Patent Application
20240000893
The present invention relates to methods for the treatment of fibrosis. The invention discloses new research which demonstrates that interferon-lambda has directly acting anti-fibrotic effects both in vitro and in vivo and may be used to provide effective new therapies for the treatment of multiple types of fibrosis.
Fibrotic diseases are diseases which are characterised by an aberrant wound healing response in which excess fibrous connective tissue is formed in an organ or tissue. The deposition and accumulation of excess extracellular matrix (ECM) components such as collagen and fibronectin results in the hardening and scarring of tissues, causing a pathological remodelling of the organ, and can ultimately lead to organ failure.
Wounding in solid organs typically starts with endothelial damage, platelet aggregation and activation initiating an inflammatory response with infiltration of neutrophils, macrophages, eosinophils and lymphocytes to the wound site. Infiltrating inflammatory cells and affected epithelial cells secrete a variety of growth factors and cytokines that serve to further amplify the inflammatory response. Molecules such as TGF-β, PDGF and IL-13 activate macrophages and lead to the recruitment, proliferation and activation of fibroblasts at the wound site. Activated fibroblasts or myofibroblasts, are characterised by the expression of α-smooth muscle actin and secrete collagen and other ECM components to stabilise the cellular substratum. This allows proliferation and migration of epithelial and endothelial cells over the temporary matrix to regenerate the damaged tissue. Once complete, the inflammatory process shuts down while fibroblasts undergo apoptosis leading to resolution of the wound response.
In fibrotic remodelling, persistent tissue insult or injury, or a disregulation of the repair pathway, leads to an inappropriate wound response. Excess deposition and hyper cross-linking of the collagen and ECM occurs, resulting in excessive accumulation of ECM above normal requirements, which is associated with persistent myofibroblast activation, damage to epithelial cells and a loss of the normal tissue architecture.
Fibrotic diseases can affect any organ or tissue, for example kidney, lung, intestine, skin, or liver. The cause of fibrotic disease can be dependent upon the organ or tissue involved and remains unknown in some diseases such as idiopathic pulmonary fibrosis. In other types of interstitial lung disease the cause is recognised, such as exposure to environmental allergens which causes hypersensitive pneumonitis. Liver fibrosis and ultimately cirrhosis results from chronic liver damage sustained through exposure to a variety of factors including environmental and dietary factors or infectious agents. Sustained over consumption of alcohol or a high fat/sugar diet can also lead to cirrhosis of the liver. Similarly, diabetes, hypertension, exposure to toxic agents and various types of autoimmune disease can damage the kidneys leading to fibrotic remodelling and loss of function. Many types of inflammatory bowel disease such as Crohn's disease or celiac sprue can lead to fibrotic remodelling causing strictures and/or malabsorption.
Treatment of progressive fibrosis is predominantly by treating the underlying disease. For example control of blood pressure, improved glucose management in diabetes, or removal of the damaging allergen or environmental cause. However, in many patients once the fibrotic remodelling has become established the disease becomes self-perpetuating and simple improved therapy of the initiating disease fails to halt the process. Some fibrotic diseases can be treated with anti-inflammatory and immunosuppressive agents, but these are only effective in subsets of patients and slow, rather than stop the disease.
To date there are only 2 validated therapies for fibrotic remodelling and both of these are solely licensed for use in idiopathic pulmonary fibrosis (IPF). Pirfenidone is a small molecule drug that was approved for use in the treatment of IPF in Japan in 2008 and Europe in 2011 which is likely to work via multiple mechanisms of action that are not completely understood but may work in part through a down-regulation of TGF-β. Nintedanib is a tri-angiokinase inhibitor blocking tyrosine kinase activity in VEGF, FGF and PDGF receptors. Both compounds slow the development of IPF, but likely only extending life by up to 2 years based on current studies. Further both are associated with significant side effects such that more than 30% of patients cannot tolerate their use long term. To date, no targeted therapies have been approved for fibrotic indications.
Therefore, there is currently an unmet medical need for improved treatment of fibrotic disease in all organs. Accordingly, it is an object of the present invention to provide a new method of treatment of fibrosis.
The discovery and initial description of the interferon-λ (IFN-λ) family in early 2003 opened an exciting new chapter in the field of IFN research. There are 4 distinct but highly related proteins denoted IFN-λ1, IFN-λ2, IFN-λ3 and IFN-λ4. These proteins are also known as interleukin-29 (IL-29), IL-28A, IL-28B and IFNL4, respectively. Collectively, these 4 cytokines comprise the type III subset of IFNs. They are distinct from both type I and type II IFNs for a number of reasons, including the fact that they signal through a heterodimeric receptor complex that is different from the receptors used by type I or type II IFNs. Although type I IFNs (IFN-a/b) and type III IFNs (IFN-1) signal via distinct receptor complexes, they activate the same intracellular signaling pathway and many of the same biological activities, including antiviral activity, in a wide variety of target cells. Consistent with their antiviral activity, expression of the IFN-λ genes and their corresponding proteins is inducible by infection with many types of viruses. Therefore, expression of the type III IFNs (IFN-λs) and their primary biological activity are very similar to the type I IFNs. However, unlike IFN-α receptors which are broadly expressed on most cell types, including leukocytes, IFN-λ receptors are largely restricted to cells of epithelial origin. The potential clinical importance of IFN-λ as a novel antiviral therapeutic agent is already apparent. In addition, preclinical studies by several groups indicate that IFN-λ may also be useful as a potential therapeutic agent for certain types of cancer.
In the present invention we disclose new research which demonstrates for the first time that cytokines of the interferon-lambda family have directly acting anti-fibrotic effects both in vitro and in vivo, and that these proteins may be used to design effective new methods for the treatment of multiple types of fibrosis.
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. All publications and patents referred to herein are incorporated by reference.
It will be appreciated that any of the embodiments described herein may be combined.
The present invention provides interferon-lambda for use in the treatment of fibrosis. The invention also provides use of interferon-lambda for the preparation of a medicament for the treatment of fibrosis. The invention further provides a method for the treatment of fibrosis comprising administering a therapeutically effective amount of interferon-lambda.
Interferons (IFNs) are an important class of cytokines with diverse roles in host protection from viral infection and immune response modulation. Three distinct types of interferons are recognized (type I, II and III) based on their structural features, receptor usage and biological activities and their roles in host defence vary between the different types.
Type I IFNs (IFN-α/β/ω/ε/κ in humans) possess strong anti-viral activity and are able to induce a potent anti-viral response in a wide variety of cell types (Huang et al 1993). IFN-α/β binds a heterodimeric transmembrane receptor termed the IFNαR (composed of IFNAR1 and IFNAR2 subunits) which, upon engagement with ligand, triggers the activation of the kinases, JAK1 and TYK2, which then phosphorylate specific tyrosines in the intracellular domain of the receptor. This creates docking sites for STAT1 and STAT2 signalling molecules, leading to their recruitment and subsequent phosphorylation. The phosphorylated STATs recruit IFN regulatory factor 9 (IRF9), which together form IFN-stimulated gene factor 3 (ISGF3), which crosses into the nucleus and drives the transcription of IFN-stimulated genes (ISGs).
The type II class includes just IFN-γ, which is classified as a Th1-type cytokine that stimulates cell-mediated immune responses that are important for host protection against pathogenic microorganisms such as Mycobacterium tuberculosis (Bach et al 1997), has a central role in tumour immunity, and amplifies the anti-viral activity of type I IFNs. Therefore, type I and type II IFNs work together to activate both innate and adaptive immune responses, with the purpose of protecting the host against both infection and tumour invasion (Biron et al 2001).
IFNs are part of the larger family of class II cytokines that includes six IL-10-related cytokines: IL-10, IL-19, IL-20, IL-22, IL-24 and IL-26 (Kotenko 2002) and are classed together because they signal via receptors that share common motifs in their extracellular domains. These receptors comprise the class II cytokine receptor family (CRF2) and are typically heterodimers composed of 2 distinct receptor chains, a and p receptor subunits (Stahl et al 1993). In general, a subunits are the primary cytokine binding proteins, and the p subunits are required for formation of high affinity binding sites and for signal transduction.
The type III IFNs or IFN-λs are the most recent addition to the CRF2 family. They demonstrate structural features of the IL-10-related cytokines and induce anti-viral activity in a more restricted cell population than the type I IFNs, namely epithelial cells and certain immune cells (Kotenko et al, 2003; Sheppard et al, 2003). In humans, the type III IFN family is composed of 4 closely related proteins, IFN-λ1, -λ2, -λ3 and -λ4 (also known as IL-29, IL-28A, IL-28B and IFNL4, respectively) whereas the mouse only has IFN-λ2 and -λ3 (IL-28A and IL-28B, respectively). They are highly homologous; the amino acid identity between IFN-λ2 and IFN-λ3 is −96% and the identity between IFN-λ1 and IFN-λ2/λ3 is ˜81%. IFN-λ4 most closely resembles IFN-λ3 but these proteins only share ˜30% identity and IFN-λ4 is predominantly intracellular and very poorly secreted in humans (Hong et al 2016).
The IFN-λ receptor complex is composed of the specific IFN-λ receptor chain 1 (IFN-λR1 or IL-28RA) and the shared IL-10 receptor chain 2 (IL-10R2 or IL-10Rβ). Engagement of the IFN-λ receptor complex by any of the four ligands leads to activation of JAK1 and TYK2, activation of the transcription factors STAT1, STAT2 and IRF9 to form the IFN-stimulated gene factor 3 (ISGF3). ISGF3 regulates gene transcription by binding to IFN-stimulated elements (ISRE) in the promoters of several hundred IFN-stimulated genes (ISGs) which include a number of genes that are associated with the antiviral phenotype, including OAS1, MX1, EIF2AK2 (double-stranded RNA-activated protein kinase) and IRF7. Comparative cDNA microarray analyses have shown that the repertoire of genes that are induced by type III IFNs (IFN-λ) is essentially the same as that induced by type I IFNs (IFN-α/β) (Doyle et al 2006). In conclusion, despite using different receptors, both type I and III IFNs activate ISGF3 (Zhou et al 2007), and therefore induce similar transcriptional responses.
Even though the pattern of gene expression induced by either type I or type III IFNs is very similar, the relative magnitude of gene expression induced by IFN-α is often greater than IFN-λ. This may reflect a difference in the relative strength of signalling through type I versus type III receptors or may simply reflect differences in expression levels of the respective receptors for these ligands.
The crystal structure of IFN-λ reveals a four-helix bundle structure typical of class II cytokines, with the closest structural homolog of IFN-λ being IL-22 (Gad et al 2009) and may suggest that IFN-λ and IL-22 possess parallel functions, protecting epithelial tissue against viral and bacterial infections, respectively. The binding site on the IFN-λR1 receptor chain is well conserved among all four IFN-λs, whereas the binding site on IL-10R2 is poorly defined (Miknis et al 2010). IFN-λR1 consists of two distinct fibronectin type III domains of approximately 100 amino acids each. The ligand-receptor interface includes helix A, loop AB and helix F on the IFN site, as well as loops primarily from the N-terminal domain and inter-domain hinge region of IFN-λR1. The binding mode between the ligand and receptor support an initial long range ionic interaction mediated via hydrogen bonding, which is then followed by hydrophobic interactions to finalize the fit.
IFN-λs are expressed by a variety of hematopoietic cells and epithelial cells in response to viral infection. Pattern-recognition receptors that detect the invading virus on the mucosal epithelial surface initiate a transcriptional response through the transcription factors NF-κB, IRF3, IRF7 and Med23 (Osterlund et al 2007, Griffiths et al 2013). The role of type I and type III IFNs in mediating anti-viral defenses at mucosal interfaces has been shown to be both redundant and unique, depending on the virus and the point of entry. Gut epithelial cells respond exclusively to type III IFN which mediates control of epitheliotropic viruses, such as rotaviruses, in a non-redundant fashion (Pott et al 2011). Reoviruses initiate their infection in the gut epithelia but can penetrate the gut epithelial layer to cause a systemic infection in mice; type III IFN restricts the initial replication in the gut epithelium but type I IFNs are indispensable for the prevention of systemic infection (Mahlakoiv et al 2015). The compartmentalisation of type I and III IFNs is less clear in the respiratory tract where there is a degree of redundancy between the two IFN systems (Mordstein et al 2008). Thus, airway epithelia express receptors for both IFN types whereas gut epithelia express only IFN-λ receptors.
IFN-λR1 receptor expression is restricted mainly to epithelial cells and certain immune cell subsets such as monocyte-derived macrophages, plasmacytoid dendritic cells and NK cells in certain scenarios. Activation of IFN-λR1 in NK cells is linked to maximal production of IFN-γ and anti-tumour activity which may be an effect mediated synergistically with other stimuli or in combination with other cell types (Souza-Fonseca-Guimaraes et al 2015). Owing to the restricted IFN-λR1 expression to immune cells and epithelial cells, the immunomodulatory effects of the type III IFNs are limited but are very effective anti-virals when a local mucosal response is sufficient to control the virus. This is in contrast to the ubiquitous activity of type I IFNs during responses to infection, and therefore more appropriate in the control of severe or systemic infection where widespread immune activation is necessary.
The following references describe differences in signalling and biological effects between type I and type III interferons: Wack et al 2015, Chow & Gale 2015, Hemann et al 2017, Broggi et al 2017, Blumer et al 2017, Chiriac et al 2017, Bhushal et al 2017, Andreakos et al 2017, and Bhushal et al 2017.
The contribution of IFN-λs to antiviral therapies is well established with data available in pre-clinical models in mice and chimpanzees and, in vitro, in primary human hepatocytes infected with hepatitis C virus (HCV) (Thomas et al 2012, Park et al 2012) and in human clinical trials (PhlI BMS data). The range of viruses that can be targeted with IFN-λ include but are not restricted to the following: influenza A/B, severe acute respiratory syndrome (SARS), coronavirus, H1 N1, human immunodeficiency virus (HIV), herpes simplex virus type 2 (HSV2), cytomegalovirus (CMV), Middle East respiratory syndrome (MERS), nororvirus, rotavirus and Ebola (Eslam and George 2015; Gresser 2015). All of these viruses target an epithelial point of entry and therapeutically could be targeted with IFN-λ, which can activate the IFN-λR1 receptor complex at this access point.
Numerous preclinical studies have demonstrated that IFN-λs have anti-tumour activity in a range of tumour types such as hepatoma, melanoma, oesophageal carcinoma, neuroendocrine tumours, colorectal carcinoma, lung adenocarcimoma and Burkitt's lymphoma (Sato et al 2006, Zitzmann et al 2006, Steen et al 2010). The anti-tumour mechanisms of IFN-λs are tumour cell induction of apoptosis and a direct effect on immune cells to boost both the innate and adaptive immune stimulatory activities. IFN-λs are induced in the tumour microenvironment and signalling through the IFN-λR1 receptor has been shown to play an anti-tumour role in IFN-λR1 knockout mice which are more susceptible to sarcoma formation and death in transplanted tumour models. IFN-λ treatment delayed lethality and reduced sarcoma development (Numasaki et al 2007). In the context of cancer treatment, IFN-λs could be envisioned to complement current IFN-α therapy, immune check-point inhibitors or as adjunctives to traditional anti-cancer agents such as bortezomib, temozolomide, cisplatin or 5-FU (Guenterberg et al 2010, Li et al 2010).
Genetic and functional evidence has suggested a protective role for IFN-λ in inflammatory diseases such as asthma, psoriasis and rheumatoid arthritis. In a mouse model of asthma, IFN-λ minimized disease severity, reduced eosinophil infiltration and demonstrated a Th1 immune skewing effect away from Th2 and Th17 cytokines (Koltsida et al 2011). In a mouse collagen-induced arthritis model, IFN-λ2 treatment resulted in abrogation of disease by suppressing IL-1 and IL-17 responses, as well as neutrophil recruitment (Blazek et al 2015). In psoriatic lesions, Th17 cells have been shown to be a source of IFN-λ, where it can suppress the Th2 cytokine, IL-13 (Wolk et al 2013). The role of IFN-λs in inflammatory and autoimmune diseases is still ambiguous.
Three landmark genome wide association studies (GWAS) on HCV have demonstrated that single nucleotide polymorphisms (SNPs) in the IFN-λ region are the strongest single predictor of both response to pegylated IFN-λ1/ribavirin (PEG-IFN/RBV) therapy and spontaneous clearance of chronic HCV infection. These SNPs map to the IFNL3/IFNL4 gene locus and have been shown to be effective for all HCV genotypes and in all geographical locations (Ge et al 2009, Suppiah et al 2009, Tanaka et al 2009). The role of IFNL3/IFNL4 genotype in predicting treatment outcomes to all anti-viral therapies (including both first and second generation direct-acting antiviral drugs) also extends to a wide list of other viral infections including CMV, HSV and Epstein-Barr virus (EBV), and also to co-infection with HCV/HIV and HCV/HBV (Rallon et 2010, Guo et al 2013). Data are consistent with a primary role for IFN-λ in controlling viral infections in different organs, particularly those of epithelial origin.
There is evidence for the role of two SNPs in liver fibrosis, namely the rs12979860 and rs8099917 ‘responder’ genotypes, and the acceleration of hepatic inflammation and fibrosis, in hepatitis C and hepatitis B virus infections and non-alcoholic fatty liver disease (Bochud et al 2012, Eslam et al 2015).
Two functional variants in the IFNL3/IFNL4 region have been described that link to the original GWAS discovery but there may be many other as yet unidentified contributory factors. The first SNP is ΔG/TT (rs368234815) that controls the production of IFN-λ4 and also predicts HCV clearance (Prokunina-Olsson et al 2013). The ancestral ‘ΔG’ allele encodes a functional IFN-λ4, which impairs HCV clearance and hence increases viral load, whereas the more recent (in evolutionary terms) ‘TT’ allele of rs368234815 disrupts the open reading frame of IFNL4 (disrupting protein expression) and is associated with improved viral clearance. This apparent paradox may be linked to the predominantly intracellular localization and poor secretion of IFNL4 and the suggestion that IFNL4 may inhibit the secretion and hence function of the other IFN-λ family members. A second SNP in the 3′UTR region of IFNL3 (rs4803217) affects the messenger RNA stability of IFNL3 (McFarland et al 2014). Although the role of these variants in predicting liver fibrosis is unknown, especially in non-HCV cohorts, they are in linkage disequilibrium with rs12979860 so more work is needed to show if there is any association.
In summary, to date IFN-λs have been described to have antiviral and anti-tumour activity, immune-inflammatory functions and homeostatic effects. In the present invention, we disclose that IFN-λs unexpectedly have directly acting anti-fibrotic effects both in vitro and in vivo. The Examples demonstrate that interferon-lambda proteins have anti-fibrotic effects in in vivo mouse chronic kidney disease models and in in vitro human fibrosis models with relevant primary cells from liver, small intestine, skin, kidney and lung organ systems. These proteins therefore have unexpected potential as anti-fibrotic agents in multiple types of fibrosis, and may be used to design effective new therapies for the treatment of fibrotic diseases.
The terms interferon-lambda (IFN-λ) and interferon-lambdas (IFN-λs) are used interchangeably herein to refer to cytokines of the interferon-lambda family. The interferon-lambda proteins and the interferon-lambda receptor are also known by a number of other synonyms as shown in Table 1.
In one embodiment, the interferon-lambda is IFN-λ1.
In one embodiment, the interferon-lambda is IFN-λ2.
In one embodiment, the interferon-lambda is IFN-λ3.
In one embodiment, the interferon-lambda is IFN-λ4.
Amino acid and DNA sequences for IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4, are shown in
The IFN-λ may be used in the form of a fusion protein, such as an Fc fusion. An Fc fusion protein is composed of an Fc domain of an antibody (e.g. an IgG) fused to the IFN-λ. Fc fusion proteins form dimers as a result of association of the Fc domains. The IFN-λ may thus be in dimeric form, as well as monomeric form.
Functional Mimetics
In one embodiment, the interferon-lambda is a functional mimetic of IFN-λ1.
In one embodiment, the interferon-lambda is a functional mimetic of IFN-λ2.
In one embodiment, the interferon-lambda is a functional mimetic of IFN-λ3.
In one embodiment, the interferon-lambda is a functional mimetic of IFN-λ4.
The term “functional mimetic” means a molecule which has the same or similar biological effects as the wild-type protein. For example an interferon-lambda functional mimetic may activate the interferon-lambda receptor and drive the transcription of IFN-stimulated genes.
In one embodiment, the functional mimetic may reduce hydroxyproline levels in an in vivo model of fibrosis as described in Example 1. In one embodiment, the functional mimetic may inhibit fibronectin and/or collagen deposition in an in vitro model of fibrosis as described in Example 2.
In one embodiment, the interferon-lambda functional mimetic is a fragment of IFN-λ1.
In one embodiment, the interferon-lambda functional mimetic is a fragment of IFN-λ2.
In one embodiment, the interferon-lambda functional mimetic is a fragment of IFN-λ3.
In one embodiment, the interferon-lambda functional mimetic is a fragment of IFN-λ4.
In one embodiment, the interferon-lambda functional mimetic is a variant of IFN-λ1.
In one embodiment, the interferon-lambda functional mimetic is a variant of IFN-λ2.
In one embodiment, the interferon-lambda functional mimetic is a variant of IFN-λ3.
In one embodiment, the interferon-lambda functional mimetic is a variant of IFN-λ4.
A variant interferon-lambda may be or may comprise a variant of one of the specific sequences shown in
A variant interferon-lambda may comprise 1, 2, 3, 4, 5, up to 10, up to 20 or more (typically up to a maximum of 50) amino acid substitutions, deletions and/or additions compared with the specific amino acid sequences in
A variant interferon-lambda may have an amino acid sequence which has more than about 60%, or more than about 70%, e.g. 75 or 80%, typically more than about 85%, e.g. more than about 90 or 95% amino acid identity to the amino acid sequences in
The term “identity” as used herein, indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. The term “similarity” as used herein, indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids which can often be substituted for one another include but are not limited to:
In one embodiment, the interferon-lambda functional mimetic is a derivative of IFN-λ1.
In one embodiment, the interferon-lambda functional mimetic is a derivative of IFN-λ2.
In one embodiment, the interferon-lambda functional mimetic is a derivative of IFN-λ3.
In one embodiment, the interferon-lambda functional mimetic is a derivative of IFN-λ4.
In one embodiment, the interferon-lambda may be or may comprise a derivative of one of the specific sequences shown in
In one embodiment the interferon-lambda of the invention is pegylated i.e. the interferon-lambda is covalently attached to poly(ethyleneglycol) (PEG). Methods for producing pegylated proteins are well known in the art, see for example Chapman A et al., 2002, Advanced Drug Delivery Reviews 54: 531-545.
In one embodiment, the pegylated interferon-lambda is Peginterferon Lambda-1a “Lambda”. (Andersen et al., 2013). “Lambda” is an investigational type III interferon therapeutic agent originally developed at ZymoGenetics (now a fully owned subsidiary of Bristol-Myers Squibb). It was licensed to Eiger Biopharmaceuticals in 2016 and is currently in clinical development for the treatment of chronic hepatitis delta virus (HDV) infection.
In one embodiment, the interferon-lambda functional mimetic is an antibody.
The antibody molecules of the present invention may comprise a complete antibody molecule having full length heavy and light chains or a fragment or antigen-binding portion thereof. The term “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that retain the ability to selectively bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibodies and fragments and antigen binding portions thereof may be, but are not limited to Fab, modified Fab, Fab′, modified Fab′, F(ab′)2, Fv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews—Online 2(3), 209-217). The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab′ fragments described in International patent applications WO 2005/003169, WO 2005/003170 and WO 2005/003171 and Fab-dAb fragments described in International patent application WO2009/040562. Multi-valent antibodies may comprise multiple specificities or may be monospecific (see for example WO 92/22853 and WO 05/113605).
These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.
The antibody of the invention is an interferon-lambda functional mimetic and elicits the same or similar biological effects as the wild-type protein. For example the antibody may bind to an epitope on the interferon-lambda receptor, activating receptor signalling and driving the transcription of IFN-stimulated genes.
In one embodiment the interferon-lambda derivative is a small molecule chemical entity.
Methods of screening chemical libraries to identify small molecule chemical entities which may be potential drug candidates are known in the art. For example, a chemical library may be tested in a ligand-receptor binding assay, or in a cell culture model of organ fibrosis, as described in Example 2.
Fibrotic Diseases
Examples of fibrotic diseases which may be treated using the interferon-lambda of the invention include, but are not limited to interstitial lung disease, pulmonary fibrosis, such as idiopathic pulmonary fibrosis, silicosis, hypersensitivity pneumonitis, non-specific interstitial pneumonia, rheumatoid lung, scleroderma, chronic obstructive pulmonary disease and cystic fibrosis; renal fibrosis (including chronic glomerulonephritis, tubulointerstitial nephropathies and genetic diseases of the kidney) such as diabetic nephropathy, hypertensive nephrosclerosis, focal segmental glomerulosclerosis, IgA nephropathy, mesangial proliferative glomerulonephritis, membranous nephropathy, renovascular disease, polycystic kidney disease, chronic allograft nephropathy and Goodpastures disease; liver fibrosis and liver cirrhosis including primary sclerosing cholangitis, primary biliary cirrhosis, alcohol induced liver diseases and nonalcoholic steatohepatitis (including other fatty acid liver diseases); as well as endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, encapsulating peritoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's disease (plus other gut stricture causing diseases), keloid, myocardial infarction, scleroderma, systemic sclerosis and arthrofibrosis.
In one embodiment, the fibrosis is kidney fibrosis (in which case the IFN-λ may be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4).
In one embodiment, the fibrosis is lung fibrosis (in which case the IFN-λ may be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4).
In one embodiment, the fibrosis is small intestine fibrosis (in which case the IFN-λ may be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4).
In one embodiment, the fibrosis is skin fibrosis (in which case the IFN-λ may be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4).
In one embodiment, the fibrosis is liver fibrosis (in which case the IFN-λ may be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4).
In one embodiment, the fibrosis is peritoneal fibrosis (in which case the IFN-λ may be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4).
In one embodiment, the fibrosis is pancreatic fibrosis (in which case the IFN-λ may be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4).
In one embodiment, the fibrosis is atherosclerosis (in which case the IFN-λ may be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4).
In one embodiment, the fibrosis is selected from the group consisting of kidney fibrosis, lung fibrosis, small intestine fibrosis, skin fibrosis, liver fibrosis, peritoneal fibrosis, pancreatic fibrosis and atherosclerosis.
In one embodiment, the fibrosis is selected from the group consisting of kidney fibrosis, lung fibrosis, small intestine fibrosis, skin fibrosis, peritoneal fibrosis, pancreatic fibrosis and atherosclerosis.
In one embodiment, the fibrosis is not associated with a microbial infection, for example a viral infection. Thus, the subject treated in the invention may be one who does not have an infection such as a viral infection. For example, the subject may be one who does not have a viral infection that is known to be treatable by IFN-λ. The subject may be one who is not infected by a hepatitis virus, such as hepatitis A, B or C; influenza virus; severe acute respiratory syndrome (SARS) virus; coronavirus; human immunodeficiency virus (HIV); herpes simplex virus type 2 (HSV2); cytomegalovirus (CMV); Middle East respiratory syndrome (MERS) virus; norovirus; rotavirus; or Ebola virus.
Pharmaceutical Compositions, Dosages and Dosage Regimes
An interferon-lambda of the invention, or a functionally active fragment or derivative thereof, may be provided in a pharmaceutical composition. The pharmaceutical composition will normally be sterile may additionally comprise a pharmaceutically acceptable adjuvant and/or carrier.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier may be suitable for parenteral, e.g. intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Alternatively, the carrier may be suitable for non-parenteral administration, such as a topical, epidermal or mucosal route of administration.
The carrier may be suitable for oral administration. Depending on the route of administration, the interferon-lambda or functionally active fragment or derivative thereof may be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it.
The pharmaceutical compositions of the invention may include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.
Pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
Pharmaceutical compositions of the invention may comprise additional active ingredients.
Also within the scope of the present invention are kits comprising an interferon-lambda of the invention and instructions for use. The kit may further contain one or more additional reagents, such as an additional therapeutic or prophylactic agent as discussed above.
The interferon-lambda of the invention, or formulations or compositions thereof may be administered for prophylactic and/or therapeutic treatment of a fibrotic disease.
In one embodiment the treatment of a fibrotic disease is a therapeutic treatment. In therapeutic applications, compounds are administered to a subject already suffering from a disorder or condition as described above, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods. An amount adequate to accomplish this is defined as a “therapeutically effective amount”.
In one embodiment the treatment of a fibrotic disease is a prophylactic treatment. In prophylactic applications, formulations are administered to a subject at risk of a disorder or condition as described above, in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. An amount adequate to accomplish this is defined as a “prophylactically effective amount”.
Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject.
A subject for administration may be a human or non-human animal. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Administration to humans is typical.
An antibody/modulator or pharmaceutical composition of the invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Examples of routes of administration for pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection. Alternatively, a pharmaceutical composition of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration. The pharmaceutical composition of the invention may be for oral administration.
A suitable dosage of a pharmaceutical composition of the invention may be determined by a skilled medical practitioner. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A suitable dose may be, for example, in the range of from about 0.01 μg/kg to about 1000 mg/kg body weight, typically from about 0.1 μg/kg to about 100 mg/kg body weight, of the patient to be treated. For example, a suitable dosage may be from about 1 μg/kg to about 10 mg/kg body weight per day or from about 10 μg/kg to about 5 mg/kg body weight per day.
Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
Administration may be in single or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different locations. Alternatively, doses can be via a sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the half-life of the antagonist in the patient and the duration of treatment desired.
As mentioned above, modulators/antibodies or pharmaceutical compositions of the invention may be co-administered with one or other more other therapeutic agents.
Combined administration of two or more agents may be achieved in a number of different ways. Both may be administered together in a single composition, or they may be administered in separate compositions as part of a combined therapy. For example, the one may be administered before, after or concurrently with the other.
In one embodiment the interferon-lambda of the invention is administered in combination with another therapeutically active compound. In one embodiment the other therapeutically active compound is another anti-fibrotic therapeutic agent.
Alternatively, the interferon-lambda may be administered not in combination with another therapeutically active compound. For example, the patient treated with the interferon-lambda may be one who is not treated with an antiviral agent, such as another interferon (e.g. a type I interferon or a type II interferon such as interferon-gamma).
In screening conditions, mouse IL-28A (ILFNL2) was introduced into the mouse by data. Normalized Hydroxyproline (Kidney collagen) show the mean with an error bar of +SEM, **** p<0.0001, *** p<0.001, * p<0.05.
Mouse IL-28A (IFNL2) was introduced into the mice by hydrodynamic transfection as a single cDNA (n=22 group size for all treatment arms) before the administration of Adriamycin by IV injection to induce chronic kidney disease. Control mice were transfected with just saline prior to Adriamycin injection. Mice were followed up for 49 days. On termination kidney hydroxyproline levels (
Figures for Hydroxyproline (μg/mg Kidney) and Serum Creatinine (μg/mL) are box and whisker plots. The central horizontal line shows the median of the data and the box extends from the 25th to the 75th percentiles. The ‘whiskers’ cover a typical range of the data. Normalized Hydroxyproline (Kidney collagen) show the mean with an error bar of +SEM, **** p<0.0001, *** p<0.001, * p<0.05.
In the primary screen, mouse IL-28B (ILFNL3) was introduced into the mouse group by hydrodynamic transfection as a cDNA pair (n=15 group size for all treatment arms) before Adriamycin, and the mice were monitored up to 49 days and on termination kidney hydroxyproline levels (
Figures for Hydroxyproline (μg/mg Kidney) and Serum Creatinine (μg/mL) are box and whisker plots. The central horizontal line shows the median of the data and the box extends from the 25th to the 75th percentiles. The ‘whiskers’ cover a typical range of the data. Normalized Hydroxyproline (Kidney collagen) show the mean with an error bar of +SEM, **** p<0.0001, *** p<0.001, * p<0.05.
Mouse IL-28B (IFNL3) was introduced into the mouse group by hydrodynamic transfection as a single clone (n=22 group size in each treatment arm) before Adriamycin, and the mice were followed up to 49 days. Control mice were transfected with just saline prior to Adriamycin injection. On termination kidney hydroxyproline levels (
Figures for Hydroxyproline (μg/mg Kidney) and Serum Creatinine (μg/mL) are box and whisker plots. The central horizontal line shows the median of the data and the box extends from the 25th to the 75th percentiles. The ‘whiskers’ cover a typical range of the data. Normalized Hydroxyproline (Kidney collagen) show the mean with an error bar of +SEM, **** p<0.0001, *** p<0.001, * p<0.05.
At termination, kidneys from the study described in
Human primary stellate cells (at 1000 cells/well) (
Human primary stellate cells in co-culture with primary hepatocytes (in a ratio of 1:1 with a final cell concentration of 1000 cells/well), stimulated with TGFβ1 (250 μg/ml) were cultured alone (TGFβ1 co-culture) or treated with IL-28A or IL-28B at 10000, 1000, 100 or 10 ng/ml for 5 days. Images shown are from 1 representative field out of 16 fields for each condition. Magnification was performed with a ×10 objective.
Human primary small intestinal fibroblasts (at 2000 cells/well) were treated with IL-28A, IL-28B or IL-29 (all at 10 ng/ml) in culture for 7 days in the presence of TGFβ (10 ng/ml). Cells were removed & the ECM proteins, Fibronectin, Collagen I&III and Collagen IV were measured by immunofluorescent labelling with imaging and quantification on a Cellomics Arrayscan. Data represents Mean Total Intensity plotted as Mean+/−SD of 4 separate wells.
Human primary dermal fibroblasts from 3 individual batches were placed in co-culture with primary human keratinocytes (in a ratio of 9:1 with a final cell concentration of 1500 cells/well) and treated with increasing amounts of IL-28A or IL-28B for 7 days. Cells were removed & the ECM proteins, Fibronectin, Collagen I&III and Collagen IV were measured by immunofluorescent labelling with imaging and quantification on a Cellomics Arrayscan. The normalized data from the 3 batches was calculated as % inhibition and plotted as Mean±SD. Cell viability was measured with PrestoBlue, with the 3 fibroblast batches in co-culture with keratinocytes normalized to control co-culture and the data averaged to give the fold change.
Human primary dermal fibroblasts in co-culture with primary human keratinocytes (in a ratio of 9:1 with a final cell concentration of 1500 cells/well) were treated with IL-29, IL-28A or IL-28B for 7 days in the concentration range of 1250-10000 ng/ml cytokine. Cells were removed & the ECM proteins, Fibronectin, Collagen I&III and Collagen IV were measured by immunofluorescent labelling, with imaging and quantification on a Cellomics Arrayscan. Data represents Mean Total Intensity plotted as Mean+/−SD of 4 separate wells. Cell viability was measured with PrestoBlue.
Human primary dermal fibroblasts from a representative donor in co-culture with primary keratinocytes (in a ratio of 9:1 with a final cell concentration of 1500 cells/well) were cultured alone (control) or treated with IL-29, IL-28A or IL-28B at 10000 or 1250 ng/ml for 7 days. The images represent the combined signal from the Fibronectin, Col I&III and Col IV ECM matrix proteins and shows 1 representative field from a total of 16 fields per condition. Magnification was performed with a ×10 objective.
Human primary dermal fibroblasts (1500 cells/well), stimulated with IL-1α (10 ng/ml) were treated with increasing amounts of IL-28A or IL-28B in culture for 7 days. Cells were removed & the ECM proteins, Fibronectin, Collagen I&III and Collagen IV were measured by immunofluorescent labelling with imaging and quantification on a Cellomics Arrayscan. Data represents Mean Total Intensity plotted as Mean+/−SD of 4 separate wells. Cell viability was measured with PrestoBlue. The data is representative of one out of two different fibroblast batches.
Human primary renal proximal tubular epithelial cells (RPTECs) were placed in co-culture with primary human kidney fibroblasts (in a ratio of 1:1 with a final seeding cell concentration of 2000 cells/well) and treated with IL-29, IL-28A or IL-28B for 7 days. Cells were removed & the ECM proteins, Fibronectin, and Collagen I&III were measured by immunofluorescent labelling with imaging and quantification on a Cellomics Arrayscan. Data represents Mean Total Intensity plotted as Mean+/−SD of 4 separate wells. Cell viability was measured with PrestoBlue. IL-29, IL-28A or IL-28B were either added to the co-culture of RPTECs and fibroblasts at day 0 (
The data presented in
Human primary renal proximal tubular cells (RPTECs) in co-culture with primary human kidney fibroblasts (in a ratio of 1:1 with a final cell concentration of 2000 cells/well at seeding) were cultured alone (control) or treated with IL-29, IL-28A or IL-28B at 1000, 100 or 10 ng/ml for 7 days. Cytokines were added to the RPTECs 24 hours before the inclusion of the fibroblasts in the culture as depicted in
Primary human small airway lung epithelial cells (SAEpithelial) in co-culture with human IPF primary lung fibroblasts (1:1 ratio at a final cell concentration of 2000 cells/well at seeding) were treated with IL-28A or IL-28B for 7 days. Cells were removed & the ECM proteins, Fibronectin, Collagen I&III and Collagen IV were measured by immunofluorescent labelling while total ECM protein was identified by flamingo pink staining. Imaging and quantification was performed on a Cellomics Arrayscan. Data represents Mean Total Intensity plotted as Mean+/−SD of 4 separate wells. Cell viability was measured with PrestoBlue with a read-out on fluorescence.
The protein sequences of IFNL1, IFNL2, IFNL3 and IFNL4 were obtained from Uniprot database and DNA sequences of IFNL1, IFNL2, IFNL3 and IFNL4 were from GenBank.
Human primary stellate cells and primary intrahepatic biliary epithelial cells were placed in co-culture at 1000 cells/well (1:1 ratio), and treated with IL-28A or IL-29 for 5 days. Cells were removed & the ECM proteins, Fibronectin, Collagen I&III and Collagen IV were measured by immunofluorescent labelling with imaging and quantification on a Cellomics Arrayscan. Data represents Mean Total Intensity plotted as Mean+/−SD of 4 separate wells.
(a) 3D glomerular spheroids were constructed from an inner core of human glomerular endothelial cells surrounded by GFP-labelled human podocytes. (b) The spheroids were either grown in media or treated with focal segmental glomerular sclerosis (FSGS) patient plasma at a 15% final concentration plus or minus the presence of human IL-28A protein. In each experiment and for each condition, at least 5 spheroids were imaged. (c) The 2D maximum intensity projections (MIPs) of these spheroids were then analysed using Definiens image analysis to define marker areas above threshold intensity in the green channel under the different culture conditions which was plotted as cell marker area as a proportion of total spheroid area. 7% of podocytes were GFP labelled
Mice were either UUO operated (UUO operated control HDT, n=10), non-operated (non-op, n=5), UUO operated but treated with IL-28B at day-1 for prophylactic dosing (IL-28B HDT Day-1, n=10) or treated with IL-28B at day 7 for therapeutic dosing (IL-28B HDT Day 7, n=10) and all animals were terminated at day 21, kidneys removed, fixed in NBF, paraffin-embedded, sectioned, and stained with picro-sirius red (PSR). The slides were then imaged with a Hamamatsu slide scanner. Representative images of the PSR-stained kidneys sections are shown in
Human primary renal proximal tubular epithelial cells were placed in co-culture at 2000 cells/well and treated with IL-29 for 5 days. Cells were removed & the ECM proteins, Fibronectin, Collagen I&III and Collagen IV were measured by immunofluorescent labelling with imaging and quantification on a Cellomics Arrayscan. Data represents Mean Total Intensity plotted as Mean+/−SD of 4 separate wells.
C57Bl/6 mice were subjected to hydrodynamic transfection with a control vector containing SEAP (n=10) or a vector containing a human IL-29 construct (n=9). 24 hours post transfection mice were subjected to unilateral ureteric obstruction (UUO) of the left kidney or mice were non-operated (n=4). After 7 days the hydrodynamic transfections were repeated and the animals culled at 19 days post UUO.
Kidneys were recovered fixed in NBF, paraffin-embedded, sectioned and stained with picro-sirius red (PSR) and the slides were then imaged with a Hamamatsu slide scanner.
SEAP=secreted alkaline phosphatase control protein
Kotenko, 2002, Cytokine Growth Factor Rev 13(3):223-240.
Kotenko et al, 2003, Nature Immunology 4(1): 69-77 Koltsida et al., 2011, EMBO Molecular Medicine 3(6): 348-361.
Li et al., 2010, European Journal of Cancer 46(1):180-190.
Liu et al., 1999, Gene Therapy 6, 1258-1266.
Mahlakoiv T. et al., 2015, PLoS Pathog. 11: e1004782.
McFarland et al., 2014, Nature Immunology 15: 72-79.
Miknis et al., 2010, Journal of Molecular Biology 404:650-664.
Mordstein M. et al., 2008, PLoS Pathog. 4: e1000151
Summary
Cytokines of the interferon-λ (IFN-λ) family, known as IFN-λ1 (IL-29), IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B), signal through a distinct receptor complex, composed of the IFN-λR1 (IL-28R1) and interleukin-10R2 (IL-10R2) receptor chains.
Using a hydrodynamic transfection in vivo screen in the mouse Adriamycin-induced model of chronic kidney disease (CKD), IFN-λ2 and IFN-λ3 were reported as hits. A hit was called based on the level of kidney fibrosis as determined by a 40% reduction in renal hydroxyproline levels (renal collagen). Both IFN-λ2 and IFN-λ3 also showed a corresponding preservation of renal function as measured by serum creatinine. This led conditional survival rates (ie. not reaching end stage kidney failure) to increase from 50% at 49 days post Adriamycin in saline transfected mice, to 85% when transfected with IFN-λ2, and 100% with IFN-λ3 To validate this anti-fibrotic effect in an alternative model of CKD with a different stimulus, both IFN-λ1 and IFN-λ3-were applied by hydrodynamic transfection in the Unilateral Ureteral Obstruction (UUO) induced kidney fibrosis model of chronic kidney disease, with both giving protection against tubulointerstitial fibrosis.
The anti-fibrotic effect of the IL-28R1 ligands has been confirmed in several human primary cell in vitro models of fibrosis that read out on mature accumulated extracellular matrix (ECM) protein. Recombinant human IFN-λ1, IFN-λ2 and IFN-λ3 each inhibited the accumulation of the fibrosis-associated ECM proteins (fibronectin and collagens I, III and IV) in primary human cell in vitro models of liver, lung, gut, skin and kidney fibrosis. This is seen in several models including liver stellate cells, liver stellate-hepatocyte co-cultures (both basal matrix and TGFβ-induced), liver stellate-epithelial cell co-cultures, TGFβ-induced small intestinal fibroblasts, IL-1α-induced dermal fibroblasts, keratinocyte-dermal fibroblast co-cultures, renal proximal tubular epithelial cell-kidney fibroblast co- and mono-cultures and lung small airway epithelial-IPF lung fibroblast co-cultures. In a more complex glomerular organoid model of glomerulosclerosis, application of plasma from a patient with relapsing nephrotic syndrome caused both a loss of podocyte number and retraction of processes and pedicels in a manner consistent with podocyte effacement and loss seen in many glomerular diseases that lead to glomerulosclerosis and renal fibrosis. Application of recombinant IFN-λ2 in this model totally prevented changes to the podocytes caused by this patient plasma.
The IL-28R1 ligands have been shown to have potent anti-fibrotic effects in both in vitro and in vivo fibrosis screens. Potent effects have been described in in vivo mouse models of renal fibrosis and in numerous in vitro human fibrosis models with relevant primary cells from liver, small intestine, skin, kidney and lung organ systems. These proteins therefore have unexpected potential as anti-fibrotic agents in multiple types of fibrosis.
Methods
Mouse In Vivo
Effect of IL-28A and IL-28B in Adriamycin-Induced Kidney Fibrosis Model of Chronic Kidney Disease (CKD)
Female Balb/c mice, at least 6 weeks old and a weight greater than 24 g, were subjected to an IV injection through the tail vein of cDNA (or pair of cDNA for paired studies) in an appropriate hydrodynamic transfection expression vector. After 7 days the mice were given adriamycin (doxorubicin) at 11 mg/kg IP and monitored for 49 days. Mice were monitored every day for weight and health; those mice that had lost greater than 40% of starting body weight or showing physical signs of end stage kidney failure were terminated and the data collected. At the end of the 49 day study period, all mice were terminated and kidneys and sera collected for analyses. Kidney hydroxyproline was measured using the QuickZyme assay method (QuickZyme Biosciences) and serum creatinine was measured using the three enzyme method and read out as μg/ml serum creatinine. Kidney fibrosis (hydroxyproline) in the saline plus adriamycin group, marked as “Saline” on the graphs, was considered to be a maximum disease response. This was set at 100% and used to normalize across the treatment groups. Naïve animals received no adriamycin and each screen experiment contained a “HGF” group which received adriamycin and the cDNA for HGF. HGF was used as an anti-fibrotic positive control, with a >40% reduction in hydroxyproline used for quality control.
Effect of IL-28 and IL-29 in Mouse Unilateral Ureteral Obstruction-Induced Kidney Fibrosis Model
DNA Construction for Hydrodynamic Transfection of Mouse IL28B and Human IL29
A DNA construct for hydrodynamic transfection was created by insertion of the DNA sequence (NM_172140.1) encoding the human IL29 precursor protein (NP_742152.1) into the pLive vector (Mir5420, Mirus Bio LLC, 545 Science Dr., Madison, WI 53711 USA) using NheI-XhoI restriction digestion followed by DNA ligation of homologous ends. A gene (mRNA Ref Seq NM_177396) construct encoding mouse IFNL3 (NP_796370) was cloned into pLive (Mirus) using a BamHI-XhoI restriction digestion strategy of both vector and insert followed by ligation. Gene insertions into pLive were verified by DNA sequencing. The gene was preceded by a CCACC sequence directly up steam of the ATG encoding the start methionine of the signal sequence. This vector harbours a kanamycin resistance gene and a pUC origin that allow for DNA amplification in Escherichia coli. Furthermore, this vector harbours mouse minimal albumin promoter to transcribe the human IFNL-1 gene flanked by introns on either side. [The pLive vector allows for long term protein expression in the liver].
The DNA construct for hydrodynamic transfection was isolated from Escherichia coli culture harbouring this construct using a plasmid isolation kit that limits the amount of endotoxin in the solution (HiSpeed Plasmid Giga EF, Qiagen). Plasmid DNA was Sanger sequenced to verify the insert.
Kanefuji et al. and Liu et al. describe typical vectors for hydrodynamic transfection.
Expression of Proteins by Hydrodynamic Transfection (HDT)
Mice were hydrodynamically transfected with 50 μg of either the pLIVE-human IL-29, pLIVE-mouse IL-28B or pLIVE SEAP Vector (Mirus Bio, Cat #MIR 5320) in TransIT-EE vehicle. The solution containing the vector was injected rapidly (less than 2.5 seconds) intravenously in a volume of 2 ml. General anaesthesia with Isoflurane was induced prior to hydrodynamic injection and maintained for at least 1 minute post injection.
Quantitation of Mouse IL-28B and Human IL-29 from Mouse Sera
Mouse IL-28B was quantitated using a meso scale discovery (MSD) quantitation assay using commercial antibody pairs (RnD systems, cat #DY1598B) and mouse sera was diluted 1 in 100 to place on the linear standard curve. Human IL-29 was quantitated using the MSD U-plex human IL-29 antibody set (cat #B21WD) and mouse sera was diluted 1 in 100 to place in the linear range.
Unilateral Ureteral Obstruction (UUO) Induced Kidney Fibrosis Model of Chronic Kidney Disease
Male C57BL/6 mice (Charles River) weighing at least 19 g were subjected to a Unilateral Ureteral Obstruction (UUO) induced kidney fibrosis model of chronic kidney disease.
Surgery was performed using Isoflurane induced general anaesthesia and under aseptic conditions as appropriate for surgical techniques. Mice underwent laparotomy followed by tying off the left ureter using 3.0 Mersilk ligature. The muscle wall was sealed using 5-0 Vicryl in a continuous pattern, with the skin being closed by subcuticular stitching using 5-0 Vicryl. Appropriate analgesia was administered pre-surgery and post-surgery. Blood samples were taken at appropriate times as detailed in the results. Mice were restrained using a recognised mouse restrainer and 30 μl of blood was taken from the tail vein via the tail prick method, using a pipette to accurately measure the blood sample. This sample was placed in a 0.2 ml PCR tube (Thermo) and spun down (20,000 g, 5 minutes) to take off as much serum as possible, which was placed in a fresh PCR tube and stored at −80° C.
On Day 19 or 21 mice were anaesthetised using Isoflurane, blood removed by cardiac puncture into serum tubes (Sarstedt) and the mice killed by cervical dislocation. The left kidney was removed and two quarters snap frozen in liquid nitrogen and stored at −80° C. The remaining half was placed into 10% neutral buffered formalin, dehydrated in a tissue processor and paraffin embedded for histological analysis. Paraffin blocks were sectioned at 4 μm on a microtome and mounted on glass slides. Slides were then de-waxed and hydrated, stained with picro-sirius red (PSR) before being washed with acidified water. Slides were then dehydrated, cleared and coverslipped. Slides were left to dry and then scanned using a Hamamatsu slide scanner.
Whole slide images were imported into Definiens Developer XD 64 and analysed using the bright-field module. Briefly, cortex areas were manually annotated to remove the medulla from analysis. Marker threshold levels (e.g. PSR stain) were determined for the individual markers on a training data set of no fewer than four images. The analysis algorithm was then run on a dedicated server to determine marker areas above threshold intensity for each individual marker and the area of each marker above the threshold intensity was determined as a proportion of the area of the cortex.
Histological assessment of the level of fibrosis was performed by blinded researchers with expertise in the field of renal fibrosis using a combination of Pico Sirius red, Masson's Trichrome and Haematoxylin and Eosin stained sections as preferred. A 10 point scale was used where 10 was very severe fibrosis equivalent to end stage renal failure and 1 normal. Parameters assessed in the score were tubular basement membrane expansion, epithelial cell flattening, brush border integrity, tubular atrophy and collapse, tubular lumen size, interstitial cell infiltrate, glomerular tuft structure, mesangial expansion, glomerular infiltrate, glomerular urinary space, glomerular basement membrane and mesangial matrix expansion.
The Definiens image analyses and the histological assessment of images also applies to the Adriamycin-treated kidney sections.
Human In Vitro Extracellular Matrix Fibrosis Assay Measurements
a) Cell Culture
All extracellular matrix (ECM) accumulation assays were performed in 384-well black clear-bottomed plates (Greiner cat #781090), where the media, cell number/well, incubation time and stimulus varied with the primary cell type used.
Human Hepatic Stellate Cells (HHSteC) (ScienCell, Cat.number sc-5300) (at passage 4) and Human Intrahepatic Biliary Epithelial Cells (ScienCell, Catalogue Number sc-5100) (at passage 3) co-culture were seeded at 1000 cells/well (1:1 ratio) in 25 uL of Epithelial Cell Medium-phenol red free (EpiCM-prf) with supplements (ScienCell, Catalogue Number sc-sc-4101-prf). Human stellate cells (ScienCell) in mono-culture were seeded at 1000 cells/well in stellate cell media (ScienCell) and human stellate cells and hepatocytes (ScienCell) co-culture at 1000 cells/well (1:1 ratio) in the mixed culture media (ScienCell).
Human small intestine fibroblasts (ScienCell) were seeded at 2000 cells/well in renal epithelial cell basal media +0.5% FCS and supplements (ATCC).
Human dermal skin fibroblasts were grown in fibroblast growth media and human keratinocytes were grown in keratinocyte media and seeded at 1500 cells/well. The fibroblast-keratinocyte co-culture (9:1 ratio) was grown in a 1:1 mixture of media and seeded at 1500 cells/well.
Human renal proximal tubular epithelial cells (RPTEC, Innoprot) and human renal fibroblasts (HRF, InnoProt) were seeded at 2000 cells per well (ratio 1:1) in co-culture using renal epithelial cell basal medium (ATCC)+0.5% FCS and supplements (ATCC).
Human small airway epithelial cells (ATCC) were grown in basal growth media plus bronchial epithelial growth kit supplements (ATCC) and IPF134 lung fibroblasts (ATCC) were grown in fibroblast growth media (Lonza). The co-culture was seeded at 2000 cells/well (1:1 ratio).
b) Measurement of Cell Growth
After 7 days incubation at 37° C., 5% CO2, cell growth was assessed using PrestoBlue® Cell Viability reagent (Thermo Fischer Scientific) following the instructions of the manufacturer.
c) Measurement of ECM Accumulation
Measurement of Individual ECM Components: Immunofluorescence
After 7 days incubation at 37° C. in 5% CO2, cells were washed in PBS and lysed with 20 μl/well of 0.25 M NH4OH/25 mM Tris (Sigma-Aldrich) for 15 min at 37° C. Matrix was then washed 3 times in PBS, fixed in 40 μl 100% methanol for 30 min at −20° C. and washed 3 times in PBS before being stained using anti-Fibronectin (eBiosciences), anti-Collagen I (Millipore), anti-Collagen III (Millipore), anti-Collagen IV (eBiosciences) and anti-Collagen V (Abcam) antibodies. Plates were scanned on the Arrayscan HC reader (Cellomics) using a 3-channel protocol under the “Cellomics CellHealth” profiling bioapplication and a 10× objective (new X1 camera) with 2×2 binning (1.104×1.104 pixels/field).
Measurement of Total ECM Components: Flamingo™ Staining
Immunofluorescence plates were washed 3 times in Flowfusor water before being stained using Flamingo™ fluorescent gel stain reagent (BioRad) following the instructions of the manufacturer. Plates were scanned on the Arrayscan HC reader (Cellomics) using a 2-channel protocol under the “Cellomics CellHealth” profiling bioapplication and a 1 Ox objective (new X1 camera) with 2×2 binning (1.104×1.104 pixels/field).
Method for Generation of 3D Human Glomerular Spheroid
Spheroid Generation and Treatment
Nano-shuttle-PL (Cat #657841) was added to T75 flasks of stably transfected GFP-tagged actin, conditionally immortalised SV40 infected podocytes and human glomerular endothelial cells (HRGECs) provided by the University of Bristol, grown in RPMI 1640 (10% FCS, 5 ML L-glutamine and 1XITS, Gibco) and EBM-2 media, with supplement kit from Lonza (Cat #CC4147), respectively. GFP transfected podocytes were confirmed by FACS. All cells were harvested using TrypLE Express (Gibco) and magnetised HRGECs were seeded at 5000 cells/well in 96-well low adhesion Greiner plates (Cat #655976). A Greiner Spheroid Drive (Cat #655830) was placed beneath the 96-well plate for 6h and spheroids were grown in EBM-2 media at 37° C./5% CO2/100% humidity. The Drive was removed, and magnetised GFP-podocytes were seeded at 5000 cells/well. The Drive was returned beneath overnight and spheroids were differentiated for 10 days feeding every 3 days using the Holding Drive. Spheroids were then treated with human FSGS plasma at 15% final concentration, plus or minus IL-28A (10,000 ng/mL), or media only for 7 days.
Fixation and Staining
Spheroids were fixed and permeabilised for 3 hours at 4° C. in 10% formalin/1% Triton X-100/PBS. After 3×10 min PBS washes spheroids were dehydrated in ascending series of methanol at 4° C. in PBS: 25%, 50%, 75%, 95% for 30 mins each and left in 100% methanol overnight. Spheroids were rehydrated in the same descending series and washed as before in PBS before blocking in PBST (0.1% Triton X-100 in PBS) containing 3% BSA overnight at 4° C. After 4×30 min PBST washes, secondary antibodies (1:500/PBST: Alexa GAR-AF647 and GAM-AF555) were added. To enhance the GFP signal, anti-GFP-AF488 was added at 1:200 for 24h. Spheroids were washed (4×30 mins/PBST) before image capture using the YokoGawa confocal quantification 1 (CQ1) at ×20 magnification.
Podocyte Cell-Marker Area Quantification
Maximum intensity projections were imported into Definiens Developer XD 64 and analysed using the immunofluorescence module. Spheroid areas were manually annotated and marker threshold levels were determined for the individual markers and an analysis algorithm was run to determine marker areas above threshold intensity for each marker. The area of each marker above the threshold intensity was determined as a proportion of the area of the spheroid.
Results
(1) Mouse Adriamycin-Induced Model of Chronic Kidney Disease (CKD)
Mouse IL-28A (IFNL2) was identified in a screen for fibrosis modifying proteins in the mouse Adriamycin CKD model initially as one member of a hydrodynamically transfected cDNA pair (n=15 mice). The transfected pair reduced kidney fibrosis as measured by total renal hydroxyproline by 51% relative to a saline transfection control group (p=0.02)(
Application of IL-28A has almost completely prevented signs of glomerulosclerosis with little evidence of glomerular basement membrane thickening. Only 2 of the 22 animals showed notable signs of epithelial flattening and the lumen expansion characteristic of untreated animals. The remainder of the animals had a tubulointerstitium that appeared near normal in greater than 80% of the cortex with little effect of the tubular basement membrane.
The 47 mouse kidney samples that were stained with PSR were subjected to high content image analysis using Definiens software and the area of PSR staining measured in the three groups was plotted as percentage PSR stained area (
Mouse IL-28B (IFNL3) was also identified in a screen for fibrosis modifying proteins in the mouse Adriamycin CKD model initially as one member of a hydrodynamically transfected cDNA pair (n=15 mice per group). The transfected pair reduced kidney fibrosis as measured by total renal hydroxyproline by 59% relative to the saline transfection control group (p=0.00086) (
(2) Primary Human Cell In Vitro Models of Organ Fibrosis
(2.1) Liver Fibrosis
Human primary stellate cells grown on plastic are self-activating and accumulate a substantial basal level of extracellular matrix as measured by the levels of the ECM proteins fibronectin, collagen IV and collagen I&III. This ECM accumulation can be inhibited by treatment of the culture with recombinant IL-28A in a dose dependent manner (
TGFβ1 stimulation of both the stellate cell monoculture and the stellate-hepatocyte co-culture induced a further increase in the ECM proteins, fibronectin, collagen IV and collagen I&III. Treatment of the TGFβ1-stimulated stellate cell mono-culture, or the stellate-hepatocyte co-culture with either IL-28A or IL-28B demonstrated a near complete inhibition of any increase in any ECM protein at the highest concentration of 10 μg/ml of IL-28A or B, with both inhibiting in a dose dependent manner. IL-28A was more potent than IL-28B across all the ECM proteins in TGFβ1-stimulated liver cells (
(2.2) Small intestine Fibrosis
Human small intestine fibroblasts stimulated with TGFβ1 (10 ng/ml) induce the extracellular matrix proteins, fibronectin and collagen I&III. The fibronectin level was significantly inhibited by IL-28A, IL-28B and IL-29 (10 ng/ml), with inhibition of 76%, 73%, and 83%, respectively relative to the control TGFβ1 stimulation. Collagen I&III was inhibited by IL-28A, IL-28B and IL-29 (10 ng/ml), by 48%, 33% and 35%, respectively. Collagen IV was only significantly inhibited by IL-28A (37% inhibition at 10 μg/ml) relative to control TGFβ1 stimulation (
(2.3) Skin Fibrosis
Human primary dermal fibroblasts in co-culture with primary keratinocytes (9:1 ratio) accumulate a substantial amount of fibronectin, collagen I&III and collagen IV just by being in co culture. Using 3 separate batches of fibroblasts in combination with a single donor's keratinocytes, cultures were treated with either IL-28A or IL-28B. The percentage inhibition in ECM proteins relative to the untreated cultures was measured with all 3 fibroblast batches and the mean inhibition plotted (
The dermal fibroblast-keratinocyte co-culture was treated with IL-29, IL-28A and IL-28B over a narrower concentration range of 1250-10000 ng/ml. This showed dose dependent inhibition of fibronectin, collagen I&III and collagen IV relative to the control co-culture (
Images of untreated dermal co-cultures compared to the IL-29, IL-28A and IL-28B treated co-cultures at both 1250 ng/ml and 10000 ng/ml, confirmed the total prevention of ECM accumulation determined by image analysis in the presence of the 10 μg/ml IFNL (
Finally human dermal fibroblasts were stimulated in mono-culture with IL-1α (10 ng/ml) to induce the ECM proteins fibronectin, collagen I&III and collagen IV. Treatment of the IL-1α-stimulated fibroblast mono-cultures with IL-28A or IL-28B demonstrated a potent inhibition of the pro matrix response for all ECM proteins with complete inhibition of matrix at the top concentration of 10 μg/ml. IL-28A was more potent than IL-28B in all batches of fibroblasts, with one representative example depicted (
(2.4) Kidney Fibrosis
Human primary renal proximal tubular epithelial cells (RPTEC) placed in co-culture with human primary renal fibroblasts (seeded with 2000 cells/well at a 1:1 ratio) generate a robust ECM, high in fibronectin and collagen I&III. This co-culture was treated with IL-29, IL-28A or IL-28B using 3 different protocols for addition of cytokines.
Firstly IL-29, IL-28A and IL-28B were added to the renal co-culture at day 0 and the culture incubated for 7 days. All 3 cytokines potently inhibited in a dose-dependent manner the accumulation of fibronectin and collagen I&III (
Secondly IL-29, IL-28A and IL-28B were added to the RPTEC cells at day 0, the fibroblasts were added 24 hours later and then the culture was allowed to continue for a total of 7 days after which the ECM was measured. Here, IL-29, IL-28A and IL-28B all again inhibited in a dose dependent manner the accumulation of fibronectin and collagen I&III (
Finally, IL-29, IL-28 and IL-28B were added to the fibroblasts on day 0. Twenty four hours later the RPTECs were added and the co-culture was continued for a total of 7 days when fibronectin and collagen I&III were measured. Adding the IFNLs to the fibroblasts first also showed significant inhibition of the ECM proteins (
Cell viability was not compromised by addition of the cytokines first to the RPTECs or fibroblasts before the alternate cell of the co-culture as read-out by presto blue (
Representative images (one out of a total of 16 fields per treatment) of the untreated co-culture compared to the IL-28A, IL-28B and IL-29 treated co-cultures show both the dose-dependent effects of IL-29, IL-28A and IL-28B and the near total inhibition observed in the presence of 1000 ng/ml IL-29 (
Renal proximal tubular tubular epithelial cells (RPTECs) in mono-culture lay down basal ECM proteins by growing on plastic, albeit at low levels. The low levels of ECM can still be inhibited in the presence of very low concentrations of IL-29 as shown for fibronectin, collagen IV and Collagen I&III in
(2.5) Lung Fibrosis
Human primary small airway lung epithelial cells in co-culture with lung fibroblasts isolated from an idiopathic pulmonary fibrosis (IPF) patient (2000 cells/well at a 1:1 ratio) generated a robust total ECM (measured by flamingo staining) after 7 days in culture and was positive for the ECM markers, fibronectin, collagen I&III and collagen IV. IL-28A or IL-28B had no significant effect on the level of fibronectin. However, IL-28B demonstrated a significant inhibition of the collagen I&III, collagen IV and flamingo stains at the top concentration of 10 μg/ml (
(3) Primary Human Cell Models of Organ Fibrosis
(3.1) Liver Fibrosis
The co-culture of human primary stellate cells with primary human intrahepatic biliary epithelial cells (1:1 ratio) leads to a basal extracellular matrix that can also be inhibited by treatment with recombinant human IL-28A and IL-29 in a dose dependent manner with fibronectin, collagen IV and collagen I&III reduced (
(3.2) 3D Glomerular Spheroid Model
Glomerular spheroids were constructed from an inner core of magnetised human primary glomerular endothelial cells with an outer core of temperature conditional human podocytes that can be maintained in stable culture for up to 3 weeks. Treatment of the glomerular spheroids with 15% relapse plasma from patients with focal segmental glomerulosclerosis (FSGS) induced a loss of GFP-labelled podocytes from the outer core, which could be protected in the presence of IL-28A. Plasma from healthy volunteers had no effect on podocytes. Quantitation of the green fluorescent podocyte cell marker area demonstrated a significant reduction in the presence of FSGS relapse plasma that was significantly increased on IL-28A treatment (
(4) Mouse Unilateral Ureteral Obstruction Model (UUO) of Kidney Fibrosis
(4.1) Mouse IL28B Delivered by Hydrodynamic Transfection (HDT) UUO kidneys in animals receiving the control SEAP vector by HDT demonstrated tubulointerstitial fibrosis in the cortex typical of this model at 21 days post surgery with widespread expansion of the tubular basement membrane, strong collagen staining with the basement membrane particularly strongly stained within the medullary ray, widespread loss of epithelial cell volume, tubular atrophy, loss of proximal tubular brush border and extensive tubulointerstitial infiltrate. Less than 15% of tubules had near normal architecture. The medulla was effectively destroyed and missing while the changes to the glomeruli were minimal (
UUO kidneys in animals undergoing HDT with mouse IL-28B 1 day before UUO and those 7 days post UUO both showed protection from cortical tubulointerstitial fibrosis at 21 days post surgery (
The picro-sirius red (PSR) stained collagen sections were quantitated using definiens image analyses algorithms, showing the statistically significant protective effect of mouse IL-28B delivered either at day-1 or at day 7 when plotted as either total collagen area (
The serum levels of mouse IL28B protein achieved at day 21 post HDT in the mouse UUO model were measured by ELISA and were between 43 and 485 ng/ml across all the groups. No mouse IL28B protein could be measured in the control SEAP HDT mice so the levels were below 4 ng/ml which was the lower limit of quantitation (LLQ) of the assay used.
(4.2) Human IL-29 Delivered by Hydrodynamic Transfection (HDT)
Human IL-29 delivered by HDT shows significant protein expression in all mice at both day 0 (24 hours post-transfection) and at day 19 when the model was terminated compared to the SEAP control plasmid treated group (
The untreated 19 day UUO shows typical histological changes for this model. There is extensive destruction and loss of the renal medulla in all of the kidneys. The cortex displays a widespread expansion of the tubular basement membrane with significant interstitial collagen accumulation as shown by the increased picro-sirius red (PSR) staining. There is also a notable infiltration of cells into the interstitium throughout the whole cortex. The tubular epithelium structure is significantly damaged. The majority of epithelial cells have lost volume and generally appear flattened including a small number of tubules with a dilated lumen. Notably, greater than 80% of tubules have undergone extensive atrophy and in many cases collapsed completely. The lumen of the tubules are difficult to distinguish in most cases due to loss of patency, with epithelial cells clearly dislodged from the nephron tubular basement membrane and residing in what would have been the lumen.
Day 19 UUO kidneys treated from the time of UUO with IL-29 have a dramatically different appearance to those receiving just SEAP vector with the structure clearly preserved. The medulla is damaged, but remains present and has a more recognisable structure than the untreated UUO in 75% of animals. In the cortex, the primary observation at lower levels of magnification is a large number of tubules that have a dilated tubular lumen compared to the SEAP UUO. However, where this would be typically associated with very flattened epithelial cells and expansion of the tubular basement membrane in an untreated UUO, with IL-29 treatment the epithelial cells have retained volume, there is minimum expansion of the tubular basement membrane and the architecture of the tubules is significant preserved with little to no evidence of atrophy and tubular collapse. There is clearly lower levels of interstitial collagen and reduced expansion of the tubular basement membrane.
Overall the application of IL-29 has reduced tubulointerstitial fibrosis caused by UUO significantly, with less interstitial collagen and a dramatic preservation of tubular architecture as shown by representative images in
All the picro-sirius red (PSR) stained collagen sections were quantitated using definiens image analyses algorithms, showing the statistically significant protective effect of human IL-29 delivered at day-1 when plotted as total stained collagen area relative to the control SEAP plasmid treated mice (
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16469236 | Jun 2019 | US |
| Child | 18343944 | US |
