CN117858726A

CN117858726A - Treatment and prevention of alcoholic liver disease

Info

Publication number: CN117858726A
Application number: CN202280052768.6A
Authority: CN
Inventors: M·埃芬伯格; H·蒂尔格
Original assignee: Boehringer Ingelheim International GmbH
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 2021-07-26
Filing date: 2022-07-26
Publication date: 2024-04-09
Also published as: GB202110862D0

Abstract

The present invention discloses methods of treating or preventing alcoholic liver disease comprising administering to a subject a therapeutically or prophylactically effective amount of an agent capable of inhibiting interleukin II (IL-ll) mediated signaling.

Description

Treatment and prevention of alcoholic liver disease

The present application claims priority from GB 2110721.4 filed on 7.26 of 2021 and GB 2110862.6 filed on 7.28 of 2021, the contents and elements of which are incorporated herein by reference for all purposes.

Technical Field

The present invention relates to diagnosis, treatment and prevention of alcoholic liver disease.

Background

Alcoholic Hepatitis (AH) reflects an acute exacerbation of Alcoholic Liver Disease (ALD), an increasing worldwide medical burden with extremely limited treatment options.

Long-term drinking can cause physical and mental disabilities, resulting in death of about 300 tens of thousands of people worldwide each year. Overall, alcohol abuse accounts for 5.1% of the global disease burden [ lancet (2018) 392): 1015-1035]. Alcoholic Liver Disease (ALD) is a common liver disease, a complex pro-inflammatory process that can lead to steatosis, alcoholic Hepatitis (AH), fibrosis, cirrhosis, and ultimately hepatocellular carcinoma [ Asrani et al, J liver disease (2019) 70:151-171]. AH reflects in particular the acute exacerbation of alcoholic liver disease and is a worldwide increasing medical burden with extremely limited treatment options.

The pathogenesis of alcoholic liver disease (especially AH) is poorly understood, which is reflected in few treatment options and patient prognosis. In addition to the direct toxicity of ethanol and acetaldehyde, ethanol also causes dysregulation of the intestinal flora, leading to altered intestinal barrier function and endotoxemia [ Avila et al, intestinal tract (2020) 69:764-780; lang et al, intestinal microorganisms (2020) 12:1785251; parlesak et al, J liver disease (2000) 32:742-747]. This disorder also induces overexpression of different pro-inflammatory liver cytokines, leading to cell necrosis and liver injury [ Tilg and Diehl, england journal of medicine (2000) 343:1467-1476]. Interleukin (IL) -6, tumor necrosis factor-alpha (TNF-alpha) and IL-1β drive ALD in experimental models [ Lopetuso et al, liver J.International molecular science (2018) 19; schmidt-Arras and Rose-John, J hepatology (2016) 64:1403-1415; barbier et al, immunological front (2019) 10: 2014).

IL-11 mediated signaling has recently been implicated in the pathology of nonalcoholic liver disease [ Widjaja et al gastroenterology (2019) 157:777-792.E714], however the role of IL-11 mediated signaling in alcoholic liver disease is not yet clear.

Summary of The Invention

In a first aspect, the invention provides a formulation capable of inhibiting interleukin 11 (IL-11) -mediated signaling, for use in a method of treating or preventing alcoholic liver disease.

Also provided is a formulation capable of inhibiting interleukin 11 (IL-11) mediated signaling for use in the manufacture of a medicament for use in a method of treating or preventing alcoholic liver disease.

Also provided is a method of treating or preventing alcoholic liver disease comprising administering to a subject a therapeutically or prophylactically effective amount of an agent capable of inhibiting interleukin 11 (IL-11) -mediated signaling.

In some embodiments, the formulation is a formulation capable of preventing or reducing the binding of interleukin 11 (IL-11) to interleukin 11 (IL-11R) receptors.

In some embodiments, the formulation is capable of binding to interleukin 11 (IL-11) or interleukin 11 receptor (IL-11R).

In some embodiments, the formulation is selected from the group consisting of: an antibody or antigen binding fragment thereof, a polypeptide, a peptide, a nucleic acid, an oligonucleotide, an aptamer, or a small molecule.

In some embodiments, the agent is an antibody or antigen binding fragment thereof.

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 antibody or antigen binding fragment comprises:

(i) A heavy chain Variable (VH) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:34 HC-CDR1

Contains the amino acid sequence of SEQ ID NO: HC-CDR2 of 35

Contains the amino acid sequence of SEQ ID NO:36 HC-CDR3; and

(ii) A light chain Variable (VL) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:37 LC-CDR1

Contains the amino acid sequence of SEQ ID NO:38 LC-CDR2

Contains the amino acid sequence of SEQ ID NO:39 LC-CDR3.

In some embodiments, the antibody or antigen binding fragment comprises:

(i) A heavy chain Variable (VH) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:40 HC-CDR1

Contains the amino acid sequence of SEQ ID NO:41 HC-CDR2

Contains the amino acid sequence of SEQ ID NO:42 HC-CDR3; and

(ii) A light chain Variable (VL) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:43 LC-CDR1

Contains the amino acid sequence of SEQ ID NO:44 LC-CDR2

Contains the amino acid sequence of SEQ ID NO:45 LC-CDR3.

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

In some embodiments, the antibody or antigen binding fragment comprises:

(i) A heavy chain Variable (VH) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:46 HC-CDR1

Contains the amino acid sequence of SEQ ID NO:47 HC-CDR2

Contains the amino acid sequence of SEQ ID NO:48 HC-CDR3; and

(ii) A light chain Variable (VL) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:49 LC-CDR1

Contains the amino acid sequence of SEQ ID NO:50 LC-CDR2

Contains the amino acid sequence of SEQ ID NO:51 LC-CDR3.

In some embodiments, the formulation is a decoy receptor.

In some embodiments, the agent is a decoy receptor for IL-11.

In some embodiments, the bait 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-11 ra.

In some embodiments, the agent is an IL-11 mutein.

In some embodiments, the IL-11 mutein is W147A.

In some embodiments, the formulation is capable of preventing or reducing the expression of interleukin 11 (IL-11) or interleukin 11 receptor (IL-11R).

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

In some embodiments, the agent is an antisense oligonucleotide capable of preventing or reducing expression of IL-11.

In some embodiments, the antisense oligonucleotide capable of preventing or reducing expression of IL-11 is an siRNA targeting IL11, comprising SEQ ID NO: 12. 13, 14 or 15.

In some embodiments, the agent is an antisense oligonucleotide capable of preventing or reducing expression of IL-11Rα.

In some embodiments, the antisense oligonucleotide capable of preventing or reducing expression of IL-11 ra is an siRNA targeting IL11 ra, comprising SEQ ID NO: 16. 17, 18 or 19.

In some embodiments, the interleukin 11 receptor is or comprises IL-11 ra.

In some embodiments, the methods comprise administering the formulation to a subject whose interleukin 11 (IL-11) or IL-11 receptor (IL-11R) expression is up-regulated.

In some embodiments, the methods comprise administering the formulation to a subject that has been determined to have up-regulated interleukin 11 (IL-11) or interleukin 11 receptor (IL-11R) expression.

In some embodiments, the methods comprise determining whether the expression of interleukin 11 (IL-11) or IL-11 receptor (IL-11R) is up-regulated in a subject, and administering the formulation to a subject in which the expression of interleukin 11 (IL-11) or IL-11 receptor (IL-11R) is up-regulated.

Description of the invention

In the present invention, the inventors determined IL-11 mediated signaling as a driver of the pathology of Alcoholic Liver Disease (ALD). In the ALD model, an up-regulation of IL-11 expression was found in mouse livers. Treatment of mice with ALD with antibody antagonists of IL-11 mediated signaling has been shown to alleviate symptoms of ALD. Thus, the present invention identifies IL-11/IL-11 receptor signaling as a therapeutic target for ALD and demonstrates that IL-11 mediated antagonism of signaling is a suitable intervention in ALD.

Interleukin 11 and IL-11 receptor

Interleukin 11 (IL-11), also known as adipogenesis inhibitory factor, is a member of the pleiotropic cytokine and IL-6 cytokine families, including IL-6, IL-11, IL-27, IL-31, oncostatin M (OSM), leukemia Inhibitory Factor (LIF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), ciliary neurotrophic factor (CNTF) and neuropeptides (NP-1).

Interleukin 11 (IL-11) is expressed in a variety of mesenchymal cell types. IL-11 genomic sequences have been mapped to the centromere region of chromosome 19 and chromosome 7 and transcribed with classical signal peptides to ensure efficient secretion by cells. The activator protein complex cJun/AP-1 of IL-11 is located within its promoter sequence and is critical for the basal transcriptional regulation of IL-11 (Du and Williams., blood 1997, vol 89:3897-3908). The immature form of human IL-11 is a 199 amino acid polypeptide, whereas the mature form of IL-11 encodes a 178 amino acid residue protein (Garers and Scheller., biochemistry 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 (olpride) is commercially available. IL-11 from other species, including mice, rats, pigs, cattle, most teleost and primate species, have also been cloned and sequenced.

In the present specification, "IL-11" refers to IL-11 from any species and includes subtypes, fragments, variants or homologs of IL-11 from any species. In a preferred embodiment, the species is human (homo sapiens). A subtype, fragment, variant or homologue of IL-11 may optionally be characterized as having at least 70% identity, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence with the amino acid sequence of immature or mature IL-11 from a given species (e.g. human). Subtypes, fragments, variants or homologs of IL-11 can be selectively characterized by binding IL-11Rα (preferably from the same species) and the ability to stimulate signal transduction in cells to express IL-11Rα and gp130 (e.g., as in Curtis et al blood, 1997, 90 (11); or Karpovich et al, molecular human reproduction 2003,9 (2): 75-80). The IL-11 fragment may be of any length (by the number of amino acids), although at least 25% of the length of the mature IL-11 may optionally be present, and one of the mature IL-11 lengths having 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of the mature IL-11. The minimum length of an IL-11 fragment may be 10 amino acids and the maximum length may be 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.

IL-11 signals via dimers of ubiquitously expressed glycoprotein 130 (gp 130, also known as glycoprotein 130, IL-6ST, IL-6-. Beta.or CD 130). Gp130 is a transmembrane protein that forms a subunit of the type I cytokine receptor with the IL-6 receptor family. Specificity is obtained by a single interleukin 11 receptor subunit α (IL-11 ra) that is not directly involved in signal transduction, whereas the initial cytokine binding event α receptor leads to the formation of the final complex gp 130.

Human gp130 (a 22 amino acid signal peptide) is a 918 amino acid protein, in mature form 866 amino acids, comprising 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 a Cytokine Binding Module (CBM) of gp 130. The gp130 CBM comprises the Ig-like domain D1 and fibronectin type III domains D2 and D3 of gp 130. The amino acid sequence of human gp130 is available under UniProt accession number P40189-1 (SEQ ID NO: 2).

Human IL-11Rα is a polypeptide consisting of 422 amino acids (UniProt Q14626; SEQ ID NO: 3) and has-85% nucleotide and amino acid sequence identity with mouse IL-11Rα. Two subtypes of IL-11Rα have been reported that differ in the cytoplasmic domain (Du and Williams, supra). The IL-11 receptor alpha chain (IL-11 Ralpha) shares many similarities in structure and function with the IL-6 receptor alpha chain (IL-6 Ralpha). The extracellular domains exhibit 24% amino acid identity, including a characteristic conserved Trp-Ser-X-Trp-Ser (WSXWS) motif. Short cytoplasmic domains (34 amino acids) lack the Box1 and 2 regions required to activate JAK/STAT signaling pathways.

The receptor binding site on mouse IL-11 has been mapped and three sites have been identified-sites I, II and III. Binding to gp130 is reduced by substitution in the site II region and substitution in the site III region. Site III mutants did not detect agonist activity and had IL-11Rα antagonist activity (Chapter 8 of cytokine inhibitors; edited by Gennaro Ciliberto and Rocco Savino, marcel Dekker, inc., 2001).

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

The IL-11 receptor may be from any species, including subtypes, fragments, variants, or homologs of the IL-11 receptor from any species. In a preferred embodiment, the species is human (homo sapiens).

In some embodiments, the IL-11 receptor may be IL-11Rα. In some embodiments, the receptor for IL-11 can be a polypeptide complex comprising IL-11Rα. In some embodiments, the IL-11 receptor can be a polypeptide complex comprising IL-11Rα and gp 130. In some embodiments, the IL-11 receptor may be gp130 or comprise a complex that binds gp130 to IL-11.

A subtype, fragment, variant or homologue of IL-11 ra may optionally be characterized as having at least 70% identity, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence with the amino acid sequence of IL-11 ra from a given species (e.g. human). The isoforms, fragments, variants or homologues of IL-11Rα may optionally be characterized by binding IL-11 (preferably from the same species) and stimulating signal transduction in cells that express IL-11Rα and gp130 (e.g., as in Curtis et al blood, 1997, 90 (11) or Karpovich et al, human molecular reproduction, 2003 9 (2): 75-80). The IL-11 receptor fragment may be of any length (number of amino acids), although it may optionally be at least 25% of the length of mature IL-11Rα and the maximum length is 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 IL-11 receptor fragment has a minimum length of 10 amino acids and a maximum length of one of 15, 20, 25, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, or 415 amino acids.

IL-11 signaling

IL-11 binds IL-11Rα with low affinity (Kd 22nM; see Metcalfe et al, JBC (2020) manuscript RA 119.012351), and the interaction between these binding partners alone is insufficient to transduce biological signals. Co-expression of IL-11Rα and gp130 is required for the production of high affinity receptors (Kd 400 to 800 pmol/L) capable of signal transduction (Curtis et al, blood 1997;90 (11): 4403-12; hilton et al, european journal of molecular biology tissue, 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 transduction and transcription activator (Jak/STAT) pathway (Garers and Scheller, supra).

In principle, soluble IL-11Rα may also form a biologically active soluble complex with IL-11 (Pflnz et al, 1999 february letters, 450, 117-122), which increases the likelihood that, like IL-6, IL-11 may in some cases bind to soluble IL-11Rα before it binds to cell surface gp130 (Garers and Scheller, supra). Curtis et al (blood 1997, 12, 1; 90 (11): 4403-12) describe the expression of the soluble mouse IL-11 receptor alpha chain (sIL-11R) and examined the signaling of gp130 in cells. In the presence of gp130 but in the absence of transmembrane IL-11R, sIL-11R mediates IL-11 dependent differentiation of M1 leukemia cells and proliferation of Ba/F3 cells and early intracellular events, including phosphorylation of gp130, STAT3 and SHP2, similar to signaling through transmembrane IL-11R. It has recently been demonstrated that cell membrane binding gp130 activates signaling through IL-11 binding to soluble IL-11Rα (Lokau et al, 2016 cell report 14, 1761-1773). This so-called IL-11 trans-signaling may be important for disease pathogenesis, but its role in human disease has not been studied.

As used herein, "IL-11 trans-signaling" is used to refer to signaling triggered by the binding of IL-11, which binds to IL-11Rα, to gp 130. The IL-11 can bind to IL-11Rα as a non-covalent complex. The gp130 is membrane bound and is found in IL-11: the IL-11Rα complex is expressed by cells that undergo signaling after binding to gp 130. In some embodiments, the IL-11Rα may be a soluble IL-11Rα. In some embodiments, the soluble IL-11Rα is a soluble (secreted) subtype of IL-11Rα (e.g., lacks a transmembrane domain). In some embodiments, the soluble IL-11Rα is a release product of proteolytic cleavage of the cell membrane extracellular domain that binds IL-11Rα. In some embodiments, the IL-11Rα can bind to a cell membrane and trigger gp130 signaling by IL-11 binding to the cell membrane, referred to as "IL-11 cis signaling". In a preferred embodiment, inhibition of IL-11 mediated signaling is achieved by disrupting IL-11 mediated cis-signaling.

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

The physiological role of interleukin 11 (IL-11) is not yet clear. IL-11 is closely related to the activation of hematopoietic cells and thrombopoiesis. IL-11 has also been shown to prevent graft versus host disease, inflammatory arthritis and inflammatory bowel disease, resulting in IL-11 being considered an anti-inflammatory cytokine (Putoczki and Ernst, journal of white blood cell biology, 2010, 88 (6): 1109-1117). However, it is believed that IL-11 has a pro-inflammatory effect with anti-inflammatory, pro-angiogenic effects and tumors are important. Recent studies have shown that IL-11 is readily detectable in virus-induced inflammation in mouse models of arthritis and cancer, suggesting that IL-11 expression may be induced by pathological stimuli. IL-11 is also associated with Stat 3-dependent activation of tumor promoting target genes in neoplastic gastrointestinal epithelial cells (Putoczki and Ernst, supra).

As used herein, "IL-11 signaling" and "IL-11 mediated signaling" refer to signaling mediated by binding IL-11 or a fragment thereof having the function of a mature IL-11 molecule to the IL-11 receptor. It will be appreciated that "IL-11 signaling" and "IL-11 mediated signaling" refer to signaling initiated by IL-11/functional fragments thereof, such as by binding to the IL-11 receptor. "Signal transduction" in turn refers to signal transduction and other cellular processes that control cellular activity.

Alcoholic liver disease

The present invention relates to the treatment and/or prevention of Alcoholic Liver Disease (ALD).

As used herein, "alcoholic liver disease" (ALD) refers to any disease/disorder associated with (e.g., caused by or characterized by) an alcohol-induced perturbation to normal (i.e., healthy, non-diseased) liver function and/or morphology. ALD may be characterized by alcohol-induced liver damage, alcohol-induced liver tissue damage, and/or alcohol-induced one or more hepatocyte damage.

ALD includes alcoholic fatty liver (AFL, also known as alcoholic liver steatosis), alcoholic hepatitis (including Alcoholic Steatohepatitis (ASH)), alcoholic liver fibrosis, alcoholic liver cirrhosis, and alcoholic liver cancer (e.g., hepatocellular carcinoma). Thus, in some embodiments according to aspects and embodiments described herein, the alcoholic liver disease is selected from: alcoholic Fatty Liver (AFL), alcoholic hepatitis, alcoholic Steatohepatitis (ASH), alcoholic liver fibrosis, alcoholic cirrhosis and alcoholic liver cancer (e.g. alcoholic hepatocellular carcinoma). In some embodiments, the alcoholic liver disease is selected from: alcoholic Fatty Liver (AFL), alcoholic hepatitis and Alcoholic Steatohepatitis (ASH).

ALD in Seitz et al, nature review-disease guide (2018) 4:16, seitz et al, journal of clinical oncology. Med (2021) 10:858 and Osna et al, alcohol research, 2017;38 (2): 147-161, all of which are incorporated by reference in their entirety. Alcoholic liver disease is caused by (excessive) drinking and may also be referred to as alcohol-related liver disease (ARLD). According to the present invention, excessive drinking may refer to a male subject regularly (e.g., 3 or more days/week) taking up to or about 40 grams of ethanol per day, and a female subject regularly taking up to or about 20 grams of ethanol per day.

Long-term (excessive) drinking can lead to accumulation of fat (mainly in the form of triglycerides, phospholipids and cholesterol esters) in hepatocytes (i.e. hepatocyte steatosis), leading to AFL.

AFL may progress to Alcoholic Steatohepatitis (ASH) characterized by liver tissue damage and liver inflammation (i.e., hepatitis). More specifically, ASH may be characterized by hepatocyte injury (accompanied by increased serum transaminase activity), hepatocyte distension, the presence of Mallory-Denk corpuscles in the hepatocytes, lobular inflammation, infiltration of activated Kupffer cells and/or granulocytes (especially neutrophils) into the liver tissue.

ASH may in turn progress to alcohol-induced liver fibrosis, alcohol-induced cirrhosis, characterized by excessive deposition of extracellular matrix components by activated, early-fibrillated Hepatic Stellate Cells (HSCs). Early alcoholic liver fibrosis is often manifested as pericellular fibrosis, characterized by extracellular matrix deposition along the antrum of the liver and around small populations of hepatocytes. Advanced fibrosis and cirrhosis are characterized by extensive fibrosis, narrowing of the hepatic vasculature (including sinuses), portal hypertension, hepatocyte death, and severe impairment of liver function (liver failure). Alcohol-induced fibrosis and cirrhosis may further cause hepatocellular carcinoma (HCC); in fact, cirrhosis is the most effective risk factor for the development of hepatocellular carcinoma.

A subject with an alcoholic liver disease may have one or more symptoms/associations of an alcoholic liver disease. In some embodiments, a subject with alcoholic liver disease may have one or more of the following: liver cell steatosis, liver cell injury, liver tissue injury, hepatitis, elevated serum aspartate Aminotransferase (AST), elevated serum alanine Aminotransferase (ALT), balloon-like enlargement of liver cells, including Mallory-Denk corpuscles, lobular inflammation, activated Kupffer cells, liver tissue including infiltration of granulocytes (e.g., neutrophils), liver fibrosis, activated hematopoietic stem cells, pericellular fibrosis, cirrhosis, stenosis of the hepatic vascular system, portal hypertension, hepatocyte death, liver failure, and hepatocellular carcinoma (HCC).

A subject with alcoholic liver disease may have been diagnosed with alcoholic liver disease. The subject may meet diagnostic criteria for the diagnosis of alcoholic liver disease. Diagnosis of alcoholic liver disease is described in Toruella et al, journal of world gastroenterology (2014) 20 (33): 11684-11699, which are incorporated herein by reference in their entirety. For patients with a history of high alcohol consumption, alcoholic liver disease can often be diagnosed based solely on clinical and laboratory characteristics, wherein other causes of chronic liver disease have been eliminated. In general, patients with a significant history of alcohol consumption and with an abnormality in serum transaminase (in particular serum AST levels higher than serum ALT levels), hepatomegaly, clinical signs of chronic liver disease, imaging evidence of liver steatosis or fibrosis/cirrhosis, or liver biopsies showing large vesicular steatosis or cirrhosis should be suspected to have ALD.

The genetic risk factors for ALD have been determined by whole genome association studies, including gene variants of PNPLA3 (e.g., rs738409-g, M148I), TM6SF2 (e.g., rs58542926-T, E167K), MBOAT7 (e.g., rs 641738-T), MARC1 (e.g., rs 2642438-C/g/T), and HNRNPUL1 (e.g., rs 15052-C). In some embodiments, a subject with alcoholic liver disease according to the present disclosure may comprise one or more copies of one or more of the following alleles: PNPLA3 containing rs738409-G, TM6SF2 containing rs58542926-T, MBOAT7 containing rs641738-T, MARC1 containing rs2642438-C/G/T and HNRNPUL1 containing rs 15052-C.

Preparation capable of inhibiting IL-11 action

Aspects of the invention relate to inhibiting/antagonizing IL-11 mediated signaling.

Herein, "inhibition" refers to a decrease, decrease or alleviation relative to control conditions. For example, inhibition of the effect of IL-11 by an agent capable of inhibiting IL-11 mediated signaling refers to a decrease, or alleviation of the extent/extent of IL-11 mediated signaling in the absence of the agent and/or in the presence of an appropriate control agent.

Inhibition may also be referred to herein as neutralization or antagonism. That is, an agent that is capable of inhibiting IL-11-mediated signaling (e.g., an interaction, signaling, or other activity mediated by IL-11 or an IL-11-containing complex) can be said to be a "neutralizing" agent or "antagonist" with respect to a related function or process. For example, an agent capable of inhibiting IL-11 mediated signaling may be referred to as an agent capable of neutralizing IL-11 mediated signaling, or may be referred to as an antagonist of IL-11 mediated signaling.

The IL-11 signaling pathway provides a variety of pathways that 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, with or necessarily through signaling of the IL-11 receptor.

For example, inhibition of IL-11 signaling can be achieved by disrupting the interaction of IL-11 (or a complex containing IL-11, e.g., a complex of IL-11 and IL-11Rα) and an IL-11 receptor (e.g., IL-11Rα, a receptor complex comprising IL-11Rβ, gp130, or a receptor complex comprising IL-11Rα and gp 130). In some embodiments, inhibiting IL-11 mediated signaling is achieved by inhibiting the expression of a gene or protein, such as one or more of IL-11, IL-11Rα and gp 130.

Inhibition of IL-11 mediated signaling may also be achieved by disrupting the interaction between the IL-11:11 receptor complex (i.e., a complex comprising IL-11 and IL-11Rα, or IL-11 and gp130, or IL-12, IL-11Rα and gp 130) to form a multimer (e.g., a hexameric complex) that is required for cells expressing the IL-11 receptor to activate downstream signaling.

In embodiments, inhibition of IL-11 mediated signaling is achieved by disrupting IL-11 mediated cis-signaling without disrupting IL-11 mediated trans-signaling, e.g., by inhibiting gp130 mediated cis-complexes of IL-11Rα that involve membrane binding. In embodiments, inhibition of IL-11 mediated signaling is achieved by disrupting IL-11 mediated trans-signaling without disrupting IL-11 mediated cis-signaling, i.e., inhibition of IL-11 mediated signaling is achieved by inhibiting gp130 mediated cross-signaling complexes, such as IL-11 binding to soluble IL-11Rα or IL-6 binding to soluble IL-6R. In embodiments, inhibition of IL-11-mediated signaling is achieved by disrupting IL-11-mediated cis-signaling and IL-11-mediated trans-signaling. Any of the agents described herein may be used to inhibit IL-11-mediated cis and/or trans signaling.

In other embodiments, inhibition of IL-11 signaling may be achieved by disrupting a signaling pathway downstream of IL-11/IL-11Rα/gp 130. That is, in some embodiments, inhibition/antagonism of IL-11 mediated signaling includes inhibition of signaling pathways/processes/factors downstream through IL-11/IL-11 receptor complex signaling.

In some embodiments, inhibition/antagonism of IL-11 mediated signaling includes inhibition of signaling through an intracellular signaling pathway activated by the IL-11/IL-11 receptor complex. In some embodiments, inhibition/antagonism of IL-11 mediated signaling includes inhibition of one or more factors whose expression/activity is up-regulated due to signaling through the IL-11/IL-11 receptor complex.

In some embodiments, the methods described herein use an agent capable of inhibiting JAK/STAT signaling. In some embodiments, an agent capable of inhibiting JAK/STAT signaling is capable of inhibiting the effects of JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT B, and/or STAT 6. For example, the formulation may be capable of inhibiting the activity of JAK/STAT proteins, inhibiting the interaction of JAK or STAT proteins with cell surface receptors such as IL-11 ra or gp130, inhibiting the phosphorylation of JAK proteins, inhibiting the interaction between JAK and STAT proteins, inhibiting the phosphorylation of STAT proteins, inhibiting dimerization of STAT-proteins, inhibiting translocation of STAT proteins to the nucleus, inhibiting binding of STAT proteins to DNA, and/or promoting degradation of JAK and/or STAT proteins. In some embodiments, the JAK/STAT inhibitor is selected from the group consisting of ruxolitinib (Jakafi/Jakavi; incyte), tofacitinib (Xejanz/Jakvinus; NIH/Pfizer; gilead Sciences), pacritinib (SB 1518; CTI), PF-04965842 (trim), upadacitinib (ABT-494; abbVie), oxcarbacetinib (apoque), baricitinib (Olumian; incyte/Eli Lilly), non-gotinib (G-146034/GLPG-0634;Galapagos NV), gan Duo tinib (LY-2784544;Eli Lilley), litatinib (CEP-701; teva), mo Meiluo tinib (GS-0387/CYT-387;Gilead Sciences), pacritinib (SB 1518; PF-04965842 (Pfiir), wu Pati tinib (ABT-494), non-peinib (AbK/ASP-36; JNI), JNI (Celite-86124; JNI-124, JNI-37124).

In some embodiments, the methods described herein employ agents capable of inhibiting MAPK/ERK signaling. In some embodiments, the agent capable of inhibiting MAPK/ERK signaling is capable of inhibiting the effects of GRB2, inhibiting the effects of RAF kinase, inhibiting the effects of MEK protein, inhibiting the activation of MAP3K/MAP2K/MAPK and/or Myc, and/or inhibiting phosphorylation of STAT protein. In some embodiments, an agent capable of inhibiting ERK signaling is capable of inhibiting ERKp42/44. In some embodiments, the ERK inhibitor is selected from the group consisting of SCH772984, SC1, VX-11e, DEL-22379, sorafenib (Nexavar; bayer/Onyx), SB590885, PLX4720, XL281, RAF265 (Novartis), enofenib (LGX 818/Braftovi; array BioPharma), semantenib (AZD 6244; array/Azikang) and tramatinib (GSK 1120212/Mejist; norhua). In some embodiments, the methods described herein use an agent capable of inhibiting c-Jun N-terminal kinase (JNK) signaling/activity. In some embodiments, an agent capable of inhibiting JNK signaling/activity is capable of inhibiting the effects and/or phosphorylation of JNK (e.g., JNK1, JNK 2). In some embodiments, the JNK inhibitor is selected from SP600125, CEP 1347, TCS JNK 6o, c-JUN peptide, SU3327, AEG 3482, TCS JNK5a, BI78D3, IQ3, SR3576, IQ1S, JIP-1 (153-163), and CC401 dihydrochloride.

In this example, the inventors demonstrated that NOX4 expression and activity is upregulated by IL-11/IL-11Rα/gp130 signaling. NOX4 is an NADPH oxidase, and is also a source of Reactive Oxygen Species (ROS). Up-regulating Nox4 expression in transgenic mice with hepatocyte-specific Il11 expression, and up-regulating Nox4 expression in primary human hepatocytes stimulated with Il 11.

In some embodiments, the invention employs agents capable of inhibiting NOX4 expression (gene or protein expression) or function. In some embodiments, the invention employs agents capable of inhibiting IL-11 mediated upregulation of NOX4 expression/function. Agents capable of inhibiting NOX4 expression or function may be referred to herein as NOX4 inhibitors. For example, a NOX4 inhibitor may be capable of reducing expression of NOX4 (e.g., gene and/or protein expression), reducing the level of RNA encoding NOX4, reducing the level of NOX4 protein, and/or reducing the level of NOX44 activity (e.g., reducing NOX 4-mediated NADPH oxidase activity and/or NOX 4-mediated ROS production).

NOx4 inhibitors include NOx4 binding molecules and molecules capable of reducing the expression of NOx 4. Inhibitors of NOX4 binding include peptide/nucleic acid aptamers, antibodies (and antibody fragments) and fragments of interacting partners to NOX4 as antagonists of NOX4 function, and inhibitors of NOX4 small molecules. Molecules capable of reducing NOX4 expression include antisense RNAs (e.g., siRNA, shRNA) to NOX 4. In some embodiments, the NOX4 inhibitor is selected from the group consisting of Altenhofer et al antioxidants and redox signals (2015) 23 (5): 406-427 or Augsburder et al journal of redox biology (2019) 26:101272, as described in GKT137831 for NOX4 inhibitors.

Binding agent

In some embodiments, an agent capable of inhibiting IL-11-mediated signaling may bind to IL-11. In some embodiments, an agent capable of inhibiting IL-11 mediated signaling may bind to a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and/or gp 130). Binding of such agents may inhibit IL-11-mediated signaling by reducing/preventing the ability of IL-11 to bind to IL-11 receptors, thereby inhibiting downstream signaling. Binding of such agents may inhibit IL-11-mediated cis and/or trans signaling by reducing/preventing the ability of IL-11 to bind to IL-11 receptors (e.g., IL-11Rα and/or gp 130), thereby inhibiting downstream signaling. The formulation can bind to trans-signaling complexes such as IL-11 and soluble IL-11Rα and inhibit gp 130-mediated signaling.

The agent capable of binding to IL-11/an IL-11 containing complex or IL-11 receptor may be of any kind, but in some embodiments the agent may be an antibody, antigen binding fragment thereof, polypeptide, peptide, nucleic acid, oligonucleotide, aptamer or small molecule. The formulations may be provided in isolated or purified form, or may be formulated as pharmaceutical compositions or medicaments.

Antibodies and antigen binding fragments

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

In some embodiments, the agent capable of binding to IL-11/IL-11-containing complex or IL-11 receptor is an antibody or antigen-binding fragment thereof. "antibody" as used herein is the broadest sense and includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit binding to a target molecule of interest.

In view of the current technology associated with monoclonal antibody technology, antibodies to most antigens can be made. The antigen binding portion may be part of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single chain Fv fragment [ ScFv ]). Monoclonal antibodies directed against the selected antigen can be prepared by known techniques, for example, as described in "monoclonal antibodies: technical manual ", H Zola (CRC press, 1988) and" monoclonal hybridoma antibodies: techniques and applications ", J G R Hurrell (CRC Press, 1982). Chimeric antibodies are discussed by Neuberger et al (1988, section 2 of the International Biotechnology conference at 8, 792-799). Monoclonal antibodies (mabs) are particularly useful in the methods of the invention and are homogeneous populations of antibodies that specifically target a single epitope on an antigen.

Polyclonal antibodies may also be used in the methods of the invention. Monospecific polyclonal antibodies are preferred. Suitable polyclonal antibodies may be prepared using methods well known in the art.

Antigen binding fragments of antibodies, e.g., fab and Fab2 fragments, such as genetically engineered antibodies and antibody fragments, may also be used/provided. The Variable Heavy (VH) and Variable Light (VL) domains of the antibodies are involved in antigen recognition, a fact that was first recognized by early protease digestion experiments. This was further confirmed by "humanization" of rodent antibodies. The variable domains of rodent origin may be fused to constant domains of human origin such that the resulting antibodies retain the antigen specificity of the rodent parent antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).

Antibodies and antigen binding fragments according to the present disclosure comprise Complementarity Determining Regions (CDRs) of an antibody capable of binding to a related target molecule (i.e., IL-11/complex containing IL-11/receptor for IL-11).

Antibodies capable of binding IL-11 include, for example, monoclonal mouse anti-human IL-11 antibody clone #22266; catalog No. MAB218 (R & D Systems, MN, USA), for example, in Bockhorn et al Nature communications (2013) 4 (0): 1393, clone 6D9A (Abbiotec), clone KT8 (Abbiotech), clone M3103F11 (BioLegend), clone 1F1 (Abnova Corporation), clone 3C6 (Abnova Corporation), clone GF1 (LifeSpan Biosciences), clone 13455 (Source BioScience), 11h3/19.6.1 (Hermann et al, arthritis and rheumatic diseases (1998) 41 (8): 1388-97), AB-218-NA (R & DSsystems), X203 (Ng et al, science conversion medicine (2019) 11 (511) pii: eaaw 1237) and anti-IL-11 antibodies disclosed in US2009/0202533A1, WO 99/59608A2, WO 2018/109174A2 and WO 9/238882A 1.

In particular, anti-IL-11 antibody clone 22626 (also known as MAB 218) has been shown to be an antagonist of IL-11 mediated signaling, such as described in Schaefer et al Nature (2017) 552 (7683): 110-115. Monoclonal antibody 11h3/19.6.1 is disclosed in Hermann et al, arthritis and rheumatology (1998) 41 (8): 1388-97 as neutralizing anti-IL-11 IgG1. AB-218-NA from the R & D system, for example in McCoy et al, BMC cancer (2013) 13:16, is another example of neutralizing anti-IL-11 antibodies. Another exemplary anti-IL-11 antibody antagonist of IL-11 mediated signaling is disclosed in WO 2018/109174A2 and WO 2019/238882 A1. X203 (also called Enx 203) is disclosed in Ng et al, "IL-11 is a therapeutic target for idiopathic pulmonary fibrosis. "bioRxiv 336537; doi: https:// doi.org/10.1101/336537 and WO 2019/238882A1 are anti-IL-11 antibody antagonists of IL-11 mediated signaling and include the VH region of SEQ ID NO:92 according to WO 2019/2238882A1 (SEQ ID NO:22 of said patent) and the SEQ ID NO according to WO2019-238882 A1: 94 (SEQ ID NO:23 of said patent). Humanized versions of X203 are described in WO 2019/238882A1, including hENX203, which hENX203 comprises the VH region of SEQ ID NO:117 according to WO 2019/2238882A1 (SEQ ID NO:30 of the present disclosure) and the VL region of SEQ ID NO:122 according to WO2019-238882A1 (SEQ ID NO:31 of the disclosure). Enx108A is another example of an anti-IL-11 antibody antagonist of IL-11 mediated signaling and includes the VH region of SEQ ID NO:8 (SEQ ID NO:6 of the present disclosure) according to WO 2019/238882A1 and the VL region of SEQ ID NO:20 according to WO2019-238882A1 (SEQ ID NO:27 of the present disclosure).

Antibodies capable of binding IL-11Rα include, for example, monoclonal antibody clone 025 (Sino Biological), clone EPR5446 (Abcam), clone 473143 (R & D Systems), clones 8E2, 8D10 and 8E4, and affinity matured variants of 8E2 described in US2014/0219919A1, monoclonal antibodies described in Blanc et al (J.Immunol. 7.31; 241 (1-2); 43-59), X209 (Widjaja et al, gastroenterology (2019) 157 (3): 777-792), antibodies disclosed in WO 2014125 A1 and US 2019/03077 A1, and IL-11 neutralization therapies targeting hepatic stellate-induced liver inflammation and fibrosis in NASH, "bioRxiv 472; doi: https:// 10.1101/470062), also published in WO 2014125 A1 and US 2019/157/21333, WO 213170 A1 and WO 2019/2229 A1 and African antibody published in WO 2018/2138/gA1.

In particular, anti-IL-11 Rα antibody clone 473143 (also known as MAB 1977) has been shown to be an antagonist of IL-11 mediated signaling, such as described in Schaefer et al Nature (2017) 552 (7683): 110-115. US2014/0219919A1 provides sequences of anti-human IL-11 ra antibody clones 8E2, 8D10 and 8E4 and discloses their ability to antagonize IL-11 mediated signaling-see, e.g., [0489] to [0490] of US2014/0219919 A1. US2014/0219919A1 also provides sequence information for another 62 affinity matured variants of clone 8E2, 61 of which are disclosed for antagonizing IL-11 mediated signaling-see table 3 of US 2014/0229919 A1. Still another exemplary anti-IL-11 Rα antibody antagonist for IL-11 mediated signaling is disclosed in WO 2018/109170 A2 and WO 2019/238884 A1. X209 (also known as Enx 209) is disclosed in Widjaja et al, "IL-11 neutralization therapy targets hepatic stellate cell-induced liver inflammation and fibrosis in NASH. "bioRxiv 470062; doi: https:// doi.org/10.1101/470062 and WO 2019/238884A1 are anti-IL-11 ra antibody antagonists of IL-11 mediated signaling and include the VH region of SEQ ID NO:7 according to WO 2019/2238884A1 (SEQ ID NO:24 of the present disclosure) and the VL region of SEQ ID NO:14 according to WO 2019-238884A1 (SEQ ID NO:25 of the present disclosure). Humanized versions of X209 are described in WO 2019/238884A1, comprising hENX209, said hENX209 comprising the VH region of SEQ ID NO:11 according to WO 2019/2238884A1 (SEQ ID NO:32 of said invention) and the VL region of SEQ ID NO:17 according to WO 2019-238884A1 (SEQ ID NO:33 of said invention).

Techniques for producing antibodies suitable for therapeutic use in a given species/subject are well known to those skilled in the art. Methods of producing antibodies suitable for therapeutic use in humans are described, for example, in park and smolen protein chemistry research progression (2001) 56:369-421, incorporated herein by reference in its entirety.

Antibodies to a given target protein (e.g., IL-11 or IL-11Rα) can be cultured in model species (e.g., rodents, lagomorpha) and subsequently engineered to increase their suitability for therapeutic use in a given species/subject. For example, one or more amino acids of a monoclonal antibody generated by immunization of a model species may be substituted to obtain an antibody sequence more similar to a human germline immunoglobulin sequence (thereby reducing the potential of an anti-xenogenous antibody immune response treated with the antibody in a human subject). Modifications in the antibody variable domains may be concentrated on the framework regions to preserve the antibody binding sites. Antibody humanization is a common practice in the antibody arts, for example, reviewed in Almagro and Franson bioscience fronts (2008) 13:1619-1633, safdari et al Biotechnology and genetic engineering reviews (2013) 29 (2): 175-186 and Lo et al microbiology Spectrum (2014) 2 (1), all of which are incorporated herein by reference in their entirety. The requirement for humanization can be circumvented by culturing antibodies to a given target protein (e.g., IL-11 or IL-11Rα) in transgenic model species expressing human immunoglobulin genes, such that the antibodies cultured in these animals are entirely human (e.g., as described in Brucgamann et al immune experimental treatment Profile (Warsz) (2015) 63 (2): 101-108), which is incorporated herein by reference in its entirety).

Phage display techniques can also be used to identify antibodies to a given target protein (e.g., IL-11 or IL-11Rα) and are well known to those skilled in the art. Phage display the use of phage display for the identification of fully human antibodies to human target proteins is reviewed in, for example, hoogenboom Nature Biotechnology (2005) 231105-1116 and Chan et al International immunology (2014) 26 (12): 649-657, the entire contents of which are incorporated herein by reference.

The antibody/fragment may be an antagonist antibody/fragment that inhibits or reduces the biological activity of IL-11. The antibody/fragment may be a neutralizing antibody that neutralizes the biological effects of IL-11, e.g., its ability to generate signaling through stimulation of the IL-11 receptor. Neutralization activity can be measured by the ability to neutralize IL-11-induced proliferation in T11 mouse plasmacytoma cell lines (Nordan, R.P. et al (1987) J.Immunol.139:813).

IL-11-or IL-11Rα -binding antibodies may be used to assess the antagonistic capacity against IL-11-mediated signaling, for example using US2014/0219919 A1 or Blanc et al (journal of immunology methods, 7 month 31; 241 (1-2); 43-59. Briefly, IL-11-and IL-11Rα -binding antibodies may be used to assess their capacity to inhibit proliferation of Ba/F3 cells expressing IL-11R a and gp130 from the appropriate species in vitro in response to stimulation of IL-11 from the appropriate species. Alternatively, by assessing αSMA expression, the capacity of IL-11-and IL-11Rα -binding antibodies to inhibit the conversion of fibroblasts to myofibroblasts after stimulation of fibroblasts with TGF β1 may be analyzed in vitro (as in WO 2018/109174 A2 (example 6) and WO 2018/1097170 A2 (example 6), ng et al conversion medicine (2019) 11 (511: eaaw1237 and Wija 1237 et al (157) 777-157).

Antibodies typically comprise six CDRs; three in the light chain variable region (VL): LC-CDR1, LC-CDR2, LC-CDR3, and three in the heavy chain variable region: HC-CDR1, HC-CDR2 and HC-CDR3. The six CDRs together define the binding site of the antibody, i.e., the portion of the antibody that binds to the target molecule. The VH and VL regions include Framework Regions (FR) on either side of each CDR, providing a scaffold for the CDRs. From N-terminal to C-terminal, the VH region comprises the following structure: n-terminal- [ HC-FR1] - [ HC-DR1] - [ HC-FR2] - [ HC-RR3] - [ HC-CR4] -C-terminal; and the VL region comprises the structure: the N-terminus- [ LC-FR1] - [ LC-CDR1] - [ LC-FR2] - [ LC-CDR 2] - [ LC-FR 3] - [ LC-CD R3] - [ LC-FR4] -C-terminus.

There are several different conventions used to define antibody CDRs and FRs, such as the fifth edition of the immunoprotein sequences of interest to Kabat et al, public health service, national institutes of health, bethesda, MD (1991), chothia et al molecular biology journal 196:901-917 (1987), and VBASE2, such as the reference et al nucleic acid study (2005) 33 (journal 1): and D671-D674. The CDRs and FRs of the VH and VL regions of the antibodies described herein are defined according to the Kabat system.

In some embodiments, an antibody or antigen-binding fragment thereof according to the invention is derived from an antibody that specifically binds IL-11 (e.g., enx108A, enx203 or hEnx 203). In some embodiments, an antibody or antigen binding fragment thereof according to the present disclosure is derived from an antibody that specifically binds IL-11 ra (e.g., enx209 or hEnx 209).

Antibodies and antigen binding fragments according to the invention preferably inhibit IL-11 mediated signaling. Such antibody/antigen binding fragments may be described as antagonists of IL-11 mediated signaling and/or may be described as having the ability to neutralize IL-11 mediated signaling.

In some embodiments, the antibody/antigen binding fragment comprises CDRs of an antibody that binds IL-11. In some embodiments, the antibody/antigen binding fragment comprises the CDRs of an IL-11-binding antibody described herein (e.g., enx108A, enx203 or hEnx 203) or CDRs derived therefrom.

In some embodiments, the antibody/antigen binding fragment comprises a VH region incorporating the following CDRs:

(1)

HC-CDR1 having the amino acid sequence of SEQ ID NO 34

HC-CDR2 having the amino acid sequence of SEQ ID NO. 35

HC-CDR3 having the amino acid sequence of SEQ ID NO. 36,

or a variant thereof, wherein one, two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are substituted with another amino acid.

In some embodiments, the antibody/antigen binding fragment comprises a VL region that incorporates the following CDRs:

(2)

LC-CDR1 having the amino acid sequence of SEQ ID NO 37

LC-CDR2 having the amino acid sequence of SEQ ID NO 38

LC-CDR3 having the amino acid sequence of SEQ ID NO 39,

or a variant thereof, wherein one, two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

(3)

HC-CDR1 having the amino acid sequence of SEQ ID NO. 40

HC-CDR2 having the amino acid sequence of SEQ ID NO. 41

HC-CDR3 having the amino acid sequence of SEQ ID NO. 42,

(4)

LC-CDR1 having the amino acid sequence of SEQ ID NO. 43

LC-CDR2 having the amino acid sequence of SEQ ID NO 44

LC-CDR3 having the amino acid sequence of SEQ ID NO. 45,

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

In some embodiments, the antibody/antigen binding fragment comprises a VH region and a VL region of an antibody that binds IL-11. In some embodiments, the antibody/antigen-binding fragment comprises the VH and VL regions, or derived VH and VL regions, of an IL-11 binding antibody described herein (e.g., enx108A, enx203 or hEnx 203).

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

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

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

In some embodiments, the antibody/antigen binding fragment comprises CDRs that bind to an IL-11 ra antibody. In some embodiments, the antibody/antigen binding fragment comprises the CDRs of or derived from an IL-11 ra binding antibody described herein (e.g., enx209 or hEnx 209).

(5)

HC-CDR1 having the amino acid sequence of SEQ ID NO. 46

HC-CDR2 having the amino acid sequence of SEQ ID NO. 47

HC-CDR3 having the amino acid sequence of SEQ ID NO. 48,

(6)

LC-CDR1 having the amino acid sequence of SEQ ID NO. 49

LC-CDR2 having the amino acid sequence of SEQ ID NO. 50

LC-CDR3 having the amino acid sequence of SEQ ID NO. 51,

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

In some embodiments, the antibody/antigen binding fragment comprises a VH region and a VL region of an antibody that binds IL-11 ra. In some embodiments, the antibody/antigen binding fragment comprises the VH and VL regions of an IL-11 ra binding antibody described herein (e.g., enx209 or hEnx 209), or VH and VL regions derived thereof.

In embodiments according to the invention wherein one or more amino acids of a reference amino acid sequence (e.g., a CDR sequence, VH region sequence, or VL region sequence as described herein) are substituted with another amino acid, the substitution may be a conservative substitution, e.g., according to the table below. In some embodiments, the amino acids in the same block in the middle are substituted. In some embodiments, the amino acids in the same row in the rightmost column are substituted:

in some embodiments, the substitutions may be function-conservative. That is, in some embodiments, the substitution may not affect (or substantially not affect) one or more functional properties (e.g., target binding) of the antibody/fragment comprising the substitution relative to an equivalent unsubstituted molecule.

In some embodiments, substitutions relative to a reference VH region or VL region sequence may be concentrated in one or more specific regions of the VH region or VL region. For example, changes in the sequence of the reference VH or VL regions may be concentrated in one or more framework regions (FR 1, FR2, FR3 and/or FR 4).

Antibodies and antigen binding fragments according to the invention may be designed and prepared using monoclonal antibody (mAb) sequences capable of binding to the relevant target molecule. Antigen binding regions of antibodies, such as single chain variable fragments (scFv), fab and Fab2 fragments, may also be used/provided. An "antigen binding region" or "antigen binding fragment" refers to any fragment of an antibody that is capable of binding to a target specific for a given antibody.

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

The VL and light chain Constant (CL) regions of the antigen binding region of the antibody together with the VH and heavy chain constant 1 (CH 1) regions constitute the Fab region. In some embodiments, the antibody/fragment comprises or consists of a Fab region of an antibody which is capable of binding to IL-11, a complex containing IL-11, or a receptor for IL-11.

In some embodiments, the antibody/fragment comprises or consists of a whole antibody capable of binding to IL-11, a complex comprising IL-11, or a receptor for IL-11. "whole antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Different classes of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini allergy and journal of clinical immunology (2010) 125 (202): S41-S52, the entire contents of which are incorporated herein by reference. The type G immunoglobulin (i.e., igG) is a glycoprotein of about-150 kDa, comprising two heavy chains and two light chains. From N-terminal to C-terminal, the heavy chain comprises VH, followed by a heavy chain constant region comprising three constant domains (CH 1, CH2, and CH 3), and similarly, the light chain comprises VL, followed by CL. Immunoglobulins can be classified as IgG (e.g., igG1, igG2, igG3, igG 4), igA (e.g., igA1, igA 2), igD, igE, or IgM, depending on the heavy chain. The light chain may be kappa or lambda.

In some embodiments, the antibodies/antigen binding fragments of the invention comprise an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence may be a human immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from an IgG (e.g., igG1, igG2, igG3, igG 4), igA (e.g., igA1, igA 2), igD, igE, or IgM, e.g., a human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG 4), hIgA (e.g., hIgG1, hIgG 2), hIgD, hIgE, or hiigm heavy chain constant sequence. In some cases, the immunoglobulin heavy chain constant sequence is or is derived from a heavy chain constant sequence of a human IgG1 isotype (e.g., G1m1, G1m2, G1m3, or G1m 17).

In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from a human immunoglobulin G1 constant region sequence (IGHG 1; uniProt: P01857-1, v 1). In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from a human immunoglobulin G1 constant region sequence (IGHG 1; uniProt: P01857-1, v 1) comprising substitutions K214R, D356E and L358M (i.e., G1M3 isoforms). In some embodiments, the antibody/antigen binding fragment comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 52, more preferably an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from a human immunoglobulin G4 constant region sequence (IGHG 4; uniProt: P01861, v 1). In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from a human immunoglobulin G4 constant region sequence comprising substitutions S241P and/or L248E (IGHG 4; uniProt: P01861, v 1). The S241P mutation is hinge stable, while the L248E mutation further reduces the ADCC effector function of already very low IgG4 (Davies and Sutton immunology comment 2015, month 11; 268 (1): 139-159; angal et al J.Molec.immunology 1993, month 1; 30 (1): 105-8). In some embodiments, the antibody/antigen binding fragment comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO. 53, more preferably an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the antibody/antigen binding fragments described in the present disclosure comprise immunoglobulin light chain constant sequences. In some embodiments, the immunoglobulin light chain constant sequence may be a human immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is or is derived from a kappa (kappa) or lambda (lambda) light chain, such as a human immunoglobulin kappa constant (IGKC; ckappa; uniProt: P09834-1, v 2), or a human immunoglobulin lambda constant (IGLC; clambda), such as IGLC1 (UniProt: P0CG004-1, v 1), IGLC2 (UniProt: P0DOY2-1, v 1), IGLC3 (UniProt: 00DOY3-1, v 1) or IGLC6 (UniProt: A0M8Q6-1, v 3).

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

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

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

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

Both Fab, fv, scFv and dAb antibody fragments can be expressed and secreted in e.coli, allowing for easy production of large amounts of the fragments.

Whole antibodies and F (ab') 2 fragments are "bivalent". By "bivalent" we mean that the antibody and F (ab') 2 fragment have two antigen binding sites. In contrast, fab, fv, scFv and dAb fragments are monovalent, having only one antigen binding site. Synthetic antibodies capable of binding IL-11, IL-11-containing complexes or IL-11 receptors may also be prepared using phage display techniques well known in the art.

Antibodies can be produced by an affinity maturation process wherein modified antibodies are produced that have increased affinity for an antigen as compared to the unmodified parent antibody. Affinity matured antibodies can be generated by procedures known in the art, such as Marks et al, rio/Technology 10:779-783 (1992); barbas et al, national academy of sciences of the United states of America 91:3809-3813 (1994); schier et al gene 169:147-155 (1995); yelton et al J.Immunol.155:1994-2004 (1995); jackson et al J.Immunol.154 (7): 3310-15 9 (1995) and Hawkins et al J.Molec.Biol.226:889-896 (1992).

Antibodies/fragments include bispecific antibodies, e.g., consisting of two different fragments of two different antibodies, such that the bispecific antibody binds to two types of antigens. The bispecific antibodies include antibodies/fragments described herein that are capable of binding to IL-11, a complex comprising IL-11, or a receptor for IL-11. The antibodies may comprise different fragments having affinity for a second antigen, which may be any desired antigen. Techniques for preparing bispecific antibodies are well known in the art, see for example Mueller, D et al (2010 biopharmaceutical 24 (2): 89-98), wozniak-Knopp G et al (2010 protein engineering design and selection 23 (4): 289-297), and Bauerle, PA et al (2009 cancer research 69 (12): 4491-44944). Bispecific antibodies and bispecific antigen-binding fragments may be provided in any suitable form, such as those described in Kontermann monoclonal antibody 2012,4 (2): 182-197, the entire contents of which are incorporated herein by reference. For example, the bispecific antibody or bispecific antigen binding fragment may be a bispecific antibody conjugate (e.g., igG2, F (ab ') 2, or CovX entity), a bispecific IgG or IgG-like molecule (e.g., igG, scFv4-Ig, igG-scFv, scFv-IgG, DVD-Ig, igG sVD, sVD IgG, diabody IgG, mAb2, or Tandemia universal LC), an asymmetric bispecific IgG or IgG-like molecule (e.g., kih-IgG universal LC, crossMab, kih-IgG-scFab, mAb-Fv, charge pair, or SEED entity), a specific antibody molecule (e.g., diabody (Db), dsDb, DART, scDb, tandAbs, tandem scFv, tandem dAb/VHH, trisomy, fab-scFv, or F (ab') 2-scFv 2), a bispecific Fc and CH3 fusion protein (e.g., taFv-Fc, diabody, scDb-CH3, scFv-Fc-HCAb, scFv-kFv-or scFv-Fv-or a fusion protein such as a diabody, a Fab-4-DNk-Fv, a fusion protein, a Fab-4-DNk-Fv, or a fusion protein. See in particular Kontermann monoclonal antibody 2012,4 (2): 182-19.

Methods of producing bispecific antibodies include chemically crosslinking the antibody or antibody fragment, for example with reducible disulfide or non-reducible thioether linkages, for example as described in Segal and Bast, 2001. Current protocol for production of bispecific antibodies 14:iv:2.13:2.13.1-2.13.16, the entire contents of which are incorporated herein by reference. For example, N-succinimidyl-3- (-2-pyridyldithio) propionate (SPDP) can be used for chemical crosslinking, e.g., fab fragments via the hinge region SH-group, to produce disulfide-linked bispecific F (ab) 2 heterodimers.

Other methods of producing bispecific antibodies include fusing antibody-producing hybridomas, e.g., with polyethylene glycol, to produce tetragenic cells capable of secreting bispecific antibodies, e.g., as described in d.m. and Bast, b.j.2001. Current protocols for production of bispecific antibodies 14:iv:2.13:2.13.1-2.13.16.

Bispecific antibodies and bispecific antigen binding fragments can also be recombinantly produced, for example, by expression from nucleic acid constructs encoding antigen binding molecule polypeptides, for example, antibody engineering: methods and protocols are described in second edition (Humana Press, 2012), chapter 40: production of bispecific antibodies: diabodies and tandem scfvs (Hornig and Schwarz), or french, how to make bispecific antibodies, molecular medicine method 2000;40:333-339.

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

Decoy receptor

Peptide or polypeptide-based formulations capable of binding to IL-11 or complexes containing IL-11 may be based on IL-11 receptors, e.g., IL-11 binding fragments of IL-11 receptors.

In some embodiments, the binding agent may comprise an IL-11-binding fragment of an IL-11Rα chain, and may preferably be soluble and/or exclude one or more or all of the transmembrane domains. In some embodiments, the binding agent may comprise an IL-11 binding fragment of gp130, and may preferably be soluble and/or exclude one or more or all of the transmembrane domains. Such molecules may be described as decoy receptors. Binding of such agents may inhibit IL-11-mediated cis and/or trans signaling by reducing/preventing the ability of IL-11 to bind to IL-11 receptors (e.g., IL-11Rα or gp 130), thereby inhibiting downstream signaling.

Curtis et al (blood 1997, 12, 1; 90 (11): 4403-12) reported that the soluble murine IL-11 receptor alpha chain (sIL-11R) was able to antagonize IL-11 activity when tested on cells expressing transmembrane IL-11R and gp 130. They suggested that the observed antagonism of IL-11 by sIL-11R depends on the limited number of gp130 molecules on cells that have expressed transmembrane IL-11R.

Soluble decoy receptors have also been reported for other signaling molecules as a basis for inhibiting signaling and therapeutic intervention: receptor pairs, such as VEGF and VEGF receptors (De Chao Yu et al molecular therapy (2012); 20, 938-947; konner and Dupont clinical cancer, month 10. 2004; 4 journal of increased 2: S81-5).

Thus, in some embodiments, the binding agent may be a decoy receptor, such as IL-11 and/or a soluble receptor for 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 action (Curtis et al, supra). Decoy IL-11 receptors are described in WO 2017/103108 A1 and WO 2018/109168 A1, which are incorporated herein by reference in their entirety.

The decoy IL-11 receptor preferably binds IL-11 and/or IL-11 containing complexes such that these substances cannot bind gp130, IL-11Rα and/or gp130:IL-11Rα receptors. Thus, they act as "decoy" receptors for IL-11 and IL-11 containing complexes, much like etanercept acts as a decoy receptor for TNFα. IL-11 mediated signal levels are reduced compared to signal levels in the absence of decoy receptors.

The decoy IL-11 receptor preferably binds IL-11 via one or more Cytokine Binding Modules (CBM). The CBM is a CBM of a naturally occurring IL-11 receptor molecule, or derived from or homologous thereto. For example, decoy IL-11 receptors can comprise or consist of one or more CBM's that are derived from, or homologous to CBM's of gp130 and/or IL-11Rα.

In some embodiments, the decoy IL-11 receptor may comprise or consist of an amino acid sequence corresponding to a cytokine binding module of gp 130. In some embodiments, decoy IL-11 receptors can comprise an amino acid sequence corresponding to a cytokine binding module of IL-11Rα. Herein, an amino acid sequence "corresponding" to a reference region or sequence of a given peptide/polypeptide has at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the reference region/sequence.

In some embodiments, the decoy receptor may be capable of binding IL-11, e.g., with a binding affinity of at least 100 μm or less, optionally one of 10 μm or less, 1 μm or less, 100nM or less, or about 1 to 100 nM. In some embodiments, the decoy receptor may comprise 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.

Inhibitors

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 IL-11Rα and/or gp130 containing complexes and inhibiting IL-11 mediated signaling.

In some embodiments, the formulation is a binding agent based on a peptide or polypeptide of IL-11, e.g., a mutant, variant, or binding fragment of IL-11. Suitable peptide or polypeptide-based formulations may bind to the receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rβ and/or gp 130) in a manner that does not result in initiation of signal transduction or in suboptimal signaling. This type of IL-11 mutant can be used as a competitive inhibitor of endogenous IL-11.

For example, W147A is an IL-11 antagonist in which amino acid 147 is mutated from tryptophan to alanine, thereby disrupting the so-called "site III" of IL-11. The mutant binds IL-11Rα but not gp130 homodimers, resulting in an effective blocking of IL-11 signaling (underwill-Day et al, 2003; endocrinology, month 8; 144 (8): 3406-14). Lee et al (journal of respiratory and molecular biology, 2008, 12; 39 (6): 739-746) also reported the generation of IL-11 antagonist mutants ("muteins") capable of specifically inhibiting IL-11 binding to IL-11Rα. IL-11 muteins are described in WO 2009/052588 A1.

Menkhorst et al (reproduction biology, 5.1.80, vol.5.920-927) describe a pegylated IL-11 antagonist PEGIL11A (CSL Co., ltd., pakeville, victoria, australia) which is effective in inhibiting IL-11 action in female mice.

Pasqualini et al cancer (2015) 121 (14): 2411-2421 describes a ligand-directed mimetic drug bone metastasis targeting mimetic peptide-11 (BMTP-11) capable of binding IL-11Rα.

In some embodiments, the binding agent capable of binding to the IL-11 receptor may be provided in the form of a small molecule inhibitor of IL-11Rα, gp130, or one of the complexes containing IL-11R a and/or gp 130. In some embodiments, the binding agent may be provided in the form of a small molecule inhibitor of IL-11 or a complex containing IL-11, such as Lay et al J.International oncology (2012); 41 (2): 759-764, the entire contents of which are incorporated herein by reference.

Aptamer

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

The aptamer of a given target (e.g., IL-11 containing complex or IL-11 receptor) may be identified and/or produced by ex-vivo ligand evolution (SELEXTM) or by methods that develop SOMMamers (slow-modifying aptamers) (Gold L et al (2010) public science library complex 5 (12): e 15004). Aptamers and SELEX are described in Tuerk and Gold science (1990) 249 (4968): 505-10 and WO 91/19813. Application of SELEX and SOMAmer techniques involves, for example, the addition of functional groups that mimic amino acid side chains to expand the chemical diversity of the aptamer. As a result, high affinity aptamers to the target can be enriched and identified.

The aptamer may be a DNA or RNA molecule, and may be single-stranded or double-stranded. The aptamer may comprise a chemically modified nucleic acid, for example, wherein the sugar and/or phosphate and/or base are chemically modified. Such modifications may increase the stability of the aptamer or make the aptamer more resistant to degradation, and may include modifications at the 2' position of ribose.

The aptamer may be synthesized by methods well known to those skilled in the art. For example, the aptamer may be chemically synthesized, e.g., on a solid supportAnd (5) synthesizing. Solid phase synthesis may use phosphoramidite chemistry. Briefly, solid supported nucleotides are demethylated and then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite-triester linkage. The closure may then be performed and the phosphite triester then oxidized with an oxidizing agent, typically iodine. This cycle can then be repeated to assemble the aptamer (see, e.g., sinha, n.d.; biennat, j.; mcManus, j.; H nucleic acids research 1984, 24539 and Beaucage, s.l.; lyer, R.P. (1992) tetrahedron 48 (12): 2223).

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

The aptamer may be a peptide selected or engineered to bind a particular target molecule. Peptide aptamers and methods for their production and identification are reviewed in Reverdato et al, current pharmaceutical chemistry topic (2015) 15 (12): 1082-101, the entire contents of which are incorporated herein by reference. The peptide aptamer may optionally have a minimum length of one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. The peptide aptamer may optionally have a maximum length of one of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. 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.

The aptamer may have a K in the nM or pM range _D For example, less than one of 500nM, 100nM, 50nM, 10nM, 1nM, 500pM, 100 pM.

Characterization of IL-11 binding agent

According to the invention, a formulation capable of binding to IL-11/IL-11 containing complexes or IL-11 receptors may exhibit one or more of the following properties:

specific binding to IL-11/IL-11 containing complexes or IL-11 receptors;

Binding to IL-11/IL-11 containing complexes or IL-11 receptors, K _D At 10 μM or less, preferably 5 μM or less 1 μM or less, 500nM or less, 100nM or less, 10nM or less, 1nM or less, or 100pM or less;

inhibit the interaction between IL-11 and IL-11Rα;

inhibit the interaction between IL-11 and gp 130;

inhibit the interaction between IL-11 and IL-11Rα: gp130 receptor complex;

inhibit the interaction between the IL-11:IL-11Rα complex and gp 130; and

inhibit the interaction between the IL-11:IL-11Rα:gp130 complex (i.e., multimerization of such complexes).

These properties may be determined by analyzing the relevant reagents in a suitable assay, which may include comparing the performance of the reagents to a suitable control reagent. Those skilled in the art will be able to identify appropriate control conditions for a given assay.

For example, a suitable negative control is used to analyze the ability of the test antibody/antigen-binding fragment to bind to IL-11/IL-11-containing complex/IL-11 receptor, and may be an antibody/antigen-binding fragment directed against a non-target protein (i.e., an antibody/antibody-binding fragment that is non-specific for IL-11/IL-11-containing complex/IL-21 receptor). Suitable positive controls may be known, validated (e.g., commercially available) IL-11-or IL-11 receptor binding antibodies. The control may have the same isotype as the analytically recognized complex containing IL-11/IL-11 receptor binding antibody/antigen binding fragment and may, for example, have the same constant region.

In some embodiments, the formulation may be capable of specifically binding 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 gp 130). Reagents that specifically bind a given target molecule preferably have greater affinity and/or longer duration for binding to the target than other non-target molecules.

In some embodiments, the formulation can bind to IL-11 or a complex containing IL-11 with an affinity that is greater than the affinity for binding to one or more other members of the IL-6 cytokine family (e.g., IL-6, leukemia Inhibitory Factor (LIF), oncology inhibin M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine (CLC)). In some embodiments, the formulation binds to a receptor for IL-11 (e.g., IL-11Rα, gp130, or a complex containing IL-11Rβ and/or gp 130) with greater affinity than to one or more other members of the IL-6 receptor family. In some embodiments, the agent binds IL-11 ra with greater affinity than one or more of IL-6 ra, leukemia Inhibitory Factor Receptor (LIFR), oncostatin M receptor (OSMR), ciliary neurotrophic factor receptor a (cntfrα), and cytokine receptor-like factor 1 (CRLF 1).

In some embodiments, the binding agent binds to less than about 10% of the binding of the agent to the target as measured, for example, by ELISA, SPR, biological Layer Interferometry (BLI), micro-scale thermal electrophoresis (MST), or by Radioimmunoassay (RIA). Alternatively, the binding specificity may be reflected in terms of binding affinity, wherein the binding agent binds IL-11IL-11-containing complexes or IL-11 receptor binding with a binding affinity that is aligned with K to another non-target molecule _D K being at least 0.1 orders of magnitude greater (i.e., 0.1X10 n, where n is an integer representing the order of magnitude) _D . 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.

The binding affinity of a given binding agent for its target is typically determined by its dissociation constant (K _D ) To describe. Binding affinity can be measured by methods known in the art, for example by ELISA, surface plasmon resonance (SPR; see, e.g., hearty et al molecular biology methods (2012) 907:411-442; or Rich et al analytical biochemistry, 1 st. 2008, 373 (1): 112-20), biological layer interferometry (see, e.g., lad et al, (2015) journal of molecular biology screening 20 (4): 498-507; or Concepciation et al combinatorial chemistry and high throughput screening, 2009, 12 (8): 791-800), micro-scale thermal electrophoresis (MST) analysis (see, e.g., jerabek Willemsen et al assay and drug development techniques 2011, 8, 9 (4): 342-353), or by radiolabeled antigen binding assay (RIA).

In some embodiments, the agent is capable of binding IL-11 or a complex containing IL-11, or IL-11 receptor, with a KD of 50 μM or less, preferably 10 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, 500nM, 100nM, 75nM, 50nM, 40nM, 30nM, 20nM, 15nM, 12.5nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 500pM, 400pM, 300pM, 200 nM or 100 pM.

In some embodiments, the agent binds to IL-11, a complex comprising IL-11, or a receptor for IL-11 with an EC 50=10000 ng/ml or less, preferably less than or equal to 5,000ng/ml, less than or equal to 1000ng/ml, less than or equal to 900ng/ml, less than or equal to 800ng/ml, less than or equal to 700ng/ml, less than or equal to 600ng/ml, less than or equal to 500ng/ml, less than or equal to 400ng/ml, less than or equal to 300ng/ml, less than or equal to 200ng/ml, less than or equal to 100ng/ml, less than or equal to 90ng/ml, less than or equal to 80ng/ml, less than or equal to 70ng/ml, less than or equal to 60ng/ml, less than or equal to 50ng/ml, less than or equal to 40ng/ml, less than or equal to 30ng/ml, less than or equal to 20ng/ml, less than or equal to 15ng/ml, less than or equal to 10ng/ml, 7.5ng/ml, less than or equal to 5ng/ml, or equal to 2.5ng/ml, or equal to 1 ng/ml. Such ELISA can be performed, for example, as described in antibody engineering volume 1 (version 2), springer Protocols, springer (2010), part V, pages 657-665.

In some embodiments, the agent binds IL-11 or IL-11-containing complex in a region where binding to an IL-11 or IL-11-containing complex receptor (e.g., gp130 or IL-11Rα) is important, thereby inhibiting the interaction between IL-11 or IL-11-containing complex and the IL-11 receptor, and/or signaling through the receptor. In some embodiments, the agent binds to an IL-11 receptor in a region important for binding to IL-11 or an IL-11-containing complex, thereby inhibiting the interaction between IL-11 or an IL-11-containing complex and the IL-11 receptor, and/or signaling through the receptor.

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

The 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-21 and IL-11Lα: gp130, or between IL-11: IL-11Rβ and gp130, or between IL-11: IL-11Rα: gp130 complex) is identified by observing a decrease/decrease in the level of interaction between the interaction partners present or below, or incubation of one or both interaction partners with the binding agent, as compared to the level of interaction in the absence of the binding agent (or the presence of an appropriate control binding agent). Suitable assays may be performed in vitro, for example using recombinant interaction partners or using cells expressing interaction partners. Cells expressing the interaction partner may be made endogenously or may be made by introducing nucleic acids into the cell. For the purposes of such assays, one or both of the interaction partner and/or binding agent may be labeled or used in conjunction with a detectable entity in order to detect and/or measure the level of interaction. For example, the reagent may be labeled with a radioactive atom or a colored molecule, a fluorescent molecule, or a molecule that can be readily detected in any other manner. Suitable detectable molecules include fluorescent proteins, luciferases, enzyme substrates and radiolabels. The binding agent may be directly or indirectly labeled with a detectable label. For example, the binding agent may be unlabeled and detected by another binding agent that is itself labeled. Alternatively, the second binding agent may bind to biotin and the binding of labeled streptavidin to biotin may be used to indirectly label the first binding agent.

The ability of a binding agent to inhibit an interaction between two binding partners can also be determined by analyzing the downstream functional results of such interactions, such as IL-11 mediated signaling. For example, IL-11 and IL-11Rα: downstream functional results of interactions between gp130, between IL-11:IL-11Rβ and gp130, or between IL-11:IL-11Lα:gp130 complex, include, for example, processes mediated by IL-11, or gene/protein expression of, for example, collagen or IL-11.

Inhibition of the interaction between IL-11 or IL-11 containing complexes and IL-11 receptors can be analyzed using assays for 3H-thymidine incorporation and/or Ba/F3 cell proliferation, such as Curtis et al blood 1997, 90 (11) and Karpovich et al molecular human reproduction 2003 9 (2): 75-80.Ba/F3 cells co-express IL-11Rα and gp130.

In some embodiments, the binding agent is capable of inhibiting the interaction between IL-11 and IL-11 ra to a level of interaction between IL-11 and IL-11 ra of less than 100%, e.g., 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, of one of the binding agent (or an appropriate control binding agent) in the absence of the binding agent. In some embodiments, the binding agent is capable of inhibiting the interaction between IL-11 and IL-11Rα to less than 1 fold, e.g., a level of interaction between IL-11 and IL-11Rα of less than 0.99 fold, less than 0.95 fold, less than 0.9 fold, less than 0.85 fold, less than 0.8 fold, less than 0.75 fold, less than 0.7 fold, less than 0.65 fold, less than 0.6 fold, less than 0.55 fold, less than 0.5 fold, less than 0.45 fold, less than 0.4 fold, less than 0.35 fold, less than 0.3 fold, less than 0.25 fold, less than 0.2 fold, less than 0.15 fold, less than 0.1 fold, in the absence of the binding agent (or the presence of an appropriate control binding agent).

In some embodiments, the binding agent is capable of inhibiting the interaction between IL-11 and gp130 to a level of interaction between IL-11 and gp130 of less than 100%, e.g., 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, of one of the binding agent in the absence of the binding agent (or in the presence of an appropriate control binding agent). In some embodiments, the binding agent is capable of inhibiting the interaction between IL-11 and gp130 to a level of 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 or less than the level of the interaction between IL-11 and gp130 of one of the binding agents in the absence of the binding agent (or in the presence of an appropriate control agent).

In some embodiments, the binding agent is capable of inhibiting the interaction between IL-11 and IL-11 ra:gp 130 to a level of interaction between IL-11 and IL-11 ra:gp 130 of less than 100%, e.g., 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, of one of the binding agent in the absence of the binding agent (or in the presence of an appropriate control binding agent). In some embodiments, the binding agent is capable of inhibiting the interaction between IL-11 and IL-11Rα: gp130 to a level of 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, or 0.1 fold, the level of interaction between IL-11 and IL-11Rα: gp130 of one of the binding agents in the absence of the binding agent (or an appropriate control binding agent).

In some embodiments, the binding agent is capable of inhibiting the interaction between the IL-11:il-11 ra complex and gp130 to a level of interaction between the IL-11:il-11 ra complex and gp130 of less than 100%, e.g., 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, of one of the IL-11:il-11 ra complex and gp130, in the absence of the binding agent (or in the presence of an appropriate control binding agent). In some embodiments, the binding agent is capable of inhibiting the interaction between IL-11:11 Rα complex and gp130 to less than 1 fold, e.g., less than 0.99 fold, less than 0.95 fold, less than 0.9 fold, less than 0.85 fold, less than 0.8 fold, less than 0.75 fold, less than 0.7 fold, less than 0.65 fold, less than 0.6 fold, less than 0.55 fold, less than 0.5 fold, less than 0.45 fold, less than 0.4 fold, less than 0.35 fold, less than 0.3 fold, less than 0.25 fold, less than 0.2 fold, less than 0.15 fold, less than 0.1 fold, the level of interaction between IL-11:11 Rα complex and gp130 of one of the binding agent in the absence of the binding agent (or in the presence of an appropriate control binding agent).

In some embodiments, the binding agent is capable of inhibiting the interaction between the IL-11:il-11 ra:gp 130 complex to a level of interaction between the IL-11:il-11 ra:gp 130 complex of less than 100%, e.g., 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, of one of the IL-11:il-11 ra:gp 130 complex in the absence of the binding agent (or in the presence of an appropriate control binding agent). In some embodiments, the binding agent is capable of inhibiting the interaction between IL-11:IL-11Rα: gp130 complex to less than 1 fold, e.g., less than 0.99 fold, less than 0.95 fold, less than 0.9 fold, less than 0.85 fold, less than 0.8 fold, less than 0.75 fold, less than 0.7 fold, less than 0.65 fold, less than 0.6 fold, less than 0.55 fold, less than 0.5 fold, less than 0.45 fold, less than 0.4 fold, less than 0.35 fold, less than 0.3 fold, less than 0.25 fold, less than 0.2 fold, less than 0.15 fold, less than 0.1 fold, the level of interaction between IL-11:IL-11Rα: gp130 complex in the absence of the binding agent (or the presence of an appropriate control binding agent).

Formulations capable of reducing IL-11 or IL-11 receptor expression

In aspects of the invention, the agent capable of inhibiting IL-11 mediated signaling may be capable of preventing or reducing expression of one or more of IL-11, IL-11Rα, or gp 130.

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

Suitable agents may be of any kind, but in some embodiments, agents capable of preventing or reducing expression of one or more of IL-11, IL-11ra, or gp130 may be small molecules or oligonucleotides.

Agents capable of preventing or reducing expression of one or more of IL-11, IL-11Rα or gp130 can reduce stability of RNA encoding IL-11, IL-11Rα or gp130, promote degradation of RNA encoding IL-11, IL-11Rα or gp130, inhibit post-translational processing of IL-11, IL-11Rα or gp130 polypeptides, reduce stability of IL-11, IL-21Rα and gp130 polypeptides, or promote degradation of IL-11, IL-1Rα or gp130 polypeptides, for example, by inhibiting transcription of genes encoding IL-11, IL-1Rα and gp 130.

Taki et al clinical laboratory immunology (4 th month 1998); 112 (1) 133-138 report reduced IL-11 expression in rheumatoid synovial cells following treatment with indomethacin, dexamethasone or interferon gamma (IFNgamma).

The present invention contemplates the use of antisense nucleic acids to prevent/reduce the expression of IL-11, IL-11Rα or gp 130. In some embodiments, an agent capable of preventing or reducing expression of IL-11, IL-11Rα, or gp130 can result in reduced expression by RNA interference (RNAi).

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

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 shRNA of one or more of IL-11, IL-11Rα, or gp 130.

Oligonucleotide molecules, particularly RNA, can be used to modulate gene expression. Including antisense oligonucleotides, small interfering RNAs (siRNAs) targeted degradation of mRNAs, post-Transcriptional Gene Silencing (PTGs), specific translational inhibition of messenger ribonucleic acid (RNA) to developmental regulatory sequences, and targeted transcriptional gene silencing.

Antisense oligonucleotides are preferably single stranded oligonucleotides that bind to target oligonucleotides, such as mRNA, through complementary sequence binding targeting. Where the target oligonucleotide is an mRNA, binding of the antisense to the mRNA blocks translation of the mRNA and expression of the gene product. Antisense oligonucleotides can be designed to bind to sense genomic nucleic acid and inhibit transcription of a target nucleotide sequence.

Given the known nucleic acid sequences for IL-11, IL-11Rα and gp130 (e.g., known mRNA sequences available from GenBank under accession numbers BC012506.1GI:15341754 (human IL-11), BC134354.1GI:1266320022 (mouse IL-11), AF347935.1GI:113549072 (rat IL-11); NM_001142784.2GI:3391353394 (human IL-1Rα), NM_001163401.1GI:254281268 (mouse IL-1Rα), NM_139116.1GI:20806172 (rat IL-1Rα), NM_001190981.1GI:300244534 (human gp 130), NM_010560.3GI:225007524 (mouse gp 130) and NM_001008725.3GI:300244 570 (rat gp 130)) oligonucleotides may be designed to inhibit or silence expression of IL-11, IL-11Rα or gp 130.

Such oligonucleotides may be of any length, but may preferably be short, e.g., less than 100 nucleotides, e.g., 10-40 nucleotides, or 20-50 nucleotides, and may include nucleotide sequences of corresponding length (e.g., IL-11Rα, or gp130 mRNA) (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) that have complete or near complementarity to the target oligonucleotide. The complementary region of 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, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.

Inhibition of IL-11, IL-11Rα or gp130 expression will preferably result in a decrease in the amount of IL-11, IL-1Rα and gp130 expressed by a cell/tissue/organ system/subject. For example, in a given cell, inhibition of IL-11, IL-11Rα or gp130 by administration of an appropriate nucleic acid will result in a decrease in the amount of IL-11, IL-1Rα and gp130 expressed by the cell relative to untreated cells. Inhibition may be local. The preferred degree of inhibition is at least 50%, more preferably at least one of 60%, 70%, 80%, 85% or 90%. The level of inhibition of 90% to 100% is considered "silencing" of expression or function.

RNAi mechanisms and small RNAs have been demonstrated to target heterochromatin complexes and epigenetic gene silencing at specific chromosomal sites. Double-stranded RNA (dsRNA) dependent post-transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. It acts as a signal that promotes the degradation of mRNA having sequence identity. The 20nt siRNA is typically long enough to induce specific gene silencing, but short enough to evade the host response. The reduction in expression of the targeted gene product can be broad, with 90% silencing induced by a small number of siRNA molecules. RNAi-based therapies have entered into a number of indications I, II and phase III clinical trials (Nature 2009, 7, 22; 457 (7228): 426-433).

These RNA sequences are referred to in the art as "short or small interfering RNAs" (sirnas) or "micrornas" (mirnas), depending on their source. Both types of sequences can down-regulate gene expression by binding to complementary RNAs and triggering mRNA elimination (RNAi) or preventing translation of the mRNA into protein. siRNA is derived by processing long double stranded RNAs, which are usually of exogenous origin when found in nature. Micro-interfering RNAs (mirnas) are endogenously encoded small non-coding RNAs obtained by processing short hairpins. Both siRNA and miRNA can inhibit translation of mRNA carrying a partially complementary target sequence without RNA cleavage and degradation of mRNA carrying the fully complementary sequence.

The siRNA ligand is 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 that the RISC complex that mediates the recognition of the mRNA target by the siRNA correctly recognizes the siRNA and to make the siRNA short enough to reduce host response.

miRNA ligands are typically single-stranded and have regions of partial complementarity such that the ligand is capable of forming a hairpin. mirnas are RNA genes transcribed from DNA but are not translated into proteins. The DNA sequence encoding the miRNA gene is longer than miRNA. The DNA sequence includes a miRNA sequence and a similar reverse complement. When the DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse complementary base pairs form a partially double-stranded RNA fragment. The design of microRNA sequences is discussed in J.PLoS biology 11 (2), 1862-1879, 2004, john et al.

Typically, the RNA ligand used to mimic the effect of an siRNA or miRNA has 10 to 40 ribonucleotides (or synthetic analogues thereof), more preferably 17 to 30 ribonucleotides, more preferably 19 to 25 ribonucleotides and most preferably 21 to 23 ribonucleotides. In some embodiments of the invention using double stranded siRNA, the molecule may have symmetrical 3 'overhangs, such as one or two (ribose) nucleotides, especially UU overhangs of dTdT 3'. Based on the disclosure provided herein, one of skill in the art can readily design suitable siRNA and miRNA sequences, for example, using Ambion siRNA finder, etc., resources. siRNA and miRNA sequences may be synthetically produced and exogenously added to cause down-regulation of genes or production using expression systems (e.g., vectors). In a preferred embodiment, the siRNA is synthetic.

Longer double stranded RNA can be processed in cells to produce siRNA (see, e.g., myers (2003) Nature Biotechnology 21:324-328). Longer dsRNA molecules may have symmetrical 3 'or 5' overhangs, such as one or two (ribose) nucleotides, or may have blunt ends. Longer dsRNA molecules may be 25 nucleotides or more. Preferably, the longer dsRNA molecule is between 25 and 30 nucleotides in length. More preferably, the longer dsRNA molecule is between 25 and 27 nucleotides in length. Most preferably, the longer dsRNA molecule is 27 nucleotides in length. The vector pDECAP may be used to express dsRNA of 30 nucleotides in length or more (Shinagawa et al Gene and development 171340-52003).

Another option is to express short hairpin RNA molecules (shRNA) in cells. shRNA is more stable than synthetic siRNA. shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complementary to the target gene. In cells, shRNA is treated by DICER to siRNA, which degrades target gene mRNA and inhibits expression. In a preferred embodiment, shRNA is produced endogenously (in the cell) by transcription from the vector. shRNAs may be produced intracellularly by transfecting a cell with a vector encoding an shRNA sequence under the control of an RNA polymerase III promoter (e.g., a human H1 or 7SK promoter or an RNA polymerase II promoter). Alternatively, the shRNA may be synthesized exogenously (in vitro) by transcription from a vector. The shRNA can then be introduced directly into the cell. Preferably, the shRNA molecule comprises a partial sequence of IL-11, IL-11 ra or gp 130. Preferably, the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length. The length of the stem of the hairpin is preferably between 19 and 30 base pairs. The stem may comprise a G-U pairing to stabilize the hairpin structure.

siRNA molecules, longer dsRNA molecules or miRNA molecules may be made recombinantly by transcription of a nucleic acid sequence, preferably comprising nucleotides within a vector. Preferably, the siRNA molecule, longer dsRNA molecule or miRNA molecule comprises a partial sequence of IL-11, IL-11 ra or gp 130.

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

Suitable vectors may be oligonucleotide vectors configured to express oligonucleotide agents capable of inhibiting IL-11, IL-11Rα or gp 130. Such a vector may be a viral vector or a plasmid vector. The therapeutic oligonucleotide may be incorporated into the genome of a viral vector and operably linked to regulatory sequences, such as promoters, that drive its expression. The term "operably linked" may include the case where the selected nucleotide sequence and the regulatory nucleotide sequence are covalently linked such that expression of the nucleotide sequence is affected or controlled by the regulatory sequence. Thus, a regulatory sequence is operably linked to a selected nucleotide sequence if it is capable of effecting the transcription of a nucleotide sequence that forms part or all of the selected nucleotide sequence.

Viral vectors encoding siRNA sequences for promoter expression are known in the art and have the benefit of long-term expression of therapeutic oligonucleotides. Examples include lentiviruses (Nature 2009, 7, 22; 457 (7228): 426-433), adenoviruses (Shen et al, february letter 2003, 3, 27; 539 (1-3) 111-4) and retroviruses (Barton and Medzhitov Proc. Natl. Acad. Sci. USA, 2002, 11, 12, 99, 23, 14943-14945).

In other embodiments, the vector may be configured to facilitate delivery of the therapeutic oligonucleotide to a site where inhibition of IL-11, IL-11Rα or gp130 expression is desired. Such carriers typically involve complexing the oligonucleotide with positively charged carriers (e.g., cationic cell penetrating peptides, cationic polymers and dendrimers, and cationic lipids); coupling oligonucleotides to small molecules (e.g., cholesterol, bile acids, and lipids), polymers, antibodies, and RNAs; or the oligonucleotides are encapsulated in nanoparticle formulations (Wang et al, journal of the American pharmaceutical sciences, 2010, 12 months; 12 (4): 492-503).

In one embodiment, the vector may comprise nucleic acid sequences in both sense and antisense directions such that when expressed as RNA, the sense and antisense portions will combine to form double stranded RNA.

Alternatively, siRNA molecules can be synthesized using standard solid or liquid phase synthesis techniques known in the art. The linkage between nucleotides may be a phosphodiester linkage or a substitute, for example, a linking group P (O) S, (thio); p (S) S, (dithiosulfate); 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), linked to the adjacent nucleotide by-O-or-S-.

Modified nucleotide bases can be used in addition to naturally occurring bases and can confer advantageous properties to siRNA molecules containing them.

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

The term "modified nucleotide base" includes nucleotides having covalently modified bases and/or sugars. For example, modified nucleotides include nucleotides having a sugar covalently linked to a low molecular weight organic group other than the hydroxyl group at the 3 'position and other than the phosphate group at the 5' position. Modified nucleotides may thus 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' -halo-, or azido ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, and sedoheptulose.

Modified nucleotides are known in the art and include alkylated purines and pyrimidines, acylated purines and purines, and other heterocycles. These pyrimidine and purine types are known in the art and include pseudoisocytosine, N4-ethylcytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6 isopentyladenine, 1-methyladenine, 1-ethylpseudouracil, 1-methylguanine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil, -D-mannosyl guanosine, 5-methoxycarbonylmethyl uracil, 5-methoxyuracil, 2-methylsulfanyl-N6-isopentenyl adenine, methyl uracil-5-oxoacetate, pseudouracil, 2-thiocytosine, 5-methyl-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxoacetate methyl uracil-5-oxoacetic acid, guanosine, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine, 5-butyluracil, pentapentylhuracil, 5-pentylcytosine, and 2,6 diaminopurine, methylsulfonylbiskei, 1-methylguanine, 1-methylcytosine.

Methods related to the use of RNAi to silence genes in caenorhabditis elegans, drosophila, plants and mammals are known in the art (Fire A et al 1998 Nature 391:806-811; fire, A genetics trend 15358-363 (1999); sharp, P.A. RNA interference 2001 genes and growth journal 15485-490 (2001); hammond, S.M. et al Nature reviewed-genetics 2,110-1119 (2001); tuschl, T. Chem. Biochemistry 2,239-245 (2001); hamilton, A. Et al science 286950-952 (1999); hammond, S.M. et al Nature 404293-296 (2000); zamore, P.D. et al cells 101, 25-33 (2000); bernstein, E. Et al Nature 409363-366 (2001); elbashi r, S.M. et al Gene development 15188-200 (2001); WO0129058; WO 3299and Elbashi 2001: SM 498 Nature).

Thus, the invention provides a nucleic acid that, when properly introduced or expressed in a mammalian (e.g., human) cell that otherwise expresses IL-11, IL-11Rα or gp130, is capable of inhibiting the expression of IL-11, IL-1Rα or gp130 by RNAi.

Nucleic acid sequences of IL-11, IL-11Rα and gp130 (e.g., known mRNA sequences available from GenBank under accession numbers: BC012506.1GI:15341754 (human IL-11), BC134354.1GI:1126632002 (mouse IL-11), AF347935.1GI:113549072 (rat IL-11), NM_001142784.2GI:3391353394 (human IL-11Rα), NM_001163404.1GI:254281268 (mouse IL-11Rα), NM_139116.1GI:20806172 (rat IL-11Rα), NM_001190981.1GI:300244534 (human gp 130), NM_010560.3GI:2250075624 (mouse gp 130) and NM_001008725.3GI:300244570 (rat gp 130)) oligonucleotides may be designed to inhibit or silence expression of IL-11, IL-11Rα or gp 130.

The nucleic acid may have substantial identity to a partial sequence of IL-11, IL-11Rα or gp130 mRNA, e.g., genBank accession No. NM-000641.3 GI:391353405 (IL-11), NM-001142784.2GI: 3913353394 (IL-11 Rα), NM-001190981.1GI:300244534 (gp 130), or as defined in the complementary sequence to the mRNA.

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.)

Alternatively, the nucleic acid may be DNA (typically double stranded DNA) that when transcribed in a mammalian cell produces RNA having two complementary portions connected by a spacer such that when the complementary portions hybridize to each other, the RNA forms a hairpin. In mammalian cells, the hairpin structure can be cleaved from the molecule by the DICER enzyme, resulting in two distinct but hybridized RNA molecules.

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

Only the single-stranded (i.e., non-self-hybridizing) region of the mRNA transcript is considered a suitable target for RNAi. It was therefore suggested that other sequences in the IL-11 or IL-11Rα mRNA transcripts that are very close to the sequences represented by one of SEQ ID NO 4-7 or 8-11 may also be suitable targets for RNAi. Such target sequences are preferably 17-23 nucleotides in length and preferably overlap with one of SEQ ID NO 4-7 or 8-11 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 NO 4-7 or 8-11).

Accordingly, the present invention provides a nucleic acid capable of inhibiting IL-11 or IL-1Rα expression by RNAi when appropriately introduced or otherwise expressed in a mammalian cell, wherein the nucleic acid is generally targeted to the sequence of one of SEQ ID NO 4-7 or 8-11.

The sequences overlapping SEQ ID NOs 4 to 7 or 8 to 11 can be targeted by "normally targeting" the nucleic acids. 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 (preferably 17-23 nucleotides in length) than one of SEQ ID NO 4-7 or 8-11, but otherwise has identity to one of SEQ ID NO 4-8 or 8-10.

It is contemplated that complete identity/complementarity between the nucleic acids of the invention and the target sequence, while preferred, is not required. Thus, the nucleic acids of the invention may include a single mismatch compared to the mRNA of IL-11 or IL-11Rα. However, it is expected that even the presence of a single mismatch may lead to reduced efficiency, and therefore no mismatch is preferred. If a 3' overhang is present, it can be excluded from consideration for the number of mismatches.

The term "complementarity" is not limited to conventional base pairing between nucleic acids consisting of naturally occurring ribonucleotides and/or deoxyribonucleotides, but also includes base pairing between mRNA and nucleic acids comprising non-natural nucleotides of the invention.

In one embodiment, the nucleic acid (referred to herein as double stranded siRNA) comprises the double stranded RNA sequence shown in SEQ ID NO 12-15. 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.

However, it is also contemplated that slightly shorter or slightly longer sequences directed to the same region of IL-11 or IL-11Rα mRNA are also effective. In particular, double stranded sequences between 17 and 23bp in length are expected to be effective as well.

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

When present, the dinucleotide overhangs may be symmetrical to one another, although this is not required. In fact, the 3' overhang of the sense (upper) strand is not related to RNAi activity, as it is not involved in the recognition and degradation of mRNA (Elbashir et al, 2001a,2001b,2001 c).

However, RNAi experiments in Drosophila have shown that antisense 3 'overhang may be involved in mRNA recognition and targeting (Elbashir et al, 2001 c), but 3' overhang does not appear to be necessary for RNAi activity of siRNA in mammalian cells. Thus, incorrect annealing of the 3' overhang is believed to have little effect on mammalian cells (Elbashir et al, 2001c; czaudena et al, 2003).

Thus, any dinucleotide overhang can be used in the antisense strand of the siRNA. Nonetheless, the dinucleotide is preferably-UU or-UG (or-TT or-TG if the overhang is DNA), more preferably-UU (or-TT.). the-UU (or-TT) dinucleotide overhang is most efficient and is consistent with (i.e., able to form part of) the RNA polymerase III transcriptional termination signal (terminator signal TTTTT). Therefore, such dinucleotides are most preferred. Dinucleotides AA, CC and GG may also be used, but are less effective and therefore less preferred.

Furthermore, the 3' overhang may be omitted entirely from the siRNA.

The invention also provides single stranded nucleic acids (referred to herein as single stranded siRNAs) each consisting of a component strand of one of the double stranded nucleic acids described above, preferably having a 3' -overhang, but optionally not. The invention also provides a kit comprising such a single-stranded nucleic acid pair, which are capable of hybridizing to each other in vitro to form the double-stranded siRNA described above, which can then be introduced into a cell.

The invention also provides DNA that when transcribed in mammalian cells produces RNA (also referred to herein as shRNA) having two complementary portions that are capable of self-hybridization to produce a double stranded motif, e.g., comprising a sequence selected from the group consisting of SEQ ID NOs 12-15 or 16-19, or a sequence other than a single base pair substitution from any of the foregoing sequences.

The complementary portions will typically be joined by a spacer of suitable length and sequence to allow the two complementary portions to hybridize to each other. The two complementary (i.e., sense and antisense) portions may be joined 5'-3' in any order. The spacer is typically a short sequence of about 4-12 nucleotides, preferably 4-9 nucleotides, more preferably 6-9 nucleotides.

Preferably, the 5 'end of the spacer (the 3' end immediately upstream of the complementary portion) consists of the nucleotide-UU-or-UG-again preferably-UU- (although again the use of these specific dinucleotides is not necessary). A suitable spacer for the pSuper system recommended for OligoEngine (Seattle, washington, U.S.A.) is UUCAAGAGAG. In this and other cases, the ends of the spacer region may hybridize to each other, e.g., such that the double stranded motif extends a fraction (e.g., 1 or 2) base pairs beyond the exact sequence of SEQ ID NOS 12-15 or 16-19.

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

Such shRNA molecules can then be cleaved in mammalian cells by DICER enzymes to produce double stranded siRNA as described above, wherein one or each strand of the hybridized dsRNA comprises a 3' overhang.

The techniques for synthesizing the nucleic acids of the invention are of course well known in the art.

Those skilled in the art are able to construct suitable transcription vectors for the DNA of the present invention using well known techniques and commercially available materials. In particular, the DNA will be related to control sequences, including promoters and transcription termination sequences.

Particularly suitable are the commercially available pSuper and psuberior systems of OligoEngine (seattle, washington, usa). They use the polymerase III promoter (H1) and T5 transcription terminator sequences that contribute two U residues at the 3 'end of the transcript (which provides a 3' uu overhang of one siRNA strand after DICER treatment).

Shin et al describe another suitable system (RNA, 5 months 2009; 15 (5): 889-910) that uses another polymerase III promoter (U6).

The double stranded siRNA of the present invention may 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.

Similarly, transcription vectors containing the DNA of the present 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 inhibiting expression of IL-11 or IL-11 receptor.

Accordingly, the present invention also provides a method of inhibiting the expression of IL-11 or IL-11 receptor in a mammalian (e.g., human) cell, comprising administering to the cell a double stranded siRNA of the invention or a transcription vector of the invention.

Similarly, the invention also provides a method of treating alcoholic liver disease comprising administering to a subject the double stranded siRNA of the invention or the transcription vector of the invention.

The invention further provides the double stranded siRNA of the invention and the transcription vector of the invention for use in a method of treatment, preferably a method of treatment of alcoholic liver disease.

The invention further provides the use of the double stranded siRNA of the invention and the transcription vector of the invention in the preparation of a formulation for the treatment of alcoholic liver disease.

The invention further provides a composition comprising a double stranded siRNA of the invention or a transcription vector of the invention in admixture with one or more pharmaceutically acceptable carriers. Suitable carriers include lipophilic carriers or vesicles, which may facilitate permeation of the cell membrane.

Materials and methods suitable for administration of the siRNA duplex and DNA vectors of the invention are well known in the art and, given the potential of RNAi technology, improved methods are being developed.

In general, a number of techniques are available for introducing nucleic acids into mammalian cells. The choice of technique will depend on whether the nucleic acid is transferred in vitro into cultured cells or in vivo in patient cells. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE, dextran and calcium phosphate precipitation. In vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein liposome mediated transfection (Dzau et al (2003) Biotechnology trends 11205-210).

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

general comments: is new hope of blocking oncogenes in malignant cells by RNA interference-highly specific cancer treatment? Cancer cells.2:167-8. Hannon, G.J.2002.RNA interference natural 418:244-51 McManus, M.T. and P.A.Sharp.2002. Small interfering RNA in mammals in gene silencing. Gene Nature comment 3:737-47.M., M.A.Morgan and M.Eder.2003b. Gene silencing mediated by small interfering RNAs in mammalian cells. Current medical chemistry 10:245-56.Shuey, D.J., D.E.McCallus and T.Giordano.2002.RNAi: gene silencing in therapeutic intervention today drug discovery 7:1040-6.

Systemic delivery using liposomes: lewis, D.L., J.E.Hagstrom, A.G.Loomis, J.A.Wolff and H.Herweijer.2002. Effective delivery of siRNA inhibiting postnatal mouse gene expression. Nature genetics 32:107-8. Efficient expression of Paul, C.P., P.D.Good, I.Winer and D.R.Engelke.2002. Small interfering RNAs in human cells Natural Biotechnology 20:505-8.Song, E. S.K.Lee, J.Wang, N.Ince, N.Ouyang, J.Min, J.Chen, P.Shankar and J.Lieberman.2003. RNA interference targeting Fas protects mice from fulminant hepatitis. Nature medicine 9:347-51.Sorensen, d.r., m.leiridal, and m.sioud.2003. Gene silencing by systemic delivery of synthetic siRNA in adult mice journal of molecular biology 327:761-6.

Virus-mediated transfer: abbas Terki, t., w.blanco Bose, N.Deglon, W.Pralong and p.aebischer.2002. Lentivirus-mediated RNA interference human gene therapy 13:2197-201. Barton, g.m. and r.medzhitov.2002. Retrovirus delivers small interfering RNA into primary cells in the national academy of sciences of the United states of America (Proc Natl Acad Sci U S A.) 99:14943-5.Devroe, e.and p.a. silver.2002. Retroviral delivery of sirna.bmc biotechnology 2:15. Gene therapy for HIV infection is described in Lori, F., P.Guallini, L.Galluzzi and J.Lisziewicz.2002. J.America. J.Pharmacol.2:245-52. Matta, H., B.Hozayev, R.Tomar, P.Chugh and P.M.Chaudhary.2003. Use of a lentiviral vector for delivery of small interfering RNA cancer biotherapy.2:206-10. Qin, X.F., D.S.An, I.S.Chen and d.baltimore.2003. Inhibition of HIV-1 infection in human T cells by lentivirus-mediated delivery of small interfering RNAs to CCR 5. National academy of sciences (Proc Natl Acad Sci U S a.) 100:183-8.Scherr, M. K.Battmer, A.Ganser and M.Eder.2003a. Regulate gene expression by lentivirus-mediated delivery of small interfering RNAs. Cell cycle. 2:251-7. Gene silencing of siRNA delivered by Shen, C. A.K.Buck, X.Liu, M.Winkler and S.N.Reske.2003. Adenovirus. February letter 539:111-4.

Peptide delivery: morris, M.C., L.Chaloin, F.Heitz and G.Divita.2000. Transporter peptides and proteins and their use in gene delivery. Contemporary Biotechnology view 11:461-6. The mechanism of peptide-based gene delivery system MPG is well understood by Simeoni, f.m.c. morris, f.heitz and g.divita.2003: the meaning of siRNA delivery into mammalian cells nucleic acid research 31:2717-24. Other techniques that may be suitable for delivering siRNA to target cells are based on nanoparticles or nanocapsules, such as described in U.S. patent nos. 6649192B and 5843509B.

Inhibition of IL-11 mediated signaling

In embodiments of the invention, an agent capable of inhibiting the action of IL-11 may have one or more of the following functional properties:

inhibition of IL-11 mediated signaling;

inhibit signaling mediated by IL-11 binding to the IL-11Rα: gp130 receptor complex;

inhibit signaling mediated by binding of the IL-11:11 Rα complex to gp130 (i.e., IL-11 trans-signaling);

inhibit signaling mediated by the multimer of the IL-11:IL-11Rα: gp130 complex;

inhibition of IL-11 mediated processes;

inhibiting gene/protein expression of IL-11 and/or IL-11Rα.

IL-11 mediated signaling and/or IL-11 mediated processes include information transduction mediated by IL-11 fragments and polypeptide complexes comprising IL-11 or fragments thereof. IL-11 mediated signaling may be signaling mediated by human IL-11 and/or mouse IL-11. IL-11 mediated signaling may occur after IL-11 or a complex containing IL-11 binds to IL-11 or a receptor to which the complex binds.

In some embodiments, the formulation is capable of inhibiting the biological activity of IL-11 or a complex containing IL-11.

In some embodiments, the agent is an antagonist of one or more signaling pathways that are activated by signaling of a receptor comprising IL-11Rα and/or gp130 (e.g., IL-11Rα: gp 130). In some embodiments, the formulation is capable of inhibiting signaling through one or more immunoreceptor complexes comprising IL-11Rα and/or gp130, such as IL-11Rα: gp130. In various aspects of the invention, the formulations 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 formulations provided herein are capable of inhibiting IL-11 mediated cis-signaling.

In some embodiments, the formulation is capable of inhibiting IL-11 mediated signaling to less than 100%, e.g., 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, 1% or less of the signal level in the absence of the formulation (or in the presence of an appropriate control agent). In some embodiments, the formulation is capable of reducing IL-11 mediated signaling 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 in the absence of the formulation (or in the presence of an appropriate control formulation).

In some embodiments, the IL-11 mediated signaling may be mediated through the binding of IL-11 to the IL-11Rα: gp130 receptor. Such signaling can be analyzed, for example, by treatment with IL-11 or by stimulating IL-11 production in cells expressing IL-11Ra and gp 130.

IC for agents that inhibit IL-11 mediated signaling ₅₀ Can be determined, for example, by culturing Ba/F3 cells expressing IL-11Rα and gp130 in the presence of human IL-11 and the formulation and measuring 3H-thymidine incorporation into DNA. In some embodiments, in such assays, the reagent may exhibit an IC of 10 μg/ml or less ₅₀ Preferably one of 5. Mu.g/ml, 4. Mu.g/ml, 3.5. Mu.g/ml, 3. Mu.g/ml, 2. Mu.g/ml, 1. Mu.g/ml, 0.9. Mu.g/ml, 0.8. Mu.g/ml, 0.7. Mu.g/ml, 0.6. Mu.g/ml or 0.5. Mu.g/ml.

In some embodiments, the IL-11 mediated signaling may be mediated through the binding of IL-11:IL-11Rα complex to gp 130. In some embodiments, the IL-11:IL-11Rα complex may be soluble, e.g., a complex of IL-11Rα and the extracellular domain of IL-11, or a complex of soluble IL-11Rα subtype/fragment and IL-11. In some embodiments, the soluble IL-11Rα is a soluble (secreted) isomer of IL-11R, or is the release product of proteolytic cleavage of the extracellular domain of a cell membrane-bound IL-11Rα.

In some embodiments, the IL-11:IL-11Rα complex may be cell-bound, e.g., a complex of cell-membrane-bound IL-11Rα and IL-11. The signaling mediated by binding of the IL-11:11 Rα complex to gp130 can be analyzed by treating gp 130-expressing cells with the IL-11:11 Rα complex, e.g., recombinant fusion proteins comprising IL-11, e.g., super IL-11, linked to the extracellular domain of IL-11Rα by a peptide linker. Super IL-11 was constructed using fragments of IL-11Rα (amino acid residues 1-317 consisting of domains 1-3; uniProtKB: Q14626) and IL-11 (UniProtKB: amino acid residues 22-199 of P20809) and 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.

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

In some embodiments, the formulation may be capable of inhibiting a process mediated by IL-11.

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

In some embodiments, the formulation is capable of inhibiting the expression of a gene/protein of IL-11 and/or IL-11 ra to less than 100%, e.g., 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or one of the expression levels or less in the absence of the agent (or in the presence of an appropriate control agent). In some embodiments, the formulation is capable of inhibiting gene/protein expression of IL-11 and/or 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 in the absence of the agent (or in the presence of an appropriate control agent).

Treatment/prevention of alcoholic liver disease

The present invention provides methods and articles (formulations and compositions) for the treatment and/or prevention of alcoholic liver disease.

Treatment is achieved by inhibiting IL-11 mediated signaling (i.e., antagonizing IL-11 mediated signaling). That is, the present invention provides for the treatment/prevention of alcoholic liver disease by inhibited IL-11 mediated signaling, e.g., in cells, tissues/organs/organ systems/subjects. In some embodiments, inhibition of IL-11 mediated signaling according to the present disclosure includes inhibition of IL-11 mediated signaling in the liver, liver tissue, and/or cells thereof. In some embodiments, inhibition of IL-11 mediated signaling according to the invention includes inhibition of IL-11 mediated signaling in hepatic stellate cells and/or hepatocytes.

Accordingly, the present invention provides an agent capable of inhibiting interleukin 11 (IL-11) -mediated signaling, for use in a method of treating or preventing alcoholic liver disease.

Also provided is the use of an agent capable of inhibiting interleukin 11 (IL-11) -mediated signaling in the preparation of a method of treating or preventing alcoholic liver disease.

Further provided is a method of treating or preventing alcoholic liver disease, 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.

The invention also provides the treatment/prevention of diseases/conditions caused or exacerbated by alcoholic liver disease. In some embodiments, the invention provides treatment/prevention of diseases/disorders in subjects that provide a poor prognosis for alcoholic liver disease.

In some embodiments, the alcoholic liver disease may be characterized by increased expression of IL-11 and/or IL-11 ra (i.e., gene and/or protein expression) in liver/liver tissue/liver cells, e.g., as compared to normal expression levels in the relevant organ/tissue/cell (i.e., in the absence of the disease/disorder).

Alcoholic liver disease according to the present disclosure may be associated with up-regulation of IL-11 gene and/or protein expression, for example in hepatocytes (e.g., hepatic stellate cells and/or hepatocytes) or liver tissue, or extracellular IL-11 or IL-11 ra.

According to various aspects disclosed herein, in some embodiments, the alcoholic liver disease is characterized by one or more of the following (relative to a healthy, non-diseased state): elevated serum ALT levels, elevated liver to body weight ratios, elevated body weight loss, elevated liver triglyceride levels, elevated serum IL-11 levels, increased expression of genes and/or proteins of IL-11 in the liver, increased expression of genes and/or proteins of one or more pro-inflammatory factors in the liver (e.g., selected from tnfα, TIMP1, IL-10, CXCL1, IL-1b, and MIP 2), increased activation of ERK in the liver (i.e., increased levels of pERK in liver tissue), increased liver tissue steatosis, increased infiltration of neutrophils (e.g., mpo+ neutrophils) into liver tissue, and/or increased infiltration of macrophages (e.g., f4/80+ macrophages) into liver tissue.

According to various aspects of the present disclosure, in some embodiments, alcoholic liver disease is characterized by reduced/impaired liver function relative to function in the absence of the disease.

The treatment is effective in reducing/delaying/preventing the development or progression of alcoholic liver disease. The treatment may be effective in reducing/delaying/preventing the exacerbation of one or more symptoms of alcoholic liver disease. The treatment may be effective in ameliorating one or more symptoms of alcoholic liver disease. Treatment may be effective to reduce the severity and/or reverse one or more symptoms of alcoholic liver disease. The treatment may effectively reverse the effects of alcoholic liver disease.

Prevention may refer to preventing the development of alcoholic liver disease and/or preventing the exacerbation of alcoholic liver disease, e.g., preventing the progression of alcoholic liver disease, e.g., to an advanced/chronic stage (e.g., fibrosis and/or cirrhosis).

In some embodiments, the intervention may be directed to slowing, stopping, and/or reversing liver function damage associated with alcoholic liver disease.

According to various aspects of the present invention, methods of treating and/or preventing alcoholic liver disease according to the present disclosure may include increasing survival of a patient suffering from alcoholic liver disease.

According to various aspects of the invention there is provided a method for or comprising (e.g. in the case of treatment/prevention of alcoholic liver disease) one or more of the following: decreasing serum ALT levels, decreasing liver to body weight ratio, increasing/maintaining body weight, decreasing liver triglyceride levels, decreasing serum IL-11 levels, decreasing gene and/or protein expression of IL-11 in the liver, decreasing gene and/or protein expression of one or more pro-inflammatory factors (e.g., selected from tnfα, TIMP1, IL-10, CXCL1, IL-1b, and MIP 2) in the liver, decreasing activation of ERK in the liver (i.e., decreasing pERK levels in liver tissue), decreasing liver tissue steatosis, decreasing infiltration of neutrophils (e.g., mpo+ neutrophils) into liver tissue, and/or decreasing infiltration of macrophages (e.g., f4/80+ macrophages) into liver tissue.

Also provided are formulations according to the present disclosure for use in such methods, and the use of agents according to the present disclosure in the preparation of compositions (e.g., medicaments) for use in such methods. It is to be understood that the method comprises administering to the subject an agent capable of inhibiting IL-11 mediated signaling.

Similarly, after a therapeutic or prophylactic intervention according to the present disclosure (e.g., as compared to the level prior to the intervention), one or more of the following phenomena may be observed in the subject: reduced serum ALT levels, reduced liver to body weight ratio, increased/maintained body weight, reduced liver triglyceride levels, reduced serum IL-11 levels, reduced expression of genes and/or proteins of IL-11 in the liver, reduced expression of genes and/or proteins of one or more pro-inflammatory factors in the liver (e.g., selected from tnfα, TIMP1, IL-10, CXCL1, IL-1b, and MIP 2), reduced ERK activity in the liver (i.e., reduced pERK levels in liver tissue), reduced hepatic tissue steatosis, reduced infiltration of neutrophils (e.g., mpo+ neutrophils) into liver tissue, and/or reduced infiltration of macrophages (e.g., f4/80+ macrophages) into liver tissue.

In some embodiments, therapeutic/prophylactic interventions according to the invention may be described as "related" to one or more of the effects described in the preceding paragraphs. Those skilled in the art can readily evaluate such characteristics using techniques conventionally practiced in the art.

In some embodiments, the treatment according to the invention may be effective to reverse one or more symptoms of alcoholic liver disease. Even this treatment can effectively reverse symptoms of established, advanced or severe disease/pathology (e.g., fibrosis and/or cirrhosis).

Application of

The administration of an agent capable of inhibiting IL-11 mediated signaling is preferably in a "therapeutically effective" or "prophylactically effective" amount sufficient to show benefit to the subject.

The actual amount, rate and time of administration will depend on the nature and severity of the disease and the nature of the formulation. Treatment prescriptions, such as dose decisions, are taken into account by general practitioners and other medical practitioners, and typically take into account the disease/condition to be treated, the condition of the individual subject, the site of distribution, the method of administration, and other factors known to the physician. Examples of the above techniques and schemes may be found in the 20 th edition of the Ramington pharmaceutical science, 2000 published Lippincott, williams & Wilkins.

Multiple doses of the formulation may be provided. One or more doses, or each dose, may be administered simultaneously or sequentially with another therapeutic agent.

The plurality of doses may be separated by a predetermined time interval, which may be selected as one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days, or as 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. For example, it may be administered once every 7 days, 14 days, 21 days, or 28 days (plus or minus 3 days, 2 days, or 1 day).

In therapeutic applications, formulations capable of inhibiting IL-11-mediated signaling are preferably formulated as a drug (medium) or pharmaceutical (medicament) 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, flavorants, and sweeteners.

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

Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example, 18 th edition of the pharmaceutical science of ramington, mack publishing company, easton, pa,1990; and handbook of pharmaceutical excipients, 2 nd edition, 1994.

The formulation may be prepared by any method known in the pharmaceutical arts. Such methods include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carriers, etc.) and then, if necessary, shaping the product.

The formulations may be prepared for suitable administration, for example, parenteral, systemic, intravenous, intra-arterial, intramuscular, intrathecal, epidermal, intraocular, intracconjunctival, subcutaneous, oral or transdermal routes of administration, which may include injection, depending on the disease/condition to be treated. The injectable formulation may contain the selected agents in sterile or isotonic media. The formulation and mode of administration may be selected according to the formulation and disease to be treated.

In some embodiments, agents capable of inhibiting IL-11-mediated signaling according to the present disclosure may be formulated and/or modified to facilitate delivery to and/or uptake by the liver, liver tissue, and/or hepatocytes (e.g., hepatic stellate cells and/or hepatocytes).

IL-11 and detection of its receptor

Aspects and embodiments of the invention relate to detecting expression of IL-11 or IL-11 receptor (e.g., IL-11Rα, gp130, or a complex containing IL-11Rβ and/or gp 130) in a sample obtained from a subject.

In some aspects and embodiments, the invention relates to upregulation (overexpression) of IL-11 or IL-11 receptor (as a protein or oligonucleotide encoding the corresponding IL-11 or IL-11 receptor), and detection of such upregulation as appropriate for use as an agent capable of inhibiting the effect of IL-11 or as a therapeutic index capable of preventing or reducing the expression of IL-11 or IL-11 receptor.

Upregulated expression includes expression at levels higher than would normally be expected for a given type of cell or tissue. Upregulation may be determined by measuring the expression level of the relevant factor in the cell or tissue. A comparison may be made between the expression level in a cell or tissue sample from the subject and a reference expression level of the relevant factor, e.g. a value or range of values representing the normal expression level of the relevant factor for the same or a corresponding cell or tissue type. In some embodiments, the reference level can be determined by detecting the expression level of IL-11 or IL-11 receptor in a control sample, for example in a corresponding cell or tissue from a healthy subject or from healthy tissue of the same subject. In some embodiments, the reference level may be obtained from a standard curve or dataset. The expression levels may be quantified for absolute comparison, or may be compared relatively.

In some embodiments, an up-regulation of IL-11 or IL-11 receptor (e.g., IL-11Rα, gp130, or a complex containing IL-11Rβ and/or gp 130) may be considered to be present when the expression level in the test sample is at least 1.1 times the reference level. More preferably, the expression level may be selected from at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.5, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, or at least 10.0 times the reference level.

Expression levels may be determined by one of a variety of known in vitro assay techniques, such as PCR-based assays, in situ hybridization assays, flow cytometry assays, immunological or immunohistochemical assays.

For example, a suitable technique involves a method of detecting the level of IL-11 or its receptor in a sample by contacting the sample with an agent capable of binding IL-11 or IL-11 receptor and detecting the formation of a complex of the agent with IL-11 or its 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, such as visualization, of the complex formed. Suitable labels and detection methods are well known to those skilled in the art and include 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, cy 5), isotopic labels, radioisotopes (e.g., 32P, 33P, 35S), chemiluminescent labels (e.g., acridinium esters, luminol, isoluminol), enzymes (e.g., peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, luciferase), antibodies, ligands and receptors. Detection techniques are well known to those skilled in the art and may be selected to correspond to labeling. Suitable techniques include PCR amplification of the oligonucleotide tag, mass spectrometry, fluorescence or colour detection, for example enzymatic conversion of the substrate by a reporter protein, or radioactive detection.

The detection can be configured to quantify the amount of IL-11 or IL-11 receptor in the sample. The amount of IL-11 or IL-11 receptor in the test sample can be compared to a reference value and used to determine whether the test sample contains an amount of IL-11 or IL-11 receptor above or below the reference value to achieve a selected statistical significance.

Quantification of IL-11 or IL-11 receptor detected can be used to determine up-or down-regulation or amplification of genes encoding IL-11 or IL-11 receptor. Where the test sample contains fibrotic cells, this up-regulation, down-regulation or amplification can be compared to a reference value to determine if there are any statistically significant differences.

The sample obtained from the subject may be of any kind. The biological sample may be taken 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 sample of blood source may be a selected portion of the patient's blood, such as a selected cell-containing portion or a plasma or serum portion. The sample may comprise a tissue sample or a biopsy; or cells isolated from a subject. The sample may be collected by known techniques, such as biopsy or puncture. The sample may be stored and/or processed for subsequent determination of IL-11 expression levels.

The sample can be used to determine the up-regulation of IL-11 or IL-11 receptor in a subject from which the sample was taken.

In some preferred embodiments, the sample may be a tissue sample taken from liver/liver tissue, such as a biopsy. Samples may be obtained from the liver. The sample may comprise liver tissue or liver cells.

According to the invention, subjects may be selected for treatment/prevention based on determining that the subject has an up-regulated level of IL-11 or IL-11 receptor (e.g., IL-11Rα, gp130, or a complex containing IL-11Rα and/or gp 130) expression. The upregulation of IL-11 or IL-11 receptor expression may be used as a marker for alcoholic liver disease and is suitable for treatment with agents capable of inhibiting IL-11 mediated signaling.

Upregulation may be present in a given organ (e.g., liver), tissue (e.g., liver tissue), or selected cells (e.g., hepatocytes, e.g., hepatic stellate cells and/or hepatocytes) from a given tissue. Upregulation of IL-11 or IL-11 receptor expression may also be measured in circulating fluids (e.g., blood) or blood-derived samples. Up-regulation may be extracellular IL-11 or IL-11 ra. In some embodiments, expression may be locally or systematically up-regulated.

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

Diagnosis and prognosis

Detection of upregulation of IL-11 or IL-11 receptor (e.g., IL-11Rα, gp130, or a complex comprising IL-11Rα and/or gp 130) expression may also be useful in methods of diagnosing alcoholic liver disease, identifying a subject at risk for having alcoholic liver disease, and prognosing or predicting treatment of a subject with an agent capable of inhibiting IL-11 mediated signaling.

"development", "development" and other forms of "development" may refer to the onset of a condition/disease, or the progression or progression of a condition/disease.

In some embodiments, the subject may be suspected of having or suffering from an alcoholic liver disease, e.g., based on the presence of other symptoms indicative of an alcoholic liver disease in the subject or in selected cells/tissues of the subject's body (e.g., liver/liver tissue/hepatocytes). In some embodiments, the subject may be considered at risk of developing alcoholic liver disease, e.g., due to genetic susceptibility; for example, the subject may comprise one or more copies of one or more of the following alleles: PNPLA3 includes rs738409-G, TM6SF2 includes rs58542926-T, MBOAT7 includes rs641738-T, MARC1 includes rs2642438-C/G/T and HNRNPUL1 includes rs15052-C. In some embodiments, due to exposure to conditions of known risk factors for alcoholic liver disease, such as excessive alcohol consumption (e.g., men often ingest. Gtoreq.40 g ethanol/day, women. Gtoreq.20 g ethanol/day).

Assaying for an up-regulation of IL-11 or IL-11 receptor expression may confirm a diagnosis or a suspected diagnosis, or may confirm that the subject is at risk for developing alcoholic liver disease. The assay may also diagnose alcoholic liver disease or susceptibility, making it suitable for treatment with agents capable of inhibiting IL-11 mediated signaling.

Accordingly, a method of providing a prognosis for a subject having or suspected of having an alcoholic liver disease may be provided, the method comprising determining from a sample obtained from the subject whether expression of IL-11 or an IL-11 receptor is up-regulated, and based on the determination, providing a prognosis for treating the subject with an agent capable of inhibiting IL-11 mediated signaling.

In certain aspects, a diagnostic method or prognosis or a method of predicting a subject's response to an agent therapy capable of inhibiting IL-11 mediated signaling may not require determining expression of IL-11 or IL-11 receptor, but may be based on determining genetic factors that predict up-regulation of expression or activity in the subject. These genetic factors may include the determination of gene mutations, single Nucleotide Polymorphisms (SNPs) or gene amplifications in IL-11, IL-11 ra and/or gp130 that are correlated and/or predicted to express or activate and/or IL-11 mediated signaling. The use of genetic factors to predict susceptibility to disease states or response to treatment is known in the art, see for example Peter Gut 2008;57:440-442; wright et al, cell molecular biology, month 3 2010, volume 30, phase 6, 1411-1420.

Genetic factors can be determined by methods known to those of ordinary skill in the art, including PCR-based assays, such as quantitative PCR, competitive PCR. By determining the presence of a genetic factor, e.g., in a sample obtained from a subject, a diagnosis can be confirmed, and/or the subject can be classified as at risk of developing a disease/disorder described herein, and/or the subject can be determined to be suitable for treatment with an agent capable of inhibiting IL-11 mediated signaling.

Some methods may include determining that the presence of one or more SNPs is associated with secretion of IL-11 or susceptibility to alcoholic liver disease. SNPs are typically biallelic and thus can be readily determined using one of a variety of conventional assays known to those skilled in the art (see, e.g., anthony J. Brooks. SNPs, vol. 234, 2, 7, 8, 1999, pages 177-186; fan et al, highly parallel SNP genotyping, cold spring harbor quantitative BioInd. 2003.68:69-78; matsuzaki et al, parallel genotyping of more than 10,000 SNPs on a high density oligonucleotide array using a single primer assay).

The methods can include determining the presence of a SNP allele 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 alcoholic liver disease.

Accordingly, in one aspect the invention provides a method of screening a subject, the method comprising:

obtaining a nucleic acid sample from a subject;

determining alleles present in a sample at polymorphic nucleotide positions of one or more SNPs listed in fig. 33, fig. 34 or fig. 35 of WO 2017/103108 A1 (incorporated herein by reference), or SNPs in linkage disequilibrium with one of the listed SNPs, r ² ≥0.8

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

The screening method may be or constitute a method of determining a subject's susceptibility to the development of alcoholic liver disease, or a diagnostic or prognostic method as described herein.

The method may further comprise the step of identifying a subject's susceptibility to or increased risk of developing alcoholic liver disease, e.g., if it is determined that the subject has a minor allele at a polymorphic nucleotide position. The method may further comprise the step of selecting the subject for treatment, the agent capable of inhibiting IL-11 mediated signaling and/or administering an agent capable of inhibiting IL-11 mediated signaling to the subject, so as to provide the subject with a treatment for alcoholic liver disease or to prevent the development or progression of alcoholic liver disease in the subject.

In some embodiments, methods of diagnosing alcoholic liver disease, identifying a subject at risk for alcoholic liver disease, and predicting or predicting a subject's response to treatment with an agent capable of inhibiting IL-11-mediated signaling employ an index that is not a measure of IL-11 or of upregulation of IL-11 receptor expression, or genetic factors.

In some embodiments, the methods of diagnosing alcoholic liver disease, identifying a subject at risk for alcoholic liver disease, and the methods of predicting 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 indicators of liver function and/or liver tissue/cell damage.

The diagnostic or prognostic method can be performed in vitro on a sample obtained from the subject, or can be performed after processing the sample obtained from the subject. Once the sample is collected, the patient need not be on-site for in vitro diagnostic or prognostic methods, and thus the method may not be one employed on humans or animals. As described above, the sample obtained from the subject may be of any kind.

Other diagnostic or prognostic assays can be used in conjunction with the assays described herein to improve the accuracy of the diagnosis or prognosis or to confirm the results obtained using the assays described herein.

A subject

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

The patient may have alcoholic liver disease as described herein. The subject may be diagnosed with an alcoholic liver disease, may be suspected of having an alcoholic liver disease, or may be at risk of having an alcoholic liver disease.

In an embodiment according to the invention, the subject is preferably a human subject. In embodiments according to the invention, subjects may be selected for treatment according to methods based on certain markers of alcoholic liver disease.

Sequence identification

Pairwise and multisequence alignments for determining the percentage of identity between two or more amino acid or nucleic acid sequences may be accomplished in various ways known to those skilled in the art, e.g.using well known computer software such as ClustalOmega @J.2005, biological information 21951-960), T-offe (Notrendame et al 2000, journal of molecular biology (2000) 302205-217), kalign (Lassmann and Sonnhammer2005, BMC bioinformatics, 6 (298)) and MAFFT (Katoh and Starley 2013, molecular biology and evolution, 30 (4) 772-780) software. When using these software, default parameters, such as a preferred gap penalty and an extended penalty, are preferably used. / >

Sequence(s)

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***

The invention includes combinations of aspects and preferred features described unless such combinations are clearly not permitted or explicitly avoided.

The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

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

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

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

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" 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 values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and means, for example, +/-10%.

The methods disclosed herein can be performed in vitro, ex vivo, or in vivo, or the product can be present. The term "in vitro" is intended to include experiments performed on materials, biological substances, cells and/or tissues under laboratory conditions or in culture, while the term "in vivo" is intended to include experiments and procedures performed on whole multicellular organisms. In some embodiments, the method performed in vivo may be performed on a non-human animal. "ex vivo" refers to something that exists or occurs outside of an organism, for example outside of a human or animal body, possibly on a tissue (e.g., whole organ) or cells taken from an organism.

In the case of the disclosed nucleic acid sequences, their reverse complementarity is also explicitly contemplated.

For standard molecular biology techniques, see Sambrook, j., russel, d.w. molecular cloning, laboratory manual 3 rd edition 2001, cold spring harbor, new york: cold spring harbor laboratory Press.

Aspects and embodiments of the invention will now be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference in their entirety. While the invention has been described in conjunction with the exemplary embodiments described below, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are to be considered as illustrative and not limiting. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.

Brief Description of Drawings

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

Fig. 1A to 1E: schematic, histogram and graph showing that therapeutic administration of anti-IL-11 RA antibodies ameliorates liver injury in a mouse model of alcoholic liver disease. The time of the program of (1A) represents the intention. C57BL/6J female mice were pair fed ("pairing fed") or EtOH fed for 15 days. The EtOH-group received an anti-IL-11 RA antibody ("IL-11 RA") or an IgG control ("IgG") intraperitoneally. (1B) Administration of EtOH-fed mice with anti-IL-11 RA antibodies resulted in a significant decrease in ALT levels compared to IgG control treatment. (1C, 1D, 1E) EtOH-fed mice treated with anti-IL-11 RA antibody showed a decrease in (1C) liver mass ratio, (1D) weight loss and (1E) liver triglyceride accumulation compared to EtOH-fed mice treated with IgG control. * p <0.05, < p <0.01; n is more than or equal to 5 per group. IL-11RA = anti-interleukin 11 receptor alpha antibody, etOH = ethanol, igG = IgG isotype matched control antibody, ALT = alanine transferase, IL-11 = interleukin 11.

Fig. 2A to 2R: antagonism of IL-11 mediated signaling was shown to reduce IL-11 protein levels in the liver and improve images and histograms of liver inflammation and fibrosis in alcoholic liver disease. (2A) Representative images of liver tissue sections harvested from mice in the treatment group and stained for IL-11. (2B) Administration of the anti-IL-11 RA antibodies reduced the level of IL-11 in liver tissue of EtOH-fed mice compared to EtOH-fed mice treated with IgG controls. (2C to 2H) EtOH fed mice administered anti-IL-11 RA antibody showed reduced gene expression of the pro-inflammatory cytokines (2C) TNFα, (2D) TIMP1, (2E) IL-10, (2F) CXCL1, (2G) IL-1b and (2H) MIP2 compared to EtOH fed mice administered with the IgG control. (2I) Representative immunoblots of pERK and ERK in liver tissue of mice of different treatment groups. (2J) The glycogen score of EtOH fed mice treated with anti-IL-11 RA antibodies tended to increase compared to EtOH fed mice administered IgG control. (2K to 2R) shows gene expression of (2K) Col1A2, (2L) Col3A1, (2M) Col1A 1, (2N) CCL5, (2O) MCP1, (2P) PPARα and (2Q and 2R) PPARγ in liver tissues of mice of different treatment groups. In (2Q), the results of analysis of the data by T-test, and in (2R), the results of analysis of the data by ANOVA are shown. * p <0.05, < p <0.01, ns = no significance; n is more than or equal to 5 per group. IL-11RA = anti-interleukin 11 receptor alpha antibody, etOH = ethanol, igG = IgG isotype matched control antibody, IL-11 = interleukin 11, timp1 = metalloprotease tissue inhibitor 1, IL-10 = interleukin-10, tnfα = tumor necrosis factor alpha, CXCL-1 = chemokine (C-X-C motif) ligand 1, IL-1b = interleukin 1b, mip2-macrophage inflammatory protein 2.

Fig. 3A to 3F: images and bar graphs showing that antagonism of IL-11 mediated signaling can improve histological inflammation of alcoholic liver disease. (3A and 3B) representative (3A) images and (3B) quantification of hematoxylin and eosin staining of liver tissue sections harvested from mice in the indicated treatment groups. EtOH fed mice treated with anti-IL-11 RA antibodies had significantly lower hepatic steatosis scores than EtOH fed mice administered with IgG control. (3C and 3D) representative (3C) images and (3D) quantification of MPO+neutrophils in liver tissue sections collected from mice in the indicated treatment groups. EtOH fed mice treated with anti-IL-11 RA antibodies had significantly less mpo+ neutrophils than EtOH fed mice given IgG control. (3E and 3F) representative (3E) images and (3F) quantification of F4/80+ macrophages in liver tissue sections harvested from mice in the indicated treatment groups. EtOH-fed mice treated with anti-IL-11 RA antibodies had significantly fewer F4/80+ macrophages than EtOH-fed mice administered with IgG controls. * p < 0.05, p < 0.01, p < 0.001, n.gtoreq.5/group. IL-11RA = anti-interleukin 11 receptor alpha antibody, etOH = ethanol, igG = IgG isotype matched control antibody, HPF = high power field, mpo+=myeloperoxidase positive, f4/80+=f4/80 positive.

Fig. 4A to 4E: therapeutic administration of anti-IL-11 RA antibodies improves the schematic, histogram and graph of liver injury in a mouse model of alcoholic liver disease at the onset of treatment following EtOH injury. The time of the program of (4A) represents the intention. C57BL/6J female mice were pair fed ("pairing fed") or EtOH fed for 15 days. The EtOH-group received an anti-IL-11 RA antibody ("IL-11 RA") or an IgG control ("IgG" or "IgG Ab") intraperitoneally from day 7. (4B) Administration of EtOH-fed mice with anti-IL-11 RA antibodies resulted in a significant decrease in ALT levels compared to IgG control treatment. (4C, 4D, 4E) EtOH-fed mice treated with anti-IL-11 RA antibody showed a decrease in (4C) liver mass ratio, (4D) weight loss and (4E) liver triglyceride accumulation compared to EtOH-fed mice treated with IgG control. * p <0.05, ns = no significance; n is more than or equal to 5 per group. IL-11RA = anti-interleukin 11 receptor alpha antibody, etOH = ethanol, igG = IgG isotype matched control antibody, ALT = alanine transferase, IL-11 = interleukin 11.

Examples

In the following examples, the inventors demonstrate that IL-11 mediated signaling drives alcohol-related liver disease, and that inhibiting IL-11 mediated signaling ameliorates symptoms of alcoholic liver disease.

Example 1 materials and methods

Animal study

C57BL/6 mice purchased from Jacksons Laboratories (Bar Harbor, ME) were co-housed in university of medical university animal facility, geneticica for one week before the start of the experiment. All mice were fed the Lieber-deculi pair for five days to accommodate a fluid diet. Female wild type (wt) mice (7-8 weeks old) were then fed with Lieber-DeCarli diet (BioServ, flemington, N.J.) for 15 days (EtOH fed) [ Bertola et al, nat-Protoc (2013) 8:627-637]. The control diet was supplemented with isocaloric maltose (paired feeding). Matched mice were heat matched with ethanol-fed mice. Mice were weighed every other day. After 8 hours of gastric lavage, mice were euthanized. All mice received xylan 5 mg/kg body weight (Intervet, vienna, austria) and ketamine 100mg/kg body weight (AniMedica, senden, germany) for anesthesia. Both liver and intestinal blood and tissue samples were negative.

In vivo administration of anti-IL-11 RA antibodies

For antibody treatment, mice were intraperitoneally injected with 20mg/kg of anti-IL-11 RA antibody X209 or the same amount of IgG control (11E10, alrvron; produced by 1.10E+11 cells (ATCC, accession No. CRL-1907)). X209 is mouse anti-mouse IL-11Rα IgG and is described, for example, in Widjaja et al gastroenterology (2019) 157 (3): 777-792. X209 is also referred to as "Enx209" and includes the VH region according to SEQ ID NO:7 of WO 2019/238884A1 (SEQ ID NO:32 of the present disclosure) and the VL region according to SEQ ID NO:14 of WO 2019/238884A1 (SEQ ID NO 0:33 of the present disclosure).

All anti-IL-11 RA experiments were conducted in accordance with the Oldhamiia law (BMWFW-2020.0.547.764) following ethical principles and in animal facilities at university of medical science, studies.

ALT analysis

Mouse body weight and liver weight were measured. Serum ALT levels were analyzed using the BQ kit, enzyme assay kit from stock limited (san diego, california) according to the manufacturer's instructions.

Triglyceride measurement

To assess liver triglyceride levels, frozen liver samples were homogenized in PBS. The volume was adjusted according to the liver tissue weight. The samples were then incubated at 60℃for 30 minutes and then centrifuged (12000 g,10 minutes, room temperature). Supernatants were collected and triglycerides isolated in fat free BSA (Sigma, st Louis, MO) coated vials. The concentration of triglycerides was measured using TG reagent (Roche, basel, switzerland).

Isolation of RNA from tissue

UsingSamples were homogenized in reagents (Thermo Fisher Scientific, waltham, MA) to purify tissue RNA. A reverse transcription system (Thermo Fisher Scientific, waltham, mass.) was used to complete reverse transcription. qPCR was then performed using qPCR-SybrGreen (Eurogentec, sering, belgium) and Mx3000qPCR cycler (Stratagene, san Diego, calif.) and primers shown in the following table.

Western blot

Liver proteins were extracted using T-PER tissue protein extraction reagent supplemented with HALT protease inhibitor cocktail (Thermo Fisher Scientific, waltham, mass., USA). Protein concentration was measured by BCA protein assay (Pierce, thermo Fisher Scientific, waltham, MA, USA), then separated by SDS-PAGE (Hercules, bio-Rad, CA, USA) and blotted onto Hybond-P PVDF membrane (GE Healthcare, chicago, IL, USA). SNAP (social network access point)Protein detection system(Millipore, burlington, mass., USA) was used for blocking, washing and ERK and pERK incubations. Immunoreactivity was observed by using chemiluminescence on Amersham Hyperfilms (GE Healthcare, chicago, IL, USA). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is used as a reference protein. Western blot signals were quantified using a Biorad ChemiDoc MP imaging system (Hercules, calif., USA).

Histological examination

For histological analysis, liver sections were stained with hematoxylin and eosin (H & E) or with IL-11, myeloperoxidase or F4/80 specific antibodies. The pathologist analyzed H & E-IL-11-, myeloperoxidase-and F4/80-stained liver sections for liver steatosis, inflammation, inflammatory cell infiltration and IL-11, myeloperoxidase and F4/80% positive cells in a blind manner. Liver steatosis was quantified as the percentage of cells showing lipid accumulation with a maximum steatosis score of 300. Glycogen is also analyzed by an independent pathologist as relative content in liver tissue.

IL-11 immunohistochemistry

Formalin-fixed paraffin-embedded sections were deparaffinized and rehydrated. The slide is blocked by peroxidase. Primary antibodies (anti-IL-11, 1:200 dilution; R & D systems, minneapolis, MN) were incubated overnight at 4 ℃. The secondary biotinylated antibody (Vector, DAKO, santa Clara, CA) was incubated for 1 hour at room temperature. The antibody incubation step is performed in a chamber in a humid atmosphere. Immunoreactivity was observed with horseradish peroxidase (HRP) -driven 3,3' -diaminobenzidine (DAKO, santa Clara, CA). Stained sections were scanned and analyzed by a expert pathologist.

Myeloperoxidase immunohistochemistry

Mouse liver sections were dewaxed in xylene and gradient dehydrated in ethanol. Antigens were exposed in a conventional steamer with 2% citrate buffer (ph= 6;Vector Laboratories,Burlingame,CA). Endogenous peroxidase activity was blocked with peroxidase (Dako, santa Clara, calif.) and protein blocking was performed using a ready-to-use kit (MP-740, dako, santa Clara, calif.). Rabbit anti-myeloperoxidase (Dako, santa Clara, calif.) and a second anti-rabbit antibody (Vector Laboratories, burlingame, calif.) were used to observe myeloperoxidase. Tissue samples were stained with DAB (Dako, santa Clara, calif.) and counterstained with hematoxylin (Dako). MPO positive cells were counted by the skilled person in a blind method in 10 randomly selected high power fields.

F4/80 immunohistochemistry

Specific anti-F4/80 rabbit monoclonal antibody (CellSignaling, cambridge, UK) and a second anti-rabbit antibody (Vector Laboratories, burlingame, calif.) stained F4/80. Immunoreactivity was observed with DAB and the samples were counterstained with hematoxylin (Dako, santa Clara, CA). The technician counted F4/80 positive cells in 10 randomly selected high power fields in a blind manner.

Data are expressed as mean ± standard error of the mean or median of the first and third quartiles. For comparison of quantitative variables, student t test or nonparametric Mann-Whitney U or Wilcoxon signed rank test or ANOVA were used as appropriate. The normalization of the distribution was determined by the Kolmogorov-Smirnov test. Correlation analysis was estimated using the Spearman p-coefficient. Outliers are identified using the ROUT method or Grubbs test. p-values <0.05 are considered statistically significant. All statistical analyses were performed using SPSS Statistics v.22 (IBM, chicago, IL) and GraphPad PRISM 5 (La Jolla, calif.).

Example 2: results

Inhibition of IL-11 mediated signaling can prevent experimental ALD

Female C57BL/6J mice were fed either a Lieber-DeCarli diet with 5% ethanol or an isocaloric paired diet for 15 days. In this experimental setup, etOH-fed mice received an antagonist anti-IL-11 RA antibody X209 or IgG control intraperitoneally, as shown in fig. 1A. Control IgG-treated EtOH-fed mice showed signs of liver injury, which was reversed by administration of anti-IL-11 RA antibodies (fig. 1B). Furthermore, administration of anti-IL-11 RA antibody resulted in a decrease in liver mass ratio compared to IgG control (fig. 1C), and anti-IL-12 RA antibody also prevented EtOH-induced weight loss (fig. 1D). Treatment with anti-IL-11 RA antibody also inhibited EtOH-induced accumulation of liver triglycerides observed in IgG control treated EtOH-fed mice (fig. 1E).

Inhibition of IL-11 mediated signaling can prevent alcohol-induced liver inflammation

EtOH-fed mice treated with anti-IL-11 RA antibodies had significantly lower IL-11 protein expression in liver tissue compared to EtOH-fed mice administered with IgG control (fig. 2A and 2B). The inventors studied whether up-regulated IL-11 expression in the liver of EtOH-fed mice up-regulated the expression of other pro-inflammatory mediators and found that the liver gene expression of tnfα, metalloproteinase tissue inhibitor 1 (Timp 1), IL10, cxcl1, IL1 β and macrophage inflammatory protein 2 (mip2) was up-regulated in EtOH-fed mice administered IgG controls (fig. 2C to 2H). Treatment with anti-IL-11 RA antibodies was found to significantly reduce gene expression in each of these pro-inflammatory mediators. Fig. 2J to 2R show the results of analysis of several other markers of fibrosis and inflammation.

Liver protection following treatment with anti-IL-11 RA antibody was associated with reduced pErk activation (FIG. 2I), consistent with the findings in the mouse non-alcoholic fatty liver disease (NAFLD) model [ Widjaja et al, gastroenterology (2019) 157:777-792.E714], where IL-11 driven ERK phosphorylation was found to be central to Hepatic Stellate Cell (HSC) transformation and fibrosis.

IL-11RA treatment reduces infiltration of pro-inflammatory cells into the liver

After hematoxylin and eosin staining of liver tissue, 20 High Power Fields (HPF) were analyzed and the steatosis score was calculated. The fat score of EtOH-fed mice was significantly reduced after treatment with anti-IL-11 RA antibodies compared to IgG control-treated animals (fig. 3A and 3B).

Myeloperoxidase (MPO) staining indicated that anti-IL-11 RA antibody treatment strongly inhibited neutrophil infiltration into liver tissue (figures 3C and 3D), and significantly fewer F4/80 positive macrophages were observed in liver tissue of EtOH fed mice treated with anti-IL-11 RA antibody compared to IgG control treated mice (figures 3E and 3F).

Inhibiting IL-11 mediated signaling in a therapeutic model reduces experimental ALD

Female C57BL/6J mice were fed either a Lieber-DeCarli diet with 5% ethanol or an isocaloric paired diet for 15 days. In this experimental setup, etOH-fed mice received an antagonist anti-IL-11 RA antibody X209 or IgG control intraperitoneally from day 7 as shown in fig. 4A. Control IgG-treated EtOH-fed mice showed signs of liver injury, which was reversed by administration of anti-IL-11 RA antibodies (fig. 4B). Furthermore, administration of anti-IL-11 RA antibody resulted in a decrease in liver mass ratio (fig. 4C), and anti-IL-11 RA antibody also reduced EtOH-induced weight loss (fig. 4D) compared to IgG control. Treatment with anti-IL-11 RA antibody also inhibited EtOH-induced accumulation of liver triglycerides observed in IgG control treated EtOH-fed mice (fig. 4E).

Example 3: discussion of the invention

The inventors hypothesize that IL-11 may play an important role in the pro-inflammatory process of matrix immunization and that inhibition of IL-11 mediated signaling by treatment with neutralizing antibodies to the IL-11 receptor may have a beneficial effect on the inflammatory and pathological relevance of alcoholic liver disease.

Mesenchymal infiltration of neutrophils and macrophages is a significant feature of alcoholic liver disease and may be due to ethanol-mediated activation of innate immunity and subsequent induction of pro-inflammatory cytokines and chemokines [ Gao et al, gastroenterology (2011) 141:1572-1585; mandrekar et al, hepatology (2016) 64:1343-1355; seitz et al, nat Rev Dis Primers (2018) 4:16; louvet al, natural review: gastroenterology and hepatology (2015) 12:231-242]. Hepatocytes, when linked, express the IL-11 receptor and secrete cytokines such as transforming growth factor beta (TGF beta 1). IL-11 mediated activation of hepatocytes is unexpectedly cytotoxic, and in the present model of alcoholic liver disease, hepatocytes exhibit autocrine and maladaptive circulation of IL-11 activity.

It was found that administration of anti-IL-11 RA antibodies significantly reduced inflammation of alcoholic liver disease. The results described above demonstrate that antagonism of IL-11 mediated signaling (e.g., using IL-11 mediated signaling antibody antagonists) is effective for the treatment/prevention of alcohol-induced liver disease, particularly alcoholic hepatitis.

Claims

1. An agent capable of inhibiting interleukin 11 (IL-11) mediated signaling, for use in a method of treating or preventing alcoholic liver disease.

2. Use of an agent capable of inhibiting interleukin 11 (IL-11) -mediated signaling in the manufacture of a medicament for use in a method of treating or preventing alcoholic liver disease.

3. A method of treating or preventing alcoholic liver disease comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting interleukin 11 (IL-11) -mediated signaling.

4. The formulation for use according to claim 1, the use according to claim 2 or the method according to claim 3, wherein the formulation is a formulation capable of preventing or reducing the binding of interleukin 11 (IL-11) to interleukin 11 receptor (IL-11R).

5. The formulation for use according to claim 1 or 4, the use according to claim 2 or 4 or the method according to claim 3 or 4, wherein the formulation is capable of binding to interleukin 11 (IL-11) or interleukin 11 receptor (IL-11R).

6. The formulation for use according to any one of claims 1, 4 or 5, the use according to any one of claims 2, 4 or 5 or the method according to any one of claims 3 to 5, wherein the formulation is selected from the group consisting of: an antibody or antigen binding fragment thereof, a polypeptide, a peptide, a nucleic acid, an oligonucleotide, an aptamer, or a small molecule.

7. The formulation, use or method for use according to claim 5 or 6, wherein the formulation is an antibody or antigen-binding fragment thereof.

8. The formulation, use or method for use according to claim 7, wherein said formulation is an anti-IL-11 antibody antagonist of IL-11 mediated signaling or an antigen-binding fragment thereof.

9. The formulation, use or method for use according to claim 7 or 8, wherein said antibody or antigen-binding fragment thereof comprises:

(i) A heavy chain Variable (VH) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:34 HC-CDR1

Contains the amino acid sequence of SEQ ID NO: HC-CDR2 of 35

Contains the amino acid sequence of SEQ ID NO:36 HC-CDR3; and

(ii) A light chain Variable (VL) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:37 LC-CDR1

Contains the amino acid sequence of SEQ ID NO:38 LC-CDR2

Contains the amino acid sequence of SEQ ID NO:39 LC-CDR3.

10. The formulation, use or method for use according to claim 7 or 8, wherein said antibody or antigen-binding fragment thereof comprises:

(i) A heavy chain Variable (VH) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:40 HC-CDR1

Contains the amino acid sequence of SEQ ID NO:41 HC-CDR2

Contains the amino acid sequence of SEQ ID NO:42 HC-CDR3; and

(ii) A light chain Variable (VL) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:43 LC-CDR1

Contains the amino acid sequence of SEQ ID NO:44 LC-CDR2

Contains the amino acid sequence of SEQ ID NO:45 LC-CDR3.

11. The formulation for use, use or method according to claim 7, wherein said formulation is an anti-IL-11 ra antibody antagonist of IL-11 mediated signaling, or an antigen-binding fragment thereof.

12. The formulation, use or method for use according to claim 7 or 11, wherein said antibody or antigen-binding fragment thereof comprises:

(i) A heavy chain Variable (VH) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:46 HC-CDR1

Contains the amino acid sequence of SEQ ID NO:47 HC-CDR2

Contains the amino acid sequence of SEQ ID NO:48 HC-CDR3; and

(ii) A light chain Variable (VL) region comprising the following CDRs:

contains the amino acid sequence of SEQ ID NO:49 LC-CDR1

Contains the amino acid sequence of SEQ ID NO:50 LC-CDR2

Contains the amino acid sequence of SEQ ID NO:51 LC-CDR3.

13. A formulation for use, use or method according to claim 5 or 6 wherein the formulation is a decoy receptor.

14. The formulation for use, use or method according to claim 13, wherein the formulation is a decoy receptor for IL-11.

15. The formulation for use, use or method according to claim 14, wherein 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-11 ra.

16. The formulation, use or method for use according to claim 5 or 6, wherein the formulation is an IL-11 mutein.

17. The formulation, use or method for use according to claim 16, wherein said IL-11 mutein is W147A.

18. The formulation for use according to claim 1, the use according to claim 2 or the method according to claim 3, wherein the formulation is capable of preventing or reducing the expression of interleukin 11 (IL-11) or interleukin 11 receptor (IL-11R).

19. The formulation, use or method for use according to claim 18, wherein the formulation is an oligonucleotide or a small molecule.

20. The formulation for use, use or method according to claim 19, wherein said formulation is an antisense oligonucleotide capable of preventing or reducing expression of IL-11.

21. The formulation, use or method for use according to claim 20, wherein said antisense oligonucleotide capable of preventing or reducing expression of IL-11 is an siRNA targeting IL11 comprising the amino acid sequence of SEQ ID NO: 12. 13, 14 or 15.

22. The formulation for use, use or method according to claim 19, wherein said formulation is an antisense oligonucleotide capable of preventing or reducing expression of IL-11 ra.

23. The formulation, use or method for use according to claim 22, wherein said antisense oligonucleotide capable of preventing or reducing expression of IL-11 ra is an siRNA targeting IL11 ra, comprising SEQ ID NO: 16. 17, 18 or 19.

24. A formulation for use, use or method according to any of claims 4 to 23 wherein the interleukin 11 receptor is or comprises IL-11 ra.

25. The formulation for use according to any one of claims 1 or 4 to 24, the use according to any one of claims 2 or 4 to 24 or the method according to any one of claims 3 to 24, wherein the method comprises administering the formulation to a subject whose expression of interleukin 11 (IL-11) or IL-11 receptor (IL-11R) is up-regulated.

26. The formulation for use according to any one of claims 1 or 4 to 25, the use according to any one of claims 2 or 4 to 25 or the method according to any one of claims 3 to 25, wherein the method comprises administering the formulation to a subject who has been determined to have up-regulated interleukin 11 (IL-11) or interleukin 11 receptor (IL-11R) expression.

27. The formulation for use according to any one of claims 1 or 4 to 26, the use according to any one of claims 2 or 4 to 26 or the method according to any one of claims 3 to 26, wherein the method comprises determining whether the expression of interleukin 11 (IL-11) or IL-11 receptor (IL-11R) is up-regulated in a subject and administering the formulation to a subject in which the expression of interleukin 11 (IL-11) or IL-11 receptor (IL-11R) is up-regulated.