Treatment and prevention of disease by il-7r inhibition

Abstract

Agents capable of inhibiting interleukin 7 receptor (IL-7R)-mediated signalling and their use in methods of treating or preventing diseases or conditions characterised by fibrosis and / or inflammation are disclosed herein.

Description

[0001] Treatment and Prevention of Disease by IL-7R Inhibition

[0002] This application claims priority from SG 10202400682T filed 12 March 2024, the contents and elements of which are incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present invention relates to the diagnosis, treatment, and prophylaxis of a disease or condition characterised by fibrosis and / or inflammation.

[0005] Background

[0006] Metabolic dysfunction-associated fatty (or steatotic) liver disease (MAFLD / MASLD), also known as nonalcoholic fatty liver disease (NAFLD), is a prevalent chronic liver disease affecting a significant portion (31 %) of the global population. In Asian countries such as Singapore, the prevalence of MAFLD is even higher (34-51%). Metabolic dysfunction-associated steatohepatitis (MASH), also known as non-alcoholic steatohepatitis (NASH), is a more severe form of MAFLD that is clinically characterized by the presence of hepatic fat, inflammation, and fibrosis. MASH is a leading cause of cirrhosis and hepatocellular carcinoma and is highly associated other metabolic co-morbidities in skeletal muscle, heart, fat, and kidney.

[0007] Addressing MAFLD / MASH is crucial due to its public health impact and the lack of FDA-approved therapies. Current treatments focus on lifestyle modifications, but there is a need for effective pharmacological options. The complex nature of MAFLD / MASH poses significant hurdles for therapeutic development; therefore, developing effective pharmacological treatments requires a thorough understanding of the underlying mechanisms of MASH progression. The ‘multiple-hit’ hypothesis emphasizes the role of lipotoxic species in MASH development, particularly saturated long-chain free fatty acids, in disease progression.

[0008] It is likely that an overload of saturated lipids within hepatocytes causes the over-production of reactive oxygen species (ROS) from mitochondria, leading to oxidative stress and ER stress. The combination of lipotoxic metabolites, ROS-induced oxidative stress, and ER stress initiates inflammation, and causes the release of cytokines and chemokines from the injured hepatocytes. This, in turn, attracts immune cells such as macrophages and lymphocytes to the affected area of the liver. These immune cells infiltrate the liver to remove the injured cells and initiate a defence response. Over time, chronic lipotoxic exposure and inflammation cause fibrosis and MAFLD / MASH progression.

[0009] Interleukin 7 (IL-7) and interleukin 7 receptor (IL-7R) are broadly expressed across both lymphoid and non-lymphoid tissues, including liver. The primary signaling pathway for IL-7R signaling involves JAK1 / JAK3 and STAT5. However, there are non-canonical pathways that involve STAT1 , STAT3, PI3K / AKT and MEK / ERK. Drugs that block the IL-7 signal transduction pathway are being tested for the treatment of autoimmune diseases and acute lymphoblastic leukemia (ALL). IL-7R deficiency has been shown to reduce obesity and insulin resistance in mice. W02007 / 130636 discloses around 40 genes, including IL-7R, whose expression is upregulated in MASH patients having bridging fibrosis compared to normal liver histology. Studies have also pointed to an anti- fibrotic effect of IL-7 (Huang et al., J Clin Invest. (2002) 109(7): 931-937; Zhang et al., J Biol Chem. (2004) 279(27):28315-9), indicating that the upregulation of IL-7R in subjects having bridging fibrosis could be a hepatoprotective response to limit MASH pathology. However, the role of the IL-7 and IL-7R- mediated signalling in MAFLD / MASH, and other diseases characterised by inflammation and / or fibrosis, still remains unclear.

[0010] Summary

[0011] In a first aspect the present disclosure provides an agent capable of inhibiting interleukin 7 receptor (IL- 7R)-mediated signalling for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation.

[0012] The present disclosure also provides the use of an agent capable of inhibiting interleukin 7 receptor (IL- 7R)-mediated signalling in the manufacture of a medicament for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation.

[0013] The present disclosure also provides a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation, comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting interleukin 7 receptor (IL-7R)-mediated signalling to a subject.

[0014] In some embodiments, the agent is an agent capable of reducing the expression or activity of interleukin 7 (IL-7) or a constituent polypeptide of IL-7R, or an agent capable of inhibiting interaction between IL-7 and IL-7R or a constituent polypeptide of IL-7R.

[0015] In some embodiments, the agent is selected from the group consisting of: a polypeptide complex capable of binding to IL-7, a polypeptide complex capable of binding to IL-7R, and a polypeptide complex capable of binding to interleukin 7 receptor subunit alpha (IL-7Ra).

[0016] In some embodiments, the agent is an antibody or antigen-binding fragment thereof capable of binding to IL-7, IL-7R or IL-7Ra.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof is capable of binding to IL-7Ra.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a bispecific antibody, or a multispecific antibody, or an antigen-binding fragment thereof.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof is a sweeping antibody.

[0020] In some embodiments, the agent is an aptamer capable of binding to IL-7, IL-7R, or IL-7Ra. In some embodiments, the agent is a sequence-specific nuclease (SSN) targeting a gene encoding IL-7, IL-7R, or lL-7Ra.

[0021] In some embodiments, the SSN is a CRISPR-Cas9 nuclease, a transcription activator-like effector nuclease (TALEN), or zinc-finger nuclease (ZFN).

[0022] In some embodiments, the agent is a nucleic acid or oligonucleotide capable of reducing the expression of IL-7, IL-7R, or lL-7Ra.

[0023] In some embodiments, the agent is a nucleic acid capable of reducing expression of IL-7, IL-7R, or IL- 7Ra by RNA interference (RNAi).

[0024] In some embodiments, the agent is capable of modifying a gene encoding IL-7, IL-7R or IL-7Ra to reduce its expression.

[0025] In some embodiments, the agent is a small molecule capable of binding to IL-7, IL-7R, or IL-7Ra and / or a small molecule capable of reducing the activity of IL-7, IL-7R, or IL-7Ra.

[0026] In some embodiments, the disease or condition is characterised by fibrosis and / or inflammation of the liver.

[0027] In some embodiments, the disease or condition is selected from: chronic liver disease, liver fibrosis, bridging fibrosis, liver cancer, hepatocellular carcinoma (HCC), cirrhosis, hepatitis, alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, steatohepatitis, alcoholic steatohepatitis (ASH), primary biliary cirrhosis (PBC), schistosomal liver disease, metabolic dysfunction-associated fatty or steatotic liver disease (MAFLD / MASLD) and metabolic dysfunction-associated steatohepatitis (MASH).

[0028] In some embodiments, the disease or condition characterised by fibrosis and / or inflammation of the liver is metabolic dysfunction-associated fatty liver disease or Metabolic dysfunction-associated steatotic liver disease (MAFLD / MASLD).

[0029] In some embodiments, the disease or condition characterised by fibrosis and / or inflammation of the liver is metabolic dysfunction-associated steatohepatitis (MASH).

[0030] In some embodiments, the disease or condition characterised by fibrosis and / or inflammation of the liver is cirrhosis.

[0031] Description

[0032] The present invention is based on the inventors’ unexpected finding that elevated IL-7R expression is associated with disease progression in diseases associated with inflammation and / or fibrosis, such as MALFD / MASLD and MASH, thus demonstrating the utility of inhibition of IL-7R-mediated signalling in the treatment of such diseases.

[0033] Interleukin 7 (IL-7) and receptors for IL-7

[0034] Interleukin 7 (IL-7), also known as lymphopoietin, belongs to the IL-2 / IL-15 family of cytokines, which includes IL-2, IL-4, IL-9, IL-15, and IL-21. IL-7 is secreted from a range of cells / tissues, including liver epithelial cells, gut epithelial cells, endothelial cells, bone marrow and thymus stromal cells, and is a key regulator of B- and T-cell differentiation. IL-7 genomic sequences have been mapped onto chromosome 8. The human IL-7 amino acid sequence is available under UniProt accession no. P13232.

[0035] The canonical isoform of human IL-7 (isoform 1) has the amino acid sequence shown in SEQ ID NO: 1. Alternative splicing of mRNA encoded by the human IL7 gene yields three main IL-7 isoforms: isoform 1 (SEQ ID NO: 1), isoform 2 (SEQ ID NO: 2) and isoform 3 (SEQ ID NO: 3). Isoform 2 differs from isoform 1 in that positions 77-120 of SEQ ID NO: 1 are different. Isoform 3 differs from isoform 1 in that positions 1-51 of SEQ ID NO: 1 are different and positions 77-138 of SEQ ID NO: 1 are missing.

[0036] In this specification ‘IL-7’ refers to IL-7 from any species, and includes isoforms, fragments, variants, or homologues from any species. In some embodiments IL-7 is IL-7 from a mammal (e.g. a therian, placental, epitherian, preptotheria, archontan, primate (rhesus, cynomolgous, non-human primate or human)). In some embodiments, the IL-7 is human IL-7, rhesus IL-7, mouse IL-7, rat IL-7 or canine IL-7. In some embodiments, the IL-7 is human IL-7 or mouse IL-7.

[0037] The receptor for IL-7, Interleukin 7 receptor (IL-7R), is a heterodimer comprising a high-affinity a-subunit (IL-7Ra; also known as CD127) and the common y-chain (yc; also known as interleukin-2 receptor (IL-2R) subunit gamma (IL2RG); also known as p64 or CD132). IL-7Ra is identified by UniProt accession no. P16871 , and yc is identified by UniProt accession no. P31785. The yc subunit functions as an activating receptor for the IL-2 / IL-15 cytokine family (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21).

[0038] The structure and functions of IL-7 and IL-7R is described in, e.g., Wang et al. Int J Mol Sci. 2022;23(1): 10412, which is hereby incorporated by reference in its entirety.

[0039] IL-7, IL-7Ra and yc form a ternary complex which signals through several signalling cascades. It is thought that IL-7 binds to IL-7Ra, and then recruits yc to form the ternary complex, which activates downstream signalling. IL-7 and IL-7R predominantly signal through the Janus kinase (JAK), signal transduction factor and transcription activator 5 (STAT5) and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-mediated signal pathways. IL-7 and IL-7R also induce the activation of the mitogen- activated protein kinases (MAPK) pathway. An 8 amino acid motif in the membrane proximal region of IL- 7Ra mediates the recruitment of JAK1 , whereas JAK3 is linked to yc. Tyrosine 449 in the cytoplasmic domain is part of a YXXM motif, which mediates docking of STAT5 proteins and the p85-binding subunit of PI3K. IL-7Ra also cross-reacts to form a complex with thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR), which play a role in T cell / dendritic cell proliferation in humans (see McElroy et al. Structure 2009;17(1):54-56, which is hereby incorporated by reference in its entirety).

[0040] Full-length human IL-7Ra contains an extracellular domain of 219 amino acids in length. The transmembrane domain is 25 amino acids in length. The intracellular domain of IL-7Ra is 195 amino acids in length and provides docking sites for signalling molecules.

[0041] Human IL-7Ra has a membrane-bound form and a soluble form. Soluble IL-7Ra competes with its membrane-bound form, thereby antagonizing IL-7-mediated signalling. Membrane-bound IL-7Ra promotes cell growth and proliferation and inhibits apoptosis (see e.g. Wang et al. Int J Mol Sci. 2022;23(1): 10412, which is hereby incorporated by reference).

[0042] The canonical isoform of human IL-7Ra (isoform 1) has the amino acid sequence shown in SEQ ID NO: 4. Alternative splicing of mRNA encoded by the human IL7R gene yields four main IL-7Ra isoforms: isoform 1 (SEQ ID NO: 4), isoform 2 (H1 ; SEQ ID NO: 5), isoform 3 (H6, secreted; SEQ ID NO: 6) and isoform 4 (secreted; SEQ ID NO: 7). Isoform 2 differs from isoform 1 in that positions 293-459 of SEQ ID NO: 4 are different. Isoform 3 differs from isoform 1 in that positions 237-459 of SEQ ID NO: 4 are different. Isoform 4 differs from isoform 1 in that positions 237-252 of SEQ ID NO: 4 are different, and positions 253-459 of SEQ ID NO: 4 are missing.

[0043] The canonical isoform of human yc (isoform 1) has the amino acid sequence shown in SEQ ID NO: 8. Alternative splicing of mRNA encoded by the human yc gene (IL2RG) yield two main yc isoforms: isoform 1 (SEQ ID NO: 8) and isoform 2 (SEQ ID NO: 9). Isoform 2 differs from isoform 1 in that positions 1-8 of SEQ ID NO: 8 are different, and positions 9-198 of SEQ ID NO: 8 are missing.

[0044] In this specification ‘interleukin 7 receptor' (‘IL-7 receptor’, ‘receptor for IL-7', or ‘IL-7R’) refers to a polypeptide or polypeptide complex capable of binding IL-7. In some embodiments, the IL-7 receptor is capable of binding IL-7 and inducing signal transduction in cells expressing the receptor. In some embodiments, the IL-7 receptor is IL-7Ra. In some embodiments, the IL-7 receptor is a polypeptide complex comprising IL-7Ra. In some embodiments, the IL-7 receptor is a polypeptide complex comprising IL-7Ra and yc (‘IL-7Ra:yc’). In some embodiments, the IL-7 receptor is a polypeptide complex comprising yc to which IL-7 binds. In some embodiments, one yc polypeptide associates with one IL-7Ra polypeptide to form the IL-7 receptor.

[0045] In this specification ‘interleukin 7 receptor' (‘IL-7 receptor’, ‘receptor for IL-7', or IL-7R’) refers to IL-7R from any species, and includes isoforms, fragments, variants, or homologues from any species. In some embodiments IL-7R is IL-7R from a mammal (e.g. a therian, placental, epitherian, preptotheria, archontan, primate (rhesus, cynomolgous, non-human primate or human)). In some embodiments, the IL- 7R is human IL-7R, rhesus IL-7R, mouse IL-7R, rat IL-7R or canine IL-7R. In some embodiments, the IL- 7R is human IL-7R or mouse IL-7R. As used herein, isoforms, fragments, variants or homologues of a given reference protein (e.g. IL-7R, IL- 7Ra, yc or IL-7) may be characterised as having at least 70% sequence identity, preferably one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein.

[0046] A ‘fragment’ generally refers to a fraction of the reference protein. A ‘variant’ generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of the reference protein but retaining a considerable degree of sequence identity (e.g., at least 60%) to the amino acid sequence of the reference protein. An ‘isoform’ generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein. A ‘homologue’ generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein. Homologues include orthologues. Homologues of human IL-7Ra include e.g. mouse IL-7Ra (UniProt accession no. P16872). Homologues of human yc include e.g. mouse yc (UniProt accession no. P34902).

[0047] Isoforms, fragments, variants or homologues of a given reference protein may optionally be characterised as having at least 70%, preferably one of >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature (i.e. after processing to remove signal peptide) form of a specified isoform of the relevant protein from a given species, e.g. human. Isoforms, fragments, variants, or homologues of IL-7R (e.g. IL-7Ra) may optionally be characterised by ability to bind IL-7 (preferably from the same species) and stimulate signal transduction in cells expressing IL-7R.

[0048] Isoforms, fragments, variants, or homologues of IL-7R according to the present disclosure may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature IL-7R isoform from a given species, e.g. human.

[0049] Isoforms, fragments, variants, or homologues may optionally be functional isoforms, fragments, variants, or homologues, e g. having a functional property / activity of the reference IL-7R (e.g. human IL-7Ra isoform 1), as determined by analysis by a suitable assay for the functional property / activity.

[0050] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 4.

[0051] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 5. In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 6.

[0052] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 7.

[0053] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 4, and an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 8.

[0054] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 5, and an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 8.

[0055] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 6, and an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 8.

[0056] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 7, and an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 8.

[0057] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 4, and an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 9.

[0058] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 5, and an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 9.

[0059] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 6, and an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 9.

[0060] In some embodiments, the IL-7R comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 7, and an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 9.

[0061] A ‘fragment’ of a reference protein may be of any length (by number of amino acids), although may optionally be at least 25% of the length of the reference protein (that is, the protein from which the fragment is derived) and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

[0062] A fragment of IL-7R may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800 or 900 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800 or 900 amino acids.

[0063] IL-7R-mediated signalling

[0064] In this specification ‘IL-7R-mediated signalling’ refers to signalling mediated by IL-7R, multimeric receptor complexes comprising IL-7R and / or a fragment thereof having the function of IL-7R. It will be appreciated that IL-7R-mediated signalling’ and ‘IL-7R signalling' refer to signalling initiated by IL-7R or a functional fragment thereof, e.g. through binding to IL-7. ‘Signalling’ in turn refers to signal transduction and other cellular processes governing cellular activity.

[0065] IL-7R-mediated signalling may be mediated by IL-7Ra or a polypeptide complex comprising IL-7Ra (e.g. a polypeptide complex comprising one or more IL-7Ra polypeptides). I L-7R- mediated signalling may be mediated by a polypeptide complex comprising one or more IL-7Ra polypeptides and one or more yc polypeptides. IL-7R-mediated signalling may be mediated by a polypeptide complex comprising one or more yc polypeptides which is capable of binding to IL-7.

[0066] Polypeptide complexes according to the present disclosure may be characterised by non-covalent, protein: protein interaction between constituent polypeptide(s) / peptide(s). In some embodiments, the association comprises electrostatic interaction (e.g. ionic bonding, hydrogen bonding) and / or Van der Waals forces. Agents capable of inhibiting the action of IL-7R

[0067] Aspects of the present invention involve inhibition / antagonism of IL-7R-mediated signalling.

[0068] Herein, ‘inhibition’ refers to a reduction, decrease or lessening relative to a control condition. For example, inhibition of the action of IL-7R by an agent capable of inhibiting IL-7R-mediated signalling refers to a reduction, decrease or lessening of the extent / degree of IL-7R-mediated signalling in the absence of the agent, and / or in the presence of an appropriate control agent.

[0069] Inhibition may herein also be referred to as neutralisation or antagonism. That is, an agent capable of inhibiting IL-7R-mediated signalling {e.g. interaction, signalling or other activity mediated by IL-7R or an IL-7R-containing complex) may be said to be a ‘neutralising’ or ‘antagonist’ agent with respect to the relevant function or process. For example, an agent which is capable of inhibiting IL-7R-mediated signalling may be referred to as an agent which is capable of neutralising IL-7R-mediated signalling or may be referred to as an antagonist of IL-7R-mediated signalling.

[0070] The IL-7R signalling pathway offers multiple routes for inhibition of IL-7R signalling. An agent capable of inhibiting IL-7R-mediated signalling may do so e.g. through inhibiting the action of one or more factors involved in, or necessary for, signalling through a receptor for IL-7R.

[0071] For example, inhibition of IL-7R signalling may be achieved by disrupting interaction between IL-7R (e.g. IL-7Ra, a polypeptide complex comprising IL-7Ra, or a polypeptide complex comprising IL-7Ra and yc) and IL-7 (or an IL-7 containing complex, e.g. a complex of IL-7 and IL-7Ra). In some embodiments, inhibition of IL-7R-mediated signalling is achieved by inhibiting / reducing the gene or protein expression of one or more of e.g. IL-7, IL-7R, IL-7Ra and yc.

[0072] Inhibition of IL-7R-mediated signalling may also be achieved by disrupting interaction between IL-7JL-7R complexes {i.e. complexes comprising IL-7 and IL-7Ra, or IL-7 and yc, or IL-7, IL-7Ra and yc) to form multimers (e.g. hexameric complexes) required for activation of downstream signalling by cells expressing IL-7 receptors.

[0073] In some embodiments, inhibition of IL-7R-mediated signalling is achieved by disrupting IL-7R-mediated cis signalling but not disrupting IL-7R-mediated trans signalling, e.g. inhibition of IL-7R-mediated signalling is achieved by inhibiting cis complexes involving membrane-bound IL-7Ra. In some embodiments, inhibition of IL-7R-mediated signalling is achieved by disrupting IL-7R-mediated trans signalling but not disrupting IL-7R-mediated cis signalling, i.e. inhibition of IL-7-mediated signalling is achieved by inhibiting trans signalling complexes such as IL-7 bound to soluble IL-7Ra or IL-7Ra bound to soluble IL-7. In some embodiments, inhibition of IL-7R-mediated signalling is achieved by disrupting IL- 7R-mediated cis signalling and IL-7R-mediated trans signalling. Any agent as described herein may be used to inhibit IL-7R-mediated cis and / or trans signalling. In other examples, inhibition of IL-7R-mediated signalling may be achieved by disrupting signalling pathways downstream of IL-7R. That is, in some embodiments, inhibition / antagonism of IL-7R-mediated signalling comprises inhibition of a signalling pathway / process / factor downstream of signalling through an IL-7JL-7R complex, an IL-7:IL-7Ra complex, or an IL-7:yc complex.

[0074] In some embodiments, inhibition / antagonism of IL-7R-mediated signalling comprises inhibition of signalling through an intracellular signalling pathway which is activated by the IL-7 / IL-7R complex. In some embodiments, inhibition / antagonism of IL-7R-mediated signalling comprises inhibition of one or more factors whose expression / activity is upregulated as a consequence of signalling through the I L-7 / IL- 7R receptor complex.

[0075] In some embodiments, the methods of the present invention employ agents capable of inhibiting JAK / STAT signalling. In some embodiments, the methods of the present invention employ agents capable of inhibiting MAPK / ERK signalling. In some embodiments, the methods of the present invention employ agents capable of inhibiting PI3K signalling.

[0076] Binding agents

[0077] In some embodiments, agents capable of inhibiting IL-7R-mediated signalling may bind to IL-7R (e.g. IL- 7Ra, or a complex containing IL-7Ra and / or yc). In some embodiments, agents capable of inhibiting IL- 7R-mediated signalling may bind to IL-7. Binding of such agents may inhibit IL-7R-mediated signalling by reducing / preventing the ability of IL-7 to bind to IL-7R, thereby inhibiting downstream signalling. Binding of such agents may inhibit IL-7R-mediated cis and / or frans-signalling by reducing / preventing the ability of IL-7 to bind to IL-7R, thereby inhibiting downstream signalling. Agents may bind to trans-signalling complexes such as IL-7 and soluble IL-7Ra and inhibit IL-7R-mediated signalling.

[0078] Agents capable of binding to IL-7R (e.g. IL-7Ra, or a polypeptide complex comprising IL-7Ra and / or yc) or IL-7 may be of any kind, but in some embodiments the agent may be an antibody, an antigen-binding fragment thereof, a polypeptide, a peptide, a nucleic acid, an oligonucleotide, an aptamer, or a small molecule. The agents may be provided in isolated or purified form or may be formulated as a pharmaceutical composition or medicament.

[0079] Antibodies and antigen-binding fragments

[0080] In some embodiments, an agent capable of binding to IL-7R (e.g. IL-7Ra, or a polypeptide complex comprising IL-7Ra and / or yc) is an antibody, or an antigen-binding fragment thereof. In some embodiments, an agent capable of binding to IL-7 is an antibody, or an antigen-binding fragment thereof.

[0081] An ‘antibody’ is used herein in the broadest sense, and encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they display binding to the relevant target molecule. In view of today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982). Chimeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799). Monoclonal antibodies (mAbs) are particularly useful in the methods of the invention and are a homogenous population of antibodies specifically targeting a single epitope on an antigen.

[0082] Polyclonal antibodies are also useful in the methods of the invention. Monospecific polyclonal antibodies are preferred. Suitable polyclonal antibodies can be prepared using methods well known in the art.

[0083] Antigen-binding fragments of antibodies, such as Fab and Fab2 fragments may also be used / provided as can genetically engineered antibodies and antibody fragments. The variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81 , 6851-6855).

[0084] Antibodies and antigen-binding fragments according to the present disclosure comprise the complementarity-determining regions (CDRs) of an antibody which is capable of binding to the relevant target molecule (e.g. IL-7R, IL-7Ra, yc, a receptor for IL-7, or IL-7).

[0085] Antibodies capable of binding to IL-7R include e g. anti-IL-7Ra monoclonal antibody GSK2618960 (GlaxoSmithKline), anti-human-IL-7Ra monoclonal antibodies disclosed in Belarif et al. Nat Commun. 2018; 9:4483, hereby incorporated by reference in its entirety, and anti-IL-7Ra monoclonal antibody clone B12 disclosed in Akkapeddi et al. Leukemia. 2019; 33: 2155-2168, hereby incorporated by reference in its entirety.

[0086] The skilled person is well aware of techniques for producing antibodies suitable for therapeutic use in a given species / subject. For example, procedures for producing antibodies suitable for therapeutic use in humans are described in Park and Smolen Advances in Protein Chemistry (2001) 56: 369-421 (hereby incorporated by reference in its entirety).

[0087] Antibodies to a given target protein (e.g. IL-7R, IL-7Ra, a receptor for IL-7, or IL-7) can be raised in model species (e.g. rodents, lagomorphs), and subsequently engineered in order to improve their suitability for therapeutic use in a given species / subject. For example, one or more amino acids of monoclonal antibodies raised by immunisation of model species can be substituted to arrive at an antibody sequence which is more similar to human germline immunoglobulin sequences (thereby reducing the potential for anti-xenogenic antibody immune responses in the human subject treated with the antibody). Modifications in the antibody variable domains may focus on the framework regions in order to preserve the antibody paratope. Antibody humanisation is a matter of routine practice in the art of antibody technology, and is reviewed e.g. in Almagro and Fransson, Frontiers in Bioscience (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 hereby incorporated by reference in their entirety. The requirement for humanisation can be circumvented by raising antibodies to a given target protein (e.g. IL-7R, IL-7Ra, a receptor for IL-7, or IL-7) in transgenic model species expressing human immunoglobulin genes, such that the antibodies raised in such animals are fully-human (described e.g. in Briiggemann et al., Arch Immunol Ther Exp (Warsz) (2015) 63(2) : 101 —108, which is hereby incorporated by reference in its entirety).

[0088] Phage display techniques may also be employed to the identification of antibodies to a given target protein (e.g. IL-7R, IL-7Ra, a receptor for IL-7, or IL-7), and are well known to the skilled person. The use of phage display for the identification of fully human antibodies to human target proteins is reviewed e.g. in Hoogenboom, Nat. Biotechnol. (2005) 23, 1105-1116 and Chan et al., International Immunology (2014) 26(12): 649-657, which are hereby incorporated by reference in their entirety.

[0089] The antibodies / fragments may be antagonist antibodies / fragments that inhibit or reduce a biological activity of IL-7R. The antibodies / fragments may be neutralising antibodies that neutralise the biological effect of IL-7, e.g. its ability to stimulate productive signalling via an IL-7 receptor.

[0090] IL-7R-, IL-7Ra-, or IL-7-binding antibodies can be evaluated for the ability to antagonise IL-7R-mediated signalling, e.g. using the western blotting assay described in Belarif et al. Nat Commun. 2018; 9:4483.

[0091] Antibodies generally 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 (VH): HC-CDR1 , HC-CDR2 and HC- CDR3. The six CDRs together define the paratope of the antibody, which is the part of the antibody which binds to the target molecule. The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VH regions comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC- CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FR1]-[LC-CDR1]- [LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C term.

[0092] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671-D674, and the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In some embodiments, the antibody or antigen-binding fragment thereof comprises the CDRs of an antibody that binds to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL- 7. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises the FRs of an antibody that binds to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL- 7. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises the CDRs and the FRs of an antibody that binds to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL-7. That is, in some embodiments, the antibody, or antigen-binding fragment thereof, comprises the VH region and the VL region of an antibody that binds to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL-7.

[0093] Antibodies and antigen-binding fragments according to the present disclosure preferably inhibit IL-7R- mediated signalling. Such antibodies / antigen-binding fragments may be described as being antagonists of IL-7R-mediated signalling, and / or may be described as having the ability to neutralise IL-7R-mediated signalling.

[0094] Antibodies and antigen-binding fragments according to the present disclosure may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to the relevant target molecule. Antigen-binding regions of antibodies, such as single chain variable fragment (scFv), Fab and Fab2 fragments may also be used / provided. An ‘antigen-binding region’ or ‘antigen binding fragment’ is any fragment of an antibody which is capable of binding to the target for which the given antibody is specific.

[0095] In some embodiments, the antibodies / fragments comprise the VL and VH regions of an antibody which is capable of binding to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL-7. The VL and VH region of an antigen-binding region of an antibody together constitute the Fv region. In some embodiments the antibodies / fragments comprise or consist of the Fv region of an antibody which is capable of binding to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL-7. The Fv region may be expressed as a single chain wherein the VH and VL regions are covalently linked, e.g. by a flexible oligopeptide. Accordingly, antibodies / fragments may comprise or consist of an scFv comprising the VL and VH regions of an antibody which is capable of binding to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL-7.

[0096] The VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1) region of an antigen-binding region of an antibody together constitute the Fab region. In some embodiments, the antibodies / fragments comprise or consist of the Fab region of an antibody which is capable of binding to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL-7.

[0097] In some embodiments, antibodies / fragments comprise, or consist of, whole antibody capable of binding to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL-7. A whole antibody’ refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety. Immunoglobulins of type G (i.e. IgG) are ~150 kDa glycoproteins comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1 , CH2, and CH3), and similarly the light chain comprises a VL followed by a CL. Depending on the heavy chain, immunoglobulins may be classed as IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM. The light chain may be kappa (K) or lambda (A).

[0098] In some embodiments, the agent capable of inhibiting IL-7R-mediated signalling is an antibody or fragment thereof comprising CDRs and / or FRs of an antibody that binds to IL-7Ra. In some embodiments, the agent capable of inhibiting IL-7R-mediated signalling is an antibody or fragment thereof comprising the VL and VH regions of an antibody which is capable of binding to IL-7Ra. In some embodiments, the agent capable of inhibiting IL-7R-mediated signalling is an antibody comprising or consisting of a whole antibody capable of binding to IL-7Ra. In some embodiments, the agent capable of inhibiting IL-7R-mediated signalling is an antibody or fragment thereof that binds to IL-7Ra.

[0099] In some embodiments, the agent capable of inhibiting IL-7R-mediated signalling is an antibody or fragment thereof comprising CDRs and / or FRs of an antibody that binds to IL-7. In some embodiments, the agent capable of inhibiting IL-7R-mediated signalling is an antibody or fragment thereof comprising the VL and VH regions of an antibody which is capable of binding to IL-7. In some embodiments, the agent capable of inhibiting IL-7R-mediated signalling is an antibody comprising or consisting of a whole antibody capable of binding to IL-7. In some embodiments, the agent capable of inhibiting IL-7R-mediated signalling is an antibody or fragment thereof that binds to IL-7.

[0100] In some embodiments, the agent capable of inhibiting IL-7R-mediated signalling is not an antibody capable of binding to IL-7R.

[0101] In some embodiments, the antibody, or antigen-binding fragment thereof, is or comprises a monoclonal antibody, or an antigen-binding fragment thereof.

[0102] In some embodiments, the antibody, or antigen-binding fragment thereof, is or comprises a fully human antibody / antibody fragment. A fully human antibody / antibody fragment may be encoded by human nucleic acid sequence(s). A fully human antibody / antibody fragment may be devoid of non-human amino acid sequences. Commonly employed techniques for the production of fully human antibodies include (i) phage display, in which human antibody genes are expressed in phage display libraries, and (ii) production of antibodies in transgenic mice engineered to have human antibody genes (described in Park and Smolen, Advances in Protein Chemistry (2001) 56: 369-421). Briefly, in the human antibody genephage display technique, genes encoding the VH and VL chains are generated by PCR amplification and cloning from ‘naive’ human lymphocytes, and assembled into a library from which they can be expressed either as disulfide-linked Fab fragments or as single-chain Fv (scFv) fragments. The Fab- or scFv- encoding genes are fused to a surface coat protein of filamentous bacteriophage and Fab or scFv capable of binding to the target of interest can then be identified by screening the library with antigen. Molecular evolution or affinity maturation procedures can be employed to enhance the affinity of the Fab / scFv fragment. In the transgenic mouse technique, mice in which the endogenous murine Ig gene loci have been replaced by homologous recombination with their human homologues are immunized with antigen, and monoclonal antibody is prepared by conventional hybridoma technology, to yield a fully human monoclonal antibody.

[0103] In some embodiments, the antibody, or antigen-binding fragment thereof, of the present disclosure is a mouse antibody / antibody fragment. In some embodiments, the antibody / antibody fragment is obtained from phage display using a human naive antibody gene library.

[0104] In some embodiments, the antibody, or antigen-binding fragment thereof, is a mouse / human chimeric antibody / antibody fragment ( / .e. an antibody, or antigen-binding fragment thereof, comprising mouse antibody variable domains and human antibody constant regions). In some embodiments, the antibody, or antigen-binding fragment thereof, is a humanised antibody / antibody fragment. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises mouse antibody CDRs and human antibody framework and constant regions.

[0105] Mouse / human chimeric anti bod ies / anti body fragments can be prepared from mouse antibodies by the process of chimerisation, e.g. as described in Human Monoclonal Antibodies: Methods and Protocols, Michael Steinitz (Editor), Methods in Molecular Biology 1060, Springer Protocols, Humana Press (2014), in Chapter 8 thereof, in particular section 3 of Chapter 8.

[0106] Humanised antibodies / antibody fragments can be prepared from mouse antibodies by the process of humanisation, e.g. as described in Human Monoclonal Antibodies: Methods and Protocols, Michael Steinitz (Editor), Methods in Molecular Biology 1060, Springer Protocols, Humana Press (2014), in Chapter 7 thereof, in particular section 3.1 of Chapter 7 entitled Antibody Humanization’. Techniques for antibody humanisation are also described e.g. in Safdari et al., Biotechnol Genet Eng Rev (2013) 29:175- 86.

[0107] Antibodies may be produced by a process of affinity maturation in which a modified antibody is generated that has an improvement in the affinity of the antibody for antigen, compared to an unmodified parent antibody. Affinity-matured antibodies may be produced by procedures known in the art, e.g., Marks et al., Rio / Technology 10:779-783 (1992); Barbas et al. Proc Nat. Acad. Sci. USA 91 :3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.

[0108] Immunol. 154(7):331 0-15 9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0109] Antibodies / fragments include bispecific antibodies, e.g. composed of two different fragments of two different antibodies, such that the bispecific antibody binds two types of antigen. The bispecific antibody comprises an antibody / fragment as described herein capable of binding to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL-7. The antibody may contain a different fragment having affinity for a second antigen, which may be any desired antigen. Techniques for the preparation of bi-specific antibodies are well known in the art, e.g. see Mueller, D et al., (2010 Biodrugs 24 (2): 89-98), Wozniak-Knopp G et al., (2010 Protein Eng Des 23 (4): 289-297), and Baeuerle, PA et al., (2009 Cancer Res 69 (12): 4941-4944). Bispecific antibodies and bispecific antigen-binding fragments may be provided in any suitable format, such as those formats described in Kontermann MAbs 2012, 4(2): 182-197, which is hereby incorporated by reference in its entirety. For example, a bispecific antibody or bispecific antigenbinding fragment may be a bispecific antibody conjugate (e.g. an lgG2, F(ab’)2 or CovX-Body), a bispecific IgG or IgG-like molecule (e.g. an IgG, scFv4-lg, IgG-scFv, scFv-IgG, DVD-lg, IgG-sVD, sVD- IgG, 2 in 1 -IgG , mAb2, or Tandemab common LC), an asymmetric bispecific IgG or IgG-like molecule (e.g. a kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair or SEED-body), a small bispecific antibody molecule (e.g. a Diabody (Db), dsDb, DART, scDb, tandAbs, tandem scFv (taFv), tandem dAbA / HH, triple body, triple head, Fab-scFv, or F(ab’)2-scFv2), a bispecific Fc and CH3 fusion protein (e.g. a taFv-Fc, Di-diabody, scDb-CH3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv- kih-CH3), or a bispecific fusion protein (e.g. a scFv2-albumin, scDb-albumin, taFv-toxin, DNL-Fab3, DNL- Fab4-lgG, DNL-Fab4-lgG-cytokine2). See in particular Figure 2 of Kontermann MAbs 2012, 4(2): 182-19.

[0110] Methods for producing bispecific antibodies include chemically crosslinking antibodies or antibody fragments, e.g. with reducible disulphide or non-reducible thioether bonds, for example as described in Segal and Bast, 2001 . Production of Bispecific Antibodies. Current Protocols in Immunology.

[0111] 14:IV:2.13:2.13.1 - 2.13.16, which is hereby incorporated by reference in its entirety. For example, N- succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically crosslink e.g. Fab fragments via hinge region SH- groups, to create disulfide-linked bispecific F(ab)2 heterodimers.

[0112] Other methods for producing bispecific antibodies include fusing antibody-producing hybridomas e.g. with polyethylene glycol, to produce a quadroma cell capable of secreting bispecific antibody, for example as described in D. M. and Bast, B. J. 2001. Production of Bispecific Antibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.

[0113] Bispecific antibodies and bispecific antigen-binding fragments can also be produced recombinantly, by expression from e.g. a nucleic acid construct encoding polypeptides for the antigen binding molecules, for example as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antibodies: Diabodies and Tandem scFv (Hornig and Farber-Schwarz), or French, How to make bispecific antibodies, Methods Mol. Med. 2000; 40:333-339.

[0114] For example, a DNA construct encoding the light and heavy chain variable domains for the two antigen binding domains (i.e. the light and heavy chain variable domains for the antigen binding domain capable of binding to IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra, a receptor for IL-7, or IL-7, and the light and heavy chain variable domains for the antigen binding domain capable of binding to another target protein), and including sequences encoding a suitable linker or dimerization domain between the antigen binding domains can be prepared by molecular cloning techniques. Recombinant bispecific antibody can thereafter be produced by expression (e.g. in vitro) of the construct in a suitable host cell (e.g. a mammalian host cell) and expressed recombinant bispecific antibody can then optionally be purified.

[0115] In some embodiments of the present invention, the antibody or antigen-binding fragment is a sweeping antibody. Sweeping antibodies are reviewed in e.g. Igawa et al. Immunol Rev. 2016;270(1):132-51 and Igawa et al. PLoS One. 2013;8(5): e63236, which are hereby incorporated by reference. Sweeping antibodies are capable of eliminating soluble antigens from circulation, by binding to the soluble antigen in plasma and then dissociating from the soluble antigen in the endosome, where the antigen undergoes lysosomal degradation.

[0116] In some embodiments, the sweeping antibody comprises a variable region capable of binding to IL-7R, IL-7Ra or IL-7. In some embodiments, the sweeping antibody comprises a variable region capable of binding to soluble IL-7R, IL-7Ra or IL-7. The sweeping antibody may additionally comprise a constant region, which is optionally capable of increasing the cellular uptake of the antigen-antibody complex into the endosome.

[0117] Decoy receptors

[0118] Peptide or polypeptide-based agents capable of binding to IL-7 or IL-7 containing complexes may be based on the IL-7 receptor, e.g. an IL-7 binding fragment of an IL-7 receptor.

[0119] In some embodiments, the binding agent may comprise an IL-7-binding fragment of IL-7Ra, and may preferably be soluble and / or exclude one or more, or all, of the transmembrane domain(s). In some embodiments, the binding agent may comprise an IL-7-binding fragment of yc, and may preferably be soluble and / or exclude one or more, or all, of the transmembrane domain(s). Such molecules may be described as decoy receptors. Binding of such agents may inhibit IL-7 mediated cis and / or trans- signalling by reducing / preventing the ability of IL-7 to bind to a receptor for IL-7, e.g. IL-7Ra, yc, or IL-7R, thereby inhibiting downstream signalling.

[0120] The use of soluble decoy receptors as the basis for inhibition of signal transduction and therapeutic intervention has been reported for other signalling molecule:receptor pairs, e.g. VEGF and the VEGF receptor (De-Chao Yu et al., Molecular Therapy (2012); 20 5, 938-947; Konner and Dupont Clin Colorectal Cancer 2004 Oct;4 Suppl 2:S81-5).

[0121] As such, in some embodiments a binding agent may be a decoy receptor, e.g. a soluble receptor for IL-7 and / or IL-7 containing complexes. Competition for IL-7 and / or IL-7 containing complexes provided by a decoy receptor may lead to IL-7 or IL-7R antagonist action.

[0122] Decoy IL-7 receptors preferably bind IL-7 and / or IL-7 containing complexes, and thereby make these species unavailable for binding to a receptor for IL-7, e.g. IL-7R, IL-7Ra, or a polypeptide complex comprising IL-7Ra. As such, they act as ‘decoy’ receptors for IL-7 and IL-7 containing complexes, much in the same way that etanercept acts as a decoy receptor for TNFa. IL-7 / IL-7R-mediated signalling is reduced as compared to the level of signalling in the absence of the decoy receptor.

[0123] Decoy IL-7 receptors preferably bind to IL-7 through one or more cytokine binding modules (CBMs). The CBMs are, or are derived from or homologous to, the CBMs of naturally occurring receptor molecules for IL-7. For example, decoy IL-7 receptors may comprise, or consist of, one or more CBMs which are from, are derived from or homologous to the CBM of yc and / or IL-7Ra.

[0124] In some embodiments, a decoy IL-7 receptor may comprise, or consist of, an amino acid sequence corresponding to the cytokine binding module of IL-7Ra. Herein, an amino acid sequence which ‘corresponds’ to a reference region or sequence of a given peptide / polypeptide has at least 60%, e.g. one of 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.

[0125] In some embodiments a decoy receptor may be able to bind IL-7 or IL-7-containing complexes, e.g. with binding affinity of at least 10OpM or less, optionally one of 10pM or less, 1 pM or less, 10OnM or less, or about 1 to 100nM. In some embodiments a decoy receptor may comprise all or part of the IL-7 binding domain and may optionally lack all or part of the transmembrane domains. The decoy receptor may optionally be fused to an immunoglobulin constant region, e.g. IgG Fc region.

[0126] Inhibitors

[0127] The present invention contemplates the use of inhibitor molecules capable of binding to one or more of IL-7R, an IL-7R containing complex, IL-7Ra, yc, or a polypeptide complex containing IL-7Ra and / or yc, and inhibiting IL-7R-mediated signalling.

[0128] In some embodiments the agent is a peptide- or polypeptide-based binding agent based on IL-7, e.g. mutant, variant, or binding fragment of IL-7. Suitable peptide or polypeptide-based agents may bind to a receptor for IL-7 (e.g. IL-7R, IL-7Ra, a polypeptide complex comprising IL-7Ra and / or yc) in a manner that does not lead to initiation of signal transduction, or which produces sub-optimal signalling. IL-7 mutants of this kind may act as competitive inhibitors of endogenous IL-7.

[0129] In some embodiments, a binding agent capable of binding to a receptor for IL-7 may be provided in the form of a small molecule inhibitor of one of IL-7R, IL-7Ra, yc, or a polypeptide complex containing IL-7Ra and / or yc. In some embodiments, a binding agent may be provided in the form of a small molecule inhibitor of IL-7R, an IL-7R-containing complex, IL-7, or an IL-7-containing complex.

[0130] Aptamers

[0131] In some embodiments, an agent capable of binding to IL-7R, an IL-7R containing complex, IL-7Ra, a polypeptide complex comprising IL-7Ra and / or yc, or IL-7 is an aptamer. Aptamers, also called nucleic acid / peptide ligands, are nucleic acid or peptide molecules characterised by the ability to bind to a target molecule with high specificity and high affinity. Almost every aptamer identified to date is a non-naturally occurring molecule.

[0132] Aptamers to a given target (e.g. IL-7R, an IL-7R containing complex, a receptor for IL-7, or IL-7) may be identified and / or produced by the method of Systematic Evolution of Ligands by Exponential enrichment (SELEXTM), or by developing SOMAmers (slow off-rate modified aptamers) (Gold L et al. (2010) PLoS ONE 5(12):e15004). Aptamers and SELEX are described in Tuerk and Gold, Science (1990) 249(4968):505-10, and in WO 91 / 19813. Applying the SELEX and the SOMAmer technology includes for instance adding functional groups that mimic amino acid side chains to expand the aptamer's chemical diversity. As a result, high affinity aptamers for a target may be enriched and identified.

[0133] Aptamers may be DNA or RNA molecules and may be single stranded or double stranded. The aptamer may comprise chemically modified nucleic acids, for example in which the sugar and / or phosphate and / or base is chemically modified. Such modifications may improve the stability of the aptamer or make the aptamer more resistant to degradation and may include modification at the 2' position of ribose.

[0134] Aptamers may be synthesised by methods which are well known to the skilled person. For example, aptamers may be chemically synthesised, e.g. on a solid support. Solid phase synthesis may use phosphoramidite chemistry. Briefly, a solid supported nucleotide is detritylated, then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester linkage. Capping may then occur, followed by oxidation of the phosphite triester with an oxidant, typically iodine. The cycle may then be repeated to assemble the aptamer (e.g., see Sinha, N. D.; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539; and Beaucage, S. L.; Lyer, R. P. (1992). Tetrahedron 48 (12): 2223).

[0135] 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,

[0136] 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,

[0137] 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,

[0138] 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 ,

[0139] 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.

[0140] Aptamers may be peptides selected or engineered to bind specific target molecules. Peptide aptamers and methods for their generation and identification are reviewed in Reverdatto et al., Curr Top Med Chem. (2015) 15(12): 1082-101 , which is hereby incorporated by reference in its entirety. Peptide aptamers may optionally have a minimum length of one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Peptide aptamers may optionally have a maximum length of one of 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26,

[0141] 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.

[0142] Aptamers may have KD’S in the nM or pM range, e.g. less than one of 500nM, 100nM, 50nM, 10nM, 1 nM, 500pM, 100pM.

[0143] Properties oflL-7R binding agents

[0144] Agents capable of binding to IL-7R, an IL-7R containing complex, IL-7Ra, a polypeptide complex comprising IL-7Ra and / or yc, or IL-7 according to the present invention may exhibit one or more of the following properties:

[0145] • Specific binding to IL-7R, an IL-7R containing complex, IL-7Ra, a polypeptide complex comprising IL-7Ra and / or yc, or IL-7;

[0146] • Binding to IL-7R / IL-7R containing complex, a receptor for IL-7, or IL-7, with a KD of 10pM or less, preferably one of < 5pM < 1 pM, <500nM, < 100nM, <10nM, <1 nM or <100pM;

[0147] • Inhibition of interaction between IL-7 and IL-7Ra;

[0148] • Inhibition of interaction between IL-7 and yc;

[0149] • Inhibition of interaction between IL-7 and IL-7Ra:yc receptor complex;

[0150] • Inhibition of interaction between IL-7:IL-7Ra complex and yc; and

[0151] • Inhibition of interaction between IL-7:IL-7Ra:yc complexes ( / .e. multimerisation of such complexes).

[0152] These properties can be determined by analysis of the relevant agent in a suitable assay, which may involve comparison of the performance of the agent to suitable control agents. The skilled person is able to identify appropriate control condition(s) for a given assay.

[0153] For example, a suitable negative control for the analysis of the ability of a test antibody / antigen-binding fragment to bind to IL-7R / an IL-7R containing complex / a receptor for IL-7 / IL-7 may be an antibody / antigen-binding fragment directed against a non-target protein (j.e. an antibody / antigen-binding fragment which is not specific for IL-7R / an IL-7R containing complex / a receptor for I L-7 / IL-7) . A suitable positive control may be a known, validated (e.g. commercially available) IL-7R- or IL-7-binding antibody. Controls may be of the same isotype as the putative IL-7R / IL-7R containing complex / IL-7 receptor / IL-7- binding antibody / antigen-binding fragment being analysed and may e.g. have the same constant regions.

[0154] In some embodiments, the agent may be capable of binding specifically to IL-7R or an IL-7R containing complex, a receptor for IL-7 (e.g. IL-7Ra, or a complex containing IL-7Ra and / or yc), or IL-7. An agent which specifically binds to a given target molecule preferably binds the target with greater affinity, and / or with greater duration than it binds to other, non-target molecules.

[0155] In some embodiments, the extent of binding of a binding agent to a non-target is less than about 10% of the binding of the agent to the target as measured, e.g., by ELISA, SPR, Bio-Layer Interferometry (BLI), MicroScale Thermophoresis (MST), or by a radioimmunoassay (RIA). Alternatively, the binding specificity may be reflected in terms of binding affinity, where the binding agent binds to IL-7R, an IL-7R containing complex, a receptor for IL-7, or IL-7 with a Ko that is at least 0.1 order of magnitude (i.e. 0.1 x 10r, where n is an integer representing the order of magnitude) greater than the KD towards another, non-target molecule. This may optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, or 2.0.

[0156] Binding affinity for a given binding agent for its target is often described in terms of its dissociation constant (KD). Binding affinity can be measured by methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e g. Hearty et al., Methods Mol Biol (2012) 907:411-442; or Rich et al., Anal Biochem. 2008 Feb 1 ; 373(1 ):112-20), Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507; or Concepcion et al., Comb Chem High Throughput Screen. 2009 Sep;

[0157] 12(8)791-800), MicroScale Thermophoresis (MST) analysis (see e.g. Jerabek-Willemsen et al., Assay Drug Dev Technol. 2011 Aug; 9(4): 342-353), or by a radiolabelled antigen binding assay (RIA).

[0158] In some embodiments, the agent is capable of binding to IL-7R, an IL-7R containing complex, a receptor for IL-7, or IL-7 with a KD of 50 pM or less, preferably one of <10 pM, <5 pM, <4 pM, <3 pM, <2 pM, <1 pM, <500 nM, <100 nM, <75 nM, <50 nM, <40 nM, <30 nM, <20 nM, <15 nM, <12.5 nM, <10 nM, <9 nM, <8 nM, <7 nM, <6 nM, <5 nM, <4 nM <3 nM, <2 nM, <1 nM, <500 pM, <400 pM, <300 pM, <200 pM, or <100 pM.

[0159] In some embodiments, the agent binds to IL-7R, an IL-7R containing complex, a receptor for IL-7, or IL-7 with an affinity of binding (e.g. as determined by ELISA) of EC50 = 10,000 ng / ml or less, preferably one of <5,000 ng / ml, <1000 ng / ml, <900 ng / ml, <800 ng / ml, <700 ng / ml, <600 ng / ml, <500 ng / ml, <400 ng / ml, <300 ng / ml, <200 ng / ml, <100 ng / ml, <90 ng / ml, <80 ng / ml, <70 ng / ml, <60 ng / ml, <50 ng / ml, <40 ng / ml, <30 ng / ml, <20 ng / ml, <15 ng / ml, <10 ng / ml, <7.5 ng / ml, <5 ng / ml, <2.5 ng / ml, or <1 ng / ml. Such ELISAs can be performed e.g. as described in Antibody Engineering, vol. 1 (2nd Edn), Springer Protocols, Springer (2010), Part V, pp657-665.

[0160] In some embodiments, the agent binds to IL-7 or an IL-7-containing complex in a region which is important for binding to a receptor for the IL-7 or IL-7-containing complex, e.g. IL-7R or IL-7Ra, and thereby inhibits interaction between IL-7 or an IL-7-containing complex and a receptor for IL-7, and / or signalling through the receptor. In some embodiments, the agent binds to a receptor for IL-7 in a region which is important for binding to IL-7 or an IL-7-containing complex, and thereby inhibits interaction between IL-7 or an IL-7-containing complex and a receptor for IL-7, and / or signalling through the receptor.

[0161] The ability of a given binding agent (e.g. an agent capable of binding IL-7R / an IL-7R containing complex / a receptor for IL-7 / IL-7) to inhibit interaction between two proteins can be determined for example by analysis of interaction in the presence of, or following incubation of one or both of the interaction partners with, the binding agent. An example of a suitable assay to determine whether a given binding agent is capable of inhibiting interaction between two interaction partners is a competition ELISA. A binding agent which is capable of inhibiting a given interaction (e.g. between IL-7 and IL-7R, or between IL-7 and IL-7Ra, or between IL-7 and yc, or between IL-7 and IL-7RCCYC, or between IL-7:IL-7Ra and yc, or between IL-7:IL-7Ra:yc complexes) is identified by the observation of a reduction / decrease in the level of interaction between the interaction partners in the presence of - or following incubation of one or both of the interaction partners with - the binding agent, as compared to the level of interaction in the absence of the binding agent (or in the presence of an appropriate control binding agent). Suitable analysis can be performed in vitro, e.g. using recombinant interaction partners, or using cells expressing the interaction partners. Cells expressing interaction partners may do so endogenously or may do so from nucleic acid introduced into the cell. For the purposes of such assays, one or both of the interaction partners and / or the binding agent may be labelled or used in conjunction with a detectable entity for the purposes of detecting and / or measuring the level of interaction. For example, the agent may be labelled with a radioactive atom or a coloured molecule or a fluorescent molecule or a molecule which can be readily detected in any other way. Suitable detectable molecules include fluorescent proteins, luciferase, enzyme substrates, and radiolabels. The binding agent may be directly labelled with a detectable label, or it may be indirectly labelled. For example, the binding agent may be unlabelled, and detected by another binding agent which is itself labelled. Alternatively, the second binding agent may have bound to it biotin and binding of labelled streptavidin to the biotin may be used to indirectly label the first binding agent.

[0162] Ability of a binding agent to inhibit interaction between two binding partners can also be determined by analysis of the downstream functional consequences of such interaction, e.g. IL-7R-mediated signalling. For example, downstream functional consequences of interaction between IL-7 and IL-7Ra:yc or between IL-7:IL-7Ra and yc, or between IL-7:IL-7Ra:yc complexes may include e.g. a process mediated by IL-7 / 1 L- 7R, or gene / protein expression of e.g. or IL-7.

[0163] Inhibition of interaction between IL-7 or an IL-7 containing complex and a receptor for IL-7 (e.g. IL-7Ra, yc, or IL-7R) can be analysed using radioligand binding assays and nanoanalytical tools like atomic force microscopy (AFM) and optical tweezers.

[0164] In some embodiments, the binding agent may be capable of inhibiting interaction between IL-7 and IL- 7Ra to less than 100% , e.g. one of 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1 % or less of the level of interaction between IL-7 and IL-7Ra in the absence of the binding agent (or in the presence of an appropriate control binding agent). In some embodiments, the binding agent may be capable of inhibiting interaction between IL-7 and IL-7Ra to less than 1 times, e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times the level of interaction between IL-7 and IL-7Ra in the absence of the binding agent (or in the presence of an appropriate control binding agent). In some embodiments, the binding agent may be capable of inhibiting interaction between IL-7 and IL-7R to less than 100%, e.g. one of 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less of the level of interaction between IL-7 and IL-7R in the absence of the binding agent (or in the presence of an appropriate control binding agent). In some embodiments, the binding agent may be capable of inhibiting interaction between IL-7 and IL-7R to less than 1 times, e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times the level of interaction between IL-7 and IL-7R in the absence of the binding agent (or in the presence of an appropriate control binding agent).

[0165] In some embodiments, the binding agent may be capable of inhibiting interaction between IL-7 and yc to less than 100%, e.g. one of 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less of the level of interaction between IL-7 and yc in the absence of the binding agent (or in the presence of an appropriate control binding agent). In some embodiments, the binding agent may be capable of inhibiting interaction between IL-7 and yc to less than 1 times, e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times the level of interaction between IL-7 and yc in the absence of the binding agent (or in the presence of an appropriate control binding agent).

[0166] In some embodiments, the binding agent may be capable of inhibiting interaction between IL-7 and IL- 7Ra:yc to less than 100%, e g. one of 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1 % or less of the level of interaction between IL-7 and IL-7Ra: yc in the absence of the binding agent (or in the presence of an appropriate control binding agent). In some embodiments, the binding agent may be capable of inhibiting interaction between IL-7 and IL-7Rccyc to less than 1 times, e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times the level of interaction between IL-7 and IL-7Ra:yc in the absence of the binding agent (or in the presence of an appropriate control binding agent).

[0167] In some embodiments, the binding agent may be capable of inhibiting interaction between IL-7:IL-7Ra complex and yc to less than 100%, e.g. one of 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1 % or less of the level of interaction between IL-7:IL-7Ra complex and yc 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 interaction between IL-7:IL-7Ra complex and yc to less than 1 times, e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times the level of interaction between IL-7:IL-7Ra complex and yc in the absence of the binding agent.

[0168] In some embodiments, the binding agent may be capable of inhibiting interaction between IL-7:IL-7Ra:yc complexes ( / .e. multimerisation of such complexes) to less than 100%, e.g. one of 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1 % or less of the level of interaction between IL-7:IL-7Ra:yc complexes 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 interaction between IL-7:IL-7Rccyc complexes to less than 1 times, e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times the level of interaction between IL-7:IL-7Ra:yc complexes in the absence of the binding agent.

[0169] Agents capable of reducing expression of IL-7 or IL-7R

[0170] In aspects of the present invention the agent capable of inhibiting IL-7R-mediated signalling may be capable of preventing or reducing the expression of one or more of IL-7, IL-7R, IL-7Ra or yc.

[0171] Expression may be gene or protein expression and may be determined as described herein or by methods in the art that will be well known to a skilled person. Expression may be by a cell / tissue / organ / organ system of a subject.

[0172] Suitable agents may be of any kind, but in some embodiments an agent capable of preventing or reducing the expression of one or more of IL-7, IL-7R, IL-7Ra or yc may be a small molecule or an oligonucleotide.

[0173] An agent capable of preventing or reducing of the expression of one or more of IL-7 or IL-7R may do so e.g. through inhibiting transcription of a gene encoding IL-7, IL-7R, or IL-7Ra, inhibiting post- transcriptional processing of RNA encoding IL-7, IL-7R, or IL-7Ra, reducing the stability of RNA encoding IL-7, IL-7R, or IL-7Ra, promoting degradation of RNA encoding IL-7, IL-7R, or IL-7Ra, inhibiting post- translational processing of IL-7, IL-7R, or IL-7Ra polypeptide, reducing the stability of an IL-7, IL-7R, or IL-7Ra polypeptide or promoting degradation of an IL-7, IL-7R, or IL-7Ra polypeptide.

[0174] The present invention contemplates the use of antisense nucleic acid to prevent / reduce expression of IL- 7, IL-7R, or IL-7Ra. In some embodiments, an agent capable of preventing or reducing the expression of IL-7, IL-7R, or IL-7Ra may cause reduced expression by RNA interference (RNAi). RNAi uses small double-stranded RNA molecules to cause degradation of target mRNA.

[0175] In some embodiments, the agent capable of inhibiting IL-7R-mediated signalling is: an siRNA (or an siRNA derivative or pre-cursor), a short hairpin RNA (shRNA), a micro RNA (miRNA), a long primary miRNA transcript (pri-miRNA), a partially-processed 60-70 base pair hairpin miRNA transcript (pre- miRNA), a small activating RNA (saRNA) or a small nucleolar RNA (snoRNA) that prevents / reduces expression of IL-7, IL-7R, or IL-7Ra.

[0176] In some embodiments, the agent may be an inhibitory nucleic acid, such as antisense or small interfering RNA, including but not limited to shRNA or siRNA.

[0177] In some embodiments the inhibitory nucleic acid is provided in a vector. For example, in some embodiments the agent may be a lentiviral vector encoding shRNA for one or more of IL-7, IL-7R, or IL- 7Ra.

[0178] Oligonucleotide molecules, particularly RNA, may be employed to regulate gene expression. These include antisense oligonucleotides, targeted degradation of mRNAs by small interfering RNAs (siRNAs), post transcriptional gene silencing (PTGs), developmentally regulated sequence-specific translational repression of mRNA by micro-RNAs (miRNAs) and targeted transcriptional gene silencing.

[0179] An antisense oligonucleotide is an oligonucleotide, preferably single-stranded, that targets and binds, by complementary sequence binding, to a target oligonucleotide, e.g. mRNA. 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 may be designed to bind sense genomic nucleic acid and inhibit transcription of a target nucleotide sequence.

[0180] In view of the known nucleic acid sequences for IL-7, IL-7Ra and yc (e.g. the known mRNA sequences available from GenBank under Accession No.s: BC047698.1 Gl: 29126904 (human IL-7), BC110553.2 Gl: 115527463 (mouse IL-7), NM_013110.3 Gl: 1937918433 (rat IL-7), BC069999.1 Gl: 47122838 (human IL-7Ra), BC089571.1 Gl: 58476797 (mouse IL-7Ra), NM_001106418.1 Gl: 157823236 (rat IL- 7Ra), NM 000206.3 Gl : 1780222514 (human yc), BC014720.1 Gl: 15928483 (mouse yc), BC079343.1 Gl: 51260770 (rat yc)) oligonucleotides may be designed to repress or silence the expression of IL-7, IL- 7Ra or yc.

[0181] Such oligonucleotides may have any length, but may preferably be short, e g. less than 100 nucleotides, e.g. 10-40 nucleotides, or 20-50 nucleotides, and may comprise a nucleotide sequence having complete- or near-complementarity (e.g. 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity) to a sequence of nucleotides of corresponding length in the target oligonucleotide, e.g. the IL-7 or IL-7Ra mRNA. The complementary region of the nucleotide sequence may have any length, but is preferably at least 5, and optionally no more than 50, nucleotides long, e.g. 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.

[0182] Repression of expression of IL-7, IL-7R, or IL-7Ra will preferably result in a decrease in the quantity of IL- 7, IL-7R, or IL-7Ra expressed by a cell / tissue / organ / organ system / subject. For example, in a given cell the repression of IL-7, IL-7R, or IL-7Ra by administration of a suitable nucleic acid will result in a decrease in the quantity of IL-7, IL-7R, or IL-7Ra expressed by that cell relative to an untreated cell. Repression may be partial. Preferred degrees of repression are at least 50%, more preferably one of at least 60%, 70%, 80%, 85% or 90%. A level of repression between 90% and 100% is considered a ‘silencing’ of expression or function.

[0183] A role for the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci has been demonstrated. Double-stranded RNA (dsRNA)- dependent post transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. It acts as a signal to promote degradation of mRNA with sequence identity. A 20-nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA. RNAi based therapeutics have been progressed into Phase I, II and III clinical trials for a number of indications (Nature 2009 Jan 22; 457(7228):426-433).

[0184] In the art, these RNA sequences are termed ‘short or small interfering RNAs’ (siRNAs) or ‘microRNAs’ (miRNAs) depending on their origin. Both types of sequence may be used to down-regulate gene expression by binding to complementary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNA are derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin. Micro-interfering RNAs (miRNA) are endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences. siRNA ligands are typically double stranded and, in order to optimise the effectiveness of RNA mediated down-regulation of the function of a target gene, it is preferred that the length of the siRNA molecule is chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of the mRNA target and so that the siRNA is short enough to reduce a host response. miRNA ligands are typically single stranded and have regions that are partially complementary enabling the ligands to form a hairpin. miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. A DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse- complement base pair to form a partially double stranded RNA segment. The design of microRNA sequences is discussed in John et al, PLoS Biology, 1 1 (2), 1862-1879, 2004.

[0185] Typically, the RNA ligands intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogues thereof), more preferably between 17 and 30 ribonucleotides, more preferably between 19 and 25 ribonucleotides and most preferably between 21 and 23 ribonucleotides. In some embodiments of the invention employing double-stranded siRNA, the molecule may have symmetric 3' overhangs, e.g. of one or two (ribo)nucleotides, typically a UU of dTdT 3' overhang. Based on the disclosure provided herein, the skilled person can readily design suitable siRNA and miRNA sequences, for example using resources such the Ambion siRNA finder. siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors). In a preferred embodiment, the siRNA is synthesized synthetically.

[0186] Longer double stranded RNAs may be processed in the cell to produce siRNAs (see for example Myers (2003) Nature Biotechnology 21 :324-328). The longer dsRNA molecule may have symmetric 3' or 5' overhangs, e.g. of one or two (ribo)nucleotides, or may have blunt ends. The longer dsRNA molecules may be 25 nucleotides or longer. Preferably, the longer dsRNA molecules are between 25 and 30 nucleotides long. More preferably, the longer dsRNA molecules are between 25 and 27 nucleotides long. Most preferably, the longer dsRNA molecules are 27 nucleotides in length. dsRNAs 30 nucleotides or more in length may be expressed using the vector pDECAP (Shinagawa et al. , Genes and Dev., 17, 1340-5, 2003).

[0187] Another alternative is the expression of a short hairpin RNA molecule (shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. A shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target. In the cell the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression. In a preferred embodiment the shRNA is produced endogenously (within a cell) by transcription from a vector. shRNAs may be produced within a cell by transfecting the cell with a vector encoding the shRNA sequence under control of a RNA polymerase III promoter such as the human H1 or 7SK promoter or a RNA polymerase II promoter. Alternatively, the shRNA may be synthesised exogenously (in vitro) by transcription from a vector. The shRNA may then be introduced directly into the cell. Preferably, the shRNA molecule comprises a partial sequence of IL-7, IL-7R, or IL-7Ra. Preferably, the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length. The stem of the hairpin is preferably between 19 and 30 base pairs in length. The stem may contain G-U pairings to stabilise the hairpin structure. siRNA molecules, longer dsRNA molecules or miRNA molecules may be made recombinantly by transcription of a nucleic acid sequence, preferably contained within a vector. Preferably, the siRNA molecule, longer dsRNA molecule or miRNA molecule comprises a partial sequence of IL-7, IL-7R, or IL- 7Ra. In one embodiment, the siRNA, longer dsRNA, or miRNA is produced endogenously (within a cell) by transcription from a vector. The vector may be introduced into the cell in any of the ways known in the art. Optionally, expression of the RNA sequence can be regulated using a tissue specific (e.g. liver specific) promoter. In a further embodiment, the siRNA, longer dsRNA, or miRNA is produced exogenously (in vitro) by transcription from a vector.

[0188] Suitable vectors may be oligonucleotide vectors configured to express the oligonucleotide agent capable of IL-7, IL-7R, or IL-7Ra repression. Such vectors may be viral vectors or plasmid vectors. The therapeutic oligonucleotide may be incorporated in the genome of a viral vector and be operably linked to a regulatory sequence, e.g. promoter, which drives its expression. The term ‘operably linked’ may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide sequence under the influence or control of the regulatory sequence. Thus, a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide sequence which forms part or all of the selected nucleotide sequence.

[0189] Viral vectors encoding promoter-expressed siRNA sequences are known in the art and have the benefit of long-term expression of the therapeutic oligonucleotide. Examples include lentiviral (Nature 2009 Jan 22; 457(7228) :426-433), adenovirus (Shen et a!., FEBS Let 2003 Mar 27;539(1-3)111-4) and retroviruses (Barton and Medzhitov PNAS November 12, 2002 vol.99, no.23 14943-14945).

[0190] In other embodiments, a vector may be configured to assist delivery of the therapeutic oligonucleotide to the site at which repression of IL-7, IL-7R, or IL-7Ra expression is required. Such vectors typically involve complexing the oligonucleotide with a positively charged vector (e.g., cationic cell penetrating peptides, cationic polymers and dendrimers, and cationic lipids); conjugating the oligonucleotide with small molecules (e.g., cholesterol, bile acids, and lipids), polymers, antibodies, and RNAs; or encapsulating the oligonucleotide in nanoparticulate formulations (Wang et al., AAPS J. 2010 Dec; 12(4): 492-503).

[0191] In one embodiment, a vector may comprise a nucleic acid sequence in both the sense and antisense orientation, such that when expressed as RNA the sense and antisense sections will associate to form a double stranded RNA.

[0192] Alternatively, siRNA molecules may be synthesized using standard solid or solution phase synthesis techniques which are known in the art. Linkages between nucleotides may be phosphodiester bonds or alternatives, for example, linking groups of the formula P(O)S, (thioate); P(S)S, (dithioate); P(O)NR'2; P(O)R'; P(O)OR6; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through-O-or-S-.

[0193] Modified nucleotide bases can be used in addition to the naturally occurring bases and may confer advantageous properties on siRNA molecules containing them. For example, modified bases may increase the stability of the siRNA molecule, thereby reducing the amount required for silencing. The provision of modified bases may also provide siRNA molecules which are more, or less, stable than unmodified siRNA.

[0194] The term ‘modified nucleotide base’ encompasses nucleotides with a covalently modified base and / or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3'position and other than a phosphate group at the 5'position. Thus, modified nucleotides may also include 2'substituted sugars such as 2'-O-methyl- ; 2'-O-alkyl ; 2'-O-ally I ; 2'-S-alky I; 2'-S-allyl; 2'-fluoro- ; 2'-halo or azidoribose, carbocyclic sugar analogues, a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.

[0195] Modified nucleotides are known in the art and include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles. These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4,N4-ethanocytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine,5- (carboxyhydroxylmethyl) uracil, 5 fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1 -methyladenine, 1- methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3- methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5- methoxy amino methyl-2-thiouracil, -D-mannosylqueosine, 5-methoxycarbonylmethyluracil, 5methoxyuracil, 2 methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, pseudouracil, 2-thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil, 4-thiouracil, 5methyluracil, N-uracil-5-oxyacetic acid methylester, uracil 5-oxyacetic acid, queosine, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5- ethy luracil, 5ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine, and 2, 6, diaminopurine, methylpsuedouracil, 1-methylguanine, 1 -methylcytosine.

[0196] Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and mammals are known in the art (Fire A, et al., 1998 Nature 391 :806-811 ; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001 . Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101 , 25-33 (2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001); W00129058; WO9932619, and Elbashir S M, et al., 2001 Nature 411 :494-498).

[0197] Accordingly, the invention provides nucleic acid that is capable, when suitably introduced into or expressed within a mammalian, e.g. human, cell that otherwise expresses IL-7, IL-7R, or IL-7Ra, or suppressing IL-7, IL-7R, or IL-7Ra by RNAi.

[0198] Nucleic acid sequences for IL-7, IL-7R, or IL-7Ra (e.g. the known mRNA sequences available from GenBank under Accession No s: BC047698.1 Gl: 29126904 (human IL-7), BC110553.2 Gl: 115527463 (mouse IL-7), NM_013110.3 Gl: 1937918433 (rat IL-7), BC069999.1 Gl: 47122838 (human IL-7Ra), BC089571.1 Gl: 58476797 (mouse IL-7Ra), NM_001106418.1 Gl: 157823236 (rat lL-7Ra), NM_000206.3 Gl: 1780222514 (human yc), BC014720.1 Gl: 15928483 (mouse yc), BC079343.1 Gl: 51260770 (rat yc)) oligonucleotides may be designed to repress or silence the expression of IL-7, IL-7R, or IL-7Ra.

[0199] The nucleic acid may have substantial sequence identity to a portion of IL-7, IL-7R, or IL-7Ra mRNA, or the complementary sequence to said mRNA.

[0200] The nucleic acid may be a double-stranded siRNA. As the skilled person will appreciate, a siRNA molecule may include a short 3’ DNA sequence also.

[0201] Alternatively, the nucleic acid may be a DNA (usually double-stranded DNA) which, when transcribed in a mammalian cell, yields an RNA having two complementary portions joined via a spacer, such that the RNA takes the form of a hairpin when the complementary portions hybridise with each other. In a mammalian cell, the hairpin structure may be cleaved from the molecule by the enzyme DICER, to yield two distinct, but hybridised, RNA molecules.

[0202] Accordingly, the invention provides nucleic acid that is capable, when suitably introduced into or expressed within a mammalian cell that otherwise expresses IL-7, IL-7R, or IL-7Ra, of suppressing IL-7 or IL-7Ra expression by RNAi.

[0203] It is expected that perfect identity / complementarity between the nucleic acid of the invention and the target sequence, although preferred, is not essential. Accordingly, the nucleic acid of the invention may include a single mismatch compared to the mRNA of IL-7, IL-7R, or IL-7Ra. It is expected, however, that the presence of even a single mismatch is likely to lead to reduced efficiency, so the absence of mismatches is preferred. When present, 3’ overhangs may be excluded from the consideration of the number of mismatches.

[0204] The term “complementarity” is not limited to conventional base pairing between nucleic acid consisting of naturally occurring ribo- and / or deoxyribonucleotides, but also includes base pairing between mRNA and nucleic acids of the invention that include non-natural nucleotides.

[0205] However, it is also expected that slightly shorter or longer sequences directed to the same region of IL-7, IL-7R, or IL-7Ra mRNA will also be effective. In particular, it is expected that double-stranded sequences between 17 and 23 bp in length will also be effective.

[0206] The strands that form the double-stranded RNA may have short 3’ dinucleotide overhangs, which may be DNA or RNA. The use of a 3’ DNA overhang has no effect on siRNA activity compared to a 3’ RNA overhang but reduces the cost of chemical synthesis of the nucleic acid strands (Elbashir et al., 2001 c). For this reason, DNA dinucleotides may be preferred. When present, the dinucleotide overhangs may be symmetrical to each other, though this is not essential. Indeed, the 3’ overhang of the sense (upper) strand is irrelevant for RNAi activity, as it does not participate in mRNA recognition and degradation (Elbashir et al., 2001a, 2001 b, 2001c).

[0207] While RNAi experiments in Drosophila show that antisense 3’ overhangs may participate in mRNA recognition and targeting (Elbashir et al. 2001 c), 3' overhangs do not appear to be necessary for RNAi activity of siRNA in mammalian cells. Incorrect annealing of 3’ overhangs is therefore thought to have little effect in mammalian cells (Elbashir et al. 2001c; Czauderna et al. 2003).

[0208] Any di nucleotide overhang may therefore be used in the antisense strand of the siRNA. Nevertheless, the dinucleotide is preferably -UU or -UG (or -TT or -TG if the overhang is DNA), more preferably -UU (or - TT). The -UU (or -TT) dinucleotide overhang is most effective and is consistent with (j.e. capable of forming part of) the RNA polymerase III end of transcription signal (the terminator signal is TTTTT).

[0209] Accordingly, this dinucleotide is most preferred. The dinucleotides AA, CC and GG may also be used, but are less effective and consequently less preferred.

[0210] Moreover, the 3’ overhangs may be omitted entirely from the siRNA.

[0211] The invention also provides single-stranded nucleic acids (herein referred to as single-stranded siRNAs) respectively consisting of a component strand of one of the aforementioned double-stranded nucleic acids, preferably with the 3’-overhangs, but optionally without. The invention also provides kits containing pairs of such single-stranded nucleic acids, which are capable of hybridising with each other in vitro to form the aforementioned double-stranded siRNAs, which may then be introduced into cells.

[0212] The invention also provides DNA that, when transcribed in a mammalian cell, yields an RNA (herein also referred to as an shRNA) having two complementary portions which are capable of self-hybridising to produce a double-stranded motif.

[0213] The complementary portions will generally be joined by a spacer, which has suitable length and sequence to allow the two complementary portions to hybridise with each other. The two complementary ( / .e. sense and antisense) portions may be joined 5’-3’ in either order. The spacer will typically be a short sequence, of approximately 4-12 nucleotides, preferably 4-9 nucleotides, more preferably 6-9 nucleotides.

[0214] Preferably the 5’ end of the spacer (immediately 3’ of the upstream complementary portion) consists of the nucleotides -UU- or -UG-, again preferably -UU- (though, again, the use of these particular dinucleotides is not essential). A suitable spacer, recommended for use in the pSuper system of OligoEngine (Seattle, Washington, USA) is UUCAAGAGA. In this and other cases, the ends of the spacer may hybridise with each other, e.g. elongating the double-stranded motif beyond the exact sequences by a small number (e.g. 1 or 2) of base pairs. Similarly, the transcribed RNA preferably includes a 3’ overhang from the downstream complementary portion. Again, this is preferably -UU or -UG, more preferably -UU.

[0215] Such shRNA molecules may then be cleaved in the mammalian cell by the enzyme DICER to yield a double-stranded siRNA as described above, in which one or each strand of the hybridised dsRNA includes a 3’ overhang.

[0216] Techniques for the synthesis of the nucleic acids of the invention are of course well known in the art.

[0217] The skilled person is well able to construct suitable transcription vectors for the DNA of the invention using well-known techniques and commercially available materials. In particular, the DNA will be associated with control sequences, including a promoter and a transcription termination sequence.

[0218] Of particular suitability are the commercially available pSuper and pSuperior systems of OligoEngine (Seattle, Washington, USA). These use a polymerase-lll promoter (H1) and a T5 transcription terminator sequence that contributes two U residues at the 3’ end of the transcript (which, after DICER processing, provide a 3’ UU overhang of one strand of the siRNA).

[0219] Another suitable system is described in Shin et al. (RNA, 2009 May; 15(5): 898-910), which uses another polymerase-lll promoter (U6).

[0220] The double-stranded siRNAs of the invention may be introduced into mammalian cells in vitro or in vivo using known techniques, as described below, to suppress expression of IL-7 or a receptor for IL-7.

[0221] Similarly, transcription vectors containing the DNAs of the invention may be introduced into tumour cells in vitro or in vivo using known techniques, as described below, for transient or stable expression of RNA, again to suppress expression of IL-7 or a receptor for IL-7.

[0222] Accordingly, the invention also provides a method of suppressing expression of IL-7 or a receptor for IL-7 in a mammalian, e.g. human, cell, the method comprising administering to the cell a double-stranded siRNA of the invention or a transcription vector of the invention.

[0223] Similarly, the invention further provides a method of treating a disease or condition characterised by inflammation and / or fibrosis, comprising administering to a subject a double-stranded siRNA of the invention or a transcription vector of the invention.

[0224] The invention further provides the double-stranded siRNAs of the invention and the transcription vectors of the invention, for use in a method of treatment, preferably a method of treating a disease or condition characterised by inflammation and / or fibrosis. The invention further provides the use of the double-stranded siRNAs of the invention and the transcription vectors of the invention in the preparation of a medicament for the treatment of a disease or condition characterised by inflammation and / or fibrosis.

[0225] 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 assist in penetration of the cell membrane.

[0226] Materials and methods suitable for the administration of siRNA duplexes and DNA vectors of the invention are well known in the art and improved methods are under development, given the potential of RNAi technology.

[0227] Generally, many techniques are available for introducing nucleic acids into mammalian cells. The choice of technique will depend on whether the nucleic acid is transferred into cultured cells in vitro or in vivo in the cells of a patient. Techniques suitable for the transfer of nucleic acid 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) Trends in Biotechnology 11 , 205-210).

[0228] In particular, suitable techniques for cellular administration of the nucleic acids of the invention both in vitro and in vivo are disclosed in the following articles:

[0229] General reviews: Borkhardt, A. 2002. Blocking oncogenes in malignant cells by RNA interference-new hope for a highly specific cancer treatment? Cancer Cell. 2:167-8. Hannon, G.J. 2002. RNA interference. Nature. 418:244-51. McManus, M.T., and P.A. Sharp. 2002. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet. 3:737-47. Scherr, M., M.A. Morgan, and M. Eder. 2003b. Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem. 10:245-56. Shuey, D.J., D.E. McCallus, and T. Giordano. 2002. RNAi: gene-silencing in therapeutic intervention. Drug Discov Today. 7:1040-6.

[0230] Systemic delivery using liposomes: Lewis, D.L., J.E. Hagstrom, A.G. Loomis, J.A. Wolff, and H. Herweijer. 2002. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat Genet. 32:107-8. Paul, C.P., P.D. Good, I. Winer, and D.R. Engelke. 2002. Effective expression of small interfering RNA in human cells. Nat Biotechnol. 20:505-8. Song, E., S.K. Lee, J. Wang, N. Ince, N.

[0231] Ouyang, J. Min, J. Chen, P. Shankar, and J. Lieberman. 2003. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 9:347-51 . Sorensen, D.R., M. Leirdal, and M. Sioud. 2003. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol. 327:761-6. Virus mediated transfer: Abbas-Terki, T., W. Blanco-Bose, N. Deglon, W. Pralong, and P. Aebischer.

[0232] 2002. Lentiviral-mediated RNA interference. Hum Gene Ther. 13:2197-201. Barton, G.M., and R. Medzhitov. 2002. Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A. 99:14943-5. Devroe, E., and P.A. Silver. 2002. Retrovirus-delivered siRNA. BMC Biotechnol. 2:15. Lori, F., P. Guallini, L. Galluzzi, and J. Lisziewicz. 2002. Gene therapy approaches to HIV infection. Am J Pharmacogenomics. 2:245-52. Matta, H., B. Hozayev, R. Tomar, P. Chugh, and P.M. Chaudhary. 2003. Use of lentiviral vectors for delivery of small interfering RNA. Cancer Biol Ther. 2:206-10. Qin, X.F., D.S. An, LS. Chen, and D. Baltimore. 2003. Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A. 100:183-8. Scherr, M., K.

[0233] Battmer, A. Ganser, and M. Eder. 2003a. Modulation of gene expression by lentiviral-mediated delivery of small interfering RNA. Cell Cycle. 2:251-7. Shen, C., A.K. Buck, X. Liu, M. Winkler, and S.N. Reske.

[0234] 2003. Gene silencing by adenovirus-delivered siRNA. FEBS Lett. 539:111-4.

[0235] Peptide delivery: Morris, M.C., L. Chaloin, F. Heitz, and G. Divita. 2000. Translocating peptides and proteins and their use for gene delivery. Curr Opin Biotechnol. 11 :461-6. Simeoni, F., M.C. Morris, F. Heitz, and G. Divita. 2003. Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. Nucleic Acids Res. 31 :2717-24. Other technologies that may be suitable for delivery of siRNA to the target cells are based on nanoparticles or nanocapsules such as those described in US patent numbers 6,649,192B and 5,843,509B.

[0236] In some embodiments the methods employ target nucleic acid editing using SSNs. Gene editing using SSNs is reviewed e.g. in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10): e265, which is hereby incorporated by reference in its entirety. Enzymes capable of creating site-specific double strand breaks (DSBs) can be engineered to introduce DSBs to target nucleic acid sequence(s) of interest. DSBs may be repaired by either error-prone non-homologous end-joining (NHEJ), in which the two ends of the break are rejoined, often with insertion or deletion of nucleotides. Alternatively, DSBs may be repaired by highly homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is supplied and introduced at the site of the DSB.

[0237] SSNs capable of being engineered to generate target nucleic acid sequence-specific DSBs include zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats / CRISPR-associated-9 (CRISPR / Cas9) systems.

[0238] ZFN systems are reviewed e.g. in Umov et al., Nat Rev Genet. (2010) 11 (9):636-46, which is hereby incorporated by reference in its entirety. ZFNs comprise a programmable Zinc Finger DNA-binding domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). The DNA-binding domain may be identified by screening a Zinc Finger array capable of binding to the target nucleic acid sequence.

[0239] TALEN systems are reviewed e.g. in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is hereby incorporated by reference in its entirety. TALENs comprise a programmable DNA-binding TALE domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). TALEs comprise repeat domains consisting of repeats of 33-39 amino acids, which are identical except for two residues at positions 12 and 13 of each repeat which are repeat variable di-residues (RVDs). Each RVD determines binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: “HD” binds to C, “Nl” binds to A, “NG” binds to T and “NN” or “NK” binds to G (Moscou and Bogdanove, Science (2009) 326(5959): 1501 .).

[0240] CRISPR / Cas9 and related systems e.g. CRISPR / Cpf1 , CRISPR / C2c1 , CRISPR / C2c2 and CRISPR / C2c3 are reviewed e.g. in Nakade et al., Bioengineered (2017) 8(3):265-273, which is hereby incorporated by reference in its entirety. These systems comprise an endonuclease (e.g. Cas9, Cpf1 etc.) and the singleguide RNA (sgRNA) molecule. The sgRNA can be engineered to target endonuclease activity to nucleic acid sequences of interest.

[0241] In some embodiments the SSN system is a ZFN system, a TALEN system, CRISPR / Cas9 system, a CRISPR / Cpf1 system, a CRISPR / C2c1 system, a CRISPR / C2c2 system or a CRISPR / C2c3 system.

[0242] SSN-mediated knockout of IL-7R, IL-7Ra, or IL-7 (e.g. CRISPR / Cas9-mediated knockout of IL-7Ra or IL- 7) and agents for achieving the same are described e.g. in Choi etal., eLife (2022) 11 :e81559, which is hereby incorporated by reference in its entirety.

[0243] Delivery

[0244] Agents capable of inhibiting IL-7R-mediated signalling according to the present disclosure may be designed / modified and / or formulated to facilitate delivery to, and / or uptake by, a cell type / tissue / organ of interest. For example, the agent may be designed / formulated for delivery to, and / or uptake by, a liver cell (hepatocyte) or hepatic tissue.

[0245] Strategies for targeted delivery of bioactive molecules are reviewed e.g. in Li etal., Int. J. Mol. Sci. (2015) 16: 19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9): 1602-1608, which are hereby incorporated by reference in their entirety.

[0246] In some embodiments, an agent capable of inhibiting IL-7R-mediated signalling according to the present disclosure may be encapsulated in a nanoparticle or a liposome. In some embodiments, an agent capable of inhibiting IL-7R-mediated signalling may be (covalently or non-covalently) associated with a cell-penetrating peptide (e.g. a protein transduction domain, trojan peptide, arginine-rich peptide, vectocell peptide), a cationic polymer, a cationic lipid or a viral carrier.

[0247] Nanoparticles may be organic, e.g. micelles, liposomes, proteins, solid-lipid particles, solid polymer particles, dendrimers, and polymer therapeutics. Nanoparticles may be inorganic, e.g. such as nanotubes or metal particles, optionally with organic molecules added. In some embodiments, a nanoparticle is a nanoparticle described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3:16023, which is hereby incorporated by reference in its entirety. In some embodiments, a nanoparticle is a PLGA, polypeptide, poly(p-amino ester), DOPE, p-cyclodextrin-containing polycation, linear PEI, PAMAM dendrimer, branched PEI, chitosan or polyphosophoester nanoparticle.

[0248] In some embodiments, a nucleic acid according to the present disclosure (e.g. an inhibitory nucleic acid, e.g. an siRNA) comprises one or more moieties facilitating delivery to, and / or uptake by, a cell type or tissue of interest (e.g. a hepatocyte or hepatic tissue). In some embodiments, a nucleic acid according to the present disclosure is linked (e.g. chemically conjugated to) one or more moieties facilitating delivery to, and / or uptake by, a cell type or tissue of interest (e.g. a hepatocyte or hepatic tissue).

[0249] Modification to, and formulation of, nucleic acids to facilitate targeted delivery to cell types and / or tissues of interest is described e.g. in Lorenzer et al., J Control Release (2015) 203:1-15, which is hereby incorporated by reference in its entirety. A moiety facilitating delivery to, and / or uptake by, a cell type or tissue of interest may bind selectively to the target cell type / tissue of interest. The moiety may facilitate traversal of the cell membrane of the target cell type and / or of cells of the tissue of interest. The moiety may bind to a molecule expressed at the cell surface of the target cell type / tissue of interest. The moiety may facilitate internalisation of the nucleic acid by the target cell type / tissue of interest (e.g. by endocytosis).

[0250] Moieties facilitating delivery to, and / or uptake by, cell types or tissues of interest are described e.g. in Benizri et al., Bioconjug Chem. (2019) 30(2): 366-383, which is hereby incorporated by reference in its entirety. Such moieties include e.g. / V-acetylgalactosamine (GalNAc), a-tocopherol, cell-penetrating peptide, nucleic acid aptamer, antibody and antigen-binding fragments / derivatives thereof, cholesterol, squalene, polyethylene glycol (PEG), fatty acid (e.g. palmitic acid) and nucleolipid moieties.

[0251] In some embodiments, a nucleic acid according to the present disclosure (e.g. an inhibitory nucleic acid, e.g. an siRNA) comprises a moiety facilitating delivery to, and / or uptake by, a liver cell (e.g. a hepatocyte) and / or hepatic tissue. In such embodiments, the moiety may facilitate traversal of the hepatocyte cell membrane. The moiety may bind to a molecule expressed at the cell surface of hepatocytes. In some embodiments, a molecule expressed at the cell surface of hepatocytes is an asialoglycoprotein receptor (ASGPR), e.g. ASGR1 or ASGR2. The moiety may facilitate internalisation of a nucleic acid by hepatocytes (e.g. by endocytosis).

[0252] In some embodiments, the moiety is, or comprises, GalNAc ( / V-acetylgalactosamine). In some embodiments, a nucleic acid according to the present disclosure (e.g. an inhibitory nucleic acid, e.g. an siRNA) is conjugated to GalNAc or a GalNAc-containing moiety. GalNAc interacts with asialoglycoprotein receptors expressed by hepatocytes. Nucleic acids conjugated to GalNAc are efficiently internalised by hepatic cells via receptor-mediated endocytosis following binding of GalNAc to ASGPR (see e.g. Nair et al., J. Am. Chem. Soc. (2014) 136(49): 16958-16961). In some embodiments, a nucleic acid is conjugated to one or more (e.g. 1 , 2, 3, 4 or more) GalNAc moieties. In some embodiments, one or more GalNAc moieties may be covalently associated to the 5’ or 3’ end of one or more strands of a nucleic acid. In some embodiments, a nucleic acid is conjugated to a triantennary GalNAc carbohydrate moiety (such moieties are described e.g. in Nair et al., supra).

[0253] In some embodiments, the moiety is, or comprises, a-tocopherol (i.e. vitamin E). In some embodiments, a nucleic acid is conjugated to a-tocopherol. Nucleic acid-a-tocopherol conjugates have been employed for targeted delivery of nucleic acids to the liver (see e.g. Nishina et al., Mol Ther. (2008) 16(4):734-740). In some embodiments, a nucleic acid is conjugated to one or more (e.g. 1 , 2, 3, 4 or more) a-tocopherol moieties. In some embodiments, one or more a-tocopherol moieties may be covalently associated to the 5’ or 3’ end of one or more strands of a nucleic acid.

[0254] Conjugates of biomolecules may be produced utilising ‘click chemistry’, as described e.g. in Nwe and Brechbiel, Cancer Biother Radiopharm. (2009) 24(3):289-302 and Astakhova et al., Mol Pharm. (2018) 15(8): 2892-2899, both of which are hereby incorporated by reference in their entirety. In some embodiments, conjugation may employ akyne-azide or thio-maleimide approaches. In some embodiments, a nucleic acid may be conjugated to a moiety facilitating delivery to, and / or uptake by, a cell type or tissue of interest e.g. at the 3’ and / or 5' end of one or more strands of the nucleic acid.

[0255] Inhibition of IL-7R-mediated signalling

[0256] In embodiments of the present invention, agents capable of inhibiting the action of IL-7R may possess one or more of the following functional properties:

[0257] • Inhibition of signalling mediated by IL-7R;

[0258] • Inhibition of signalling mediated by IL-7Ra;

[0259] • Inhibition of signalling mediated by binding of IL-7 to IL-7R;

[0260] • Inhibition of signalling mediated by binding of IL-7 to IL-7Ra;

[0261] • Inhibition of signalling mediated by binding of IL-7 to IL-7Ra:yc receptor complex;

[0262] • Inhibition of signalling mediated by binding of IL-7:IL-7Ra complex to yc (i.e. IL-7 / IL-7R trans signalling);

[0263] • Inhibition of signalling mediated by multimerisation of IL-7:IL-7Ra:yc complexes;

[0264] • Inhibition of a process mediated by IL-7;

[0265] • Inhibition of gene / protein expression of IL-7 and / or IL-7Ra.

[0266] These properties can be determined by analysis of the relevant agent in a suitable assay, which may involve comparison of the performance of the agent to suitable control agents. The skilled person is able to identify appropriate control conditions for a given assay.

[0267] IL-7R-mediated signalling and / or processes mediated by IL-7R includes signalling mediated by fragments of IL-7R and polypeptide complexes comprising IL-7R or fragments thereof. IL-7R-mediated signalling may be signalling mediated by human IL-7R and / or mouse IL-7R. Signalling mediated by IL-7R may occur following binding of IL-7 or an IL-7 containing complex to IL-7R or a receptor to which IL-7 or said complex binds. In some embodiments, an agent may be capable of inhibiting the biological activity of IL-7R or an IL-7R- containing complex.

[0268] In some embodiments, an agent may be capable of inhibiting the biological activity of IL-7 or an I L-7- containing complex.

[0269] In some embodiments, the agent is an antagonist of one or more signalling pathways which are activated by signal transduction through receptors comprising IL-7Ra and / or yc, e g. IL-7Ra:yc. In some embodiments, the agent is capable of inhibiting signalling through one or more immune receptor complexes comprising IL-7Ra and / or yc, e.g. IL-7Ra:yc. In various aspects of the present invention, an agent provided herein is capable of inhibiting IL-7R-mediated cis and / or trans signalling. In some embodiments in accordance with the various aspects of the present invention an agent provided herein is capable of inhibiting IL-7R-mediated cis signalling.

[0270] In some embodiments, the agent may be capable of inhibiting IL-7R-mediated signalling 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, or 1% or less of the level of signalling in the absence of the agent (or in the presence of an appropriate control agent). In some embodiments, the agent is capable of reducing IL-7R-mediated signalling to less than 1 times, e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times the level of signalling in the absence of the agent (or in the presence of an appropriate control agent).

[0271] In some embodiments, the IL-7R-mediated signalling may be signalling mediated by binding of IL-7 to IL- 7Ra:yc receptor. Such signalling can be analysed e.g. by treating cells expressing IL-7Ra and yc with IL- 7, or by stimulating IL-7 production in cells which express IL-7Ra and yc.

[0272] The IC50 for an agent for inhibition of IL-7R-mediated signalling may be determined, e.g. by culturing cells expressing IL-7Ra and yc in the presence of human IL-7 and the agent. In some embodiments, the agent may exhibit an IC50 of 10 pg / ml or less, preferably one of < 5 pg / ml, < 4 pg / ml, < 3.5 pg / ml, < 3 pg / ml, < 2 pg / ml, < 1 pg / ml, < 0.9 pg / ml, < 0.8 pg / ml, < 0.7 pg / ml, < 0.6 pg / ml, or < 0.5 pg / ml in such an assay.

[0273] In some embodiments, the IL-7R-mediated signalling may be signalling mediated by binding of an IL-7JL- 7Ra complex to yc. In some embodiments, the IL-7:IL-7Ra complex may be soluble, e.g. complex of an extracellular domain of IL-7Ra and IL-7, or a complex of a soluble IL-7Ra isoform / fragment and IL-7. In some embodiments, the soluble IL-7Ra is a soluble (secreted) isoform of IL-7Ra or is the liberated product of proteolytic cleavage of the extracellular domain of cell membrane bound IL-7Ra. In some embodiments, the IL-7:IL-7Ra complex may be cell-bound, e.g. complex of cell-membrane bound IL-7Ra and IL-7. Signalling mediated by binding of IL-7:IL-7Ra complex to yc can be analysed by treating cells expressing yc with IL-7:IL-7Ra complex, e g. recombinant fusion protein comprising IL-7 joined by a peptide linker to the extracellular domain of IL-7Ra.

[0274] In some embodiments, the agent may be capable of inhibiting signalling mediated by binding of IL-7JL- 7Ra complex to yc and is also capable of inhibiting signalling mediated by binding of IL-7 to IL-7Ra:yc receptor.

[0275] In some embodiments, the agent may be capable of inhibiting a process mediated by IL-7R.ln some embodiments, the agent may be capable of inhibiting a process mediated by IL-7. A process mediated by IL-7 and / or IL7R may be the activation of inflammatory genes (e.g. IL1B, ILS) and / or fibrotic genes (e.g. COL1A1, ACTA2, CTGF). Therefore, in some embodiments, the agent may be capable of inhibiting IL-7 and / or IL7R mediated activation of inflammatory and / or fibrotic genes.

[0276] In some embodiments, the agent may be capable of inhibiting gene / protein expression of IL-7 and / or IL- 7Ra. Gene and / or protein expression can be measured as described herein or by methods in the art that will be well known to a skilled person.

[0277] In some embodiments, the agent may be capable of inhibiting gene / protein expression of IL-7 and / or IL- 7Ra 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, or 1 % or less of the level of expression in the absence of the agent (or in the presence of an appropriate control agent). In some embodiments, the agent is capable of inhibiting gene / protein expression of IL-7 and / or IL-7Ra to less than 1 times, e g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times the level of expression in the absence of the agent (or in the presence of an appropriate control agent).

[0278] Treatment / prevention of a disease / condition characterised by fibrosis and / or inflammation

[0279] The present invention provides methods and articles (agents and compositions) for the treatment and / or prevention of a disease or condition characterised by fibrosis and / or inflammation.

[0280] Treatment is achieved by inhibition of IL-7R-mediated signalling (i.e. antagonism of IL-7R-mediated signalling). That is, the present invention provides for the treatment / prevention of a disease or condition characterised by fibrosis and / or inflammation through inhibition of IL-7R mediated signalling, in e.g. a cell, tissue / organ / organ system / subject. Accordingly, the present invention provides an agent capable of inhibiting IL-7R-mediated signalling for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation.

[0281] Also provided is the use of an agent capable of inhibiting IL-7R-mediated signalling for use in the manufacture of a medicament for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation.

[0282] Further provided is a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation, comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting IL-7R-mediated signalling to a subject.

[0283] The methods may be effective to reduce the development or progression of a disease / condition, alleviation of the symptoms of a disease / condition or reduction in the pathology of a disease / condition. The methods may be effective to prevent progression of the disease / condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease / condition. In some embodiments, the methods may lead to an improvement in the disease / condition, e.g. a reduction in the symptoms of the disease / condition or reduction in some other correlate of the severity / activity of the disease / condition. In some embodiments, the methods may prevent development of the disease / condition a later stage (e.g. a chronic stage or metastasis).

[0284] It will be appreciated that the agents of the present disclosure may be used for the treatment / prevention of any disease / condition that would derive therapeutic or prophylactic benefit from a reduction in the level / activity of signalling mediated by IL-7R, IL-7Ra, or an IL-7Ra-containing complex (e.g. IL-7Ra:yc or IL-7R), or of signalling mediated by a cytokine that signals through IL-7R, IL-7Ra, or an IL-7Ra-containing complex (e.g. IL-7).

[0285] It will also be appreciated that the agents of the present disclosure may be used for the treatment / prevention of any disease / condition that would derive therapeutic or prophylactic benefit from a reduction in the number or activity of cells comprising / expressing IL-7R, IL-7Ra, or an IL-7Ra-containing complex comprising IL-7Ra (e.g. IL-7Ra:yc or IL-7R), or IL-7.

[0286] For example, the disease / condition may be a disease / condition in which IL-7R, IL-7Ra, an IL-7Ra- containing complex and / or a cytokine that signals through IL-7R, IL-7Ra, or an IL-7Ra-containing complex are pathologically-implicated, e.g. a disease / condition in which an increased level / activity of IL- 7R, IL-7Ra, a an IL-7Ra-containing complex and / or a cytokine that signals through IL-7R, IL-7Ra, or an IL-7Ra-containing complex is positively-associated with the onset, development or progression of the disease / condition, and / or severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of lL-7R, IL-7Ra, or an IL-7Ra-containing complex and / or a cytokine that signals through IL-7R, IL-7Ra, or an IL-7Ra-containing complex may be a risk factor for the onset, development, or progression of the disease / condition. In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by an increase in the level of expression or activity of IL- 7R, IL-7Ra, or an IL-7Ra-containing complex and / or a cytokine that signals through IL-7R, IL-7Ra, or an IL-7Ra-containing complex, e.g. as compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition to be treated / prevented is a disease / condition characterised by an increase in the number / proportion / activity of cells expressing IL- 7R, IL-7Ra, or an IL-7Ra-containing complex and / or a cytokine that signals through IL-7R, IL-7Ra, or an IL-7Ra-containing complex, e.g. as compared to the level / number / proportion / activity in the absence of the disease / condition (e.g. in a healthy subject, or in equivalent non-diseased tissue).

[0287] Treatment in accordance with the methods of the present disclosure may achieve a reduction in the activity of IL-7R, IL-7Ra, or an IL-7Ra-containing complex and / or a cytokine that signals through IL-7R, IL-7Ra, or an IL-7Ra-containing complex in a subject (compared to an equivalent untreated subject, or a subject treated with an appropriate control).

[0288] In some aspects and embodiments, the agents of the present disclosure are provided for the treatment / prevention of disease / condition selected from: a disease / condition in which IL-7R-mediated signalling is pathologically-implicated, a disease / condition in which signalling through a IL-7Ra-containing complex (e.g. an IL-7Ra:yc complex) is pathologically implicated, a disease / condition in which signalling mediated by a cytokine that signals through IL-7R, IL-7Ra, or an IL-7Ra-containing complex is pathologically-implicated, pathological inflammation, fibrosis, a disease / condition characterised by inflammation or a disease / condition characterised by fibrosis.

[0289] In some aspects and embodiments, the agents of the present disclosure are provided for the treatment / prevention of inflammation, e.g. pathological inflammation. In some aspects and embodiments, the agents of the present disclosure are provided for the treatment / prevention of a disease or condition characterised by inflammation.

[0290] Inflammation and its role in heath and disease is reviewed e.g. in Chen et al., Oncotarget (2018) 9(6): 7204-7218, which is hereby incorporated by reference in its entirety. Inflammation refers to the bodily response to cellular / tissue injury, and is characterised by edema, erythema (redness), heat, pain, and loss of function (stiffness and immobility) resulting from local immune, vascular, and inflammatory cell responses to infection or injury. The injury may result from e.g. of physical (e.g. mechanical) or chemical insult, trauma, infection, cancer, or overactive / aberrant immune responses (e.g. autoimmune disease). Inflammation forms part of the innate immune response and plays an important physiological role in wound healing and the control of infection and contributes to the restoration of tissue homeostasis.

[0291] However, many diseases are associated with an overactive inflammatory response (j.e. excessive inflammation and / or aberrantly activated inflammation), and / or chronic (prolonged) inflammation. Herein, excessive and / or chronic inflammation may be referred to as ‘pathological inflammation’. Pathological inflammation may refer to inflammation which is implicated in ( / .e. which positively contributes to) the pathology of a disease.

[0292] Inflammation to be treated / prevented in accordance with the present disclosure can be of any tissue / organ of the body. In some embodiments, the inflammation is of the lung (e.g. bronchioles, alveoli), airways (e.g. nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi), heart, kidney, liver, skeletal muscle, blood vessels, eye, skin, pancreas, bowel, small intestine, large intestine, colon joints, brain, or bone marrow. Inflammation may also occur in multiple tissues / organs at once.

[0293] In some embodiments, inflammation may be of an organ or tissue of the respiratory system, e.g. the lung (e.g. bronchioles, alveoli), or airways (e.g. nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi). In some embodiments, inflammation may be of an organ or tissue of the cardiovascular system, e.g. the heart or blood vessels. In some embodiments, inflammation may be of an organ or tissue of the gastrointestinal system, e.g. of the liver, bowel, small intestine, large intestine, colon, or pancreas. In some embodiments, inflammation may be of the eye. In some embodiments, inflammation may be of the skin. In some embodiments, inflammation may be of an organ or tissue of the nervous system, e.g. the brain. In some embodiments, inflammation may be of the bone marrow. In some embodiments, inflammation may be of the joints. In some embodiments, inflammation may be of an organ or tissue of the urinary system, e.g. the kidneys. In some embodiments, inflammation may be of an organ or tissue of the musculoskeletal system, e.g. muscle tissue. In some embodiments, inflammation may be of an organ or tissue of one or more organ systems.

[0294] Inflammatory reactions play an important part in triggering fibrosis in many different organ systems. Inflammation can lead to excess in deposition of ECM components in the affected tissues. Low-grade but persistent inflammation is also thought to contribute to the progression of fibrosis in cardiovascular disease and hypertension. In many fibrotic disorders, a persistent inflammatory trigger is crucial to upregulation of production of growth factors, proteolytic enzymes, angiogenic factors and fibrogenic cytokines, which stimulate the deposition of connective tissue elements that progressively remodel and destroy normal tissue architecture.

[0295] In some aspects and embodiments, the articles of the present disclosure are provided for the treatment / prevention of fibrosis. In some aspects and embodiments, the agents of the present disclosure are provided for the treatment / prevention of a disease or condition characterised by fibrosis.

[0296] Fibrosis is a form of pathologic tissue remodelling characterised by the formation of excess connective tissue as a consequence of the excess deposition of extracellular matrix (ECM) components (including collagen). ‘Excess connective tissue’ refers to an amount of connective tissue at a given location (e.g. a given tissue / organ, or part of a given tissue / organ) which is greater than the amount of connective tissue present at that location under normal, non-pathological conditions. Similarly, ‘excess deposition of ECM components’ refers to a level of deposition of one or more ECM components which is greater than the level of deposition under normal, non-pathological conditions. The cellular and molecular mechanisms of fibrosis are described in Wynn, J. Pathol. (2008) 214(2): 199- 210, and Wynn and Ramalingam, Nature Medicine (2012) 18:1028-1040, both of which are hereby incorporated by reference in their entirety.

[0297] Damage to tissues can result from various stimuli, including infections, autoimmune reactions, toxins, radiation, and mechanical injury. Repair typically involves replacement of injured cells by cells of the same type, and replacement of normal parenchymal tissue with connective tissue. Repair processes become pathologic when they are not controlled properly, resulting in excess deposition of ECM components in which normal parenchymal tissue is replaced with connective tissue. In diseases such as idiopathic pulmonary fibrosis, liver cirrhosis, cardiovascular fibrosis, systemic sclerosis and nephritis, extensive tissue remodelling and fibrosis can ultimately lead to organ failure and death.

[0298] The main cellular effectors of fibrosis are myofibroblasts. In response to tissue injury, damaged cells and leukocytes produce pro-fibroinflammatory factors such as TGFp, IL-13 and PDGF, which activate fibroblasts (and other myofibroblast precursor cells) to become aSMA-ex pressing myofibroblasts, and recruit myofibroblasts to the site of injury. Myofibroblasts produce large amounts of extracellular matrix components such as collagen and periostin for wound contracture and closure, and also produce proinflammatory cytokines such as IL-6, and tissue remodelling factors such as MMP2 and TIMP1. Persistent / chronic infection and / or inflammation can result in the generation of too many myofibroblasts, and consequently the over-production of extracellular matrix, resulting in fibrosis. In many diseases and conditions characterised by fibrosis, a persistent inflammatory trigger is crucial to upregulation of production of growth factors, proteolytic enzymes, angiogenic factors and fibrogenic cytokines, which stimulate the deposition of connective tissue elements that progressively remodel and destroy normal tissue architecture.

[0299] Fibrosis can be triggered by pathological conditions, e.g. conditions, infections or disease states that lead to production of pro-fibrotic factors (e.g. as TGFpl). Fibrosis may be caused by physical i njury / stimuli, chemical injury / stimuli , or environmental injury / stimuli. Physical injury / stimuli may occur during surgery, e.g. iatrogenic causes. Chemical injury / stimuli may include drug-induced fibrosis, e.g. following chronic administration of drugs such as bleomycin, cyclophosphamide, amiodarone, procainamide, penicillamine, gold and nitrofurantoin (Daba et al., Saudi Med J. (2004) 25(6): 700-706). Environmental injury / stimuli may include exposure to asbestos fibres or silica.

[0300] Fibrosis can be of any tissue / organ of the body. In some embodiments, fibrosis is of the lung (e.g. bronchioles, alveoli), airways (e g. nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi), heart, kidney, liver, skeletal muscle, blood vessels, eye, skin, pancreas, bowel, small intestine, large intestine, colon, joints, brain, or bone marrow. Fibrosis may also occur in multiple tissues / organs at once.

[0301] In some embodiments, fibrosis may be of an organ or tissue of the respiratory system, e.g. the lung (e.g. bronchioles, alveoli), or airways (e.g. nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi). In some embodiments, fibrosis may be of an organ or tissue of the cardiovascular system, e.g. the heart or blood vessels. In some embodiments, fibrosis may be of an organ or tissue of the gastrointestinal system, e.g. of the bowel, small intestine, large intestine, colon, or pancreas. In some embodiments, fibrosis may be of the liver. In some embodiments, fibrosis may be of the eye. In some embodiments, fibrosis may be of the skin. In some embodiments, fibrosis may be of an organ or tissue of the nervous system, e.g. the brain. In some embodiments, fibrosis may be of the bone marrow. In some embodiments, fibrosis may be of the joints. In some embodiments, fibrosis may be of an organ or tissue of the urinary system, e.g. the kidneys. In some embodiments, fibrosis may be of an organ or tissue of the musculoskeletal system, e.g. muscle tissue. In some embodiments, fibrosis may be of an organ or tissue of one or more organ systems.

[0302] In some embodiments, the disease / condition to be treated in accordance with the present disclosure is a disease / condition characterised by inflammation. In some embodiments, the disease / condition is a disease / condition characterised by fibrosis. In some embodiments, the disease / condition is a disease / condition characterised by inflammation and fibrosis.

[0303] As used herein, a disease / condition which is ‘characterised by inflammation’ is a disease / condition in which inflammation is a symptom of the disease / condition. Diseases / conditions characterised by inflammation include, but are not limited to:

[0304] Diseases / conditions of the respiratory system, such as sinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonitis, pleuritis and mediastinitis;

[0305] Diseases / conditions of the accessory digestive organs such as hepatitis, ascending cholangitis, cholecystitis, pancreatitis and peritonitis;

[0306] Diseases / conditions of the cardiovascular system such as carditis, endocarditis, myocarditis, pericarditis, vasculitis, arteritis, phlebitis, capillaritis and cardiogenic shock;

[0307] Diseases / conditions of the urinary system such as nephritis, glomerulonephritis, pyelonephritis, ureteritis, cystitis and urethritis;

[0308] Diseases / conditions of the nervous system such as encephalitis, myelitis, meningitis, arachnoiditis and neuritis;

[0309] Diseases / conditions of the musculoskeletal system such as arthritis, dermatomyositis, soft tissue, myositis, synovitis / tenosynovitis, bursitis, enthesitis, fasciitis, capsulitis, epicondylitis, tendinitis, panniculitis, osteochondritis: osteitis / osteomyelitis, spondylitis, periostitis and chondritis;

[0310] Diseases / conditions of the oral cavity and throat such as stomatitis, gingivitis, gingivostomatitis, periodontitis, glossitis, tonsillitis, sialadenitis, parotitis, cheilitis, pulpitis and gnathitis;

[0311] Diseases / conditions of the gastrointestinal system such as esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, duodenitis, ileitis, caecitis, appendicitis, proctitis and Peutz-Jeghers syndrome;

[0312] Diseases / conditions of the skin such as dermatitis, folliculitis, cellulitis and hidradenitis;

[0313] Diseases / conditions of the eye such as dacryoadenitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, uveitis and dry eye syndrome;

[0314] Diseases / conditions of the ear such as otitis externa, otitis media, labyrinthitis and mastoiditis; Diseases / conditions of the reproductive system such as oophoritis, salpingitis, endometritis, endometriosis, parametritis, cervicitis, vaginitis, vulvitis, mastitis, orchitis, epididymitis, prostatitis, seminal vesiculitis, balanitis, posthitis, balanoposthitis, chorioamnionitis, funisitis and omphalitis;

[0315] Diseases / conditions of the endocrine system such as insulitis, hypophysitis, thyroiditis, parathyroiditis and adrenalitis;

[0316] Diseases / conditions of the lymphatic system such as lymphangitis and lymphadenitis;

[0317] Cancers, including inflammation-induced and inflammation-associated cancers, such as lung cancer (e.g. lung adenocarcinoma, lung squamous cell carcinoma), prostate cancer, hematological malignancies (e.g. multiple myeloma), pancreatic cancer, cervical cancer, stomach cancer, oesophageal cancer, head and neck cancer, colorectal cancer, colon cancer, liver cancer (e.g. hepatocellular carcinoma) and bile duct cancer;

[0318] Diseases / conditions of the liver, such as chronic liver disease, liver fibrosis, bridging fibrosis, liver cancer, hepatocellular carcinoma (HCC), cirrhosis, hepatitis, metabolic and alcohol related / associated liver disease (MetALD; also known as alcoholic liver disease / ALD or alcoholic fatty liver / AFL), alcoholic hepatitis, steatohepatitis, alcoholic steatohepatitis (ASH), primary biliary cirrhosis (PBC), schistosomal liver disease, metabolic dysfunction-associated liver disease (MAFLD / MASLD; also known as nonalcoholic fatty liver disease or NAFLD) or metabolic dysfunction-associated steatohepatitis (MASH; also known as non-alcoholic steatohepatitis or NASH).

[0319] As used herein, a disease / condition which is ‘characterised by fibrosis’ is a disease / condition in which fibrosis is a symptom of the disease / condition. Diseases and conditions characterised by fibrosis include, but are not limited to:

[0320] Diseases / conditions affecting the respiratory system such as pulmonary fibrosis, fibrothorax, radiation-induced lung injury, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, obliterative bronchiolitis, Hermansky-Pudlak syndrome, asbestosis, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis and asthma;

[0321] Diseases / conditions of the liver, such as chronic liver disease, liver fibrosis, bridging fibrosis, liver cancer, hepatocellular carcinoma (HCC), cirrhosis, hepatitis, metabolic and alcohol related / associated liver disease (MetALD; also known as alcoholic liver disease / ALD or alcoholic fatty liver / AFL), alcoholic hepatitis, steatohepatitis, alcoholic steatohepatitis (ASH), primary biliary cirrhosis (PBC), schistosomal liver disease, metabolic dysfunction-associated liver disease (MAFLD / MASLD; also known as nonalcoholic fatty liver disease or NAFLD) or metabolic dysfunction-associated steatohepatitis (MASH; also known as non-alcoholic steatohepatitis or NASH);

[0322] Diseases / conditions affecting the cardiovascular system such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), fibrosis of the atrium, atrial fibrillation, fibrosis of the ventricle, ventricular fibrillation, myocardial fibrosis, interstitial fibrosis, replacement fibrosis Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrotic vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), atherosclerosis, arterial stiffness, chronic pulmonary hypertension, AIDS-associated pulmonary hypertension, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), varicose veins and cerebral infarcts;

[0323] Diseases / conditions affecting the kidneys such as tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport's syndrome, HIV-associated nephropathy, polycystic kidney disease, Fabry's disease, diabetic nephropathy, chronic glomerulonephritis and nephritis associated with systemic lupus;

[0324] Diseases / conditions affecting the pancreas such as pancreatic fibrosis, cystic fibrosis and chronic pancreatitis;

[0325] Diseases / conditions affecting the nervous system such as gliosis, Alzheimer's disease and multiple sclerosis;

[0326] Diseases / conditions affecting the musculoskeletal system such as muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker’s muscular dystrophy (BMD) and fibrotic myopathy;

[0327] Diseases / conditions affecting the gastrointestinal system such as inflammatory bowel disease (IBD), Crohn’s disease, microscopic colitis and primary sclerosing cholangitis (PSC);

[0328] Diseases / conditions affecting the skin such as scleroderma, nephrogenic systemic fibrosis, Dupuytren’s contracture and cutis keloid;

[0329] Diseases / conditions affecting the eye such as Grave's ophthalmopathy, epiretinal fibrosis, retinal fibrosis, subretinal fibrosis, subretinal fibrosis associated with macular degeneration (e.g. wet age-related macular degeneration (AMD)), diabetic retinopathy, glaucoma, corneal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or of the bleb following trabeculectomy for glaucoma), conjunctival fibrosis and subconjunctival fibrosis;

[0330] Diseases / conditions affecting the joints such as arthrofibrosis, arthritis and adhesive capsulitis; Diseases / conditions affecting multiple tissues / organ systems, including progressive systemic sclerosis (PSS), chronic graft versus host disease (GVHD); fibrotic pre-neoplastic and fibrotic neoplastic disease, and fibrosis induced by chemical or environmental insult (e.g., cancer chemotherapy, pesticides, radiation / cancer radiotherapy);

[0331] Cancers, such as hepatocellular carcinoma, gastric cancer, oesophageal cancer, head and neck cancer, colorectal cancer, pancreatic cancer, cervical cancer, and vulvar cancer;

[0332] Mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis and Peyronie’s disease.

[0333] In some embodiments, the disease / condition to be treated in accordance with the present disclosure is liver disease. In some embodiments, the disease / condition to be treated in accordance with the present disclosure is chronic liver disease. In some embodiments, the disease / condition to be treated in accordance with the present disclosure is liver fibrosis. In some embodiments, the disease / condition to be treated in accordance with the present disclosure is bridging fibrosis. In some embodiments, the disease / condition to be treated in accordance with the present disclosure is liver cancer. In some embodiments, the disease / condition to be treated in accordance with the present disclosure is hepatocellular carcinoma (HCC). In some embodiments, the disease / condition to be treated in accordance with the present disclosure is cirrhosis. In some embodiments, the disease / condition to be treated in accordance with the present disclosure is hepatitis. In some embodiments, the disease / condition to be treated in accordance with the present disclosure is metabolic and alcohol related / associated liver disease (MetALD; also known as alcoholic liver disease / ALD). In some embodiments, the disease / condition to be treated in accordance with the present disclosure is alcoholic fatty liver (AFL). In some embodiments, the disease / condition to be treated in accordance with the present disclosure is alcoholic hepatitis. In some embodiments, the disease / condition to be treated in accordance with the present disclosure is steatohepatitis. In some embodiments, the disease / condition to be treated in accordance with the present disclosure is alcoholic steatohepatitis (ASH). In some embodiments, the disease / condition to be treated in accordance with the present disclosure is primary biliary cirrhosis (PBC). In some embodiments, the disease / condition to be treated in accordance with the present disclosure is schistosomal liver disease. In some embodiments, the disease / condition to be treated in accordance with the present disclosure is metabolic dysfunction-associated liver disease (MASLD; also known as non-alcoholic fatty liver disease or NAFLD). In some embodiments, the disease / condition to be treated in accordance with the present disclosure is metabolic dysfunction- associated steatohepatitis (MASH; also known as non-alcoholic steatohepatitis or NASH).

[0334] In some embodiments, the disease / condition to be treated in accordance with the present disclosure is not chronic inflammation, acute lymphoblastic leukemia, or type I diabetes.

[0335] In some embodiments, the disease / condition to be treated in accordance with the present disclosure is cancer e.g. inflammation induced or inflammation-associated cancer. In some embodiments, the cancer is not acute lymphoblastic leukemia.

[0336] Diseases / conditions contemplated to be treated / prevented in accordance with the present disclosure include e.g. cancer (e.g. liver cancer or HCC), steatosis (e.g. of the liver), MALFD / MASLD and MASH.

[0337] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by an increased level of signalling mediated by a cytokine that signals through IL-7R, IL-7Ra, or an IL-7Ra-containing complex. An ‘increased’ level of signalling in accordance with the present disclosure may by a level of signalling that is greater than a reference level of signalling, which may be the level of signalling detected in the absence of the disease / condition. For example, the reference level of signalling may be the level of signalling detected in a healthy (e.g. nondiseased) subject. In some embodiments, the increased level of signalling may be in cells of a tissue / organ affected by the disease / condition (e.g. cells of a tissue / organ in which one or more symptoms of the disease / condition manifest), or in cells of a cancer (e.g. cells of a tumor). In accordance with such embodiments, an increased level of signalling may be a level of signalling that is greater than the level of signalling detected in cells of the same type (e.g. cells from the same organ / tissue) in the absence of the disease / condition (e.g. the level of signalling detected in cells of the same type from a healthy (e.g. non-diseased) subject), or a level of signalling that is greater than the level of signalling detected in equivalent non-cancerous cells (e.g. equivalent non-tumor tissue). Accordingly, in some embodiments, a disease / condition in which IL-7R-mediated signalling is pathologically implicated, or in which IL-7Ra-mediated signalling is pathologically implicated, or in which IL-7-mediated signalling is implicated, may be selected from: a disease / condition characterised by inflammation, a disease / condition characterised by fibrosis, and a disease / condition characterised by inflammation and fibrosis.

[0338] IL-7R signalling in health and disease is reviewed e.g. in Barata et al. Nat Immunol. 2019;20(12): 1584- 1593. For example, IL-7R / IL-7Ra / IL-7-mediated signalling is implicated in the pathology of cancer. Diseases / conditions in which IL-7R-, IL-7Ra- or IL-7-mediated signalling is pathologically implicated and / or characterised by an increased level of IL-7R-, IL-7Ra- or IL-7-mediated signalling also include diseases / conditions characterised by pathological inflammation and / or fibrosis, e.g. as described herein.

[0339] In some embodiments, inhibition of IL-7R-mediated signalling in accordance with the present disclosure comprises inhibition of IL-7R-mediated signalling in the liver, liver tissue and / or cells thereof. In some embodiments, inhibition of IL-7R-mediated signalling in accordance with the present disclosure comprises inhibition of IL-7R-mediated signalling in hepatic stellate cells and / or hepatocytes.

[0340] Accordingly, the present invention also provides an agent capable of inhibiting IL-7R-mediated signalling for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation of the liver. The present disclosure also provides an agent capable of inhibiting IL-7Ra- mediated signalling for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation of the liver. The present disclosure also provides an agent capable of inhibiting IL-7-mediated signalling for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation of the liver.

[0341] Also provided is the use of an agent capable of inhibiting IL-7R-mediated signalling for use in the manufacture of a medicament for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation of the liver. The present disclosure also provides the use of an agent capable of inhibiting IL-7Ra-mediated signalling for use in the manufacture of a medicament for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation of the liver. The present disclosure also provides the use of an agent capable of inhibiting IL-7-mediated signalling for use in the manufacture of a medicament for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation of the liver.

[0342] Further provided is a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation of the liver, comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting IL-7R-mediated signalling to a subject. The present disclosure also provides a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation of the liver, comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting IL-7Ra-mediated signalling to a subject. The present disclosure also provides a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation of the liver, comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting IL-7-mediated signalling to a subject.

[0343] In some embodiments, the disease / condition characterised by fibrosis and / or inflammation of the liver is selected from: chronic liver disease, liver fibrosis, bridging fibrosis, liver cancer, hepatocellular carcinoma (HCC), cirrhosis, hepatitis, metabolic and alcohol related / associated liver disease (MetALD; also known as alcoholic liver disease / ALD or alcoholic fatty liver / AFL), alcoholic hepatitis, steatohepatitis, alcoholic steatohepatitis (ASH), primary biliary cirrhosis (PBC), schistosomal liver disease, metabolic dysfunction- associated liver disease (MAFLD / MASLD; also known as non-alcoholic fatty liver disease or NAFLD) and metabolic dysfunction-associated steatohepatitis (MASH; also known as non-alcoholic steatohepatitis or NASH).

[0344] In some embodiments, the present invention provides an agent capable of inhibiting IL-7R-mediated signalling for use in a method of treating or preventing MAFLD / MASLD. In some embodiments, the present invention provides an agent capable of inhibiting IL-7R-mediated signalling for use in a method of treating or preventing MASH. In some embodiments, the present invention provides an agent capable of inhibiting IL-7R-mediated signalling for use in a method of treating or preventing MAFLD / MASLD or MASH.

[0345] In some embodiments, the present invention provides the use of an agent capable of inhibiting IL-7R- mediated signalling for use in the manufacture of a medicament for use in a method of treating or preventing MAFLD / MASLD. In some embodiments, the present invention provides the use of an agent capable of inhibiting IL-7R-mediated signalling for use in the manufacture of a medicament for use in a method of treating or preventing MASH. In some embodiments, the present invention provides the use of an agent capable of inhibiting IL-7R-mediated signalling for use in the manufacture of a medicament for use in a method of treating or preventing MAFLD / MASLD or MASH.

[0346] In some embodiments, the present invention provides a method of treating or preventing MAFLD / MASLD, comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting IL-7R-mediated signalling to a subject. In some embodiments, the present invention provides a method of treating or preventing MASH, comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting IL-7R-mediated signalling to a subject. In some embodiments, the present invention provides a method of treating or preventing MAFLD / MASLD or MASH, comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting IL-7R-mediated signalling to a subject.

[0347] The present invention also provides for the treatment / prevention of diseases / conditions that are exacerbated by MAFLD / MASLD or MASH. In some embodiments, the present invention provides for the treatment / prevention of diseases / conditions in a subject for which MAFLD / MASLD or MASH provides a poor diagnosis. In some embodiments, the MAFLD / MASLD or MASH may be characterised by an increase in the expression of IL-7R, IL-7Ra and / or IL-7 (i.e. gene and / or protein expression) in the liver / hepatic tissue / liver cells e.g. as compared to the normal level of expression in the relevant organ / tissue / cells (i.e. in the absence of the disease / condition).

[0348] MAFLD / MASLD or MASH according to the present disclosure may be associated with an upregulation of IL-7R, IL-7Ra and / or IL-7 gene and / or protein expression, e.g. in hepatic cells (e.g. hepatic stellate cells and / or hepatocytes) or in hepatic tissue, or upregulation of extracellular IL-7, IL-7R or IL-7Ra.

[0349] MAFLD / MASLD (also known as NAFLD) is reviewed e.g. in Benedict and Zhang, World J Hepatol. (2017) 9(16): 715-732 and Albhaisi et al., Version 1. FIOOORes. (2018) 7: F1000 Faculty Rev-720, both of which are hereby incorporated by reference in their entirety. MAFLD / MASLD is characterised by steatosis of the liver, and in particular of hepatocytes. MAFLD / MASLD may progress to MASH (also known as NASH), which is steatosis combined with inflammation and / or fibrosis (steatohepatitis).

[0350] In accordance with the various aspects described herein, in some embodiments the disease or condition is characterised by reduced / impaired liver function relative to function in the absence of the disease.

[0351] Treatment may be effective to reduce / delay / prevent the development or progression of the disease / condition. Treatment may be effective to reduce / delay / prevent the worsening of one or more symptoms of the disease / condition. Treatment may be effective to improve one or more symptoms of the disease / condition. Treatment may be effective to reduce the severity of and / or reverse one or more symptoms of the disease / condition. Treatment may be effective to reverse the effects of the disease / condition.

[0352] Prevention may refer to prevention of development of the disease / condition, and / or prevention of worsening of the disease / condition, e.g. prevention of progression of MAFLD / MASLD / MASH, e.g. to a later / chronic stage (e.g. fibrosis and / or cirrhosis).

[0353] In some embodiments, the intervention may be aimed at slowing, stopping and / or reversing impairment of liver function associated with the disease / condition.

[0354] In accordance with various aspects of the present invention, a method of treating and / or preventing a disease / condition according to the present disclosure may comprise increasing survival of a subject having the disease / condition.

[0355] In accordance with various aspects of the present disclosure, methods are provided which are for, or which comprise (e.g. in the context of treatment / prevention of a disease / condition characterised by inflammation and / or fibrosis of the liver), one or more of the following: reducing serum ALT level; reducing liver-to-body weight ratio; increasing / maintaining bodyweight; reducing liver triglyceride level; reducing serum IL-7R level; reducing serum IL-7 level; reducing gene and / or protein expression of IL-7R or IL-7 in the liver; reducing gene and / or protein expression of one or more proinflammatory factors (e.g. selected from TNFa, TIMP1 , IL-10, CXCL1 , IL-1 b, IL-6, MIP2 and MMP2) in the liver; reducing gene and / or protein expression of one or more profibrotic factors (e.g. selected from COL1A1, ACTA2 and CTGF) in the liver; reducing activation of ERK in the liver (i.e. reducing the level of pERK in hepatic tissue); reducing steatosis of hepatic tissue; reducing infiltration of neutrophils (e.g. MPO+ neutrophils) into hepatic tissue; and / or reducing infiltration of macrophages (e.g. F4 / 80+ macrophages) into hepatic tissue.

[0356] Also provided are agents according to the present disclosure for use in such methods, and the use of agents according to the present disclosure in manufacture of compositions (e.g. medicaments) for use in such methods. It will be appreciated that the methods comprise administering an agent capable of inhibiting IL-7R-mediated signalling to a subject.

[0357] Similarly, one or more of the following may be observed in a subject following therapeutic or prophylactic intervention in accordance with the present disclosure (e.g. compared to the level prior to intervention): reduced serum ALT level; reduced liver-to-body weight ratio; increased / maintained bodyweight; reduced liver triglyceride level; reduced serum IL-7R level; reduced gene and / or protein expression of IL-7R in the liver; reduced gene and / or protein expression of one or more proinflammatory factors (e.g. selected from TNFa, TIMP1 , IL-10, CXCL1 , IL-1 b, IL-6, MIP2 and MMP2) in the liver; reducing gene and / or protein expression of one or more profibrotic factors (e.g. selected from COL1A1, ACTA2 and CTGF) in the liver; reduced activation of ERK in the liver (i.e. reducing the level of pERK in hepatic tissue); reduced steatosis of hepatic tissue; reduced infiltration of neutrophils (e.g. MPO+ neutrophils) into hepatic tissue; and / or reduced infiltration of macrophages (e.g. F4 / 80+ macrophages) into hepatic tissue.

[0358] In some embodiments, therapeutic / prophylactic intervention in accordance with the present disclosure may be described as being ‘associated with’ one or more of the effects described in the preceding paragraph. The skilled person is readily able to evaluate such properties using techniques that are routinely practiced in the art.

[0359] In some embodiments, treatment in accordance with the present disclosure may be effective to reverse one or more symptoms of a disease or condition associated with inflammation and / or fibrosis of the liver. Such treatment may be effective to reverse symptoms even in the case of established, advanced or severe disease / pathology (e.g. fibrosis and / or cirrhosis).

[0360] Administration

[0361] Administration of an agent capable of inhibiting IL-7R-mediated signalling is preferably in a "therapeutically effective” or “prophylactically effective” amount, this being sufficient to show benefit to the subject.

[0362] The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease and the nature of the agent. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / condition to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0363] Multiple doses of the agent may be provided. One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.

[0364] Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months. By way of example, doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

[0365] In therapeutic applications, agents capable of inhibiting IL-7R-mediated signalling are preferably formulated as a medicament or pharmaceutical together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.

[0366] The term ‘pharmaceutically acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) 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.

[0367] Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0368] The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

[0369] The formulations may be prepared for suitable administration in accordance with the disease / condition to be treated, e.g. parenteral, systemic, intravenous, intra-arterial, intramuscular, intrathecal, topical, intraocular, intra-conjunctival, subcutaneous, oral, or transdermal routes of administration which may include injection. Injectable formulations may comprise the selected agent in a sterile or isotonic medium. The formulation and mode of administration may be selected according to the agent and disease to be treated.

[0370] In some embodiments, agents capable of inhibiting IL-7R-mediated signalling according to the present disclosure may be formulated and / or modified to facilitate delivery to, and / or uptake by, the liver, hepatic tissue, and / or a liver cell (e.g. hepatic stellate cells and / or hepatocytes).

[0371] Detection of IL-7 and receptors for IL-7

[0372] Some aspects and embodiments of the present invention concern detection of expression of IL-7 or a receptor for IL-7 (e.g. IL-7R, IL-7Ra, or a complex containing IL-7Ra and / or yc) in a sample obtained from a subject.

[0373] In some aspects and embodiments the present invention concerns the upregulation of expression (overexpression) of IL-7 or a receptor for IL-7 (as a protein or oligonucleotide encoding the respective IL-7 or receptor for IL-7) and detection of such upregulation as an indicator of suitability for treatment with an agent capable of inhibiting the action of IL-7 or with an agent capable of preventing or reducing the expression of IL-7 or a receptor for IL-7.

[0374] Upregulated expression comprises expression at a level that is greater than would normally be expected for a cell or tissue of a given type. Upregulation may be determined by measuring the level of expression of the relevant factor in a cell or tissue. Comparison may be made between the level of expression in a cell or tissue sample from a subject and a reference level of expression for the relevant factor, e.g. a value or range of values representing a normal level of expression of the relevant factor for the same or corresponding cell or tissue type. In some embodiments reference levels may be determined by detecting expression of IL-7 or a receptor for IL-7 in a control sample, e.g. in corresponding cells or tissue from a healthy subject or from healthy tissue of the same subject. In some embodiments reference levels may be obtained from a standard curve or data set. Levels of expression may be quantitated for absolute comparison, or relative comparisons may be made.

[0375] In some embodiments upregulation of IL-7 or a receptor for IL-7 (e.g. IL-7R, IL-7Ra, or a complex containing IL-7Ra and / or yc) may be considered to be present when the level of expression in the test sample is at least 1 .1 times that of a reference level. More preferably, the level of expression may be selected from one of at least 1 .2, at least 1 .3, at least 1 .4, at least 1 .5, at least 1 .6, at least 1 .7, at least 1 .8, at least 1 .9, at least 2.0, at least 2.1 , at least 2.2, at least 2.3, at least 2.4 at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.5, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, or at least 10.0 times that of the reference level.

[0376] Expression levels may be determined by one of a number of known in vitro assay techniques, such as PCR based assays, in situ hybridisation assays, flow cytometry assays, immunological or immunohistochemical assays. By way of example suitable techniques involve a method of detecting the level of IL-7 or a receptor for IL- 7 in a sample by contacting the sample with an agent capable of binding IL-7 or a receptor for IL-7 and detecting the formation of a complex of the agent and IL-7 or receptor for IL-7. The agent may be any suitable binding molecule, e.g. an antibody, polypeptide, peptide, oligonucleotide, aptamer or small molecule, and may optionally be labelled to permit detection, e.g. visualisation, of the complexes formed. Suitable labels and means for their detection are well known to those in the art and include fluorescent labels (e.g. fluorescein, rhodamine, eosine and NDB, green fluorescent protein (GFP), chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4- methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, Cy5), isotope markers, radioisotopes (e.g. 32P, 33P, 35S), chemiluminescence labels (e.g. acridinium ester, luminol, isoluminol), enzymes (e.g. peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, luciferase), antibodies, ligands and receptors. Detection techniques are well known to those of skill in the art and can be selected to correspond with the labelling agent. Suitable techniques include PCR amplification of oligonucleotide tags, mass spectrometry, detection of fluorescence or colour, e.g. upon enzymatic conversion of a substrate by a reporter protein, or detection of radioactivity.

[0377] Assays may be configured to quantify the amount of IL-7 or receptor for IL-7 in a sample. Quantified amounts of IL-7 or receptor for IL-7 from a test sample may be compared with reference values, and the comparison used to determine whether the test sample contains an amount of IL-7 or receptor for IL-7 that is higher or lower than that of the reference value to a selected degree of statistical significance.

[0378] Quantification of detected IL-7 or receptor for IL-7 may be used to determine up- or down-regulation or amplification of genes encoding IL-7 or a receptor for IL-7. In cases where the test sample contains fibrotic cells, such up-regulation, down-regulation or amplification may be compared to a reference value to determine whether any statistically significant difference is present.

[0379] A sample obtained from a subject may be of any kind. A biological sample may be taken from any tissue or bodily fluid, e.g. a blood sample, blood-derived sample, serum sample, lymph sample, semen sample, saliva sample, synovial fluid sample. A blood-derived sample may be a selected fraction of a patient’s blood, e.g. a selected cell-containing fraction or a plasma or serum fraction. A sample may comprise a tissue sample or biopsy; or cells isolated from a subject. Samples may be collected by known techniques, such as biopsy or needle aspirate. Samples may be stored and / or processed for subsequent determination of IL-7, IL-7R, or IL-7Ra expression levels.

[0380] Samples may be used to determine the upregulation of IL-7 or receptor for IL-7 in the subject from which the sample was taken.

[0381] In some preferred embodiments a sample may be a tissue sample, e.g. biopsy, taken from the tissue (e.g. liver / hepatic tissue). A sample may be obtained from the liver. A sample may comprise hepatic tissue or liver cells. A subject may be selected for therapy / prophylaxis in accordance with the present invention based on determination that the subject has an upregulated level of expression of IL-7 or of a receptor for IL-7 (e.g. IL-7R, IL-7Ra, or a complex containing IL-7Ra and / or yc). Upregulated expression of IL-7 or of a receptor for IL-7 may serve as a marker of a disease or condition characterised by inflammation and / or fibrosis suitable for treatment with an agent capable of inhibiting IL-7R-mediated signalling.

[0382] Upregulation may be in a given organ (e.g. the liver), tissue (e.g. hepatic tissue) or in selected cells from a given tissue (e.g. hepatic cells, e.g. hepatic stellate cells and / or hepatocytes). Upregulation of expression of IL-7 or of a receptor for IL-7 may also be determined in a circulating fluid, e.g. blood, or in a blood derived sample. Upregulation may be of extracellular IL-7, IL-7R or IL-7Ra. In some embodiments expression may be locally or systemically upregulated.

[0383] Following selection, a subject may be administered with an agent capable of inhibiting IL-7R-mediated signalling.

[0384] Diagnosis and prognosis

[0385] Detection of upregulation of expression of IL-7 or a receptor for IL-7 (e.g. IL-7R, IL-7Ra, or a complex containing IL-7Ra and / or yc) may also be used in a method of diagnosing a disease or condition characterised by inflammation and / or fibrosis, identifying a subject at risk of developing a disease or condition characterised by inflammation and / or fibrosis, and in methods of prognosing or predicting a subject’s response to treatment with an agent capable of inhibiting IL-7R-mediated signalling.

[0386] “Developing", “development” and other forms of “develop” may refer to the onset of a disorder / disease, or the continuation or progression of a disorder / disease.

[0387] In some embodiments, a subject may be suspected of having or suffering from a disease or condition characterised by inflammation and / or fibrosis, e.g. based on the presence of other symptoms indicative of a disease or condition characterised by inflammation and / or fibrosis in the subject's body or in selected cells / tissues of the subject’s body (e.g. the liver / hepatic tissue / liver cells). In some embodiments, the disease or condition characterised by inflammation and / or fibrosis is MAFLD / MASLD or MASH. In some embodiments, a subject may be considered at risk of developing MAFLD / MASLD or MASH, e g. because of age or obesity.

[0388] Determination of upregulation of expression of IL-7 or a receptor for IL-7 may confirm a diagnosis or suspected diagnosis or may confirm that the subject is at risk of developing MAFLD / MASLD or MASH. The determination may also diagnose MAFLD / MASLD / MASH or predisposition as one suitable for treatment with an agent capable of inhibiting IL-7R-mediated signalling.

[0389] As such, a method of providing a prognosis for a subject having, or suspected of, having a disease or condition characterised by inflammation and / or fibrosis may be provided, the method comprising determining whether the expression of IL-7 or a receptor for IL-7R is upregulated in a sample obtained from the subject and, based on the determination, providing a prognosis for treatment of the subject with an agent capable of inhibiting IL-7R-mediated signalling.

[0390] In some aspects, methods of diagnosis or methods of prognosing or predicting a subject’s response to treatment with an agent capable of inhibiting IL-7R-mediated signalling may not require determination of the expression of IL-7 or a receptor for IL-7 but may be based on determining genetic factors in the subject that are predictive of upregulation of expression or activity. Such genetic factors may include the determination of genetic mutations, single nucleotide polymorphisms (SNPs) or gene amplification in IL-7, IL-7R, IL-7Ra and / or yc which are correlated with and / or predictive of upregulation of expression or activity and / or IL-7R-mediated signalling. The use of genetic factors to predict predisposition to a disease state or response to treatment is known in the art, e.g. see Peter Starkel Gut 2008;57:440-442; Wright et al., Mol. Cell. Biol. March 2010 vol. 30 no. 6 1411-1420.

[0391] Genetic factors may be assayed by methods known to those of ordinary skill in the art, including PCR based assays, e.g. quantitative PCR, competitive PCR. By determining the presence of genetic factors, e.g. in a sample obtained from a subject, a diagnosis may be confirmed, and / or a subject may be classified as being at risk of developing a disease / condition described herein, and / or a subject may be identified as being suitable for treatment with an agent capable of inhibiting IL-7R-mediated signalling.

[0392] Some methods may comprise determination of the presence of one or more SNPs linked to secretion of I L-7R / IL-7 or susceptibility to development of a disease or condition characterised by inflammation and / or fibrosis. SNPs are usually bi-allelic and therefore can be readily determined using one of a number of conventional assays known to those of skill in the art (e.g. see Anthony J. Brookes. The essence of SNPs. Gene Volume 234, Issue 2, 8 July 1999, 177-186; Fan et al., Highly Parallel SNP Genotyping.

[0393] Cold Spring Harb Symp Quant Biol 2003. 68: 69-78; Matsuzaki et al., Parallel Genotyping of Over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array. Genome Res. 2004. 14: 414- 425).

[0394] The methods may comprise determining which SNP allele is present in a sample obtained from a subject. In some embodiments determining the presence of the minor allele may be associated with increased IL- 7R / IL-7 secretion or susceptibility to development of a disease or condition characterised by inflammation and / or fibrosis.

[0395] Accordingly, in one aspect of the present invention a method for screening a subject is provided, the method comprising: obtaining a nucleic acid sample from the subject; determining which allele is present in the sample at the polymorphic nucleotide position of one or more SNPs linked to secretion of I L-7R / I L-7 or a SNP in linkage disequilibrium with the one or more SNPs with an r2> 0.8. The determining step may comprise determining whether the minor allele is present in the sample at the selected polymorphic nucleotide position. It may comprise determining whether 0, 1 or 2 minor alleles are present.

[0396] The screening method may be, or form part of, a method for determining susceptibility of the subject to development of a disease or condition characterised by inflammation and / or fibrosis, or a method of diagnosis or prognosis as described herein.

[0397] The method may further comprise the step of identifying the subject as having susceptibility to, or an increased risk of, developing a disease or condition characterised by inflammation and / or fibrosis, e.g. if the subject is determined to have a minor allele at the polymorphic nucleotide position. The method may further comprise the step of selecting the subject for treatment with an agent capable of inhibiting IL-7R- mediated signalling and / or administering an agent capable of inhibiting IL-7R-mediated signalling to the subject in order to provide a treatment for a disease or condition characterised by inflammation and / or fibrosis in the subject or to prevent development or progression of a disease or condition characterised by inflammation and / or fibrosis in the subject.

[0398] In some embodiments, a method of diagnosing a disease or condition characterised by inflammation and / or fibrosis, identifying a subject at risk of developing a disease or condition characterised by inflammation and / or fibrosis, and methods of prognosing or predicting a subject’s response to treatment with an agent capable of inhibiting IL-7R-mediated signalling employs an indicator that is not detection of upregulation of expression of IL-7 or a receptor for IL-7, or genetic factors.

[0399] In some embodiments, a method of diagnosing a disease or condition characterised by inflammation and / or fibrosis (e.g. of the liver), identifying a subject at risk of developing a disease or condition characterised by inflammation and / or fibrosis (e.g. of the liver), and methods of prognosing or predicting a subject’s response to treatment with an agent capable of inhibiting IL-7R-mediated signalling is based on detecting, measuring and / or identifying one or more indicators of organ function (e.g. hepatic function) and / or damage to tissue / cells (e.g. hepatic tissue / cells).

[0400] Methods of diagnosis or prognosis may be performed in vitro on a sample obtained from a subject or following processing of a sample obtained from a subject. Once the sample is collected, the patient is not required to be present for the in vitro method of diagnosis or prognosis to be performed and therefore the method may be one which is not practised on the human or animal body. The sample obtained from a subject may be of any kind, as described herein above.

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

[0402] Subjects may be animal or human. Subjects are preferably mammalian, more preferably human. The subject may be a non-human mammal but is more preferably human. The subject may be male or female. The subject may be a patient.

[0403] The patient may have a disease or condition characterised by inflammation and / or fibrosis as described herein. A subject may have been diagnosed with a disease or condition characterised by inflammation and / or fibrosis, may be suspected of having a disease or condition characterised by inflammation and / or fibrosis, or may be at risk from developing a disease or condition characterised by inflammation and / or fibrosis.

[0404] In embodiments according to the present invention the subject is preferably a human subject. In embodiments according to the present invention, a subject may be selected for treatment according to the methods based on characterisation for certain markers of a disease or condition characterised by inflammation and / or fibrosis.

[0405] Kits

[0406] In some aspects of the present disclosure a kit of parts is provided. In some embodiments, the kit may have at least one container having a predetermined quantity of an inhibitor or composition described herein.

[0407] In some embodiments, the kit may comprise materials for producing an inhibitor or composition described herein.

[0408] The kit may provide the inhibitor or composition together with instructions for administration to a patient in order to treat a specified disease / condition.

[0409] In some embodiments the kit may further comprise at least one container having a predetermined quantity of another therapeutic agent (e.g. as described herein). In such embodiments, the kit may also comprise a second medicament or pharmaceutical composition such that the two medicaments or pharmaceutical compositions may be administered simultaneously or separately such that they provide a combined treatment for the specific disease or condition.

[0410] Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

[0411] Sequence identity

[0412] As used herein, ‘sequence identity’ refers to the percent of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences.

[0413] Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity

[0414] 5 between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Sdding, J. 2005, Bioinformatics 21 , 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When using such 0 software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0415] Sequences ***

[0416] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0417] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0418] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0419] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0420] It must be noted that, as used in the 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 the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

[0421] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0422] Methods described herein may preferably be performed in vitro. The term 'in vitro’ is intended to encompass procedures performed with cells in culture whereas the term 'in vivo’ is intended to encompass procedures with / on intact multi-cellular organisms.

[0423] Brief Description of the Figures

[0424] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

[0425] Figures 1 A to 1 E. (1 A) Volcano plot showing RNA-Seq analysis, representing significantly up- and down- regulated genes in palmitate-treated human primary hepatocytes. (1 B-1 E). Dot plots representing significantly high hepatic IL-7R gene expression analyzed by RT-qPCR in palmitate-treated human primary hepatocytes (1B), HepG2 cells (human hepatic cell line) (1 C), AML12 cells (mouse hepatic cell line) (1D), and human primary hepatic stellate cells (HSCs) (1E). Figures 2A to 2E. (2A) MASH / Cirrhosis patient population distribution (126 samples). (2B) SNP IDs (IL- 7R gene mutations) and their occurrence in the patient population shown in Figure 2A. (2C) Violin plots representing normalized hepatic IL-7R gene expression in healthy controls / obese, steatosis, and MASH patients. The databases (GSE48452, GSE89632, GSE37031) are publicly available at Gene Expression Omnibus (GEO, a database repository of high throughput gene expression data). (2D) and (2E) Box plots representing normalized hepatic IL-7R gene expression in mice (D) and rat (E) fed with different MASLD / MASH-inducing diets. The databases (GSE67679, GSE93819, GSE83596, and GSE65220) are publicly available at GEO.

[0426] Figure 3A and 3B. (3A) Dot plots representing hepatic IL-7R gene expression analyzed by RT-qPCR in mice fed the WDF diet (Western diet + Fructose w / v in drinking water) for 8 or 30 weeks to induce steatosis or MASH, respectively, or high fat methionine-choline deficient (HFMCD) diet for 1 , 4, and 6 weeks to progressively induce MASH. (3B) Correlation plots representing the association between hepatic IL-7R gene expression and collagen content (analyzed by HPA measurement) in mice fed the WDF diet for 8 or 30 weeks to induce steatosis or MASH, respectively, or a high fat methionine-choline deficient (HFMCD) diet for 1 , 4, and 6 weeks to progressively induce MASH.

[0427] Figures 4A to 4C. (4A) Dot plots representing inflammatory and fibrotic gene expression analysis by RT- qPCR in HepG2 cells overexpressing hlL-7R (left-hand figure) or with siRNA knockdown of IL-7R (righthand figure). (4B and 4C) RT-qPCR analysis of key inflammatory and fibrosis gene expression in HepG2 cells following palmitate-induced toxicity (0.5 mM, 24 h) with hlL-7R overexpression (B) or siRNA IL-7R knockdown (C).

[0428] Figure 5A and 5B. IL-7R enhanced palmitate-induced cell death in human hepatic cell line HepG2. (5A and 5B). Representative western blots, and corresponding dots plots, showing Caspase-3 cleavage (C- CASP3), a hallmark of PA-induced lipotoxicity, under hlL-7R overexpression (A) or siRNA IL-7R knockdown (B). The dot blots show densitometric measurements of the western blots.

[0429] Figures 6A to 6D. (6A) Representative western blots showing IL-7R expression in mice liver tissues injected with either AAV8-Null (Null), AAV8-Alb-mlL7R (LOE), or AAV8-Alb-shMirlL7R (LKO), and fed a normal chow diet (NCD) or a high fat methionine-choline deficient (HFMCD) diet as indicated (6B) Representative micrographs (n=5-6 mice per group) showing histopathological analysis using Hematoxylin and Eosin (H&E) for gross histology (indications of steatosis and lobular inflammation), and Sirius Red (indicative of collagen deposition for hepatic fibrosis). Scale bar is 100 pm. (6C and 6D). Dot plots representing IL-7R, inflammatory (IL1 B and IL6) and fibrotic (COL1A1 and CTGF) gene expression analysis by RT-qPCR in the indicated groups.

[0430] Figure 7. RNAseq analysis showing expression of Il7r, 117 and Il2rg in liver tissues from MASH mice fed WDF diet for 8, 16 and 30 weeks to develop MASH progressively. Figures 8A to 8C. I L7 / IL7R neutralization rescues saturated fatty acid PA-induced expression of key inflammatory and fibrotic genes in vitro. (8A) RT-qPCR analysis of key inflammatory (IL1B, IL6) and fibrosis (COL1A 1, ACTA2) genes when stimulated with recombinant IL7 protein (5 ng / ml, 24 h), with or without IL7 and IL7R neutralizing antibodies in AML12 cells. (8B) RT-qPCR analysis of key inflammatory and fibrosis genes during palmitate (PA)-induced toxicity (0.75 mM, 24 h) with or without IL7 and IL7R neutralizing antibodies in AML12 cells. (8C) RT-qPCR analysis of key inflammatory and fibrosis genes during palmitate (PA)-induced toxicity (0.5 mM, 24 h) with or without IL7 neutralizing antibodies in primary human hepatocytes (PHHeps).

[0431] Figures 9A to 9C. Palmitate (PA)-induced IL7R expression in primary human hepatic stellate cells (HSCs), IL7 neutralization rescues PA-induced expression of key inflammatory and fibrotic genes in vitro. (9A) Dot plots representing significantly high IL7R gene expression analyzed by RT-qPCR in PA-treated human primary hepatic stellate cells (HSCs), (9B) RT-qPCR analysis of key inflammatory (IL1B, ILS') and fibrosis (COL1A 1, ACTA2) genes when stimulated with recombinant IL7 protein (5 ng / ml, 24 h), with or without IL7 and IL7R neutralizing antibodies in HSCs. (9C) ) RT-qPCR analysis of key inflammatory and fibrosis genes during palmitate (PA)-induced toxicity (0.5 mM, 24 h) with or without IL7 neutralizing antibodies in HSCs.

[0432] Figures 10A to 10D. IL7 and IL7R blocking antibodies regressed hepatic inflammation and fibrosis in HFMCD diet-induced NASH mice. (10A) Schematic showing NASH treatment strategy using antibodies to block IL7 and IL7R signalling (n=4-5 mice per group). Antibodies were injected (20 pg / g bodyweight) i.p. every other day for two weeks after four weeks of HFMCD diet (in pre-established NASH mice). Isotype IgG was used as control for antibodies. (10B) Dot plots showing percentage body weight change (during antibody treatment for two weeks) and liver index. (10C) Representative micrographs (n=4-5 mice per group) showing histo-pathological analysis using Hematoxylin and Eosin (H&E) for gross histology (indications of steatosis and lobular inflammation) and Sirius Red (indicative of collagen deposition for hepatic fibrosis) Scale bar is 100 pm. (10D) Dot plots representing H7r, inflammatory (111b and 116) and fibrotic Col1a1, Acta2 and Ctgf) gene expression analysis by RT-qPCR in indicated groups.

[0433] Figures 11A and 11 B. Anti-IL7r mAb inhibited hyperactivation of immune cells and improved hepatocyte regeneration in NASH. (11 A) UMAP plots from Single nucleus RNAseq (snRNAseq) analysis of liver tissues from the mice as described in Figure 10A. (11 B) Percent population of indicated cell types in the liver tissues collected from NCD control mice, HFMCD-fed NASH mice, and HFMCD-fed NASH followed by anti-IL7R mAb-treated mice.

[0434] Examples

[0435] Example 1 : IL-7R expression is significantly elevated an in vitro lipotoxic model

[0436] To identify novel modifiers of MASH, RNA-Seq analysis was performed in an in vitro model of MASH. Primary human hepatocytes were treated with saturated long chain free fatty acid palmitate (0.5 mM for 24 h) to induce lipotoxicity (Figure 1 A). The results show that IL-7R expression was highly induced in primary human hepatocytes when subjected to lipotoxic conditions.

[0437] Hepatic IL-7R gene expression was analyzed by RT-qPCR in several palmitate-treated hepatocyte cell lines. Palmitate-induced IL-7R expression was observed in primary human hepatocytes, HepG2 cells (human hepatic cell line), AML12 cells (mouse hepatic cell line), and human primary hepatic stellate cells (HSCs) in a dose-dependent manner (Figure 1 B-E).

[0438] Taken together, these results demonstrate that IL-7R is significantly upregulated in vitro under lipotoxic conditions, in both human and mouse liver cell lines.

[0439] Example 2: IL-7R expression is associated with MASH / cirrhosis progression in patients and animal models

[0440] Genomic data analysis was performed using whole genome sequences from tissues isolated from MASH / Cirrhosis patients. The samples were collected from a cohort of 126 HCC Asian patients with MASH / Cirrhosis (Figure 2A). The analysis revealed five risk loci / germline SNPs within the IL-7R gene at genome-wide significance (Figure 2B; SNP IDs rs1494558, rs1494555, rs194051 , rs2228141 and rs6897932). The five SNPs were identified in a high percentage of MASH patients (about 60%) and an even higher percentage of Cirrhosis patients (about 80%). This suggests a strong association between IL- 7R expression and disease progression.

[0441] IL-7R expression was confirmed in several publicly available human, mouse, and rat hepatic RNA- Seq / transcriptome databases for MASH. IL-7R expression was significantly higher in human MASH subjects when compared to control, healthy obese, or hepatosteatotic subjects (Figure 2C). Hepatic IL-7R expression also was significantly increased in three independent mouse models of MASH when compared to control mice fed with a normal chow (NC) diet (Figure 2D). The three mouse models were as follows: 1) high fat high sugar (HFHS) diet; 2) high fat, cholesterol, and choline deficient (HFCC) diet; and 3) 6 weeks or 12 weeks on HF diet for steatosis or MASH, respectively, in STAM™ mice.

[0442] Moreover, hepatic IL-7R expression increased in a dietary rat model of MASH (Control / CT fed NCD = normal chow diet; Steatotic / ST fed HFD = high fat diet; and NASH fed MCD = methionine and choline deficient diet) (Figure 2E).

[0443] Taken together, these results show that increased IL-7R expression is associated with MASH / Cirrhosis progression in human patients, and in mouse and rat models of these diseases.

[0444] Example 3: Hepatic IL-7R expression is significantly upregulated in dietary mouse models of MASH Hepatic IL-7R expression was confirmed in two dietary mouse models of steatosis and MASH (J Hepatol. 2022 Nov;77(5):1246-1255; Nat Commun. 2022 Sep 3; 13(1 ) :5202; Gastroenterology. 2019 Sep;157(3)777-792. e14). Hepatic IL-7R expression increased significantly in mice fed the WDF diet (Western diet + 15% Fructose w / v in drinking water) for 16 and 30 weeks (to induce MASH progression) (Figure 3A) compared with mice fed the control diet (normal chow diet = NCD). In contrast, hepatic IL-7R expression did not increase in mice fed the WDF diet for 8 weeks (to cause hepatosteatosis) or in mice fed the control diet (normal chow diet = NCD).

[0445] Hepatic IL-7R expression also increased during MASH progression in mice fed a high fat methioninecholine deficient (HFMCD) diet for 4 to 6 weeks (to induce MASH progression) (Figure 3A).

[0446] IL-7R expression also correlated significantly with hepatic collagen (hydroxyproline) content in mice fed WDF or HFMCD diets (Figure 3B).

[0447] Taken together, these results show that hepatic IL-7R expression is strongly associated with MASH progression.

[0448] Example 4: IL-7R directly regulates pro-inflammatorv / fibrogenic gene expression and lipotoxicitv- mediated cell death

[0449] To determine whether elevated IL-7R expression had a causal role in fibrosis, human IL-7R was overexpressed, or downregulated (using siRNA knockdown), in HepG2 cells (Figure 4A). IL-7R overexpression increased the expression of MASH-associated inflammatory genes (IL-1 B and IL-6) and fibrogenic genes (COL1 A1 , ACTA2 and CTGF), whereas IL-7R siRNA knockdown decreased their expression. siRNA knockdown was performed using SMART pool mouse and human siRNAs from Horizon.

[0450] Significantly, IL-7R overexpression further increased the expression of proinflammatory (IL-1 B, IL-6 CCL2, and CCL5) and fibrogenic (COL1A, ACTA2 and CTGF) genes induced by palmitate (PA, 0.5 mM) (Figure 4B). In contrast, IL-7R siRNA knockdown attenuated PA-induced expression of these genes (Figure 4C).

[0451] IL-7R overexpression also increased PA-induced cleavage of CASP3 (Figure 5A), a marker of lipotoxicity in MASH and hepatic cells. In contrast, siRNA knockdown of IL-7R significantly inhibited PA-induced CASP3 cleavage (Figure 5B).

[0452] Taken together, these findings show that IL-7R directly regulates pro-inflammatory and fibrogenic gene expression, and lipotoxicity-mediated cell death.

[0453] Example 5: IL-7R expression correlates with inflammation and fibrosis in vivo

[0454] To investigate whether IL-7R expression correlates with inflammation and fibrosis in vivo, IL-7R was specifically overexpressed or silenced in the hepatocytes of a mouse model of MASH induction (using the high fat methionine-choline deficient (HFMCD) diet). AAV8-mediated gene transduction and shRNA under the control of the Alb promoter were used to overexpress and silence IL-7R expression, respectively (Figure 6A). Liver-specific IL-7R overexpression (LOE) induced inflammatory cell infiltration (H&E staining) and fibrosis (Sirius Red staining showed collagen accumulation) in mice on a normal chow diet (NCD), which was exacerbated in HFMCD diet-fed mice (Figure 6B). Conversely, liver specific IL-7R knockdown (LKO) decreased inflammatory cell infiltration and collagen deposition, aligning with the in vitro data of Example 4.

[0455] Gene expression analysis confirmed that IL-7R overexpression in mice fed the NCD or HFMCD diet exacerbated inflammatory (IL-1 B and IL-6) and fibrogenic (COL1A1 and CTGF) gene expression, while IL-7R silencing completely inhibited the expression of these genes (Figures 6C and 6D).

[0456] In summary, the inventors identified an interleukin receptor (IL-7R) that is highly expressed in MAFLD / MASLD and MASH. IL-7R shows increased expression in human primary hepatocytes treated with long chain free fatty acids and in liver tissues of MASH mice. These findings were confirmed in three hepatic RNAseq databases of control healthy obese, steatosis, and MASH human subjects and two independent dietary MASH mouse models. Further, the inventors confirmed that IL-7R is causatively associated with inflammatory and fibrotic gene regulation by overexpression gene knockdown analyses. Considering the higher prevalence of MASH in older individuals, significantly higher expression of IL-7R was observed in older mice, especially when fed a MAFLD / MASLD-inducing diet.

[0457] Thus, the data in the above Examples highlight a crucial role of IL-7R in the pathogenesis of MASH. The significant induction of IL-7R in response to lipotoxic conditions and its association with genetic risk factors in human MASH and cirrhosis tissues positions IL-7R as a pivotal player in MASH development and progression, offering a promising target for therapeutic intervention. Further, neutralizing or sweeping antibodies against IL-7 / IL-7R, liver-targeted mRNA-based therapies to inhibit IL-7 / IL-7R could be therapeutically beneficial for treating MASH.

[0458] Example 6: Expression of Il7r, 117 and / / 2rq in liver tissues from MASH mice fed a WDF diet develop MASH progressively

[0459] RNAseq analysis of liver samples was performed as described previously (Widjaja et aL, Gastroenterology (2019) 157(3):777-792.e14).

[0460] RNAseq analysis was performed on liver tissues collected from mice fed a western diet supplemented with fructose (WDF) for 8, 16 and 30 weeks to progressively develop hepatosteatosis, mild MASH, and severe MASH respectively. The data demonstrate in an unbiased manner that the expression of Il7r, its ligand 117, as well heterodimerization partner receptor Il2rg, increased significantly as MASH progressed from the mild to severe form (Figure 7). Example 7: IL7 / IL7R neutralization rescues IL7- and PA-induced expression of key inflammatory and fibrotic genes in vitro.

[0461] Mouse hepatic cell line AML12, primary human hepatocytes (PHHeps) and primary human hepatic stellate cells (PH HSCs) were treated either with ligand IL7 or lipotoxic fatty acid palmitic acid (PA) with or without IL7 and IL7R neutralizing antibodies for 24h. RNA was collected, and RT-qPCR was performed to analyze mRNA expression of inflammatory (JL1B and IL6) and fibrotic (C0L1A 1 and ACTA2) genes. The antibodies used in the experiments were: anti-l L7 monoclonal antibody (Bio X Cell Cat# BE0048, RRID:AB_1107711); anti-IL7Ra monoclonal antibody (Bio X Cell Cat# BE0065, RRID:AB_1107590); Mouse lgG2b isotype control antibody (Bio X Cell Cat# BE0086, RRID:AB_1107791).

[0462] RT-qPCR analysis of mouse and human hepatic cells showed that IL7R activation through its ligand IL7 or saturated fat PA, activated the expression of inflammatory (JL1B and ILS) and fibrotic (COL1A1 and ACTA2) genes (Figure 8A, Figure 8B, Figure 8C, Figure 9B, Figure 9C). Interestingly, IL7 and IL7R neutralizing antibodies significantly inhibited the expression of inflammatory (JL1B and IL6) and fibrotic (COL1A1 and ACTA2) genes in AML12 (Figure 8A, Figure 8B), PHHeps (Figure 8C) and PH HSCs (Figure 9B, Figure 9C).

[0463] Example 8: IL7 and IL7R blocking antibodies regressed hepatic inflammation and fibrosis in HFMCD diet- induced NASH mice.

[0464] High fat, methionine and choline deficient (HFMCD) diet fed mice (a dietary-mice model of MASH) was used to test the efficacy of IL7 and IL7R neutralizing antibodies (20 pg / g body weight; injected i.p.) to prevent and regress liver fibrosis. Figure 10A shows the experimental strategy. Body weight change and liver index were recorded. Histological analysis was performed for gross hepatic steatosis, inflammation and fibrosis. RNA was collected from the liver tissues and RT-qPCR was performed to analyze mRNA expression of inflammatory (IL1B and ILS) and fibrotic (COL1A 1, ACTA2 and CTGF) genes, including IL7R expression.

[0465] Body weight and liver index analysis showed that IL7R neutralizing antibody treatment prevented change in body weight and improved liver index caused by a MASH-inducing HFMCD diet (Figure 10B).

[0466] Hematoxylin and eosin (H&E) analysis showed mild improvements in hepatic steatosis and a significant reduction in lobular inflammation with both IL7 and IL7R neutralizing antibody treatment (Figure 10C). Similarly, fibrosis was significantly reduced with both IL7 and IL7R neutralizing antibody treatment when compared to HFMCD fed mice alone. RT-qPCR analysis of these liver tissues showed an increase in IL7R expression, inflammatory (IL1B and IL6) and fibrotic (COL1A 1, ACTA2 and CTGF) genes upon HFMCD diet feeding (Figure 10D). Interestingly, both IL7 and IL7R neutralizing antibody treatment was able to reduce the expression of inflammatory (IL1B and ILS) and fibrotic (COL1A 1, ACTA2 and CTGF) genes upon HFMCD diet feeding, when compared to HFMCD fed mice who received control IgG treatment (Figure 10D). Example 9: IL7R neutralization reduces inflammatory cells and increases hepatocytes in HFMCD diet- induced NASH mice.

[0467] Materials & Methods

[0468] Single nuclei RNA sequencing library preparation and sequencing: Single nuclei RNAseq (snRNAseq) was performed on the livers collected from mice fed normal chow diet (NCD), high fat methionine and choline deficient diet (HFMCD), or HFMCD diet treated with IL7R neutralizing antibody.

[0469] Single nuclei were isolated from frozen mouse liver samples. The single nuclei RNA-seq libraries were constructed using GEXSCOPE™ Single Nuclei RNAseq Library Kit (Singleron Biotechnologies) according to the manufacturer's instructions. Briefly, for each library, the nuclei suspension of specified concentration was loaded onto a microfluidic chip for capture. The single-nuclei capture, lysis and mRNA capture steps were automated using Singleron Matrix NEOTM system. The final single-cell RNA sequencing libraries were sequenced on Illumina NovaSeqX plus 25B flowcell with paired-end 150 bp.

[0470] Transcriptome data pre-processing: Fastq files were preprocessed using the CeleScope® tools (version 2.0.7; www.github.com / singleron-RD / CeleScope, Singleron Biotechnologies), to generate raw data using default parameters. Briefly, cellular barcodes in Read 1 were used to demultiplex and identify reads of the same cell origin. Low quality and adapter sequences were removed using Cutadapt (version 3.7). The mapping was done using STARSOLO (https: / / github.com / alexdobin / STAR / blob / master / docs / STARsolo.md) against mouse genome Mus musculus ensembl 99). The reads were assigned to genes using the feature Count tool (https: / / subread.sourceforge.net) and the cell calling was performed by fitting a negative bimodal distribution and determining the threshold between empty wells and cell-associated wells. The gene count matrix was then generated, providing the number of unique molecular identifier (UMI) for each gene and cell (https: / / cutadapt.readthedocs.io / en / stable / installation.html).

[0471] Single-nuclei data analysis: Downstream analysis was done using the snRNA analysis pipeline CeleScoot 1.2.2 (https: / / github.com / singleron-RD / scrna). QC metrics, such as the number of genes detected per cell (nFeature_RNA) and the percentage of mitochondrial UMI (percent_mt) were extracted from the gene count matrix with the function calculate_qc_metrics. Nuclei with high percentage of mitochondrial counts (>20%) were filtered out. The cells with a high number of detected genes (>5000) were excluded to remove possible doublets. Cell debris characterized by low detected genes (<200) was also excluded from the data analysis. The datasets were then integrated and scaled before performing principal component analysis. Uniform manifold approximation and projection (UMAP) was used to calculate a 2D embedding and to display the data in a 2D scatter plot. Cell type annotation was performed using Cell-ID algorithms and UMAP using the default parameters and the SynEcoSys database V1 .2.0 (https: / / www.synecosys.com / , Singleron Biotechnologies).

[0472] Results snRNAseq on the livers collected from mice fed a normal chow diet (NCD), high fat methionine and choline deficient diet (HFMCD), or HFMCD diet and treated with IL7R neutralizing antibody, showed that MASH induction by a HFMCD diet caused an increase in liver resident inflammatory cells, such as mononuclear phagocytes (MPs: Kupffer cells / Macrophages) and neutrophils. These inflammatory cells were reduced alter treatment with IL7R neutralizing antibody (Figure 11 B). Furthermore, a HFMCD diet caused a reduction in hepatocytes, suggesting loss of healthy liver function. Interestingly, IL7R neutralizing antibody treatment increased liver epithelial cells (cells responsible for regenerating hepatocytes) that corresponds with the increase in hepatocytes observed after IL7R neutralizing antibody treatment (Figure 11 B).

Claims

Claims:1 . An agent capable of inhibiting interleukin 7 receptor (IL-7R)-mediated signalling for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation.

2. Use of an agent capable of inhibiting interleukin 7 receptor (IL-7R)-mediated signalling in the manufacture of a medicament for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation.

3. A method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation, comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting interleukin 7 receptor (IL-7R)-mediated signalling to a subject.

4. The agent for use according to claim 1 , the use according to claim 2, or the method according to claim 3, wherein the agent is an agent capable of reducing the expression or activity of interleukin 7 (IL-7) or a constituent polypeptide of IL-7R, or an agent capable of inhibiting interaction between IL-7 and IL-7R or a constituent polypeptide of IL-7R.

5. The agent for use according to claim 1 or claim 4, the use according to claim 2 or claim 4, or the method according to claim 3 or claim 4, wherein the agent is selected from the group consisting of: a polypeptide complex capable of binding to IL-7, a polypeptide complex capable of binding to IL-7R and a polypeptide complex capable of binding to interleukin 7 receptor subunit alpha (IL-7Ra).

6. The agent for use according to claim 1 , claim 4 or claim 5, the use according to claim 2, claim 4 or claim 5, or the method according to claim 3, claim 4 or claim 5, wherein the agent is an antibody or antigen-binding fragment thereof capable of binding to IL-7, IL-7R or IL-7Ra.

7. The agent for use according to any one of claims 1 or 4-6, the use according to any one of claims 2 or 4-6, or the method according to any one of claims 3-6, wherein the antibody or antigen-binding fragment thereof is capable of binding to IL-7Ra.

8. The agent for use, the use, or the method according to claim 6 or claim 7, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, a bispecific antibody, or a multispecific antibody, or an antigen-binding fragment thereof.

9. The agent for use, the use, or the method according to any one of claims 6-8, wherein the antibody or antigen-binding fragment thereof is a sweeping antibody.

10. The agent for use according to claim 1 or claim 4, the use according to claim 2 or claim 4, or the method according to claim 3 or claim 4, wherein the agent is an aptamer capable of binding to IL-7, IL-7R, or IL-7Ra.11 . The agent for use according to claim 1 or claim 4, the use according to claim 2 or claim 4, or the method according to claim 3 or claim 4, wherein the agent is a sequence-specific nuclease (SSN) targeting a gene encoding IL-7, IL-7R, or IL-7Ra.

12. The agent for use, the use, or the method according to claim 11 , wherein the SSN is a CRISPR-Cas9 nuclease, a transcription activator-like effector nuclease (TALEN), or zinc-finger nuclease (ZFN).

13. The agent for use according to claim 1 or claim 4, the use according to claim 2 or claim 4, or the method according to claim 3 or claim 4, wherein the agent is a nucleic acid or oligonucleotide capable of reducing the expression of IL-7, IL-7R, or IL-7Ra.

14. The agent for use, the use, or the method according to claim 4 or 13, wherein the agent is a nucleic acid capable of reducing expression of IL-7, IL-7R, or IL-7Ra by RNA interference (RNAi).

15. The agent for use, the use, or the method according to claim 4, wherein the agent is capable of modifying a gene encoding IL-7, IL-7R or IL-7Ra to reduce its expression.

16. The agent for use according to claim 1 or claim 4, the use according to claim 2 or claim 4, or the method according to claim 3 or claim 4, wherein the agent is a small molecule capable of binding to IL-7, IL-7R, or IL-7Ra and / or a small molecule capable of reducing the activity of IL-7, IL-7R, or IL-7Ra.

17. The agent for use, the use, or the method according to any of the preceding claims, wherein the disease or condition is characterised by fibrosis and / or inflammation of the liver.

18. The agent for use, the use, or the method according to claim 16, wherein the disease or condition is selected from: chronic liver disease, liver fibrosis, bridging fibrosis, liver cancer, hepatocellular carcinoma (HCC), cirrhosis, hepatitis, alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, steatohepatitis, alcoholic steatohepatitis (ASH), primary biliary cirrhosis (PBC), schistosomal liver disease, metabolic dysfunction-associated liver disease (MAFLD / MASLD) and metabolic dysfunction- associated steatohepatitis (MASH).

19. The agent for use, the use, or the method according to any of the preceding claims, wherein the disease or condition characterised by fibrosis and / or inflammation of the liver is Metabolic dysfunction- associated steatotic liver disease (MAFLD / MASLD).

20. The agent for use, the use, or the method according to any of the preceding claims, wherein the disease or condition characterised by fibrosis and / or inflammation of the liver is metabolic dysfunction- associated steatohepatitis (MASH). 21 . The agent for use, the use, or the method according to any of the preceding claims, wherein the disease or condition characterised by fibrosis and / or inflammation of the liver is cirrhosis.

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