Compositions and methods for treating or preventing fibrosis

Abstract

The present application relates to compositions and methods comprising polypeptides derived from MFG-E8 for the treatment and prevention of fibrotic diseases and other diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Korean Application No. 10-2018-0128033 / KR, filed October 25, 2018, Korean Application No. 10-2018-0128625 / KR, filed October 26, 2018, and Korean Application No. 10-2018-0128204 / KR, filed October 25, 2018, the entire contents of each of which are incorporated herein by this reference in their entirety. [Background technology]

[0002] The process of tissue repair as part of wound healing has two phases. The first phase is the regenerative phase, which replaces damaged cells with the same type of cells. The second phase is the formation of fibrous tissue, also called desmoplasia or fibrosis, in which connective tissue replaces normal parenchymal tissue. If the fibrosis phase continues unchecked, the tissue repair process can become pathogenic, resulting in extensive tissue remodeling and the formation of permanent scar tissue.

[0003] Liver fibrosis is characterized by the excessive accumulation of extracellular matrix proteins such as collagen. In the progression of liver fibrosis, damaged and dead hepatocytes due to liver injury recruit Kupffer cells to their lesion sites. These Kupffer cells secrete large amounts of cytokines, such as transforming growth factor β1 (TGF-β1), to control liver inflammation. Elevated TGF-β1 triggers the activation of quiescent hepatic stellate cells (HSCs), which proliferate and become myofibroblast-like cells that produce extracellular matrix (ECM). Activated HSCs further accumulate excess collagen-rich ECM in the liver, resulting in distortion of normal liver architecture.

[0004] Liver fibrosis results from chronic inflammatory liver disease or recurrent liver injury. Causes of recurrent liver damage and fibrosis include viral infections (hepatitis B and C), alcohol abuse, and non-alcoholic steatohepatitis (NASH). Liver fibrosis can progress to cirrhosis and cancer, which has a higher mortality rate than other major cancers (lung, colon, gastric, or breast cancer). Therefore, liver fibrosis remains a major cause of death with few therapeutic strategies, and there is a great need for treatments to reduce and prevent fibrosis. Summary of the Invention

[0005] The present invention is based, at least in part, on the discovery that a fragment of MFG-E8, hereinafter NP-011, is surprisingly effective in treating and / or preventing diseases such as, but not limited to, fibrosis, cirrhosis, steatosis, and non-alcoholic steatohepatitis (NASH).

[0006] In certain embodiments, provided herein are polypeptides for treating and / or preventing diseases, including, but not limited to, fibrosis, cirrhosis, steatosis, and NASH. In some embodiments, the polypeptide comprises an MFG-E8 polypeptide comprising an epidermal growth factor (EGF)-like domain, a C1 domain, and optionally a signal peptide, but lacking a functional C2 domain. In some embodiments, the polypeptide comprises an MFG-E8 polypeptide comprising an epidermal growth factor (EGF)-like domain, a C1 domain, and optionally a signal peptide, but lacking a medin polypeptide or a fragment thereof.

[0007] In certain embodiments, the MFG-E8 polypeptide lacks at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 amino acids within a C-terminal domain that includes amino acids 226 to 335 of the MFG-E8 polypeptide. In some embodiments, the MFG-E8 polypeptide contains at least 180, 190, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, or 280 amino acids of the MFG-E8 polypeptide. In some embodiments, the MFG-E8 polypeptide comprises at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% of amino acids 1-225 of SEQ ID NO:10 or SEQ ID NO:12. In some embodiments, the MFG-E8 polypeptide comprises at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% of amino acids 24-225 of SEQ ID NO:10 or SEQ ID NO:12.

[0008] In a preferred embodiment, the MFG-E8 polypeptide is aglycosylated.

[0009] In certain embodiments, the polypeptide further comprises a heterologous sequence, such as a FLAG tag, a HIS tag, and / or an Fc portion of an immunoglobulin. Such heterologous sequences can increase the half-life of the polypeptide in vivo.

[0010] In some embodiments, the polypeptides enable one or more biological activities including, but not limited to, a decrease in the expression level of TGF-β; a decrease in TGF-β signaling; a decrease in phosphorylation of SMADs, e.g., SMAD2, ERK, and / or any other phosphorylated proteins downstream of TGF-β; an increase in NOTCH signaling; a decrease in fibrosis-related gene expression, such as, but not limited to, Col1a1, Col1a2, or Acta2; a decrease in the interaction between TGF-β and one or more integrins, e.g., integrin β3 and / or integrin β5; a decrease in hepatic stellate cell (HSC) proliferation; a decrease in the expression levels of matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 12 (MMP12), TMP2, ERK, and / or SMAD2; an increase in collagenase activity; and / or an increase in collagen uptake by macrophages.

[0011] In some embodiments, the polypeptide is in a pharmaceutical composition comprising the polypeptide and one or more pharma- ceutically acceptable carriers and / or diluents.

[0012] Also provided herein are kits that include the polypeptides.

[0013] In certain embodiments, provided herein is an isolated nucleic acid molecule encoding the above polypeptide. In some embodiments, the nucleic acid comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to nucleotides 61-735 of SEQ ID NO:9 or SEQ ID NO:11. In some embodiments, the nucleic acid comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to nucleotides 130-735 of SEQ ID NO:9 or SEQ ID NO:11. Also provided herein is a vector comprising the nucleic acid molecule, optionally the vector is an expression vector or a viral vector. In some embodiments, the nucleic acid molecule is operably linked to a promoter and / or other regulatory sequences. In some embodiments, the nucleic acid molecule and / or vector is in a pharmaceutical composition suitable for administration to a subject. Such pharmaceutical compositions may include one or more agents (e.g., liposomes, polymers) that aid in endocytosis and / or longer half-life in vivo and / or reduce immune response to the composition in a subject.

[0014] In certain embodiments, the present specification provides a viral particle comprising the above-mentioned vector.In some embodiments, the viral particle is AAV.In certain embodiments, the AAV comprising the viral vector is administered to a subject, for example, a human, to treat and / or prevent disease, for example, fibrosis, liver cirrhosis, steatosis, NASH, myocardial infarction, pulmonary fibrosis, idiopathic pulmonary fibrosis and / or Alzheimer's disease.

[0015] In certain embodiments, provided herein is a host cell comprising the vector. In some such embodiments, the host cell is a mammalian cell. In other embodiments, the host cell is a yeast cell, such as Pichia pastoris.

[0016] In certain aspects, provided herein are methods of producing a polypeptide by culturing a host cell in a medium, e.g., the host cell secretes the polypeptide into the medium, hi some embodiments, the method further comprises isolating the polypeptide from the medium and, optionally, further purifying the polypeptide to produce a substantially pure polypeptide.

[0017] In some aspects, provided herein is a method of reducing or inhibiting fibrosis in a subject by contacting cells of the subject with a polypeptide. Also provided herein is a method of reducing or inhibiting proliferation of HSCs by contacting the HSCs with a polypeptide. Further provided herein is a method of reducing or inhibiting steatosis in a subject by contacting cells of the subject with a polypeptide. In some embodiments, the subject in these methods is a mammal, such as a rat, a mouse, or a human, preferably a human.

[0018] In certain aspects, provided herein are methods of increasing macrophage activity (e.g., uptake of collagen or fibrous tissue) by contacting macrophages with a polypeptide, which increase in macrophage activity contributes to the reversal of fibrosis in various tissues (e.g., liver, lung, etc.).

[0019] In certain aspects, provided herein are methods of treating or preventing a disorder in a subject in need thereof by administering a polypeptide to the subject. In some embodiments, the disorder is fibrosis (chronic or acute), liver cirrhosis, steatosis, NASH, and / or pulmonary fibrosis.

[0020] In some embodiments, the polypeptide of the present disclosure treats or prevents idiopathic pulmonary fibrosis (IPF). In addition to reducing or eliminating fibrotic tissue, the polypeptide may also modify the expression level of IPF biomarkers. In some cases, the polypeptide may reduce the expression level of at least one biomarker selected from αSMA, collagen (Col1a1), TMP2, MMP2, MMP12, phosphorylated ERK, ERK, phosphorylated SMAD2, and SMAD2.

[0021] In other embodiments, the polypeptides of the present disclosure treat or prevent myocardial infarction. The polypeptides can improve or increase cardiac function after myocardial infarction, for example, compared to the improvement seen in corresponding untreated subjects. For example, the polypeptides can increase left ventricular ejection fraction and / or fractional shortening. Furthermore, the polypeptides can inhibit or reduce fibrosis associated with myocardial infarction.

[0022] The present invention also contemplates a method of treating or preventing Alzheimer's disease (AD). The subject may have a genetic mutation associated with AD. For example, the subject may have (i) at least one mutation in amyloid precursor protein (APP) selected from K670N / M671L, I716V, and V717I, and / or (ii) at least one mutation in PSEN1 selected from M146L and L286V. The polypeptides of the present disclosure may improve memory loss and / or behavior associated with Alzheimer's disease. Furthermore, the polypeptides may modify physiological and histopathological changes in the brain. For example, the polypeptides may reduce the amount of amyloid plaques, the amount of amyloid beta, the number of microglia, the level of neuroinflammation (or brain inflammation), and / or the amount of glial fibrillary acidic protein (GFAP) in the subject's brain, preferably in the hippocampus and / or brain cortex of the subject's brain.

[0023] The subject can also be treated with an additional agent to treat the disorder.

[0024] In some embodiments, the method does not induce amyloid formation in the subject.

[0025] The polypeptide can be administered to the subject via any of a variety of routes, for example, intravenous, subcutaneous, intraarterial, intraperitoneal, or intramuscular.

[0026] In some embodiments, the subject in these methods is a mammal, such as a rat, a mouse, or a human, preferably a human. [Brief description of the drawings]

[0027] [Figure 1A] Figure 1A-1F show that NP-011 inhibits liver fibrosis and the benefits of NP-011 for treating liver disease. (Figure 1A) Overall schematic timeline for testing the efficacy of human recombinant protein in liver fibrosis models. [Figure 1B] (FIG. 1B) Representative images of histological analysis (H&E and Sirius red staining) of normal mouse liver, TAA-induced liver fibrosis model (sham), and liver fibrosis models administered proteins (MFG-E8, NP-011, NP-012, NP-013). Scale bar, 200 μm. [Figure 1C-D] (FIG. 1C) Comparison of quantitative fibrotic area in mouse liver. Bar graphs represent the mean ± SD from three replicates in each group. *P<0.05, **P<0.01. (FIG. 1D) Comparison of mRNA expression of fibrotic markers (Col1a1, Col1a2, and Acta2) in livers of normal mice, TAA-induced liver fibrosis model (sham), and liver fibrosis models administered proteins (MFG-E8, NP-011, NP-012, and NP-013). Bar graphs represent the mean ± SD from three replicates in each group. *P<0.05, **P<0.01. [Figure 1E-F](Fig. 1E) Luciferase reporter assay to confirm TGF-β signaling pathway transcription activity from human HEK-293FT cells after treatment with NP-011 (500 ng / mL) or NP-013 (500 ng / mL) for 0.5, 1, and 2 hours. Bar graphs represent the mean ± SD from three replicates in each group. *P<0.05, **P<0.01. (Fig. 1F) Gene Set Enrichment Assay (GSEA) data showed significant enrichment of Notch signaling gene sets in the NP-011-treated group, but not in the MFG-E8-treated group, compared with the sham group. [Figure 2A-C] 2A-2F show that NP-011 inhibits fibrosis at low doses. Abolition of fibrosis by NP-011 at low doses. (Fig. 2A) Overall schematic schedule for testing the efficacy of NP-011 in liver fibrosis models. (Fig. 2B) Representative images comparing fibrotic areas (Sirius red stained areas) in the livers of normal mice, TAA-induced liver fibrosis models, and liver fibrosis models administered NP-011 at various dose ranges (20 μg / kg-160 μg / kg). Scale bar, 200 μm. (Fig. 2C) Quantitative analysis of fibrotic areas in the livers of test mice in Fig. 2B. Bars indicate mean ± SD of 4 mice per group. **P<0.01. [Fig. 2D-F] (Fig. 2D) Comparison of α-SMA (Acta2) expression in the livers of normal mice, TAA-induced liver fibrosis models, and NP-011-treated liver fibrosis models by quantitative PCR and immunostaining. Scale bar, 100 μm. Bar graphs represent the mean ± SD from three replicates in each group. **P<0.01. (Fig. 2E) Heat map data show fibrosis-related genes observed in the livers of normal mice, TAA-induced mice, and NP-011-treated liver fibrosis mice. (Fig. 2F) Comparison of TGF-β1 mRNA expression in advanced liver fibrosis models. Bar graphs represent the mean ± SD from three replicates in each group. **P<0.01. [Figure 3A-C]3A-3E are diagrams showing that NP-011 inhibits and prevents liver fibrosis. Therapeutic effects of the minimum dose of NP-011 in various model inductions. (FIG. 3A) Overall schematic schedule for testing the efficacy of NP-011 in a progressive liver fibrosis model. (FIG. 3B) Representative images for analyzing the fibrotic area of ​​the liver of normal mice, TAA-induced liver fibrosis model, and 40 μg / kg NP-011-administered liver fibrosis model with different administration frequencies (1, 2, 4, and 6 times). Scale bar, 50 μm. Quantitative analysis of the fibrotic area of ​​the liver of normal mice, TAA-induced liver fibrosis model, and 40 μg / kg NP-011-administered liver fibrosis model is shown in the left panel. Bars indicate the mean value ± SD of 4 mice in each group. **P<0.01. (FIG. 3C) Overall schematic schedule for testing the efficacy of NP-011 in a progressive liver fibrosis model. [Fig. 3D-E] (Fig. 3D) Representative images for analyzing the fibrotic area of ​​the liver in normal mice, TAA-induced liver fibrosis model, and TAA-injected liver fibrosis model with co-administration of NP-011. Scale bar, 100 μm. Quantitative analysis of the liver fibrotic area in normal mice, TAA-induced liver fibrosis model, and TAA-injected liver fibrosis model with co-administration of NP-011. Bars indicate the mean ± SD of 5 mice in each group. **P<0.01. (Fig. 3E) Left panel: Graphic description represents the human liver fibrosis model using human embryonic stem cell (hES)-derived hepatocytes and human primary HSCs treated with APAP. Center panel: Representative images for analyzing hepatocytes and activated myofibroblasts with ALB (albumin, red) and α-SMA (a biomarker of fibrosis, Acta2, green). Scale bar, 50 μm. Right panel: Quantitative analysis of the percentage of α-SMA-positive cells in the human liver fibrosis model. Bar graphs represent the mean ± SD from five replicates for each group. **P<0.01. [Figure 4A-C]Figures 4A-4D show that NP-011 inhibits and prevents liver cirrhosis. Efficacy study of DMN-induced liver cirrhosis model in rats. (Figure 4A) Schematic diagram of efficacy study of NP-011 in DMN-induced liver cirrhosis model. (Figure 4B) Survival curve of DMN-induced liver cirrhosis model is shown for 10 cases per group. (Figure 4C) Representative images of H&E staining and immunohistochemical analysis of rat liver tissue labeled with alpha smooth muscle actin. [Figure 4D] (FIG. 4D) Quantification scores of fibrosis area, segmental necrosis, and lobular necrosis, and quantification of α-smooth muscle actin positive area in the livers of normal, DMN-induced cirrhosis model, and NP-011-treated cirrhosis model. [Figure 5A-C] Figures 5A-5D show that NP-011 inhibits and prevents NASH. Efficacy study in MCD diet NASH model. (Figure 5A) Overall schematic diagram of efficacy study of NP-011 in MCD diet NASH model. (Figure 5B) Representative images of Oil Red O staining, and (Figure 5C) quantification of relative Oil Red O positive area in liver of normal mice, MCD diet NASH model, and NP-011-treated hepatic NASH model show significant therapeutic effect of secreted NP-011. [Figure 5D] (FIG. 5D) Quantification of the extent of macrodroplet and microdroplet steatosis and hypertrophy in the liver of each group. Scale bar, 100 μm. Bars indicate the mean ± SD of five mice per group. **P<0.01, ANOVA followed by Tukey's multiple comparison test. [Figure 6] Figure 6 shows the interaction of NP-011 with activated HSCs and macrophages. Representative images of immunostaining for human MFG-E8 (NP-011), mouse α-SMA, and mouse F4 / 80 (a macrophage marker) in liver tissue from a TAA-induced liver fibrosis model treated with NP-011. Scale bar = 20 μm. [Figure 7A-B]7A-7D show that NP-011 inhibits the proliferation of HSCs. Effects of NP-011 on HSCs. (FIG. 7A and FIG. 7B) PLA assay to study the physical interaction between TGFBRI and integrin αvβ3 / αvβ5. Red signals (white arrowheads) indicate the interaction of TGFBRI with integrin β3 (FIG. 7A) and β5 (FIG. 7B). Quantitative analysis of the number of red signals in each cell. Bar graphs represent the mean ± SD from five replicates of each group. *P<0.05, **P<0.01. [Figure 7C-D] (FIG. 7C) Western blot (WB) and immunoprecipitation (IP) analysis of the regulation of TGF-β signaling in human HSCs in the presence of TGF-β1 (10 ng / mL) and / or NP-011 (500 ng / mL) and the physical interaction of TGFBRI with integrin β3 / β5. (FIG. 7D) EdU incorporation assay for cell proliferation of human HSCs in the presence of TGF-β1, NP-011, and / or cilengitide trifluoroacetate (CT, an inhibitor of integrins νβ3 and νβ5). Bar graphs represent the mean ± SD from five replicates for each group. **P<0.01. [Figure 8] 8A-8C show that NP-011 decreases MMP2 expression and increases collagenase activity. Regulation of profibrotic MMP2 and collagenase activity in HSCs. (FIG. 7A) Comparison of MMP2 mRNA expression in human HSCs in the presence of TGF-β1 and NP-011. Bar graphs represent the mean ± SD from three replicates in each group. **P<0.01. (FIGS. 7A and 7B) Collagenase activity assay of cell lysates (FIG. 7B) and conditioned media (FIG. 7C) from cultures of control HSCs and TGF-β1-treated HSCs with and without NP-011 treatment. Bar graphs represent the mean ± SD from three replicates in each group. **P<0.01. [Figure 9A-C]9A-9E show that NP-011 increases macrophage-mediated collagen uptake. (FIG. 9A) Overall schematic timeline of macrophage differentiation from monocytes by treatment with PMA. (FIG. 9B) Representative images of differentiated macrophages (upper panel) and FITC-labeled bead uptake by differentiated macrophages (lower panel). Scale bar=10 μm (FIG. 9C). Flow cytometry results showed that FITC beads were taken up by differentiated macrophages. [Fig. 9D-E] (FIG. 9D) Graphical depiction of fibrillar collagen uptake assay by differentiated macrophages with / without NP-011. (FIG. 9E) Quantitative analysis of relative CUI (collagen uptake index) for control and NP-011 treatment. Bar graphs represent mean values ​​± SD from three replicates in each group. **P<0.01. [Figure 10] Figure 10 shows that NP-011 interacts with activated HSCs and macrophages. Representative images of immunostaining for mouse collagen and mouse F4 / 80 (a marker for macrophages) in liver tissues from a TAA-induced liver fibrosis model and a TAA-induced liver fibrosis model administered NP-011. Arrowheads indicate collagen engulfment by macrophages (F4 / 80 positive cells). [Figure 11A] 11A-11C show the biodistribution and safety profile of NP-011. (FIG. 11A) Organ distribution 30 and 60 minutes after intravenous injection of NP-011 (160 μg / kg) in healthy male mice. [Figure 11B-C] (FIG. 11B and FIG. 11C) Quantitative analysis of inflammation-related cell counts and blood biochemistry in male (FIG. 11B) and female (FIG. 11C) rats was compared between normal and the two NP-011-treated groups. [Figure 12] FIG. 12 is a diagram of a model of idiopathic pulmonary fibrosis (IPF). [Figure 13] FIG. 13 shows images of lung tissue stained to show collagen and αSMA distribution on days 3 (D3), 5 (D5), 7 (D7), and 14 (D14) after bleomycin administration. [Figure 14] FIG. 14 shows changes in expression levels of IPF biomarkers (Col1a1, MMP2, MMP12, and TIMP1) on days 3 (D3), 5 (D5), 7 (D7), and 14 (D14) after bleomycin administration. [Figure 15] Figure 15 shows the change in expression levels of subfactors of IPF-related signaling pathways at days 3 (D3), 5 (D5), 7 (D7), and 14 (D14) after bleomycin treatment. The subfactors include αSMA, pERK, tERK, pSMAD2 (phosphorylated SMAD2), and tSMAD2 (total SMAD2). Actin is shown as a loading control. [Figure 16] Figures 16A-B show diagrams of a model of IPF for testing the in vivo efficacy of NP-011. Two cases with different time points are presented: Case 1 (Figure 16A) and Case 2 (Figure 16B). [Figure 17] Figure 17 shows that NP-011 is effective against IPF. After NP-011 injection, lung tissue was stained to show the distribution of collagen and αSMA. Mice were injected with NP-011 on day 3 (Case 1 in Figure 16A). [Figure 18] Figure 18 shows that NP-011 is effective against IPF. After NP-011 injection, lung tissue was stained to show the distribution of collagen and αSMA. Mice were injected with NP-011 on day 5 (case 2 in Figure 16B). [Figure 19] Figure 19 shows that NP-011 is effective against IPF. NP-011 reverses bleomycin-induced changes in the expression levels of signaling pathway subfactors related to IPF. Mice were injected with NP-011 on day 3 (case 1 in Figure 16A). The subfactors include pERK, tERK, pSMAD2, and tSMAD2. Actin is shown as a loading control. The histogram in the right panel shows quantification of the expression levels of pSMAD2 normalized to actin levels. [Figure 20]Figure 20 shows that NP-011 is effective against IPF. NP-011 completely reverses bleomycin-induced changes in the expression levels of signaling pathway subfactors related to IPF. Mice were injected with NP-011 on day 5 (case 1 in Figure 16B). The subfactors include αSMA, pERK, and tERK. Actin is shown as a loading control. The histogram in the right panel shows the quantification of the expression levels of pERK normalized to actin levels. [Figure 21] Figure 21 shows that NP-011 is effective against IPF. NP-011 alters the expression levels of IPF biomarkers (e.g., collagen (Cola1), MMP2, MMP12, and TIMP1 as shown herein). Specifically, NP-011 reverses the bleomycin-induced increase in the expression levels of IPF biomarkers. [Figure 22] FIG. 22 shows a diagram of the LAD ligation model for myocardial infarction. The left anterior descending artery (LAD) of a rat is ligated with a single suture, creating an almost immediately visible ischemia. By closing the LAD, no further blood flow is allowed in the area, but the surrounding myocardial tissue is largely unaffected. This surgical procedure mimics the pathobiological and pathophysiological aspects that occur in infarction-related myocardial ischemia (e.g., Kolk et al. (2009) J Vis Exp, 21:pii 1438). In addition to the LAD ligation model, FIG. 22 also presents the details of the study shown in FIG. 23-FIG. 26. [Figure 23A] Figures 23A-C show that NP-011 improves symptoms of myocardial infarction and increases cardiac function. Left ventricular ejection fraction (EF) and fractional shortening (FS) measurements are shown for mice 2 weeks (Figure 23A), 4 weeks (Figure 23B), or 8 weeks (Figure 23C) after LAD ligation. Measurements are shown for mice treated with none (AMI, acute myocardial infarction), PBS (phosphate buffered saline), MFG-E8, or NP-011. [Figure 23B]Left ventricular ejection fraction (EF) and fractional shortening (FS) measurements are shown for mice 2 (FIG. 23A), 4 (FIG. 23B), or 8 (FIG. 23C) weeks after LAD ligation. Measurements are shown for mice treated with none (AMI, acute myocardial infarction), PBS (phosphate buffered saline), MFG-E8, or NP-011. [Figure 23C] Left ventricular ejection fraction (EF) and fractional shortening (FS) measurements are shown for mice 2 (FIG. 23A), 4 (FIG. 23B), or 8 (FIG. 23C) weeks after LAD ligation. Measurements are shown for mice treated with none (AMI, acute myocardial infarction), PBS (phosphate buffered saline), MFG-E8, or NP-011. [Figure 24] Figures 24A-B show that NP-011 improves symptoms of myocardial infarction and increases cardiac function. A time course analysis of the heart from day 1 to day 56 after LAD ligation is shown. In particular, cardiac functional improvement is assessed by measuring left ventricular ejection fraction (EF, Figure 24A) and fractional shortening (FS, Figure 24B). Unlike MFG-E8, NP-011 shows significant improvement in cardiac function over time. [Diagram 25] 25 shows the change in body weight over time. No significant change in body weight is observed from day 1 to day 56. [Figure 26] Figures 26A-B show that NP-011 ameliorates symptoms of myocardial infarction. NP-011 reverses fibrosis associated with myocardial infarction. Figure 26A shows images of H&E stained cross sections of the hearts of rats treated with none (AMI), PBS, MFG-E8, or NP-011. Figure 26B shows measurements of the fibrotic area compared to the total cross-sectional area of ​​the heart from Figure 26A. [Figure 27A]Figures 27A-27E show that NP-011 reverses behavior and memory loss associated with Alzheimer's disease. Figure 27A shows a diagram of Alzheimer's disease (AD) model 5XFAD mice and details of in vivo testing. 5XFAD mice express human APP and PSEN1 transgenes with a total of five AD-associated mutations: Swedish (K670N / M671L), Florida (I716V), and London (V717I) mutations in APP, and M146L and L286V mutations in PSEN1. NP-011 was injected into 5XFAD mice, followed by behavior and memory testing. [Fig. 27B-C] Figure 27B shows the behavioral analysis of the AD model immediately after NP-011 injection (3.5 months after the start of the study, as shown in Figure 27A). Figure 27C shows the behavioral analysis of the AD model 3 months after NP-011 injection (6.5 months after the start of the study, as shown in Figure 27A). [Fig. 27D-E] Figure 27D shows the results of the passive avoidance test in the AD model immediately after injection of NP-011 (3.5 months after the start of the study, as shown in Figure 27A). Figure 27E shows the results of the passive avoidance test in the AD model 3 months after injection of NP-011 (6.5 months after the start of the study, as shown in Figure 27A). [Figure 28] Figures 28A-B show that NP-011 reverses neuropathological changes in Alzheimer's disease brains. Specifically, NP-011 reduces the amount of amyloid plaques in AD. Figure 28A shows the hippocampus and cortex of an AD brain stained with thioflavin S to detect amyloid plaques, and Figure 28B shows the quantification of amyloid plaques stained by thioflavin S. [Figure 29] Figure 29A-B shows that NP-011 reduces the amount of amyloid beta in Alzheimer's disease. Figure 29A shows the hippocampus and cortex of AD brain stained with 6E10 antibody that specifically recognizes amyloid beta, and Figure 29B shows the quantification of amyloid beta in the hippocampus and cortex of AD brain. [Diagram 30]Figures 30A-B show that NP-011 reduces microglial cell numbers in Alzheimer's disease. Figure 30A shows the hippocampus and cortex of AD brains stained with IBA-1 antibody that recognizes microglia, and Figure 30B shows quantification of microglial cells in the hippocampus and cortex of AD brains. [Diagram 31] Figures 31A-B show that NP-011 reduces the amount of glial fibrillary acidic protein (GFAP) upregulated in Alzheimer's disease astrocytes. Figure 31A shows the hippocampus and cortex of AD brains stained for GFAP, and Figure 30B shows quantification of GFAP in the hippocampus and cortex of AD brains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present invention relates, in part, to compositions and methods for treating and / or preventing diseases associated with fibrosis.

[0029] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. The headings provided herein are not limitations of the various embodiments, but may be had by reference to the specification as a whole. Additionally, the terms defined immediately below are more fully defined by reference to the entire specification.

[0030] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an" element means one element or more than one element.

[0031] The terms "about" and "approximately" used in reference to numerical values ​​throughout the specification and claims indicate an interval of accuracy that is familiar and acceptable to those skilled in the art. Typically, such an interval of accuracy is ±10%. Alternatively, particularly in biological systems, the terms "about" and "approximately" can mean values ​​within an order of magnitude of a given value, preferably ≦5-fold, and more preferably ≦2-fold.

[0032] The term "coding region" refers to the region of a nucleotide sequence that contains codons that are translated into amino acid residues, while the term "non-coding region" refers to the regions of a nucleotide sequence that are not translated into amino acids (e.g., the 5' and 3' untranslated regions).

[0033] The term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region can form specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region that is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand can base pair with a residue of a second nucleic acid strand that is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or different nucleic acid if at least one nucleotide residue of the first region can base pair with a residue of the second region when the two regions are arranged antiparallel. Preferably, the first region comprises a first portion and the second region comprises a second portion such that when the first and second portions are arranged in antiparallel, at least about 50%, preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues of the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues of the second portion.

[0034] The term "treating" includes prophylactic and / or therapeutic treatments. The term "prophylactic or therapeutic" treatment is art-recognized and includes administration of one or more compositions of the invention to a host. When administered prior to clinical manifestation of an undesirable condition (e.g., a disease or other undesirable condition in a host animal), the treatment is prophylactic (i.e., protects the host from the onset of the undesirable condition), whereas when administered after manifestation of the undesirable condition, the treatment is therapeutic (i.e., intended to reduce, ameliorate, or stabilize an existing undesirable condition or its side effects).

[0035] The term "preventing," when used in reference to a condition, such as local recurrence (e.g., pain), a disease, such as cancer, a complex syndrome, such as heart failure, or any other medical condition, is art-recognized and well understood in the art and includes administration of a composition that reduces the frequency of or delays the onset of symptoms of a medical condition in a subject compared to subjects not receiving the composition. Thus, preventing cancer includes, for example, reducing the number of detectable cancerous growths in a patient population receiving a prophylactic treatment compared to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population compared to an untreated control population, for example, by a statistically and / or clinically significant amount.

[0036] An amount (e.g., of TGF-β expression, HSC proliferation, NOTCH signaling) is "higher" or "lower" or "increased" or "decreased" than a control amount if the amount is greater than the standard error of the assay used to assess the amount, and preferably at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% greater or greater than the control level, respectively. Such "significance" can be assessed from any desired or known point of comparison, such as a ratio of measurements after a particular treatment to before treatment (e.g., 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, etc.). Alternatively, the amount of a growth factor in a subject can be considered "significantly" higher or lower than a control amount if the amount is at least about 2-fold, 3-fold, 4-fold, or 5-fold higher or lower, respectively, than the normal amount of the biomarker. Such "significance" can also be applied to any other measured parameter described herein.

[0037] For example, a "therapeutically effective amount" of a polypeptide is an amount that can produce medically desirable results in a treated patient, e.g., reduction in fibrosis, steatosis, reduction in TGF-β expression, preferably in a human or non-human mammal, with an acceptable benefit:risk ratio.

[0038] The term "subject" refers to any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, non-human primates, domestic animals, livestock, rodents, and the like, which are the recipients of a particular treatment.

[0039] "Increased serum half-life" or "increased in vivo half-life" refers to a positive change in the circulating half-life of a modified biologically active molecule relative to its unmodified form. In some embodiments, serum half-life is measured by taking blood samples at various time points after administration of a biologically active molecule and determining the concentration of the molecule in each sample. By measuring the change in serum concentration over time, serum half-life can be calculated. By comparing the serum half-life of a modified molecule, e.g., a conjugated molecule, with the unmodified molecule, the relative increase in serum half-life or t1 / 2 can be determined.

[0040] A "kit" is any article of manufacture (e.g., a package or container) that includes at least one reagent, such as a polypeptide described herein. The kit may also include additional reagents, carriers, or diluents. The kit may be promoted, distributed, or sold as a unit for carrying out the method of the invention. The kit may include one or more reagents required to express a composition useful in the method of the invention. In certain embodiments, the kit may further include a reference standard. One of skill in the art can envision many such controls. The reagents in the kit may be provided in individual containers or as a mixture of two or more reagents in a single container. Additionally, materials describing the use of the composition in the kit may be included.

[0041] Numeric ranges are inclusive of the numbers defining the range.

[0042] Polypeptides and Representative Biological Activities Polypeptides provided herein for the treatment of various diseases include MFG-E8 polypeptides that contain an epidermal growth factor (EGF)-like domain, a C1 domain, and optionally a signal peptide, but lack a functional C2 domain and / or a medin polypeptide or fragment thereof. The MFG-E8 polypeptide may lack at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 amino acids within the C-terminal domain of MFG-E8, including amino acids 226-335. The MFG-E8 polypeptide can comprise at least 180, 190, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, or 280 amino acids. The MFG-E8 polypeptide can have at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 1-225 of SEQ ID NO:10. The polypeptide can also have at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% sequence identity to amino acids 2-225 of SEQ ID NO: 10. The MFG-E8 polypeptide can be of human origin or any ortholog, such as, for example, from mouse, rat, chimpanzee, horse, etc.

[0043] MFG-E8 is also called lactadherin. Representative human MFG-E8 cDNA and human BRD7 protein sequences are well known in the art and publicly available from the National Center for Biotechnology Information (NCBI). For example, four different human MFG-E8 isoforms are known. Human MFG-E8 isoform B (NP 0001108086.1) can be encoded by transcript variant 2 (NM 001114614.3), which lacks an alternative in-frame exon compared to variant 1. The resulting isoform B has the same N-terminus and C-terminus, but is shorter compared to isoform A. Human MFG-E8 isoform D (NP 001297248.1) is encoded by transcript variant 4 (NM 001310319.2), which lacks an alternative in-frame exon in the 5' coding region compared to transcript variant 1. The encoded isoform D has the same N-terminus and C-terminus, but is shorter compared to isoform A. Human MFG-E8 isoform C (NP 001297249.1) can be encoded by transcript variant 3 (NM 001310320.2), which contains an alternative internal exon that results in the use of an alternative translation start site compared to variant 1. The encoded isoform C has a shorter distinct N-terminus compared to isoform A. Human MFG-E8 isoform E (NP 001297250.1) can be encoded by transcript variant 5 (NM 001310321.2), which contains an alternative 5' terminal exon that results in the use of a downstream in-frame translation start site compared to variant 1. The encoded isoform E has a shorter N-terminus than isoform A. Human MFG-E8 isoform A (NP 005919.2) is encoded by transcript variant 1 (NM 005928.4), which encodes the longest isoform A.Nucleic acid and polypeptide sequences of MFG-E8 orthologues in organisms other than humans are known, e.g., chimpanzee MFG-E8 (XM 016927386.2 → XP 016782875.1; and XM 001165898.6 → XP 001165898.1), dog MFG-E8 (XM 022416924.1 → XP 022272632.1), horse MFG-E8 (XM 023650241.1 → XP 023506009.1; XM 023650222.1 → XP 023505990.1; and XM 023650232.1 → XP 023506000.1), pig MFG-E8 (XM 021098306.1 → XP 020953965.1; and XM 013996945.2 → XP 013852399.1), cat MFG-E8 (XM 011282749.2 → XP 011281051.1; and XM 011282750.2 → XP 011281052.1), sheep MFG-E8 (XM 027957063.1 → XP 027812864.1), goat MFG-E8 (XM 018065819.1 → XP 017921308.1), rat MFG-E8 (NM 001040186.2 → NP 001035276.1; and NM 012811.3 → NP 036943.1), and mouse MFG-E8 (NM 001045489.1 → NP 001038954.1; and NM 008594.2 → NP 032620.2).

[0044] An exemplary polypeptide is NP-011. NP-011 and other polypeptides described herein have multiple biological activities useful for inhibiting fibrosis. In some embodiments, the polypeptides described herein, such as NP-011, reduce the expression of multiple genes, such as TGF-β1, MMP2, and / or fibrosis-related genes, such as Col1a1, Col1a2, Acta2, etc.

[0045] In some embodiments, the polypeptides described herein modulate signal transduction pathways, for example, decreasing TGF-β signaling or increasing NOTCH signaling. TGF-β signaling controls a diverse set of cellular processes, including cell proliferation, recognition, differentiation, apoptosis, and developmental fate specification, during embryogenesis and in mature tissues in species ranging from flies and nematodes to mammals. TGF-β ligands initiate signal transduction by binding to serine / threonine kinases of type I and type II receptors and bringing them to the cell surface. This allows receptor II to phosphorylate the receptor I kinase domain, which transmits the signal via phosphorylation of Smad proteins. Activated Smad complexes translocate into the nucleus and, together with other nuclear cofactors, regulate the transcription of target genes.

[0046] TGF-β and its signaling pathway regulate cell proliferation. In the progression of liver fibrosis, damaged and dead hepatocytes due to liver injury recruit Kupffer cells to the lesion site. These Kupffer cells secrete large amounts of cytokines, such as transforming growth factor β1 (TGF-β1), to control liver inflammation. Elevated TGF-β1 causes activation of quiescent HSCs, which proliferate and become extracellular matrix (ECM)-producing myofibroblast-like cells. Activated HSCs further accumulate excess collagen-rich ECM in the liver, resulting in distortion of normal liver architecture. Thus, decreasing the expression of TGF-β1 or decreasing TGF-β signaling inhibits HSC activation and / or proliferation, leading to inhibition of fibrosis. Similarly, NOTCH signaling is important for liver regeneration, and increased NOTCH signaling demonstrates the role of NP-011 in promoting liver regeneration.

[0047] The reduction of TGF-β signaling can be monitored through a number of methods known in the art, such as the expression levels of downstream target genes (e.g., ATF4, CDKN1A (p21CIP1, WAF1), CDKN1B (P27KIP1), CDKN2B (p15INK4b), COL1A1, COL1A2, DCN, EMP1, FOS, GADD45B, GSC, HERPUD1, IFRD1, IGF1, IGFBP3, IL6, JUN, JUNB, MYC, PDGFB, SERPINE1 (PAI-1), TGFB1I1, TNFSF10 (TRAIL), TSC22D1 (TGFB1I4), TGFBI, TGIF1) and / or the phosphorylation levels of downstream proteins (e.g., SMADs). Additionally, there are a number of commercially available kits that allow for monitoring of the TGF-β signaling pathway, e.g., Qiagen Cat# PAHS-035Z, PAMM-035Z, PARN-035Z, PAHS-235Z, PAMM-235Z, PARN-235Z, CRHS-00035Z-100, CRHS-00245Z-100, CRMM-00035Z-100, CRMM-00235Z-100, CCS-017L, CCS-017G, CLS-017L, EAHS-251Z, GH-035A, SEH00508A, SEM02991A. Similarly, the levels of downstream target genes (e.g., CDKN1A (p21CIP1, WAF1), CFLAR (Casper), FOSL1 (fra-1), ID1, IL2RA (CD25), NFKB1, PTCRA, CD44, ERBB2 (HER-2, NEU), DTX1, HES1, HES5, HEY1, HEY2, HEYL, JAG1, KRT1, LFNG, LOR, NOTCH1, PPARG, CHUK (IKKα), IFNG, IL17B, IL2RA (CD25), NFKB1, NFKB2, STAT6) and / or the levels of phosphorylation of downstream proteins can be monitored to assess the Notch signaling pathway.For example, there are a number of commercially available kits that allow for monitoring of the Notch signaling pathway, e.g., Qiagen Cat# PAHS-059Z, PAMM-059Z, PARN-059Z, CRHS-00059Z-100, CRMM-00059Z-100, CCS-014L, CCS-1014G, CLS-014L, CLS-014G, EAHS-611Z, GH-059A.

[0048] In some embodiments, as described above, the polypeptides described herein reduce phosphorylation of SMADs, proteins in the TGF-β signaling pathway.

[0049] In some embodiments, the polypeptides described herein disrupt and / or reduce the interaction between TGF-β receptor 1 (TGFBR1) and one or more integrins, such as integrin β3 and / or integrin β5.

[0050] In some embodiments, the polypeptides described herein reduce the proliferation of HSCs.

[0051] In some embodiments, the polypeptides described herein increase collagenase activity and / or collagen uptake by macrophages.

[0052] Amyloid formation Certain polypeptides provided herein lack the Medin polypeptide or a fragment thereof, which represents about 50 amino acids of the C2 domain. Medin is the main component of amyloid found in the aorta. Medin and amyloid are implicated in the pathogenesis of diseases such as Alzheimer's disease and type 2 diabetes. Repeated administration of a polypeptide containing a Medin polypeptide may increase the risk of inducing amyloid formation. Polypeptides provided herein lacking the Medin polypeptide or a fragment thereof reduce the risk of inducing amyloid formation.

[0053] Polypeptide Modifications The present invention further provides modified polypeptides in which the polypeptides described herein are fused in frame with a heterologous polypeptide and / or one or more chemical moieties. Such modifications may be useful, for example, to extend the serum half-life of the polypeptide, to serve as tags during protein purification, or to enhance immunogenicity during the production of antibodies against the polypeptide.

[0054] As used herein, a "chimeric protein" or "fusion protein" comprises all or a portion (preferably a biologically active portion) of a polypeptide of the invention linked to a heterologous polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the respective polypeptide. Within the fusion protein, the polypeptide of the invention and the heterologous polypeptide are preferably fused to each other in such a way that they maintain the function of each exhibited when expressed independently of the fusion. The heterologous polypeptide can be fused to the amino or carboxyl terminus of the polypeptide of the invention.

[0055] In some embodiments, the fusion protein comprises a heterologous signal sequence, an immunoglobulin fusion protein, a tag (e.g., FLAG, GST, etc.), a toxin, or other useful protein sequence. The chimeric and fusion proteins of the present invention can be produced by standard recombinant DNA techniques. In other embodiments, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that generate complementary overhangs between two consecutive gene fragments, followed by annealing and re-amplification to generate chimeric gene sequences. In addition, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the present invention can be cloned into such an expression vector such that the fusion moiety is linked in frame to the polypeptide of the present invention.

[0056] The heterologous peptide may optionally correspond to a moiety that modifies the solubility, affinity, stability or valency of the polypeptide (e.g., NP-011). For example, the polypeptide, e.g., NP-011, may be operably linked to an immunoglobulin constant region, e.g., a human C1 domain or a C4 domain (e.g., the hinge, CH2 and CH3 regions of human IgC1 or human IgC4, see, e.g., Capon et al., U.S. Pat. Nos. 5,116,964, 5,580,756, 5,844,095, which are incorporated herein by reference). Such constant regions may retain regions that mediate effector functions (e.g., Fc receptor binding) or may be modified to reduce effector functions. Such constant regions may also extend the serum half-life of the fusion protein. The resulting fusion protein may have modified solubility, binding affinity, stability and / or valency (i.e., the number of binding sites available per polypeptide) compared to the independently expressed first peptide, and may increase the efficiency of protein purification. In preferred embodiments, the fusion protein retains the biological activity of the polypeptide.

[0057] In other embodiments, the polypeptide may be covalently or non-covalently modified with one or more chemical moieties, such as polyethylene glycol (PEG), lipids, or PEG-modified lipids. Such modifications may, for example, increase the solubility, stability, and / or serum half-life of the polypeptide.

[0058] Treatment methods - exemplary conditions The compositions and methods provided herein are particularly useful for treating and / or preventing disorders, including those set forth below.

[0059] Liver fibrosis In liver fibrosis, excess connective tissue accumulates in the liver. This tissue scars in response to chronic and repeated hepatocellular injury. Generally, fibrosis progresses and destroys liver architecture, eventually leading to the regenerative hepatocytes functioning as they attempt to replace and repair the damaged tissue. When such destruction becomes widespread, the condition is diagnosed as cirrhosis.

[0060] Various types of chronic liver injury can cause fibrosis. Even fulminant, self-limited acute liver injury (e.g., acute viral hepatitis A) does not necessarily distort the scaffold structure and therefore does not cause fibrosis despite loss of liver cells. In the early stages, liver fibrosis may regress if the cause is reversible (e.g., with viral clearance). With chronic injury or repeated injury over months to years, fibrosis occurs permanently. With mechanical biliary obstruction, fibrosis progresses even more rapidly.

[0061] Activation of hepatic perivascular stellate cells (fat-storing Ito cells) initiates fibrosis. These cells and adjacent cells proliferate and become contractile cells called myofibroblasts. These cells produce excessive amounts of abnormal matrix (composed of collagen, other glycoproteins, and glycans) and matricellular proteins. Kupffer cells (resident macrophages), damaged hepatocytes, platelets, and leukocytes aggregate. As a result, reactive oxygen species and inflammatory mediators (e.g., platelet-derived growth factor, transforming growth factor, connective tissue growth factor) are released. Thus, stellate cell activation results in an abnormal extracellular matrix, both in quantity and composition.

[0062] Myofibroblasts stimulated by endothelin-1 contribute to increased portal resistance and increase the density of the abnormal matrix. Fibrous tracts connect to the branches of the afferent portal vein and efferent hepatic veins, bypassing hepatocytes and limiting their blood supply. Thus, fibrosis contributes to both hepatocellular ischemia (leading to hepatocellular dysfunction) and portal hypertension. The degree of ischemia and portal hypertension determines how the liver is affected. For example, congenital hepatic fibrosis affects the portal vein branches and primarily spares the parenchyma. The result is portal hypertension with sparing of hepatocellular function.

[0063] Cirrhosis Cirrhosis is the later stage of liver fibrosis, resulting in widespread distortion of normal liver architecture. Cirrhosis is characterized by regenerative nodules surrounded by dense fibrous tissue. Symptoms may not appear for years and are often nonspecific (e.g., anorexia, fatigue, weight loss). Late symptoms include portal hypertension, ascites, and, if decompensation occurs, liver failure. Diagnosis often requires a liver biopsy. Cirrhosis is usually considered irreversible. Treatment is symptomatic.

[0064] According to the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), cirrhosis is the seventh leading cause of death in the United States. Cirrhosis is pathologically defined by the loss of normal microscopic lobular architecture with fibrosis (i.e., the proliferation of scar tissue resulting from infection, inflammation, injury, or even healing) and nodular regeneration. As the liver becomes chronically damaged, scar tissue gradually replaces normally functioning liver tissue, and blood flow through the liver gradually decreases. With the loss of normal liver tissue, nutrients, hormones, drugs, and toxins are not effectively processed by the liver. In addition, proteins and other substances produced by the liver are inhibited.

[0065] Symptoms of cirrhosis vary in severity and from person to person. Symptoms may include abnormal neurological function, ascites (accumulation of fluid in the abdominal cavity), enlarged breasts in men, coughing up blood or vomiting blood, curled fingers (Dupuytren's contractions of the palms), gallstones, hair loss, itching, jaundice, kidney failure, hepatic encephalopathy, muscle loss, loss of appetite, portal hypertension, redness of the palms, enlarged salivary glands in the cheeks, shrinkage of the testes, small spider veins in the skin, weakness, weight loss, etc. Symptoms of cirrhosis may resemble other conditions or medical problems. Mild cirrhosis may not have any symptoms at all.

[0066] The most common cause of cirrhosis is alcohol abuse. Other causes include hepatitis and other viruses (e.g., HCV, described in Section 2.2 below), use of certain drugs, exposure to chemicals, bile duct blockage, autoimmune diseases, blockage of blood outflow from the liver (i.e., Budd-Chiari syndrome), heart and vascular disorders, alpha-1-antitrypsin deficiency, high blood galactose levels, high blood tyrosine levels, glycogen storage diseases, diabetes, malnutrition, and genetic accumulation of excess copper (Wilson's disease) or iron (hemochromatosis).

[0067] Cirrhosis is a progressive liver disease, and the damage sustained by the liver is irreversible.However, proper nutrition, avoidance of certain toxins (i.e., alcohol), vitamin supplementation, and management of cirrhosis complications often slow or stop further liver damage.In severe cases of cirrhosis, liver transplantation may be considered.

[0068] Nonalcoholic steatohepatitis (NASH) Nonalcoholic steatohepatitis (NASH), also known as nonalcoholic fatty liver disease, typically represents a liver disorder characterized by alcoholic etiology without alcohol intake. Fatty deposits in hepatocytes are primarily triglycerides, and the severity of NASH is directly related to the amount of fat in the liver. Histologically, steatohepatitis can be diagnosed when 50% of hepatocytes are steatotic (fatty liver accumulation) or when the total weight of fat is greater than 5% of the total liver. NASH is further characterized by elevated serum aminotransferase activity accompanied by hepatic steatosis, inflammation, and fibrosis that sometimes progresses to cirrhosis.

[0069] The prevalence of NASH ranges from 3 to 19% in most parts of the world. NASH has many possible causes but no clear origin. The most likely causes are obesity due to poor diet, diabetes, long-term use of steroids, and tetracycline use. Some studies have shown signs of reversal of adiposity after weight loss.

[0070] Currently, no established treatment exists for this potentially serious disorder. Treatment of patients with nonalcoholic fatty liver disease has typically focused on managing associated conditions, such as obesity, diabetes mellitus, and hyperlipidemia, and discontinuing potentially hepatotoxic drugs.

[0071] Fibrosis-related diseases Fibrosis-related disorders that may be suitable for treatment with the methods provided herein include, but are not limited to, collagen diseases, interstitial lung diseases, human fibrotic lung diseases (e.g., obstructive bronchitis, idiopathic pulmonary fibrosis, pulmonary fibrosis from known etiology, tumor stroma in pulmonary diseases, systemic sclerosis affecting the lungs, Hermansky-Pudlak syndrome, coal workers' pneumonia, asbestosis, silicosis, chronic pulmonary hypertension, AIDS-associated pulmonary hypertension, sarcoidosis, moderate to severe asthma, etc.), fibrotic vascular diseases, arteriosclerosis, atherosclerosis, varicose veins, coronary artery infarction, cerebral infarction, myocardial fibrosis, musculoskeletal fibrosis, postoperative adhesions, human kidney diseases (e.g., nephritic syndrome, Alport syndrome, HIV-associated nephropathy, polycystic kidney disease, Fabry disease, diabetic nephropathy, chronic glomerulonephritis, systemic lupus associated nephritis due to nephritis), progressive systemic sclerosis (PSS), primary sclerosing cholangitis (PSC), liver fibrosis, liver cirrhosis, renal fibrosis, pulmonary fibrosis, cystic fibrosis, chronic graft-versus-host disease, sclerosing dermopathy (localized and systemic), Graves' ophthalmopathy, diabetic retinopathy, glaucoma, Peyronie's disease, penile fibrosis, urethral stricture after cystoscopy, internal attachments after surgery, scarring, myelofibrosis, idiopathic retroperitoneal fibrosis, peritoneal fibrosis of known etiology, drug-induced In some embodiments, the fibrosis-associated disorder includes ergotine toxicity, fibrosis associated with benign or malignant cancer, fibrosis associated with microbial infection (e.g., viral, bacterial, parasitic, fungal, etc.), Alzheimer's disease, fibrosis associated with inflammatory bowel disease (including stricture formation in Crohn's disease and microscopic colitis), stromal cell tumors, mucositis, fibrosis induced by chemical or environmental insults (e.g., cancer chemotherapy, pesticides, radiation (e.g., cancer radiotherapy), etc.). In some embodiments, the fibrosis-associated disorder is selected from systemic or localized scleroderma, keloids, hypertrophic scars, atherosclerosis, restenosis, pulmonary inflammation and fibrosis, idiopathic pulmonary fibrosis, liver cirrhosis, fibrosis as a result of chronic hepatitis B or C infection, kidney disease, cardiac disease due to scar tissue, macular degeneration, and retinal and vitreoretinopathy. In some embodiments, the fibrosis-associated disorder is due to chemotherapy drugs, radiation-induced fibrosis, and injury and burns.

[0072] Genetic diseases associated with liver fibrosis include Wilson's disease, hereditary hemochromatosis, non-HFE hereditary hemochromatosis ferroportin, transferrin receptor 2, hepcidin, hemojuvelin, sitosterolemia / hepatobiliary cholesterol transporters 5 and 8, progressive familial intrahepatic cholestasis type 3, hereditary fructose intolerance, hypertyrosinemia type I, argininosuccinate lyase deficiency, citrin deficiency, cholesteryl ester storage disease and Wolman's disease, alpha 1-antitrypsin deficiency, cystic fibrosis, Alstrom syndrome, and congenital hepatic fibrosis.

[0073] Pulmonary fibrosis Pulmonary fibrosis ("lung scarring") is a respiratory disease in which scarring forms in lung tissue, resulting in serious breathing problems. Scarring, the accumulation of excess fibrous connective tissue (a process called fibrosis), leads to thickening of the walls and reduced oxygen delivery in the blood. The result is permanent shortness of breath.

[0074] Pulmonary fibrosis involves the slow replacement of normal lung parenchyma with fibrous tissue. The replacement of normal lung with scar tissue irreversibly reduces oxygen diffusing capacity, and the resulting stiffness or reduced compliance makes pulmonary fibrosis a restrictive lung disease. Pulmonary fibrosis is sustained by abnormalities in wound healing, not chronic inflammation. It is the primary cause of restrictive lung disease that is intrinsic to the lung parenchyma. In some cases, pulmonary fibrosis is associated with smoking. Treatment options for pulmonary fibrosis are limited and include immunosuppressive therapy, such as corticosteroids.

[0075] Idiopathic pulmonary fibrosis Idiopathic pulmonary fibrosis (IPF), the most common form of idiopathic interstitial pneumonia, is a chronic, progressive, irreversible, usually fatal lung disease of unknown cause. IPF occurs in middle-aged and older adults (median age at diagnosis 66 years, range 55-75 years), is confined to the lungs, and is associated with histopathological or radiological patterns that are typical of usual interstitial pneumonias.

[0076] The main histopathological feature of usual interstitial pneumonia is a heterogeneous appearance, best seen in low-power positive ion, with areas of subpleural and paraseptal fibrosis and honeycombing (i.e., cystic fibrous airspaces lined by bronchiolar epithelium and often filled with mucin and variable numbers of inflammatory cells) alternating with areas of less affected or normal parenchyma (spatial heterogeneity). Small areas of active fibrosis (fibroblastic foci) are present in the background of collagen deposition, reflecting the temporal heterogeneity of this process and indicating ongoing disease. Inflammation is usually mild and consists of a patchy lymphoplasmacytic interstitial infiltrate. The presence of a usual interstitial pneumonia pattern on high-resolution CT is characterized by reticular opacities, often associated with traction bronchiectasis, and little or no ground-glass opacities. Honeycombing, which manifests as subpleural clustered cystic air spaces with well-defined walls (typically 3-10 mm in diameter), is common and important in making a definite diagnosis.

[0077] Patients with IPF usually seek medical attention because they suffer from chronic, progressive exertional dyspnea and cough. Bibasilar inspiratory crackles are heard on chest auscultation, and clubbing is often present. The natural history of IPF has been characterized as persistent or slowly progressive lung damage, and most patients follow this pattern. However, recent findings indicate that IPF is a heterogeneous disease, and new clinical phenotypes with different patterns of survival have been reported. Although the pathogenetic mechanism is unclear, increasing evidence indicates that the disease is the result of abnormal behavior of alveolar epithelial cells that induce migration, proliferation, and activation of mesenchymal cells with the formation of fibroblasts and myofibroblasts. Activated myofibroblasts secrete excessive amounts of extracellular matrix molecules, which subsequently destroy the lung architecture. No treatment has been clearly shown to extend survival.

[0078] Myocardial infarction Myocardial infarction, also commonly known as a heart attack, occurs when part of the heart is deprived of oxygen due to a blockage in a coronary artery. The coronary arteries supply oxygen-rich blood to the heart muscle (myocardium). Without oxygen, muscle cells in the blocked artery begin to die (infarction).

[0079] The most common cause of myocardial infarction is the rupture of atherosclerotic plaque in an artery that supplies the heart muscle. The plaque becomes unstable and ruptures, promoting the formation of a clot that further occludes the artery. This can occur within minutes. When an artery is occluded, tissue supplied by that artery can die. Atherosclerotic plaques can often be present for decades before symptoms appear.

[0080] The gradual buildup of cholesterol and fibrous tissue in plaques within the walls of coronary or other arteries, typically over decades, is called atherosclerosis. Atherosclerosis is characterized by progressive inflammation of the arterial wall. Inflammatory cells, particularly macrophages, migrate into the affected arterial wall. Over time, they deposit cholesterol products, particularly LDL, and become foam cells. As the foam cells die, a cholesterol core forms. In response to growth factors secreted by the macrophages, smooth muscle and other cells migrate into the plaque and act to stabilize it. Stable plaques may have a thick fibrous cap with calcification. If inflammation continues, the cap may be thin or ulcerated. When exposed to the blood pressure associated with blood flow, plaques, especially those with a thin inner layer, may rupture, inducing the formation of a blood clot (thrombus).

[0081] Alzheimer's Disease Alzheimer's disease is a cognitive disorder characterized by progressive impairment of memory and cognition. It typically develops later in life and is associated with a wide variety of structural, chemical and functional abnormalities involving brain regions involved in cognition and memory. Epidemiological studies suggest that dementia currently affects up to 10% of people over the age of 65, and that as many as 4 million people in the United States alone may be affected by Alzheimer's disease. The cost of caring for these individuals is well over $80 billion annually and is rising rapidly.

[0082] The causes of Alzheimer's disease are not fully understood. Approximately 70% of risk is thought to be inherited from parents, with many genes usually involved. Other risk factors include a history of head trauma, depression, and high blood pressure. The course of the disease is associated with plaques and neurofibrillary tangles in the brain. A possible diagnosis is based on a history of the disease and cognitive testing with medical imaging and blood tests to rule out other possible causes. Early symptoms are often mistaken for normal aging. A definitive diagnosis requires an examination of brain tissue. No drugs or supplements have been shown to reduce risk.

[0083] It has been hypothesized that extracellular amyloid beta (Aβ) deposition is the underlying cause of the disease. Support for this hypothesis comes from the location of the gene for amyloid precursor protein (APP) on chromosome 21 and the fact that individuals with trisomy 21 (Down syndrome), who have an extra copy of the gene, almost universally exhibit at least the earliest AD symptoms by age 40. Also, a specific isoform of apolipoprotein, APOE4, is a major genetic risk factor for AD. Although apolipoproteins promote the breakdown of beta-amyloid, some isoforms (e.g., APOE4) are less effective at this task, leading to the accumulation of excess amyloid in the brain. Further evidence comes from the finding that transgenic mice expressing a mutant form of the human APP gene develop fibrillar amyloid plaques and Alzheimer's-like brain pathology accompanied by spatial learning impairments.

[0084] Additional drugs The compositions provided herein can be administered to subjects in combination with any known drug that treats disorders, such as liver fibrosis.Similarly, the methods provided herein can be administered to subjects in combination with any known drug that treats disorders, such as liver fibrosis, with the compositions disclosed herein (e.g., NP-011).Various treatments for fibrosis-related disorders are known to those skilled in the art.

[0085] Treatments for fibrotic diseases include anti-inflammatory drugs, corticosteroids, penicillamine, and colchicine. See, e.g., The Merck Manual. 20th Edition, Merck Research Laboratories, 2018.

[0086] Because fibrosis is a response to liver injury, primary treatment should focus on the cause (removing the cause of liver injury). Such treatments include removal of hepatitis B or C viruses in chronic viral hepatitis, withdrawal from alcohol in alcoholic liver disease, removal of heavy metals, such as iron or copper, in hemochromatosis in Wilson's disease, and decompressing the bile ducts in biliary obstruction. Such treatments halt the progression of fibrosis and, in some patients, may even reverse the fibrotic changes.

[0087] Treatments aimed at reversing fibrosis are usually too toxic for long-term use (e.g., corticosteroids, penicillamine) or have no proven effectiveness (e.g., colchicine). Other anti-fibrotic treatments are also under investigation. Silymarin, present in milk thistle, is a common alternative medicine used to treat liver fibrosis. It appears safe (except when combined with certain drugs to treat hepatitis C), but lacks efficacy.

[0088] In some embodiments, the anti-fibrotic therapy comprises administration of a pro-fibrotic factor antagonist and / or an anti-fibrotic agent.

[0089] Profibrotic factor antagonists Antifibrotic therapies include agents that inhibit or antagonize profibrotic factors, such as agents that antagonize one or more growth factors or cytokines involved in the formation and maintenance of fibrotic tissue. In this manner, antifibrotic therapies target fibrocyte, fibrocyte precursor, myofibroblast precursor, and / or hematopoietic monocyte precursor differentiation and the formation and maintenance of fibrotic tissue.

[0090] Profibrotic factors that may be targeted by antagonists as part of the therapeutic methods of the invention include, but are not limited to, transforming growth factor beta (TGF-β, e.g., TGF-β1-5), VEGF, EGF, RANTES, members of the interleukin family (e.g., IL-1, IL-4, IL-5, IL-6, IL-8, and IL-13), tumor necrosis factor type alpha (TNF-a), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and / or fibroblast growth factor (GFGF). GF), monocyte chemotactic protein type 1 (MCP-1), macrophage inflammatory proteins (e.g., MIP-1a, MIP-2), connective tissue growth factor (CTGF), endothelin-1, angiotensin-11, rennin, leptin, chemokines (e.g., CCL2, CCL12, CXCL12, CXCR4, CCR3, CCR5, CCR7), SLC / CCL21, and other factors known to promote or be associated with the formation, growth or maintenance of fibrous tissue.

[0091] Antifibrotic therapy may include antagonists of the corresponding receptors of one or more profibrotic factors. Such antagonists may include inactive forms of one or more profibrotic factors and / or cytokines, such as fragments thereof. Such forms at appropriate concentrations may compete with the corresponding profibrotic factors and / or cytokines for binding to their receptors. Similarly, certain antibodies against receptors can be used to prevent or block the binding of the corresponding profibrotic factors and / or cytokines to them.

[0092] Antifibrotic therapies may also include soluble forms of receptors of one or more profibrotic factors and / or cytokines, such that the soluble receptors compete with their corresponding natural cellular receptors for targeting ligands. Therapies may further include compounds that compete with or otherwise interfere with the binding of one or more profibrotic factors and / or cytokines to their receptors. For example, the proteoglycan decorin is known to bind to TGF-β, thereby reducing its availability for binding to its receptor. Mannose-6-phosphate is also known to compete with TGF-β for binding to its corresponding receptor. Other known binding inhibitors of TGF-β include latent transforming growth factor-β binding protein (LTBP) and latency associated peptide (LAP), both of which naturally bind to the intracellular precursor of TGF-β.

[0093] In certain embodiments, the antifibrotic therapy may comprise one or more oligonucleotides that contain at least one sequence that is antisense with respect to one or more profibrotic factors and / or cytokines. Such components may also comprise one or more expression plasmids with appropriate transcription control sequences that produce the antisense sequences. In other selected embodiments, the antifibrotic therapy may comprise one or more double-stranded oligonucleotides, or expression plasmids that encode them, that are suitable for degrading the transcripts of one or more profibrotic factors and / or cytokines via RNA-mediated interference. In other selected embodiments, the antifibrotic therapy may comprise one or more single-stranded oligonucleotide aptamers, or expression plasmids that encode them, that are suitable for inhibiting or interfering with the binding of profibrotic factors to their cognate receptors.

[0094] Suitable profibrotic factor antagonists may include components known to inhibit, attenuate, or prevent one or more components of the intracellular signaling pathway activated by one or more profibrotic factors when binding to their corresponding receptors. For example, antifibrotic therapy may include components that inhibit or attenuate downstream signaling pathway molecules, such as SMAD family members and SARA. Suitable antifibrotic therapy may include one or more molecules suitable for inhibiting or preventing cell adhesion required for fibrosis. For example, suitable components may include interference antibodies against ICAM-1 and / or CD11, CD49, or CD18 molecules, thereby interfering with the adhesive interaction between them.

[0095] In other selected embodiments, suitable profibrotic factor antagonists may include inhibitors of collagen synthesis, such as proline analogs that interfere with post-translational processing of collagen precursors. For example, pirfenidone is an orally active small molecule drug that can inhibit collagen synthesis, downregulate the production of multiple cytokines, and block fibroblast proliferation.

[0096] TGF-β antagonists Transforming growth factor (TGF) beta family cytokines play a central role in wound healing and tissue repair and are found in all tissues. TGF-β is produced by many parenchymal cell types as well as infiltrating cells, such as lymphocytes, monocytes / macrophages, and platelets. After wounding or inflammation, such cells are potential sources of TGF-β. In general, TGF-β stimulates the production of various extracellular matrix proteins, inhibits the degradation of these matrix proteins, and promotes tissue fibrosis, all of which contribute to the repair and restoration of diseased tissues. In many diseases, excess TGF-β contributes to pathological excess tissue fibrosis that may impair normal organ function.

[0097] Examples of TGF-β antagonists include, but are not limited to, monoclonal and polyclonal antibodies against one or more isoforms of TGF-β (Dasch et al., U.S. Pat. No. 5,571,714; see also WO 97 / 13844 and WO 00 / 66631); TGF-β receptors, soluble forms of such receptors (preferably soluble TGF-P type III receptors) or antibodies against TGF-receptors (Segarini et al., U.S. Pat. No. 5,693,607; Lin et al., U.S. Pat. No. 6,001,969, U.S. Pat. No. 6,010,872, U.S. Pat. No. 6,086,86, U.S. Pat. No. 6,201,108; WO 98 / 48024; WO 95 / 10610; WO 93 / 09228; WO 92 / 00330; latency associated peptide (WO 91 / 08291); big latency TGF-β (WO 94 / 09812); fetuin (U.S. Pat. No. 5,821,227); decorin and other proteoglycans such as biglycan, fibromodulin, lumican and endoglin (WO 91 / 110727; Ruoslahti et al., U.S. Pat. Nos. 5,654,270, 5,705,609, 5,726,149; Border, U.S. Pat. No. 5,824,655; WO 91 / 04748; Letarte et al., U.S. Pat. No. 5,830,847, 6,015,693; WO 91 / 110727; WO 93 / 09800; and WO 94 / 110187); somatostatin (WO 98 / 08529); mannose-6-phosphate or mannose-1-phosphate (Ferguson et al., U.S. Pat. No. 5,520,926); prolactin (WO 97 / 40848); insulin-like growth factor II (WO 98 / 117304); IP-10 (WO 97 / 00691); arg-gly-asp containing peptides (Pfeffer, U.S. Pat. No. 5,958,411; WO 93 / 10808); plant, fungal and bacterial extracts (European Patent Application Publication No. 813 875; Patent 8119984; and Matsunaga et al., U.S. Pat. No. 5,693,610);Antisense oligonucleotides (Chung, U.S. Pat. No. 5,683,988; Fakhrai et al., U.S. Pat. No. 5,772,995; Dzau, U.S. Pat. No. 5,821,234, U.S. Pat. No. 5,869,462; and WO 94 / 25588); proteins involved in TGF-β signaling, such as SMADs and MADs (European Patent Application Publication No. 874 046; WO 97 / 31020; WO 97 / 38729; WO 98 / 03663; WO 98 / 07735; WO 98 / 07849; WO 98 / 45467; WO 98 / 53068; WO 98 / 55512; WO 98 / 56913; WO 98 / 53830; WO 99 / 50296; Falb, U.S. Pat. No. 5,834,248; Falb et al., U.S. Pat. No. 5,807,708; and Gimeno et al., U.S. Pat. No. 5,948,639), Ski and Sno (Vogel, 1999, Science, 286:665; and Stroschein et al., 1999, Science, 286:771-774); one or more single-stranded oligonucleotide aptamers suitable for inhibiting or disrupting the binding of TGF-β to its cognate receptor, or expression plasmids encoding same, and any mutant, fragment or derivative of the above defined molecules that retain the ability to inhibit the activity of TGF-β;

[0098] In some embodiments, the TGF-β antagonist is a human or humanized monoclonal antibody (or a fragment thereof, such as an F(ab)2 fragment, an Fv fragment, a single chain antibody, and other forms or fragments of an antibody that retain the ability to bind to TGF-β, e.g., the monoclonal antibody from hybridoma 1D 11.16 (ATCC Accession No. HB 9849, described in Dasch et al., U.S. Patent No. 5,783,185)) that blocks TGF-β binding to its receptor.

[0099] Antifibrotic Agents In certain embodiments, the profibrotic factor antagonist may be replaced or augmented by cytokines known to have antifibrotic effects, such as IL-12, IL-10, IFN-g, or BMP-7 (OP-1). Nucleic acid sequences encoding IFN-y polypeptides may be accessed, for example, from public databases, such as Genbank, journal publications, and the like. Although a variety of mammalian IFN-g polypeptides are of interest, for the treatment of human disease, the human protein is generally used. Human IFN-g coding sequences may be found in Genbank, Accession Nos. P01579 and CAA00375. Corresponding genomic sequences may be found in Genbank, Accession Nos. 100219, M37265, and V00536. See, for example, Gray et al. (1982) Nature 295:501 (Genbank Xl3274); and Rinderknecht et al. (1984) J. Biol. Chern. 259:6790. IFN-g1b (Actimmune®, human interferon) is a single polypeptide chain of 140 amino acids. It is produced recombinantly in E. coli and is not glycosylated. Rinderknecht et al. (1984) J. Biol. Chern. 259:6790-6797. The IFN-g used in antifibrotic therapy can be either natural IFN-g, recombinant IFN-g, or derivatives thereof, so long as it has IFN-g activity, particularly human IFN-g activity.

[0100] Antifibrotic therapies include one or more serum amyloid P (SAP) agonists, one or more C-reactive protein (CRP) antagonists, or a combination thereof. SAP agonists are useful in the treatment of various fibrotic diseases. SAP agonists include all compounds and compositions that increase or otherwise mimic endogenous SAP signaling, including compounds that increase SAP activity.

[0101] Serum amyloid P (SAP) or pentraxin-2, a member of the pentraxin family, is a 27 kDa protein that is produced by the liver, secreted into the blood, and circulates as a stable 135 kDa pentamer. SAP reduces neutrophil adhesion to ECM proteins, inhibits monocyte differentiation into fibrocytes, reduces profibrotic macrophages, activates the complement pathway, and promotes phagocytosis of cellular debris. SAP reduces bleomycin-induced pulmonary fibrosis. Representative agonists of SAP include human SAP protein or its active fragments, anti-FcyR antibodies, agglutinating antibodies, SAP peptidomimetics, FcyR crosslinkers, and aptamers. CRP antagonists encompass all compounds and compositions that reduce, block, or inhibit CRP signaling. Representative antagonists include antisense nucleic acids that target the expression of CRP, RNA interference molecules, such as small interfering RNA, dsRNA, and locked nucleic acids (LNA).

[0102] Nucleic Acid Molecules, Vectors, and Host Cells Nucleic acid molecules encoding the polypeptides disclosed herein, such as NP-011 proteins, are provided.

[0103] The nucleic acid molecules disclosed herein may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand and / or the non-coding (antisense) strand. In certain embodiments, the nucleic acid molecule is isolated. In further embodiments, the nucleic acid molecule is substantially pure. In some embodiments, the nucleic acid is cDNA or derived from cDNA. In some embodiments, the nucleic acid is recombinantly produced.

[0104] The nucleic acid molecules of the present invention can be amplified using cDNA, mRNA, or genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecules thus amplified can be cloned into a suitable vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or part of the nucleic acid molecules of the present invention can be prepared using standard synthesis techniques, for example, an automated DNA synthesizer.

[0105] In some embodiments, the nucleic acid molecule comprises a protein coding sequence operably linked to a control sequence that controls expression of the coding sequence in a host cell or in vitro (see below). In certain embodiments, the coding sequence is a cDNA. The present disclosure also relates to vectors containing the nucleic acid molecule comprising a protein (e.g., NP-011) coding sequence operably linked to a control sequence that controls expression of the coding sequence in a host cell or in vitro.

[0106] In some embodiments, the nucleic acid molecule comprises a coding sequence for a protein fused in the same reading frame to a heterologous polynucleotide sequence. In some embodiments, the heterologous polynucleotide sequence encodes a leader peptide sequence that facilitates secretion of the expressed protein from a host cell transformed with a nucleic acid molecule encoding a polypeptide (e.g., NP-011). Proteins that include a leader sequence are called preproteins, and can have a leader sequence that is cleaved by the host cell to form a processed form of the protein. Such leader peptide sequences and their use to facilitate secretion of recombinant proteins in host cells are generally known in the art. In further embodiments, the heterologous polynucleotide sequence encodes additional 5' amino acid residues that can function, for example, to facilitate purification, add or improve protein stability and / or therapeutic properties of the recombinantly expressed protein. In preferred embodiments, NP-011 and / or similar proteins are secreted from the host cell. In certain preferred embodiments, NP-011 and / or similar proteins lack an N-terminal methionine and do not include heterologous sequences. In other preferred embodiments, NP-011 includes heterologous sequences that extend its half-life in vivo.

[0107] In some embodiments, the nucleic acid sequence encoding the protein, or the vector containing the nucleic acid sequence, is constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and codon optimization based on the selection of the host cell. Standard methods can be routinely applied to synthesize and isolate polynucleotide sequences encoding a polypeptide (e.g., NP-011).

[0108] A vector can be a construct that can deliver, and in some embodiments, express, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0109] Once assembled (synthetically or otherwise), the nucleic acid sequence encoding the polypeptide may be routinely operably linked to control sequences suitable for expression in a desired host. In some embodiments, the nucleic acid sequence encoding the polypeptide is inserted into one or more expression vectors and operably linked to control sequences suitable for expression of the protein in a desired host. To obtain high expression levels of the transfected coding sequence in the host, the coding sequence may be operably linked to or associated with transcriptional and translational expression control sequences that are functional in the selected expression host.

[0110] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding a polypeptide. A recombinant expression vector is a replicable DNA construct that has a synthetic or cDNA-derived DNA fragment encoding a polypeptide (e.g., NP-011) operably linked to appropriate transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally comprises an assembly of: (1) a genetic element or elements that have a regulatory role in gene expression, such as a transcriptional promoter or enhancer; (2) a structural or coding sequence that is transcribed into mRNA and translated into protein; and (3) appropriate transcriptional and translational initiation and termination sequences, as described in more detail below. Such regulatory elements may include operator sequences to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene that facilitates recognition of transformants may further be incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned to allow translation. Structural elements intended for use in yeast expression systems include leader sequences that allow extracellular secretion of translated protein by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, the protein may include an N-terminal methionine residue. This residue can then be cleaved from the expressed recombinant protein to provide the final protein. In certain embodiments, the disclosure provides compositions, e.g., pharmaceutical compositions, comprising a nucleic acid or vector as described above or elsewhere herein, optionally further comprising one or more carriers, diluents, excipients, or other additives.

[0111] Also provided are host cells transformed with the nucleic acid molecules or cDNA molecules and / or vectors disclosed herein. The disclosure also provides host cells transformed with the disclosed nucleic acid molecules, or molecules operably linked to a control sequence and optionally inserted into a vector.

[0112] In further embodiments, the present disclosure provides a method of making a polypeptide provided herein, comprising culturing a host cell transformed herein under conditions suitable for production of the polypeptide. The present disclosure optionally provides isolating the polypeptide secreted from the host cell. The present disclosure also optionally provides a polypeptide produced using this method, as well as a pharmaceutical composition comprising the polypeptide and a pharma- ceutically acceptable carrier.

[0113] The choice of expression control sequence and expression vector depends on the choice of host. A wide variety of expression host / vector combinations can be used (see also below for further examples). Useful expression vectors for eukaryotic hosts include, for example, vectors that contain expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts also include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, as well as broader host range plasmids, such as M13 and filamentous single-stranded DNA phages.

[0114] A host cell can be any cell or population of cells that carries or can carry a recombinant nucleic acid. A host cell can be a prokaryotic cell (e.g., E. coli) or a eukaryotic cell. Prokaryotes include gram-negative or gram-positive bacteria, such as E. coli or Bacillus. A host cell can be a fungal cell, including yeast, such as Saccharomyces cerevisiae, Pichia pastoris, or Schizosaccharomyces pombe. A host cell can also be any of a variety of animal cells, such as insect cells (e.g., Sf-9) or established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include HEK-293, HEK293F, and HEK-293T, the COS-7 line of monkey kidney cells described by Gluzman (Cell 23:175 (1981)), and, for example, L cell, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors can include non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Baculovirus systems for producing heterologous proteins in insect cells are reviewed in Luckow and Summers, BioTechnology 6:47. Cell-free translation systems can also be used. In a preferred embodiment, the host cell is Pichia pastoris.

[0115] Delivery of Nucleic Acid Molecules, Vectors and Hosts In some embodiments, the nucleic acid molecules, vectors, and host cells are provided in a pharmaceutical composition, hi some embodiments, components of the pharmaceutical composition aid in delivery of the nucleic acid to an intended recipient.

[0116] In some embodiments, the DNA construct is delivered to cells by transfection, i.e., by delivery of "naked" DNA, or in a complex with a colloidal dispersion system. Colloidal systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the present invention is lipid-complexed or liposomally formulated DNA. In the former approach, a plasmid containing a transgene with the desired DNA construct can first be experimentally optimized for expression (e.g., inclusion of introns in the 5' untranslated region and removal of unnecessary sequences (Felgner et al., Ann NY Acad Sci pp. 126-139, 1995)) before formulating the DNA with, for example, lipids. The DNA can then be formulated with, for example, various lipid or liposomal materials, using known methods and materials, and delivered to the recipient mammal. See, e.g., Canonico et al., Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al., Am J Physiol 268; Alton et al., Nat Genet 5:135-142, 1993, and U.S. Patent No. 5,679,647 by Carson et al.

[0117] Liposome targeting can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, e.g., organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be differentiated based on whether it is passive or active. Passive targeting exploits the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) of organs, including sinusoidal capillaries. On the other hand, active targeting involves modifying liposomes by binding them to specific ligands, e.g., monoclonal antibodies, sugars, glycolipids, or proteins, or by changing the composition or size of the liposomes, to achieve targeting to organs and cell types other than their naturally occurring localization sites.

[0118] The surface of the targeted delivery system can be modified in various ways. In the case of liposome targeted delivery system, lipid groups can be incorporated into the lipid bilayer of liposome to stably associate targeting ligand with the liposome bilayer. Various linking groups can be used to connect lipid chains to targeting ligand. Delivery vehicle, such as naked DNA or DNA associated with liposome, can be administered to several sites of a subject.

[0119] The nucleic acid can be delivered in any desired vector. These include viral or non-viral vectors, such as adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno-associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). The nucleic acid can be administered in any desired format that provides a sufficiently efficient level of delivery, including complexed to viral particles, liposomes, nanoparticles, and polymers.

[0120] The nucleic acid encoding the protein or nucleic acid of interest may be in a plasmid or viral vector, or other vectors known in the art. Such vectors are well known and one can be selected for a particular application. In one embodiment of the present invention, the gene delivery vehicle comprises a promoter and a demethylase coding sequence. Preferred promoters are tissue-specific promoters and promoters activated by cell proliferation, such as thymidine kinase and thymidylate synthase promoters. Other preferred promoters include promoters that can be activated by viral infection, such as α- and β-interferon promoters, and promoters that can be activated by hormones, such as estrogen. Other promoters that can be used include Moloney virus LTR, CMV promoter, and mouse albumin promoter. The promoter can be constitutive or inducible.

[0121] In another embodiment, naked polynucleotide molecules are used as gene delivery vehicles as described in WO 90 / 11092 and U.S. Patent No. 5,580,859. Such gene delivery vehicles can be either growth factor DNA or RNA, and in certain embodiments are linked to killed adenovirus. Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other vehicles that may optionally be used include inducible DNA-ligands (Wu et al., J. Biol. Chem. 264:16985-16987, 1989), ligand-DNA combinations (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989), liposomes (Wang et al., Proc. Natl. Acad. Sci. 84:7851-7855, 1987), and microprojectiles (Williams et al., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).

[0122] The gene delivery vehicle can optionally include viral sequences, such as a viral origin of replication or a packaging signal. These viral sequences can be selected from viruses, such as astroviruses, coronaviruses, orthomyxoviruses, papovaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, retroviruses, togaviruses, or adenoviruses. In a preferred embodiment, the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and their various uses are described in numerous references, including, for example, Mann et al., Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci. USA 81:6349, 1984, Miller et al., Human Gene Therapy 1:5-14, 1990, U.S. Pat. Nos. 4,405,712, 4,861,719, and 4,980,289, PCT Publication Nos. WO 89 / 02468, WO 89 / 05349, and WO 90 / 02806. A number of retroviral gene delivery vehicles can be used in the present invention, for example those described in EP 0415731; WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; U.S. Pat. No. 5,219,740; WO 93 / 11230; WO 93 / 10218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993; Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg. 79:729-735, 1993 (U.S. Pat. No. 4,777,127, British Patent No. 2200651, European Patent No. 0345242, and International Publication No. WO 91 / 02805).

[0123] Other viral vector systems which can be used to deliver the polynucleotides of the invention include herpes viruses, e.g., herpes simplex virus (U.S. Pat. No. 5,631,236, issued May 20, 1997 to Woo et al., and WO 00 / 08191 to Neurovex), vaccinia virus (U.S. Pat. No. 5,631,236, issued May 20, 1997 to Woo et al., and WO 00 / 08191 to Neurovex), vaccinia virus (Ridgeway (1988) Ridgeway, "Mammalian expression vectors", In: Rodriguez RL, Denhardt DT, ed. Vectors: A survey of molecular cloning vectors and their uses. Stoneham: Butterworth,; Baichwal and Sugden (1986) "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes", In: Kucherlapati R, ed. New New York: Plenum Press; Coupar et al., (1988) Gene, 68:1-10, and several RNA viruses. Preferred viruses include alphaviruses, poxyviruses, arenaviruses, vaccinia viruses, and polioviruses. They offer several attractive characteristics for a variety of mammalian cells (Friedmann (1989) Science, 244:1275-1281; Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra; Coupar et al., 1988; Horwich et al., (1990) J. Virol., 64:642-650).

[0124] Methods for Producing Polypeptides In some embodiments, a vector containing a nucleic acid encoding a polypeptide (e.g., NP-011) can be transfected into a host cell. As disclosed herein, the host cell can be cultured in a medium to produce the polypeptide. In some embodiments, the polypeptide is secreted into the medium. The polypeptide can be isolated from the medium using any suitable technique.

[0125] The polypeptides of the invention (e.g., NP-011) produced by transformed host cells can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags, such as hexahistidine, maltose binding domain, influenza envelope sequence, and glutathione-S-transferase, can be attached to the protein and easily purified by passage over an appropriate affinity column. Polypeptides can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0126] For example, supernatants from systems that secrete polypeptides into the medium can be first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be used, such as a matrix or substrate with pendant diethylaminoethyl (DEAE) groups. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation or anion exchange step can be used. Suitable cation exchangers include various insoluble matrices that contain sulfopropyl or carboxymethyl groups. Finally, the polypeptide can be further purified using one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel with pendant methyl or other aliphatic groups.

[0127] Polypeptides produced in cell culture can be isolated, for example, by initial extraction from a cell pellet followed by one or more concentration, salting out, aqueous ion exchange, or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be used for final purification steps. Cells used to express recombinant proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

[0128] An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or, if chemically synthesized, is substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). When a protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% by volume of the volume of the protein preparation. When a protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.

[0129] Pharmaceutical Compositions In another aspect, the present invention provides a pharma- ceutically acceptable composition comprising a therapeutically effective amount of a polypeptide disclosed herein, formulated with one or more pharma- ceutically acceptable carriers (additives) and / or diluents. The pharmaceutical compositions of the present invention can be specifically formulated for storage and use by combining the purification agents of the present invention encompassed by the present invention with a pharma- ceutically acceptable vehicle (e.g., carrier or excipient). Those skilled in the art generally consider pharma- ceutically acceptable carriers, excipients, and / or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.

[0130] Suitable pharma- ceutically acceptable vehicles include, but are not limited to, non-toxic buffers, such as phosphate, citric acid, and other organic acids; salts, such as sodium chloride; antioxidants, such as ascorbic acid and methionine; preservatives, such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzenemethonium chloride, benzethonium chloride, phenol or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanediol, ethyl ketone ... Examples of suitable surfactants include ethanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophobic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates, such as monosaccharides, disaccharides, glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes, such as Zn-protein complexes; non-ionic surfactants, such as TWEEN® or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London). Formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filtration membranes.

[0131] Examples of pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffers; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0132] Pharmaceutically acceptable salts can be part of pharmaceutical compositions that include the polypeptides of the present invention. Salts can be the addition of relatively non-toxic inorganic and organic acids to the formulation. Salts can be added to the polypeptide at any stage of purification, including after purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulfonate, and the like (see, e.g., Berge et al., (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0133] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as release agents, preservatives and antioxidants can also be present in the composition.Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc., (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc., and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0134] Pharmaceutical compositions of the invention suitable for parenteral administration can comprise one or more pharma- ceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted into sterile injectable solutions or dispersions immediately prior to use and can contain antioxidants, buffers, bacteriostats, solutes, which render the formulation isotonic with the blood of the intended recipient, or which can contain suspending agents or thickening agents.

[0135] Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, etc. in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0136] In certain embodiments, the pharmaceutical formulation comprises a polypeptide complexed with liposomes. Methods for producing liposomes are known to those skilled in the art. For example, some liposomes can be produced by reverse phase evaporation using a lipid composition that includes phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to produce liposomes with desired diameter.

[0137] In certain embodiments, sustained release formulations containing the polypeptide of the present invention can be produced.Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, the matrices being in the form of shaped articles (e.g., films or microcapsules).Examples of sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0138] The compositions described herein can be administered by any suitable means, such as parenterally, subcutaneously, intraperitoneally, intrapulmonary, and intranasally. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Additionally, the compositions can be suitably administered by pulse infusion.

[0139] In some embodiments, the polypeptides of the present invention can be conjugated or modified according to pharmacological methods well known in the art (e.g., PEGylation, glycosylation, oligomerization, etc.) to further enhance the desired biological activity, e.g., increased bioavailability and reduced proteolysis.

[0140] In some embodiments, the polypeptide is lyophilized and / or stored in lyophilized form, hi some embodiments, formulations comprising the polypeptides described herein are lyophilized.

[0141] kit A "kit" is any article of manufacture (e.g., package or container) that contains at least one composition, e.g., a polypeptide, of the invention. The kit may also include additional diluents. The kit may include one or more agents necessary to resuspend the lyophilized composition and / or filtration units to remove aggregates. The kit may be promoted, distributed, or sold as a unit for performing the methods of the invention. The kit may include one or more reagents necessary to express a composition useful in the methods of the invention. The reagents in the kit may be provided in individual containers or as a mixture of two or more reagents in a single container. Additionally, the kit may include materials describing the use of the composition in the kit.

[0142] array The term "sequence identity or homology" refers to the sequence similarity between two polypeptide molecules or two nucleic acid molecules. If both positions of two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percentage of homology or sequence identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, x 100. For example, if 6 / 10 positions in two sequences are the same, then the two sequences have 60% homology or 60% sequence identity. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, the comparison is performed when the two sequences are aligned to give the maximum homology. Unless otherwise specified, "loop-out regions", e.g., those resulting from deletions or insertions in one of the sequences, are counted as mismatches. The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm.

[0143] In addition to naturally occurring allelic variants of the nucleic acid molecules of the present invention that may exist in a population, those skilled in the art will further recognize that sequence changes can be introduced by mutation, thereby leading to changes in the amino acid sequence of the encoded protein without altering the biological activity of the encoded protein. For example, nucleotide substitutions can be made that result in amino acid substitutions at "non-essential" amino acid residues. "Non-essential" amino acid residues are residues that can be altered from the wild-type sequence without altering the biological activity, whereas "essential" amino acid residues are necessary for biological activity. For example, amino acid residues that are not conserved or are only semi-conserved among homologs of various species are not essential for activity and therefore can be targets for modification. Alternatively, amino acid residues that are conserved among homologs of various species (e.g., mouse and human) are essential for activity and therefore cannot be targets for modification.

[0144] Thus, another aspect of the invention pertains to nucleic acid molecules encoding the polypeptides of the invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from the naturally occurring proteins corresponding to the markers of the invention, yet retain biological activity. In one embodiment, the polypeptides have an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 75%, 80%, 83%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the polypeptides described herein.

[0145] Nucleic acid variants may include modifications in coding regions, non-coding regions, or both. In some embodiments, nucleic acid variants include modifications that result in silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, nucleic acid variants are generated by silent substitutions due to the degeneracy of the genetic code. Nucleic acid variants can be generated for a variety of reasons, such as optimizing codon expression for a particular host (changing codons in human mRNA to those preferred by bacterial hosts, such as E. coli). Vectors and cells containing the nucleic acids described herein are also provided.

[0146] An isolated nucleic acid molecule encoding a mutant protein can be generated by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of the nucleic acid of the present invention, such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of a coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0147] Non-conservative substitutions include (a) a residue having a positive charge (e.g., Arg, His, or Lys) is replaced by or with a negatively charged residue (e.g., Glu or Asp), (b) a hydrophilic residue (e.g., Ser or Thr) is replaced by or with a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val), (c) Cys or Pro is replaced by or with another residue, or (d) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) is replaced by or with one having a smaller side chain (e.g., Ala or Ser).

[0148] Other substitutions can be easily identified. For example, for the amino acid alanine, the substitution can be any one of D-Ala, Gly, beta-Ala, L-Cys, and D-Cys. For lysine, the substitution can be any one of D-Lys, Arg, D-Arg, homo-Arg, Met, D-Met, ornithine, or D-ornithine. In general, substitutions in functionally important regions that are expected to induce changes in the properties of the isolated polypeptide are (a) a polar residue (e.g., Ser or Thr) replaced with (or by) a hydrophobic residue (e.g., Leu, Ile, Phe, or Ala), (b) a Cys residue replaced with (or by) any other residue, (c) a residue with a positively charged side chain (e.g., Lys, Arg, or His) replaced with (or by) a residue with a negatively charged side chain (e.g., Glu or Asp), or (d) a residue with a bulky side chain (e.g., Phe) replaced with (or by) one without such a side chain (e.g., Gly). The likelihood that one of the above non-conservative substitutions may alter the functional properties of a protein also correlates with the location of the substitution with respect to the functionally important regions of the protein. Thus, some non-conservative substitutions may have little or no effect on biological properties.

[0149] There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequence which can encode that protein, as defined by the genetic code (shown below).Similarly, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

[0150] Genetic code Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp, D) GAC, GAT Cysteine ​​(Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAG Glutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGT Histidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAG Methionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P) CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT Threonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAC, TAT Valin (Val, V) GTA, GTC, GTG, GTT Termination signals (stop) TAA, TAG, TGA

[0151] An important and well-known feature of the genetic code is its degeneracy, whereby more than one coding nucleotide triplet can be used for most amino acids used to make proteins (see above). Thus, many different nucleotide sequences can code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (some organisms may translate some sequences more efficiently than others). Furthermore, sometimes methylated variants of purines or pyrimidines can be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0152] In view of the foregoing, a DNA or RNA nucleotide sequence (or any portion thereof) encoding a polypeptide of the invention can be used to derive a fusion protein or polypeptide amino acid sequence using the genetic code to translate the DNA or RNA into an amino acid sequence. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence capable of encoding the fusion protein or polypeptide can be deduced from the genetic code (which, due to its degeneracy, generates multiple nucleic acid sequences for any given amino acid sequence). Thus, any description and / or disclosure herein of a nucleotide sequence encoding a fusion protein or polypeptide should be considered to also include a description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, any description and / or disclosure herein of a fusion protein or polypeptide amino acid sequence should be considered to also include a description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0153] Finally, nucleic acid and amino acid sequence information for nucleic acid and polypeptide molecules useful in the present invention is well known in the art and is readily available in publicly available databases, such as the National Center for Biotechnology Information (NCBI). For example, exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases are provided in Table 1 below.

[0154] [Table 1] TIFF0007659499000002.tif255159TIFF0007659499000003.tif255158TIFF000 7659499000004.tif255161TIFF0007659499000005.tif255156TIFF00076594990 00006.tif255160TIFF0007659499000007.tif255162TIFF0007659499000008.t if255162TIFF0007659499000009.tif255162TIFF0007659499000010.tif222170

[0155] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, within the skill of the art.

[0156] The present invention includes, for example, the following embodiments: [Embodiment 1] A polypeptide comprising an MFG-E8 polypeptide comprising an epidermal growth factor (EGF)-like domain, a C1 domain, and optionally a signal peptide, (a) lacks a functional C2 domain, and / or (b) A polypeptide lacking a medin polypeptide or a fragment thereof. [Embodiment 2] (a) The polypeptide is deleted of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 amino acids within a C-terminal domain that includes amino acids 226 to 335 of the MFG-E8 polypeptide; (b) the polypeptide comprises at least 180, 190, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, or 280 amino acids of an MFG-E8 polypeptide; (c) the polypeptide comprises at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of amino acids 1 to 225 of SEQ ID NO: 10 or SEQ ID NO: 12; and / or (d) The polypeptide of embodiment 1, wherein the polypeptide comprises at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of amino acids 24 to 225 of SEQ ID NO: 10 or SEQ ID NO: 12. [Embodiment 3] A polypeptide described in embodiment 1 or 2, further comprising a heterologous sequence, optionally comprising a FLAG tag, a HIS tag, and / or an Fc portion of an immunoglobulin. [Embodiment 4] The polypeptide described in embodiment 3, wherein the heterologous sequence increases the half-life of the polypeptide in vivo. [Embodiment 5] A polypeptide described in any one of embodiments 1 to 4, wherein the polypeptide is not glycosylated. [Embodiment 6] The polypeptide, (a) It can reduce the expression level of TGF-β1; (b) can reduce TGF-β signaling; (c) reducing phosphorylation of SMAD2 and / or ERK; (d) capable of increasing NOTCH signaling; (e) reducing fibrosis-associated gene expression, optionally including fibrosis-associated genes Col1a1, Col1a2, and / or Acta2; (f) reducing the interaction between TGF-β receptor 1 (TGFBR1) and one or more integrins, optionally including integrin β3 and / or integrin β5; (g) can reduce hepatic stellate cell (HSC) proliferation; (h) It can reduce the expression levels of matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 12 (MMP12), TMP2, ERK, and SMAD2; (i) capable of increasing collagenase activity; and / or (j) A polypeptide according to any one of embodiments 1 to 5, which is capable of increasing collagen uptake by macrophages. [Embodiment 7] A pharmaceutical composition comprising the polypeptide according to any one of embodiments 1 to 6, and one or more pharma- ceutically acceptable carriers and / or diluents. [Embodiment 8] An isolated nucleic acid molecule encoding the polypeptide according to any one of embodiments 1 to 6. [Embodiment 9] The nucleic acid molecule comprises: (i) nucleotides 61 to 735 of SEQ ID NO: 9 or SEQ ID NO: 11, and / or (ii) Nucleotides 130 to 735 of SEQ ID NO: 9 or SEQ ID NO: 11 9. The nucleic acid molecule of embodiment 8, having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to [Embodiment 10] A vector comprising the nucleic acid molecule described in embodiment 8 or 9, which is optionally an expression vector. [Embodiment 11] A host cell comprising the vector described in embodiment 10. [Embodiment 12] The host cell of embodiment 11, wherein the host cell is Pichia pastoris. [Embodiment 13] A method for producing a polypeptide, comprising the step of culturing a host cell described in embodiment 11 or 12 in a medium to produce the polypeptide. [Embodiment 14] A method for increasing macrophage activity, comprising a step of contacting macrophages with a polypeptide described in any one of embodiments 1 to 6, wherein the activity optionally includes uptake of collagen or fibrous tissue. [Embodiment 15] A method for reducing or inhibiting proliferation of HSCs, comprising a step of contacting HSCs with a polypeptide described in any one of embodiments 1 to 6. [Embodiment 16] A method for reducing or inhibiting fibrosis in a subject, comprising the step of contacting cells of the subject with a polypeptide described in any one of embodiments 1 to 6. [Embodiment 17] A method for reducing or inhibiting adiposity in a subject, comprising the step of contacting the subject's cells with a polypeptide described in any one of embodiments 1 to 6. [Embodiment 18] A method for treating or preventing a disorder in a subject in need thereof, comprising administering to the subject a polypeptide described in any one of embodiments 1 to 6. [Embodiment 19] The method of embodiment 18, wherein the disorder is fibrosis (chronic or acute), liver cirrhosis, steatosis, nonalcoholic steatohepatitis (NASH), and / or pulmonary fibrosis. [Embodiment 20] The method of embodiment 18, wherein the disorder is idiopathic pulmonary fibrosis (IPF). [Embodiment 21] The method of embodiment 20, wherein the polypeptide alters the expression level of an IPF biomarker, and optionally the polypeptide reduces the expression level of at least one biomarker selected from αSMA, collagen (Col1a1), TMP2, MMP2, MMP12, phosphorylated ERK, ERK, phosphorylated SMAD2, and SMAD2. [Embodiment 22] The method of embodiment 18, wherein the disorder is myocardial infarction. [Embodiment 23] The method of embodiment 22, wherein the polypeptide increases cardiac function, and optionally the polypeptide increases left ventricular ejection fraction and / or fractional shortening. [Embodiment 24] The method of embodiment 22 or 23, wherein the polypeptide inhibits or reduces fibrosis associated with myocardial infarction. [Embodiment 25] The method of embodiment 18, wherein the disorder is Alzheimer's disease. [Embodiment 26] The method of embodiment 25, wherein the subject has (i) at least one mutation in amyloid precursor protein (APP) selected from K670N / M671L, I716V, and V717I, and / or (ii) at least one mutation in PSEN1 selected from M146L and L286V. [Embodiment 27] The method of embodiment 25 or 26, wherein the polypeptide improves memory loss and / or behavior associated with Alzheimer's disease.

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[0049] (i) the amount of amyloid plaques; (ii) the amount of amyloid beta; (iii) the number of microglia; (iv) neuroinflammation, and / or (v) Glial fibrillary acidic protein (GFAP) levels 28. The method of embodiment 25, 26 or 27, wherein [Embodiment 29] The method of any of embodiments 16 to 28, wherein the polypeptide is administered via an intravenous, subcutaneous, intraarterial, intraperitoneal, or intramuscular route. [Embodiment 30] The method of any one of embodiments 16 to 29, wherein the subject is a mammal, and optionally the mammal is a human, a mouse, or a rat. [Embodiment 31] The method of any one of embodiments 16 to 30, further comprising administering to the subject an additional agent for treating the disorder. [Embodiment 32] The method of any one of embodiments 16 to 31, wherein the method does not induce amyloid formation in the subject. [Embodiment 33] A kit comprising a polypeptide described in any one of embodiments 1 to 6. The following examples are offered by way of illustration and not by way of limitation.

[0157] [Example] The foregoing description of specific embodiments fully reveals the general nature of the present disclosure, such that such specific embodiments may be easily modified and / or adapted for various applications without undue experimentation by applying knowledge within the scope of the art without departing from the general concept of the present disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It should be understood that the phrases or terms used herein are for purposes of description and not limitation. Thus, the terms or terms used herein should be interpreted by those skilled in the art in light of the teachings and guidance.

[0158] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0159] All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was indicated to be incorporated by reference for all purposes.

[0160] [Example 1] Materials and Methods Induction of rodent fibrosis model and efficacy test of NP-011 To compare the efficacy of different derivatives of MFG-E8 (NP-011, NP-012, and NP-013) and full-length protein MFG-E8, 200 mg / kg of TAA (thioacetamide, Sigma-Aldrich, St. Louis, MO, USA) was administered intraperitoneally to 5-6 week-old male C57BL / 6 mice (n=4 in each group, 3 times / week, 8 weeks) with 160 μg / kg of protein each. For analysis, mice were sacrificed 3 days after administration of protein. Various doses of NP-011 (20 μg / kg-160 μg / kg, n=5 in each group) were further tested in the same TAA-induced liver fibrosis model. To test the antifibrotic effect of multiple doses of NP-011, TAA was injected into mice for 12 weeks, then 40 μg / kg NP-011 was administered intraperitoneally 1 to 6 times at 5-day intervals in the TAA-induced liver fibrosis model (n=4 in each group). In the progressive liver fibrosis model, TAA was injected into mice for 4 weeks, then 40 μg / kg NP-011 and TAA were co-administered intraperitoneally to mice 3 times a week for another 4 weeks (n=5 in each group). In all animal experiments, mice were sacrificed 3 days after the last NP-011 administration, and all experimental procedures were approved by the Animal Care and Use Committee of Korea University (KOREA-2016-0254).

[0161] DMN-induced liver cirrhosis model induction and efficacy test of NP-011 An animal model of liver cirrhosis was designed and performed by the National Center of Efficacy Evaluation for the Development of Health Products Targeting Digestive Disorders (NCEED, Incheon, Korea). Liver cirrhosis was induced in 6-week-old male Sprague-Dawley rats (Orient Bio, Gapyeong, Korea) by intraperitoneal injection of dimethylnitrosamine (DMN, 10 mg / ml / kg in saline) for 3 consecutive days every week for 6 weeks. To minimize the weight deviation between groups, on the first day and the third week of the experiment, rats were randomly divided into seven groups: G1 (vehicle control, n=10), G2 (fibrosis control, n=12), G3 (NP-011 treatment, once a week, n=10), G4 (NP-011 treatment, twice a week, n=10), G5 (NP-011 treatment, three times a week, n=10), and G6 (NP-011 treatment, daily, n=10) twice. Administration of NP-011 was started from the third week using 40 μg / kg intravenously (G3-G6). General conditions were observed daily and body weights were measured twice a week. At the end of the sixth week, all groups of surviving rats were sacrificed for necropsy, and the survival rate of each group was calculated. Rats were anesthetized with isoflurane, and blood samples and major organs were collected. After fixing the liver tissues in 10% neutral buffered formalin solution, histological evaluation and immunohistochemistry were performed using hematoxylin and eosin staining and α-smooth muscle actin antibody from PostBio Inc. (Guri, Korea).

[0162] MCD diet-induced NASH mice Six-week-old male C57BL / 6 mice were fed a standard diet or a methionine- and choline-deficient (MCD) diet for 10 weeks. The procedures used for animal handling and care were approved by the Korea University Animal Care and Use Committee (KUIACUC-2018-3). After 5 weeks, mice were fed the MCD diet for an additional 5 weeks and injected intraperitoneally with saline or NP-011 (20 μg, 40 μg, 80 μg, or 160 μg / kg body weight, once a week, n ​​= 5). Body weight was measured once a week during the experimental period.

[0163] Immunofluorescence assay Liver tissues were fixed in 4% paraformaldehyde (PFA) and dehydrated in a graded ethanol series. Tissues were then cleared in xylene and embedded in paraffin. Paraffin-embedded tissue sections were stained with Sirius Red (American MasterTech Scientific, Lodi, CA, USA) to assess liver fibrosis. For immunofluorescence staining, sections were subjected to antigen retrieval with citric acid, blocked with 10% donkey serum in PBS, and probed overnight at 4°C with primary antibodies against α-smooth muscle actin (α-SMA) or albumin (ALB). For visualization of staining, sections were washed with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and stained with fluorescently labeled secondary antibodies (Invitrogen / Thermo Fisher Scientific, Carlsbad, CA, USA). Digital images were captured using a microscope (Nikon, Tokyo, Japan) and analyzed using ImageJ software.

[0164] Histopathological analysis and NAS scores Liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) according to standard procedures. Histological scoring of liver lesions was assessed by the grading system for rodent NAFLD (1): macrodroplet steatosis (0–3), microdroplet steatosis (0–3), hypertrophy (0–3), and number of inflammatory foci / field (0–3). All cells in five randomly selected images were counted for each histological feature and calculated. For Oil Red O staining, OCT-embedded frozen livers were sectioned at 4 μm and stained with Oil Red O staining kit (Lifeline cell technology, Frederick, MD, USA, Cat. LL-0052). Oil Red O stained areas were measured using Image J. For immunostaining of frozen sections, OCT-embedded frozen livers were sectioned at 4 μm, slides were permeabilized and masked with 0.1% Triton X-100 and 10% donkey serum. Liver sections were incubated with primary and secondary antibodies identified in Table 2.

[0165] TGF-β luciferase signaling reporter assay Luciferase signaling reporter arrays were performed according to the manufacturer's protocol (Qiagen, Hilden, Germany). Detailed methods for whole transcriptome analysis are described in Supplementary Materials and Methods.

[0166] Whole-transcriptome analysis of mouse liver Normal mouse livers (n = 3), TAA-induced fibrotic livers (n = 3), and NP-011-treated livers (n = 3) were homogenized in cold Trizol (Sigma, USA), and next-generation sequencing (NGS) analysis was performed by BGI Tech Solutions (Hong Kong). Gene set amplification analysis (GSEA) was performed using GSEAv17 (Broad Institute, Cambridge, MA, USA). Detailed methods for whole-transcriptome analysis were described in Supplementary Materials and Methods.

[0167] Quantitative reverse transcription PCR (RT-qPCR) Quantitative polymerase chain reaction (qPCR) was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with iQ™ SYBR® Green Supermix (Bio-Rad). The specific primers used are shown in Table 3. The mRNA levels were normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels.

[0168] Cell culture and reagents The human hepatic stellate cell (HSC) line hTERT-HSC was cultured in Dulbecco's modified Eagle's medium (DMEM, GE Healthcare Life Sciences, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco / Thermo Fisher Scientific, Waltham, MA, USA), 100 U / mL penicillin, and 100 mg / mL streptomycin (Gibco). Human HEK-293FT cells were kindly provided by Prof. Hyunggee Kim (Korea University) and cultured in Dulbecco's modified Eagle's medium (DMEM, GE Healthcare Life Sciences, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, NY, USA), 100 U / mL penicillin, and 100 mg / mL streptomycin (Gibco).

[0169] Western blot and immunoprecipitation (IP) analysis Protein samples were prepared by solubilizing HSCs in RIPA lysis buffer (LPS solution) containing proteinase inhibitors (Roche, Basel, Switzerland). A total of 40 μg of protein from cells was separated by SDS-PAGE (Bio-Rad) and transferred to a PVDF transfer membrane (Pall Corporation, Port Washington, NY, USA). The membrane was incubated with 5% skim milk in TBS-T (10 mM Tris-HCl pH 7.9, 150 mM NaCl, and 0.05% Tween®-20) for 60 min to block nonspecific antibody binding sites. After blocking, the membrane was immunoblotted with primary antibodies overnight at 4°C. The antibodies used in this study are listed in Table 2. For immunoprecipitation (IP) analysis, a total of 400 μg of protein was incubated with 1 μg of TGFβRI antibody conjugated to protein A / G Sepharose beads (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 12 h at 4°C, washed in lysis buffer, and then separated on an SDS-PAGE gel. To detect each band in the Western blot and IP analysis, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Fisher Scientific) for 2 h at room temperature. After rinsing with TBS-T, the membrane was developed using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) to detect the bands using a chemiluminescence imaging system (GE Healthcare Life Sciences).

[0170] Efficacy Study of NP-011 in a 3D Human Liver Fibrosis Model To establish a human liver fibrosis model, liver spheroids were formed by a mixture of hepatocytes (Hepatosight-S®, NEXEL, Seoul, South Korea) and hTert-HSCs (kindly provided by Dr. David Brenner, University of California at San Diego, Ca, USA) in ultra-low attachment 96-well plates (Corning) with a cell density ratio of 2:1, respectively. The liver spheroids were cultured for 21 days and treated with 50 mM acetaminophen (APAP) to induce fibrosis. To test the efficacy of NP-011 on the APAP-induced 3D liver fibrosis model, 500 ng / ml NP-011 was treated for 48 h.

[0171] In vitro studies on HSC activation To examine the effect of NP-011 on TGF-β1-mediated HSC activation, a human HSC line (hTERT-HSC) was grown in the presence of serum and starved in DMEM containing 0.2% FBS for 24 h before TGF-β1 treatment. Serum-starved HSC were pretreated with 10 ng / mL TGF-β1 for 1 h, and HSC were exposed to 100–1500 ng / mL NP-011 for 6 h. To block integrin avβ3 / β5 in HSC, HSC were pretreated with 1 μM CT (Selleck Chemicals, Houston, TX, USA) for 2 h and then treated with TGF-β1. Activation and inactivation of HSC were quantitatively determined by 5-ethynyl-2'-deoxyuridine (EdU) assay.

[0172] Proximity Ligation Assay (PLA) For PLA uptake assays, 2 × 10 cells were plated per well on 18 mm circular coverslips in 12-well plates. 4Human HSCs were seeded with cells and cultured for 24 h. After 24 h of serum starvation, cells were treated with 10 ng / ml TGF-β1 and / or 500 ng / ml NP-011 and then incubated for an additional 30 min. PLA uptake was assessed using the Duolink In Situ Red Starter Kit (Merck) according to the manufacturer's instructions. Digital images of PLA-positive cells were captured using a microscope (Nikon) and analyzed using ImageJ software.<https: / / imagej.nih.gov / ij / > The analysis was carried out using

[0173] EdU incorporation assay For EdU incorporation assays, 2 × 10 cells per well in a 12-well plate 4 Human HSCs were seeded with cells and cultured for 24 h. After treatment with TGF-β1 and / or NP-011, serum-starved HSCs were incubated with EdU (10 μM) for an additional 6 h and EdU incorporation was assessed using the Click-iT EdU Imaging Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Digital images of EdU-positive cells were captured using a microscope (Nikon) and analyzed using ImageJ software.

[0174] Collagenase activity assay Collagenase activity assay was performed according to the manufacturer's protocol (Chondrex, WA, USA). The enzyme source was mixed with 180 μl of solution B. The enzyme reaction was started by mixing with 200 μl of 1.0 mg / mL FITC-labeled bovine collagen I substrate and then incubated at 37°C for 1 h. For negative controls, the enzyme reaction was performed without collagen substrate or enzyme source. To stop the enzyme reaction, 10 μl of 10 mM o-phenanthroline and 10 μl of 38.5 μM elastase were added to each sample, followed by incubation at 37°C for 10 min. Finally, a 400 μl aliquot of extraction buffer was thoroughly mixed with the reaction solution, which was then centrifuged at 10,000 rpm for 5 min. The supernatant (200 μl) was used to measure the FL intensity at 520 nm using a spectrofluorometer during 490 nm excitation in a black 96-well plate.

[0175] THP-1 differentiation and fluorescent bead phagocytosis assay THP-1 cells were purchased from ATCC and cultured in RPMI-1640 (Gibco, 31800-022) containing 10% heat-inactivated fetal bovine serum (Gibco, 16000-044) and 50 μM β-mercaptoethanol at 37 °C in a 5% CO2 incubator. THP-1 cells were differentiated into macrophages using 200 ng / ml phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) for 48 h, followed by 72 h in PMA-free medium. For phagocytosis assays, THP-1 cells were cultured at 1.05 × 10 5 cells / cm 2 Carboxylate-modified microspheres, 2.0 μm, yellow-green fluorescent beads (Invitrogen, F8827) were washed in THP-1 cell culture medium and resuspended in serum-free RPMI-1640 at a final dilution of 1:500. Cells were incubated with the fluorescent beads for 4 hours at 37°C in a 5% CO2 incubator. Cells were separated with TrypLE (Gibco, 12604-021) and measured by flow cytometry (Accuri C6 Plus).

[0176] Biodistribution studies Six-week-old male C57BL / 6 mice were randomly assigned into two groups: control (saline) and NP-011-treated (NP-011 in saline, 160 μg / kg body weight) groups, with three animals at each time point. Mice were intravenously injected and maintained at different time points. Mice were sacrificed by CO2 inhalation, and organs (brain, heart, lungs, liver, kidneys, and spleen) were directly collected in PBS to remove fat and blood traces. Organs were immediately stored at -80 °C. The procedures used for handling and care of animals were approved by the Korea University Animal Care and Use Committee (KUIACUC-2018-0027 and KUIACUC-2018-0040). Whole organs were homogenized in Lysis buffer (RayBiotech, Norcross, GA, USA, Cat. EL-LYSIS) with cOmplete™ protease inhibitor cocktail (Sigma-Aldrich Cat. 11697498001) at 4°C. After homogenization, samples were centrifuged at 13,000 rpm at 4°C for 20 min and the supernatant was collected for ELISA analysis. Protein concentration of each sample was calculated using Bio-Rad Bradford. To detect NP-011, Human MFG-E8 Quantikine ELISA kit (R&D. Cat. DFGE80) was performed according to the manufacturer's instructions. Results were measured at 450 nm, 540 nm and 570 nm by SpectraMax® iD3 (Molecular Devices. San Jose, CA, USA).

[0177] 28-day repeated dose chronic toxicity study A 4-week repeated toxicity study of NP-011 was conducted by ChemOn Inc. (a non-clinical CRO company, Gyeonggi-DO, Korea). A total of 30 SD rats (15 males and 15 females, 6 weeks old) were randomly divided into three groups, G1 (vehicle control), G2 (NP-011 0.2 mg / kg, daily), and G3 (NP-011 2.2 mg / kg, daily), with 5 male rats and 5 female rats per group, respectively. Vehicle and NP-011 were administered via the tail vein of the rats. Body weight, food consumption, and water consumption were measured in all groups before the start of the study and once a week during the study. After the completion of the study, all rats were sacrificed and blood was collected for ophthalmology, urinary, hematology, biochemistry, and histopathology examinations. Histopathological evaluation was performed on all major organs and regions (brain, liver, heart, spleen, lungs, kidneys, bone marrow-sternum, injection site, testis / uterus, and thymus).

[0178] statistical analysis Values ​​were expressed as the mean ± SEM of at least three independent experiments performed in triplicate for in vitro testing. To evaluate the antifibrotic effect of NP-011 in vivo, at least three animals per group were used in each experiment, and data were obtained from two to three independent experiments unless indicated. The percentage of positive areas of Sirius red staining or immunostaining in the total image area was measured using ImageJ software and expressed as relative values ​​compared with those in normal liver or control cell cultures, which were arbitrarily set as 1. The Student's t-test was used to analyze the statistical significance of differences between corresponding groups. One-way analysis of variance (ANOVA) was used to test the statistical significance of differences between multiple groups (more than two groups). Data were expressed as the mean and 95% confidence interval (CI). All statistical tests were two-sided, and data with P<0.05 or P<0.01 were assumed to be statistically significant.

[0179] [Table 2]

[0180] [Table 3]

[0181] [Example 2] NP-011 inhibits liver fibrosis Human MFG-E8 contains three domains: an N-terminal signal peptide, an epidermal growth factor (EGF)-like domain with an arginine-glycine-aspartic acid (RGD) motif, and C domains (C1 and C2). It is well known that MFG-E8 regulates inflammatory responses through RGD motif-mediated binding to immune cells and engulfment of phosphatidylserine (PS)-expressing apoptotic cells, but how MFG-E8 is involved in antifibrotic activity is unclear. A recent report showed that the C2 domain of MFG-E8 plays an important role in recognizing PS in apoptotic cells, suggesting that the C2 domain may be important for the overall function of MFG-E8, including its antifibrotic activity. To test this, two different truncated forms of MFG-E8 (NP-011: EGF-C1 domain and NP-012: EGF-C2 domain) were synthesized in this example along with the EGF domain to maintain the RGD motif for cell binding. As a control, full-length MFG-E8 (NP-013) was also synthesized.

[0182] Subsequent efficacy testing in a thioacetamide (TAA)-induced liver fibrosis mouse model (Figure 1A) revealed that administration of commercial MFG-E8 or NP-013 effectively reduced the fibrotic area (Sirius red stained area, Figures 1B and 1C) and downregulated the expression levels of liver fibrosis-related genes (Col1a1, Col1a2, and Acta2) (Figure 1D). However, it was more convincing that administration of NP-011 substantially eliminated the fibrotic area and significantly downregulated the expression levels of fibrotic genes in the injured liver than administration of MFG-E8 and NP-013 (Figures 1B-1C). This was surprising because NP-011 lacks the C2 domain, which was thought to be important for the functions of MFG-E8, including the antifibrotic action of MFG-E8. Unlike the strong efficacy of NP-011, administration of NP-012 was less effective in reducing fibrotic area than MFG-E8 and NP-013 (Figure 1B and 1C), and significantly upregulated the expression of Acta2 (alpha smooth muscle actin, α-SMA) in injured liver (Figure 1D). Signaling reporter assay revealed that NP-011 rapidly suppressed TGF-β signaling within 30 min, whereas MFG-E8 could suppress TGF-β signaling 2 h after treatment (Figure 1E). GSEA further identified that administration of NP-011 significantly enhanced Notch signaling in whole transcriptome analysis of injured liver (Figure 1F), which is associated with the regenerative process of injured liver (15). These results indicate that deletion of the C2 domain of MFG-E8 unexpectedly results in a stronger antifibrotic effect compared to the full-length protein.

[0183] [Example 3] NP-011 significantly reverses liver fibrosis at the lowest dose To investigate the effective dosage of NP-011, different doses of NP-011 (20 μg / kg, 40 μg / kg, 80 μg / kg, and 160 μg / kg) were administered in a TAA-induced liver fibrosis model, and fibrosis factors were analyzed 3 days after NP-011 administration (Figure 2A). Administration of TAA significantly increased the fibrotic area in mouse liver (Figure 2B and Figure 2C, sham). In contrast to sham-treated liver tissue, all NP-011-administered groups ranging from 20 to 160 μg / kg showed a significant decrease in the fibrotic area (Figure 2B and Figure 2C). Consistent with the reduction in fibrosis, the important fibrosis marker Acta2 (α-SMA, a marker of myofibroblast-like cells differentiated from HSCs), and other fibrosis-related genes were notably and significantly downregulated at both the mRNA and protein levels after NP-011 administration (Figure 2D and Figure 2E). Of note, administration of doses higher than 40 μg / kg resulted in stable and significant suppression of TGF-β mRNA expression with little individual variation (Figure 2F). Collectively, these results imply that NP-011 exhibits significant therapeutic effects at low dosage ranges, and most effectively, administration of 40 μg / kg of NP-011 may be the minimally effective dose, resulting in a consistent reduction in TGF-β expression in the injured liver.

[0184] [Example 4] The lowest dose of NP-011 shows therapeutic efficacy in different models related to fibrosis The minimal effective dose of NP-011 (40 μg / kg) was further tested in different liver fibrosis models. First, the efficacy of repeated administration of NP-011 was tested in a chronic model of liver fibrosis. For this, TAA injections were extended to 8–12 weeks (3 times per week), after which 40 μg / kg of NP-011 was administered 1–6 times at 5-day intervals to mice (Figure 3A). Despite the TAA injections being stopped 30 days prior, 12 weeks of TAA injections led to the development of persistent fibrotic areas in the liver, after which the mice were sacrificed (Figure 3B, Sham). In contrast, administration of NP-011 significantly disappeared fibrotic areas in the injured liver, and the regression of fibrosis showed a positive correlation with the administration time (Figure 3B).

[0185] Liver fibrosis is a progressive disease, therefore, evaluation of the efficacy of NP-011 in progressive liver fibrosis will provide a better translation of animal studies to the clinical domain. To confirm the therapeutic effect of NP-011 in progressive liver fibrosis, NP-011 (40 μg / kg, 3 times a week) was combined with TAA and administered for the final 4 weeks of model induction (Figure 3C). As expected, co-administration of TAA and NP-011 significantly reduced the fibrotic area of ​​the liver compared to livers that received TAA alone (Figure 3D). In addition, using an APAP-induced in vitro human fibrosis model, we found that NP-011 significantly reduced APAP-induced HSC activation in 3D liver spheroids composed of hepatocytes and hepatic stellate cells (Figure 3E).

[0186] The efficacy of NP-011 was further tested in a dimethylnitrosamine (DMN)-induced liver cirrhosis model (Figure 4A) (16). Injection of rats with DMN for 6 weeks resulted in 75% mortality and severe fibrosis in the liver (Figure 4B and Figure 4C). Histopathological analysis showed that significant necrotic (piecemeal and lobular) and fibrotic events occurred in the liver of the DMN-induced liver cirrhosis model (Figure 4D). Importantly, daily administration of NP-011 reduced animal mortality by 50% and markedly attenuated liver fibrosis (Figure 4B and Figure 4C), as well as reduced fragment necrosis (~20%), lobular necrosis (~30%), fibrosis score (~24.3%), and HSC activation (~17.7%) (Figure 4D).

[0187] Finally, the efficacy of NP-011 in a methionine-choline deficient (MCD) diet-induced NASH model was evaluated (Figure 5A). As reported in various studies, the MCD diet led to fat accumulation in the liver, but administration of NP-011 significantly reduced fat accumulation at all doses tested (Figures 5B and C). Furthermore, a significant reduction in macrodroplet / microdroplet steatosis and hypertrophy was observed after NP-011 administration (Figure 5D). In summary, NP-011 showed therapeutic effects not only in severe liver fibrosis but also in NASH at the lowest dose.

[0188] [Example 5] NP-011 reverses liver fibrosis by interacting with hepatic stellate cells (HSCs) and macrophages in the injured liver. In this example, we investigated the mechanism of action of NP-011 on the rapid and effective suppression of TGF-β signaling observed in diseased liver (Figures 1E and 2F). One day after administration of NP-011 in TAA-induced fibrotic liver, NP-011 preferentially bound to α-SMA-positive HSCs, and a portion of NP-011 interacted with hepatic macrophages (Figure 6). Integrins αvβ3 and αvβ5 on the HSC surface have been previously implicated in regulating fibrosis. Furthermore, crosstalk between integrins and TGF-β signaling may be important in regulating pathological epithelial to mesenchymal transition (EMT) and myofibroblast differentiation. Proximity ligation assays (PLA) revealed a direct physical interaction between TGFBRI and integrin β3 / β5, and the interaction was stronger upon TGF-β1 treatment in HSCs (Figures 7A and 7B). However, NP-011 treatment in TGF-β1-treated HSCs significantly reduced the interaction between TGFBRI and integrin β3 / β5 (Figure 7A and Figure 7B). Western blot analysis showed that NP-011 treatment significantly attenuated the phosphorylation of Smad2, a downstream molecule of TGF-β signaling, in TGF-β1-treated HSCs, and the interaction between TGFBRI and integrin β3 / β5 was further confirmed by immunoprecipitation assay (Figure 7C). As a result of NP-011 treatment in TGF-β1-treated HSCs, proliferative HSCs returned to a quiescent state (Figure 7D). However, treatment with integrin β3 / β5 inhibitor in the presence of NP-011 abolished the suppressive role of NP-011 in the proliferation of TGF-β1-treated HSCs (Figure 7D). Thus, these results indicate that NP-011 directly binds to integrin β3 / β5 and prevents its interaction with TGFβRI, resulting in inhibition of the TGF-β cascade and reduced HSC proliferation.

[0189] The effect of NP-011 on the expression of profibrotic MMP2 and collagenase activity in HSCs was further tested based on previous findings showing the secretion of profibrotic MMP2 and expression of collagenase mRNA in rat HSCs. The expression of MMP2 mRNA in HSCs was increased in HSCs after TGF-β1 treatment. However, NP-011 treatment in TGF-β1-treated HSCs significantly downregulated the increased expression of MMP2 (Figure 8A). In contrast, collagenase activity was downregulated in HSCs after TGF-β1 treatment, and the decreased collagenase activity was restored in TGF-β1-treated HSCs after NP-011 treatment in both cell lysates and conditioned medium (Figures 8B and C).

[0190] Removal of accumulated collagen by immune response is another important factor for resolving fibrosis, as seen in the pulmonary fibrosis model. To evaluate this, we differentiated THP-1 monocytes into macrophages by PMA treatment (Figure 9A), and confirmed the phagocytic ability of differentiated macrophages by fluorescence and flow cytometry analysis (Figures 9B and C). Collagen uptake assays revealed that green fluorescently labeled collagen was significantly taken up by macrophages differentiated in the presence of NP-011, unlike control cultures (Figures 9D and E). Indeed, collagen engulfment by macrophages was indicated by immunostaining for collagen and F4 / 80 after NP-011 administration in the TAA-induced liver fibrosis model (Figure 10, arrowheads in the lower panel). Taken together, these results indicate that NP-011 inhibits and resolves liver fibrosis by suppressing TGF-β signaling in the injured liver, inactivating activated HSCs, and interacting with macrophages.

[0191] [Example 6] Biodistribution and Safety Profile of NP-011 The biodistribution and safety studies of NP-011 were further evaluated in rodent models. When NP-011 was administered intravenously to mice, the administered NP-011 was preferentially delivered to the liver, with approximately 48% and 58% of the administered NP-011 being detected in the liver within 30 and 60 minutes, respectively (Figure 11). Furthermore, when NP-011 was administered intravenously to rats at 0.2 mg / kg and 2 mg / kg daily for 4 weeks, no adverse effects were observed in hematological and blood biochemistry tests in both males and females (Figure 11). Thus, these results indicate that NP-011 preferentially targets the liver after administration and has an excellent safety profile.

[0192] NP-011 exhibits potent antifibrotic effects that inhibit, prevent, and reverse liver fibrosis. NP-011 is superior in clinical application compared to full-length MFG-E8 (lactadherin) by cleaving the C2 domain for the following reasons: 1) Structural cleavage of MFG-E8 removes the medin site in NP-011. Medin is an aortic medial amyloid that develops in people over 60 years old, and accumulated medin is involved in Alzheimer's disease and type 2 diabetes. MFG-E8 (lactadherin) has medin as its component, which is located within the C2 domain. Therefore, NP-011, through structural cleavage of the C2 domain from MFG-E8, eliminates concerns about inducing amyloid formation in patients upon administration, as well as concerns about side effects that may cause Alzheimer's disease and diabetes upon repeated administration to patients. Nonalcoholic fatty liver disease (NAFLD), the most common liver disease, is closely associated with type 2 diabetes, and is therefore an important factor for treating liver disease. 2) NP-011 shows better binding affinity to collagen by removing the glycosylation site of MFG-E8. MFG-E8 contains a glycosylation site in the C2 domain. By removing the C2 domain, NP-011 does not have glycosylation that may prevent the binding of the discoidin domain (C1 domain) of MFG-E8 to collagen accumulated in liver fibrosis. The well-known discoidin domain receptor (DDR) has a discoidin domain, a stalk region, and a transmembrane domain in its structure. Although the mechanism of collagen binding of DDR has not been identified, binding of the discoidin domain of DDR to collagen has been reported. Interestingly, the discoidin domain of DDR does not have glycosylation (glycosylation site is located only in the stalk region of DDR), therefore, the discoidin domain of NP-011 may be similar to the discoidin domain of DDR, which may promote the binding of NP-011 to collagen accumulated in the liver.

[0193] NP-011 has therapeutic efficacy in various liver disease models, including nonalcoholic steatohepatitis (NASH). Clinically, nonalcoholic steatohepatitis (NASH) is frequently seen in patients with liver fibrosis, as NASH patients are more likely to progress to fibrosis and cirrhosis, and NASH patients have a nearly 10-fold higher risk of liver disease-related death than patients with simple steatosis. Thus, the therapeutic potential of NP-011 in both NASH and fibrosis represents a novel therapeutic approach to inhibit and reverse complex progressive liver disease.

[0194] High manufacturability is another advantage of NP-011 for clinical applications. NP-011 is produced using a yeast system, which has several advantages including easy genetic manipulation and rapid growth, as well as eukaryotic features including a secretory pathway for correct protein processing and post-translational modification. Currently, the production yield of NP-011 from yeast is 40 mg / L, which provides reliable production for clinical applications. For example, by producing NP-011 at a 200 L GMP (General Good Manufacturing Practice) run scale, approximately 3,400 injections can be made at an effective dosage of 40 μg / kg for a 60 kg adult. Furthermore, NP-011 is produced as a secreted protein (authentic NP-011) without methionine, any tag (e.g., FLAG-tag or its tags) or random glycosylation (which can introduce significant problems when administering pharmaceutical products) at the N-terminus of the protein. In conclusion, therefore, NP-011 provides a highly effective and reliable novel protein therapy for treating, inhibiting, preventing, and reversing liver fibrosis as well as other liver diseases disclosed herein.

[0195] [Example 7] NP-011 reverses, inhibits, and prevents idiopathic pulmonary fibrosis (IPF) As shown in Figures 12-21, NP-011 is highly effective against IPF, reversing and inhibiting IPF in vivo. A model of IPF was induced by injection of bleomycin. Reversal of IPF by NP-011 is evident in histological studies of lung tissue (Figures 17 and 18, compare with Figure 13). Furthermore, NP-011 reverses bleomycin-induced changes in the expression levels of IPF biomarkers, such as αSMA, collagen (Col1a1), TMP2, MMP2, MMP12, pERK, tERK, pSMAD2 (phosphorylated SMAD2), and tSMAD2 (total SMAD2) (Figures 12-21). Thus, NP-011 provides a highly effective therapy for the treatment, inhibition, prevention, and reversal of IPF.

[0196] [Example 8] NP-011 reverses, inhibits and prevents symptoms of myocardial infarction and improves cardiac function As shown in Figures 22-26, NP-011 is highly effective against myocardial infarction in vivo. A rat model of myocardial infarction was induced by LAD ligation. Injection of NP-011 increases cardiac function as evidenced by increased left ventricular ejection fraction (EF) and fractional shortening (FS) in rats with myocardial infarction (Figures 23A-24B). Similar body weights of rats injected with NP-011 compared to control rats demonstrate the safety of NP-011 (Figure 25). Histological studies of cardiac cross sections show that NP-011 also reverses and inhibits fibrosis associated with myocardial infarction (Figures 26A-26B). Thus, NP-011 provides a highly effective therapy for the treatment, inhibition, prevention, and reversal of myocardial infarction.

[0197] [Example 9] NP-011 reverses, inhibits, and prevents Alzheimer's disease As shown in Figures 27-31, NP-011 is highly effective against Alzheimer's disease in vivo. 5XFAD mice expressing human APP and PSEN1 transgenes with a total of five AD-linked mutations: Swedish (K670N / M671L), Florida (I716V) and London (V717I) mutations in APP, and M146L and L286V mutations in PSEN1, were used as models of Alzheimer's disease. NP-011 reverses behavioral and memory loss associated with Alzheimer's disease (Figures 27A-E). NP-011 also reverses and inhibits neuropathological changes in AD brains. For example, NP-011 specifically reduces the amount of amyloid plaques (Figures 28A-B) and amyloid beta (Figures 29A-B). Furthermore, NP-011 reduces the number of microglial cells, the brain's innate immune system, whose presence suggests neuroinflammation (Figures 30A-B). Furthermore, NP-011 reduces the amount of glial fibrillary acidic protein (GFAP), which is upregulated in astrocytes in Alzheimer's disease (Figures 31A-B). Thus, NP-011 provides a highly effective therapy for treating, inhibiting, preventing, and reversing Alzheimer's disease.

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Claims

1. (a) an amino acid sequence having at least 90% sequence identity to amino acid residues 1 to 225 of SEQ ID NO:10 or SEQ ID NO:12; or (b) an amino acid sequence having at least 90% sequence identity to amino acid residues 24 to 225 of SEQ ID NO:10 or SEQ ID NO:

12.

10. A pharmaceutical composition comprising a polypeptide comprising the formula (I) of SEQ ID NO: 10 or SEQ ID NO: 12, wherein amino acid residues 226 to 387 are deleted, and the polypeptide increases macrophage activity.

2. The pharmaceutical composition of claim 1 , wherein the polypeptide further comprises a heterologous sequence.

3. The pharmaceutical composition of claim 2, wherein the heterologous sequence comprises a FLAG tag, a HIS tag, and / or GST.

4. The pharmaceutical composition of claim 2, wherein the heterologous sequence increases the half-life of the polypeptide in vivo.

5. The pharmaceutical composition of claim 2 or 4, wherein the heterologous sequence comprises an Fc portion of an immunoglobulin.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the polypeptide is non-glycosylated.

7. The polypeptide is (a) It can reduce the expression level of TGF-β1; (b) can reduce TGF-β signaling; (c) reducing phosphorylation of SMAD2 and / or ERK; (d) reducing fibrosis-associated gene expression, optionally including fibrosis-associated genes Col1a1, Col1a2, and / or Acta2; (e) reducing the interaction between TGF-β receptor 1 (TGFBR1) and one or more integrins, optionally including integrin β3 and / or integrin β5; (f) can reduce hepatic stellate cell (HSC) proliferation; (g) It can reduce the expression levels of matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 12 (MMP12), TMP2, ERK, and SMAD2; (h) capable of increasing collagenase activity; and / or (i) The pharmaceutical composition according to any one of claims 1 to 6, which is capable of increasing collagen uptake by macrophages.

8. The pharmaceutical composition according to any one of claims 1 to 7, further comprising one or more pharma- ceutically acceptable carriers and / or diluents.

9. 9. The pharmaceutical composition of any one of claims 1 to 8 for use in reducing or inhibiting fibrosis in a subject, increasing macrophage activity in a subject, or reducing or inhibiting proliferation of HSCs in a subject.

10. 9. A pharmaceutical composition according to any one of claims 1 to 8 for use in reducing or inhibiting adiposity in a subject.

11. A pharmaceutical composition according to any one of claims 1 to 8 for use in the treatment or prevention of a disorder in a subject in need thereof.

12. 12. The pharmaceutical composition of claim 11, wherein the disorder is fibrosis (chronic or acute), liver cirrhosis, steatosis, non-alcoholic steatohepatitis (NASH), and / or pulmonary fibrosis.

13. 12. The pharmaceutical composition of claim 11, wherein the disorder is idiopathic pulmonary fibrosis (IPF).

14. The pharmaceutical composition of claim 13, wherein the polypeptide or pharmaceutical composition alters the expression level of an IPF biomarker, and optionally the polypeptide or pharmaceutical composition reduces the expression level of at least one biomarker selected from αSMA, collagen (Col1a1), TMP2, MMP2, MMP12, phosphorylated ERK, ERK, phosphorylated SMAD2, and SMAD2.

15. 12. The pharmaceutical composition of claim 11, wherein the disorder is myocardial infarction.

16. 16. The pharmaceutical composition of claim 15, wherein the polypeptide or pharmaceutical composition increases cardiac function, and optionally the polypeptide or pharmaceutical composition increases left ventricular ejection fraction and / or fractional shortening.

17. 17. The pharmaceutical composition of claim 15 or 16, wherein the polypeptide or pharmaceutical composition inhibits or reduces fibrosis associated with myocardial infarction.

18. 12. The pharmaceutical composition of claim 11, wherein the disorder is Alzheimer's disease.

19. 19. The pharmaceutical composition of claim 18, wherein the subject has (i) at least one mutation in amyloid precursor protein (APP) selected from K670N / M671L, I716V, and V717I, and / or (ii) at least one mutation in PSEN1 selected from M146L and L286V.

20. 20. The pharmaceutical composition of claim 18 or 19, wherein the polypeptide or pharmaceutical composition ameliorates memory loss and / or behavior associated with Alzheimer's disease.

21. wherein the polypeptide or pharmaceutical composition is in the brain of a subject, and optionally in the hippocampus and / or brain cortex of the subject; (i) the amount of amyloid plaques; (ii) the amount of amyloid beta; (iii) the number of microglia; (iv) neuroinflammation, and / or (v) Glial fibrillary acidic protein (GFAP) levels The pharmaceutical composition according to any one of claims 18 to 20, which reduces

22. The pharmaceutical composition according to any one of claims 9 to 21, administered via intravenous, subcutaneous, intraarterial, intraperitoneal or intramuscular routes.

23. The pharmaceutical composition according to any one of claims 9 to 22, wherein the subject is a mammal, optionally wherein the mammal is a human, a mouse, or a rat.

24. The pharmaceutical composition of any one of claims 9 to 23, administered in combination with an additional agent for treating a disorder.

25. The pharmaceutical composition according to any one of claims 9 to 24, wherein the polypeptide or pharmaceutical composition does not induce amyloid formation in a subject.

26. An isolated nucleic acid molecule encoding a polypeptide of the pharmaceutical composition according to any one of claims 1 to 8.

27. The nucleic acid molecule, (i) nucleotides 61 to 735 of SEQ ID NO: 9 or SEQ ID NO: 11, and / or (ii) nucleotides 130 to 735 of SEQ ID NO:9 or SEQ ID NO:11 27. The nucleic acid molecule of claim 26, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to

28. 28. A vector comprising a nucleic acid molecule according to claim 26 or 27, optionally an expression vector.

29. 29. A host cell comprising the vector of claim 28.

30. 30. The host cell of claim 29, wherein the host cell is Pichia pastoris.

31. A method for producing a polypeptide, comprising the step of culturing a host cell according to claim 29 or 30 in a medium to produce the polypeptide.

32. 10. A method for increasing macrophage activity comprising contacting macrophages in vitro with a pharmaceutical composition according to any one of claims 1 to 8, wherein the activity optionally comprises uptake of collagen or fibrous tissue.

33. A method for decreasing or inhibiting proliferation of HSCs, comprising the step of contacting HSCs with the pharmaceutical composition according to any one of claims 1 to 8 (excluding the case where the contact is performed in vivo in humans).

34. 10. Use of a pharmaceutical composition according to any one of claims 1 to 8 in the manufacture of a medicament for reducing or inhibiting fibrosis or steatosis in a subject.

35. 10. Use of the pharmaceutical composition according to any one of claims 1 to 8 in the manufacture of a medicament for treating a disorder in a subject in need thereof.

36. 36. The use according to claim 35, wherein the disorder is fibrosis (chronic or acute), idiopathic pulmonary fibrosis (IPF), liver cirrhosis, steatosis, non-alcoholic steatohepatitis (NASH), myocardial infarction, Alzheimer's disease, and / or pulmonary fibrosis.

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