .Graphs show that control and CKO mice have similar APAP and APAP-glutathione serum levels 24 h after APAP administration. LC-MS / MS quantification of (A) APAP and (B) APAP-glutathione in serum of control and CKO mice. Data are shown as whisker plots of median (center line), 25-75 percentiles (boxes), and minimum-maximum values ​​(whiskers); two-tailed Student's t-test.

[0452] Figures 25A to 25C .Graphs show that anti-IL11Ra antibody (X209) reduces serum AST after APAP injury. (A) 16 h before APAP ( Fig.13A ), received a preventive dose of X209 (10 mg kg -1 ) Serum AST levels in saline and APAP mice. (B) Dose-dependent effects of X209 on serum AST levels in APAP mice receiving a therapeutic dose of X209 at 3 h after APAP administration ( Fig.13D , the values ​​of saline were the same as those used in S11A). (C) 3 h after APAP injury, NAC (500 mg / kg -1 ) or with X209 (5 mg kg -1 ) Serum AST levels in mice treated with the combination of Figure 13G ). (AC) Data are shown as whisker plots of median (center line), 25-75 percentiles (boxes), and minimum-maximum values ​​(whiskers); two-tailed, Tukey-corrected Student's t-test.

[0453] Fig.26A and 26B .Graphs show APAP and APAP-glutathione levels in mouse serum 24 h after APAP OD. LC-MS / MS quantification of (A) APAP and (B) APAP-glutathione in saline control mice, and IgG and X209 treated mice 24 h after APAP administration. Data are shown as whisker plots of median (center line), 25-75 percentiles (boxes), and minimum-maximum values ​​(whiskers); two-tailed, Student's t-test with Tukey correction.

[0454] Fig.27A and 27B .Graphs and images show that X209 reversed APAP-induced liver damage. (A) Serum AST levels and (B) Western blot showed the total ERK, JNK, CASP3 and RB levels in the liver of mice in the reversal experimental group. Fig.14A shown.

[0455] Fig.28A and 28B Images and graphs show the recovery of X209-treated mice after administration of a lethal dose of APAP. (A) Representative H&E images (bar, 500 μm) of livers from IgG (24 h after APAP) and X209-treated mice (day 8 after APAP). (B) Serum ALT levels in saline control and X209-treated mice (day 8 after APAP).

[0456] Fig.29 Schematic representation of the proposed mechanism and role of IL11 in APAP-induced hepatotoxicity. APAP is metabolized intrahepatically to generate ROS via NAPQI, which triggers IL11 secretion. The autocrine IL11 signaling loop on hepatocytes and continued generation of ROS via NOX4 drives continued cell death and limits liver regeneration, independent of APAP and its metabolites. If the IL11 pathway is blocked genetically or therapeutically, hepatocyte death can be prevented and liver regeneration can be restored.

[0457] Fig. 30A and 30B .Box plots show that anti-IL11 treatment given 16 h before APAP overdose protects against acute liver injury. Mice were intraperitoneally injected with 20 mg / kg anti-IL11 antibody (ENx203) or IgG control antibody and then given a severe overdose of APAP (400 mg / kg) 16 h later. Mice were euthanized 24 h later. (30A) Serum alanine aminotransferase (ALT) and (30B) aspartate aminotransferase (AST) were measured as correlates of acute liver injury and hepatocyte death.

[0458] Figures 31A to 31K . Scatter plots, box plots, histograms, and images related to IL-11 and IL-6 receptor expression and IL-11 and IL-6 signaling. (31A) Representative flow cytometry forward scatter (FSC) and fluorescence intensity plots of hepatocyte IL11RA, IL6R, and gp130 staining. (31B) Abundance of IL11RA1 and IL6R in hepatocytes at baseline readout based on RNA-seq (left) and Ribo-seq (right) (transcripts per million, TPM). (31C and 31D) (31C) IL11RA1 and (31D) IL6R transcript coverage based on human hepatocyte RNA-seq and Ribo-seq (n=3). (31E and 31F) (31E) Western blots showing ERK, JNK, and STAT3 activation status and (31F) ALT secretion by hepatocytes after hyperIL11 or hyperIL6 stimulation over a dose range. (31G) ALT levels in supernatants of hepatocytes stimulated with hyperIL11 alone or in the presence of increasing amounts of soluble gp130 (sgp130). (31H and 31I) Western blots of hepatocyte lysates showing (31H) phosphorylated ERK and JNK and their respective total expression in response to hyperIL11 stimulation alone or with sgp130, and (31I) phosphorylated STAT3 and total STAT3 in response to hyperIL6 with or without sgp130 stimulation. (31J) Representative FSC plots of propidium iodide (PI) staining of IL11-stimulated hepatocytes in the presence of sgp130 or soluble IL11RA (sIL11RA). (31K) Western blots 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 sILRA. (31A-31K) Primary human hepatocytes; (31E-31K) 24 hours stimulation; (31E-31K) hyperIL11, hyperIL6, IL11 (20 ng / ml), sgp130, sIL11RA (1 μg / ml). (31B, 31F-31G) Data are shown as whisker plots of median (center line), 25-75 percentiles (boxes), and minimum-maximum values ​​(whiskers).

[0459] Figures 32A to 32H. Scatter plots, box plots, histograms and images and graphs related to IL-11 and IL-6 receptor expression and IL-11 and IL-6 signaling. (32A) Representative FSC plots of IL11RA, IL6R and gp130 staining of activated THP-1 cells. (32B) gp130 transcripts in primary human hepatocytes based on RNA-seq and Ribo-seq (transcripts per million, TPM). (32C) gp130 transcript read coverage based on RNA-seq and Ribo-seq in primary human hepatocytes (n=3). (32D) Immunofluorescence images of IL11RA, IL6R, gp130 and albumin expression in primary human hepatocytes and activated THP-1 cells (scale bar, 100 μm). (32E) Baseline levels of soluble IL6R in hepatocytes. (32F) Quantification of PI staining of primary human hepatocytes (PI+VE cells) stimulated with IL11 in the presence of sgp130 or sIL11RA. (32G) Dose-dependent effects of increasing IL11 concentrations on ALT secretion levels from primary human hepatocytes in the presence of 1 μg / ml sgp130 or sIL11RA. (32H) Dose-dependent effects of increasing sgp130 or sIL11RA concentrations on IL11-induced ALT secretion. (32B, 32G-32H) Data are shown as whisker plots of median (midline), 25-75 percentiles (boxes), and minimum-maximum values ​​(whiskers); (32E-32F) Data are shown as mean ± SEM; (32F-32H) Student's t-test with Tukey correction. Example

[0460] In the following examples, the inventors demonstrate that IL-11 directly impairs hepatocyte survival and that anti-IL-11 therapy can ameliorate hepatotoxicity. The inventors demonstrate the ability of IL-11 antagonists administered before DILI to protect hepatocyte death and liver function, and also show that IL-11 antagonists administered after DILI can reverse liver damage symptoms and restore liver function.

[0461] Example 1: Effects of IL-11 on hepatocytes

[0462] To explore the effect of IL-11 on hepatocytes, primary human hepatocytes were used for cell culture experiments.

[0463] Human hepatocytes (5200, ScienCell) were grown and maintained at 37°C and 5% CO2. Hepatocyte medium (5201, ScienCell) supplemented with 2% fetal bovine serum and 1% penicillin-streptomycin was refreshed every 2-3 days, and cells were passaged at 80% confluence using a standard trypsin method. All experiments were performed at low cell passages (P2-P3), and cells were serum starved for 16 hours before the corresponding stimulation (24 hours) in serum-free hepatocyte medium. Stimulated cells were compared with unstimulated cells, which were grown under the same conditions for the same time (serum-free hepatocyte medium) but were not stimulated.

[0464] Immunofluorescence staining was used to detect the expression of IL-11Rα in human hepatocytes. Human hepatocytes were seeded on 8-well chamber slides (1.5×104 cells per well) 24 hours before staining. Cells were fixed in 4% PFA for 20 minutes, washed with PBS, and nonspecific sites were blocked with blocking buffer (PBS containing 5% BSA) for 2 hours. Cells were incubated with anti-IL-11Rα antibody [EPR5446] (ab125015, Abcam, 1:100) overnight (4C) and then incubated with goat anti-rabbit IgG H&L (Alexa Fluor488) (ab150077, Abcam, 1:200) for 1 hour. The slide chambers were dried in the dark, and 5 drops of mounting medium with DAPI were added to the slides for 15 minutes before imaging with a fluorescence microscope (Leica). Negative control samples were treated in the same way except for the anti-IL-11Rα antibody incubation step.

[0465] IL-11-mediated hepatocyte death was detected by measuring the level of alanine aminotransferase (ALT) in the supernatant of hepatocytes after treatment with a dose range of IL-11 (0.019-10 ng / ml). ALT levels were determined using an ALT activity assay kit (ab105134, Abcam) according to the manufacturer's procedures. Rhodamine-phalloidin staining was used to detect the number of concurrent stress fibers 24 h after IL-11 stimulation.

[0466] The effects of reactive oxygen species (ROS; stimulated by hydrogen peroxide (0.2 mM H2O2, 24 h, 31642, Sigma)) on primary human hepatocytes were studied.

[0467] The results are as follows Figures 1A to 1C As shown. IL-11 was found to directly impair hepatocyte survival.

[0468] Primary human hepatocytes were found to highly express the IL-11Rα receptor (1A). IL-11 stimulation was found to induce dose-dependent hepatocyte cell death, as evidenced by a gradual increase in alanine aminotransferase (ALT) over a physiologically relevant dose range (1B). Furthermore, over the dose range, IL-11 gradually stimulated an increase in actin stress fibers in hepatocytes (1B; micrographs from different dose ranges), which reflects a partial epithelial to mesenchymal transition of hepatocytes known to contribute to hepatocyte dysfunction (Grant Rowe et al. Mol Cell Biol 2011;31(12):2392–2403).

[0469] Because APAP is known to induce ROS-dependent liver injury, we stimulated human hepatocytes with H2O2 and found that IL-11 was upregulated 10-fold in the supernatant (1C). Thus, IL-11 directly causes hepatocyte death and drives hepatocytes into a dysfunctional partial epithelial-mesenchymal transition (EMT) state, which is known to limit the regenerative capacity of the liver (Grant Rowe et al., supra).

[0470] Example 2: Effect of anti-IL-11 therapy on hepatotoxicity

[0471] The acetaminophen (APAP)-induced mouse liver injury model was used to investigate the effect of anti-IL-11 therapy on hepatotoxicity.

[0472] The 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 ad libitum, except during starvation periods.

[0473] Briefly, 12-14 week old male mice were starved and injected intraperitoneally (IP) with anti-IL-11Rα antibody 10 mg / kg or IgG isotype control 16 hours before APAP (A3035, Sigma) (ip, 400 mg / kg). Mice were sacrificed 24 hours after APAP administration.

[0474] The levels of IL-11 in mouse serum and hepatocyte supernatant were quantified using mouse IL-11 DuoSet (DY418 and DY008, R&D Systems) and human IL-11 Quantikine ELISA kit (D1100, R&D Systems), respectively, according to the manufacturer's instructions.

[0475] Liver samples were fixed in 10% neutral buffered formalin (NBF) at room temperature for 48 h, dehydrated, embedded in paraffin blocks, and sliced ​​at 7 μm. The slices were stained with hematoxylin and eosin (H&E) according to standard methods and examined under an optical microscope.

[0476] The results are as follows Figures 2A to 2E shown. Figure 2A A schematic diagram of the treatment regimen is shown.

[0477] As shown in the figure above, serum IL-11 was found to be significantly elevated after APAP toxicity (2B), mean ± SD, control, n = 2; APAP, n = 3. Mice treated with a single dose of anti-IL-11Rα antibody were found to have significantly reduced ALT levels (55% reduction compared to the IgG control group; 2C), which significantly reduced the extent of liver damage. Anti-IL-11 treatment was also found to prevent APAP-induced liver mass loss, which reflects the destruction of hepatocytes, with a liver mass loss of 24% compared to the IgG control antibody (liver index; 2D). (E) H&E staining of liver histology showed severe centrilobular necrosis in IgG-treated mice, which is a typical histological feature of APAP toxicity and was found to be alleviated by anti-IL11Rα treatment.

[0478] The mobility and activity of IgG control or anti-IL-11Rα antibody treated mice were observed 24 hours after APAP treatment. The control IgG treated mice were found to be immobile / near death with obvious signs of poor health (such as erect hair and hunched posture), while the mobility and activity of anti-IL-11Rα antibody treated mice were normal.

[0479] Therefore, inhibition of IL-11 signaling by blocking IL-11Rα can prevent hepatotoxicity in an acceptable translational model of APAP-induced liver injury (DILI).

[0480] Example 3: Antagonism of IL-11-mediated signaling protects hepatocytes from drug-induced cell death In vitro analysis of the effects of IL-11-mediated signaling antagonism on hepatocyte viability

[0481] Human hepatocytes (5200, ScienCell) were cultured at 37°C and 5% CO2 in a hepatocyte culture medium (5201, ScienCell) supplemented with 2% fetal bovine serum and 1% penicillin-streptomycin. The culture medium was replaced every 2-3 days, and the cells were passaged at 80% confluence using a standard trypsin method. All experiments were performed at low cell passages (P2-P3). Cells were cultured in serum-free hepatocyte culture medium before being used in experiments, and serum starved cells for 16 hours.

[0482] In the first experiment, hepatocytes were treated with APAP (A3035, Sigma) at a final concentration of 20 mM for 24 h in the absence (baseline, BL) or presence of antagonist anti-IL11Ra antibody (X209, 2 μg / ml) or isotype-matched IgG control antibody (IgG, 2 μg / ml).

[0483] Hepatocytes were then stained with the FITC Annexin V / Dead Cell Apoptosis Kit (V13242, Thermo Fisher Scientific) according to the manufacturer's instructions, and Annexin V-FITC / PI-stained cells were analyzed by flow cytometry using a BD LSRFortessa flow cytometer (BD Biosciences). 10,000 cells were analyzed for each sample. Data were analyzed using FlowJo (version 7) software.

[0484] The results are as follows Figure 3 It was found that treatment of hepatocytes with an antibody inhibitor, an antagonist of IL-11-mediated signaling, significantly reduced the proportion of dead hepatocytes.

[0485] In a separate experiment, hepatocytes were treated with APAP (A3035, Sigma) at a final concentration of 10 mM for 24 h in the absence (baseline, BL) or presence of antagonist anti-IL11Ra antibody (X209, 2 μg / ml) or isotype-matched IgG control antibody (IgG, 2 μg / ml).

[0486] Protein extracts were prepared from hepatocytes using radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific), and the lysates were clarified by centrifugation. Protein concentrations were determined using the Bradford assay (Bio-Rad). Equal amounts of protein lysates were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting with the indicated primary antibodies (ERK, pERK, pJNK). Proteins were visualized using the ECL detection system (Pierce) and appropriate secondary antibodies.

[0487] The results are as follows Figure 4 As shown. It was found that APAP treatment of hepatocytes significantly increased p-ERK and pJNK levels (comparing BL with IgG). It was found that treatment of hepatocytes with an antagonist antibody inhibitor of IL-11-mediated signaling significantly reduced p-ERK and pJNK levels (comparing IgG with X209).

[0488] Example 4: Antagonizing IL-11-mediated signal transduction to protect against drug-induced liver injury

[0489] Male mice aged 12 to 14 weeks were intraperitoneally injected with 20 mg / kg antagonist anti-IL11Ra antibody (X209) or isotype-matched IgG control antibody, and then injected intraperitoneally with a severe overdose of APAP (400 mg / kg) or an equal volume of saline 16 hours later.

[0490] 24 hours after APAP administration, mice were euthanized. Serum ALT levels were determined using an ALT activity assay kit (ab105134, Abcam) according to the manufacturer's instructions, and livers were harvested, fixed in 10% neutral buffered formalin (NBF) at room temperature for 48 h, dehydrated, embedded in paraffin blocks, and sliced ​​at 7 μm. The sections were stained with hematoxylin and eosin (H&E) according to standard methods and examined under an optical microscope.

[0491] The results are as follows Figure 5A and 5B Pretreatment with anti-IL-11Rα antibody inhibitor, an antagonist of IL-11-mediated signaling, was shown to significantly protect mice from DILI-associated liver function suppression, as determined by a significant decrease in serum ALT levels ( Figure 5A The livers of mice pretreated with an antibody inhibitor, an antagonist of IL-11-mediated signaling, also showed significantly less hepatocellular necrosis compared with livers of IgG-treated controls ( Figure 5B ).

[0492] In further experiments, 12-14 week old male mice were given a severe overdose of APAP (400 mg / kg) by intraperitoneal injection 16 hours after 20 mg / kg of antagonist anti-IL11 antibody (X203) or isotype-matched IgG control antibody.

[0493] 24 h after APAP administration, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were measured using ALT activity (ab105134, Abcam) and AST (ab105135, Abcam) kits according to the manufacturer's method.

[0494] The results are as follows Fig. 30A and 30B Pretreatment with an anti-IL-11 antibody inhibitor, an antagonist of IL-11-mediated signaling, showed significant protection of mice from DILI-associated liver function suppression, as evidenced by significant reductions in serum ALT and AST levels.

[0495] Example 5: Antagonism of IL-11-mediated signaling reverses liver damage symptoms and restores liver function after drug-induced liver injury able

[0496] Male mice aged 12-14 weeks were intraperitoneally injected with a severe overdose of APAP (400 mg / kg) or an equal volume of saline, and 10 hours later were intraperitoneally injected with 20 mg / kg of antagonist anti-IL11Ra antibody (X209), isotype-matched IgG control antibody, or left untreated.

[0497] Mice were euthanized at 24, 36, and 48 h. Serum ALT levels were analyzed as described in Example 4. Livers were harvested and fixed as described in Example 4, and digital photographs were taken.

[0498] The results are as follows Fig. 6A and 6B As shown. When an IL-11-mediated signaling antagonist antibody inhibitor was administered 10 hours after severe APAP overdose, the gross morphology of the liver of mice was restored to the state when the mice were not treated with APAP ( Fig. 6A ). Administration of an antagonistic antibody inhibitor of IL-11-mediated signaling 10 h after severe APAP overdose was further demonstrated to protect mice from DILI-associated liver function suppression, as determined by a significant decrease in serum ALT levels ( Figure 6B ).

[0499] Western immunoblotting was also performed on protein extracts prepared from mouse liver. Liver tissue was homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific), and the lysates were subsequently separated by SDS-PAGE and analyzed for protein as described in Example 3.

[0500] The results are as follows Figure 7 As shown. APAP overdose significantly upregulated p-ERK, pJNK1, and pJNK2 levels (comparing control with 10h). Subsequent treatment with IL-11-mediated signaling antagonist antibody inhibitors significantly reduced p-ERK, pJNK1, and pJNK2 levels (comparing IgG with X209).

[0501] In further experiments, 12-14 week old male mice were intraperitoneally injected with a lethal overdose of APAP (550 mg / kg) or an equal volume of saline, and 10 hours later were intraperitoneally injected with 20 mg / kg of antagonist anti-IL11Ra antibody (X209), isotype-matched IgG control antibody, or left untreated.

[0502] The survival of mice was monitored for 8 days after APAP / saline administration, and the results were as follows Fig. 8A Treatment with an antibody inhibitor, an antagonist of IL-11-mediated signaling, significantly increased the survival of mice administered a lethal dose of APAP compared with IgG-treated controls.

[0503] Mice were euthanized at 24 hours and 192 hours (8 days). Serum ALT levels were analyzed as described in Example 4. Livers were harvested and fixed as described in Example 4, and digital photographs were taken.

[0504] The results are as follows Figure 8B and 8CAs shown. Administration of an IL-11-mediated signaling antagonist antibody inhibitor 10 hours after lethal APAP overdose showed that the gross morphology of the liver of mice recovered to that of mice not treated with APAP after 8 days ( Figure 8B ). Administration of an antagonist antibody inhibitor of IL-11-mediated signaling 10 h after lethal APAP overdose was further demonstrated to rescue mice from DILI-associated liver function suppression; after 8 days, serum ALT levels were not significantly different from those in normal (saline-administered) control mice ( Figure 8C ).

[0505] The ability of an IL-11-mediated signaling antagonist administered 10 hours after hepatotoxic insult to reverse DILI-associated hepatotoxicity and prevent death in subjects with severe / lethal APAP overdose is a truly remarkable result. 10 hours after overdose in mice is considered equivalent to approximately 24 hours after overdose in humans.

[0506] These results suggest that antagonizing IL-11-mediated signaling is a highly promising therapeutic strategy to reduce liver damage and associated morbidity / mortality following hepatotoxic insult.

[0507] Example 6: Antagonism of IL-11-mediated signaling reverses liver damage symptoms and restores liver function after drug-induced liver injury able

[0508] 6.1 Overview

[0509] Acetaminophen (APAP) overdose is a major cause of liver failure. Administration of recombinant human interleukin 11 (rhIL11) has a protective effect in the APAP-induced liver injury (AILI) model in mice.

[0510] This study demonstrates that the beneficial effects of rhIL11 in mouse AILI are due to an unexpected inhibitory effect of exogenous rhIL11 on endogenous mouse IL11 activity. In contrast to the accepted paradigm, IL11 is secreted by damaged hepatocytes to drive apoptosis and inhibit liver regeneration.

[0511] Hepatocyte-specific IL11-expressing mice spontaneously developed liver damage, whereas those with IL11RA1 deletion were robustly protected from AILI. Neutralizing anti-IL11R antibodies given to moribund mice 10 h after lethal APAP overdose resulted in a 90% survival rate.

[0512] The data disclosed herein overturn misconceptions, indicate disease mechanisms, and identify therapeutic targets.

[0513] 6.2 Introduction

[0514] Acetaminophen (N-acetyl-p-aminophenol, APAP) is an over-the-counter analgesic that is often overdosed (OD) and leads to APAP-induced liver injury (AILI), a leading cause of acute liver failure (1). The antioxidant N-acetylcysteine ​​(NAC) is beneficial for patients who present early (2), but there is no drug-based treatment within 8 hours of OD, and if liver transplantation is not possible, death may occur (3, 4).

[0515] In hepatocytes, APAP is metabolized to N-acetyl-p-benzoic acid imide (NAPQI), which depletes cellular glutathione (GSH) levels, damages mitochondrial proteins, and leads to reactive oxygen species (ROS) production and JNK activation (5). ROS-dependent JNK activation leads to a combination of necrosis, apoptosis, and other forms of hepatocyte death, resulting in liver failure (1,6,7). JNK and ASK1 inhibitors have shown partial protection against AILI in mouse models, but this has not yet translated to the clinic (8,9).

[0516] Liver regeneration has fascinated humans since the story of Prometheus and can be quite profound, as seen following partial liver resection in rodents and humans (10, 11). However, in the setting of AILI, liver regeneration is persistently suppressed, leading to permanent damage and patient death. Targeting pathways that impede the liver’s extraordinary regenerative capacity could potentially trigger natural regeneration, which could be particularly useful in AILI (12, 13).

[0517] Interleukin 11 (IL11) is a little-studied cytokine that is essential for the activation of cardiac fibroblasts and fibrosis in the heart, kidney, lung, and liver (14-16). IL11 is known to be secreted by damaged hepatocytes and is detected at high levels in the serum of AILI mouse models, where its expression is thought to be compensatory and cytoprotective (17). On this basis, administration of recombinant human IL11 (rhIL11) can effectively treat AILI mouse models and has protective effects against liver ischemia, endotoxemia, or inflammation (17-22). As recently as 2016, rhIL11 has been proposed as a treatment for AILI patients (23).

[0518] In studies of liver fibrosis, the inventors unexpectedly observed that IL11 could be detrimental to hepatocyte function in the context of some models of fibro-inflammatory liver disease (14). This apparent discrepancy with previous literature prompted the inventors to look in more detail at the effects of IL11 on hepatocytes rather than fibrosis, and they chose to do so in a mouse model of AILI, in which IL11 is largely upregulated (17).

[0519] IL11 drives APAP-induced hepatocyte death

[0520] As previously reported (17), AILI was shown to be characterized by elevated serum levels of IL11 in injured mice. Fig.9A The inventors then sought to address whether the elevated serum levels of IL11 in the mouse AILI model originate in the liver. APAP induced a strong upregulation of IL11 transcripts in hepatocytes (35-fold, P < 0.0001). Bioluminescent imaging of reporter mice in which luciferase was cloned into the IL11 start codon indicated that IL11 was expressed throughout the liver ( Fig. 9B and 9C ,as well as Fig.15 Western blot confirmed that IL11 was upregulated at the protein level during a time course of AILI ( Fig.9D ). Experiments using a second reporter mouse that had the EGFP reporter construct inserted into the 3'UTR of IL11 ( Fig.16 ), showing that after APAP, IL11 protein was highly expressed in necrotic centrilobular hepatocytes, which is a pathological feature of AILI, and occurred simultaneously with cleaved caspase3 (Cl.CASP3) ( Fig.9E ).

[0521] Having identified the source of Il11 upregulation in AILI in vivo, the inventors performed in vitro experiments to investigate the underlying mechanism. Exposure of primary human hepatocytes to APAP resulted in a dose-dependent secretion of IL11 ( Fig.9F Hepatocytes express interleukin 11 receptor subunit α (IL11RA), and IL11 is known to activate ERK in certain cell types (14), so the inventors explored the effects of IL11 on ERK and JNK, which are important in AILI and activated in hepatocytes. IL11 induced late (>6h) and sustained ERK and JNK activation, which occurred simultaneously with CASP3 cleavage ( Figure 9G ). FACS-based analysis revealed dose-dependent IL11-induced hepatocyte death ( Figure 9H and Fig.17A To explore the role of IL11 signaling in APAP-challenged hepatocytes, the inventors used an IL11RA neutralizing antibody (X209) (14), which inhibits CASP3 cleavage and cell death, as well as ERK and JNK activation ( Fig.9I and 9J , Fig. 17B ). Although these data confirm the upregulation of IL11 in AILI, they challenge the prevailing view that this effect is compensatory and protective in the injured liver.

[0522] 6.4 Species-specific effects of recombinant human IL11

[0523] rhIL11 has been reported to have a protective effect in rodent liver injury models (17-20, 23), however, the results described in Example 6.3 showed that rhIL11 had completely opposite effects on human hepatocytes in vitro (Figure 9). This prompted the inventors to test the potential inconsistency of rhIL11 protein when used in foreign species, because human and mouse IL11 have only 82% protein sequence homology. First, they compared the effects of rhIL11 and recombinant mouse IL11 (rmIL11) on mouse hepatocytes. Species-matched rmIL11 stimulated ERK and JNK phosphorylation in mouse hepatocytes and induced CASP3 cleavage, while rhIL11 had no effect ( Fig. 10A Likewise, while rmIL11 induced mouse hepatocyte cell death, rhIL11 did not. In fact, at higher doses, rhIL11 tended to inhibit mouse hepatocyte cell death ( Fig. 10A In corresponding experiments with human hepatocytes, the inventors found that rhIL11 stimulated ERK and JNK signaling and hepatocyte death, whereas rmIL11 did not ( Fig.18A and 18B ).

[0524] This suggests that the role of IL11 signaling in hepatocyte death is conserved across species, but that recombinant IL11 protein has species-specific effects and does not activate this signaling pathway in foreign species. This hypothesis was tested in vivo by injecting mice with either rmIL11 or rhIL11 ( Fig. 10C ). Injection of rmIL11 resulted in gradual ERK and immediate JNK activation. In contrast, rhIL11 had no effect on ERK or JNK phosphorylation ( Fig. 10D ). Injection of rmIL11 also caused liver damage, accompanied by elevated ALT and AST ( Fig.10E and Fig.18C In sharp contrast, 24 h after rhIL11 injection, ALT and AST levels in naive mice were slightly decreased (ALT, P = 0.018; AST, P = 0.0017).

[0525] To investigate the potential protective effects of rhIL11 in mice, a protocol similar to the AILI study of 2001 (20) was performed in which mice were injected with rhIL11 after APAP OD ( Fig.10F ). This confirmed that rhIL11 reduced the severity of AILI in mice (reduction: ALT, 52%, P = 0.0001; AST, 39%, P < 0.0001), while species-matched rmIL11 had no protective effect on mice ( Figure 10G and Fig.18D The therapeutic effect of rhIL11 was accompanied by a decrease in hepatic ERK and JNK activation ( Fig. 10H ), suggesting that rhIL11 blocks IL11-driven signaling pathways in the liver, similar to the IL11RA antibody ( Fig.9I ).

[0526] Using surface plasmon resonance (SPR), it was found that rhIL11 binds to mouse interleukin 11 receptor alpha chain 1 (mIL11RA1) with a KD of 72nM, which is slightly stronger than the rmIL11:mIL11RA1 interaction (94nM) and close to the previously reported rhIL11:hIL11RA (50nM), a result that was reconfirmed ( Fig.10I and Fig.18E )(24). The inventors subsequently performed a competitive ELISA analysis and found that rhIL11 competed with rmIL11 for binding to mIL11RA1 and was a very effective blocker, as shown by its higher affinity for mIL11RA1 ( Fig.10J In mouse hepatocytes, rhIL11 is a potent, dose-dependent inhibitor of rmIL11-induced signaling and cytotoxic activity ( Figure 10K and 10L , Fig.18F ). Thus, paradoxically, exogenous rhIL11 acts as a neutralizer of mouse IL11 both in vitro and in vivo, and these observations pose challenges to understanding the role of IL11 in liver injury and in disease more broadly.

[0527] 6.5 Hepatocyte-specific expression of IL11 causes spontaneous liver failure

[0528] To examine the role of endogenous IL11 secretion by mouse hepatocytes in vivo, ROSA26 cells were injected with AAV8 virus encoding a Cre construct driven by the albumin promoter. IL11 / + In mice, the IL11 transgene is specifically expressed in hepatocytes (15, 16) (IL11-Tg mice, Fig.11A ). Three weeks after transgenic induction, the liver of IL11-Tg mice was severely abnormal and smaller (38%, P < 0.0001), accompanied by increased serum ALT and AST levels, while other organs were not affected ( Figures 11A to 11D and Fig.19A and 19B Histologically, there is marked portal venous dilatation and blood accumulation within the sinusoids—suggesting sinus obstruction syndrome—as well as periportal infiltration of the portal triad ( Fig.11E and Fig.19C ). Molecular analysis of Il11-Tg livers revealed activation of ERK, JNK, and CASP3 cleavage and increased expression of pro-inflammatory genes ( Fig.11F and Fig.19D and 19E). Therefore, secretion of IL11 by hepatocytes, as seen in APAP toxicity (Figure 9), is hepatotoxic.

[0529] 6.6IL11 promotes NOX4-mediated reactive oxygen species production

[0530] APAP-driven JNK activation requires IL11 signaling in vitro ( Fig.9I and 9J ), which is known to occur with the generation of ROS and depletion of GSH. Hepatic GSH levels in IL11-Tg mice were examined and found to be reduced (62%, P < 0.0001), suggesting that IL11 signaling - either directly or indirectly - induces ROS ( Fig.11G ).

[0531] In fibroblasts, expression of NOX4, a NADPH oxidase and source of ROS, correlates with IL11 expression (15, 25), and hepatocyte-specific NOX4 deletion prevents pathological activation of JNK (26). Therefore, the inventors investigated the relationship between IL11, NOX4, and ROS in more detail. In IL11-Tg mice, hepatic NOX4 expression was upregulated ( Fig.11H In primary human hepatocytes, IL11 stimulated a dose-dependent GSH depletion over time, which was reflected by ERK and JNK activation and was accompanied by NOX4 upregulation ( Figure 9G and Figures 11I-11K As expected, only species-specific IL11 induced NOX4 upregulation and decreased GSH levels ( Fig.11L and Figures 20A to 20D ).

[0532] APAP stimulation also resulted in upregulation of NOX4 in hepatocytes, concurrent with depletion of hepatocyte GSH levels, which was blocked by the anti-IL11RA antibody X209 ( Fig.11M and 11N The inventors reconsidered the role of rhIL11 in inhibiting endogenous IL11-induced mouse hepatocyte death ( Fig.10J and 10K ) and found that rhIL11 had a significant dose-dependent effect in restoring GSH levels in rmIL11-stimulated mouse cells ( Fig.21A ). Similarly, rhIL11 restored APAP-induced GSH depletion in mice, whereas rmIL11 did not ( Fig. 21B GKT-13781, a specific NOX4 inhibitor, prevented IL11-stimulated GSH depletion, CASP3 activation, and cell death in a dose-dependent manner ( Fig.11O and Fig.22A and 22B). The specificity of pharmacological inhibition of NOX4 was confirmed using siRNA, thereby preventing IL11-induced hepatotoxicity ( Figure 11P and 11Q , Fig.23A and 23B These data suggest that IL11-stimulated NOX4 activity, which can also affect mitochondrial ROS, is important for GSH depletion in the context of AILI.

[0533] 6.7 Hepatocyte-specific deletion of IL11RA1 prevents APAP-induced liver failure

[0534] To specifically delete L11RA1 in adult mouse hepatocytes, L11RA1 conditional knockout (CKO) was generated by injecting AAV8-ALB-Cre virus into mice homozygous for the LoxP-flanked Il11ra1 allele as well as wild-type controls. Three weeks after viral infection, control mice and CKO were given APAP (400 mg kg -1 )( Fig. 12A ). On the second day after APAP administration, gross autopsy showed that the livers of control mice were small and discolored, while the livers of CKO mice looked normal ( Fig. 12B Histology showed extensive centrilobular necrosis typical of normal mice, which was not observed in CKO ( Fig. 12C ).

[0535] Strikingly, ALT and AST levels in CKO mice were 99% and 95% lower than those in controls, respectively, while GSH levels were similar to baseline. The levels of APAP and APAP-glutathione (APAP metabolites) in serum were similar in both groups, indicating that IL11RA1 deficiency does not affect APAP metabolism ( Figures 12D to 12F , Fig.24A and 24B ). Activation of ERK and JNK was observed in control mice but not in CKO ( Figure 12G ). Loss of this receptor in hepatocytes also significantly reduced inflammatory markers, suggesting that inflammation in AILI is secondary to parenchymal damage. ( Fig.12H ). Taken together, these data suggest that hepatocyte-specific IL11 signaling plays a dominant role in the pathogenesis of AILI. Indeed, hepatocyte IL11RA1 depletion is sufficient to protect against APAP OD, suggesting that free soluble Il11RA1 in serum or receptor shedding from other cellular sources does not contribute to disease pathogenesis via trans-signaling.

[0536] 6.8 Effect of early anti-IL11Ra administration in APAP-induced liver injury

[0537] The inventors next tested whether therapeutic inhibition of IL11 signaling by administration of an anti-IL11RA (X209) antibody (14) could effectively alleviate AILI. Initially, X209 or a control antibody (10 mg kg -1 This approach reduced serum liver damage markers by more than 70%, greatly restored hepatic GSH levels, and limited histologic evidence of centrilobular necrosis ( Figures 13A to 13D and Fig.25A ).

[0538] Next, anti-IL11RA treatment was administered in a therapeutically relevant fashion by administering the antibody 3 h after APAP, a time point at which APAP metabolism and toxicity are established and after which most interventions have no effect in the AILI mouse model ( Fig.13E )(9). X209 in different dose ranges (2.5-10 mg kg -1 ) inhibited AILI and had dose-dependent improvements in liver injury markers and hepatic GSH levels. The reduction in JNK and ERK activation confirmed dose-dependent target coverage ( Figures 13F to 13H and Fig.25B ).

[0539] Finally, we determined whether inhibition of IL11 signaling has added value when combined with the current standard of care, NAC, 3 h after APAP administration ( Fig.13I ). NAC alone reduced serum ALT and AST levels. However, NAC combined with X209 was even more effective than NAC or X209 alone (ALT reduction: NAC, 38%, P = 0.0007; X209, 47%, P < 0.0001; NaC + x209, 75%; P < 0.0001) ( Fig.13F , 13J and Fig.25C At the molecular level, the extent to which NAC or NAC together with X209 inhibited ERK and JNK was reflected by the extent to which ALT in serum was reduced and GSH levels in the liver were restored ( Figure 13K and 13L ). Therefore, anti-IL11RA therapy could have additive benefit when combined with current standard of care.

[0540] 6.9 Anti-IL11Ra therapy for liver regeneration

[0541] There is no effective treatment for patients who present to the ED 8 h or later after APAP OD. This prompted us to administer APAP (400 mg kg -1 ) 10h after the detection of anti-IL11RA ( Fig.14A). Given the accelerated metabolism of APAP in mice, 10 h of treatment in this model is equivalent to 24 h of treatment in humans after APAP OD. Quantification of APAP and APAP-glutathione in serum by mass spectrometry revealed elevated levels compared to saline-treated controls, with comparable levels between experimental groups, as expected ( Fig.26A and 26B ). Analysis of gross anatomy, histology, and serum IL11, ALT, and AST levels showed that X209 significantly reversed liver injury on the second day after APAP, whereas IgG-treated mice had severe and persistent liver injury ( Figures 14B-14E and Fig.27A Throughout the experiment, the therapeutic antibody effectively blocked the activation of ERK and JNK, which resulted in a decrease in cleaved CASP3 at 24 h ( Fig.14F and Fig.27B ).

[0542] Interventions that promote liver regeneration have great potential and may provide a new approach for the treatment of AILI (12). Therefore, the status of genes important for liver regeneration was evaluated (10). Inhibition of IL11 signaling was associated with a robust regenerative signature, with strong upregulation of PCNA, cyclin D1 / D3 / E1, and phosphorylation of RB, as seen during regeneration after partial hepatectomy (10). EdU injection and histological analysis revealed that X209-treated mice had a larger number of nuclei and evidence of recent DNA synthesis compared with controls ( Figure 14G ). The effect of X209 given 3 hours after APAP was re-evaluated ( Figures 13I to 13L ) to see if regeneration was associated with early IL11 signaling inhibition. This turned out to be the case, with the combination of X209 and NAC increasing molecular markers of regeneration more effectively than NAC alone, particularly for cyclins D1 and D3 ( Fig.14H ).

[0543] Finally, X209 (20 mg kg -1 ) at a higher and lethal dose of acetaminophen (550 mg kg -1 ) 10 hours after the onset of the attack, the mice were moribund and the liver was undergoing fulminant necrotic inflammation ( Fig.14I By the end of the study, X209-treated mice recovered and had a 90% survival rate. In contrast, IgG-treated mice did not recover and died within 48 h, with a 100% mortality rate ( Fig.14J On day 8 after a lethal dose of APAP, X209-treated mice appeared healthy, with normal liver morphology and ALT levels comparable to those of controls that did not receive APAP ( Figure 14K and Fig.28A and 28B).

[0544] 6.10 Discussion

[0545] APAP OD is common, with up to 50,000 people presenting to emergency departments in the UK each year, some of whom develop liver failure and require transplantation (1). Here, IL11, which has previously been reported to have a protective role in APAP-induced liver failure (17, 20), hepatic ischemia (18, 21), endotoxemia (22), and inflammation (19), was shown to be actually hepatotoxic and critical for liver failure after APAP OD.

[0546] The observation that endogenous IL11 is hepatotoxic was most surprising, given that more than 30 articles have reported cytoprotective and / or anti-inflammatory effects of rhIL11 in rodent models of human disease (Tables 1 and 2). rhIL11 was shown to be a competitive inhibitor of mouse IL11 binding to IL11RA1, which overturned previous understandings of the role of IL11 in AILI and liver disease more generally. This also implies that anti-IL11 therapy may be effective in other diseases in which rhIL11 has protective effects in mouse models, such as rheumatoid arthritis (27) and colitis (28) (Table 2). Many clinical trials using rhIL11 have been conducted in patients (Table 3) based on the erroneous assumption that the effects of rhIL11 in mice represent a beneficial IL11 gain of function.

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555] The inventors proposed a well-established mechanism of APAP toxicity, namely that NAPQI damages mitochondria to produce ROS, which stimulates IL11-dependent NOX4 upregulation and further maintains ROS production ( Fig.29This results in a dual pathology: killing of hepatocytes through JNK and caspase activation and prevention of hepatocyte regeneration, through mechanisms that are not yet clearly understood. Mouse models of AILI closely resemble human disease, and thus therapeutics targeting IL11 signaling hold promise for the treatment of patients with APAP-induced hepatotoxicity. Since IL11 neutralization therapy does not rely on altering APAP metabolism ( Fig.12F ), and specifically stimulate tissue regeneration, they are much more effective than the current standard of care and may be particularly useful for patients who present late to the emergency department.

[0556] 6.11 Materials and Methods of Example 6

[0557] Antibody

[0558] Cleaved caspase3 (9664, CST), caspase3 (9662, CST), cyclin D1 (55506, CST), cyclin D3 (2936, CST), cyclin E1 (20808, CST), p-ERK1 / 2 (4370, CST), ERK1 / 2 (4695, CST), GAPDH (2118, CST), GFP (ab6673, Abcam), IgG (Adevron), p-JNK (4668, CST), JNK (9258, CST), neutralizing anti-IL11RA (X209, Aldevron; in vivo study), IL11RA (130920, Santa Cruz; WB), NOX4 (110-58849, Novus Biologicals), PCNA (13110, CST), p-RB (8516, CST), RB (9313, CST), anti-rabbit HRP (7074, CST), anti-mouse HRP (7076, CST), anti-rabbit Alexa Fluor488 (ab150077, Abcam), anti-rabbit Alexa Fluor647 (ab150079, Abcam), anti-mouse Alexa Fluor488 (ab150113, Abcam), anti-goat Alexa Fluor488 (ab150129, Abcam).

[0559] Recombinant protein

[0560] Recombinant human IL11 (rhIL11, UniprotKB: p20809, Genscript), recombinant mouse IL11 (rmIL11, UniprotKB: P47873, Genscript), human IL11RA (10252-H08H, SinoBiological), mouse IL11RA (50075-M08H, SinoBiological).

[0561] Chemicals

[0562] Acetaminophen (APAP, A3035, Sigma), DAPI (D1306, Thermo Fisher Scientific), D-luciferin (L6882, Sigma), GKT-137831 (17764, Cayman Chemical Company), N-acetyl-L-cysteine ​​(NAC, A7250, Sigma).

[0563] LC-MS / MS Reagents

[0564] Reference standard acetaminophen (APAP, P030000, Sigma), internal standard (IS) acetaminophen-D4 (APAP-D4, A161222, Toronto Research Chemicals), IS acetaminophen glutathione (APAP GLUT, A161223, Toronto Research Chemicals), acetonitrile (900667, Sigma), ammonium formate (A115-50, Sigma), formic acid (F0507, Sigma), mouse serum (IGMSCD1SER50ML, i-DNA Biotechnologies). All chemicals, reagents, and solvents were of LC-MS grade quality.

[0565] Animal models

[0566] 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 ad libitum, except during the fasting period, during which only water was provided ad libitum.

[0567] Acetaminophen poisoning mouse model

[0568] Prior to APAP, 12-14 week old male mice (C57BL6 / NTAC, unless otherwise stated) were fasted overnight. Mice were then given severe (400 mg kg-1) or lethal (550 mg kg-1) doses of APAP by intraperitoneal (IP) administration. Anti-IL11RA (X209) or IgG isotype control antibodies were administered to mice at different times and doses as described above or in the figure legends. Mice were euthanized at different time points after APAP, from 10 hours to 8 days, as described above or in the figure legends.

[0569] IL11-luciferase mice

[0570] The mouse IL11 gene consists of five exons, with the ATG start codon in exon 1 and the TGA stop codon in exon 5. Three transcripts of mouse IL11 have been identified (ENSMUSG00000004371): transcript IL11-201 is the longest, encoding a 199aa propeptide, while transcripts IL11-202 and IL11-203 contain an alternative first exon and are both predicted to encode a shorter 140aa isoform lacking a signal peptide. Using CRISPR / Cas9 technology, a Kozak-Luciferase-WPRE-polyA sequence was introduced to replace the ATG start codon in exon 1 of IL11-201 (ENSMUST00000094892.11), resulting in a disruption of translation of this particular transcript. A single guide RNA (sgRNA) with a recognition site in exon 1 was microinjected into fertilized zygotes along with Cas9 and a targeting construct containing the Kozak-luciferase-WPRE-polyA sequence and then transferred into pseudopregnant mice (Shanghai Model Organism Center). Insertion of the luciferase cassette into the Il11 locus was verified by sequencing. Mutant Il11-luciferase progeny were generated on a C57BL / 6 background and characterized by genotyping to detect insertion of the luciferase construct in exon 1 using primers that amplified an 818 bp region corresponding to the wild-type Il11 allele (5'-GGAGGGAGGGGACGCCAATGACC-3' (SEQ ID NO:22) and 5'-TCTGCCTCCCCTGCCTGTTTCTCG-3' (SEQ ID NO:23)) and a second set of primers that amplified a 928 bp region corresponding to the targeted allele containing the luciferase construct (5'-AATTCCGTGGTGTTGTCG-3' (SEQ ID NO:24) and 5'-TCTGCCTCCCCTGCCTGTTTCTCG-3' (SEQ ID NO:25)).

[0571] Hybrid Il11-luciferase was subjected to APAP-induced liver injury as described above. 24 h later, mice were injected intraperitoneally with 150 mg kg -1 D-luciferin in PBS, and bioluminescent images of the liver were then acquired using the IVIS Lumina system (PerkinElmer) according to the manufacturer's instructions.

[0572] Il11-EGFP mice

[0573] Transgenic mice with EGFP constitutively knocked into the Il11 gene were generated by Sagan Biosciences. Briefly, knock-in mice were generated to contain a 2A-EGFP cassette inserted into exon 5, which replaced the TGA stop codon sequence, and translation of the targeted transcript will produce the full-length IL11 propeptide and EGFP separated by a 2A self-cleaving peptide linker. Targeting vector homology arms of the IL11 gene, containing a Neo cassette inserted into intron 4 (flanked by SDA: self-deletion anchor sites) and a 2A-EGFP cassette inserted into exon 5, were generated by PCR using BAC clones from a C57BL / 6 library. Gene targeting was performed using C57BL / 6 ES cells, and targeted clones were successfully injected into C57BL / 6 albino embryos, which were then reimplanted into CD-1 pseudopregnant females. Elite transgenic animals were identified by coat color, and germline transmission was confirmed by breeding with C57BL / 6 females and subsequent genotyping of the offspring. Genotyping primers were designed to amplify selected regions of intron 4, spanning the Neo box SDA site, according to the following primer sequences: (5'-GAAATGAGAGCCTAGAGTCCAGAG-3' (SEQ ID NO: 26) and 5'-GAGGCTTGGAAGAATGCACAATTA-3' (SEQ ID NO: 27)).

[0574] Hepatocyte-specific Il11 overexpression mice (Il11-Tg)

[0575] Mice in which mouse Il11 cDNA was introduced into the Rosa26 locus under the control of loxP-Stop-loxP sites to allow cell type-specific overexpression of Il11 following Cre recombinase-mediated excision have been described previously (15). These animals are available at the Jackson Laboratory (C57BL / 6N-Gt(ROSA)26SortM1(CAG-Il11)Cook / J). To induce specific expression of Il11 in hepatocytes, heterozygous Il11-Rosa26 mice were injected intravenously (IV) with 4x10 11 The genome copies of AAV8-ALB-Null (control) or AAV8-ALB-Cre (Il11-Tg) were measured in the liver and serum three weeks later.

[0576] Hepatocyte-specific Il11ra1-deficient mice

[0577] Il11ra1-floxed mice were recently generated and validated in which exons 4 to 7 of the Il11ra1 gene are flanked by loxP sites, allowing for spatial and temporal deletion of Il11ra1 following Cre recombinase-mediated excision (Ng et al., Sci Transl Med. (2019) 11(511) pii:eaaw1237). To induce specific deletion of Il11ra1 in hepatocytes, homozygous Il11ra1-floxed mice were injected intravenously with AAV8-ALB-Cre virus (4x10 in PBS / mouse) via the tail vein. 11 A similar amount of AAV8-ALB-Null virus was injected into homozygous Il11ra1-floxed mice as a control. Mice treated with AAV8 were allowed to recover for three weeks before APAP injury. Western blot was used to detect the knockdown efficiency of hepatic IL11RA.

[0578] Cell culture

[0579] Both human and mouse primary hepatocytes were grown and maintained at 37°C and 5% CO2. Growth medium was refreshed every 2-3 days and cells were passaged at 80% confluence using a standard trypsin method. All experiments were performed at low cell passages (P1-P3). Stimulated cells were compared to unstimulated cells that were grown for the same amount of time under the same conditions but without stimulation.

[0580] Primary human hepatocytes

[0581] Human hepatocytes (5200, ScienCell) were maintained in Hepatocyte Medium (520, ScienCell) supplemented with 2% fetal bovine serum and 1% penicillin-streptomycin. Cells were serum starved for 16 hours before the respective stimulation, as described above or in the figure legends, in serum-free Hepatocyte Medium for 24 hours.

[0582] Primary mouse hepatocytes

[0583] Mouse hepatocytes (ABC-TC3928, AcceGen Biotech) were maintained in mouse hepatocyte culture medium (ABC-TM3928, AcceGen Biotech) supplemented with 1% penicillin-streptomycin. Cells were stimulated with different treatment conditions for 24 h, as described above or indicated in the figure legends.

[0584] siRNA transfection

[0585] 16 h before transfection, primary human hepatocytes were seeded in 6-well plates at 60-70% confluence. Cells were transfected with 50 nm NOX4 siRNA (ON-TARGETplus SMARTpool siRNA, L-010194-00-0005, Dharmacon) or control siRNA (D-001810-10-05, Dharmacon) in OptiMEM (31985070, Thermo Fisher) containing Lipofectamine RNAiMAX transfection reagent (13778150, Thermo Fisher) at 37°C for 24 h. Transfected cells were then stimulated with rhIL11 for 24 h. Knockdown efficiency was determined by NOX4 immunoblotting.

[0586] Flow cytometry

[0587] Human primary hepatocytes (5×10 5 ) were stained with FITC Annexin V / Dead Cell Apoptosis Kit (V13242, Thermo Fisher Scientific) according to the manufacturer's instructions. PI was analyzed by flow cytometry (Fortessa, BD Biosciences). +VE Cells were quantified and analyzed using FlowJo (version 7) software (TreeStar).

[0588] Colorimetric analysis

[0589] ALT activity (ab105134, Abcam) and AST (ab105135, Abcam) assay kits were used to measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum and hepatocyte supernatant. Liver glutathione sulfhydryl (GSH) was measured using a glutathione colorimetric assay kit (EIAGSHC, Thermo Fisher Scientific). All colorimetric analyses were performed according to the manufacturer's methods.

[0590] ELISA

[0591] The levels of IL-11 in mouse serum and hepatocyte supernatant were quantified using mouse IL-11 DuoSet (DY418 and DY008, R&D Systems) and human IL-11 Quantikine ELISA kit D1100, R&D Systems, respectively.

[0592] Competitive ELISA

[0593] Mouse IL11RA (1 μg ml -1The cells were coated onto 96-well plates (overnight at 4°C) with 1% BSA in PBS and then blocked with blocking buffer (1% BSA in PBS containing 0.05% Tween 20). Biotinylated mouse Il11 was prepared using the Lightning-Link Rapid Biotin Type A Kit (Expedeon) according to the manufacturer's instructions. RhIL11 or rmIl11 was serially diluted 2-fold in blocking buffer (from 5 μg ml -1 start), and with 0.01 μg ml -1 The mixture of biotinylated mouse IL11 and rhIL11 or rmIL11 was added to the coated plate and incubated for 1 h at room temperature. Color development was performed by adding streptavidin-HRP (1:1000 in blocking buffer) and TMB chromogen solution (002023, Thermo Fisher Scientific).

[0594] Immunoblotting

[0595] Western blotting was performed on hepatocyte and liver tissue lysates. Hepatocytes and liver tissues were homogenized in radioimmunoprecipitation (RIPA) buffer (Thermo Fisher) containing protease and phosphatase inhibitors, and the lysates were clarified by centrifugation. Protein concentration was determined by Bradford method (Bio-Rad). Equal amounts of protein lysates were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting with the indicated primary antibodies. Proteins were visualized using ECL detection system (Pierce) and appropriate secondary antibodies.

[0596] Quantitative polymerase chain reaction (qPCR)

[0597] Total RNA was extracted from snap-frozen liver tissue or hepatocyte lysate using Trizol (Invitrogen) and purified using RNeasy columns (Qiagen). TM cDNA was synthesized using a cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. Gene expression analysis was performed on duplicate samples using TaqMan (Applied Biosystems) or fast SYBRgreen (Qiagen) technology using StepOnePlusTM (Applied Biosystems) for 40 cycles. Expression data were normalized to GAPDH mRNA expression and were expressed using 2 -ΔΔCt Methods Fold changes were calculated. Sequences of specific TaqMan probes and SYBR green primers are available upon request.

[0598] Surface plasmon resonance (SPR)

[0599] SPR measurements were performed on a BIAcore T200 (GE Healthcare) at 25°C. Buffers were degassed and sterilized by filtration through a 0.2 μm filter before use. rhIL11 or rmIl11 was immobilized on a carboxymethylated dextran (CM5) sensor chip using standard amine coupling chemistry. For kinetic analysis, a series of concentrations (3.125 nM to 100 nM) of human IL11RA or mouse Il11ra were added at 40 μL min -1 rhIL11, rmIl11 and the reference surface were injected at a flow rate of 1.5 %. All analytes were dissolved in a solution containing 1 mg ml -1 BSA in HBS-EP+ (BR100669, GE Healthcare). Binding and dissociation were measured for 150 s and 200 s, respectively. After each analyte injection, the surface was regenerated by injecting glycine at pH 2.5 for 30 s, followed by a 5-minute stabilization period. All sensorgrams were aligned and double referenced. Affinity and kinetic constants were determined by fitting the corrected sensorgrams to a 1:1 Langmuir model using BIAevaluation v3.0 software (GE Healthcare). The equilibrium binding constant KD was determined by the ratio of the binding rate constants kd / ka.

[0600] Histology

[0601] Hematoxylin & Eosin (H&E) staining

[0602] The liver was fixed in 10% neutral buffered formalin (NBF) at room temperature for 48 h, dehydrated, embedded in paraffin blocks, and sectioned at 7 μm. The sections were stained with H&E according to standard methods and examined under a light microscope.

[0603] EdU staining

[0604] The liver was rinsed in cold PBS and patted dry with lint-free paper and then incubated in OCT compound (4583, Tissue- ). After freezing the OCT compound, the liver specimens were wrapped in aluminum foil and stored at -80°C. Before EdU detection using Baseclick's EdU IV Imaging Kit 488L (BCK488-IV-IM-L) according to the manufacturer's method, the cryo-embedded livers were cryosectioned at 7 μm thickness (-20°C) and dried on slides for 1 h.

[0605] Immunofluorescence staining

[0606] The liver was processed and frozen as described above (EdU stained sections). Frozen liver tissue was sliced ​​at 7 μm at -20°C and dried for 1 h (room temperature). Liver sections were first fixed in cold acetone for 15 min, then permeabilized with 0.1% TritonX-100 (T8787, Sigma) and blocked with 2.5% normal goat serum (S-1012, Vector Labs) for 1 h (room temperature). Liver sections were incubated with GFP (1:500) and Caspase3 (1:1000) primary antibodies overnight (4°C), followed by incubation with appropriate Alexa Fluor488 / 647 secondary antibodies (1:250) for 1 h (room temperature). Cell nuclei were stained with DAPI before fluorescence microscopy (Leica).

[0607] LC-MS / MS

[0608] Mouse serum samples (20 μL), calibrators, and QCs were transferred to a 96-deep-well plate and 50 μL of 10 μg of -1 APAP-D4 heavy isotope standard. After treatment with 360 μL of ice-cold acetonitrile containing 0.1% formic acid, the plate was mixed (rpm min -1 , 10 min) followed by centrifugation (2270 g, 50 min, 4 °C). 140 μL of the supernatant was carefully transferred to a 96-well microplate and loaded into an autosampler for analysis by LC-MS / MS. Ion counts were then normalized to heavy isotope standards before quantification using a standard curve. Liquid chromatography (LC) separation of biomarkers was performed on an Agilent 1290 Infinity II LC system (Agilent Technologies) using PEEK-coated -cHILIC 3mm, A 100x2.1 mm HPLC column (Merck Pte Ltd) was maintained at 40°C. The organic solvent was acetonitrile containing 0.1% formic acid (solvent A) and the aqueous solvent was 20 mM ammonium formate pH 4.0 (solvent B). A linear LC gradient on a binary pump A (G7120A, Agilent Technologies) was set up with the following percentage of solvent B: 10% at 0 min, 70% at 9 min, 70% at 11 min, 10% between 11.1 and 11.5 min, with a flow rate of 0.4 ml min -1 The column was further equilibrated with 10% solvent B for 11.5 min. Another high-speed pump, binary pump B and a quick-change valve head, 2 positions / 10 ports, 1300 bar (5067-4240, Agilent) was used to reduce the cycle time by automatic alternating column regeneration. The percentage of solvent B on binary pump B was maintained at 10% with a flow rate of 0.3 ml min -1For mass detection, the LC eluent was connected to an Agilent 6495 triple quadrupole mass spectrometer system (G6495A, Agilent Technologies) with an electrospray source operating in positive or negative ionization mode. The electrospray source conditions were as follows: capillary voltage 4.0 kV, nozzle voltage 500 V, iFunnel parameters high / low RF 90 V, nebulizer pressure 60 psi, gas temperature 290 °C, sheath gas temperature 350 °C, nebulizer pressure 35 psi, sheath gas flow 12 l min -1 The multiple reaction monitoring (MRM) conditions for APAP and APAP-D4 were 152.1→110, collision energy (CE) of 16 eV, collision accelerator voltage (CAV) of 5 V and 156→114, collision energy (CE) of 8 eV, CAV of 5 V, respectively. The MRM used for APAP-glutathione was 457.1→140, collision energy (CE) of 42 eV, collision accelerator voltage (CAV) of 5 V.

[0609] Calibration and Linearity

[0610] A nine-point calibration curve was obtained by fortifying drug-free mouse serum with APAP and APAP-glutathione working solutions. The final APAP concentrations were 0.32, 0.46, 2.6, 5.2, 10.3, 20.6, 41.3, 82.5, and 330 mg l -1 (Low QC: 1.29mgl -1 ; High QC: 165mg l -1 The final concentrations of APAP-glutathione were 0.244, 0.49, 0.98, 1.95, 3.91, 7.81, 15.6, 62.5, 125, and 25 mg l -1 (Low QC: 1.95mg l -1 ; High QC: 31.3 mg l -1 APAP and APAP-glutathione were linearly regressed using weighted linear least squares regression (1 / x2) and (1 / x), respectively, to obtain standard curves for the peak area ratios of each analyte to IS, and the linear coefficients (r2) were 0.97807145 and 0.99655914, respectively. The precision and accuracy of the assay in mouse serum samples were as described previously (32).

[0611] Statistical analysis

[0612] Statistical analysis was performed using GraphPad Prism software (version 6.07). P values ​​were corrected for multiple testing according to Dunnett (several experimental groups compared under one condition), Tukey (several conditions compared with each other in one experiment), and Sidak (several conditions of two different genotypes compared with each other). Two parameters used to compare two different groups were analyzed by two-way analysis of variance. Survival curves were analyzed using the Gehan-Breslow-Wilcoxon test. Statistical significance was determined at P < 0.05.

[0613] 6.12 References for Example 6

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[0641] 28. BS Qiu, CJPfeiffer, JC Keith, Protective effect of recombinant human interleukin-11 on experimental TNB-induced colitis in rats, Digestive Diseases and Sciences. 41 (1996), pp. 1625-1630.

[0642] 29.Murray, , RP Brandes, RD Hughes, AM Shah, AC Brewer, NADPH oxidase 4 regulates homocysteine ​​metabolism and protects against acetaminophen-induced liver injury in mice, Free Radic. Biol. Med. 89, 918-930 (2015).

[0643] 30. L. Hecker, R. Vittal, T. Jones, R. Jagirdar, TRL Luckhardt, JC Horowitz, S. Pennathur, FJ Martinez, VJ Thannickal. NADPH oxidase-4 mediates myofibroblast activation and fibrogenic response in lung injury. Nat. Med. 15, 1077-1081 (2009).

[0644] 31. PJ Wermuth, FA Mendoza, SA Jimenez, Abolition of transforming growth factor-β-induced tissue fibrosis in NOX4-deficient mice, Lab. Invest. 99, 470-482 (2019).

[0645] 32. T. Gicquel, J. Aubert, S. Lepage, B. Fromenty, I. Morel, Quantitative analysis of acetaminophen and its major metabolites in small volumes of plasma by liquid chromatography-tandem mass spectrometry, Journal of Analytical Toxicology, 37 (2013), pp. 110-116.

[0646] Example 7: Expression and signal transduction of IL-11 and IL-6 receptors in primary human hepatocytes

[0647] 7.1 Introduction

[0648] IL11 is a member of the interleukin 6 (IL6) cytokine family and, like IL6, binds to the membrane-bound alpha receptor (IL11RA) and glycoprotein 130 (gp130) to signal in cis. IL6 itself has been implicated in liver function and publications suggest that it is generally beneficial (Klein et al., 2005; Kroy et al., 2010; Matthews et al., 2010; Schmidt-Arras and Rose-John, 2016; Wuestefeld et al., 2003). However, it has also been suggested that IL6 can bind to the soluble IL6 receptor (sIL6R) and signal in trans, which is considered maladaptive (Schmidt-Arras and Rose-John, 2016). IL11 may, like IL6, signal in trans in a pathogenic mode, but experiments to date have not found evidence for this in tumor or reproductive tissues (Agthe et al., 2017; Balic et al., 2017).

[0649] 7.2 Results

[0650] The inventors first evaluated the expression levels of IL6R, IL11RA and gp130 in primary human hepatocytes by flow cytometry. Most cells were observed to strongly express IL11RA and gp130 (92.6% and 91.9%, respectively), but only a few hepatocytes (3.0%) expressed IL6R at a low level ( Fig.31A and 32A ). Based on this result, RNA-seq and Ribo-seq studies found that IL11RA and gp130 were highly expressed and actively translated in hepatocytes. In contrast, few IL6R transcripts were observed and IL6R translation was almost undetectable ( Figures 31B-31D , 32B, and 32C). Immunofluorescence staining of hepatocytes confirmed the results of the Ribo-seq data: IL11RA was highly expressed, but IL6R expression was not detected ( Fig.32D The inventors also did not detect significant levels of IL6R in the culture medium (levels were just above the limit of detection), so they ruled out the possibility that IL6R was shed ( Fig.32E ). Taken together, these data indicate that IL6R is expressed at very low levels in primary human hepatocytes, suggesting that IL6 cis-signaling plays a limited role in these cells. However, these cells showed strong co-expression of IL11RA and gp130.

[0651] Given the lack of IL6R expression in human hepatocytes, the inventors used a synthetic IL6 trans-signaling construct, hyperIL6 (hyperIL6), to activate IL6 signaling in these cells and compared it with a synthetic IL11 trans-signaling complex (hyperIL11). HyperIL11, like IL11 itself (see Example 6), activated ERK and JNK in a dose-dependent manner (2.5 ng / ml to 20 ng / ml). In contrast, IL6 trans-signaling did not activate non-classical signaling pathways, but instead dose-dependently induced STAT3 activation ( Fig.31E ). Therefore, it is a novel and interesting finding that the synthetic complexes formed by IL11 or IL6 with their cognate receptors upon binding to hepatocyte gp130 activated different intracellular pathways.

[0652] HyperIL11 caused a dose-dependent increase in alanine aminotransferase (ALT) in primary human hepatocyte cultures, while hyperIL6 (20 ng / ml) was found to have a limited but significant cytoprotective effect (fold change (FC) = 0.9; P = 0.0468) ( Fig.31F ). Soluble gp130 (sgp130) is an inhibitor of the trans-signaling complex that acts through gp130 (Schmidt-Arras and Rose-John, 2016). Consistent with the reported decoy effect, sgp130 blocked the activation of downstream signaling pathways of hyperIL11 (p-ERK / p-JNK) and hyperIL6 (p-STAT3) and inhibited the hepatotoxic effects of hyperIL11 ( Figures 31G-31I ).

[0653] The inventors then performed experiments to examine IL-11 trans-signaling in the absence of the artificial protein complexes hyperIL6 or HyperIL11. Cells were stimulated with IL11 in the presence of soluble gp130 (sgp130, to inhibit putative trans-signaling) or soluble IL11RA (sIL11RA, to enhance putative trans-signaling). sgp130 or sIL11RA did not affect IL11-induced hepatocyte death and signaling ( Figures 31J-31K Moreover, IL11 induced hepatocyte death in a dose-dependent manner (0.625 ng / ml to 20 ng / ml), whereas the addition of sgp130 (1 μg / ml) or sIL11RA (1 μg / ml) had no effect ( Figure 32G Reciprocally, increasing doses of sgp130 or sIL11RA had no effect on IL11-stimulated ALT release from hepatocytes ( Fig.32H ). These data suggest that IL11 trans-signaling may be absent in the absence of the synthetic construct.

[0654] 7.3 Materials and methods of Example 7

[0655] Antibody

[0656] Albumin (ab207327, Abcam), Alexa Fluor488 secondary antibody (ab150077, Abcam), p-ERK1 / 2 (4370, CST), ERK1 / 2 (4695, CST), gp130 (PA5-28932, Thermo Fisher), IL6 (AF506, R&D Systems), IL6R (for flow cytometry, ab222101, Abcam), IL6R (for immunofluorescence staining, MA1-80456, Thermo Fisher), IL11 ( Aldevron), IL11RA (flow cytometry and immunofluorescence staining, ab125015, Abcam), IL11RA (Western blot, 130920, Santa Cruz), p-JNK (4668, CST), JNK (9258, CST), p-STAT3 (4113, CST), STAT3 (4904, CST), mouse HRP (7076, CST), rabbit HRP (7074, CST).

[0657] Recombinant protein

[0658] Commercial recombinant proteins: human hyperIL6 (IL6R:IL6 fusion protein, 8954-SR, R&D Systems), human soluble gp130 Fc (671-GP-100, R&D Systems), human IL11RA (8895-MR-050, R&D Systems). Custom recombinant proteins: human IL11 (UniprotKB: P20809, Genscript). Human hyperIL11 (IL11RA:IL11 fusion protein) mimics the trans-signaling complex and is constructed using an IL11RA fragment (amino acid residues 1-317; UniprotKB: Q14626) and IL11 (amino acid residues 22-199, UniprotKB: P20809) with a 20-amino acid linker (SEQ ID NO: 20; Schafer et al., 2017).

[0659] Chemicals

[0660] Paraformaldehyde (PFA, 28908; Thermo Fisher), phorbol 12-myristate 13-acetate (PMA, P1585, Sigma), Triton X-100 (T8787, Sigma), and 4′,6-diamino-2-phenylindole (D1306; Thermo Fisher).

[0661] Primary human hepatocyte culture

[0662] Primary human hepatocytes (5200, ScienCell) were maintained in Hepatocyte Medium (520, ScienCell) supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin at 37°C and 5% CO2. Unless otherwise specified in the methods, hepatocytes (P2-P3) were serum starved overnight before stimulation with various doses of various recombinant proteins for 24 hours as described in the text and / or figure legends.

[0663] THP-1 culture

[0664] THP-1 (ATCC) cells were cultured in RPMI1640 (A1049101, Thermo Fisher Scientific) supplemented with 10% FBS and 0.05 mM β-mercaptoethanol. 10 ng / ml PMA was added to induce THP-1 cell differentiation in RPMI1640 for 48 hours.

[0665] Flow cytometry

[0666] For surface IL11RA, IL6R and gp130 analysis, primary human hepatocytes and THP-1 cells were stained with IL11RA, IL6R or gp130 antibodies and the corresponding AlexaFluor488 secondary antibodies. Cell death analysis was performed using a dead cell apoptosis kit and primary human hepatocytes were stained with Annexin V FITC and PI (V13242, Thermo Fisher Scientific). PI+ve cells were quantified by flow cytometry (Fortessa, BD Biosciences) and analyzed using FlowJo Version X software (TreeStar).

[0667] Immunofluorescence

[0668] Primary human hepatocytes were seeded in 8-well chamber slides (1.5×104 cells / well) 24 hours before staining. Cells were fixed in 4% PFA for 20 minutes, washed with PBS, and nonspecific sites were blocked with PBS containing 5% BSA for 2 hours. Cells were incubated with IL11RA, IL6R, gp130, or albumin antibodies overnight (4°C) and then incubated with appropriate Alexa Fluor488 secondary antibodies for 1 hour. The slide chambers were dried in the dark, and 5 drops of mounting medium with DAPI were added to the slides for 15 minutes before imaging with a fluorescence microscope (Leica).

[0669] RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq)

[0670] RNA-seq and Ribo-Seq library preparation were performed as described previously ( Chothani et al., 2019 ).

[0671] Generation of RNA-seq libraries

[0672] Total RNA was extracted from human hepatocytes using RNeasy columns (Qiagen). RNA was quantified using the Qubit RNA High Sensitivity Assay Kit (Life Technologies) and its quality was assessed based on its RNA integrity number using the LabChip GX RNA Assay Kit (Perkin Elmer). The TruSeq Stranded mRNA Library Preparation Kit (Illumina) was used to measure transcript abundance following the manufacturer's standard instructions.

[0673] Generation of Ribo-seq libraries

[0674] Hepatocytes were grown to 90% confluence in a 10 cm dish and lysed in 1 ml of cold lysis buffer (eg, The lysate was then lysed using the Ribo Profile Mammalian Kit, RPHMR12126, Illumina recipe. The homogenized and clarified lysate was then blotted with Truseq nuclease (Illumina) according to the manufacturer's instructions. Ribosomes were purified using EXPLUATA Sephacryl S400 columns (GE Healthcare) and the protected RNA fragments were extracted using standard phenol:chloroform:isoamyl alcohol techniques. After ribosomal RNA removal (mammalian RiboZero magnetic gold, Illumina), the RNA was then purified using Sequencing libraries were prepared from footprinted RNA using the Ribo Profile Mammalian Kit according to the manufacturer’s protocol. Final RNA-seq and ribosome profiling libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) on a StepOnePlus Real-Time PCR System (Applied Biosystems) according to the manufacturer’s protocol.

[0675] The quality and average fragment size of the final library were determined using the LabChip GX DNA High Sensitivity Kit (Perkin Elmer). Libraries with unique barcodes were pooled and sequenced on a NextSeq500 benchtop sequencer (Illumina) using the NextSeq500 High Output v2 Kit and paired-end 75 bp sequencing chemistry.

[0676] Data processing and analysis of RNA sequencing and ribosome profiling

[0677] The raw sequencing data were demultiplexed using bcl2fastq V2.19.0.316, and the aptamers were trimmed using Trimmomatic (Bolger et al., 2014) V0.36, retaining reads longer than 20 nt after trimming. Ribo-seq reads were aligned to known mtRNA, rRNA, and tRNA sequences (RNACentral (The RNACentral Consortium, 2017), Release 5.0) using bowtie (Langmead et al., 2009), and only unaligned reads were retained as ribosome protected fragments (RPFs). Alignment to the human genome (hg38) was performed using STAR (Dobin et al., 2012). Gene expression was quantified for CDS (coding sequence) regions for Ribo-seq and exonic regions for RNA-seq using uniquely mapped reads with feature counts (Ensembl database version GRCh38 v86) (Liao et al., 2014). TPM calculation and visualization Box plots were used to compare baseline expression of IL11RA (ENSG00000137070), IL6R (ENSG00000160712), and gp130 (ENSG00000134352). Read coverage of IL11RA, IL6R, and gp130 using Ribo-seq and RNA-seq was visualized using the Gviz R package (Hahne and Ivanek, 2016) and strand-specific alignment files.

[0678] Colorimetric analysis

[0679] The ALT activity in the cell culture supernatant was measured using an ALT activity assay kit (ab105134, Abcam) according to the manufacturer's method.

[0680] Immunoblotting

[0681] Western blotting was performed on total protein extracts from hepatocytes. Hepatocyte lysates were homogenized in RIPA lysis and extraction buffer (89901, Thermo Scientific) containing protease and phosphatase inhibitors (Roche). Protein lysates were separated by SDS-PAGE and transferred to PVDF membranes. Protein bands were visualized using the ECL detection system (Pierce) and appropriate secondary antibodies: anti-rabbit HRP or anti-mouse HRP.

[0682] Statistical analysis

[0683] All statistical analyses were performed using GraphPad Prism software (version 6.07). When several conditions were compared with each other in one experiment, P values ​​were corrected for multiple testing according to Tukey. The criterion for statistical significance was P < 0.05.

[0684] 7.4 References for Example 7

[0685] Agthe, M., Garbers, Y., Putoczki, T. and Garbers, C. (2017), Interleukin-11 classical but not trans signaling is essential for fertility in mice, Placenta 57, 13-16.

[0686] Balic, J. J., Garbers, C., Rose-John, S., Yu, L. and Jenkins, B. J. (2017), Interleukin-11-driven gastric tumorigenesis is independent of trans-signaling, Cytokine 92, 118-123.

[0687] Bolger, A. M., Lohse, M. and Usadel, B. (2014), Trimmomatic: a flexible trimmer for Illumina sequence data, Bioinformatics 30, 2114-2120.

[0688] Chothani,S., , S., Adami, E., Viswanathan, S., Widjaja, A. A., Langley, S. R., Tan, J., Wang, M., Quaife, N. M., Jian Pua, C., et al., (2019), Extensive translational control of human cardiac fibrosis by RNA-binding proteins, Circulation 140, 937-951.

[0689] Dobin, A., Davis, CA, Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M. and Gingeras, TR (2012), STAR: an ultrafast universal RNA sequence aligner, Bioinformatics 29, 15-21.

[0690] Hahne, F., and Ivanek, R. (2016), Visualizing Genomic Data with Gviz and Bioconductor, InStatistical Genomics, (Humana Press, New York, NY), pp. 335-351.

[0691] Klein, C., Wüstefeld, T., Assmus, U., Roskams, T., Rose-John, S., Müller, M., Manns, MP, Ernst, M. and Trautwein, C. (2005), The IL-6-gp130-STAT3 pathway in hepatocytes triggers liver protection in T cell-mediated liver injury, J. Clin. Invest. 115, 860-869.

[0692] Kroy, D.C., Beraza, N., Tschaharganeh, D.F., Sander, L.E., Erschfeld, S., Giebeler, A., Liedtke, C., Wasmuth, H.E., Trautwein, C., and Streetz, K.L. (2010). Lack of interleukin-6 / glycoprotein 130 / signal transducer and activator of transcription 3 signaling in hepatocytes predisposes to hepatic steatosis and liver damage. Hepatology 51, 463-473.

[0693] Langmead, B, Trapnell, C, Pop, M, Salzberg, SL (2009), Ultrafast and memory-efficient alignment of short DNA sequences to the human genome, Genome Biol. 10, R25.

[0694] Liao, Y., Smyth, G. K., and Shi, W. (2014)., featurecounts: a general program for efficiently assigning sequence reads to genomic features, Bioinformatics 30, 923-930.

[0695] Matthews, VB, Allen, TL, Risis, S., Chan, MHS, Henstridge, DC, Watson, N., Zaffino, LA, Babb, JR, Boon, J., Meikle, PJ, et al. (2010). Interleukin-6-deficient mice develop hepatic inflammation and systemic insulin resistance. Diabetologia 53, 2431-2441.

[0696] Schafer, S., Viswanathan, S., Widjaja, A. A., Lim, W.-W., Moreno-Moral, A., Delaut her, D. M., Ng, B., Patone, G., Chow, K., Khin, E., et al., (2017), IL-11 is an important determinant of cardiovascular fibrosis, Nature 552, 110-115.

[0697] Schmidt-Arras, D., and Rose-John, S. (2016). Research on the IL-6 pathway in the liver: from physiology and pathology to therapy. J. Hepatol. 64: 1403-1415.

[0698] The RNAcentral Consortium (2017). RNAcentral: a comprehensive database of noncoding RNA sequences. Nucleic Acids Res. 45, D128–D134.

[0699] Wuestefeld, T., Klein, C., Streetz, KL, Betz, U., Lauber, J., Buer, J., Manns, MP, Müller, W. and Trautwein, C. (2003). An interleukin-6 / glycoprotein 130-dependent pathway is protective during liver regeneration. J. Biol. Chem. 278: 11281-11288.

Claims

1. Use of an agent capable of inhibiting interleukin 11 (IL-11)-mediated signal transduction in the preparation of a medicament or composition for treating or preventing acetaminophen-induced liver injury, wherein the agent capable of inhibiting IL-11-mediated signal transduction is an antagonist of IL-11-mediated signal transduction selected from: (i) an anti-IL-11 antibody or an antigen-binding fragment thereof, or (ii) an anti-IL-11Rα antibody or an antigen-binding fragment thereof.

2. The use according to claim 1, characterized in that The composition also includes N-acetylcysteine.

3. The use according to claim 1 or 2, characterized in that The acetaminophen-induced liver injury is characterized by upregulated expression of interleukin 11 (IL-11) or interleukin 11 receptor (IL-11R) in subjects suffering from the acetaminophen-induced liver injury.

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