Medical Use of Interferon-Lambda for the Treatment of Fibrosis

Abstract

The present invention relates to methods for treating fibrosis. The present invention discloses new studies demonstrating that interferon-lambda has direct-acting antifibrotic effects both in vitro and in vivo and can be used to provide effective new therapies for the treatment of multiple types of fibrosis.

Description

[Technical field]

[0001] The present invention relates to a method for treating fibrosis. The present invention discloses novel studies demonstrating that interferon-lambda has direct acting antifibrotic effects both in vitro and in vivo and can be used to provide effective new therapeutic approaches for the treatment of multiple types of fibrosis. [Background technology]

[0002] Fibrotic diseases are diseases characterized by an abnormal 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 (e.g., collagen and fibronectin) can result in tissue stiffening and scarring that causes pathological remodeling of the organ, ultimately leading to organ failure.

[0003] Wounds in solid organs generally begin with endothelial injury, platelet aggregation and activation that trigger an inflammatory response with infiltration of neutrophils, macrophages, eosinophils and lymphocytes into the wound site. Infiltrating inflammatory cells and affected epithelial cells secrete various growth factors and cytokines that serve to further amplify this inflammatory response. Molecules such as TGF-β, PDGF and IL-13 activate macrophages, leading to the recruitment, proliferation and activation of fibroblasts at the wound site. Activated fibroblasts or myofibroblasts are characterized by the expression of α-smooth muscle actin and secrete collagen and other ECM components to stabilize the cellular substratum. This allows the proliferation and migration of epithelial and endothelial cells on this temporary matrix, regenerating the damaged tissue. Once complete, the inflammatory process is stopped and fibroblasts undergo apoptosis leading to the resolution of the wound response.

[0004] In fibrotic remodeling, persistent tissue injury or damage or dysregulation of repair pathways leads to an inappropriate wound response, with excessive deposition and cross-linking of collagen and ECM, resulting in excessive accumulation of ECM beyond normal requirements, which is associated with persistent myofibroblast activation, damage to epithelial cells, and loss of normal tissue architecture.

[0005] Fibrotic diseases can affect any organ or tissue (e.g., kidney, lung, intestine, skin, or liver). The cause of fibrotic diseases may depend on the organ or tissue involved and remains unknown for some diseases, such as idiopathic pulmonary fibrosis. Other types of interstitial lung disease have recognized causes, such as exposure to environmental allergens causing hypersensitivity pneumonitis. Liver fibrosis, and ultimately cirrhosis, results from chronic liver damage sustained through exposure to various factors, such as environmental and dietary factors or infectious agents. Persistent excessive alcohol intake or a high fat / carbohydrate diet can also cause cirrhosis of the liver. Similarly, diabetes, hypertension, exposure to toxic substances, and various types of autoimmune diseases can damage the kidneys, causing fibrotic remodeling and loss of function. Many types of inflammatory bowel disease (e.g., Crohn's disease or celiac disease) can cause fibrotic remodeling leading to stenosis and / or malabsorption.

[0006] Treatment of progressive fibrosis is primarily by treating the underlying disease, such as controlling blood pressure, improving glucose management in diabetes, or removing harmful allergens or environmental factors. However, in many patients, once fibrotic remodeling is established, the disease becomes self-perpetuating, and simple treatment of the underlying disease cannot stop the process. Some fibrotic diseases can be treated with anti-inflammatory and immunosuppressive drugs, but these are only effective in a subset of patients and slow rather than stop the disease.

[0007] To date, there are only two effective therapies for fibrotic remodeling, both of which are approved for use only in idiopathic pulmonary fibrosis (IPF). Pirfenidone is a small molecule drug approved for use in the treatment of IPF in Japan in 2008 and in Europe in 2011 that may act through multiple mechanisms of action that are not fully understood but may act in part through downregulation of TGF-β. Nintedanib is a triangiokinase inhibitor that inhibits tyrosine kinase activity at VEGF, FGF and PDGF receptors. Both compounds delay the onset of IPF, but may only extend lifespan by up to 2 years based on current studies. Furthermore, both are associated with significant side effects, resulting in over 30% of patients being unable to tolerate their use over the long term. To date, there is no standard treatment approved for fibrotic indications. Summary of the Invention

[0008] Thus, there is currently an unmet medical need for improved treatment of fibrotic diseases in all organs.It is therefore an object of the present invention to provide a novel method for the treatment of fibrosis.

[0009] The discovery and initial description of the interferon-lambda (IFN-lambda) family in early 2003 opened an exciting new chapter in the field of IFN research. There are four distinct but highly related proteins, designated IFN-lambda1, IFN-lambda2, IFN-lambda3, and IFN-lambda4. These proteins are also known as interleukin-29 (IL-29), IL-28A, IL-28B, and IFNL4, respectively. Collectively, these four cytokines constitute the type III subset of IFNs. They differ 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 distinct from the receptors used by type I or type II IFNs. Although type I IFNs (INF-a / b) and type III IFNs (IFN-1) signal through separate receptor complexes, they activate the same intracellular signaling pathways and many of the same biological activities, such as antiviral activity, in a variety of target cells. Consistent with these antiviral activities, expression of the IFN-λ gene and its corresponding protein can be induced by infection with many types of viruses. Thus, the expression and major biological activity of type III IFN (IFN-λ) are very similar to type I IFN. However, unlike the IFN-α receptor, which is widely expressed on most cell types such as leukocytes, the IFN-λ receptor is primarily restricted to cells of epithelial origin. The potential clinical importance of IFN-λ as a novel antiviral therapeutic agent is already evident. 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.

[0010] Here, we disclose novel studies that demonstrate for the first time that cytokines of the interferon-lambda family have direct acting antifibrotic effects both in vitro and in vivo, and that these proteins can be used to design effective new treatment methods for multiple types of fibrosis. In particular embodiments, for example, the following items are provided: (Item 1) Interferon-lambda for use in the treatment of fibrosis. (Item 2) 2. The interferon-lambda according to item 1, wherein the interferon-lambda is IFN-lambda 1, IFN-lambda 2, IFN-lambda 3 or IFN-lambda 4 or a functional mimetic thereof. (Item 3) 3. The interferon-lambda according to item 2, wherein the functional mimetic is a fragment, variant or derivative of IFN-λ1, IFN-λ2, IFN-λ3 or IFN-λ4. (Item 4) 4. The interferon-lambda of item 3, wherein the functional mimetic is pegylated. (Item 5) The interferon-lambda according to item 4, wherein the functional mimetic is peg-interferon lambda-1a. (Item 6) The interferon-lambda according to item 2, wherein the functional mimetic is an antibody. (Item 7) The interferon-lambda of item 2, wherein the functional mimetic is a small molecule chemical. (Item 8) 8. Interferon-lambda according to any one of items 1 to 7, wherein said interferon-lambda or functional mimetic activates the interferon-lambda receptor. (Item 9) 9. The interferon-lambda of any one of items 1 to 8, wherein the fibrosis is renal fibrosis. (Item 10) 9. The interferon-lambda of any one of items 1 to 8, wherein the fibrosis is pulmonary fibrosis. (Item 11) 9. The interferon-lambda of any one of items 1 to 8, wherein the fibrosis is small intestinal fibrosis. (Item 12) 9. The interferon-lambda of any one of items 1 to 8, wherein the fibrosis is dermal fibrosis. (Item 13) 9. The interferon-lambda of any one of items 1 to 8, wherein the fibrosis is liver fibrosis. (Item 14) Use of interferon-lambda for the preparation of a medicament for the treatment of fibrosis. (Item 15) 15. Use of interferon-lambda according to item 14, wherein the interferon-lambda is IFN-lambda1, IFN-lambda2, IFN-lambda3 or IFN-lambda4 or a functional mimetic thereof. (Item 16) 16. Use of interferon-lambda according to item 15, wherein the functional mimetic is a fragment, variant or derivative of IFN-λ1, IFN-λ2, IFN-λ3 or IFN-λ4. (Item 17) 17. The use of interferon-lambda according to item 16, wherein the functional mimetic is pegylated. (Item 18) 18. Use of interferon-lambda according to item 17, wherein the functional mimetic is peg-interferon lambda-1a. (Item 19) 16. The use of interferon-lambda according to item 15, wherein the functional mimetic is an antibody. (Item 20) 16. The use of interferon-lambda according to item 15, wherein the functional mimetic is a small molecule chemical. (Item 21) 21. Use of interferon-lambda according to any one of items 14 to 20, wherein said interferon-lambda or functional mimetic activates the interferon-lambda receptor. (Item 22) 22. Use of interferon-lambda according to any one of items 14 to 21, wherein the fibrosis is renal fibrosis. (Item 23) 22. Use of interferon-lambda according to any one of items 14 to 21, wherein the fibrosis is pulmonary fibrosis. (Item 24) 22. Use of interferon-lambda according to any one of items 14 to 21, wherein the fibrosis is small intestinal fibrosis. (Item 25) 22. Use of interferon-lambda according to any one of items 14 to 21, wherein the fibrosis is dermal fibrosis. (Item 26) 22. The use of interferon-lambda according to any one of items 14 to 21, wherein the fibrosis is liver fibrosis. (Item 27) 20. A method of treating fibrosis comprising administering a therapeutically effective amount of interferon-lambda. (Item 28) 28. The method of claim 27, wherein the interferon-lambda is IFN-λ1, IFN-λ2, IFN-λ3 or IFN-λ4 or a functional mimetic thereof. (Item 29) 29. The method of claim 28, wherein the functional mimetic is a fragment, variant or derivative of IFN-λ1, IFN-λ2, IFN-λ3 or IFN-λ4. (Item 30) 30. The method of claim 29, wherein the functional mimetic is pegylated. (Item 31) 31. The method of claim 30, wherein the functional mimetic is pegylated interferon lambda-1a. (Item 32) 29. The method of claim 28, wherein the functional mimetic is an antibody. (Item 33) 29. The method of claim 28, wherein the functional mimetic is a small molecule chemical. (Item 34) 34. The method according to any one of items 27 to 33, wherein the interferon-lambda or functional mimetic activates an interferon-lambda receptor. (Item 35) 35. The method of any one of items 27 to 34, wherein the fibrosis is renal fibrosis. (Item 36) 35. The method of any one of items 27 to 34, wherein the fibrosis is pulmonary fibrosis. (Item 37) 35. The method according to any one of items 27 to 34, wherein the fibrosis is small intestinal fibrosis. (Item 38) 35. The method according to any one of items 27 to 34, wherein the fibrosis is dermal fibrosis. (Item 39) 35. The method of any one of items 27 to 34, wherein the fibrosis is liver fibrosis. (Item 40) 40. The method according to any one of items 27 to 39, wherein the interferon-lambda or functional mimetic is administered in combination with another therapeutically active compound. (Item 41) 41. The method of item 40, wherein the other therapeutically active compound is another antifibrotic therapeutic agent. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 shows the identification of IL-28A as an antifibrotic hit in the adriamycin model of kidney disease. In the screening condition, mouse IL-28A (ILFNL2) was introduced into mice by hydrodynamic transfection as a pair of cDNAs (i.e., IL-28A cDNA and another undisclosed cDNA) (n=15 group size for all treatment groups) prior to administration of adriamycin by IV injection to induce chronic kidney disease. Control mice were transfected with saline only prior to adriamycin injection. Mice were monitored for up to 49 days and kidney hydroxyproline levels (Figure 1(a) and Figure 1(b)) and serum creatinine levels (Figure 1(c)) were measured at the end (no conditional survival data for primary screen). Figures for hydroxyproline (μg per mg kidney) and serum creatine (μg / mL) are box and whisker plots. The central horizontal line indicates the median of the data and the boxes extend from the 25th to the 75th percentile. Whiskers cover the typical range of the data.Normalized hydroxyproline (kidney collagen) shown as mean + SEM error bars, ****p<0.0001, ***p<0.001, *p<0.05. [Diagram 2]Figure 2: Confirmation of IL-28A as an antifibrotic hit in the Adriamycin model of kidney disease. Mouse IL-28A (IFNL2) was introduced into mice by hydrodynamic transfection as a single cDNA (n=22 group size for all treatment groups) prior to administration of Adriamycin by IV injection to induce chronic kidney disease. Control mice were transfected with saline only prior to Adriamycin injection. Mice were followed for 49 days. At termination, kidney hydroxyproline levels (Figure 2(a) and Figure 2(b)) and serum creatine levels (Figure 2(c)) were measured and conditional survival data were recorded over the entire study period (Figure 2(d)). Figures for hydroxyproline (μg per mg kidney) and serum creatine (μg / mL) are box and whisker plots. The central horizontal line indicates the median of the data and the box extends from the 25th to the 75th percentile. The "whiskers" cover the typical range of the data. Normalized hydroxyproline (kidney collagen) is shown as mean and +SEM error bars, ****p<0.0001, ***p<0.001, *p<0.05. [Diagram 3]Figure 3: Identification of IL-28B as an anti-fibrotic hit in the adriamycin model of kidney disease. In the primary screen, murine IL-28B (ILFNL3) was introduced into groups of mice by hydrodynamic transfection as a cDNA pair (n=15 group size for all treatment groups) prior to adriamycin and the mice were monitored for up to 49 days, measuring kidney hydroxyproline (Figure 3(a) and Figure 3(b)) and serum creatinine (Figure 3(c)) levels at the end (no conditional survival data for this primary screen). Control mice were transfected with saline only prior to adriamycin injection. Figures for hydroxyproline (μg per mg kidney) and serum creatine (μg / mL) are box and whisker plots. The central horizontal line indicates the median of the data and the boxes extend from the 25th to the 75th percentile. The "whiskers" cover the typical range of the data. Normalized hydroxyproline (kidney collagen) is shown as mean and +SEM error bars, ****p<0.0001, ***p<0.001, *p<0.05. [Figure 4]Figure 4: Confirmation of IL-28B as an anti-fibrotic hit in the adriamycin model of kidney disease. Mouse IL-28B (IFNL3) was introduced into groups of mice by hydrodynamic transfection as a single clone (n=22 group size in each treatment group) prior to adriamycin and the mice were followed up for up to 49 days. Control mice were transfected with saline alone prior to adriamycin injection. At termination, kidney hydroxyproline levels (Figure 4(a) and Figure 4(b)) and serum creatine levels (Figure 4(c)) were measured and conditional survival data were recorded over the duration of the study (Figure 4(d)). Figures for hydroxyproline (μg per mg kidney) and serum creatine (μg / mL) are box and whisker plots. The central horizontal line indicates the median of the data and the boxes extend from the 25th to the 75th percentile. The "whiskers" cover the typical range of the data. Normalized hydroxyproline (kidney collagen) is shown as mean and +SEM error bars, ****p<0.0001, ***p<0.001, *p<0.05. [Diagram 5]Figure 5. Histological confirmation of IL-28B and IL-28A as antifibrotic in the adriamycin model of renal disease. At termination, kidneys from the studies described in Figures 2 and 4 were fixed in NBF, paraffin embedded, sectioned, and stained with either hematoxylin and eosin (H&E), picrosirius red (PSR), or Massons trichrome (MT). Slides were then imaged on a Hamamatsu slide scanner. Representative images of PSR-stained kidney sections are shown for IL-28B in Figure 5(a) for one untreated mouse, three saline adriamycin-treated mice, and three adriamycin-treated mice that also received IL-28B, and for IL-28A in Figure 5(f) for one untreated mouse, three saline adriamycin-treated mice, and three adriamycin-treated mice that also received IL-28A. All final mouse kidney slices from both IL-28B (10 untreated, 18 saline, and 22 IL-28B-treated) and IL-28A (8 untreated, 20 saline, and 19 IL-28A-treated) groups were imaged and the total area covered by PSR staining was estimated using the Definiens image analysis platform and is shown in Figure 5(b) and Figure 5(g), respectively. In addition, all slides were manually scored for fibrosis by separate experts in the field of fibrosis (2 scientists and 1 clinical pathologist) and pathology scores were calculated for IL-28B from H&E staining (Figure 5(c)) and PSR staining (Figure 5(d) and Figure 5(e)), and for IL-28A from PSR staining (Figure (h)) and MT staining (Figure (i)). Magnification = ×100. [Figure 6a]Figure 1 shows the effect of IL-28A in liver in an in vitro ECM fibrosis model. Human primary astrocytes (seeded at 1000 cells / well) were treated with IL-28A for 5 days in culture. Cells were removed and ECM proteins, fibronectin, collagen I&III, and collagen IV were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of 4 separate wells. [Figure 6b] Figure 1 shows the effect of IL-28A in liver in an in vitro ECM fibrosis model. Human primary stellate cells in co-culture with human primary hepatocytes (1000 cells / well, seeded at 1:1 ratio) were treated with IL-28A for 5 days in culture. Cells were removed and ECM proteins, fibronectin, collagen I&III, and collagen IV were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of 4 separate wells. [Figure 7a] Figure 1 shows the effect of IL28A and IL-28B in liver in vitro ECM fibrosis model and TGFβ. Human primary astrocytes (1000 cells / well) were treated with IL-28A or IL-28B in the presence of TGFβ at 250pg / ml for 5 days in culture. Cells were removed and ECM proteins, fibronectin, collagen I&III, and collagen IV were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data shows the mean total intensity plotted as the mean ± SD of 4 separate wells. [Figure 7b]Figure 1 shows the effect of IL28A and IL-28B in liver in an in vitro ECM fibrosis model and TGFβ. Human primary stellate cells in co-culture with human primary hepatocytes (1000 cells / well, 1:1 ratio) were treated with IL-28A or IL-28B in the presence of TGFβ at 250pg / ml for 5 days in culture. Cells were removed and ECM proteins, fibronectin, collagen I&III, and collagen IV were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of 4 separate wells. [Figure 8] High content images showing the effect of IL-28A and IL-28B on stellate cell-hepatocyte co-culture ECM stimulated with TGFβ1. Human primary stellate cells in co-culture with primary hepatocytes (1:1 ratio with a final cell concentration of 1000 cells / well) stimulated with TGFβ1 (250 pg / 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 one representative field out of 16 for each condition. Magnification was performed with a ×10 objective. [Figure 9] Figure 1 shows the effect of IL-28A, IL-28B and IL-29 in a TGFβ1 stimulated small intestinal fibroblast ECM in vitro model. Human primary small intestinal fibroblasts (2000 cells / well) were treated with IL-28A, IL-28B or IL-29 (all at 10 ng / ml) in the presence of TGFβ (10 ng / ml) for 7 days in culture. Cells were removed and ECM proteins, fibronectin, collagen I&III and collagen IV were measured by immunofluorescence labelling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of 4 separate wells. [Figure 10]Figure 1 shows the % inhibition of IL-28A and IL-28B in a skin keratinocyte-fibroblast co-culture ECM in vitro model. Human primary dermal fibroblasts from three individual batches were co-cultured with primary human keratinocytes (9:1 ratio 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 and ECM proteins, fibronectin, collagen I&III, and collagen IV were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Normalized data from the three batches were calculated as % inhibition and plotted as mean ± SD. Cell viability was measured with PrestoBlue and the three fibroblast batches in co-culture with keratinocytes were normalized to the control co-culture and data were averaged to obtain fold change. [Figure 11] Figure 1. Effect of IL-28A, IL-28B and IL-29 in a skin keratinocyte-fibroblast co-culture ECM in vitro model. Human primary dermal fibroblasts in co-culture with primary human keratinocytes (9:1 ratio with a final cell concentration of 1500 cells / well) were treated with IL-29, IL-28A or IL-28B for 7 days at concentrations ranging from 1250 to 10000ng / ml of cytokines. Cells were removed and ECM proteins, fibronectin, collagen I&III, and collagen IV were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of 4 separate wells. Cell viability was measured with PrestoBlue. [Figure 12]High content images showing the effect of IL-28A, IL-28B and IL-29 in a skin keratinocyte-fibroblast co-culture ECM in vitro model. Human primary dermal fibroblasts from a representative donor in co-culture with primary keratinocytes (9:1 ratio 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. Images show a combination of signals from fibronectin, collagen I&III, and collagen IV ECM matrix proteins and show one representative field out of a total of 16 fields per condition. Magnification was performed with a ×10 objective. [Figure 13] Figure 1 shows the effect of IL-28A and IL-28B in an IL-1α stimulated dermal fibroblast ECM in vitro model. 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 over 7 days of culture. Cells were removed and ECM proteins, fibronectin, collagen I&III, and collagen IV were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of four separate wells. Cell viability was measured with PrestoBlue. Data represents one of two different fibroblast batches. [Figure 14a]Figure 1 shows the effect of IL-28A, IL-28B and IL-29 in a renal RPTEC-fibroblast co-culture ECM in vitro model. Human primary renal proximal tubular epithelial cells (RPTEC) were co-cultured with primary human renal fibroblasts (1:1 ratio with a final seeding cell concentration of 2000 cells / well) for 7 days and treated with IL-29, IL-28A or IL-28B. Cells were removed and ECM proteins, fibronectin and collagen I&III were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of four separate wells. Cell viability was measured with PrestoBlue. Either IL-29, IL-28A or IL-28B was added to RPTEC and fibroblast co-cultures on day 0. [Figure 14b] Figure 1 shows the effect of IL-28A, IL-28B and IL-29 in a renal RPTEC-fibroblast co-culture ECM in vitro model. Human primary renal proximal tubular epithelial cells (RPTEC) were co-cultured with primary human renal fibroblasts (1:1 ratio with a final seeding cell concentration of 2000 cells / well) for 7 days and treated with IL-29, IL-28A or IL-28B. Cells were removed and ECM proteins, fibronectin and collagen I&III were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of four separate wells. Cell viability was measured with PrestoBlue. Either IL-29, IL-28A or IL-28B was added to RPTECs 24 hours prior to combination with fibroblasts. [Figure 14c]Figure 1 shows the effect of IL-28A, IL-28B and IL-29 in a renal RPTEC-fibroblast co-culture ECM in vitro model. Human primary renal proximal tubular epithelial cells (RPTEC) were co-cultured with primary human renal fibroblasts (1:1 ratio with a final seeding cell concentration of 2000 cells / well) for 7 days and treated with IL-29, IL-28A or IL-28B. Cells were removed and ECM proteins, fibronectin and collagen I&III were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of four separate wells. Cell viability was measured with PrestoBlue. Either IL-29, IL-28A or IL-28B was added to the fibroblasts 24 hours prior to combination with RPTEC. [Figure 15a] Figure 15 shows a comparison of the addition format to the effect of IL-28A, IL-28B, and IL-29 in a renal RPTEC-fibroblast co-culture ECM in vitro model. The data shown in Figure 14 was replotted to show the effect of IL-28A, IL-28B, and IL-29 in a renal co-culture cell line using three different addition protocols in one graph, where IL-28A, IL-28B, or IL-29 was added to a co-culture of RPTEC and HRF together (referred to as co-culture), added to RPTEC first and HRF introduced 24 hours later (referred to as RPTEC added first), added to HRF first and RPTEC introduced 24 hours later (referred to as HRF added first), and the co-culture continued for 7 days, respectively, with the fibronectin data replotted in Figure 15(a). ECM markers were analyzed using analysis of variance accounting for differences between addition order (co-cultures, RPTEC first, and HRF first) and dose (0-10000 ng / ml) for each cytokine. Post-analytical comparisons were performed and reported p-values ​​were adjusted using Bonferroni correction. All analyses were performed using SAS v9.4 (SAS Institute). On the graphs, cytokine doses reaching significance are indicated with ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05. [Figure 15b] Figure 15(b) shows a comparison of the addition formats to the effect of IL-28A, IL-28B, and IL-29 in a renal RPTEC-fibroblast co-culture ECM in vitro model. The data shown in Figure 14 was replotted to show the effect of IL-28A, IL-28B, and IL-29 in a renal co-culture cell line using three different addition protocols in one graph, where IL-28A, IL-28B, or IL-29 was added to a co-culture of RPTEC and HRF together (referred to as co-culture), added to RPTEC first and HRF introduced 24 hours later (referred to as RPTEC added first), added to HRF first and RPTEC introduced 24 hours later (referred to as HRF added first), and the co-culture continued for 7 days, respectively, with the collagen I&III data replotted in Figure 15(b). ECM markers were analyzed using analysis of variance accounting for differences between addition order (co-cultures, RPTEC first, and HRF first) and dose (0-10000 ng / ml) for each cytokine. Post-analytical comparisons were performed and reported p-values ​​were adjusted using Bonferroni correction. All analyses were performed using SAS v9.4 (SAS Institute). On the graphs, cytokine doses reaching significance are indicated with ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05. [Figure 16] Figure 14(b) shows high content images showing the effect of IL-28A, IL-28B, and IL-29 on renal RPTEC-fibroblast co-culture ECM. Human primary renal proximal tubular epithelial cells (RPTEC) in co-culture with primary human renal fibroblasts (1:1 ratio 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 RPTEC 24 hours prior to inclusion of fibroblasts in the culture as shown in Figure 14(b). Images shown are from one representative field out of a total of 16 fields for each condition. Magnification was performed with a ×10 objective. [Figure 17] Figure 2: Effect of IL-28A and IL-28B in a lung SA epithelial-fibroblast co-culture ECM in vitro model. Primary human small airway lung epithelial cells (SA epithelium) in co-culture with human IPF primary lung fibroblasts (1:1 ratio with a final cell concentration of 2000 cells / well at seeding) were treated with IL-28A or IL-28B for 7 days. Cells were removed and ECM proteins, fibronectin, collagen I&III, and collagen IV were measured by immunofluorescence labeling and total ECM proteins were identified by flamingo pink staining. Imaging and quantification were performed on a Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of 4 separate wells. Cell viability was measured by PrestoBlue and fluorescence was read. [Figure 18] Figure 1 shows the protein and DNA sequences of IFNL1, IFNL2, IFNL3, and IFNL4. The protein sequences of IFNL1, IFNL2, IFNL3, and IFNL4 were obtained from the Uniprot database and the DNA sequences of IFNL1, IFNL2, IFNL3, and IFNL4 were from GenBank. [Figure 19] Figure 1 shows the effect of IL-28A and IL-29 in an in vitro human hepatic stellate cell and epithelial cell co-culture model of liver fibrosis. Human primary stellate cells and primary intrahepatic bile duct epithelial cells were co-cultured at 1000 cells / well (1:1 ratio) and treated with IL-28A or IL-29 for 5 days. Cells were removed and ECM proteins, fibronectin, collagen I&III, and collagen IV were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of 4 separate wells. [Figure 20]Figure 1 shows the effect of IL-28A on podocyte survival in an in vitro 3D glomerular spheroid model of glomerulosclerosis. (a) 3D glomerular spheroids were composed of an inner core of human glomerular endothelial cells surrounded by GFP-labeled human podocytes. (b) Spheroids were grown in culture medium or treated with focal segmental glomerulosclerosis (FSGS) patient plasma in the presence of 15% final concentration plus or minus human IL-28A protein. At least five spheroids were imaged in each experiment and for each condition. (c) 2D maximum intensity projections (MIPs) of these spheroids were then analyzed using Definiens image analysis to define marker areas above a threshold intensity in the green channel under various culture conditions and plotted as cell marker area as a percentage of the total spheroid area. 7% of podocytes were GFP-labeled. [Figure 21]Figure 14. Hydrodynamic transfection of mouse IL-28B prevents fibrosis in a mouse unilateral ureteral obstruction (UUO) kidney fibrosis model. Mice were UUO operated (UUO operation control HDT, n=10), not operated (no-op, n=5), UUO operated but treated with IL-28B on day -1 for prophylactic administration (IL-28B HDT -1 day, n=10), or treated with IL-28B on day 7 for therapeutic administration (IL-28B HDT 7 days, n=10). All animals were sacrificed on day 21, kidneys were removed, fixed in NBF, paraffin embedded, sectioned, and stained with picrosirius red (PSR). Slides were then imaged on a Hamamatsu slide scanner. Representative images of PSR stained kidney slices are shown in Figure 21(a) for one normal mouse, two UUO surgery control mice, two UUO surgery and IL-28B treated on day -1 mice, or two UUO surgery and IL-28B treated on day 7 mice by hydrodynamic transfection. All final mouse kidney slices (5 x non-operated, 10 x UUO control, 10 x IL-28B on UUO day -1, and 10 x IL-28B treated on UUO day 7) were imaged and the total area covered by PSR staining was estimated using the Definiens image analysis platform reading either the total stained area (Figure 21(b)) or the area of ​​high intensity staining (Figure 21(c)). Percentage of collagen area is shown as mean ± SEM by treatment group compared to SEAP control using Dunnett's multiple comparison test, **=p<0.01, ***p<0.001, and ****=P<0.0001. SEAP=secreted alkaline phosphatase control protein. [Figure 22] Figure 1 shows the effect of IL-29 in an in vitro human renal proximal tubule epithelial cell monoculture model of renal fibrosis. Human primary renal proximal tubule epithelial cells were co-cultured at 2000 cells / well and treated with IL-29 for 5 days. Cells were removed and ECM proteins, fibronectin, collagen I&III, and collagen IV were measured by immunofluorescence labeling with imaging and quantification by Cellomics Arrayscan. Data show the mean total intensity plotted as the mean ± SD of 4 separate wells. [Figure 23] FIG. 23(a): Treatment with human IL-29 by hydrodynamic transfection prevents tubulointerstitial fibrosis in a mouse unilateral ureteral obstruction model of renal fibrosis. C57BI / 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 after transfection, mice were subjected to unilateral ureteral obstruction (UUO) of the left kidney or were not operated on (n=4). After 7 days, hydrodynamic transfection was repeated and animals were sacrificed 19 days after UUO. FIG. 23(a): Serum samples were taken from mice on day 0 (24 hours after transfection) and again on day 19 and analyzed for human IL-29 protein. Human IL-29 levels were plotted compared to IL-29 levels in the day 0 UUO IL-29 group, the final D19 UUO SEAP group, and the final UUO IL-29 group. Kidneys were harvested, fixed in NBF, paraffin embedded, sectioned, stained with picrosirius red (PSR), and slides were then imaged on a Hamamatsu slide scanner. Figure 23(b) shows representative images from normal kidneys, UUO kidneys transfected with either a control SEAP-containing vector or a human IL-29-containing vector, shown at 100x (upper row) and 200x (lower row) magnification. All final mouse kidney slices (4 x non-operated, 10 x UUO SEAP control, 9 x UUO IL-29 on day -1) were imaged and the total area covered by PSR staining was estimated using the Definiens image analysis platform reading the total stained area (Figure 23(c)). Percentage of collagen area is shown as mean ± SEM by IL-29 treatment group compared to UUO+SEAP control group using uncorrected Fisher's LSD comparison test, **=p<0.01. SEAP=secreted alkaline phosphatase control protein DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents referenced herein are incorporated by reference.

[0013] It will be appreciated that any of the embodiments described herein may be combined.

[0014] The present invention provides interferon-lambda for use in the treatment of fibrosis. The present invention also provides the use of interferon-lambda for the preparation of a medicament for the treatment of fibrosis. The present invention further provides a method for the treatment of fibrosis, comprising administering a therapeutically effective amount of interferon-lambda.

[0015] Interferons (IFNs) are an important class of cytokines with diverse roles in host protection from viral infections and in regulating immune responses. Three different types of interferons are recognized based on their structural features, receptor usage, and biological activity (types I, II, and III), and their roles in host defense differ between the different types.

[0016] Type I IFNs (IFN-α / β / ω / ε / κ in humans) have strong antiviral activity and can induce potent antiviral responses in a wide variety of cell types (Huang et al 1993). IFN-α / β bind to a heterodimeric transmembrane receptor called IFNαR (composed of IFNAR1 and IFNAR2 subunits) and upon ligand association, triggers the activation of kinases, JAK1 and TYK2, which then phosphorylate specific tyrosines in the intracellular domain of the receptor. This creates docking sites for STAT1 and STAT2 signaling molecules, leading to their recruitment and subsequent phosphorylation. Phosphorylated STATs recruit IFN regulatory factor 9 (IRF9), which together form IFN-stimulated gene factor 3 (ISGF3), which enters the nucleus and drives the transcription of IFN-stimulated genes (ISGs).

[0017] The type II class contains only IFN-γ, which stimulates cell-mediated immune responses that are important for host defense against pathogenic microorganisms such as Mycobacterium tuberculosis (Bach et al. 1997), and is classified as a Th1-type cytokine that plays a central role in tumor immunity and amplifies the antiviral activity of type I IFNs. Thus, type I and type II IFNs work together to activate both innate and adaptive immune responses to defend the host against both infection and tumor invasion (Biron et al. 2001).

[0018] IFNs are part of a larger family of type II cytokines that includes six IL-10-related cytokines: IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26 (Kotenko 2002), which are grouped together because they signal through receptors that share a common motif in the extracellular domain. These receptors comprise the type II cytokine receptor family (CRF2) and are typically heteromers composed of two different receptor chains, the α-receptor subunit and the β-receptor subunit (Stahl et al 1993). In general, the α-subunit is the major cytokine-binding protein, while the β-subunit is required for the formation of high-affinity binding sites and signal transduction.

[0019] Type III IFNs, or IFN-lambda, are the most recent additions to the CRF2 family. They display structural characteristics of IL-10-related cytokines and induce antiviral activity in restricted cell populations (i.e., epithelial cells and certain immune cells) compared to type I IFNs (Kotenko et al., 2003; Sheppard et al., 2003). In humans, the type III IFN family consists of four closely related protein species: IFN-lambda1, -lambda2, -lambda3 and -lambda4 (also known as IL-29, IL-28A, IL-28B and IFNL4, respectively), whereas mice possess only IFN-lambda2 and -lambda3 (IL-28A and IL-28B, respectively). They are highly homologous, with approximately 96% amino acid identity between IFN-lambda2 and IFN-lambda3 and approximately 81% identity between IFN-lambda1 and IFN-lambda2 / lambda3. IFN-λ4 is most closely similar to IFN-λ3, but these proteins share only about 30% identity, and IFN-λ4 is primarily intracellular and very poorly secreted in humans (Hong et al., 2016).

[0020] The IFN-λ receptor complex is composed of a unique IFN-λ receptor chain 1 (IFN-λR1 or IL-28RA) and a shared IL-10 receptor chain 2 (IL-10R2 or IL-10Rβ). Engagement of any of the four ligands to the IFN-λ receptor complex activates JAK1 and TYK2, which activate the transcription factors STAT1, STAT2, and IRF9 to form IFN-stimulated gene factor 3 (ISGF3). ISGF3 regulates gene transcription by binding to IFN-stimulated elements (ISREs) in the promoters of hundreds of IFN-stimulated genes (ISGs), including many genes associated with antiviral phenotypes, such as OAS1, MX1, EIF2AK2 (double-stranded RNA-activated protein kinase), and IRF7. Comparative cDNA microarray analysis has shown that the repertoire of genes induced by type III IFNs (IFN-lambda) is essentially identical to that induced by type I IFNs (IFN-alpha / beta) (Doyle et al. 2006). In conclusion, despite the use of different receptors, both type I and type III IFNs activate ISGF3 (Zhou et al. 2007) and therefore induce similar transcriptional responses.

[0021] The patterns of gene expression induced by either type I or type III IFNs are very similar, although the relative magnitude of gene expression induced by IFN-α is often greater than that of IFN-λ, which may reflect differences in the relative strength of signaling through type I versus type III receptors, or may simply reflect differences in the expression levels of the respective receptors for these ligands.

[0022] The crystal structure of IFN-lambda reveals a four-helix bundle structure typical of type II cytokines, and the closest structural analogue of IFN-lambda is IL-22 (Gad et al. 2009), which may suggest that IFN-lambda and IL-22 have parallel functions in protecting epithelial tissues against viral and bacterial infections, respectively. The binding site on the IFN-lambda R1 receptor chain is well conserved among all four IFN-lambda, whereas the binding site on IL-10R2 is poorly defined (Miknis et al. 2010). IFN-lambda R1 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 and loops mainly derived from the N-terminal domain and interdomain hinge region of IFN-lambda R1. The binding mode between the ligand and receptor favors initial long-range ionic interactions mediated through hydrogen bonds, followed by hydrophobic interactions to complete the fit.

[0023] IFNλ is expressed by various hematopoietic and epithelial cells in response to viral infection. Pattern recognition receptors that detect invading viruses on mucosal epithelial surfaces initiate transcriptional responses via the transcription factors NF-κB, IRF3, IRF7, and Med23 (Osterlund et al. 2007, Griffiths et al. 2013). The roles of type I and type III IFNs in mediating antiviral defense at the mucosal interface have been shown to be both redundant and unique, depending on the virus and the point of entry. Intestinal epithelial cells respond exclusively to type III IFNs, which nonredundantly mediate the control of epithelial-tropic viruses such as rotavirus (Pott et al. 2011). Reovirus initiates infection in the intestinal epithelium but can cross the intestinal epithelial layer to cause systemic infection in mice, and type III IFNs limit initial replication in the intestinal epithelium, whereas type I IFNs are essential for preventing systemic infection (Mahlakoiv et al. 2015). The division between type I and type III IFNs is less clear in the respiratory tract, where some redundancy exists between the two IFN systems (Mordstein et al. 2008), such that airway epithelia express receptors for both IFN types, whereas intestinal epithelia express only the IFN-λ receptor.

[0024] IFN-λR1 receptor expression is restricted in certain scenarios mainly to epithelial cells and to certain immune cell subsets (e.g., monocyte-derived macrophages, plasmacytoid dendritic cells, and NK cells). Activation of IFN-λR1 in NK cells is associated with maximal IFN-λ production and antitumor activity, effects that may be mediated synergistically with other stimuli or in combination with other cell types (Souza-Fonseca-Guimaraes et al. 2015). Although the immunomodulatory effect of type III IFNs is limited because IFN-λR1 expression is restricted to immune and epithelial cells, they are highly effective antivirals when local mucosal responses are sufficient to control the virus. This contrasts with the ubiquitous activity of type I IFNs during responses to infections and is therefore more relevant in the control of severe or systemic infections where broad immune activation is required.

[0025] The following references describe the differences in signaling 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.

[0026] The contribution of IFN-λ to antiviral therapy is well established with data available in preclinical models in mice and chimpanzees, in vitro in primary human stem cells infected with Hepatitis C virus (HCV) (Thomas et al. 2012, Park et al. 2012), and in human clinical trials (PhII BMS data). The range of viruses that can be targeted by IFN-λ includes, but is not limited to: influenza A / B, severe acute respiratory syndrome (SARS), coronavirus, H1N1, human immunodeficiency virus (HIV), herpes simplex virus type 2 (HSV2), cytomegalovirus (CMV), Middle East respiratory syndrome (MERS), norovirus, rotavirus, and Ebola (Eslam and George 2015; Gresser 2015). All of these viruses target epithelial entry points and could be therapeutically targeted with IFN-λ, which can activate the IFN-λR1 receptor complex at this access point.

[0027] Numerous preclinical studies have demonstrated that IFN-λ has antitumor activity in various tumor types (e.g., hepatocellular carcinoma, melanoma, esophageal cancer, neuroendocrine tumors, colorectal cancer, lung adenocarcinoma, and Burkitt's lymphoma) (Sato et al., 2006; Zitzmann et al., 2006; Steen et al., 2010). The antitumor mechanism of IFN-λ is tumor cell induction of apoptosis and direct effects on immune cells to boost both innate and adaptive immune stimulatory activities. IFN-λ is induced in the tumor microenvironment, and signaling through the IFN-λR1 receptor has been shown to play an antitumor role in IFN-λR1 knockout mice, which are more susceptible to sarcoma formation and death in transplanted tumor models. IFN-λ treatment delayed lethality and suppressed sarcoma development (Numasaki et al., 2007). In the context of cancer treatment, IFN-λ could be envisaged to complement current IFN-α therapy, which is an immune checkpoint inhibitor, or as an adjuvant to conventional anticancer drugs (e.g., bortezomib, temozolomide, cisplatin or 5-FU) (Guenterberg et al., 2010, Li et al., 2010).

[0028] Genetic and functional evidence suggests a protective role for IFN-λ in inflammatory diseases (e.g., 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 abrogated 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, and IFN-λ can suppress the Th2 cytokine IL-13 (Wolk et al., 2013). The role of IFN-λ in inflammatory and autoimmune diseases remains unclear.

[0029] Three landmark genome-wide association studies (GWAS) for HCV have demonstrated that a single nucleotide polymorphism (SNP) in the IFN-λ region is the strongest single predictor of both response to pegylated IFN-λ1 / ribavirin (PEG-IFN / RBV) therapy and spontaneous clearance of chronic HCV infection. This SNP is located in the IFNL3 / IFNL4 locus and has been shown to be effective against all HCV genotypes and in all geographic locations (Ge et al. 2009, Suppiah et al. 2009, Tanaka et al. 2009). The role of IFNL3 / IFNL4 genotypes in predicting treatment outcome to all antiviral therapies (including both first- and second-generation direct-acting antivirals) also extends to an extensive list of other viral infections such as CMV, HSV, and Epstein-Barr virus (EBV), as well as co-infection with HCV / HIV and HCV / HBV (Rallon et al. 2010, Guo et al. 2013). The data are consistent with a major role for IFN-λ in controlling viral infections in various organs, particularly those of epithelial origin.

[0030] There is evidence for a role for two SNPs (i.e., rs12979860 and rs8099917 "responder" genotypes) in liver fibrosis and promoting liver inflammation and fibrosis in hepatitis C and B virus infections and non-alcoholic fatty liver disease (Bochud et al., 2012; Eslam et al., 2015).

[0031] Although two functional variants in the IFNL3 / IFNL4 region have been described leading to the initial GWAS findings, there may be many other contributing factors yet to be identified. The first SNP is ΔG / TT (rs368234815), which controls the production of IFN-λ4 and also predicts HCV clearance (Prokunina-Olsson et al. 2013). The ancestral “ΔG” allele encodes functional IFN-λ4 and impairs HCV clearance and thus 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 primarily related to the subcellular localization and poor secretion of IFNL4, as well as the suggestion that IFNL4 may inhibit secretion and thus function of other IFN-λ family members. A second SNP (rs4803217) in the 3'UTR region of IFNL3 affects the stability of IFNL3 messenger RNA (McFarland et al., 2014). The role of these variants in predicting liver fibrosis is unknown, especially in non-HCV cohorts, but they are in linkage disequilibrium with rs12979860, and therefore further studies are needed to show if there is any association.

[0032] In summary, to date, IFN-lambda has been described to have antiviral and antitumor activity, immune inflammatory function, and homeostatic effect. In this invention, we disclose that IFN-lambda unexpectedly has direct acting antifibrotic effect both in vitro and in vivo. The examples demonstrate that interferon-lambda protein has antifibrotic effect in a mouse chronic kidney disease model in vivo and in a human fibrosis model in vitro with relevant primary cells derived from liver, small intestine, skin, kidney and lung organ systems. Thus, this protein has unexpected potential as an antifibrotic agent in multiple types of fibrosis and can be used to design novel therapeutics that are effective in treating fibrotic diseases.

[0033] The terms interferon-lambda (IFN-λ) (singular) and interferon-lambda (IFN-λ) (plural) are used interchangeably herein to refer to cytokines of the interferon-lambda family. Interferon-lambda protein and interferon-lambda receptor are also known by many other synonymous names, as shown in Table 1. [Table 1]

[0034] In one embodiment, the interferon-lambda is IFN-lambda 1. In one embodiment, the interferon-lambda is IFN-lambda 2. In one embodiment, the interferon-lambda is IFN-lambda 3. In one embodiment, the interferon-lambda is IFN-lambda 4.

[0035] The amino acid and DNA sequences of IFN-λ1, IFN-λ2, IFN-λ3 and IFN-λ4 are shown in FIG.

[0036] IFN-λ may be used in the form of a fusion protein, such as an Fc fusion. An Fc fusion protein is composed of the Fc domain of an antibody (e.g., IgG) fused to IFN-λ. The Fc fusion protein forms a dimer as a result of the association of the Fc domains. Thus, IFN-λ may be in dimeric and monomeric form.

[0037] Functional mimics In one embodiment, the interferon-lambda is a functional mimic of IFN-lambda 1. In one embodiment, the interferon-lambda is a functional mimic of IFN-lambda 2. In one embodiment, the interferon-lambda is a functional mimic of IFN-lambda 3. In one embodiment, the interferon-lambda is a functional mimic of IFN-lambda 4.

[0038] The term "functional mimetic" refers to a molecule that has the same or similar biological effect as the wild-type protein. For example, a functional mimetic of interferon-lambda can activate the interferon-lambda receptor and drive the transcription of IFN stimulatory factors.

[0039] 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.

[0040] In one embodiment, the functional mimetic of interferon-lambda is a fragment of IFN-lambda 1. In one embodiment, the functional mimetic of interferon-lambda is a fragment of IFN-lambda 2. In one embodiment, the functional mimetic of interferon-lambda is a fragment of IFN-lambda 3. In one embodiment, the functional mimetic of interferon-lambda is a fragment of IFN-lambda 4.

[0041] In one embodiment, the functional mimetic of interferon-lambda is a variant of IFN-lambda 1. In one embodiment, the functional mimetic of interferon-lambda is a variant of IFN-lambda 2. In one embodiment, the functional mimetic of interferon-lambda is a variant of IFN-lambda 3. In one embodiment, the functional mimetic of interferon-lambda is a variant of IFN-lambda 4.

[0042] A variant interferon-lambda can be or include a variant of one of the specific sequences shown in Figure 18. For example, the variant can be a substitution, deletion or addition variant of any of the amino acid sequences in Figure 18. A fragment of IFN-lambda can have a size of, for example, more than 50 amino acids, more than 100 amino acids or more than 150 amino acids.

[0043] Variant interferon-lambda may include substitutions, deletions and / or additions of 1, 2, 3, 4, 5, up to 10, up to 20 or more (generally up to 50) amino acids compared to the specific amino acid sequence of FIG. 18. "Deletion" variants may include deletion of individual amino acids, deletion of small groups of amino acids (e.g., 2, 3, 4 or 5 amino acids), or deletion of larger regions of amino acids (e.g., deletion of a particular amino acid domain or other feature). "Substitution" variants generally include replacing one or more amino acids with the same number of amino acids, and making conservative amino acid substitutions. For example, an amino acid may be replaced with a replacement amino acid with similar properties (e.g., another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid). Some properties of the 20 main amino acids that can be used to select appropriate replacements are shown in Table 2. [Table 2]

[0044] A variant interferon-lambda may have an amino acid sequence having greater than about 60%, or greater than about 70%, such as 75 or 80%, typically greater than about 85%, such as greater than about 90 or 95% amino acid identity to the amino acid sequence of Figure 18. Variants may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of Figure 18. Variants generally retain from about 60% to about 99% identity, from about 80% to about 99% identity, from about 90% to about 99% identity, or from about 95% to about 99% identity. This level of amino acid identity can be found over the entire length of the sequence of the relevant SEQ ID NO: or over a portion of this sequence, for example over about 20, 30, 50, 75, 100, 150, 200 or more amino acids.

[0045] The term "identity" as used herein indicates that the amino acid residues are the same between the sequences at any particular position in the aligned sequences. The term "similarity" as used herein indicates that the amino acid residues are of similar type between the sequences at any particular position in the aligned sequences. For example, leucine can be substituted for isoleucine or valine. Other amino acids that can often be substituted for one another include, but are not limited to: Phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains) · Lysine, arginine, and histidine (amino acids with basic side chains); Aspartic acid and glutamic acid (amino acids with amide side chains); and · Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0046] In one embodiment, the functional mimetic of interferon-lambda is a derivative of IFN-lambda 1. In one embodiment, the functional mimetic of interferon-lambda is a derivative of IFN-lambda 2. In one embodiment, the functional mimetic of interferon-lambda is a derivative of IFN-lambda 3. In one embodiment, the functional mimetic of interferon-lambda is a derivative of IFN-lambda 4.

[0047] In one embodiment, interferon-lambda may be or may include a derivative of one of the specific sequences shown in Figure 18. In one example, the derivative may include a structural analog of a naturally occurring amino acid. Alternatively, the amino acid may be modified, for example, labeled.

[0048] In one embodiment, the interferon-lambda of the present invention is pegylated, i.e., the interferon-lambda is covalently attached to poly(ethylene glycol) (PEG).Methods for producing pegylated proteins are known in the art, see, for example, Chapman A et al., 2002, Advanced Drug Delivery Reviews 54:531-545.

[0049] In one embodiment, the pegylated interferon-lambda is pegylated interferon lambda-1a "lambda" (Andersen et al., 2013). "Lambda" is an investigational type III interferon therapeutic originally developed at ZymoGenetics (now a wholly owned subsidiary of Bristol-Myers Squibb). The therapeutic was licensed to Eiger Biopharmaceuticals in 2016 and is currently in clinical development for the treatment of chronic hepatitis delta virus (HDV) infection.

[0050] In one embodiment, the interferon-lambda functional mimetic is an antibody.

[0051] The antibody molecule 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 is understood that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. 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, bivalent, trivalent or tetravalent antibodies, bis-scFv, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3),209-217). Methods for making and producing these antibody fragments are 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 Fab and Fab' fragments as described in WO 2005 / 003169, WO 2005 / 003170 and WO 2005 / 003171, and Fab-dAb fragments as described in WO 2009 / 040562. Multivalent antibodies may contain 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.

[0052] The antibodies of the invention are functional mimetics of interferon-lambda and induce the same or similar biological effects as the wild-type protein, for example, the antibodies can bind to an epitope on the interferon-lambda receptor and activate receptor signaling to drive transcription of IFN-stimulated genes.

[0053] In one embodiment, the interferon-lambda derivative is a small molecule chemical entity (eg, a chemical entity having a molecular weight of less than 900 daltons).

[0054] Methods for screening chemical libraries to identify small molecule chemicals that may be potential drug candidates are known in the art. For example, chemical libraries can be tested in ligand-receptor binding assays or cell culture models of organ fibrosis, as described in Example 2.

[0055] Fibrotic diseases Examples of fibrotic diseases that may be treated using the interferon-lambda of the present invention include, but are not limited to, interstitial lung disease, pulmonary fibrosis, such as idiopathic pulmonary fibrosis, silicosis, hypersensitivity pneumonitis, nonspecific interstitial pneumonia, rheumatoid lung, scleroderma, chronic obstructive pulmonary disease, and cystic fibrosis; renal fibrosis (e.g., chronic glomerulonephritis, tubulointerstitial nephropathy, and genetic diseases of the kidney), such as diabetic nephropathy, hypertensive nephrosclerosis, focal segmental glomerulosclerosis, IgA nephropathy, glomerular mesangial fibrosis, and pulmonary fibrosis (e.g., chronic glomerulonephritis, tubulointerstitial nephropathy, and genetic diseases of the kidney). Proliferative glomerulonephritis, membranous nephropathy, renovascular disease, polycystic kidney disease, chronic allograft nephropathy, and Goodpasture's disease; hepatic fibrosis and cirrhosis, e.g., primary sclerosing cholangitis, primary biliary cirrhosis, alcohol-induced liver disease, and nonalcoholic steatohepatitis (e.g., other fatty acid liver diseases); and endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, encapsulating peritoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's disease (and other diseases in which intestinal stenosis occurs), keloids, myocardial infarction, scleroderma, systemic sclerosis, and arthrofibrosis.

[0056] In one embodiment, the fibrosis is renal fibrosis (in which case the IFN-λ can be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4). In one embodiment, the fibrosis is pulmonary fibrosis (in which case the IFN-λ can be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4). In one embodiment, the fibrosis is small intestinal fibrosis (in which case the IFN-λ can be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4). In one embodiment, the fibrosis is skin fibrosis (in which case the IFN-λ can be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4). In one embodiment, the fibrosis is liver fibrosis (in which case the IFN-λ can be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4). In one embodiment, the fibrosis is peritoneal fibrosis (wherein the IFN-λ can be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4). In one embodiment, the fibrosis is pancreatic fibrosis (wherein the IFN-λ can be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4). In one embodiment, the fibrosis is atherosclerosis (wherein the IFN-λ can be IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4).

[0057] In one embodiment, the fibrosis is selected from the group consisting of renal fibrosis, pulmonary fibrosis, small intestinal fibrosis, skin fibrosis, liver fibrosis, peritoneal fibrosis, pancreatic fibrosis, and atherosclerosis.

[0058] In one embodiment, the fibrosis is selected from the group consisting of renal fibrosis, pulmonary fibrosis, small intestinal fibrosis, skin fibrosis, peritoneal fibrosis, pancreatic fibrosis, and atherosclerosis.

[0059] In one embodiment, the fibrosis is not associated with a microbial infection (e.g., a viral infection). Thus, the subject treated according to the present invention may not be suffering from an infection, such as a viral infection. For example, the subject may not be suffering from a viral infection known to be treated by IFN-λ. The subject may not be infected with a hepatitis virus, such as hepatitis A, hepatitis B, or hepatitis 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.

[0060] Pharmaceutical Compositions, Dosages, and Administration Regimens The interferon-lambda of the present invention or a functionally active fragment or derivative thereof may be included in a pharmaceutical composition, which is usually sterile and may further include a pharma- ceutical acceptable adjuvant and / or carrier.

[0061] As used herein, a "pharmaceutical 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 administration, for example, intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. Alternatively, the carrier may be suitable for parenteral administration, for example, topical, epidermal, or mucosal administration routes. The carrier may be suitable for oral administration. Depending on the route of administration, interferon-lambda or its functionally active fragment or derivative may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it.

[0062] The pharmaceutical composition of the present invention may contain one or more pharma- ceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0063] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffered water and saline. Other carriers include ethanol, polyols (e.g., 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, such as sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride in the composition.

[0064] Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0065] The pharmaceutical compositions of the invention may contain additional active ingredients.

[0066] Also within the scope of the present invention is a kit comprising an interferon-lambda of the present invention and instructions for use. The kit may further include one or more additional reagents (e.g., additional therapeutic or prophylactic agents as discussed above).

[0067] Interferon-lambda or a formulation or composition thereof of the present invention may be administered for the prophylactic and / or therapeutic treatment of fibrotic diseases.

[0068] In one embodiment, the treatment of fibrotic disease is a therapeutic treatment. In therapeutic use, the compound is administered to a subject already suffering from the above-described disorder or condition in an amount sufficient to cure, alleviate or partially stop this condition or one or more of its symptoms. Such therapeutic treatment may result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this is defined as a "therapeutically effective amount".

[0069] In one embodiment, the treatment of fibrotic disease is a prophylactic treatment. In prophylactic use, the formulation is administered to a subject at risk of the above-described disorder or condition in an amount sufficient to prevent or reduce the subsequent effects of this condition or one or more of its symptoms. An amount sufficient to achieve this is defined as a "prophylactically effective amount".

[0070] 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.

[0071] The subject of administration can be a human or a non-human animal. The term "non-human animal" includes all vertebrates, including, for example, mammals and non-mammals (e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.). Administration to humans is typical.

[0072] The antibody / modifier or pharmaceutical composition of the present 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 those of skill in the art, the route and / or mode of administration will vary depending on the desired outcome. Examples of routes of administration of the pharmaceutical composition of the present invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as parenteral routes of administration by injection or infusion. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection. Alternatively, the pharmaceutical composition of the present invention may be administered via parenteral administration (e.g., topical, epidermal, or mucosal routes of administration). The pharmaceutical composition of the present invention may be for oral administration.

[0073] The appropriate dosage of the pharmaceutical composition of the present invention can be determined by a skilled physician. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the particular patient. The dosage level selected will depend on various pharmacokinetic factors, such as the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and past medical history of the patient being treated, and similar factors known in the medical field.

[0074] Suitable doses may range, for example, from about 0.01 μg to about 1000 mg per kg of body weight of the patient to be treated, typically from about 0.1 μg to about 100 mg per kg of body weight. For example, suitable dosages may be from about 1 μg to about 10 mg per kg of body weight per day, or from about 10 μg to about 5 mg per kg of body weight per day.

[0075] The dosage regimen can be adjusted to produce the optimum desired response (e.g., therapeutic response). For example, a single dose can be administered, or multiple divided doses can be administered over time, or the dose can be relatively decreased or increased as indicated by the exigencies of the therapeutic situation. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for the subjects to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0076] Administration may be in a single dose or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different locations. Alternatively, the dose may be by 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 desired duration of treatment.

[0077] As noted above, the modulators / antibodies or pharmaceutical compositions of the invention may be co-administered with one or more other therapeutic agents.

[0078] The co-administration of two or more drugs can be achieved in many different ways. Both can be administered together in a single composition, or they can be administered in separate compositions as part of a combination therapy. For example, one can be administered before, after, or simultaneously with the other.

[0079] In one embodiment, the interferon-lambda of the present invention is administered in combination with another therapeutically active compound, which in one embodiment is another anti-fibrotic therapeutic agent.

[0080] Alternatively, the interferon-lambda may be administered without combination with another therapeutically active compound. For example, a patient treated with interferon-lambda may not be treated with an antiviral agent, such as another interferon (e.g., a type I interferon or a type II interferon, such as interferon-gamma).

[0081] References JPEG0007609556000003.jpg24149 JPEG0007609556000004.jpg214131 JPEG0007609556000005.jpg220143 JPEG0007609556000006.jpg226143 JPEG0007609556000007.jpg87131 EXAMPLES

[0082] overview The interferon-lambda (IFN-lambda) family of cytokines, known as IFN-lambda1 (IL-29), IFN-lambda2 (IL-28A), and IFN-lambda3 (IL-28B), signal through separate receptor complexes composed of the IFN-lambdaR1 (IL-28R1) and interleukin-10R2 (IL-10R2) receptor chains.

[0083] Using a hydrodynamic transfection in vivo screen in a mouse adriamycin-induced model of chronic kidney disease (CKD), IFN-λ2 and IFN-λ3 were reported as hits. Hits were called based on the level of renal fibrosis determined by a 40% reduction in renal hydroxyproline levels (renal collagen). Both IFN-λ2 and IFN-λ3 also showed corresponding preservation of renal function as measured by serum creatinine. This resulted in a conditional survival rate (i.e., no end-stage renal failure) of 50% at 49 days post-adriamycin in saline-transfected mice, to 85% when transfected with IFN-λ2 and 100% when transfected with IFN-λ3. To validate this antifibrotic effect in surrogate models of CKD with different stimuli, both IFN-λ1 and IFN-λ3 were applied by hydrodynamic transfection in a unilateral ureteral obstruction (UUO)-induced renal fibrosis model of chronic kidney disease, and both conferred protection against tubulointerstitial fibrosis.

[0084] The antifibrotic effect of IL-28R1 ligand has been confirmed in several human primary cell in vitro models of fibrosis read out for mature and accumulated extracellular matrix (ECM) proteins. Recombinant human IFN-λ1, IFN-λ2 and IFN-λ3, respectively, inhibited the accumulation of fibrosis-associated ECM proteins (fibronectin, and collagens I, III and IV) in primary human cell in vitro models of liver fibrosis, lung fibrosis, intestinal fibrosis, skin fibrosis and kidney fibrosis. This has been seen in several models including hepatic stellate cells, hepatic stellate cell-hepatocyte cocultures (basement matrix and TGFβ-induced), hepatic stellate cell-epithelial cell cocultures, TGFβ-induced small intestinal fibroblasts, IL-1α-induced dermal fibroblasts, keratinocyte-dermal fibroblast cocultures, renal proximal tubule epithelial cell-renal fibroblast cocultures and monocultures, and lung small airway epithelium-IPF lung fibroblast cocultures. In a more complex glomerular organoid model of glomerulosclerosis, application of plasma from a patient with relapsing nephrotic syndrome caused both a reduction in podocyte numbers and a retraction of processes and stalks consistent with podocyte demise and disappearance, seen in many glomerular diseases leading to glomerulosclerosis and renal fibrosis. Application of recombinant IFN-λ2 in this model completely prevented the changes to podocytes caused by this patient's plasma.

[0085] IL-28R1 ligand has been found to have strong anti-fibrotic effects in both in vitro and in vivo fibrosis screening.Strong effects have been demonstrated in in vivo mouse models of renal fibrosis, and in multiple in vitro human fibrosis models with relevant primary cells from organ systems of liver, small intestine, skin, kidney and lung.Therefore, this protein has unexpected potential as an anti-fibrotic agent in multiple types of fibrosis.

[0086] method Mouse in vivo Effects of IL-28A and IL-28B in an adriamycin-induced renal fibrosis model of chronic kidney disease (CKD) Female Balb / c mice (at least 6 weeks old and weighing more than 24 g) were injected IV via the tail vein with cDNA (or pairs of cDNA in the case of paired studies) in the appropriate hydrodynamic transfection expression vector. Seven days later, mice were given adriamycin (doxorubicin) at 11 mg / kg IP and monitored for 49 days. Mice were monitored daily for weight and health status, and mice that lost more than 40% of their starting weight or showed physical signs of end-stage renal failure were sacrificed to collect data. At the end of the 49-day study period, all mice were sacrificed and kidneys and serum were collected for analysis. Renal hydroxyproline was measured using the QuickZyme assay method (QuickZyme Biosciences), and serum creatine was measured using a three-enzyme method and read as μg / ml serum creatinine. Renal fibrosis (hydroxyproline) in the saline + adriamycin group (shown as "Saline" in the graph) was considered to be the maximum disease response. This was set to 100% and used for normalization across treatment groups. Untreated animals did not receive adriamycin, and each screening experiment included an "HGF" group that received adriamycin and HGF cDNA. HGF was used as an anti-fibrotic positive control, and a >40% reduction in hydroxyproline was used for quality control.

[0087] Effects of IL-28 and IL-29 on renal fibrosis induced by unilateral ureteral obstruction in mice DNA constructs for hydrodynamic transfection of mouse IL28B and human IL29 A DNA construct for hydrodynamic transfection was generated 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 and subsequent DNA ligation of the homologous ends. A gene (mRNA reference sequence NM_177396) encoding mouse IFNL3 (NP_796370) construct was cloned into pLive (Mirus) using a BamHI-XhoI restriction digestion strategy of both the vector and the insert, followed by ligation. Gene insertion into pLive was verified by DNA sequencing. The gene was preceded by a CCACC sequence immediately upstream of the ATG encoding the initiating methionine of the signal sequence. This vector contains a kanamycin resistance gene and a pUC origin that allows DNA amplification in Escherichia coli. In addition, this vector contains the mouse minimal albumin promoter to transcribe the human IFNL-1 gene flanked on both sides by introns. [This pLive vector allows long-term protein expression in the liver].

[0088] The DNA construct for hydrodynamic transfection was isolated from E. coli cultures containing the construct using a plasmid isolation kit (HiSpeed ​​Plasmid Giga EF, Qiagen), which limits the amount of endotoxin in the solution. The plasmid DNA was Sanger sequenced to verify the insert.

[0089] Kanefuji et al. and Liu et al. describe exemplary vectors for hydrodynamic transfection.

[0090] Protein expression by hydrodynamic transfection (HDT) Mice were hydrodynamically transfected with 50 μg of either pLIVE-human IL-29, pLIVE-mouse IL-28B or pLIVE SEAP Vector (Mirus Bio, Cat. No. MIR 5320) in TransIT-EE vehicle. The vector-containing solution was rapidly (<2.5 seconds) injected intravenously in a volume of 2 ml. General anesthesia with isoflurane was induced prior to hydrodynamic injection and maintained for at least 1 minute after injection.

[0091] Quantification of mouse IL-28B and human IL-29 from mouse serum Mouse IL-28B was quantified using a Mesoscale Discovery (MSD) quantification assay using a commercially available antibody pair (RnD systems, Cat# DY1598B) with mouse serum diluted 100-fold to lie on a linear standard curve. Human IL-29 was quantified using the MSD U-plex human IL-29 antibody set (Cat# B21WD) with mouse serum diluted 100-fold to lie in the linear range.

[0092] Unilateral ureteral obstruction (UUO)-induced renal 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 renal fibrosis model of chronic kidney disease.

[0093] Surgery was performed using isoflurane-induced general anesthesia and under aseptic conditions appropriate for surgical technique. Mice were opened laparotomy followed by tying off the left ureter using a 3.0 Mersilk ligature. The muscle wall was closed using 5-0 Vicryl in a continuous pattern and the skin was closed with subcuticular sutures using 5-0 Vicryl. Appropriate analgesia was administered pre- and post-operatively. Blood samples were taken at appropriate times as detailed in the results. Mice were restrained using a recognized mouse restrainer and 30 μl of blood was taken from the tail vein by tail prick method using a pipette to accurately measure blood samples. The sample was placed in a 0.2 ml PCR tube (Thermo) and centrifuged (20,000 g, 5 min) to remove as much serum as possible and stored at -80°C in a new PCR tube.

[0094] On day 19 or 21, mice were anesthetized using isoflurane, blood was removed by cardiac puncture into serum tubes (Sarstedt), and the mice were killed by cervical dislocation. The left kidney was removed and two quarters were flash frozen in liquid nitrogen and stored at -80°C. The other half was placed in 10% neutral buffered formalin, dehydrated in a tissue processor, and embedded in paraffin for histological analysis. Paraffin blocks were sectioned at 4 μm on a microtome and mounted on glass slides. Slides were then dewaxed, hydrated, and washed in acidified water after staining with picrosirius red (PSR). Slides were then dehydrated, cleared, and coverslipped. Slides were dried and then scanned using a Hamamatsu slide scanner.

[0095] Whole slide images were imported into Definiens Developer XD 64 and analyzed using the brightfield module. Briefly, cortical areas were manually annotated and the medulla was removed from the analysis. Marker threshold levels (e.g. PSR staining) were determined for individual markers on a training dataset of 4 or more images. Analysis algorithms were then run on a dedicated server to determine marker area above a threshold intensity for each individual marker and the area of ​​each marker above the threshold intensity as a percentage of the area of ​​the cortex.

[0096] Histological assessment of the level of fibrosis was performed by blinded investigators with expertise in the field of renal fibrosis using a combination of thin sections stained with the preferred picrosirius red, Masson's trichrome, and hematoxylin and eosin. A 10-point scale was used, with 10 being very severe fibrosis corresponding to end-stage renal failure and 1 being normal. The parameters assessed in this score were expansion of the tubular basement membrane, flattening of epithelial cells, brush border integrity, tubular atrophy and collapse, tubular lumen size, infiltration of interstitial cells, structure of the glomerular tuft, expansion of the mesangium, infiltration of the glomerulus, expansion of the glomerular lumen, glomerular basement membrane, and mesangial matrix.

[0097] Definiens image analysis and histological evaluation of images were also applied to slices of kidneys treated with adriamycin.

[0098] Human in vitro extracellular matrix fibrosis assay measurements a) Cell culture All extracellular matrix (ECM) accumulation assays were performed in 384-well black clear bottom plates (Greiner Cat# 781090) and media, cell numbers / well, incubation times, and stimuli varied depending on the primary cell type used.

[0099] Human hepatic stellate cell (HHSteC) (ScienCell, Catalog No. sc-5300) (passage 4) and human intrahepatic bile duct epithelial cell (ScienCell, Catalog No. sc-5100) (passage 3) co-cultures were seeded at 1000 cells / well (1:1 ratio) in 25uL of epithelial cell medium - phenol red free (EpiCM-prf) with nutritional supplements (ScienCell, Catalog No. sc-sc-4101-prf). Monocultured human stellate cells (ScienCell) were seeded at 1000 cells / well in stellate cell medium (ScienCell) and human stellate cell and hepatocyte (ScienCell) co-cultures were seeded at 1000 cells / well (1:1 ratio) in mixed culture medium (ScienCell).

[0100] Human small intestinal fibroblasts (ScienCell) were seeded at 2000 cells / well in renal epithelial cell basal medium + 0.5% FCS and nutritional supplements (ATCC).

[0101] Human dermal skin fibroblasts were grown in fibroblast growth medium, human keratinocytes were grown in keratinocyte medium and seeded at 1500 cells / well. Fibroblast-keratinocyte co-cultures (9:1 ratio) were grown in a 1:1 mixture of medium and seeded at 1500 cells / well.

[0102] Human renal proximal tubular epithelial cells (RPTEC, Innoprot) and human renal fibroblasts (HRF, InnoProt) were seeded at 2000 cells per well (1:1 ratio) in co-culture using renal epithelial cell basal medium (ATCC) + 0.5% FCS and nutritional supplements (ATCC).

[0103] Human small airway epithelial cells (ATCC) were grown in basal growth medium + bronchial epithelial growth kit supplement (ATCC) and IPF134 lung fibroblasts (ATCC) were grown in fibroblast growth medium (Lonza). The co-cultures were seeded at 2000 cells / well (1:1 ratio).

[0104] b) Measurement of cell proliferation After 7 days of incubation at 37°C and 5% CO2, cell proliferation was assessed using PrestoBlue® Cell Viability reagent (Thermo Fischer Scientific) according to the manufacturer's instructions.

[0105] c) Measurement of ECM accumulation Measurement of individual ECM components: immunofluorescence After 7 days of incubation at 37°C in 5% CO2, cells were washed with PBS and lysed with 20 μl / well of 0.25M NH4OH / 25mM Tris (Sigma-Aldrich) for 15 min at 37°C. Matrices were then washed 3 times with PBS, fixed with 40 μl of 100% methanol for 30 min at -20°C, washed 3 times with PBS, and stained using anti-fibronectin antibody (eBiosciences), anti-collagen I antibody (Millipore), anti-collagen III antibody (Millipore), anti-collagen IV antibody (eBiosciences), and anti-collagen V antibody (Abcam). Cellomics CellHealth profiling bioapplication and 2×2 binning (1.10 4 ×1.10 4 Plates were scanned with an Arrayscan HC reader (Cellomics) using a three-channel protocol under a 10× objective (novel X1 camera) with 10× resolution (pixels / field of view).

[0106] Measurement of total ECM components: Flamingo™ staining Immunofluorescence plates were washed three times with Flowfusor water and then stained using Flamingo™ fluorescent gel stain (BioRad) according to the manufacturer's instructions. "Cellomics CellHealth" profiling bioapplication and 2 × 2 binning (1.10 4 ×1.10 4 Plates were scanned with an Arrayscan HC reader (Cellomics) using a two-channel protocol under a 10× objective (novel X1 camera) with 10× resolution (pixels / field of view).

[0107] How to generate 3D human glomerular spheroids Spheroid generation and processing Stably transfected GFP-tagged actin, conditionally immortalized SV40-infected podocytes, and human glomerular endothelial cells (HRGECs) provided by University of Bristol were supplemented with Nano-shuttle-PL (cat. no. 657841) and grown in RPMI 1640 medium (10% FCS, 5ML L-glutamine, and 1XITS, Gibco) and EBM-2 medium, respectively, with a nutritional supplement kit from Lonza (cat. no. CC4147). GFP-transfected podocytes were confirmed by FACS. All cells were harvested using TrypLE Express (Gibco), and magnetized HRGECs were seeded at 5000 cells / well in 96-well low-attachment Greiner plates (cat. no. 655976). A Greiner Spheroid Drive (cat. no. 655830) was placed under the 96-well plate for 6 hours and spheroids were grown in EBM-2 medium at 37°C / 5%CO2 / 100% humidity. The Drive was removed and magnetized GFP podocytes were seeded at 5000 cells / well. The Drive was placed back under overnight and spheroids were differentiated using the Holding Drive for 10 days of feeding every 3 days. Spheroids were then treated with human FSGS plasma ± IL-28A (10,000ng / mL) at 15% final concentration or medium alone for 7 days.

[0108] Fixation and staining Spheroids were fixed and permeabilized in 10% formalin / 1% Triton X-100 / PBS for 3 hours at 4°C. After 3×10 min PBS washes, spheroids were dehydrated in ascending series of methanol in PBS at 4°C: 25%, 50%, 75%, 95% for 30 min each, and left in 100% methanol overnight. Spheroids were rehydrated in the same descending series and washed in PBS as above, then blocked in PBST (0.1% TritonX-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 24 hours. After washing the spheroids (4 × 30 min / PBST), images were captured using a YokoGawa Confocal Quantitation 1 (CQ1) at ×20 magnification.

[0109] Quantification of podocyte cell marker area Maximum intensity projections were imported into Definiens Developer XD 64 and analyzed using the Immunofluorescence module. Spheroid areas were manually annotated, marker threshold levels were determined for individual markers, and an analysis algorithm was run to determine the marker area above the threshold intensity for each marker. The area of ​​each marker above the threshold intensity was determined as a percentage of the area of ​​the spheroid.

[0110] result (1) Adriamycin-induced mouse model of chronic kidney disease (CKD) As a member of a hydrodynamically transfected cDNA pair, we first identified mouse IL-28A (IFNL2) in a screen for fibrosis-modifying proteins in the mouse adriamycin CKD model (n=15 mice). This transfected pair reduced renal fibrosis by 51% (p=0.02) as measured by total renal hydroxyproline compared to the saline transfection control group (Figure 1(a) and Figure 1(b)), and normalized renal function based on serum creatinine levels (p<0.05) (Figure 1(c)). The antifibrotic effect of IL-28A was confirmed in a repeat study independently transfecting IL-28A cDNA in the same model (n=22 mice per group). IL-28A reduced renal fibrosis by 48% (p=0.0004) compared to the saline control group, as measured by total kidney hydroxyproline (Figure 2(a) and (b)), while loss of kidney function returned to the normal range as measured by serum creatinine (mean serum creatinine=1 μg / ml in the IL-28A group vs. 4.8 μg / ml in the untreated adriamycin saline control group (p<0.0001); mean untreated group serum creatinine=0.8 μg / mL) (Figure 2(c)). Conditional survival, as measured by symptoms of end-stage renal failure, increased from 50% in the saline-treated adriamycin group to 85% in the mouse IL-28A-transfected group (Figure 2(d)). On day 49, all kidneys from surviving mice (untreated (n=8), untreated adriamycin animals (n=20), and IL-28A hydrodynamically transfected animals (n=19)) were fixed, paraffin embedded, sectioned, stained with either Masson's Trichrome (MT) or Picrosirius Red (PSR), and imaged on a Hamamatsu slide scanner. Exemplary and representative images of PSR stained kidneys for one untreated, three untreated adriamycin, and three IL-28B-treated adriamycin animals are shown in FIG. 5(f). With adriamycin treatment, approximately 50% of the glomeruli clearly show glomerulosclerosis with enhanced PSR staining, mainly in the glomerular basement membrane, but also in the mesangial matrix.In the tubulointerstitium, there is consistent expansion of the tubular basement membrane in over 70% of the cortex, and many tubules show squamous epithelium causing significant loss of tubular architecture with extensive dilation of the tubules leading to large lumens lacking brush borders. These areas have mild to moderate cellular infiltration.

[0111] Application of IL-28A almost completely prevented signs of glomerulosclerosis, with little evidence of glomerular basement membrane thickening. Only 2 of 22 animals showed prominent signs of epithelial flattening and luminal enlargement characteristic of untreated animals. The remaining animals had a tubulointerstitium that appeared nearly normal in over 80% of the cortex and with little involvement of the tubular basement membrane.

[0112] Forty-seven mouse kidney samples stained with PSR were subjected to high content image analysis using Definiens software and the areas of PSR staining measured in the three groups were plotted as percentage PSR stained area (Figure 5(g)). The same 47 mouse kidney slides were subjected to blinded pathological assessment of the extent of fibrosis by two individuals with expertise in renal pathology. A nephrology expert scored the PSR stained slides using a scoring range of 0-10 point scale (0 indicates no fibrosis and 10 indicates the highest fibrosis). IL-28A treatment showed a significant 47% reduction (p<0.0001) in pathology score compared to the untreated group (Figure 5(h)). A second nephrology expert manually scored the 47 MT stained slides using a scale of 1-10 (1 indicates no fibrosis and 10 indicates the highest fibrosis). The IL-28A-treated group again showed a clear improvement (p<0.0001) in the level of fibrotic remodeling compared to the untreated group (FIG. 5(i)).

[0113] As the first member of a hydrodynamically transfected cDNA pair, we also identified mouse IL-28B (IFNL3) in a screen for fibrosis-modifying proteins in the mouse adriamycin CKD model (n=15 mice per group). This transfected pair reduced renal fibrosis by 59% (p=0.00086) as measured by total renal hydroxyproline compared to the saline transfection control group (Figure 3(a) and Figure 3(b)), and normalized renal function based on serum creatinine levels (not significant) (Figure 3(c)). The antifibrotic effect of IL-28B was confirmed in a repeat study independently transfecting IL-28B cDNA in the same model (n=22 mice per group). Renal fibrosis, as measured by total renal hydroxyproline, was reduced by 65% ​​compared to the saline control group (p=0.0000018) (Figure 4(a) and (b)), while renal function returned to normal compared to a significant increase in the untreated saline control group (mean serum creatinine=1.2 μg / ml in the IL-28B group compared to a mean of 3.2 μg / ml in the untreated adriamycin saline control group (p<0.05); mean untreated group serum creatinine=0.8 μg / mL) (Figure 4(c)). Conditional survival increased from <50% in untreated adriamycin animals to 100% when IL-28B was administered (Figure 4(d)). On day 49, all kidneys from surviving mice (untreated (n=10), untreated adriamycin animals (n=18), and IL-28B hydrodynamically transfected animals (n=22)) were fixed, paraffin embedded, sectioned, stained with either hematoxylin and eosin (H&E) or picrosirius red (PSR), and imaged on a Hamamatsu slide scanner. Exemplary and representative images of PSR-stained kidneys for one untreated, three untreated adriamycin, and three IL-28B-treated adriamycin animals are shown in FIG. 5(a). Kidneys of untreated mice had extensive glomerulosclerosis and tubulointerstitial fibrosis with prominent tubular basement membrane expansion, flattening of the tubular epithelium, extensive tubular dilatation with loss of brush border, and tubular atrophy. In contrast, IL28B-treated mice were largely protected from both types of fibrotic remodeling.Fifty mouse kidney samples stained with PSR were subjected to high content image analysis using Definiens software and the areas of PSR staining measured in the three groups were plotted as percentage PSR stained area (Figure 5(b)). The PSR stained area in the IL-28B treated group was normalized compared to a 3-fold increase in untreated kidneys (p<0.0001). The same 50 mouse kidney slides were subjected to blinded pathological assessment of the extent of fibrosis by three individuals with expertise in renal pathology. Clinical pathologists scored the H&E stained slides using a scoring range of 0-4 point scale (0 indicates no fibrosis and 4 indicates the highest fibrosis). IL-28B treatment showed a significant 45% decrease (p<0.01) in the pathology score compared to the untreated group (Figure 5(c)). Two expert nephrologists manually scored 50 PSR-stained slides using a scale of 1 to 10 (1 indicating no fibrosis and 10 indicating excellent fibrosis). The IL-28B-treated group again showed a clear improvement (p<0.0001) in the level of fibrotic remodeling compared to the untreated group (Figure 5(d) and Figure 5(e)).

[0114] (2) Primary human cell in vitro models of organ fibrosis (2.1) Liver fibrosis Human primary astrocytes grown on plastic autoactivate and accumulate substantial basal levels of extracellular matrix as measured by the levels of the ECM proteins fibronectin, collagen IV, and collagen I&III. This ECM accumulation can be dose-dependently inhibited by treatment of the cultures with recombinant IL-28A (Figure 6(a)). Co-culture of human primary astrocytes and primary human hepatocytes (1:1 ratio) results in a basal extracellular matrix that can also be dose-dependently inhibited by treatment with recombinant human IL-28A, with fibronectin, collagen IV, and collagen I&III being reduced (Figure 6(b)).

[0115] TGFβ1 stimulation of both stellate cell monocultures and stellate-hepatocyte cocultures induced further increases in the ECM proteins fibronectin, collagen IV, and collagen I&III. Treatment of TGFβ1 stimulated stellate cell monocultures or stellate-hepatocyte cocultures with either IL-28A or IL-28B showed a near complete inhibition of any increase in all ECM proteins at the highest concentration of 10 μg / ml of IL-28A or B, both in a dose-dependent manner. IL-28A was more potent than IL-28B across all ECM proteins in TGFβ1 stimulated hepatocytes (Figure 7(a) and Figure 7(b)). Images of TGFβ1 stimulated coculture ECM in the presence of either IL-28A or IL-28B treated cocultures showed a clear decrease in ECM amount compared to TGFβ1 treatment alone, which is consistent with image analysis quantification (Figure 8).

[0116] (2.2) Small intestinal fibrosis Human small intestinal fibroblasts stimulated with TGFβ1 (10 ng / ml) induce the extracellular matrix proteins fibronectin and collagen I & III. Fibronectin levels were significantly inhibited by IL-28A, IL-28B and IL-29 (10 ng / ml) with 76%, 73% and 83% inhibition, respectively, compared to control TGFβ1 stimulation. Collagen I & III were inhibited by IL-28A, IL-28B and IL-29 (10 ng / ml) with 48%, 33% and 35%, respectively. Collagen IV was significantly inhibited only by IL-28A (37% inhibition at 10 μg / ml) compared to control TGFβ1 stimulation (Figure 9).

[0117] (2.3) Dermal fibrosis Co-cultures of human primary dermal fibroblasts and primary keratinocytes (9:1 ratio) accumulate substantial amounts of fibronectin, collagen I & III, and collagen IV simply by co-culture. Using three separate batches of fibroblasts in combination with keratinocytes from a single donor, cultures were treated with either IL-28A or IL-28B. The percentage inhibition of ECM proteins relative to untreated cultures was measured for all three fibroblast batches and the average inhibition was plotted (Figure 10). Both IL-28A and IL-28B completely inhibited all four matrix proteins at the highest concentration of 10 μg / ml with similar potency. Both IL-28A and IL-28B showed dose-dependent inhibition of collagen IV ECM marker from 10 to 10,000 ng / ml. Cell viability was tested using the prestoblue assay, showing that neither IL-28A nor IL-28B was toxic at any of the concentrations tested (Figure 10).

[0118] Dermal fibroblast-keratinocyte co-cultures were tested with IL-29, IL-28A and IL-28B over a narrower concentration range of 1250-10000ng / ml. This showed a dose-dependent inhibition of fibronectin, collagen I&III and collagen IV compared to control co-cultures (Figure 11). Collagen IV was most inhibited by IFNL at all concentrations tested (Figure 11). Cell viability was not affected by treatment with IL-29, IL-28A or IL-28B as determined by Presto Blue cell viability assay (Figure 11).

[0119] Images of untreated dermal co-cultures compared to IL-29, IL-28A and IL-28B test co-cultures at both 1250 ng / ml and 10000 ng / ml confirmed the complete prevention of ECM accumulation as determined by image analysis in the presence of 10 ug / ml IFNL (Figure 12).

[0120] Finally, human dermal fibroblasts were stimulated in monoculture with IL-1α (10 ng / ml) to induce the ECM proteins fibronectin, collagen I&III and collagen IV. Treatment of IL-1α stimulated fibroblast monocultures with IL-28A or IL-28B showed a strong inhibition of the pro-matrix response of all ECM proteins with complete inhibition of the matrix at the highest concentration of 10 μg / ml. IL-28A was more potent than IL-28B in all batches of fibroblasts, with one representative example shown (Figure 13). IL-28A or IL-28B did not affect cell viability as measured by prestoblue (Figure 13).

[0121] (2.4) Renal fibrosis Human primary renal proximal tubule epithelial cells (RPTEC) co-cultured with human primary renal fibroblasts (seeded at 2000 cells / well in a 1:1 ratio) produce a robust ECM with high levels of fibronectin and collagens I & III. The co-cultures were treated with IL-29, IL-28A or IL-28B using three different protocols for cytokine addition.

[0122] IL-29, IL-28A and IL-28B were first added to the kidney co-cultures on day 0 and the cultures were incubated for 7 days. All three cytokines potently inhibited fibronectin and collagen I & III accumulation in a dose-dependent manner (Figure 14(a)). IL-29 showed the most potent effect, with little difference between IL-28A and IL-28B. Cell viability was not compromised by cytokine treatment (Figure 14(a)).

[0123] Next, IL-29, IL-28A and IL-28B were added to RPTEC cells on day 0, fibroblasts were added 24 hours later, and the culture was continued for a total of 7 days after which ECM was measured. Here, IL-29, IL-28A and IL-28B all dose-dependently inhibited the accumulation of fibronectin and collagen I & III again (Figure 14(b)). The magnitude of inhibition of ECM proteins in the presence of IFNL was greater when cytokines were added to RPTEC 24 hours before co-culture induction of ECM was initiated by addition of fibroblasts, rather than to both cells at the same time (compare Figure 14(a) and Figure 14(b)).

[0124] Finally, IL-29, IL-28 and IL-28B were added to fibroblasts on day 0. After 24 hours, RPTECs were added and co-culture continued for a total of 7 days to measure fibronectin and collagen I&III. Adding IFNL to fibroblasts first also showed significant inhibition of ECM proteins (Figure 14(c)), but the potency of IL-29, IL-28A and IL-28B was reduced compared to both the addition of cytokines to the co-culture on day 0 and to RPTECs 24 hours before fibroblasts. This observation points to an IL-28R1-mediated signaling response in RPTECs that mediates an anti-fibrotic phenotype resulting in inhibition of ECM signals in co-cultures of both epithelial and fibroblast types. To clearly demonstrate the different effects of the order of addition of IFNL to different cell types versus simultaneous addition, the IL-28A, IL-28B and IL-29 data were replotted for fibronectin (Figure 15(a)) and for collagen I & III (Figure 15(b)) showing the relative effect of different addition protocols on the level of inhibition for each individual IL-28R1 ligand. At certain lower concentrations of IL-28A, IL-28B or IL-29, the differences between simultaneous addition of cells and cytokines compared to addition to RPTECs first (before fibroblasts) reached statistical significance and these values ​​are shown on the graphs.

[0125] Cell viability, as read by presto blue, was not compromised by the addition of cytokines first to RPTEC or fibroblast cells before each other in the co-culture (Figure 14(a), Figure 14(b) and Figure 14(c)).

[0126] Representative images (1 of 16 total fields per treatment) of untreated co-cultures compared to IL-28A, IL-28B and IL-29 test co-cultures show both a dose-dependent effect of IL-29, IL-28A and IL-28B and a near complete inhibition observed in the presence of 1000 ng / ml IL-29 (Figure 16), supporting the image analysis data.

[0127] Renal proximal tubule epithelial cells (RPTECs) in monoculture accumulate basal ECM proteins by growing on plastic, albeit at low levels, and as shown for fibronectin, collagen IV, and collagen I & III in Figure 22, low levels of ECM can still be inhibited in the presence of very low concentrations of IL-29.

[0128] (2.5) Pulmonary fibrosis Human primary small airway lung epithelial cells in co-culture with lung fibroblasts isolated from idiopathic pulmonary fibrosis (IPF) patients (2000 cells / well at 1:1 ratio) produced robust total ECM (measured by flamingo staining) after 7 days in culture and were positive for the ECM markers fibronectin, collagen I&III, and collagen IV. IL-28A or IL-28B had no significant effect on the levels of fibronectin. However, IL-28B showed significant inhibition of collagen I&III, collagen IV, and flamingo staining at the highest concentration of 10 μg / ml (Figure 17). However, the effect of IL-28A was less pronounced. There was no change in cell viability at day 7 with either IL-28A or IL-28B (Figure 17).

[0129] (3) Primary human cell models of organ fibrosis (3.1) Liver fibrosis Co-culture of human primary stellate cells and primary human intrahepatic bile duct epithelial cells (1:1 ratio) results in 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 reductions in fibronectin, collagen IV, and collagen I&III (Figure 19).

[0130] (3.2) 3D glomerular spheroid model Glomerular spheroids were constructed from an inner core of magnetized human primary glomerular endothelial cells with an outer core of temperature-conditioned human podocytes that could be maintained in stable culture for up to 3 weeks. Treatment of glomerular spheroids with 15% recurrent plasma from patients with focal segmental glomerulosclerosis (FSGS) induced a loss of GFP-labeled podocytes from the outer core that could be protected in the presence of IL-28A. Plasma from healthy volunteers had no effect on podocytes. Quantification of green fluorescent podocyte marker area showed a significant decrease in the presence of FSGS recurrent plasma that was significantly increased with IL-28A treatment (Figure 20(a), Figure 20(b) and Figure 20(c)). Clinical progression of FSGS is characterized by the disappearance and loss of podocytes. This model recapitulates the effects on podocytes from circulating factors in FSGS plasma, suggesting the clinical translatability of IL-28 therapy to prevent associated nephrotic syndrome and glomerulosclerosis.

[0131] (4) Murine unilateral ureteral obstruction (UUO) model of renal fibrosis (4.1) Mouse IL28B delivered by hydrodynamic transfection (HDT) UUO kidneys from animals receiving the control SEAP vector via HDT showed tubulointerstitial fibrosis in the cortex typical of this model at 21 days after surgery, with extensive expansion of the tubular basement membrane, intense collagen staining by the basement membrane, especially within the medulla, extensive loss of epithelial cell volume, tubular atrophy, loss of the proximal tubule brush border, and extensive tubulointerstitial infiltrates. Less than 15% of the tubules had a near-normal structure. There were minimal changes to the glomeruli, but the medulla was virtually destroyed and absent (Figure 21(a)).

[0132] Both UUO kidneys of animals receiving HDT with murine IL-28B 1 day before UUO and 7 days after UUO showed protection from cortical tubulointerstitial fibrosis at 21 days after surgery (Figure 21(a)). Tubular base enlargement and collagen staining were at least 30% or 40% lower in both groups receiving murine IL-28B with HDT compared to UUO mice receiving control vector. Both IL28B groups preserved tubular architecture and had less epithelial flattening and atrophy. Those with HDT from 1 day before HDT had 30-40% of tubules with near normal architecture and those with subsequent treatment had slightly less (20-30%). Interstitial infiltration was approximately 40% in both IL-28 groups compared to the control vector group. Neither IL28 group had significant protection of medullary architecture. Overall, both early and late delivery of IL28B by HDT had significant protection from UUO-induced fibrotic remodeling in the renal cortex.

[0133] Picrosirius Red (PSR) stained collagen sections were quantified using the definiens image analysis algorithm and show a statistically significant protective effect of murine IL-28B delivered on either day -1 or day 7 when plotted as either total collagen area (Figure 21(b)) or high intensity collagen area (Figure 21(c)) compared to control plasmid treated mice, consistent with the visual histological assessment of stained sections described above.

[0134] Serum levels of murine IL28B protein achieved 21 days after HDT in the murine UUO model were measured by ELISA and ranged from 43 to 485 ng / ml across all groups. No murine IL28B protein was measurable in control SEAP HDT mice, and therefore the levels were below the lower limit of quantification (LLQ) of the assay used, 4 ng / ml.

[0135] (4.2) Human IL-29 delivered by hydrodynamic transfection (HDT) Human IL-29 delivered by HDT shows significant protein expression in all mice both at day 0 (24 hours post-transfection) and at day 19 when the model was terminated compared to the SEAP control plasmid treated group (Figure 23(a)). No human IL-29 was detectable in the serum of non-transfected or SEAP control mice.

[0136] Untreated 19-day UUO shows histological changes typical of this model. In all of the kidneys, there is extensive destruction and loss of renal medulla. The cortex shows extensive expansion of the tubular basement membrane with significant interstitial collagen accumulation as shown by increased picrosirius red (PSR) staining. There is also marked infiltration of cells into the interstitium throughout the cortex. The tubular epithelial architecture is severely damaged. Most of the epithelial cells appear to have lost volume and are generally flattened, including a few tubules with dilated lumens. Notably, over 80% of the tubules have undergone extensive atrophy and in many cases are completely collapsed. The tubular lumen is difficult to distinguish in most cases due to loss of patency, and epithelial cells are clearly detached from the nephron tubule basement membrane and are present within what would otherwise be the lumen.

[0137] 19-day UUO kidneys treated with IL-29 from the time of UUO have a dramatically different appearance than those that received only the SEAP vector, with clearly preserved structure. The medulla remains present, although damaged, with many recognizable structures compared to untreated UUO in 75% of animals. In the cortex, the main observation is a large number of tubules with dilated tubule lumens compared to SEAP UUO, with a lower level of expansion. However, where this would generally be associated with the highly flattened epithelial cells and expansion of the tubular basement membrane in untreated UUO, with IL-29 treatment, the epithelial cells retain volume, there is minimal expansion of the tubular basement membrane, and the tubular architecture is significantly preserved, with little or no evidence of atrophy and tubular collapse. There is clearly a lower level of interstitial collagen and reduced expansion of the tubular basement membrane. Overall, application of IL-29 significantly reduced tubulointerstitial fibrosis caused by UUO, accompanied by a reduction in interstitial collagen and a dramatic preservation of tubular architecture as shown in the representative images in Figure 23(b).

[0138] All picrosirius red (PSR) stained collagen sections were quantified using the definiens image analysis algorithm and plotted as total stained collagen area compared to control SEAP plasmid treated mice, showing a statistically significant protective effect of human IL-29 delivered on day -1 (Figure 23(c)), consistent with the visual histological assessment of stained sections described above.

Claims

1. 1. A pharmaceutical composition for use in the treatment of fibrosis comprising a recombinant protein comprising the amino acid sequence of SEQ ID NO:1, or an anti-fibrotic fragment thereof, said anti-fibrotic fragment comprising more than 150 amino acids of SEQ ID NO:1, said fibrosis being renal fibrosis, pulmonary fibrosis, small intestinal fibrosis, skin fibrosis or liver fibrosis, said fibrosis not being associated with hepatitis virus infection.

2. The pharmaceutical composition of claim 1 , wherein the recombinant protein or fragment thereof is pegylated.

3. The pharmaceutical composition of claim 2 , wherein the recombinant protein is a fusion protein.

4. 4. The pharmaceutical composition of claim 3, wherein the recombinant protein comprises the amino acid sequence of SEQ ID NO:1, or an anti-fibrotic fragment thereof, comprising more than 150 amino acids of SEQ ID NO:1, fused to an Fc domain of an antibody.

5. The pharmaceutical composition according to claim 1 , wherein the fibrosis is renal fibrosis.

6. The pharmaceutical composition according to claim 1 , wherein the fibrosis is pulmonary fibrosis.

7. The pharmaceutical composition according to claim 1 , wherein the fibrosis is small intestinal fibrosis.

8. The pharmaceutical composition according to claim 1 , wherein the fibrosis is dermal fibrosis.

9. The pharmaceutical composition according to claim 1 , wherein the fibrosis is liver fibrosis.

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