Compositions and methods for treating and preventing fibrosis

Abstract

The present application relates to compositions and methods for treating and preventing fibrosis and other diseases comprising polypeptides derived from MFG-E8, wherein the polypeptides comprise an epidermal growth factor (EGF)-like domain, a C1 domain, and optionally a signal peptide, wherein the polypeptides: (a) lack a functional C2 domain; and / or (b) lack a medin polypeptide or fragment thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Korean Application No. 10-2018-0128033 / KR, filed on October 25, 2018; Korean Application No. 10-2018-0128625 / KR, filed on October 26, 2018; and Korean Application No. 10-2018-0128204 / KR, filed on October 25, 2018, the entire contents of each of which are incorporated herein by reference.

[0003] Background of the invention

[0004] Tissue repair is part of wound healing and involves two phases. The first phase is regeneration, where damaged cells are replaced by cells of the same type. The second phase is the formation of fibrous tissue, also known as fibrosis or fibrosis, where connective tissue replaces normal parenchymal tissue. If the fibrosis phase continues unchecked, the tissue repair process can become pathogenic, leading to extensive tissue remodeling and the formation of permanent scar tissue.

[0005] Liver fibrosis is characterized by the excessive accumulation of extracellular matrix proteins, including collagen. During the progression of liver fibrosis, damaged and dying hepatocytes recruit Kupffer cells at the lesion site. These Kupffer cells secrete large amounts of cytokines, including transforming growth factor-β1 (TGF-β1), to control liver inflammation. Increased TGF-β1 leads to the activation of quiescent hepatic stellate cells (HSCs), which proliferate and transform into myofibroblast-like cells that produce extracellular matrix (ECM). The activation of HSCs further leads to the excessive accumulation of collagen-rich ECM in the liver, resulting in distortion of normal liver structure.

[0006] Liver fibrosis is caused by chronic inflammatory liver disease or recurrent liver injury. Causes of recurrent liver injury and fibrosis include viral infections (hepatitis B and C), alcohol abuse, and non-alcoholic steatohepatitis (NASH). Liver fibrosis has the potential to develop into cirrhosis and liver cancer, which have a higher mortality rate than other major cancers (lung, colorectal, stomach, or breast cancer). Therefore, liver fibrosis remains a leading cause of death, and treatment options are limited, making treatments to reduce and prevent fibrosis crucial. Invention Overview

[0008] This invention is based, at least in part, on the discovery that a fragment of MFG-E8 (hereinafter referred to as NP-011) is unexpectedly effective in treating and / or preventing diseases including, but not limited to, fibrosis, cirrhosis, steatosis, and non-alcoholic steatohepatitis (NASH).

[0009] In some aspects, this document provides peptides for the treatment and / or prevention of diseases (including, but not limited to, fibrosis, cirrhosis, steatosis, and NASH). In some embodiments, the peptide includes an MFG-E8 peptide comprising an epidermal growth factor (EGF)-like domain, a C1 domain, and an optional signal peptide, but lacking a functional C2 domain. In some embodiments, the peptide includes an MFG-E8 peptide comprising an epidermal growth factor (EGF)-like domain, a C1 domain, and an optional signal peptide, but lacking a medin peptide or a fragment thereof.

[0010] In some embodiments, the MFG-E8 peptide 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 the C-terminal domain of amino acids 226-335 of the MFG-E8 peptide. In some embodiments, the MFG-E8 peptide 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 the MFG-E8 peptide. 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%, 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%, or 100% of amino acids 24-225 of SEQ ID NO:10 or SEQ ID NO:12.

[0011] In the preferred embodiment, MFG-E8 is not glycosylated.

[0012] In some embodiments, the polypeptide further comprises a heterologous sequence, such as a FLAG tag, an HIS tag, and / or the Fc portion of an immunoglobulin. Such a heterologous sequence can prolong the half-life of the polypeptide in vivo.

[0013] In some embodiments, the peptide has one or more biological activities, including but not limited to: reducing TGF-β expression levels; reducing TGF-β signaling; reducing SMAD (e.g., SMAD2, ERK, and / or any other phosphorylated protein downstream of TGF-β) phosphorylation; reducing fibrosis-related gene expression (including but not limited to Col1a1, Col1a2, or Acta2); reducing the interaction between TGF-β and one or more integrins (e.g., integrin β3 and / or integrin β5); reducing hepatic stellate cell (HSC) proliferation; reducing matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 12 (MMP12), TMP2, ERK, and / or SMAD2 expression levels; increasing collagenase activity; and / or increasing macrophage collagen uptake.

[0014] In some embodiments, the polypeptide is in a pharmaceutical composition, which includes the polypeptide and one or more pharmaceutically acceptable carriers and / or diluents.

[0015] This article also provides kits that include peptides.

[0016] In some aspects, this document provides isolated nucleic acid molecules encoding the said polypeptides. 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 identical 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%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with nucleotides 130-735 of SEQ ID NO:9 or SEQ ID NO:11. This document also provides vectors comprising nucleic acid molecules, optionally wherein the vector is an expression vector or a viral vector. In some embodiments, the nucleic acid molecule is operatively 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 facilitate endocytosis and / or a longer in vivo half-life, and / or reduce the immune response to the composition in a subject.

[0017] In some aspects, this document provides viral particles comprising the aforementioned vector. In some embodiments, the viral particles are AAVs. In certain embodiments, AAVs comprising viral vectors are administered to a subject (e.g., a human) to treat and / or prevent diseases such as fibrosis, cirrhosis, steatosis, NASH, myocardial infarction, pulmonary fibrosis, idiopathic pulmonary fibrosis, and / or Alzheimer's disease.

[0018] In some aspects, this document provides a host cell comprising the aforementioned 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.

[0019] In some aspects, this document provides methods for producing peptides by culturing host cells in a culture medium, for example, wherein the host cells secrete the peptide into the culture medium. In some embodiments, the method further includes isolating the peptide from the culture medium and, optionally, further purifying the peptide to produce a substantially pure peptide.

[0020] In some aspects, this document provides methods for reducing or inhibiting fibrosis in a subject by contacting the subject's cells with a peptide. This document also provides methods for reducing or inhibiting HSC proliferation by contacting HSCs with a peptide. Additionally, this document provides methods for reducing or inhibiting steatosis in a subject by contacting the subject's cells with a peptide. In some embodiments, the subject in these methods is a mammal, such as a rat, mouse, or human, preferably a human.

[0021] In some respects, this article provides a method for increasing macrophage activity (e.g., uptake of collagen or fibrotic tissue) by contacting macrophages with peptides. Such an increase in macrophage activity contributes to the reversal of fibrosis in various tissues (e.g., liver, lungs, etc.).

[0022] In some respects, this article provides methods for treating or preventing disorders in subjects by administering peptides to subjects in need. In some implementations, the disorder is fibrosis (chronic or acute), cirrhosis, steatosis, NASH, and / or pulmonary fibrosis.

[0023] In some embodiments, the disclosed peptides treat or prevent idiopathic pulmonary fibrosis (IPF). In addition to reducing or eliminating fibrotic tissue, the peptides can also alter the expression levels of IPF biomarkers. In some cases, the peptides can reduce the expression levels of at least one biomarker selected from αSMA, collagen (Col1a1), TMP2, MMP2, MMP12, phosphorylated ERK, ERK, phosphorylated SMAD2, and SMAD2.

[0024] In other embodiments, the disclosed peptides treat or prevent myocardial infarction. For example, the peptides can improve or enhance cardiac function after myocardial infarction relative to improvements seen in corresponding untreated subjects. For example, the peptides can increase left ventricular ejection fraction and / or fractional shortening. Furthermore, the peptides can inhibit or reduce fibrosis associated with myocardial infarction.

[0025] The present invention also prescribes a method for treating or preventing Alzheimer's disease (AD). The subject of treatment may have AD-related gene mutations. For example, the subject may have at least one mutation in (i) 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 disclosed peptides can improve memory loss and / or behavior associated with Alzheimer's disease. Additionally, the peptides can alter physiological and histopathological changes in the brain. For example, the peptides can reduce the number of amyloid plaques, the amount of β-amyloid protein, the number of microglia, neuroinflammatory (or brain inflammation), and / or the amount of glial fibrillary acidic protein (GFAP) in the subject's brain, preferably in the hippocampus and / or cortex of the subject's brain.

[0026] Subjects can also be treated with adjunctive medications to treat disorders.

[0027] In some implementations, the method does not induce amyloid formation in subjects.

[0028] The peptide can be administered to the subject via any of the various routes (e.g., intravenous, subcutaneous, intra-arterial, intraperitoneal, or intramuscular).

[0029] In some implementations, the subjects in these methods are mammals, such as rats, mice, or humans, preferably humans.

[0030] Brief description of the attached figures

[0031] Figures 1A-1F The study demonstrated that NP-011 inhibits liver fibrosis and showed its advantages in treating liver diseases. Figure 1A A general schematic diagram for testing the efficacy of human recombinant proteins in a liver fibrosis model. Figure 1B Representative images of histological analysis (H&E and Sirius redstaining) in normal mouse liver, a TAA-induced liver fibrosis model (sham), and a liver fibrosis model treated with proteins (MFG-E8, NP-011, NP-012, and NP-013). Scale bar, 200 μm. Figure 1CComparison of quantitative fibrotic regions in mouse livers. Bars represent the mean ± SD of three replicates in each group. *P<0.05, **P<0.01. Figure 1D Comparison of mRNA expression of fibrosis markers (Col1a1, Col1a2, and Acta2) in normal mouse liver, a TAA-induced liver fibrosis model (sham model), and a liver fibrosis model treated with proteins (MFG-E8, NP-011, NP-012, and NP-013). Bars represent the mean ± SD of three replicates in each group. *P < 0.05, **P < 0.01. Figure 1E This assay was used to determine the luciferase reporter gene activity of the TGF-β signaling pathway in human HEK-293FT cells after treatment with NP-011 (500 ng / mL) or NP-013 (500 ng / mL) for 0.5 h, 1 h, and 2 h. Bars represent the mean ± SD of three replicates in each group. *P < 0.05, **P < 0.01. Figure 1F Gene set enrichment assay (GSEA) data showed that the Notch signaling gene set was higher in the NP-011-treated group and the MFG-E8-treated group compared to the sham model group.

[0032] Figures 2A-2F NP-011 was shown to inhibit fibrosis at low doses. Low-dose NP-011 reduced fibrosis. Figure 2A A schematic diagram illustrating the overall efficacy testing of NP-011 in a liver fibrosis model. Figure 2B Representative maps of fibrotic regions (Sirius red staining areas) in normal mouse livers, TAA-induced liver fibrosis models, and NP-011-treated liver fibrosis models were compared across different dosage ranges (20 μg / kg–160 μg / kg). Scale bar: 200 μm. Figure 2C ) Figure 2B Quantitative analysis of fibrotic regions in the livers of tested mice. Bars represent the mean ± SD of four mice in each group. **P < 0.01. Figure 2D α-SMA (Acta2) expression in normal mouse liver, a TAA-induced liver fibrosis model, and a liver fibrosis model treated with NP-011 were compared using quantitative PCR and immunostaining, respectively. Scale bar, 100 μm. Bars represent the mean ± SD of three replicates in each group. **P < 0.01. Figure 2E Heatmap data showed that fibrosis-related genes were observed in normal mouse livers, TAA-induced mice, and mice with liver fibrosis treated with NP-011. Figure 2F Comparison of TGF-β1 mRNA expression in a model of advanced liver fibrosis. Bars represent the mean ± SD of three replicates in each group. **P < 0.01.

[0033] Figures 3A-3E NP-011 was shown to inhibit and prevent liver fibrosis. The therapeutic efficacy of the lowest dose of NP-011 in various induction models was demonstrated. Figure 3A A schematic diagram illustrating the overall efficacy testing of NP-011 in a model of advanced liver fibrosis. Figure 3A Representative maps of fibrotic regions in normal mouse livers, a TAA-induced liver fibrosis model, and a liver fibrosis model treated with 40 μg / kg NP-011 were analyzed at different administration frequencies (1, 2, 4, and 6 times). Scale bar, 50 μm. The left panel provides quantitative analysis of fibrotic regions in normal mouse livers, a TAA-induced liver fibrosis model, and a liver fibrosis model treated with 40 μg / kg NP-011. Bars represent the mean ± SD of four mice in each group. **P < 0.01. Figure 3C A schematic diagram illustrating the overall efficacy testing of NP-011 in a progressive liver fibrosis model. Figure 3D Representative maps of fibrotic regions in normal mouse livers, a TAA-induced liver fibrosis model, and a liver fibrosis model induced by TAA injection combined with NP-011 administration. Scale bar, 100 μm. Quantitative analysis of fibrotic regions in normal mouse livers, a TAA-induced liver fibrosis model, and a liver fibrosis model induced by TAA injection combined with NP-011 administration. Bars represent the mean ± SD of five mice in each group. **P < 0.01. Figure 3E Left panel: Graphical description of a human liver fibrosis model using human embryonic stem cell (hES)-derived stem cells and human primary HSCs treated with APAP. Middle panel: Representative graph of hepatocytes and activated myofibroblasts analyzed using ALB (albumin; red) and α-SMA (fibrosis biomarker Acta2, green). Scale bar, 50 μm. Right panel: Quantitative analysis of the percentage of α-SMA-positive cells in the human liver fibrosis model. Bars represent the mean ± SD of five replicates in each group. **P < 0.01.

[0034] Figures 4A-4D The efficacy of NP-011 in inhibiting and preventing liver cirrhosis was demonstrated in a rat DMN-induced liver cirrhosis model. Figure 4A A schematic diagram of the overall efficacy test of NP-011 in a DMN-induced liver cirrhosis model. Figure 4B Survival curves for a DMN-induced liver cirrhosis model of 10 rats in each group are shown. Figure 4C A representative figure of H&E staining and immunohistochemical analysis of rat liver tissue labeled with α-smooth muscle actin. Figure 4D Quantitative scoring of fibrotic areas, fragment necrosis, and lobular necrosis in normal liver, DMN-induced liver fibrosis model, and NP-011-treated liver cirrhosis model.

[0035] Figures 5A-5D This study demonstrates the efficacy of NP-011 in inhibiting and preventing NASH in a methionine and choline-deficient (MCD) diet-induced NASH model. Figure 5A A schematic diagram of the overall efficacy test of NP-011 in the MCD-fed NASH model. Representative Oil Red O staining images of normal mouse liver, MCD-fed NASH model, and NP-011-treated NASH model. Figure 5B ) and quantification of the relative Oil Red O positive region ( Figure 5C This demonstrates the significant therapeutic efficacy of secretory NP-011. Figure 5D Quantitative determination of macrovesicular steatosis, microvesicular steatosis, and hypertrophy in the liver of each group. Scale bar, 100 μm. Bars represent the mean ± SD of five mice in each group. **P < 0.01, ANOVA was performed, followed by Tukey multiple comparison test.

[0036] Figure 6 The interaction of NP-011 with activated HSCs and macrophages is shown. Representative images of immunostaining of human MFG-E8 (NP-011), mouse α-SMA, and mouse F4 / 80 (macrophage marker) in liver tissue from a TAA-induced liver fibrosis model treated with NP-011 are presented. Scale bar = 20 μm.

[0037] Figures 7A-7D The study showed that NP-011 inhibited the proliferation of HSCs. The effect of NP-011 on HSCs. Figure 7A and Figure 7B PLA measurements were used to study the physical interaction between TGFBRI and integrin αvβ3 / αvβ5. The red signal (white arrow) indicates the interaction between TGFBRI and integrin β3 (…). Figure 7A ) and β5 ( Figure 7B Interactions between [cells]. Quantitative analysis of the number of red signals in each cell. Bars represent the mean ± SD of five replicates in each group. *P < 0.05, **P < 0.01. Figure 7C Immunoblot (WB) and immunoprecipitation (IP) analysis of the regulation of TGF-β signaling and the physical interaction between TGFBRI and integrin β3 / β5 in human HSCs in the presence of TGF-β1 (10 ng / mL) and / or NP-011 (500 ng / mL). Figure 7D EdU incorporation assay for human HSC cell proliferation in the presence of TGF-β1, NP-011, and / or silengiptide trifluoroacetate (CT, an inhibitor of integrin ανβ3 and ανβ5). Bars represent the mean ± SD of five replicates in each group. **P < 0.01.

[0038] Figures 8A-8C The results showed that NP-011 reduced MMP2 expression and increased collagenase activity. It also regulated the activity of pro-fibrotic MMP2 and collagenase in HSCs. Figure 8A Comparison of MMP2 mRNA expression in human HSCs in the presence of TGF-β1 and NP-011. Bars represent the mean ± SD of three replicates in each group. **P < 0.01. Figure 8B and Figure 8C Cell lysates ( Figure 8B ) and conditioned medium from cultured control HSCs and HSCs treated with TGF-β1 with / without NP-011 treatment ( Figure 8C Collagenase activity was measured. Bars represent the mean ± SD of three replicates in each group. **P < 0.01.

[0039] Figures 9A-9E The results showed that NP-011 enhanced macrophage-mediated collagen uptake. Figure 9A A schematic diagram illustrating macrophage differentiation from monocytes via PMA treatment. Figure 9B A representative diagram of differentiated macrophages (top) and FITC-labeled bead uptake via differentiated macrophages (bottom). Scale bar = 10 μm. Figure 9C Flow cytometry results showed that FITC beads were taken up by differentiated macrophages. Figure 9D A graphical depiction of fibrotic collagen uptake by differentiated macrophages with and without NP-011. Figure 9E Quantitative analysis of CUI (collagen uptake index) relative to the control group and the NP-011 treatment group. Bars represent the mean ± SD of three replicates in each group. **P < 0.01.

[0040] Figure 10 This diagram shows the interaction of NP-011 with activated HSCs and macrophages. Representative plots of immunostaining of mouse collagen and mouse F4 / 80 (macrophage marker) in liver tissue from TAA-induced liver fibrosis models and TAA-induced liver fibrosis models treated with NP-011 are presented. Arrows indicate macrophages phagocytizing collagen (F4 / 80 positive cells).

[0041] Figures 11A-11C The biodistribution and safety profile of NP-011 are shown. Figure 11A Organ distribution in healthy male mice 30 and 60 minutes after intravenous injection of NP-011 (160 μg / kg). Figure 11B and Figure 11C Male rats in the normal group and the two groups treated with NP-011 ( Figure 11B ) and female rats ( Figure 11C Quantitative analysis of the number of inflammatory-related cells and blood biochemistry in the blood.

[0042] Figure 12 A diagram showing a model of idiopathic pulmonary fibrosis (IPF) is displayed.

[0043] Figure 13 The diagram shows the distribution of collagen and αSMA in lung tissue stained on days 3 (D3), 5 (D5), 7 (D7), and 14 (D14) after bleomycin treatment.

[0044] Figure 14 The changes in expression levels of IPF biomarkers (Col1a1, MMP2, MMP12, and TIMP1) were shown on days 3 (D3), 5 (D5), 7 (D7), and 14 (D14) after bleomycin treatment.

[0045] Figure 15 The changes in expression levels of sub-factors of IPF-related signaling pathways are shown on days 3 (D3), 5 (D5), 7 (D7), and 14 (D14) after bleomycin treatment. Sub-factors included αSMA, pERK, tERK, phosphorylated SMAD2 (pSMAD2), and total SMAD2 (tSMAD2). Actin was shown as an injection control.

[0046] Figures 16A-16B A graph showing the IPF model used to test the in vivo efficacy of NP-011 is displayed. Two examples with different time points are provided: Example 1 ( Figure 16A ) and Example 2 ( Figure 16B ).

[0047] Figure 17 NP-011 was shown to be effective against IPF. Lung tissue was stained to show the distribution of collagen and αSMA after NP-011 injection. On day 3, mice were injected with NP-011 ( Figure 16A Example 1).

[0048] Figure 18 NP-011 was shown to be effective against IPF. Lung tissue was stained to show the distribution of collagen and αSMA after NP-011 injection. On day 5, mice were injected with NP-011 ( Figure 16B Example 2).

[0049] Figure 19 NP-011 showed efficacy against IPF. NP-011 reversed the changes in expression levels of IPF-related signaling pathway subfactors after bleomycin induction. On day 3, mice were injected with NP-011 (… Figure 16AExample 1). Subfactors include pERK, tERK, pSMAD2, and tSMAD2. Actin is shown as an injection control. The histogram on the right shows the quantification of pSMAD2 expression levels normalized to actin levels.

[0050] Figure 20 NP-011 showed efficacy against IPF. NP-011 completely reversed the changes in expression levels of IPF-related signaling pathway subfactors after bleomycin induction. On day 5, mice were injected with NP-011 (… Figure 16B Example 1). Subfactors include αSMA, pERK, and tERK. Actin is shown as an injection control. The histogram on the right shows the quantification of pERK expression levels normalized to actin levels.

[0051] Figure 21 NP-011 was shown to be effective against IPF. NP-011 altered the expression levels of IPF biomarkers, such as collagen (Cola1), MMP2, MMP12, and TIMP1, as shown in this paper. Specifically, NP-011 reversed the increase in expression levels of IPF biomarkers after bleomycin induction.

[0052] Figure 22 A diagram of a left anterior descending artery (LAD) ligation model of myocardial infarction is shown. The left anterior descending artery (LAD) of rats was ligated with a single suture, creating a localized ischemia that was almost immediately visible. By closing the LAD, no further blood flow was allowed into the area, while the surrounding myocardial tissue remained largely unaffected. This surgical procedure simulates the pathobiological and pathophysiological aspects of infarction-associated myocardial ischemia (e.g., Kolk et al. (2009) J Vis Exp, 21:pii 1438). In addition to the LAD ligation model, Figure 22 Details of the study presented in Figures 23-26 are also provided.

[0053] Figures 23A-23C The study showed that NP-011 reversed the symptoms of myocardial infarction and improved cardiac function. The results were shown 2 weeks after LAD ligation. Figure 23A ), 4 weeks Figure 23B ), or 8 weeks ( Figure 23C The results of left ventricular ejection fraction (EF) and fractional shortening (FS) measurements in mice were presented. The results are shown for untreated mice (AMI; acute myocardial infarction), mice treated with PBS (phosphate-buffered saline), mice treated with MFG-E8, and mice treated with NP-011.

[0054] Figures 24A-24BThe study showed that NP-011 reversed the symptoms of myocardial infarction and improved cardiac function. A cardiac time-course analysis from day 1 to day 56 after LAD ligation is presented. Specifically, this was achieved by measuring left ventricular ejection fraction (EF). Figure 24A ) and minor axis shortening (FS; Figure 24B It assesses the improvement in cardiac function. Unlike MFG-E8, NP-011 shows a significant improvement in cardiac function over time.

[0055] Figure 25 The changes in weight over subsequent time periods are shown. No significant changes in weight were observed from day 1 to day 56.

[0056] Figures 26A-26B NP-011 showed to reverse the symptoms of myocardial infarction. NP-011 reversed fibrosis associated with myocardial infarction. Figure 26A Cross-sectional views of rat hearts after H&E staining, including untreated (AMI), treated with PBS, treated with MFG-E8, or treated with NP-011. Figure 26B Showing with Figure 26A Measurement results of the fibrotic area compared to the overall cross-sectional area of ​​the heart.

[0057] Figures 27A-27E The study showed that NP-011 reversed behavioral and memory losses associated with Alzheimer's disease. Figure 27A This image shows the 5XFAD mouse model of Alzheimer's disease (AD) and details of in vivo studies. The 5XFAD mice expressing human APP and PSEN1 transgenes have a total of five AD-related mutations: the Swedish mutation (K670N / M671L), Florida mutation (I716V), and London mutation (V717I) in APP, and the M146L and L286V mutations in PSEN1. Behavioral and memory tests were performed after 5XFAD mice were injected with NP-011. Figure 27B The image shows the result after injection of NP-011 (from...) Figure 27A The behavioral analysis of the AD model was performed immediately 3.5 months after the start of the study. Figure 27C The results showed that NP-011 was effective 3 months after injection (from... Figure 27A The behavioral analysis of the AD model was performed 6.5 months after the start of the study. Figure 27D The image shows the result after injection of NP-011 (from...) Figure 27A The results of the passive avoidance test conducted on the AD model immediately 3.5 months after the start of the study are shown. Figure 27E The results showed that NP-011 was effective 3 months after injection (from... Figure 27A The results of a passive avoidance test on the AD model were shown 6.5 months after the start of the study.

[0058] Figures 28A-28B The study showed that NP-011 reversed neuropathological changes in the brains of people with Alzheimer's disease. Specifically, NP-011 reduced the number of amyloid plaques in AD. Figure 28A The image shows the hippocampus and cortex stained with thiamine S in the AD brain, where amyloid plaques were detected. Figure 28B This demonstrates the quantification of amyloid plaques, such as those stained with thioflavone S.

[0059] Figures 29A-29B The study showed that NP-011 reduced the amount of β-amyloid protein in Alzheimer's disease. Figure 29A The hippocampus and cortex of an AD brain stained with a 6E10 antibody that specifically recognizes β-amyloid protein were shown. Figure 29B This study demonstrates the quantification of β-amyloid protein in the hippocampus and cortex of the AD brain.

[0060] Figures 30A-30B NP-011 showed a reduction in microglia in Alzheimer's disease. Figure 30A The image shows the hippocampus and cortex stained with IBA-1 antibody, in which microglia were identified in the AD brain. Figure 30B This study demonstrated the quantification of microglia in the hippocampus and cortex of the AD brain.

[0061] Figures 31A-31B The study showed that NP-011 reduced the amount of upregulated glial fibrillary acidic protein (GFAP) in Alzheimer's disease astrocytes. Figure 31A The image shows the hippocampus and cortex of the AD brain stained with GFAP, and... Figure 31B This study demonstrates the quantification of GFAP in the hippocampus and cortex of the AD brain.

[0062] Detailed description of the invention

[0063] This invention relates in part to compositions and methods for treating and / or preventing fibrosis-related diseases.

[0064] definition

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The headings provided herein are not intended to limit the various embodiments and are available through reference to the entire specification. Furthermore, the terms defined below can be defined more comprehensively through reference to the entire specification.

[0066] In this text, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical object of the article. For example, “an element” means one element or more elements.

[0067] Where numerical values ​​are used in conjunction throughout the specification and claims, the terms “about” and “approximately” refer to a range of precision familiar to and accepted by those skilled in the art. Typically, such a range of precision is ±10%. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean a range on the order of magnitude of a given value, preferably ≤5 times and more preferably ≤2 times.

[0068] The term "coding region" refers to a nucleotide sequence region that includes codons that are translated into amino acid residues, while the term "non-coding region" refers to a nucleotide sequence region that is not translated into amino acids (e.g., the 5' and 3' untranslated regions).

[0069] The term "complementary" refers to a broad sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that adenine residues in a first nucleic acid region can form specific hydrogen bonds ("base pairing") with residues in a second nucleic acid region antiparallel to the first region (if the residue is thymine or uracil). Similarly, it is known that cytosine residues in a first nucleic acid strand can base pair with residues in a second nucleic acid strand antiparallel to the first strand (if the residue is guanine). When a first region of a nucleic acid is arranged antiparallel to a second region of the same or different nucleic acid, the two regions are complementary if at least one nucleotide residue in the first region can base pair with a residue in the second region. 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 antiparallel, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues in the first portion can base pair with nucleotide residues in the second portion. More preferably, all nucleotide residues in the first part are capable of base pairing with nucleotide residues in the second part.

[0070] The term "treatment" includes disease prevention treatment and / or therapeutic treatment. The term "disease prevention or therapeutic" treatment is recognized in the art and includes the administration of one or more of the subject composition to the host. If administered before the clinical manifestation of a harmful condition (e.g., a disease or other harmful state in the host animal), the treatment is disease prevention (i.e., it protects the host from developing a harmful condition), and if administered after the manifestation of a harmful condition, the treatment is therapeutic (i.e., it aims to reduce, alleviate, or stabilize an existing harmful condition or its side effects).

[0071] The term "prevention" is recognized in the art and is well understood in the art when used in connection with a condition (such as local recurrence (e.g., pain)), a disease (such as cancer), a syndrome (such as heart failure), or any other medical condition, and includes the administration of a composition that, relative to a subject who has not received the composition, reduces the frequency or severity of one or more symptoms of a medical condition in the subject, or delays the onset of one or more symptoms of a medical condition in the subject. Thus, cancer prevention includes, for example, reducing the number of detectable cancerous growths in a patient population receiving treatment for disease prevention relative to an untreated control population, and / or, for example, delaying the appearance of detectable cancerous growths in the treated population by a statistically and / or clinically significant amount relative to an untreated control population.

[0072] If a quantity (e.g., TGF-β expression, HSC proliferation, NOTCH signaling) is higher or lower than the control level by an amount greater than the standard error of the measurement used to assess that quantity, 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%, or 1000% higher than the control, then the quantity is considered “higher” or “lower” or “increased” or “decreased” compared to the control. This “significance” can be assessed from any desired or known comparison point, such as a post-treatment comparison of a specific measurement before treatment (e.g., 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, etc.). Alternatively, if the amount of growth factor in a subject is at least about two, three, four, or five times higher or lower than the normal amount of the biomarker, the amount of growth factor in the subject can be considered "significantly" higher or lower than the control amount. This "significance" can also be applied to any other measured parameter described herein.

[0073] "Therapeutic effective amount" (e.g., therapeutic effective amount of peptide) is the amount that can produce medically desired results in the treated patient (e.g., preferably in humans or non-human mammals, with an acceptable benefit: risk ratio, reduction of fibrosis, steatosis, and reduction of TGF-β expression).

[0074] The term "subject" refers to any subject who is expected to undergo diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include, but are not limited to, humans, non-human primates, domesticated animals, livestock, rodents, etc., who will become recipients of a particular treatment.

[0075] "Increased serum half-life" or "increased in vivo half-life" refers to a positive change in the circulating half-life of a modified bioactive molecule relative to its unmodified form. In some embodiments, serum half-life is measured by collecting blood samples at various time points after administration of the bioactive molecule and determining the concentration of the molecule in each sample. The serum half-life can be calculated by measuring the change in serum concentration over time. Serum half-life or t-half-life can be determined by comparing the serum half-life of the modified molecule (e.g., conjugated molecule) with that of the unmodified molecule. 1 / 2 The relative increase.

[0076] A “kit” is any article (e.g., package or container) that includes at least one reagent (e.g., the polypeptide described herein). Kits may also include additional reagents, carriers, or diluents. Kits may be promoted, distributed, or sold as a unit for carrying out the methods of the present invention. The kit may include one or more reagents necessary to express compositions useful in the methods of the present invention. In some embodiments, the kit may also include reference standards. Many such controls will be contemplated by those skilled in the art. Reagents in the kit may be provided in separate containers or in a mixture of two or more reagents in a single container. Additionally, explanatory material describing the use of the compositions within the kit may be included.

[0077] The numerical range includes numbers within a defined range.

[0078] Peptides and representative biological activities

[0079] The peptides provided in this article for treating various diseases include the MFG-E8 peptide, which comprises an epidermal growth factor (EGF)-like domain, a C1 domain, and an optional signal peptide, but lacks a functional C2 domain and / or a medin peptide or fragment thereof. The MFG-E8 peptide 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 in the C-terminal domain of the MFG-E8 peptide, comprising amino acids 226-335. The MFG-E8 polypeptide may 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 may 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 with amino acids 1-225 of SEQ ID NO:10. The polypeptide may also 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 with amino acids 2-225 of SEQ ID NO:10. The MFG-E8 polypeptide may be human or any orthologous, such as mouse, rat, chimpanzee, horse, etc.

[0080] MFG-E8 is also known as lactadherin. Representative human MFG-E8 cDNA and human BRD7 protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). For example, four distinct human MFG-E8 isoforms are known. Human MFG-E8 isoform B (NP_001108086.1) can be encoded by transcriptome 2 (NM_001114614.3), which lacks alternating in-frame exons compared to transcriptome 1. The resulting isoform B has the same N-terminus and C-terminus as isoform A, but is shorter. Human MFG-E8 isoform D (NP_001297248.1) can be encoded by transcriptome 4 (NM_001310319.2), which lacks alternating in-frame exons in the 5' coding region compared to transcriptome 1. The encoded isotype D has the same N-terminus and C-terminus as isotype A, but is shorter. Human MFG-E8 isotype C (NP_001297249.1) is encoded by transcriptomorph 3 (NM_001310320.2), which contains alternating internal exons compared to variant 1, resulting in the use of alternating translation initiation sites. The encoded isotype C has a different and shorter N-terminus compared to isotype A. Human MFG-E8 isotype E (NP_001297250.1) is encoded by transcriptomorph 5 (NM_001310321.2), which contains alternating 5' terminal exons compared to variant 1, resulting in the use of downstream in-frame translation initiation sites. The encoded isotype E has a shorter N-terminus compared to isotype A. Human MFG-E8 isotype A (NP_005919.2) can be encoded by transcription variant 1 (NM_005928.4), which encodes the longest isotype A.Nucleic acid and polypeptide sequences that are orthologous to MFG-E8 in organisms other than humans are well known and include, for example, chimpanzee MFG-E8 (XM_016927386.2→XP_016782875.1; and XM_001165898.6→XP_001165898.1) and canine 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_01399694) 5.2→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_01806581) 9.1→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).

[0081] A representative polypeptide is NP-011. NP-011 and other polypeptides described herein possess a variety of biological activities useful in inhibiting fibrosis. In some embodiments, polypeptides described herein (such as NP-011) reduce the expression of many genes, including TGF-β1, MMP2, and / or fibrosis-related genes such as Col1a1, Col1a2, and Acta2.

[0082] In some embodiments, the peptides described herein regulate signaling pathways, for example, by reducing TGF-β signaling. TGF-β signaling controls a range of distinct cellular processes during embryogenesis and in mature tissues across a diverse range of species, from flies and worms to mammals, including cell proliferation, recognition, differentiation, apoptosis, and developmental fate. TGF-β ligands initiate signaling by binding to and aggregating type I and type II receptor serine / threonine kinases on the cell surface. This leads to phosphorylation of the receptor I kinase domain by receptor II, which then propagates the signal via phosphorylation of the Smad protein. The activated Smad complex translocates to the nucleus and binds to other nuclear cofactors to regulate the transcription of target genes.

[0083] TGF-β and its signaling pathway regulate cell proliferation. During the progression of liver fibrosis, damaged and dead hepatocytes from liver injury recruit Kupffer cells at their lesion sites. These Kupffer cells secrete large amounts of cytokines, including transforming growth factor β1 (TGF-β1), to control liver inflammation. Increased TGF-β1 leads to the activation of quiescent HSCs, which proliferate and transform into myofibroblast-like cells that produce extracellular matrix (ECM). Activated HSCs further accumulate excessive amounts of collagen-rich ECM in the liver, causing distortion of normal liver structure. Correspondingly, decreased TGF-β1 expression or reduced TGF-β signaling inhibits HSC activation and / or proliferation, leading to suppression of fibrosis.

[0084] The reduction in TGF-β signaling can be monitored through a variety of methods known in the art. For example, 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 of downstream proteins (e.g., SMAD) can be monitored. In addition, there are many commercially available kits that can monitor the TGF-β signaling pathway, such as 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, and SEM02991A. Similarly, the expression 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 phosphorylation of downstream proteins can be monitored to assess the Notch signaling pathway. For example, there are many commercially available kits that can monitor the Notch signaling pathway, such as Qiagen Cat#PAHS-059Z, PAMM-059Z, PARN-059Z, CRHS-00059Z-100, CRMM-00059Z-100, CCS-014L, CCS-1014G, CLS-014L, CLS-014G, EAHS-611Z, and GH-059A.

[0085] In some implementations, as described above, the peptides described herein reduce phosphorylation of SMAD, wherein SMAD is a protein in the TGF-β signaling pathway.

[0086] In some embodiments, the peptides described herein disrupt and / or reduce the interaction between TGF-β receptor 1 (TGFBR1) and one or more integrins (e.g., integrin β3 and / or integrin β5).

[0087] In some implementations, the peptides described herein reduce the proliferation of HSCs.

[0088] In some embodiments, the peptides described herein increase collagenase activity and / or macrophage uptake of collagen.

[0089] Formation of amyloid protein

[0090] Some peptides described in this article lack the medin peptide or a fragment thereof, which represents approximately 50 amino acids in the C2 domain. Medin is a major component of amyloid protein found in the aorta. Medin and amyloid protein are associated with the etiology of diseases including Alzheimer's disease and type II diabetes. Repeated administration of peptides containing the medin peptide may increase the risk of inducing amyloid formation. Peptides described in this article that lack the medin peptide or a fragment thereof reduce the risk of inducing amyloid formation.

[0091] Peptide modification

[0092] The present invention also provides modified peptides, wherein the peptides described herein are fused in-frame with heteropeptides and / or one or more chemical moieties. Such modifications can be useful, for example, in extending the serum half-life of the peptide, in use as a tag during protein purification, or in enhancing immunogenicity during antibody production against the peptide.

[0093] As used herein, "chimeric protein" or "fusion protein" includes all or part (preferably the bioactive portion) of the polypeptide of the present invention linked to a heteropeptide having an amino acid sequence corresponding to a protein substantially non-homologous to the corresponding polypeptide. Within the fusion protein, the polypeptide of the present invention and the heteropeptide are preferably fused to each other so that they retain their respective functions when expressed independently of the fusion. The heteropeptide may be fused to the amino or carboxyl terminus of the polypeptide of the present invention.

[0094] In some embodiments, the fusion protein contains 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 generated using standard recombinant DNA techniques. In other embodiments, the fusion protein can be synthesized using conventional techniques involving automated DNA synthesis. Alternatively, gene fragments can be amplified by PCR using anchor primers that generate complementary overhangs between two consecutive gene fragments, which are then annealed and re-amplified to generate a chimeric gene sequence. Furthermore, many expression vectors encoding fusion moieties (e.g., GST peptides) are commercially available. Nucleic acids encoding the peptides of the present invention can be cloned into such expression vectors, thereby in-frame fusing the fusion moieties to the peptides of the present invention.

[0095] Heterogeneous peptides can optionally correspond to portions that alter the solubility, affinity, stability, and valence of a peptide (e.g., NP-011). For example, a peptide (such as NP-011) can be operatively linked to an immunoglobulin constant region, such as a human Cγ1 or Cγ4 domain (e.g., the hinge, CH2, and CH3 regions of human IgCγ1 or human IgCγ4, see, for example, Capon et al., U.S. Patent 5,116,964; U.S. Patent 5,580,756; U.S. Patent 5,844,095, etc., all of which are incorporated herein by reference). Such constant regions can retain regions mediating effector functions (e.g., Fc receptor binding) or can be altered to weaken effector functions. Such constant regions can also prolong the serum half-life of the fusion protein. The resulting fusion protein may have altered solubility, binding affinity, stability, and / or valence (i.e., the number of binding sites available for each peptide) compared to the independently expressed first peptide, and may improve protein purification efficiency. In a preferred embodiment, the fusion protein retains the bioactivity of the polypeptide.

[0096] In other embodiments, the peptide may be covalently or non-covalently modified with one or more chemical motifs (e.g., polyethylene glycol (PEG), lipids, or PEG-modified lipids). Such modification may, for example, increase the peptide's solubility, stability, and / or serum half-life.

[0097] Treatment methods – Exemplary condition

[0098] The compositions and methods provided herein are particularly useful in the treatment and / or prevention of disorders including those described below.

[0099] liver fibrosis

[0100] In liver fibrosis, excessive connective tissue accumulates in the liver; this tissue represents scarring in response to chronic, repetitive hepatocellular injury. Typically, fibrosis continues to develop and eventually function as regenerating hepatocytes attempt to replace and repair damaged tissue while simultaneously destroying the liver's structure. When such destruction becomes widespread, it is diagnosed as cirrhosis.

[0101] Various types of chronic liver injury can lead to fibrosis. Self-limiting, acute liver injury (e.g., acute viral hepatitis A), even if it flares up, does not necessarily disrupt the scaffold structure and therefore, although hepatocytes are lost, fibrosis may not result. In the initial stages of liver fibrosis, if the cause is reversible (e.g., by clearing the virus), fibrosis can regress. After months or years of chronic or recurrent injury, fibrosis becomes permanent. In mechanical biliary obstruction, fibrosis develops even more rapidly.

[0102] Activation of perivascular stellate cells (Ito cells that store fat) in the liver triggers fibrosis. These cells and adjacent cells proliferate, transforming into contractile cells called myofibroblasts. These cells produce excessive amounts of abnormal matrix (composed of collagen, other glycoproteins, and glycans) and stromal cell 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. Therefore, stellate cell activation leads to abnormalities in both the quantity and composition of the extracellular matrix.

[0103] Endothelin-1-stimulated myofibroblasts contribute to increased portal vein resistance and increased density of abnormal matrix. Fiber bundles connect the afferent and efferent branches of the portal vein and hepatic vein, bypassing hepatocytes and restricting their blood supply. Therefore, fibrosis causes localized ischemia of hepatocytes (leading to hepatocyte dysfunction) and portal hypertension. The degree of ischemia and portal hypertension determines how the liver is affected. For example, congenital liver fibrosis affects the portal vein branches, severely damaging the parenchyma. This results in portal hypertension and impaired hepatocyte function.

[0104] Cirrhosis

[0105] Cirrhosis is the advanced stage of liver fibrosis, at which point fibrosis has caused extensive distortion of the normal liver structure. Cirrhosis is characterized by regenerative nodules surrounded by dense fibrotic tissue. Symptoms may not appear for years and are often nonspecific (e.g., anorexia, fatigue, weight loss). Late-stage manifestations include portal hypertension, ascites, and liver failure when compensatory dysfunction occurs. A liver biopsy is often required for diagnosis. Cirrhosis is generally considered irreversible. Treatment is supportive.

[0106] 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. Pathologically, cirrhosis is defined as the loss of normal lobular microstructure accompanied by fibrosis (i.e., scar tissue growth due to infection, inflammation, injury, or even healing) and nodular regeneration. Due to chronic liver damage, scar tissue slowly replaces normally functioning liver tissue, leading to a gradual reduction in blood flow to the liver. With the loss of normal liver tissue, the liver becomes unable to effectively process nutrients, hormones, drugs, and toxins. Furthermore, protein production and other substances produced by the liver are inhibited.

[0107] Symptoms of cirrhosis vary depending on severity and individual differences. They may include neurological abnormalities, ascites (fluid accumulation in the abdominal cavity), gynecomastia in men, coughing episodes or hemoptysis, finger curling (palmar aponeurosis contracture), gallstones, hair loss, itching, jaundice, kidney failure, hepatic encephalopathy, muscle atrophy, loss of appetite, portal hypertension, red palms, enlarged salivary glands in the cheeks, testicular atrophy, small spider veins in the skin, weakness, and weight loss. Symptoms of cirrhosis may resemble those of other conditions or medical problems. Mild cirrhosis may not present any symptoms at all.

[0108] The most common cause of cirrhosis is alcohol abuse. Other causes include hepatitis and other viruses (e.g., HCV, as described in Section 2.2 below), the use of certain medications, chemical exposure, bile duct obstruction, autoimmune diseases, obstruction of hepatic blood flow (i.e., Budd-Chiari syndrome), cardiac and vascular disorders, alpha-1 antitrypsin deficiency, hypergalactosemia, hypertyrosinemia, glycogen storage disease, diabetes, malnutrition, hereditary copper excess (Wilson's disease), or hereditary iron excess (hemochromatosis).

[0109] Cirrhosis is a progressive liver disease, and the damage to the liver is irreversible. However, with proper nutrition, avoidance of certain toxins (i.e., alcohol), vitamin supplementation, and management of cirrhosis complications, further liver damage can often be delayed or stopped. In cases of severe cirrhosis, liver transplantation may be considered.

[0110] Nonalcoholic steatohepatitis (NASH)

[0111] Nonalcoholic steatohepatitis (NASH), also known as nonalcoholic fatty liver disease, describes a liver disorder characterized by the pathogenesis of alcoholic liver disease occurring without alcohol consumption. The fat deposited in hepatocytes is primarily triglycerides, and the severity of NASH is directly related to the amount of fat in the liver. Histologically, a diagnosis of steatohepatitis may be made if 50% of hepatocytes exhibit steatosis (fatty liver accumulation), or if the total weight of fat exceeds 5% of the entire liver. NASH is further characterized by elevated serum aminotransferase activity, accompanied by hepatic steatosis, inflammation, and occasionally, fibrosis leading to cirrhosis.

[0112] In most parts of the world, the prevalence of NASH ranges from 3% to 19%. There are many possible causes of NASH, but no single cause is definitively identified. The most likely causes are obesity due to poor diet, diabetes, long-term steroid use, and tetracycline use. Some studies have shown signs of reversal in fatty degeneration after weight loss.

[0113] There is currently no established treatment for this potentially serious disorder. Treatment for patients with non-alcoholic fatty liver disease typically focuses on managing related conditions such as obesity, diabetes, and hyperlipidemia, as well as discontinuing potentially hepatotoxic medications.

[0114] Fibrosis-related disorders

[0115] Fibrosis-related disorders that may be suitable for treatment using the methods described in this article include, but are not limited to, collagenopathies, interstitial lung diseases, human fibrotic lung diseases (e.g., bronchiolitis obliterans, idiopathic pulmonary fibrosis, pulmonary fibrosis of known etiology, tumor stroma in lung diseases, systemic sclerosis affecting the lungs, Hermansky-Pudrag syndrome, coal worker's pneumoconiosis, asbestosis, silicosis, chronic pulmonary hypertension, HIV-associated pulmonary hypertension, sarcoidosis, moderate to severe asthma, etc.), fibrotic vascular diseases, arteriosclerosis, atherosclerosis, varicose veins, coronary artery occlusion, cerebral infarction, myocardial fibrosis, musculoskeletal fibrosis, postoperative adhesions, human kidney diseases (e.g., nephrotic syndrome, Allport syndrome, HIV-associated nephropathy, polycystic kidney disease, Fabry disease, diabetic nephropathy, chronic glomerulonephritis, systemic lupus erythematosus-associated nephritis, etc.), and progressive systemic sclerosis (…). Primary sclerosing cholangitis (PSS), liver fibrosis, cirrhosis, renal fibrosis, pulmonary fibrosis, cystic fibrosis, chronic graft-versus-host disease, scleroderma (local and systemic), Graves' eye disease, diabetic retinopathy, glaucoma, Peronis disease, penile fibrosis, post-cystoscopic urethral stricture, postoperative internal hyperplasia, scarring, myelofibrosis, idiopathic retroperitoneal fibrosis, peritoneal fibrosis of known etiology, drug-induced ergot poisoning, benign or malignant cancer-prone fibrosis, microbial infection-prone fibrosis (e.g., viruses, bacteria, parasites, fungi, etc.), Alzheimer's disease, inflammatory bowel disease-prone fibrosis (including stricture formation in Crohn's disease and microscopic colitis), stromal cell tumors, mucositis, chemical or environmental damage-induced fibrosis (e.g., cancer chemotherapy, pesticides, radiation (e.g., cancer radiotherapy), etc.), etc. In some implementations, fibrosis-related disorders are selected from systemic or localized scleroderma, keloids, hypertrophic scars, atherosclerosis, restenosis, pulmonary inflammation and fibrosis, idiopathic pulmonary fibrosis, cirrhosis, fibrosis caused by chronic hepatitis B or C infection, kidney disease, heart disease caused by scar tissue, macular degeneration, and fundus and vitreoretinal diseases. In some implementations, fibrosis-related disorders are caused by chemotherapy drugs, radiation-induced fibrosis, and injury and burns.

[0116] Hereditary diseases associated with liver fibrosis include Wilson's disease, hereditary hemochromatosis, non-HFE hereditary hemochromatosis, transferrin receptor 2, heparin, hemoblastin, sitosterolemia / hepatobiliary cholesterol transporters 5 and 8, type 3 progressive familial intrahepatic cholestasis, hereditary fructose intolerance, type I tyrosinemia, argininosuccinate lyase deficiency, citric acid deficiency, cholesterol ester storage disease and Wollman's disease, anti-1 antitrypsin deficiency, cystic fibrosis, Alstrom syndrome, and congenital liver fibrosis.

[0117] pulmonary fibrosis

[0118] Pulmonary fibrosis (“pulmonary scarring”) is a respiratory disease in which scarring forms in the lung tissue, leading to serious breathing problems. Scarring, the accumulation of excessive fibrotic connective tissue (a process known as fibrosis), causes the lung walls to thicken and reduces the oxygen supply to the blood. The result is permanent shortness of breath.

[0119] Pulmonary fibrosis involves the gradual exchange of normal lung parenchyma with fibrotic tissue. The replacement of normal lung tissue with scar tissue leads to an irreversible decrease in oxygen diffusion, and the resulting stiffness or reduced compliance transforms pulmonary fibrosis into a restrictive lung disease. Pulmonary fibrosis is caused by abnormal wound healing, not chronic inflammation. This is a major cause of inherently restrictive lung disease in the lung parenchyma. In some cases, pulmonary fibrosis is associated with smoking. Treatment options for pulmonary fibrosis are limited and include immunosuppressive therapies (such as corticosteroids).

[0120] Idiopathic pulmonary fibrosis

[0121] Idiopathic pulmonary fibrosis (IPF) is the most common form of idiopathic interstitial pneumonia. It is a chronic, progressive, irreversible, and often fatal lung disease of unknown etiology. 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 the typical histopathological or radiographic patterns of common interstitial pneumonia.

[0122] The main histopathological features of common interstitial pneumonia (CIP) are best observed at low magnification and exhibit a heterogeneous appearance, in which subpleural and paraseptal fibrosis and honeycomb areas (i.e., cystic fibrotic spaces lined with bronchial epithelium and often filled with mucin and varying numbers of inflammatory cells) alternate with less affected or normal parenchymal areas (spatial heterogeneity). Smaller active fibrotic areas (fibroblastic foci) exist within a collagen deposition environment, and they reflect the temporal heterogeneity of the process and indicate currently ongoing disease. Inflammation is usually mild and consists of patchy lymphoplasmacytic infiltration. The pattern of CIP present on high-resolution CT is characterized by reticular opacities, often associated with traction bronchiectasis, with little or no ground-glass opacities. The honeycomb appearance, characterized by well-defined subpleural clusters of cystic spaces (usually 3–10 mm in diameter), is relatively common and crucial for accurate diagnosis.

[0123] Patients with IPE typically seek medical help due to chronic or progressive exertional dyspnea and cough. Chest auscultation reveals bilateral inspiratory moist rales at the lung bases, and clubbing of the fingers is frequently present. The natural history of IPE has been characterized as a stable or slowly progressive lung disorder, and this pattern is followed by most patients. However, recent findings suggest that IPE is a heterogeneous disease and describe new clinical phenotypes with different survival patterns. The pathogenesis is unclear, but mounting evidence suggests that the disease results from abnormal alveolar epithelial cell behavior that prompts the migration, proliferation, and activation of mesenchymal cells, leading to the formation of fibroblastic myofibroblastic foci. Activated myofibroblasts secrete excessive amounts of extracellular matrix molecules, subsequently destroying lung structures. No treatment has been shown to prolong survival.

[0124] Myocardial infarction

[0125] A myocardial infarction, commonly known as a heart attack, occurs when a part of the heart is deprived of oxygen due to a blockage in a coronary artery. The coronary arteries supply oxygenated blood to the heart muscle (the layer of muscle). Without oxygen, the heart muscle cells served by the blocked artery begin to die (infarction).

[0126] The most common cause of myocardial infarction is the rupture of an atherosclerotic plaque on an artery supplying the heart muscle. The plaque may become unstable, rupture, and further promote the formation of a blood clot blocking the artery; this can happen within minutes. An artery blockage can lead to tissue death in the tissue supplied by the artery. Atherosclerotic plaques often exist for decades before causing symptoms.

[0127] Over decades, cholesterol and fibrous tissue gradually accumulate in plaques within the walls of coronary arteries or other arteries, a process known as atherosclerosis. Atherosclerosis is characterized by progressive inflammation of the arterial walls. Inflammatory cells, particularly macrophages, migrate to the affected arterial walls. Over time, they become loaded with cholesterol products, especially LDL, and transform into foam cells. As the foam cells die, cholesterol nuclei form. Smooth muscle cells or other cells, responding to growth factors secreted by macrophages, migrate to the plaque and stabilize it. Stable plaques may have a thick, calcified fibrous cap. If inflammation persists, the fibrous cap may thin or ulcerate. Upon exposure to blood flow-related stresses, plaques, especially those with a thinner lining, may rupture, triggering the formation of a blood clot (thrombus).

[0128] Alzheimer's disease

[0129] Alzheimer's disease is a dementia disorder characterized by progressive impairment of memory and cognition. It typically occurs late in life and is associated with multiple structural, chemical, and functional abnormalities involving brain regions related to cognition and memory. Epidemiological studies indicate that the current incidence of dementia in individuals aged 65 and older is as high as 10%, and it is estimated that up to 4 million individuals in the United States alone may have Alzheimer's disease. The cost of caring for such individuals exceeds $80 billion annually and is rapidly increasing.

[0130] Little is known about the causes of Alzheimer's disease. It is believed that about 70% of the risk is inherited from a person's parents, and this usually involves many genes. Other risk factors include a history of head injury, depression, and high blood pressure. The disease process is associated with plaques and neurofibrillary tangles in the brain. A possible diagnosis is based on a medical history and cognitive tests, as well as medical imaging and blood tests to rule out other possible causes. Initial symptoms are often mistaken for normal aging. A definitive diagnosis requires examination of brain tissue. No medications or supplements have been shown to reduce the risk.

[0131] It is speculated that extracellular β-amyloid protein (A) β β-amyloid plaque deposition is the underlying cause of the disease. This hypothesis is based on 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 the earliest symptoms of Alzheimer's disease (AD) by age 40. Similarly, the specific apolipoprotein isoform APOE4 is a major genetic risk factor for AD. While apolipoproteins enhance the breakdown of β-amyloid, some isoforms (such as APOE4) are not very effective in this task, leading to the accumulation of excess amyloid in the brain. Further evidence comes from the finding that transgenic mice expressing mutant forms of the human APP gene develop fibrous amyloid plaques and Alzheimer's-like brain symptoms, accompanied by spatial learning deficits.

[0132] Additives

[0133] The compositions provided herein can be administered to a subject in combination with any known agent for treating disorders (e.g., liver fibrosis). Similarly, the methods provided herein can be administered to a subject in combination with the compositions disclosed herein (e.g., NP-011) with any known agent for treating disorders. Various treatments for fibrosis-related disorders are known to those skilled in the art.

[0134] Treatment for fibrotic disorders includes anti-inflammatory agents, corticosteroids, penicillin, and colchicine. For example, see The Merck Manual, 20th ed. Merck Research Laboratories, 2018.

[0135] Because fibrosis represents a response to liver damage, primary treatment should focus on the underlying cause (eliminating the basis of liver damage). Such treatment may include eliminating hepatitis B or hepatitis C virus in chronic viral hepatitis, abstaining from alcohol in alcoholic liver disease, removing heavy metals (such as iron in hemochromatosis or copper in Wilson's disease), and decompressing the bile ducts in cases of biliary obstruction. Such treatment can halt the progression of fibrosis and, in some patients, even reverse some fibrotic changes.

[0136] Treatments aimed at reversing fibrosis are often too toxic (e.g., corticosteroids, penicillin) for long-term use, or have not been proven effective (e.g., colchicine). Other anti-fibrotic treatments are under investigation. Silymarin, found in milk thistle, is a popular alternative for treating liver fibrosis. It appears safer (except when combined with certain medications to treat hepatitis C) but lacks efficacy.

[0137] In some implementations, antifibrotic therapy includes the administration of profibrotic factor antagonists and / or antifibrotic agents.

[0138] fibrotic factor antagonists

[0139] Antifibrotic therapy encompasses agents that inhibit or antagonize pro-fibrotic factors, such as agents that antagonize one or more growth factors or cytokines involved in the formation and maintenance of fibrotic tissue. In this way, antifibrotic therapy targets the differentiation of fibroblasts, fibroblast precursors, myofibroblast precursors, and / or hematopoietic mononuclear cell precursors, and the formation and maintenance of fibrotic tissue.

[0140] As part of the therapy of the present invention, antagonist-targeted profibrotic factors may be employed, including, but not limited to, β-transforming growth factor (TGF-β, including TGF-β1-5), VEGF, EGF, RANTES, interleukin family members (e.g., IL-1, IL-4, IL-5, IL-6, IL-8, and IL-13), α-tumor necrosis factor (TNF-α), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and type I fibroblast growth factor (BGF). Monocyte chemotactic protein (MCP-1), macrophage inflammatory proteins (e.g., MIP-1a, MIP-2), connective tissue growth factor (CTGF), endothelin-1, angiotensin-11, rennet, 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 fibrotic tissue.

[0141] Antifibrotic therapies may contain antagonists of receptors corresponding to one or more profibrotic factors. Such antagonists may contain inactive forms of one or more profibrotic factors and / or cytokines (such as fragments thereof). These forms, at appropriate concentrations, can compete with their corresponding profibrotic factors and / or cytokines for binding to their receptors. Similarly, certain antibodies against the receptor can be used to interfere with or prevent the binding of the corresponding profibrotic factors and / or cytokines.

[0142] Antifibrotic therapies may also contain soluble forms of receptors for one or more profibrotic factors and / or cytokines, such that the soluble receptors compete with their corresponding native cellular receptors for target ligands. Therapies may further contain compounds that compete with one or more profibrotic factors and / or cytokines for their receptors or otherwise interfere with the binding of one or more profibrotic factors and / or cytokines to their receptors. For example, proteoglycan core proteoglycans are known to bind to TGF-β, thereby reducing their availability to bind to their receptors. Mannose-6-phosphate is also known to compete with TGF-β for binding to its corresponding receptor. Other known TGF-β binding inhibitors include potential transforming growth factor β-binding protein (LTBP) and potential related peptide (LAP), both of which are natively bound to intracellular precursors of TGF-β.

[0143] In some embodiments, the antifibrotic therapy may comprise one or more oligonucleotides containing at least one antisense sequence relative to one or more profibrotic factors and / or cytokines. Such a component may also comprise one or more expression plasmids having suitable transcriptional control sequences that generate the antisense sequence. In other alternative embodiments, the antifibrotic therapy may comprise one or more double-stranded oligonucleotides or expression plasmids encoding them, said double-stranded oligonucleotides being adapted to degrade transcripts of one or more profibrotic factors and / or cytokines via RNA-mediated interference. In other alternative embodiments, the antifibrotic therapy may comprise one or more single-stranded oligonucleotide aptamers or expression plasmids encoding them, said single-stranded oligonucleotide aptamers being adapted to inhibit or interfere with the binding of profibrotic factors to their homologous receptors.

[0144] Suitable profibrotic factors may contain components known to inhibit, attenuate, or interfere with one or more components of intracellular signaling pathways that are activated by the profibrotic factor upon binding to their corresponding receptors. For example, antifibrotic therapies may contain components that inhibit or attenuate downstream signaling pathway molecules such as SMAD family members and SARA. Suitable antifibrotic therapies may also contain one or more molecules suitable for inhibiting or interfering with cell adhesion required for fibrosis. For example, suitable components may contain interfering antibodies against ICAM-1 and / or CD11, CD49, or CD18, thereby interfering with their adhesive interactions.

[0145] In other alternative implementations, suitable profibrotic factor antagonists may comprise collagen synthesis inhibitors, such as proline analogs that interfere with the post-translational processing of collagen precursors. For example, pirfenidone is an orally active small molecule drug that inhibits collagen synthesis, downregulates the production of various cytokines, and prevents fibroblast proliferation.

[0146] TGF-β antagonists

[0147] Transforming growth factor (TGF)-β 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. These cells are potential sources of TGF-β after injury or inflammation. Generally, TGF-β stimulates the production of various extracellular matrix proteins, inhibits the breakdown of these matrix proteins, and promotes tissue fibrosis, all of which contribute to the repair and recovery of affected tissues. In many diseases, excessive TGF-β can cause pathological tissue fibrosis, which can impair normal organ function.

[0148] Examples of TGF-β antagonists include, but are not limited to: monoclonal and polyclonal antibodies against one or more TGF-β isotypes (Dasch et al., U.S. Patent No. 5,571,714; see also WO97 / 13844 and WO 00 / 66631); TGF-β receptors, soluble forms of such receptors (preferably soluble TGF-β type III receptors), or antibodies against TGF-β receptors (Segarini et al., U.S. Patent No. 5,693,607; Lin et al., U.S. Patent Nos. 6,001,969, 6,010,872, 6,086,867, 6,201,108; WO 98 / 48024; WO 95 / 10610; WO93 / 09228; WO 92 / 00330); potential related peptides (WO 91 / 08291); large potential TGF-β (WO 94 / 09812); fetoglobulin (US Patent No. 5,821,227); core proteoglycans and other proteoglycans such as disaccharide proteoglycans, fibrinolytic proteoglycans, luminescent proteoglycans and endothelial glycoproteins (WO 91110727; Ruoslahti et al., US Patent Nos. 5,654,270, 5,705,609, 5,726,149; Border, US Patent No. 5,824,655; WO91 / 04748; Letarte et al., US Patent Nos. 5,830,847, 6,015,693; WO 91110727; WO93 / 09800; and WO 94110187); somatostatin (WO 98 / 08529); mannose-6-phosphate or mannose-1-phosphate (Ferguson, US Patent No. 5,520,926); prolactin (WO 97 / 40848); insulin-like growth factor II (WO98117304); IP-10 (WO 97 / 00691); arg-gly-asp peptides (Pfeffer, US Patent Nos. 5,958,411; WO93 / 10808); plant, fungal, and bacterial extracts (EP-A-813 875; JP 8119984; and Matsunaga et al., U.S. Patent No. 5,693,610; antisense oligonucleotides (Chung, U.S. Patent No. 5,683,988; Fakhrai et al., U.S. Patent No. 5,772,995; Dzau, U.S. Patent No. 5,821,234; U.S. Patent No. 5,869,462; and WO 94 / 25588); proteins involved in TGF-β signaling, including SMAD and MAD (EP-A-874 046; WO 97 / 31020; WO 97 / 38729; WO98 / 03663);WO 98 / 0773; WO 98 / 07849; WO 98 / 45467; WO 98 / 53068; WO 98 / 55512; WO 98 / 56913; WO 98 / 53830; WO 99 / 50296; Falb, U.S. Patent No. 5,834,248; Falb et al., U.S. Patent No. 5,807,708; and Gimeno et al., U.S. Patent 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, or expression plasmids encoding them, suitable for inhibiting or interfering with the binding of TGF-β to its homologous receptor; and any mutant, fragment, or derivative of the above molecules that retains the ability to inhibit TGF-β activity.

[0149] In some embodiments, the TGF-β antagonist is a human or humanized monoclonal antibody (or a fragment thereof, such as the F(ab)2 fragment, the Fv fragment, a single-chain antibody, or other forms or fragments of an antibody that retain the ability to bind to TGF-β, for example, a monoclonal antibody derived from cyclomatomy 1D 11.16 (ATCC accession number HB 9849, as described by Dasch et al. in U.S. Patent No. 5,783,185).

[0150] Antifibrotic agents

[0151] In some implementations, the profibrotic factor antagonist can be replaced or enhanced by cytokines known to have antifibrotic effects, such as IL-12, IL-10, IFN-γ, or BMP-7 (OP-1). The nucleic acid sequences encoding IFN-γ peptides are accessible from public databases, such as GenBank, journal articles, etc. While there is interest in various mammalian IFN-γ peptides, human proteins are generally used to treat human diseases. Human IFN-γ coding sequences can be found in GenBank, accession numbers P01579 and CAA00375. The corresponding genomic sequences can be found in GenBank, accession numbers 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( Human interferon (IFN-g) is a single-chain polypeptide with 140 amino acids. It is prepared by recombinant synthesis in Escherichia coli (E. coli) without glycosylation. Rinderknecht et al. (1984) J. Biol. Chern (Journal of Biochemistry). 259:6790-6797. IFN-g used in antifibrotic therapy can be any of natural IFN-g, recombinant IFN-g, and their derivatives, as long as it has IFN-g activity, especially human IFN-g activity.

[0152] Antifibrotic therapy includes one or more serum amyloid P (SAP) agonists, one or more C-reactive protein (CRP) antagonists, or combinations thereof. SAP agonists can be used to treat a variety of fibrotic disorders. SAP agonists encompass all compounds and compositions that increase or otherwise mimic endogenous SAP signaling, including compounds that increase SAP activity.

[0153] Serum amyloid P (SAP), or penetrin 2, is a member of the penetrin family. It is a 27-kDa protein produced by the liver, secreted into the bloodstream, and circulates as a stable 135-kDa pentad. SAP reduces neutrophil adhesion to ECM proteins, inhibits monocyte differentiation into fibroblasts, reduces pro-fibrotic macrophages, activates the complement pathway, and promotes phagocytosis of cellular debris. SA can reduce bleomycin-induced pulmonary fibrosis. Representative SAP agonists include human SAP protein or its active fragments, anti-FcyR antibodies, aggregation antibodies, SAP peptide mimics, FcyR cross-linking agents, and aptamers. CRP antagonists encompass all compounds and compositions that reduce, block, or inhibit CRP signaling. Representative antagonists include antisense nucleic acids targeting CRP expression, RNA interference molecules (e.g., short interfering RNA, dsRNA), and locked nucleic acids.

[0154] Nucleic acid molecules, vectors, host cells

[0155] Nucleic acid molecules encoding the polypeptides disclosed herein (e.g., the NP-011 protein) are provided.

[0156] The nucleic acid molecules disclosed herein can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single-stranded, can be a coding strand or a non-coding (antisense) strand. In some embodiments, the nucleic acid molecules are isolated. In other embodiments, the nucleic acid molecules are substantially pure. In some embodiments, the nucleic acid molecules are cDNA or derived from cDNA. In some embodiments, the nucleic acid molecules are generated through recombination.

[0157] The nucleic acid molecules of the present invention can be amplified using cDNA, mRNA, or genomic DNA as templates and appropriate oligonucleotide primers according to standard PCR amplification techniques. The amplified nucleic acid molecules can then be cloned into appropriate vectors 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 synthetic techniques, for example, using an automated DNA analyzer.

[0158] In some embodiments, the nucleic acid molecule includes a protein-coding sequence operatively linked to a control sequence that controls the expression of the coding sequence in a host cell or in vitro (see below). In a particular embodiment, the coding sequence is cDNA. This disclosure also relates to vectors containing nucleic acid molecules including a protein (e.g., NP-011) coding sequence operatively linked to a control sequence that controls the expression of the coding sequence in a host cell or in vitro.

[0159] In some embodiments, the nucleic acid molecule includes a coding sequence for a protein, which is fused to a heteropolynucleotide sequence within the same reading frame. In some embodiments, the heteropolynucleotide sequence encodes a leader peptide sequence that facilitates the secretion of expressed proteins from host cells transformed with a polypeptide encoding one or more nucleic acid molecules (e.g., NP-011). Proteins containing a leader sequence are referred to as preproteins and can be cleaved by host cells to form processed forms of the protein. Such leader peptide sequences and their use in facilitating the secretion of recombinant proteins from host cells are well known in the art. In other embodiments, the heteropolynucleotide sequence encodes additional 5' amino acid residues that can be used, for example, to facilitate purification, increase or improve protein stability, and / or the therapeutic properties of recombinantly expressed proteins. In a preferred embodiment, NP-011 and / or similar proteins are secreted from host cells. In some preferred embodiments, NP-011 and / or similar proteins lack an N-terminal methionine and do not include the heterologous sequence. In other preferred embodiments, NP-011 includes a heterologous sequence that extends the in vivo half-life.

[0160] In some implementations, the nucleic acid sequence encoding the protein, or a vector comprising said nucleic acid sequence, is constructed chemically using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide, and codons can be optimized based on host cell preferences. Standard methods can be conventionally used to synthesize and isolate polynucleotide sequences encoding polypeptides (e.g., NP-011).

[0161] Vectors can be constructs capable of being delivered to and, in some embodiments, expressing 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, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (such as production cells).

[0162] Once assembled (by synthesis or another method), the nucleic acid sequence encoding the polypeptide can be operatively linked to a control sequence suitable for expression in the desired host, as is customary. In some embodiments, the nucleic acid sequence encoding the polypeptide is inserted into one or more expression vectors and operatively linked to a control sequence suitable for protein expression in the desired host. To achieve high expression levels of the transfected coding sequence in the host, the coding sequence can be operatively linked to or associated with transcriptional and translational expression control sequences that function in the selected expression host.

[0163] In some embodiments, recombinant expression vectors are used to amplify and express DNA encoding polypeptides. A recombinant expression vector is a reproducible DNA construct having a synthetic or cDNA-derived DNA fragment encoding a polypeptide (e.g., NP-011) operably linked to a suitable transcription or translation regulator derived from a mammalian, microbial, viral, or insect gene. A transcription unit generally comprises a combination of: (1) a genetic factor or a factor that plays a regulatory role in gene expression, such as a transcription promoter or enhancer; (2) a structural or coding sequence transcribed into mRNA and translated into protein; and (3) appropriate transcription and translation initiation and termination sequences, as described in detail below. Such regulators may contain operon sequences for controlling transcription. Additional inclusions may be made of the ability to replicate in the host, typically conferred by the origin of replication, and selection genes that facilitate the identification of transformants. DNA regions can be operably linked when they are functionally related to each other. For example, the DNA for the signal peptide (secretory leader) is operatively linked to the polypeptide's DNA if expressed as a precursor involved in polypeptide secretion; a promoter is operatively linked to the coding sequence if it controls transcription of the sequence; or a ribosome binding site is operatively linked to the coding sequence if it is configured to allow translation. Structural factors intended for use in yeast expression systems include leader sequences that enable extracellular secretion of translated proteins by host cells. Alternatively, in the absence of a leader or transport sequence, the recombinant protein may contain an N-terminal methionine residue. Optionally, this residue may be subsequently cleaved from the expressed recombinant protein to provide the final protein. In some embodiments, this disclosure provides a composition, such as a pharmaceutical composition, comprising a nucleic acid or vector as described above or elsewhere herein, optionally further comprising one or more vectors, diluents, excipients, or other additives.

[0164] The disclosure also provides host cells transformed with the nucleic acid molecules or cDNA molecules and / or vectors disclosed herein. The disclosure further provides host cells transformed with one or more of the disclosed nucleic acid molecules, which are operatively linked to a control sequence and optionally inserted into a vector.

[0165] In another embodiment, this disclosure provides a method for preparing the polypeptides provided herein, the method comprising culturing host cells transformed herein under suitable conditions to produce the polypeptides. Optionally, this disclosure provides the isolation of polypeptides secreted from host cells. Optionally, this disclosure also provides polypeptides produced using this method, and pharmaceutical compositions comprising the polypeptides and pharmaceutically acceptable carriers.

[0166] The choice of expression control sequence and expression vector will depend on the host selection. Various expression host / vector combinations can be used (see additional examples below). Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as those from *E. coli* (containing pCR1, pBR322, pMB9, and their derivatives), as well as broader host-scope plasmids such as M13 and filamentous single-stranded DNA bacteriophages.

[0167] The host cell can be a cell or cell population containing or capable of containing recombinant nucleic acids. The host cell can be prokaryotic (e.g., *Escherichia coli*) or eukaryotic. Prokaryotic cells include Gram-negative or Gram-positive organisms, such as *Escherichia coli* or bacilli. The host cell can be a fungal cell, including yeast, such as *Saccharomyces cerevisiae*, *Pichia pastoris*, or *Schizosaccharomyces pombe*. The host cell can also be any of various animal cells, such as insect cells (e.g., Sf-9) or established mammalian cell lines. Examples of suitable mammalian host cell lines include HEK-293, HEK293F, and HEK-293T as described by Gluzman (Cell 23:175 (1981)), the COS-7 line of monkey kidney cells, and other cell lines including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors may include non-transcriptional factors such as origin of replication, suitable promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanking non-transcriptional sequences and 5' or 3' non-translational sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Luckow and Summers (BioTechnology 6:47 (1988)) reviewed baculovirus systems for generating heterologous proteins in insect cells. Cell-free translation systems can also be used. In a preferred embodiment, the host cell is Pichia pastoris.

[0168] Delivery of nucleic acid molecules, vectors, and hosts

[0169] In some embodiments, nucleic acid molecules, vectors, and host cells are provided in the pharmaceutical composition. In some embodiments, components of the pharmaceutical composition facilitate the delivery of nucleic acids to the intended receptor.

[0170] In some embodiments, the DNA construct is delivered to cells via transfection, i.e., by delivering “naked” DNA or in a colloidal dispersion system within a complex. Colloidal systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). Preferred colloidal systems of this invention are lipid-complexed DNA or DNA formulated with liposomes. In the former approach, prior to DNA formulation, for example, a lipid-targeted plasmid containing the desired DNA construct can be experimentally optimized (e.g., including introns in the 5' untranslated region and eliminating unnecessary sequences (Felgner, et al., Ann NY Acad Sci 126-139, 1995) to express the transgene. Then, when the DNA is formulated using various lipid or liposome materials, it can be delivered to the recipient mammal using known methods and materials. See, for example, 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 to Carson et al.

[0171] Liposome targeting can be categorized based on anatomical and mechanical factors. Anatomical classification is based on levels of selectivity, such as organ specificity, cell specificity, and organelle specificity. Mechanical targeting can be distinguished based on whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute into cells of the reticuloendothelial system (RES) within organs containing sinusoidal capillaries. On the other hand, active targeting involves altering liposomes by coupling them to specific ligands (e.g., monoclonal antibodies, sugars, glycolipids, or proteins) or by modifying their composition or size to achieve targeting to organ and cell types beyond their naturally occurring sites of localization.

[0172] The surface of a targeted delivery system can be modified in various ways. In liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain stable association between the targeting ligand and the liposome bilayer. Various linker groups can be used to bind the lipid chain to the targeting ligand. Naked DNA or DNA associated with a delivery vehicle (e.g., liposomes) can be administered to multiple sites in the subject.

[0173] Nucleic acids can be delivered in any desired vector. These include viral or non-viral vectors, including adenovirus vectors, adeno-associated virus 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 (micetic leukemia virus). Nucleic acids can be administered in any desired form that provides a sufficient level of effective delivery, whether contained in viral particles, in liposomes, in nanoparticles, or in polymer complexes.

[0174] The nucleic acid encoding the protein, or the nucleic acid of interest, can be in a plasmid or viral vector, or in other vectors as known in the art. Such vectors are well known, and any vector can be selected for a specific application. In one embodiment of the invention, the gene delivery solvent comprises a promoter and a demethylase-coding sequence. Preferred promoters are tissue-specific promoters and promoters activated by cell proliferation, such as thymidine kinase promoters and thymidine synthase promoters. Other preferred promoters include promoters that can be activated by viral infection (such as α-interferon promoters 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 promoters, and mouse protein promoters. The promoter can be constitutive or inducible.

[0175] In another embodiment, naked polynucleotide molecules are used as gene delivery solvents, as described in WO 90 / 11092 and U.S. Patent No. 5,580,859. Such gene delivery solvents can be growth factor DNA or RNA, and in some embodiments are linked to extinguished adenoviruses. (Curiel et al., Human Gene Therapy 3:147-154, 1992) Other vehicles that may be used include DNA ligands (Wu et al., J. Biol. Chem. 264: 16985-16987, 1989), lipid-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).

[0176] The gene delivery medium may optionally include viral sequences, viral origins of replication, or packaging signals. These viral sequences may be selected from, for example, astroviruses, coronaviruses, orthomyxoviruses, papillomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, retroviruses, enveloped viruses, or adenoviruses. In a preferred embodiment, the growth factor gene delivery medium is a recombinant retroviral vector. Recombinant retroviruses and their various uses have been described in numerous references (e.g., 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. Patent Nos. 4,405,712, 4,861,719 and 4,980,289; and PCT Applications WO 89 / 02,468, WO89 / 05,349 and WO 90 / 02,806). Many retroviral gene delivery solvents can be used in this invention, including those described in the following documents: EP 0,415,731; WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; US Patent No. 5,219,740; WO 9311230; WO 9310218; 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. al., J. Neurosurg. 79:729-735, 1993 (US Patent Nos. 4,777,127, GB 2,200,651, EP 0,345,242 and WO91 / 02805).

[0177] Other viral vectors that can be used to deliver the polynucleotides of the present invention have been derived from herpesviruses and various RNA viruses, such as herpes simplex virus (US Patent No. 5,631,236, granted May 20, 1997, by Woo et al., and WO 00 / 08191 by 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. Gene transfer. New York: Plenum Press; Coupar et al. (1988) Gene, 68: 1-10). Preferred viruses include alphaviruses, poxviruses, arenaviruses, vaccinia viruses, and polioviruses. They possess a variety of features attractive to various 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).

[0178] Methods for generating peptides

[0179] In some embodiments, a vector comprising 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 culture medium to produce the polypeptide. In some embodiments, the polypeptide is secreted into the culture medium. The polypeptide can be isolated from the culture medium using any suitable technique.

[0180] The polypeptides of this invention, produced from transformed host cells (e.g., NP-011), can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and fractionation column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Affinity tags such as hexahistine, maltose-binding domains, influenza shell sequences, and glutathione S-transferases can be attached to the protein to facilitate purification by passing it through a suitable affinity column. The polypeptides can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0181] For example, firstly, a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit, can be used to concentrate the supernatant from the system in which the peptide is secreted into the culture medium. Following the concentration step, the concentrate can be coated onto a suitable purification matrix. Alternatively, anion exchange resins, such as matrices or substrates with diethylaminoethyl (DEAE) side groups, can be used. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, cation or anion exchange steps can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, the peptide can be further purified using one or more RP-HPLC steps employing hydrophobic reversed-phase high-performance liquid chromatography (RP-HPLC) media (e.g., silica gel with methyl side groups or other aliphatic groups).

[0182] Peptides generated in cell culture can be isolated, for example, by initial extraction from the cell pellet followed by one or more concentration, salting out, aqueous ion exchange, or size exclusion chromatography steps. The final purification step can be performed using high-performance liquid chromatography (HPLC). Cells used in recombinant protein expression can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysis agents.

[0183] "Isolated" or "purified" proteins or their biologically active portions are substantially free of cellular material or other contaminating proteins from which the protein is derived, or are substantially free of chemical precursors or other chemicals during chemical synthesis. The expression "substantially free of cellular material" includes protein formulations in which the protein is isolated from the cellular components of the cell from which it is isolated or recombined to produce the protein. Thus, proteins substantially free of cellular material comprise protein formulations having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). When the protein or its biologically active portion is recombined to produce the protein, it is preferably substantially free of culture medium, i.e., the culture medium constitutes less than about 20%, 10%, or 5% of the volume of the protein formulation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., isolated from chemical precursors or other chemicals involved in the protein synthesis. Accordingly, apart from the polypeptide of interest, such protein formulations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds.

[0184] Pharmaceutical Composition

[0185] In another aspect, the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of the polypeptide disclosed herein formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. Pharmaceutical compositions of the present invention can be specifically formulated for storage and use by combining purified pharmaceutical agents of the present invention as covered by the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those skilled in the art will generally consider pharmaceutically acceptable carriers, excipients, and / or stabilizers as inactive components of a formulation or pharmaceutical composition.

[0186] Suitable pharmaceutically acceptable solvents include, but are not limited to: non-toxic buffer solutions such as phosphates, citrates, and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives such as octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzyl ammonium chloride, benzyl chloride, phenol, butanol or benzyl alcohol, alkyl parabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; and low molecular weight solvents (less than about 1). (0 residues) Peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic 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 (e.g., zinc protein complexes); and nonionic surfactants, such as Tween or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22nd Supplement, 2012, British Medical Publishing House, London.) Preparations intended for in vivo administration must be sterile. This can be easily achieved through filtration using sterile filter membranes.

[0187] Examples of pharmaceutically 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) astragalus gum powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter or suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) Diols, such as propylene glycol; (11) Polyols, such as glycerol, 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) Alginate; (16) Atherless water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) Phosphate buffer; and (21) Other non-toxic and compatible substances used in pharmaceutical preparations.

[0188] Pharmaceutically acceptable salts may be part of a pharmaceutical composition comprising the polypeptides of the present invention. Salts may be relatively non-toxic, inorganic, and organic acids added to the formulation. Salts may be added to the polypeptide at any stage of purification (including post-purification). Representative salts include hydrobromide, hydrochloride, sulfate, hydrogen sulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, lysinate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthate, methanesulfonate, glucono-p-ethyl, lacturonate, and dodecyl sulfonate, etc. (see, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19).

[0189] The composition may also contain wetting agents, emulsifiers, and lubricants (such as sodium dodecyl sulfate and magnesium stearate), as well as release agents, preservatives, and antioxidants. 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 ascorbate palmitate, tert-butylhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0190] Pharmaceutical compositions of the present invention suitable for parenteral administration may include one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injections or dispersions prior to use. The sterile powders may contain antioxidants, buffers, antibacterial agents, solutes that make the formulation isotonic with the blood of the intended recipient, or suspending agents or thickeners.

[0191] By including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid, protection against microbial activity can be ensured. The composition may also need to contain isotonic agents, such as sugars and sodium chloride. Additionally, by including delayed-absorption agents such as aluminum monostearate and gelatin, the absorption of injectable drug formulations can be prolonged.

[0192] In some 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 back-evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through a filter defining a pore size to produce liposomes with a desired diameter.

[0193] In some embodiments, sustained-release formulations comprising the polypeptides of the present invention can be produced. Suitable examples of sustained-release formulations comprise a semi-permeable matrix of a solid hydrophobic polymer containing the polypeptide, wherein the matrix is ​​in the form of a shaped article (e.g., a film or microcapsule). Examples of sustained-release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide, 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 copolymer and leuprolide acetate), sucrose isobutyrate acetate, and poly-D-(-)-3-hydroxybutyrate.

[0194] The compositions described herein can be administered in suitable manner, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Alternatively, the compositions can be appropriately administered via pulsatile infusion.

[0195] In some embodiments, the peptides of the present invention may be conjugated or modified according to pharmacological methods known in the art (e.g., PEGylation, glycosylation, oligomerization, etc.) in order to further enhance the desired biological activity, such as improving bioavailability and reducing protein degradation.

[0196] In some embodiments, the peptide is lyophilized and / or stored in lyophilized form. In some embodiments, lyophilization includes formulations of the peptides described herein.

[0197] Reagent test kit

[0198] A "kit" is an article (e.g., package or container) comprising at least one composition (e.g., a polypeptide) of the present invention. The kit may also include additional diluents. The kit may include one or more agents necessary for resuspending the lyophilized composition and / or a filtration unit for removing aggregates. The kit may be promoted, distributed, or sold as a unit for carrying out the methods of the present invention. The kit may contain one or more reagents necessary for expressing compositions useful in the methods of the present invention. The reagents in the kit may be provided in separate containers or in a single container as a mixture of two or more reagents. Additionally, explanatory material describing the use of the compositions within the kit may be included.

[0199] sequence

[0200] The term "sequence identity or homology" refers to the sequence similarity between two polypeptide molecules or two nucleic acid molecules. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit—for example, if a position in each of two DNA molecules is occupied by adenine—then the two molecules are homologous at that position or have the same sequence. The percentage of homology or sequence identity between two sequences is determined by dividing the number of matching or homologous positions shared by the two sequences by the number of positions being compared and then multiplying by 100. For example, if six out of ten positions in two sequences are the same, then the two sequences are 60% homologous or have 60% sequence identity. For instance, the DNA sequences ATTGCC and TATGGC have 50% homology or sequence identity. Generally, comparisons are made when aligning two sequences to obtain the maximum homology. Unless otherwise specified, "loopout regions" (e.g., regions resulting from deletions or insertions in one sequence) are considered mismatches. Mathematical algorithms can be used to perform sequence alignment and determine the percentage of homology between two sequences.

[0201] In addition to naturally occurring allelic variants of the nucleic acid molecules of the present invention that can exist in a population, those skilled in the art will further recognize that sequence changes can be introduced through mutation, resulting in alterations to the amino acid sequence of the encoded protein without changing the biological activity of the protein thereby. For example, nucleotide substitutions can be performed, thereby replacing amino acid residues at “non-essential” amino acid residues. “Non-essential” amino acid residues are residues that can be altered from the wild-type sequence without changing biological activity, while “essential” amino acid residues are essential for biological activity. For example, amino acid residues that are not conserved or only partially conserved in homologs of various species may be non-essential for activity and are therefore potential targets for alteration. Alternatively, amino acid residues that are conserved in homologs of various species (e.g., mice and humans) may be essential for activity and are therefore not potential targets for alteration.

[0202] Accordingly, another aspect of the invention relates to nucleic acid molecules encoding the polypeptides of the invention, the nucleic acid molecules containing amino acid residue variations that are not essential for activity. Such polypeptides differ in amino acid sequence from naturally occurring proteins that correspond to the markers of the invention but retain biological activity. In one embodiment, the amino acid sequence of the polypeptide is at least about 40%, 50%, 60%, 70%, 75%, 80%, 83%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or identical to the amino acid sequence of the polypeptides described herein.

[0203] Nucleic acid variants can contain alterations to coding regions, non-coding regions, or both. In some embodiments, nucleic acid variants contain alterations that produce silencing substitutions, additions, or deletions, but do not change the properties or activity of the encoded polypeptide. In some embodiments, nucleic acid variants are generated through silencing substitutions due to the degeneracy of the genetic code. Nucleic acid variants are generated for a variety of reasons, such as to optimize codon expression for a specific host (changing codons in human mRNA to those preferred by bacterial hosts such as E. coli). Vectors and cells including the nucleic acids described herein are also provided.

[0204] 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 residues are substituted, added, or deleted, the encoded protein can be introduced, forming isolated nucleic acid molecules encoding variant proteins. Mutations can be introduced using standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conserved amino acid substitutions are performed on one or more predicted non-essential amino acid residues. A “conserved amino acid substitution” is the substitution of an amino acid residue 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 those 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be randomly introduced along all or part of the coding sequence, such as through saturation mutagenesis, and the resulting mutants can be screened for bioactivity to identify mutants that retain activity. After mutagenesis, the encoded protein can be recombinantly expressed, and the protein's activity can be measured.

[0205] Non-conservative substitutions include the following: (a) substitution of a residue with a positively charged side chain (e.g., Arg, His, or Lys) with or against a negatively charged residue (e.g., Glu or Asp); (b) substitution of a hydrophilic residue (e.g., Ser or Thr) with or against a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (c) substitution of Cys or Pro with or against any other residue; or (d) substitution of a residue with a large hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) with or against a residue with a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

[0206] Other substitutions can be easily identified. For example, for the amino acid alanine, any one of D-Ala, Gly, βAla, L-Cys, and D-Cys can be substituted. For lysine, any one of D-Lys, Arg, D-Arg, homologous Arg, Met, D-Met, ornithine, or D-ornithine can be substituted. Typically, substitutions in key functional regions that are expected to alter the properties of a polypeptide that can be induced to separate include: (a) substitution of a polar residue (e.g., Ser or Thr) for (or by) a hydrophobic residue (e.g., Leu, Ile, Phe, or Ala); (b) substitution of a Cys residue for (or by) any other residue; (c) substitution of a residue with a positively charged side chain (e.g., Lys, Arg, or His) for (or by) a residue with a negatively charged side chain (e.g., Glu or Asp); or (d) substitution of a residue with a large side chain (e.g., Phe) for (or by) a residue without a large side chain (e.g., Gly). The likelihood that one of these nonconservative substitutions can alter the functional properties of a protein is also related to the substitution location relative to the key functional region of the protein: correspondingly, some nonconservative substitutions may have little or no effect on biological properties.

[0207] As defined by the genetic code, there is a known and established correspondence between the amino acid sequence of a specific protein and the nucleotide sequence that can encode the protein (as shown below). Similarly, as defined by the genetic code, there is a known and established correspondence between the nucleotide sequence of a specific nucleic acid and the amino acid sequence encoded by the nucleic acid.

[0208] genetic code

[0209]

[0210]

[0211] A well-known and important feature of the genetic code is its redundancy, allowing for more than one nucleotide triplet (as shown above) to be used for most amino acids used to make proteins. Therefore, many different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the same amino acid sequence in all organisms (although some organisms can translate some sequences more efficiently than others). Furthermore, methylated variants of purines or pyrimidines can occasionally be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0212] In light of the foregoing, by translating DNA or RNA into amino acid sequences using the genetic code, the nucleotide sequence of the DNA or RNA encoding the polypeptide (or any part thereof) of the present invention can be used to derive the fusion protein or polypeptide amino acid sequence. Similarly, for the polypeptide amino acid sequence, the corresponding nucleotide sequence encoding the fusion protein or polypeptide can be inferred from the genetic code (which, due to its redundancy, will generate multiple nucleic acid sequences for any given amino acid sequence). Therefore, the description and / or disclosure herein regarding the nucleotide sequence encoding the fusion protein or polypeptide should be considered to equally include the description and / or disclosure regarding the amino acid sequence encoded by the nucleotide sequence. Likewise, the description and / or disclosure herein regarding the amino acid sequence of the fusion protein or polypeptide should be considered to equally include the description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0213] Finally, the nucleic acid and amino acid sequence information of the nucleic acid and polypeptide molecules used in this invention is well known in the art and can be easily obtained from public databases (e.g., the National Center for Biotechnology Information (NCBI)). For example, Table 1 below provides exemplary nucleic acid and amino acid sequences derived from public sequence databases.

[0214] Table 1

[0215] SEQ ID NO:1 Human milk fat globule-EGF factor 8 protein (MFGE8), transcript variant 2, cDNA (NM_ 001114614.3 (CDS 61 to 1068)

[0216]

[0217] SEQ ID NO:2 Human milk lectin isotype B (NP_001108086.1)

[0218]

[0219] SEQ ID NO:3 Human milk fat globule-EGF factor 8 protein (MFGE8), transcript variant 4, cDNA (NM_ 001310319.2, CDS 61 to 1092)

[0220]

[0221]

[0222] SEQ ID NO:4 Human milk lectin isotype D (NP001297248.1)

[0223]

[0224] SEQ ID NO:5 Human milk fat globule-EGF factor 8 protein (MFGE8), transcript variant 3, cDNA (NM_ 001310320.2 (CDS 138 to 186)

[0225]

[0226]

[0227] SEQ ID NO:6 Human milk lectin isotype C (NP_001297249.1)

[0228]

[0229] SEQ ID NO:7 Human milk fat globule-EGF factor 8 protein (MFGE8), transcript variant 5, cDNA (NM_ 001310321.2 (CDS 1005 to 1832)

[0230]

[0231]

[0232] SEQ ID NO:8 Human milk lectin isotype E (NP_001297250.1)

[0233]

[0234] SEQ ID NO:9 Human milk fat globule - epidermal growth factor 8 protein (MFGE8), transcript variant 1, cDNA (NM_005928.4, CDS 61 to 1224)

[0235]

[0236] SEQ ID NO:10 Human lactadherin isoform A (NP_005919.2)

[0237]

[0238] *The amino acid sequences corresponding to NP-011(a) (amino acids 24-225) are underlined. Amino acids 1-23 correspond to the signal peptide.

[0239] SEQ ID NO:11 Nucleic acid encoding human NP - 011

[0240]

[0241] SEQ ID NO:12 Human NP - 011

[0242]

[0243] *The amino acid sequence corresponding to NP-011 (amino acids 24-225) is indicated by underscores.

[0244] Amino acids 1-23 correspond to the signal peptide.

[0245] SEQ ID NO:13 Rat milk fat globule - epidermal growth factor 8 protein (Mfge8), transcript variant 1, cDNA (NM_ 001040186.2, CDS 82 to 1533)

[0246]

[0247] SEQ ID NO:14 Rat lactadherin isoform 1 (NP_001035276.1)

[0248]

[0249] SEQ ID NO:15 Rat milk fat globule - epidermal growth factor 8 protein (Mfge8), transcript variant 2, cDNA (NM_ 012811.3, CDS 82 to 1365)

[0250]

[0251] SEQ ID NO:16 Rat lactadherin isoform 2 (NP_036943.1)

[0252]

[0253] SEQ ID NO:17 Mouse milk fat globule - epidermal growth factor 8 protein (Mfge8), transcript variant 2, cDNA (NM_ 001045489.1, CDS 94 to 1374)

[0254]

[0255]

[0256] SEQ ID NO:18 Mouse lactadherin isoform 2 (NP_001038954.1)

[0257]

[0258] SEQ ID NO:19 Mouse milk fat globule - epidermal growth factor 8 protein (Mfge8), transcript variant 1, cDNA (NM_ 008594.2, CDS 94 to 1485)

[0259]

[0260]

[0261] SEQ ID NO:20 Mouse lactadherin isoform 1 (NP_032620.2)

[0262]

[0263] *Table 1 contains RNA nucleic acid molecules (e.g., uridine-substituted thymine) and nucleic acid molecules encoding orthologs of encoded proteins.

[0264] *Table 1 also contains orthologs of proteins.

[0265] Unless otherwise stated, implementations of this disclosure employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, all of which are within the scope of the art.

[0266] The following examples are provided in an illustrative manner, not in a limiting manner. Example

[0267] The foregoing description of specific embodiments will fully reveal the general nature of this disclosure, enabling others to easily modify and / or adapt these specific embodiments for various applications by applying knowledge of the art without departing from the general concepts of this disclosure, without excessive experimentation. Therefore, based on the teachings and guidance presented herein, such modifications and alterations are intended to be within the meaning and scope of equivalent forms of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive rather than limiting purposes, and therefore the terminology or terminology in this specification should be interpreted by those skilled in the art in accordance with the teachings and guidance.

[0268] The breadth and scope of this disclosure should not be limited by any of the exemplary embodiments described above, but should be defined solely by the appended claims and their equivalents.

[0269] 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 that each individual publication, patent, patent application and / or other document is individually indicated for all purposes.

[0270] Example 1: Materials and Methods

[0271] Rodent fibrosis model induction and NP-011 efficacy testing

[0272] To compare the efficacy of different derivatives of MFG-E8 (NP-011, NP-012, and NP-013) and full-length MFG-E8 protein, 5-6 week old male C57BL / 6 mice (n=4 per group, 3 times per week for 8 weeks) were administered 200 mg / kg TAA (thioacetamide, Sigma-Aldrich, St. Louis, Missouri, USA) and 160 μg / kg protein was administered intraperitoneally per mouse. Mice were sacrificed 3 days after protein administration for analysis. Various doses of NP-011 (20 μg / kg–160 μg / kg, n=5 per group) were further tested in the same TAA-induced liver fibrosis model. To test the antifibrotic effect of repeated administrations of NP-011, mice were injected with TAA for 12 consecutive weeks, followed by administration to a TAA-induced liver fibrosis model. For the fibrotic liver model, mice were administered NP-011 intraperitoneally once to six times, with each administration 5 days apart (n=4 per group). For the progressive liver fibrosis model, mice were injected with TAA for 4 weeks, followed by intraperitoneal administration of a combination of NP-011 and TAA 3 times a week for 4 weeks (n=5 per group). In all animal experiments, mice were sacrificed three days after the last administration of NP-011; all experimental procedures were approved by the Animal Care and Use Committee of Korea University (KOREA-2016-0254).

[0273] DMN-induced liver cirrhosis model induction and NP-011 efficacy testing

[0274] Animal models of liver cirrhosis were designed and implemented by the National Center for Evaluation and Research on Digestive Disorders (NCEED, Incheon, South Korea). Six-week-old male Sprague-Dawley rats (Orient Bio, Gapyeong, South Korea) were injected intraperitoneally with dimethylnitrosamine (DMN saline solution, 10 mg / ml / kg) for three consecutive days each week for a six-week period to induce liver cirrhosis. To minimize weight variability between groups, rats were randomly assigned to seven groups twice, on week one and week three: G1 (transporter control, n=10), G2 (fibrosis control, n=12), G3 (NP-011 treatment, once weekly, n=10), G4 (NP-011 treatment group, twice weekly, n=10), G5 (NP-011 treatment group, three times weekly, n=10), and G6 (NP-011 treatment group, daily, n=10). Intravenous administration of NP-011 (G3-G6) began in week 3. Routine observations were performed daily, and body weight was measured twice weekly. At the end of week 6, surviving rats in all groups were sacrificed for necropsy, and survival rates for each group were calculated. Rats were anesthetized with isoflurane, and blood and major organs were collected. Liver tissue was fixed in 10% formalin neutral buffer and then subjected to histological evaluation and immunohistochemical analysis using hematoxylin and eosin staining, along with α-smooth muscle actin antibody from PostBio Inc. (Guri, Korea).

[0275] MCD-induced NASH mice

[0276] Six-week-old male C57BL / 6 mice were fed a standard diet or a diet lacking methionine and choline (MCD) for 10 weeks. Procedures for animal handling and care were approved by the Animal Care and Use Committee of Korea University (KUIACUC-2018-3). After 5 weeks, mice were intraperitoneally injected with saline or NP-011 (20 μg, 40 μg, 80 μg, or 160 μg / kg body weight, once weekly, n=5) and fed an MCD diet for at least 5 weeks. Body weight was measured weekly during the experiment.

[0277] Immunofluorescence assay

[0278] Liver tissue was fixed in 4% paraformaldehyde (PFA) and dehydrated using a gradient series of ethanol. The tissue was then washed with xylene and embedded in paraffin. Paraffin-embedded tissue sections were stained with Sirius red (American MasterTech Scientific, Lody, CA) for liver fibrosis assessment. For immunofluorescence staining, tissue sections were antigen-retrievaled with citric acid and probed overnight at 4°C with primary antibodies against α-smooth muscle actin (α-SMA) or albumin (ALB). Tissue blocked with 10% donkey serum containing PBS was then stained. For visualization of the staining, sections were washed with 0.1% bovine serum albumin (BSA) containing phosphate-buffered saline (PBS) and stained with fluorescently labeled secondary antibody (Invitrogen / Thermo Fisher Scientific, Carlsbad, CA). Digital images were captured using a microscope (Nikon, Tokyo, Japan) and analyzed using ImageJ software.

[0279] Histopathological analysis and NASH score

[0280] Liver tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) according to standard procedures. Histological scores of liver lesions were assessed using the rodent NAFLD scoring system as described in (1): macrovesicular steatosis (0-3), microvesicular steatosis (0-3), hypertrophy (0-3), and number of inflammatory foci / field of view (0-3). Five images were randomly selected, and all cells were counted and calculated for each histological feature. For Oil Red O staining, frozen liver tissue embedded in OCT was sectioned into 4 μm sections and stained with an Oil Red O staining kit (Lifeline Cell Technology, Frederick, MD, LL-0052). The area of ​​Oil Red O staining was measured using Image J. For immunostaining of frozen sections, frozen liver tissue embedded in OCT was sectioned into 4 μm sections, slides were permeated, and masked with 0.1% Triton X-100 and 10% donkey serum. Liver sections were incubated with the primary and secondary antibodies specified in Table 2.

[0281] TGF-β luciferase signal transduction reporter gene assay

[0282] The luciferase signal transduction reporter gene assay was performed according to the manufacturer's protocol (Qiagen, Hilden, Germany). Detailed methods for the complete transcriptome analysis are described in the Supplementary Materials and Methods.

[0283] Whole transcriptome analysis of mouse liver

[0284] Liver samples from normal mice (n=3), TAA-induced fibrotic livers (n=3), and NP-011-treated livers (n=3) were homogenized in cold Trizol (Sigma, USA) and subjected to next-generation sequencing (NGS) by BGI Tech Solutions (Hong Kong, China). Gene set enrichment analysis (GSEA) was performed using GSEAv17 (Broad Institute, Cambridge, MA, USA). Detailed methodologies for the complete transcriptome analysis are described in Supplementary Materials and Methods.

[0285] Quantitative reverse transcription PCR (RT-qPCR)

[0286] Using the CFX96 real-time PCR detection system (Bio-Rad, Hercules, California, USA) with iQ TM Quantitative polymerase chain reaction (qPCR) was performed using Green Supermix (Bio-Rad). Table 3 lists the specific primers used. mRNA levels were normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels.

[0287] Cell culture and reagents

[0288] Human hepatic stellate cell (HSC) line hTERT-HSC was cultured in Delbecco's modified Eagle medium (DMEM; GE Healthcare Life Sciences, Marburg, Massachusetts) supplemented with 10% fetal bovine serum (FBS; Gibco / Thermo Fisher Scientific, Waltham, MATLAB, USA), 100 U / mL penicillin, and 100 mg / mL streptomycin (Gibco). Human HEK-293FT cells were kindly provided by Professor Hyunggee Kim (Korea University) and cultured in Delbecco's modified Eagle medium (DMEM; GE Healthcare Life Sciences, Illinois, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, New York, USA), 100 U / mL penicillin, and 100 mg / mL streptomycin (Gibco).

[0289] Western blot and immunoprecipitation (IP) analysis

[0290] HSCs were dissolved in RIPA lysis buffer (LPS solution) containing a protease inhibitor (Roche, Basel, Switzerland) to prepare protein samples. A total of 40 μg of protein was isolated from cells 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 overnight at 4 °C with primary antibody. Table 2 lists the antibodies used in this study. For immunoprecipitation (IP) analysis, a total of 400 μg of protein was incubated with 1 μg TGFβRI antibody at 4 °C for 12 h, conjugated to Protein A / G agarose beads (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), 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 for 2 hours at room temperature with a secondary antibody conjugated with horseradish peroxidase (HRP) (Thermo Fisher Scientific). After washing with TBS-T, the membrane was analyzed using Pierce. TM The membrane was developed using ECL protein blot substrate (Thermo Fisher Scientific), and the bands were detected using a chemiluminescence imaging system (GE Healthcare Life Sciences).

[0291] NP-011 efficacy test in 3D human liver fibrosis model

[0292] To establish a human liver fibrosis model, hepatocytes were mixed at a cell density ratio of 2:1 in ultra-low adsorption 96-well plates (Corning). NEXEL (Seoul, South Korea) and hTert-HSC (provided by Dr. David Brenner, University of California, San Diego, USA) were used to form hepatic spheroids. The hepatic spheroids were cultured for 21 days and treated with 50 mM acetaminophen (APAP) to induce fibrosis. To test the efficacy of NP-011 in the APAP-induced 3D liver fibrosis model, treatment with 500 ng / ml NP-011 for 48 hours was performed.

[0293] In vitro studies of HSC activation

[0294] To investigate the effect of NP-011 on TGF-β1-mediated HSC activation, human HSC line (hTERT-HSC) was grown in the presence of serum and then starved in DMEM containing 0.2% FBS for 24 hours before TGF-β1 treatment. The serum-starved HSCs were pretreated with 10 ng / mL TGF-β1 for 1 hour and then exposed to 100–1500 ng / mL NP-011 for 6 hours. To block integrin aνβ3 / β5 in HSCs, they were pretreated with 1 μM CT (Selleck Chemicals, Houston, Texas, USA) for 2 hours before TGF-β1 treatment. Activation and inactivation of HSCs were quantitatively determined by 5-ethynyl-2′-deoxyuridine (EdU) assay.

[0295] Adjacent connectivity determination (PLA)

[0296] For PLA doping determination, 2 × 10⁻⁶ ppm was used per well. 4 Human HSCs were seeded onto 18 mm round coverslips in 12-well plates and cultured for 24 hours. After serum starvation for 24 hours, cells were treated with 10 ng / ml TGF-β1 and / or 500 ng / ml NP-011, followed by a further incubation for 30 minutes. PLA incorporation was assessed using the Duolink In Situ Red Initiation 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 / ).

[0297] EdU Incorporation Determination

[0298] For EdU doping determination, 2 × 10⁻⁶ ppm was used per well. 4 Human HSCs were seeded into 12-well plates and cultured for 24 hours. After treatment with TGF-β1 and / or NP-011, serum-starved HSCs were incubated with EdU (10 μM) for 6 hours. 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.

[0299] Collagenase activity assay

[0300] Collagenase activity was determined according to the manufacturer's protocol (Chondrex, Washington, USA). The enzyme source was mixed with 180 μl of solution B. The enzyme reaction was initiated by mixing with 200 μl of 1.0 mg / mL FITC-labeled bovine collagen I substrate and incubated at 37 °C for 1 hour. For the negative control, 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 minutes. Finally, 400 μl aliquots of extraction buffer were thoroughly mixed with the reaction solution and centrifuged at 10,000 rpm for 5 minutes. FL intensity was measured at 520 nm using the supernatant (200 μl), with excitation at 490 nm in a black 96-well plate using a fluorescence spectrometer.

[0301] Assay of THP-1 differentiation and fluorescent bead phagocytosis.

[0302] THP-1 cells were purchased from ATCC and cultured in RPMI-1640 (Gibco, 31800-022) medium 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 by culturing in 200 ng / ml phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) for 48 hours, followed by 72 hours in PMA-free medium. Phagocytosis was measured at 1.05 × 10⁻⁶ cells / mL. 5 cells / cm 2 THP-1 cells were seeded. 2.0 μm carboxylate-modified microspheres (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 at 37°C for 4 hours in a 5% CO2 incubator. Cells were isolated using a TrypLE (Gibco, 12604-021) and measured by flow cytometry (Accuri C6 Plus).

[0303] Biological distribution study

[0304] Six-week-old male C57BL / 6 mice were randomly divided into two groups: a control group (saline) and an NP-011 treatment group (NP-011, 160 μg / kg body weight, saline), with three animals used at each time point. Mice were administered intravenously at different time points. Mice were sacrificed by CO2 inhalation, and organs (brain, heart, lungs, liver, kidneys, and spleen) were harvested directly in PBS to remove fat and blood traces. Organs were immediately stored at -80°C. Procedures for animal handling and care were approved by the Korea University Animal Care and Use Committee (KUIACUC-2018-0027 and KUIACUC-2018-0040). Lysis buffer (Ray Biotech, Norcross, Georgia, USA, Classification No. EL-LYSIS) and cycloplegic acid were used. TM The entire organ was homogenized using a Protease Inhibitor Cocktail (Sigma-Aldrich, Classification No. 11697498001) at 4°C. After homogenization, the samples were centrifuged at 13,000 rpm for 20 minutes at 4°C, and the supernatant was collected for ELISA analysis. Protein concentrations for each sample were calculated using Bio-Rad Bradford. For the detection of NP-011, the Human MFG-E8 Quantikine ELISA Kit (R&D, Classification No. DFGE80) was used according to the manufacturer's instructions. Measurements taken at 450 nm, 540 nm, and 570 nm using the iD3 (Molecular Device, San Jose, California, USA).

[0305] Repeated-dose 28-day chronic toxicity study

[0306] A 4-week repeated toxicity study of NP011 was conducted by ChemOn Inc. (a non-clinical CRO located in Gyeonggi Province, South Korea). A total of 30 SD rats (15 males and 15 females, 6 weeks old) were randomly assigned to three groups: G1 (solvent control), G2 (0.2 mg / kg NP-011, daily), and G3 (2.2 mg / kg NP-011, daily), with 5 males and 5 females in each group. The solvent and NP-011 were administered via the tail vein. Body weight, food intake, and water intake were measured in all groups before the start of the study and weekly during the study period. At the end of the study, all rats were sacrificed, and blood was collected for ophthalmic, urinary, hematological, biochemical, and histopathological examinations. Histopathological evaluation was performed on all major organs and regions (brain, liver, heart, spleen, lung, kidney, sternal bone marrow, injection site, testes / uterus, and thymus).

[0307] Statistical analysis

[0308] Numerical values ​​are expressed as the mean ± SEM of at least three replicated independent experiments in in vitro studies. Unless otherwise stated, for evaluating the in vivo antifibrotic effect of NP-011, at least three animals were used per group per experiment, and data were obtained from 2 to 3 independent experiments. The percentage of the total image area positive for Sirius red staining or immunostaining was measured using ImageJ and expressed as a relative value compared to normal liver or control cell cultures (arbitrarily set to 1). The statistical significance of differences between paired groups was analyzed using the Student's t-test. The statistical significance of differences between multiple groups (more than 2 groups) was detected using one-way ANOVA. Data are expressed as mean and 95% confidence intervals (CI). All statistical tests were two-way, and data were assumed to be statistically significant with P < 0.05 or P < 0.01.

[0309] Table 2

[0310]

[0311] Table 3

[0312]

[0313]

[0314] Example 2: NP-011 inhibits liver fibrosis

[0315] Human MFG-E8 comprises three domains: an N-terminal signal peptide, an epidermal growth factor (EGF)-like domain with an arginine-glycine-aspartate (TGD) motif, and C domains (C1 and C2). While MFG-E8 is known to modulate inflammatory responses via RGD motif-mediated binding to immune cells and phagocytosis of apoptotic cells expressing phosphatidylserine (PS), the mechanism by which MFG-E8 exerts its antifibrotic effect remains unclear. A recent report indicated that the C2 domain of MFG-E8 plays a crucial 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 effect. To investigate this, two different truncated forms of MFG-E8, along with the EFG domain, were synthesized to maintain the RGD motif for cell binding (NP-011: EGF-C1 domain and NP-012: EGF-C2 domain). As a control, the full-length MFG-E8 (NP-013) was also synthesized.

[0316] Subsequent efficacy testing was conducted in a mouse model of liver fibrosis induced by thioacetamide (TAA). Figure 1A The study revealed that the application of commercially available MFG-E8 or NP-013 effectively reduced fibrotic areas (Sirius red staining areas). Figure 1B and Figure 1CFurthermore, it downregulated the expression levels of liver fibrosis-related genes (Col1a1, Col1a2, and Acta2). Figure 1D However, it is convincing that the application of MFG-E8 and NP-013 ( Figure 1B and Figure 1C Compared to NP-011, the elimination of fibrotic regions was significantly increased, and the downregulation of fibrosis gene expression levels in damaged liver was much more pronounced, which is quite surprising, given that NP-011 lacks the C2 domain, which is considered important for the function of MFG-E8 (including its anti-fibrotic effects). Unlike the potent efficacy of NP-011, compared to MFG-E8 and NP-013 (…),… Figure 1B and Figure 1C The administered NP-012 was less efficient in reducing fibrotic areas and significantly upregulated the expression of Acta2 (α-smooth muscle actin, α-SMA) in the damaged liver. Figure 1D ). Reporter gene assays revealed that NP-011 rapidly inhibited TGF-β signaling within 30 minutes, while MFG-E8 could inhibit TGF-β signaling up to 2 hours after treatment. Figure 1E These results indicate that, compared to the full-length protein, the absence of the C2 domain of MFG-E8 unexpectedly enhances antifibrotic activity.

[0317] Example 3: NP-011 significantly reverses liver fibrosis at the lowest dose

[0318] To explore the effective dose of NP-011, different doses of NP-011 (20 μg / kg, 40 μg / kg, 80 μg / kg, and 160 μg / kg) were administered to 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 the liver of mice. Figure 2B and Figure 2C (Sham module). Compared with liver tissue treated with the sham module, all groups treated with NP-011 (20-160 ug / kg) showed a significant reduction in fibrotic areas. Figure 2B and Figure 2C Similar to the reduction in fibrosis, upon administration of NP-011, the mRNA and protein levels of the key fibrosis marker Acta2 (α-SMA, a marker of myofibroblast-like cells differentiated from HSCs) and other fibrosis-related genes were significantly and substantially downregulated. [[ID= / / ID=]]Figure 2D and Figure 2E Notably, doses above 40 μg / kg stably and potently inhibited TGF-β mRNA expression with minimal inter-individual variability. Figure 2FIn summary, these results indicate that NP-011 exhibits significant therapeutic efficacy in the low-dose range; most effectively, 40 μg / kg NP-011 may be the lowest effective dose administered, resulting in a continuous decrease in TGF-β expression in the damaged liver.

[0319] Example 4: Lowest doses of NP-011 demonstrate therapeutic efficacy in different fibrosis-related models.

[0320] The efficacy of the lowest 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 liver fibrosis model. For this purpose, TAA injections were extended from 8 weeks to 12 weeks (3 times per week), followed by administration of 40 μg / kg NP-011 to mice one to six times at 5-day intervals. Figure 3A TAA injections over 12 weeks resulted in persistent fibrotic areas in the liver, despite TAA injections being discontinued 30 days prior to mouse sacrifice. Figure 3B (Blank control group). In contrast, NP-011 administration significantly reduced fibrotic areas in the damaged liver, and the reduction in fibrosis was positively correlated with the number of administrations. Figure 3B ).

[0321] Liver fibrosis is a progressive disease, therefore evaluating the efficacy of NP-011 in progressive liver fibrosis can better translate animal studies into clinical applications. To confirm the therapeutic efficacy of NP-011 in progressive liver fibrosis, during the last 4 weeks of model induction, NP-011 (40 μg / kg, three times a week) and TAA were administered simultaneously. Figure 3C As expected, concurrent administration of TAA and NP-011 significantly reduced the area of ​​fibrosis in the liver compared to livers receiving TAA alone. Figure 3D We also used an APAP-induced in vitro human fibrosis model and found that NP-011 significantly reduced APAP-induced HSC activation in 3D hepatic spheroids composed of stem cells and hepatic stellate cells. Figure 3E ).

[0322] The efficacy of NP-011 was further tested in a dimethylnitrosamine (DMN)-induced liver cirrhosis model. Figure 4A (16). Continuous injection of DMN into rats for 6 weeks resulted in a mortality rate of 75% and severe fibrosis in the liver. Figure 4B and 4C Histopathological analysis revealed significant necrosis (fragmented and lobular) and fibrosis events in the liver of the DMN-induced cirrhosis model. [[ID=ID=43]]Figure 4DImportantly, daily administration of NP-011 reduced mortality in animals by up to 50% and significantly alleviated liver fibrosis. Figure 4B and Figure 4C It also reduced fragmented necrosis (~20%), lobular necrosis (~30%), fibrosis score (~24.3%), and HSC activation (~17.7%). Figure 4D ).

[0323] Subsequently, the efficacy of NP-011 was evaluated in a NASH model induced by a diet lacking methionine and choline (MCD). Figure 5A As reported in various studies, MCD diets lead to fat accumulation in the liver, but NP-011 administered at all tested doses significantly reduced fat accumulation. Figure 5B and 5C Furthermore, a significant reduction in macrovesicle / microvesicle fatty degeneration and hypertrophy was observed after NP-011 administration. Figure 5D In summary, the lowest dose of NP-011 has shown therapeutic efficacy not only in severe liver fibrosis but also in NASH.

[0324] Example 5: NP-011 reduced liver fibrosis in damaged liver through interaction with hepatic stellate cells (HSCs) and macrophages.

[0325] This article investigates the mechanism by which NP-011 rapidly and effectively inhibits TGF-β signaling in diseased liver. Figure 1E and 2F One day after administration of NP-011 to TAA-induced fibrotic liver, NPP-011 preferentially binds to α-SMA-positive HSCs, and some NP-011 interacts with macrophages in the liver. Figure 6 Integrins αvβ3 and αvβ5 on the surface of HSCs have previously been involved in the regulation of fibrosis. Furthermore, crosstalk between integrins and TGF-β signaling may play an important role in the regulation of pathological epithelial-mesenchymal transition (EMT) and myofibroblast differentiation. Ortho-joint assays (PLA) revealed a direct physical interaction between TGFBRI and integrin β3 / β5, and this interaction became stronger after TGF-β1 treatment in HSCs. Figure 7A and Figure 7B However, NP-011 treatment in TGF-β1-treated HSCs significantly reduced the interaction between TGFBRI and integrin β3 / β5. Figure 7A and Figure 7BWestern blot analysis showed that NP-011 treatment significantly reduced phosphorylation of Smad2, a downstream molecule of TGF-β signaling, in TGF-β1-treated HSCs, and immunoprecipitation further confirmed the interaction between TGFBRI and integrin β3 / β5. Figure 7C The result of NP-011 treatment in HSCs treated with TGF-β1 was that the proliferating HSCs returned to a quiescent state. Figure 7D However, treatment with integrin β3 / β5 inhibitors in the presence of NP-011 eliminated the inhibitory effect of NP-011 on the proliferation of TGF-β1-treated HSCs. Figure 7D Therefore, these results indicate that NP-011 directly binds to integrin β3 / β5 and interferes with its interaction with TGFβRI, thereby inhibiting the TGF-β cascade and reducing HSC proliferation.

[0326] Based on previous findings indicating the secretion of pro-fibrotic MMP2 and expression of collagenase mRNA in rat HSCs, the effects of NP-011 on the expression of pro-fibrotic MMP2 and collagenase activity in HSCs were further tested. Following TGF-β1 treatment, MMP2 mRNA expression increased in HSCs. However, NP-011 treatment in TGF-β1-treated HSCs significantly downregulated the increased MMP2 expression. Figure 8A In contrast, TGF-β1 treatment downregulated collagenase activity in HSCs, and the reduced collagenase activity in TGF-β1-treated HSCs was restored after NP-011 treatment of cell lysates and conditioned medium. Figure 8B and 8C ).

[0327] Eliminating accumulated collagen through an immune response is a key factor in the fibrosis observed in models of regressive pulmonary fibrosis. To evaluate this, THP-1 monocytes were differentiated into macrophages via PMA treatment. Figure 9A ), and the phagocytic capacity of differentiated macrophages was confirmed by fluorescence and flow cytometry analysis. Figure 9B and 9C Collagen uptake assays revealed that, unlike the control culture, green fluorescently labeled collagen was significantly uptaken by differentiated macrophages in the presence of NP-011. Figure 9D and 9E In fact, in a TAA-induced liver fibrosis model, macrophage phagocytosis of collagen was demonstrated by immunostaining of collagen and F4 / 80 after administration of NP-011. Figure 10(See arrow in the image below). In summary, these results indicate that NP-011 inhibits TGF-β signaling in damaged liver, and then suppresses and regresses liver fibrosis by inactivating active HSCs and interacting with macrophages.

[0328] Example 6: Biodistribution and safety of NP-011

[0329] The biodistribution and safety of NP-011 were further evaluated in rodent models. Upon intravenous administration of NP-011 to mice, the administered NP-011 was preferentially delivered to the liver, with approximately 48% and 58% of the administered NP-011 detected in the liver at 30 and 60 minutes, respectively (Figure 11). Furthermore, after continuous intravenous administration of 0.2 mg / kg and 2 mg / kg NP-011 to rats daily for 4 weeks, no adverse effects were observed in serum hematological and biochemical analyses of male and female rats (Figure 11). Therefore, these results indicate that NP-011 preferentially targets the liver after administration and has excellent safety.

[0330] NP-011 exhibits potent antifibrotic activity for inhibiting, preventing, and reversing liver fibrosis. Compared to full-length MFG-E8 (lactohemagglutinin), NP-011, by truncating the C2 domain, demonstrates superior performance in clinical applications for the following reasons: 1) By structurally truncating MFG-E8, the Medin site in NP-011 is eliminated. Medin is a type of amyloid protein found in the medial aorta in individuals over 60 years of age, and its accumulation is involved in Alzheimer's disease and type 2 diabetes. Medin is present in the components of MFG-E8 (lactohemagglutinin) and is located within the C2 domain. Therefore, by structurally truncating the C2 domain from MFG-E8, NP-011 eliminates the problem of inducing amyloid formation in patients after administration, as well as the potential side effects of Alzheimer's disease and diabetes after repeated administration. This is an important factor in the treatment of liver disease because the most common liver disease, non-alcoholic fatty liver disease (NAFLD), is closely associated with type 2 diabetes. 2) By removing the glycosylation site of MFG-E8, NP-011 exhibits a better binding affinity for collagen. MFG-E8 contains a glycosylation site in its C2 domain. By removing the C2 domain, NP-011 avoids glycosylation that could interfere with the binding of the discoidal domain (C1 domain) of MFG-E8 to collagen accumulated in liver fibrosis. The well-known discoidal domain receptor (DDR) structurally possesses a discoidal domain, a rod region, and a transmembrane domain. Although the collagen-binding mechanism of DDR is not yet fully understood, the binding of the DDR's discoidal domain to collagen has been reported. Interestingly, the DDR's discoidal domain is unglycosylated (glycosylation sites are only located in the rod region of the DDR), therefore, the NP-011's discoidal domain may be similar to that of DDR and may facilitate the binding of NP-011 to collagen accumulated in the liver.

[0331] NP-011 has demonstrated therapeutic efficacy in various liver disease models, including non-alcoholic steatohepatitis (NASH). Clinically, NASH is common in patients with liver fibrosis because NASH patients are highly likely to develop fibrosis and cirrhosis, and their risk of liver disease-related death is almost 10 times higher than that of patients with simple steatosis. Therefore, the therapeutic potential of NP-011 in NASH and fibrosis represents a novel therapy for inhibiting and reversing complex and progressive liver diseases.

[0332] High manufacturability is another advantage of NP-011 for clinical application. NP-011 is produced using a yeast system, which offers several advantages: simple genetic manipulation and rapid growth; and it also possesses eukaryotic characteristics: a secretory pathway for proper protein processing and post-translational modifications. Currently, the yield of NP-011 from yeast is 40 mg / L, providing a reliable production basis for clinical applications. For example, at an effective dose of 40 μg / kg, production of NP-011 at a 200L GMP scale would provide approximately 3,400 injections for a 60kg adult. Furthermore, NP-011 is produced as a secretory protein (true NP-011), lacking methionine residues at the N-terminus, any tags (e.g., FLAG or his tags), or random glycosylation, which could potentially induce significant problems after drug administration. Therefore, in summary, NP-011 offers a highly effective and reliable novel protein therapy for the treatment, inhibition, prevention, and reversal of liver fibrosis and other liver diseases disclosed herein.

[0333] Example 7: NP-011 reverses, inhibits, and prevents idiopathic pulmonary fibrosis (IPF)

[0334] like Figures 12 - 21 As confirmed in the study, NP-011 is highly effective against IPF, and can reverse and inhibit IPF in vivo. The IPF model was induced by injection of bleomycin. In histological studies of lung tissue, NP-011 significantly reversed IPF. Figure 17 and Figure 18 ,and Figure 13 In addition, NP-011 reversed bleomycin-induced changes in the expression levels of IPF markers such as αSMA, collagen (Col1a1), TMP2, MMP2, MMP12, pERK, tERK, pSMAD2 (phosphorylated SMAD2), and tSMAD2 (total SMAD2) (compared to αSMA). Figures 12 - 21 Therefore, NP-011 provides a highly effective therapy for treating, inhibiting, preventing, and reversing IPF.

[0335] Example 8: NP-011 reverses, inhibits, and prevents symptoms of myocardial infarction and improves cardiac function.

[0336] like Figure 22 As demonstrated in Figure 26, NP-011 is highly effective in treating myocardial infarction in vivo. The rat model of myocardial infarction was induced by LAD ligation. Injection of NP-011 enhanced cardiac function; as evidenced by increased left ventricular ejection fraction (EF) and fractional shortening (FS) in rats with myocardial infarction. Figures 23A - 24BCompared to the control group, rats injected with NP-011 had similar body weight, which confirms the safety of NP-011. Figure 25 Histological studies of the cross-section of the heart confirmed that NP-011 also reversed and inhibited fibrosis associated with myocardial infarction. Figure 26A and Figure 26B Therefore, NP-011 provides a highly effective therapy for treating, inhibiting, preventing, and reversing myocardial infarction.

[0337] Example: NP-011 reverses, inhibits, and prevents Alzheimer's disease

[0338] As demonstrated in Figures 27-31, NP-011 is highly effective against Alzheimer's disease in vivo. Using 5XFAD mice as an Alzheimer's disease model, these mice, expressing human APP and PSEN1 transgenes, possessed a total of five AD-related mutations: the Swedish mutation (K670N / M671L), Florida mutation (I716V), and London mutation (V717I) in APP, and the M146L and L286V mutations in PSEN1. NP-011 reversed Alzheimer's disease-related behavioral and memory losses. Figures 27A - 27E NP-011 also reversed and inhibited neuropathological changes in the AD brain. For example, NP-011 specifically reduced amyloid plaques (…). Figures 28A - 28B ) and β-amyloid protein ( Figures 29A - 29B The number of microglia. Additionally, NP-011 reduced the number of microglia (i.e., the brain's innate immune system), the presence of which suggests neuroinflammatory activity. Figures 30A - 30B Furthermore, NP-011 reduced the amount of upregulated glial fibrillary acidic protein (GFAP) in Alzheimer's disease astrocytes. Figures 31A - 31B Therefore, NP-011 offers a highly effective therapy for treating, inhibiting, preventing, and reversing Alzheimer's disease.

[0339] References

[0340] 1. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005; 115:209-218.

[0341] 2. Chung W, Jo C, Chung WJ, Kim DJ. Liver cirrhosis and cancer: comparison of mortality. Hepatol Int (International Journal of Hepatology) 2018; 12: 269-276.

[0342] 3. Wynn TA, Ramalingam TR. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 2012; 18:1028-1040.

[0343] 4. Dooley S,ten Dijke P. TGF-β in progression of liver disease. Cell Tissue Res. 2012; 347:245-256.

[0344] 5. Gabbiani G. The role of myofibroblasts in wound healing and fibrocontractive diseases. J Pathol 2003; 200:500-503. 6. Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer 2006; 6:392-401.

[0345] 7. Troeger JS, Mederacke I, Gwak GY, Dapito DH, Mu X, Hsu CC, Pradere JP, Friedman RA, Schwabe RF. Deactivation of hepatic stellate cells during liver fibrosis resolution in mice. Gastroenterology 2012; 143:1073-1083. 8. Uchiyama A, Yamada K, Ogino S, Yokoyama Y, Takeuchi Y, Udey MC, Ishikawa O, Motegi S. MFG-E8 regulates sangiogenesis in cutaneous wound healing. Am J Pathol 2014; 184:1981-1990.

[0346] 9.Yoshida H,Kawane K,Koike M,Mori Y,Uchiyama Y,Nagata S.

[0347] Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells. (Nature)

[0348] (Nature) 2005; 437:754-758.

[0349] 10. Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S. Identification of a factor that links apoptotic cells to phagocytes. Nature 2002; 417:182-187.

[0350] 11.Yi YS.Functional Role of Milk Fat Globule-Epidermal Growth FactorVIII inMacrophage-Mediated Inflammatory Responses and Inflammatory / AutoimmuneDiseases

[0351] (The functional role of milk fat globulin-epidermal growth factor VIII in macrophage-mediated inflammatory responses and inflammatory / autoimmune diseases). Mediators Inflamm 2016; 2016:5628486.

[0352] 12. An SY, Jang YJ, Lim HJ, Han J, Lee J, Lee G, Park JY, Park SY, Kim JH, DoBR, Han C, Park HK, Kim OH, Song MJ, Kim SJ, Kim JH. Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice. Gastroenterology, 2017; 152:1174-1186.

[0353] 13. Atabai K, Jame S, Azhar N, Kuo A, Lam M, McKleroy W, Dehart G, Rahman S, Xia DD, Melton AC, Wolters P, Emson CL, Turner SM, Werb Z, Sheppard D. Mfge8 diminishes the severity of tissue fibrosis in mice by binding and targeting collagen for uptake by macrophages. J Clin Invest 2009; 119:3713-3722.

[0354] 14. Ye H, Li B, Subramanian V, Choi BH, Liang Y, Harikishore A, Chakraborty G, Baek K, Yoon HS. NMR solution structure of C2 domain of MFG-E8 and insights into its molecular recognition with phosphatidylserine. Biochim Biophys Acta 2013; 1828: 1083-1093.

[0355] 15. C, Bell AW, Bowen WC, Monga SP, Fleig W, Michalopoulos GK. Expression of Notch-1 and its ligand Jagged-1 in rat liver during liver regeneration. Hepatology, 2004; 39: 1056-1065.

[0356] 16. Ahmad R, Ahmed S, Khan NU, Hasnain AU. Operculina turpethum attenuates N-nitrosodimethylamine induced toxic liver injury and clastogenicity in rats. Chem Biol Interact, 2009; 181:145-153.

[0357] 17. Sarrazy V, Koehler A, Chow ML, Zimina E, Li CX, Kato H, Caldarone CA, Hinz B. Integrins αvβ5 and αvβ3 promote latent TGF-β1 activation by human cardiac fibroblast contraction. Cardiovascular Research Res 2014; 102:407-417.

[0358] 18. Théret N, Musso O, L'Helgoualc'h A, Clément B. Activation of matrix metalloproteinase-2 from hepatic stellate cells requires interactions with hepatocytes. Am J Pathol 1997; 150:51-58.

[0359] 19. Iredale JP, Benyon RC, Arthur MJ, Ferris WF, Alcolado R, Winwood PJ, Clark N, Murphy G. Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis. Hepatology 1996; 24:176-184.

[0360] 20. Liu B, Shi D, Chang S, Gong X, Yu Y, Sun Z, Wu J. Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast. Sci Rep. 2015; 5:7678. 21. Westermark P, Johnson KH, O'Brien TD, Betsholtz C. Islet amyloid polypeptide—an anovulent controversy in diabetes research. Diabetologia. 1992; 35:297-303.

[0361] 22. Westermark P, Mucchiano G, Marthin T, Johnson KH, Sletten K. Apolipoprotein A1-derived amyloid in human aortic atherosclerotic plaques. Am J Pathol 1995; 147:1186-1192.23. B, J,Sletten K,Westermark GT,MucchianoG,Tjernberg LO,Nordstedt C, U, Westermark P. Medin: an integral fragment of aortic smooth muscle cell-produced lactadherin forms the most common human amyloid. Proc Natl Acad Sci USA (Proceedings of the National Academy of Sciences of the United States of America) 1999; 96: 8669-8674.

[0362] 24. Ong JP, Younossi ZM. Epidemiology and natural history of NAFLD and NASH. Clin Liver Dis 2007; 11:1-16, vii.

[0363] 25. Assy N, Kaita K, Mymin D, Levy C, Rosser B, Minuk G. Fatty infiltration of liver in hyperlipidemic patients. Dig Dis Sci (Digestive Diseases and Sciences) 2000; 45: 1929-1934.

[0364] 26. Dorison A, Dussaule JC, Chatziantoniou C. The Role of Discoidin Domain Receptor 1 in Inflammation, Fibrosis and Renal Disease. Nephron 2017; 137:212-220.

[0365] 27. Borza CM, Pozzi A. Discoidin domain receptors in disease. Matrix Biol, 2014; 34:185-192.

[0366] 28. Anstee QM, Targher G, Day CP. Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat Rev Gastroenterol Hepatol 2013; 10:330-344. 29. Solá RJ, Griebenow K. Effects of glycosylation on the stability of protein pharmaceuticals. J Pharm Sci 2009; 98:1223-1245. sequence list <110> Nihil Ltd. <120> Compositions and methods for treating and preventing fibrosis <130> NXH-00325 <140> PCT / IB2019 / 001136 <141> 2019-10-24 <150> KR 10-2018-0128625 / KR <151> 2018-10-26 <150> KR 10-2018-0128033 / KR <151> 2018-10-25 <150> KR 10-2018-0128204 / KR <151> 2018-10-25 <160> 41 <170> PatentIn version 3.5 <210> 1 <211> 1780 <212> DNA <213> Homo sapiens <400> 1 agaaccccgc ggggtctgag cagcccagcg tgcccattcc agcgcccgcg tccccgcagc 60 atgccgcgcc cccgcctgct ggccgcgctg tgcggcgcgc tgctctgcgc ccccagcctc 120 ctcgtcgccc tggatatctg ttccaaaaac ccctgccaca acggtggttt atgcgaggag 180 atttcccaag aagtgcgagg agatgtcttc ccctcgtaca cctgcacgtg ccttaagggc 240 tacgcgggca accactgtga gacgaaatgt gtcgagccac tgggcctgga gaatgggaac 300 attgccaact cacagatcgc cgcctcgtct gtgcgtgtga ccttcttggg tttgcagcat 360 tgggtcccgg agctggcccg cctgaaccgc gcaggcatgg tcaatgcctg gacacccagc 420 agcaatgacg ataacccctg gatccaggtg aacctgctgc ggaggatgtg ggtaacaggt 480 gtggtgacgc agggtgccag ccgcttggcc agtcatgagt acctgaaggc cttcaaggtg 540 gcctacagcc ttaatggaca cgaattcgat ttcatccatg atgttaataa aaaacacaag 600 gagtttgtgg gtaactggaa caaaaacgcg gtgcatgtca acctgtttga gacccctgtg 660 gaggctcagt acgtgagatt gtaccccacg agctgccaca cggcctgcac tctgcgcttt 720 gagctactgg gctgtgagct gaacggatgc gccaatcccc tgggcctgaa gaataacagc 780 atccctgaca agcagatcac ggcctccagc agctacaaga cctggggctt gcatctcttc 840 agctggaacc cctcctatgc acggctggac aagcagggca acttcaacgc ctgggttgcg 900 gggagctacg gtaacgatca gtggctgcag atcttccctg gcaactggga caaccactcc 960 cacaagaaga acttgtttga gacgcccatc ctggctcgct atgtgcgcat cctgcctgta 1020 gcctggcaca accgcatcgc cctgcgcctg gagctgctgg gctgttagtg gccacctgcc 1080 acccccaggt cttcctgctt tccatgggcc cgctgcctct tggcttctca gcccctttaa 1140 atcaccatag ggctggggac tggggaaggg gagggtgttc agaggcagca ccaccacaca 1200 gtcacccctc cctccctctt tcccaccctc cacctctcac gggccctgcc ccagccccta 1260 agccccgtcc cctaaccccc agtcctcact gtcctgtttt cttaggcact gagggatctg 1320 agtaggtctg ggatggacag gaaagggcaa agtagggcgt gtggtttccc tgcccctgtc 1380 cggaccgccg atcccaggtg cgtgtgtctc tgtctctcct agcccctctc tcacacatca 1440 cattcccatg gtggcctcaa gaaaggcccg gaagcgccag gctggagata acagcctctt 1500 gcccgtcggc cctgcgtcgg ccctggggta ccatgtggcc acaactgctg tggccccctg 1560 tccccaagac acttcccctt gtctccctgg ttgcctctct tgccccttgt cctgaagccc 1620 agcgacacag aagggggtgg ggcgggtcta tggggagaaa gggagcgagg tcagaggagg 1680 gcatgggttg gcagggtggg cgtttggggc cctctatgct ggcttttcac cccagaggac 1740 acaggcagct tccaaaatat atttatcttc ttcacgggaa 1780 <210> 2 <211> 335 <​​​​​​​​​​​​​​​​​​​​​​​​​Ile Ala Asn Ser Gln Ile Ala Ala Ser Ser Val Arg Val Thr Phe Leu 85 90 95 Gly Leu Gln His Trp Val Pro Glu Leu Ala Arg Leu Asn Arg Ala Gly 100 105 110 Met Val Asn Ala Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro Trp Ile 115 120 125 Gln Val Asn Leu Leu Arg Arg Met Trp Val Thr Gly Val Val Thr Gln 130 135 140 Gly Ala Ser Arg Leu Ala Ser His Glu Tyr Leu Lys Ala Phe Lys Val 145 150 155 160 Ala Tyr Ser Leu Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn 165 170 175 Lys Lys His Lys Glu Phe Val Gly Asn Trp Asn Lys Asn Ala Val His 180 185 190 Val Asn Leu Phe Glu Thr Pro Val Glu Ala Gln Tyr Val Arg Leu Tyr 195 200 205 Pro Thr Ser Cys His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly 210 215 220 Cys Glu Leu Asn Gly Cys Ala Asn Pro Leu Gly Leu Lys Asn Asn Ser 225 230 235 240 Ile Pro Asp Lys Gln Ile Thr Ala Ser Ser Ser Tyr Lys Thr Trp Gly 245 250 255 Leu His Leu Phe Ser Trp Asn Pro Ser Tyr Ala Arg Leu Asp Lys Gln 260 265 270 Gly Asn Phe Asn Ala Trp Val Ala Gly Ser Tyr Gly Asn Asp Gln Trp 275 280 285 Leu Gln Ile Phe Pro Gly Asn Trp Asp Asn His Ser His Lys Lys Asn 290 295 300 Leu Phe Glu Thr Pro Ile Leu Ala Arg Tyr Val Arg Ile Leu Pro Val 305 310 315 320 Ala Trp His Asn Arg Ile Ala Leu Arg Leu Glu Leu Leu Gly Cys 325 330 335 <210> 3 <211> 1804 <212> DNA <213> Homo sapiens <400> 3 agaaccccgc ggggtctgag cagcccagcg tgcccattcc agcgcccgcg tccccgcagc 60 atgccgcgcc cccgcctgct ggccgcgctg tgcggcgcgc tgctctgcgc ccccagcctc 120 ctcgtcgccc tggaatgtgt cgagccactg ggcctggaga atgggaacat tgccaactca 180<00!1103cagatcgccg cctcgtctgt gcgtgtgacc ttcttgggtt tgcagcattg ggtcccggag 240 ctggcccgcc tgaaccgcgc aggcatggtc aatgcctgga cacccagcag caatgacgat aacccctgga tccaggtgaa cctgctgcgg aggatgtggg taacaggtgt ggtgacgcag 360 ggtgccagcc gcttggccag tcatgagtac ctgaaggcct tcaaggtggc ctacagcctt 420 aatggacacg aattcgattt catccatgat gttaataaaa aacacaagga gtttgtgggt 540. aactggaaca aaaacgcggt gcatgtcaac ctgtttgaga cccctgtgga ggctcagtac gtgagattgt accccacgag ctgccacacg gcctgcactc tgcgctttga gctactgggc 600 tgtgagctga acggatgcgc caatcccctg ggcctgaaga fathercagcat ccctgacaag 720. cgatcacgg cctccagcag ctacaagacc tggggcttgc atctcttcag ctggaacccc tcctatgcac ggctggacaa gcagggcaac ttcaacgcct gggttgcggg gagctacggt 780 aacgatcagt ggctgcaggt ggacctgggc tcctcgaagg aggtgacagg catcatcacc 840 cagggggccc gtaactttgg ctctgtccag tttgtggcat cctacaaggt tgcctacagt 900 aatgacagtg cgaactggac tgagtaccag gaccccagga ctggcagcag taagatcttc 960 cctggcaact gggacaacca ctcccacaag aagaacttgt ttgagacgcc catcctggct 1020 cgctatgtgc gcatcctgcc tgtagcctgg cacaaccgca tcgccctgcg cctggagctg 1080 ctgggctgtt agtggccacc tgccaccccc aggtcttcct gctttccatg ggcccgctgc 1140 ctcttggctt ctcagcccct ttaaatcacc atagggctgg ggactgggga aggggagggt 1200 gttcagaggc agcaccacca cacagtcacc cctccctccc tctttcccac cctccacctc 1260 tcacgggccc tgccccagcc cctaagcccc gtcccctaac ccccagtcct cactgtcctg 1320 ttttcttagg cactgaggga tctgagtagg tctgggatgg acaggaaagg gcaaagtagg 1380 gcgtgtggtt tccctgcccc tgtccggacc gccgatccca ggtgcgtgtg tctctgtctc 1440 tcctagcccc tctctcacac atcacattcc catggtggcc tcaagaaagg cccggaagcg 1500 ccaggctgga gataacagcc tcttgcccgt cggccctgcg tcggccctgg ggtaccatgt 1560 ggccacaact gctgtggccc cctgtcccca agacacttcc ccttgtctcc ctggttgcct 1620 ctcttgcccc ttgtcctgaa gcccagcgac acagaagggg gtggggcggg tctatgggga 1680 gaaagggagc gaggtcagag gagggcatgg gttggcaggg tgggcgtttg gggccctcta 1740 tgctggcttt tcaccccaga ggacacaggc agcttccaaa atatatttat cttcttcacg 1800 ggaa 1804 <210> 4 <211> 343 <212> PRT <213> Homo sapiens <400> 4 Met Pro Arg Pro Arg Leu Leu Ala Ala Leu Cys Gly Ala Leu Leu Cys 1 5 10 15 Ala Pro Ser Leu Leu Val Ala Leu Glu Cys Val Glu Pro Leu Gly Leu 20 25 30 Glu Asn Gly Asn Ile Ala Asn Ser Gln Ile Ala Ala Ser Ser Val Arg 35 40 45 Val Thr Phe Leu Gly Leu Gln His Trp Val Pro Glu Leu Ala Arg Leu 50 55 60 Asn Arg Ala Gly Met Val Asn Ala Trp Thr Pro Ser Ser Asn Asp Asp 65 70 75 80 Asn Pro Trp Ile Gln Val Asn Leu Leu Arg Arg Met Trp Val Thr Gly 85 90 95 Val Val Thr Gln Gly Ala Ser Arg Leu Ala Ser His Glu Tyr Leu Lys 100 105 110 Ala Phe Lys Val Ala Tyr Ser Leu Asn Gly His Glu Phe Asp Phe Ile 115 120 125 His Asp Val Asn Lys Lys His Lys Glu Phe Val Gly Asn Trp Asn Lys 130 135 140 Asn Ala Val His Val Asn Leu Phe Glu Thr Pro Val Glu Ala Gln Tyr 145 150 155 160 Val Arg Leu Tyr Pro Thr Ser Cys His Thr Ala Cys Thr Leu Arg Phe 165 170 175 Glu Leu Leu Gly Cys Glu Leu Asn Gly Cys Ala Asn Pro Leu Gly Leu 180 185 190 Lys Asn Asn Ser Ile Pro Asp Lys Gln Ile Thr Ala Ser Ser Ser Tyr 195 200 205 Lys Thr Trp Gly Leu His Leu Phe Ser Trp Asn Pro Ser Tyr Ala Arg 210 215 220 Leu Asp Lys Gln Gly Asn Phe Asn Ala Trp Val Ala Gly Ser Tyr Gly 225 230 235 240 Asn Asp Gln Trp Leu Gln Val Asp Leu Gly Ser Ser Lys Glu Val Thr 245 250 255 甘氨酸-异亮氨酸-异亮氨酸-苏氨酸-谷氨酰胺-甘氨酸-丙氨酸-精氨酸-天冬酰胺-苯丙氨酸-甘氨酸-丝氨酸-缬氨酸-谷氨酰胺-苯丙氨酸-缬氨酸 260 265 270 丙氨酸-丝氨酸-酪氨酸-赖氨酸-缬氨酸-丙氨酸-酪氨酸-丝氨酸-天冬酰胺-天冬氨酸-丝氨酸-丙氨酸-天冬酰胺-色氨酸-苏氨酸-谷氨酸 275 280 285 酪氨酸-谷氨酰胺-天冬氨酸-脯氨酸-精氨酸-苏氨酸-甘氨酸-丝氨酸-丝氨酸-赖氨酸-异亮氨酸-苯丙氨酸-脯氨酸-甘氨酸-天冬酰胺-色氨酸 290 295 300 天冬氨酸-天冬酰胺-组氨酸-丝氨酸-组氨酸-赖氨酸-赖氨酸-天冬酰胺-亮氨酸-苯丙氨酸-谷氨酸-苏氨酸-脯氨酸-异亮氨酸-亮氨酸-丙氨酸 305 310 315 320 精氨酸-酪氨酸-缬氨酸-精氨酸-异亮氨酸-亮氨酸-脯氨酸-缬氨酸-丙氨酸-色氨酸-组氨酸-天冬酰胺-精氨酸-异亮氨酸-丙氨酸-亮氨酸 325 330 335 精氨酸-亮氨酸-谷氨酸-亮氨酸-亮氨酸-甘氨酸-半胱氨酸 340 <210> 5 <211> 1989 <212> DNA <213> Homo sapiens <400> 5 agaaccccgc ggggtctgag cagcccagcg tgcccattcc agcgcccgcg tccccgcagc 60 atgccgcgcc cccgcctgct ggccgcgctg tgcggcgcgc tgctctgcgc ccccagcctc 120 ctcgtcgccc tggggtgatg tggccttttc cagaaggagg aaacaccata cctatcttac 180 acacagatat ctgttccaaa aacccctgcc acaacggtgg tttatgcgag gagatttccc 240 aagaagtgcg aggagatgtc ttcccctcgt acacctgcac gtgccttaag ggctacgcgg 300 gcaaccactg tgagacgaaa tgtgtcgagc cactgggcct ggagaatggg aacattgcca 360 actcacagat cgccgcctcg tctgtgcgtg tgaccttctt gggtttgcag cattgggtcc 420 cggagctggc ccgcctgaac cgcgcaggca tggtcaatgc ctggacaccc agcagcaatg 480 acgataaccc ctggatccag gtgaacctgc tgcggaggat gtgggtaaca ggtgtggtga 540 cgcagggtgc cagccgcttg gccagtcatg agtacctgaa ggccttcaag gtggcctaca 600 gccttaatgg acacgaattc gatttcatcc atgatgttaa taaaaacac aaggagtttg 660 tgggtaactg gaacaaaaac gcggtgcatg tcaacctgtt tgagacccct gtggaggctc 720 agtacgtgag attgtacccc acgagctgcc acacggcctg cactctgcgc tttgagctac 780 tgggctgtga gctgaacgga tgcgccaatc cctgggcct gaagaataac agcatccctg 840 acaagcagat cacggcctcc agcagctaca agacctgggg cttgcatctc ttcagctgga 900 acccctccta tgcacggctg gacaagcagg gcaacttcaa cgcctgggtt gcggggagct 960 acggtaacga tcagtggctg caggtggacc tgggctcctc gaaggaggtg acaggcatca 1020 tcacccaggg ggcccgtaac tttggctctg tccagtttgt ggcatcctac aaggttgcct 1080 acagtaatga cagtgcgaac tggactgagt accaggaccc caggactggc agcagtaaga 1140 tcttccctgg caactgggac aaccactccc acaagaagaa cttgtttgag acgcccatcc 1200 tggctcgcta tgtgcgcatc ctgcctgtag cctggcacaa ccgcatcgcc ctgcgcctgg 1260 agctgctggg ctgttagtgg ccacctgcca cccccaggtc ttcctgcttt ccatgggccc 1320 gctgcctctt ggcttctcag cccctttaaa tcaccatagg gctggggact ggggaagggg 1380 agggtgttca gaggcagcac caccacacag tcacccctcc ctccctcttt cccaccctcc 1440 acctctcacg ggccctgccc cagcccctaa gccccgtccc ctaaccccca gtcctcactg 1500 tcctgttttc ttaggcactg agggatctga gtaggtctgg gatggacagg aaagggcaaa 1560 gtagggcgtg tggtttccct gcccctgtcc ggaccgccga tcccaggtgc gtgtgtctct 1620 gtctctccta gcccctctct cacacatcac attcccatgg tggcctcaag aaaggcccgg 1680 aagcgccagg ctggagataa cagcctcttg cccgtcggcc ctgcgtcggc cctggggtac 1740 catgtggcca caactgctgt ggccccctgt ccccaagaca cttccccttg tctccctggt 1800 tgcctctctt gccccttgtc ctgaagccca gcgacacaga agggggtggg gcgggtctat 1860 ggggagaaag ggagcgaggt cagaggaggg catgggttgg cagggtgggc gtttggggcc 1920 ctctatgctg gcttttcacc ccagaggaca caggcagctt ccaaaatata tttatcttct 1980 tcacgggaa 1989 <210> 6 <211> 379 <212> PRT <213> Homo sapiens <400> 6 Met Trp Pro Phe Pro Glu Gly Gly Asn Thr Ile Pro Ile Leu His Thr 1 5 10 15 Asp Ile Cys Ser Lys Asn Pro Cys His Asn Gly Gly Leu Cys Glu Glu 20 25 30 Ile Ser Gln Glu Val Arg Gly Asp Val Phe Pro Ser Tyr Thr Cys Thr 35 40 45 Cys Leu Lys Gly Tyr Ala Gly Asn His Cys Glu Thr Lys Cys Val Glu 50 55 60 Pro Leu Gly Leu Glu Asn Gly Asn Ile Ala Asn Ser Gln Ile Ala Ala 65 70 75 80 Ser Ser Val Arg Val Thr Phe Leu Gly Leu Gln His Trp Val Pro Glu 85 90 95 Leu Ala Arg Leu Asn Arg Ala Gly Met Val Asn Ala Trp Thr Pro Ser 100 105 110 Ser Asn Asp Asp Asn Pro Trp Ile Gln Val Asn Leu Leu Arg Arg Met 115 120 125 Trp Val Thr Gly Val Val Thr Gln Gly Ala Ser Arg Leu Ala Ser His 130 135 140 Glu Tyr Leu Lys Ala Phe Lys Val Ala Tyr Ser Leu Asn Gly His Glu 145 150 155 160 Phe Asp Phe Ile His Asp Val Asn Lys Lys His Lys Glu Phe Val Gly 165 170 175 Asn Trp Asn Lys Asn Ala Val His Val Asn Leu Phe Glu Thr Pro Val 180 185 190 Glu Ala Gln Tyr Val Arg Leu Tyr Pro Thr Ser Cys His Thr Ala Cys 195 200 205 Thr Leu Arg Phe Glu Leu Leu Gly Cys Glu Leu Asn Gly Cys Ala Asn 210 215 220 Pro Leu Gly Leu Lys Asn Asn Ser Ile Pro Asp Lys Gln Ile Thr Ala 225 230 235 240 Ser Ser Ser Tyr Lys Thr Trp Gly Leu His Leu Phe Ser Trp Asn Pro 245 250 255 Ser Tyr Ala Arg Leu Asp Lys Gln Gly Asn Phe Asn Ala Trp Val Ala 260 265 270 Gly Ser Tyr Gly Asn Asp Gln Trp Leu Gln Val Asp Leu Gly Ser Ser 275 280 285 Lys Glu Val Thr Gly Ile Ile Thr Gln Gly Ala Arg Asn Phe Gly Ser 290 295 300 Val Gln Phe Val Ala Ser Tyr Lys Val Ala Tyr Ser Asn Asp Ser Ala 305 310 315 320 Asn Trp Thr Glu Tyr Gln Asp Pro Arg Thr Gly Ser Ser Lys Ile Phe 325 330 335 Pro Gly Asn Trp Asp Asn His Ser His Lys Lys Asn Leu Phe Glu Thr 340 345 350 Pro Ile Leu Ala Arg Tyr Val Arg Ile Leu Pro Val Ala Trp His Asn 355 360 365 Arg Ile Ala Leu Arg Leu Glu Leu Leu Gly Cys 370 375 <210> 7 <211> 2544 <212> DNA <213> Homo sapiens <400> 7 acctccactg ttgacaaact tagacaaagc cccggggacc gggctgggca gaggggcggc 60 ttcttccgct gcgccctggc gggacagggg gatgcggccc tgctgtctct gcgctggggc 120 ttttgggctg ggactcggga catcgggtga cagccctgcc gcccccaggg atgcggctta 180 cagataatga caaaggaatc cgctgtgtcg ggcctctctt ttccctggtg aaaaatgagg 240 ccagggaact gcgtttgact ttcgaacccc ttccacctgg gagattctag gactctagta 300 tggataagtc ttgtctggat aactttgtcc tggccatctc cctgccaact ccagttggct 360 ggacagttca ttggattttt gcgctcccaa ttgtccgtgc ctggtcacat aagggaaggg 420 ccggggagtc ggtgcaatgg acgcaggccg taagtggggc ccgggagggg acccagaggc 480 ttcgaggagc ttggaagagg gctgcctgct gatgggagtc tcctgactcc ctccctcccg 540 cggccttggc cggctgctgt atcttccccg gtcctcctcc gcctcccagg aggcctccgg 600 aggccagctg ggccccttgc aggctggact tgcggatgcc ccgtgccatt caccgtggag 660 cgctgggagg gagtcagggc caggactctt taggtggccc ctccatcatt ttctcataga 720 aatgggattg actgaagcaa gatatctgtt ccaaaaaccc ctgccacaac ggtggtttat gcgaggagat ttcccaagaa gtgcgaggag atgtcttccc ctcgtacacc tgcacgtgcc 840 ttaagggcta cgcgggcaac cactgtgaga cgaaatgtgt cgagccactg ggcctggaga atgggaacat tgccaactca cagatcgccg cctcgtctgt gcgtgtgacc ttcttgggtt tgcagcattg ggtcccggag ctggcccgcc tgaaccgcgc aggcatggtc aatgcctgga 1020 cacccagcag caatgacgat aacccctgga tccaggtgaa cctgctgcgg aggatgtggg taacaggtgt ggtgacgcag ggtgccagcc gcttggccag tcatgagtac ctgaaggcct tcaaggtggc ctacagcctt aatggacacg aattcgattt catccatgat gttaataaaa 1260. aacacaagga gtttgtgggt aactggaaca aaaacgcggt gcatgtcaac ctgtttgaga cccctgtgga ggctcagtac gtgagattgt accccacgag ctgccacacg gcctgcactc 1320 tgcgctttga gctactgggc tgtgagctga acggatgcgc caatcccctg ggcctgaaga 1380. ataacagcat ccctgacaag cagatcacgg cctccagcag ctacaagacc tggggcttgc atctcttcag ctggaacccc tcctatgcac ggctggacaa gcagggcaac ttcaacgcct 1500 gggttgcggg gagctacggt aacgatcagt ggctgcaggt ggacctgggc tcctcgaagg 1560 aggtgacagg catcatcacc cagggggccc gtaactttgg ctctgtccag tttgtggcat 1620 cctacaaggt tgcctacagt aatgacagtg cgaactggac tgagtaccag gaccccagga 1680 ctggcagcag taagatcttc cctggcaact gggacaacca ctcccacaag aagaacttgt 1740 ttgagacgcc catcctggct cgctatgtgc gcatcctgcc tgtagcctgg cacaaccgca 1800 tcgccctgcg cctggagctg ctgggctgtt agtggccacc tgccaccccc aggtcttcct 1860 gctttccatg ggcccgctgc ctcttggctt ctcagcccct ttaaatcacc atagggctgg 1920 ggactgggga aggggagggt gttcagaggc agcaccacca cacagtcacc cctccctccc 1980 tctttcccac cctccacctc tcacgggccc tgccccagcc cctaagcccc gtcccctaac 2040 ccccagtcct cactgtcctg ttttcttagg cactgaggga tctgagtagg tctgggatgg 2100 acaggaaagg gcaaagtagg gcgtgtggtt tccctgcccc tgtccggacc gccgatccca 2160 ggtgcgtgtg tctctgtctc tcctagcccc tctctcacac atcacattcc catggtggcc 2220 tcaagaaagg cccggaagcg ccaggctgga gataacagcc tcttgcccgt cggccctgcg 2280 tcggccctgg ggtaccatgt ggccacaact gctgtggccc cctgtcccca agacacttcc 2340 ccttgtctcc ctggttgcct ctcttgcccc ttgtcctgaa gcccagcgac acagaagggg 2400 gtggggcggg tctatgggga gaaagggagc gaggtcagag gagggcatgg gttggcaggg 2460 tgggcgtttg gggccctcta tgctggcttt tcaccccaga ggacacaggc agcttccaaa 2520 atatatttat cttcttcacg ggaa 2544 <210> 8 <211> 275 <212> PRT <213> Homo sapiens <400> 8 Met Val Asn Ala Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro Trp Ile 1 5 10 15 Gln Val Asn Leu Leu Arg Arg Met Trp Val Thr Gly Val Val Thr Gln 20 25 30 Gly Ala Ser Arg Leu Ala Ser His Glu Tyr Leu Lys Ala Phe Lys Val 35 40 45 Ala Tyr Ser Leu Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn 50 55 60 Lys Lys His Lys Glu Phe Val Gly Asn Trp Asn Lys Asn Ala Val His 65 70 75 80 Val Asn Leu Phe Glu Thr Pro Val Glu Ala Gln Tyr Val Arg Leu Tyr 85 90 95 Pro Thr Ser Cys His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly 100 105 110 Cys Glu Leu Asn Gly Cys Ala Asn Pro Leu Gly Leu Lys Asn Asn Ser 115 120 125 Ile Pro Asp Lys Gln Ile Thr Ala Ser Ser Ser Tyr Lys Thr Trp Gly 130 135 140 Leu His Leu Phe Ser Trp Asn Pro Ser Tyr Ala Arg Leu Asp Lys Gln 145 150 155 160 Gly Asn Phe Asn Ala Trp Val Ala Gly Ser Tyr Gly Asn Asp Gln Trp 165 170 175 Leu Gln Val Asp Leu Gly Ser Ser Lys Glu Val Thr Gly Ile Ile Thr 180 185 190 Gln Gly Ala Arg Asn Phe Gly Ser Val Gln Phe Val Ala Ser Tyr Lys 195 200 205 Val Ala Tyr Ser Asn Asp Ser Ala Asn Trp Thr Glu Tyr Gln Asp Pro 210 215 220 Arg Thr Gly Ser Ser Lys Ile Phe Pro Gly Asn Trp Asp Asn His Ser 225 230 235 240 His Lys Lys Asn Leu Phe Glu Thr Pro Ile Leu Ala Arg Tyr Val Arg 245 250 255 Ile Leu Pro Val Ala Trp His Asn Arg Ile Ala Leu Arg Leu Glu Leu 260 265 270 Leu Gly Cys 275 <210> 9 <211> 1936 <212> DNA <213> Homo sapiens <400> 9 agaaccccgc ggggtctgag cagcccagcg tgcccattcc agcgcccgcg tccccgcagc 60 atgccgcgcc cccgcctgct ggccgcgctg tgcggcgcgc tgctctgcgc ccccagcctc 120 ctcgtcgccc tggatatctg ttccaaaaac ccctgccaca acggtggttt atgcgaggag 180 atttcccaag aagtgcgagg agatgtcttc ccctcgtaca cctgcacgtg ccttaagggc 240 tacgcgggca accactgtga gacgaaatgt gtcgagccac tgggcctgga gaatgggaac 300 attgccaact cacagatcgc cgcctcgtct gtgcgtgtga ccttcttggg tttgcagcat 360 tgggtcccgg agctggcccg cctgaaccgc gcaggcatgg tcaatgcctg gacacccagc 420 agcaatgacg ataacccctg gatccaggtg aacctgctgc ggaggatgtg ggtaacaggt 480 gtggtgacgc agggtgccag ccgcttggcc agtcatgagt acctgaaggc cttcaaggtg 540 gcctacagcc ttaatggaca cgaattcgat ttcatccatg atgttaataa aaaacacaag 600 gagtttgtgg gtaactggaa caaaaacgcg gtgcatgtca acctgtttga gacccctgtg 660 gaggctcagt acgtgagatt gtaccccacg agctgccaca cggcctgcac tctgcgcttt 720 gagctactgg gctgtgagct gaacggatgc gccaatcccc tgggcctgaa gaataacagc 780 atccctgaca agcagatcac ggcctccagc agctacaaga cctggggctt gcatctcttc 840 agctggaacc cctcctatgc acggctggac aagcagggca acttcaacgc ctgggttgcg 900 gggagctacg gtaacgatca gtggctgcag gtggacctgg gctcctcgaa ggaggtgaca 960 ggcatcatca cccagggggc ccgtaacttt ggctctgtcc agtttgtggc atcctacaag 1020 gttgcctaca gtaatgacag tgcgaactgg actgagtacc aggaccccag gactggcagc 1080 agtaagatct tccctggcaa ctgggacaac cactcccaca agaagaactt gtttgagacg 1140 cccatcctgg ctcgctatgt gcgcatcctg cctgtagcct ggcacaaccg catcgccctg 1200 cgcctggagc tgctgggctg ttagtggcca cctgccaccc ccaggtcttc ctgctttcca 1260 tgggcccgct gcctcttggc ttctcagccc cttaaatca catagggct ggggactggg 1320 gaaggggagg gtgttcagag gcagcaccac cacacagtca cccctccctc cctctttccc 1380 acctccacc tctcacgggc cctgccccag ccctaagcc ccgtcccta acccccagtc 1440 ctcactgtcc tgttttctta ggcactgagg gatctgagta ggtctgggat ggacaggaaa 1500 gggcaaagta gggcgtgtgg tttccctgcc cctgtccgga ccgccgatcc caggtgcgtg 1560 tgtctctgtc tctcctagcc cctctcac acatcacatt cccatggtgg cctcaagaaa 1620 ggcccggaag cgccaggctg gagataacag cctcttgccc gtcggccctg cgtcggccct 1680 ggggtaccat gtggccacaa ctgctgtggc cccctgtccc caagacactt ccccttgtct 1740 ccctggttgc ctctcttgcc ccttgtcctg aagcccagcg acacagaagg gggtggggcg 1800 ggtctatggg gagaaaggga gcgaggtcag aggagggcat gggttggcag ggtgggcgtt 1860 tggggccctc tatgctggct tttcacccca gaggacacag gcagcttcca aaatatattt 1920 atcttcttca cgggaa 1936 <210> 10 <211> 387 <212> PRT <213> Homo sapiens <400> 10 Met Pro Arg Pro Arg Leu Leu Ala Ala Leu Cys Gly Ala Leu Leu Cys 1 5 10 15 Ala Pro Ser Leu Leu Val Ala Leu Asp Ile Cys Ser Lys Asn Pro Cys<C 20 25 30 His Asn Gly Gly Leu Cys Glu Glu Ile Ser Gln Glu Val Arg Gly Asp 35 40 45 Val Phe Pro Ser Tyr Thr Cys Thr Cys Leu Lys Gly Tyr Ala Gly Asn [[ID=I32]]50 55 60 His Cys Glu Thr Lys Cys Val Glu Pro Leu Gly Leu Glu Asn Gly Asn 65 70 75 80 Ile Ala Asn Ser Gln Ile Ala Ala Ser Ser Val Arg Val Thr Phe Leu 85 90 95 It should be noted that there may be some inaccuracies in the original text's format and content arrangement. This translation is based on the best understanding of the given text. If you have any further questions or need more accurate translations, please provide more context or clarify the requirements.Gly Leu Gln His Trp Val Pro Glu Leu Ala Arg Leu Asn Arg Ala Gly 100 105 110 Met Val Asn Ala Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro Trp Ile 115 120 125 Gln Val Asn Leu Leu Arg Arg Met Trp Val Thr Gly Val Val Thr Gln 130 135 140 Gly Ala Ser Arg Leu Ala Ser His Glu Tyr Leu Lys Ala Phe Lys Val 145 150 155 160 Ala Tyr Ser Leu Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn 165 170 175 Lys Lys His Lys Glu Phe Val Gly Asn Trp Asn Lys Asn Ala Val His 180 185 190 Val Asn Leu Phe Glu Thr Pro Val Glu Ala Gln Tyr Val Arg Leu Tyr 195 200 205 Pro Thr Ser Cys His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly 210 215 220 Cys Glu Leu Asn Gly Cys Ala Asn Pro Leu Gly Leu Lys Asn Asn Ser 225 230 235 240 Ile Pro Asp Lys Gln Ile Thr Ala Ser Ser Ser Tyr Lys Thr Trp Gly 245 250 255 Leu His Leu Phe Ser Trp Asn Pro Ser Tyr Ala Arg Leu Asp Lys Gln 260 265 270 Gly Asn Phe Asn Ala Trp Val Ala Gly Ser Tyr Gly Asn Asp Gln Trp 275 280 285 Leu Gln Val Asp Leu Gly Ser Ser Lys Glu Val Thr Gly Ile Ile Thr 290 295 300 Gln Gly Ala Arg Asn Phe Gly Ser Val Gln Phe Val Ala Ser Tyr Lys 305 310 315 320 Val Ala Tyr Ser Asn Asp Ser Ala Asn Trp Thr Glu Tyr Gln Asp Pro 325 330 335 Arg Thr Gly Ser Ser Lys Ile Phe Pro Gly Asn Trp Asp Asn His Ser 340 345 350 His Lys Lys Asn Leu Phe Glu Thr Pro Ile Leu Ala Arg Tyr Val Arg 355 360 365 Ile Leu Pro Val Ala Trp His Asn Arg Ile Ala Leu Arg Leu Glu Leu 370 375 380 Leu Gly Cys 385 <210> 11 <211> 1936 <212> DNA <213> Homo sapiens <400> 11 agaaccccgc ggggtctgag cagcccagcg tgcccattcc agcgcccgcg tccccgcagc 60 atgccgcgcc cccgcctgct ggccgcgctg tgcggcgcgc tgctctgcgc ccccagcctc 120 ctcgtcgccc tggatatctg ttccaaaaac ccctgccaca acggtggttt atgcgaggag 180 atttcccaag aagtgcgagg agatgtcttc ccctcgtaca cctgcacgtg ccttaagggc 240 tacgcgggca accactgtga gacgaaatgt gtcgagccac tgggcatgga gaatgggaac 300 attgccaact cacagatcgc cgcctcgtct gtgcgtgtga ccttcttggg tttgcagcat 360 tgggtcccgg agctggcccg cctgaaccgc gcaggcatgg tcaatgcctg gacacccagc 420 agcaatgacg ataacccctg gatccaggtg aacctgctgc ggaggatgtg ggtaacaggt 480 gtggtgacgc agggtgccag ccgcttggcc agtcatgagt acctgaaggc cttcaaggtg 540 gcctacagcc ttaatggaca cgaattcgat ttcatccatg atgttaataa aaaacacaag 600 gagtttgtgg gtaactggaa caaaaacgcg gtgcatgtca acctgtttga gacccctgtg 660 gaggctcagt acgtgagatt gtaccccacg agctgccaca cggcctgcac tctgcgcttt 720 gagctactgg gctgtgagct gaacggatgc gccaatcccc tgggcctgaa gaataacagc 780 atccctgaca agcagatcac ggcctccagc agctacaaga cctggggctt gcatctcttc 840 agctggaacc cctcctatgc acgggctggac aagcagggca acttcaacgc ctgggttgcg 900 gggagctacg gtaacgatca gtggctgcag gtggacctgg gctcctcgaa ggaggtgaca 960 ggcatcatca cccaggggc ccgtaacttt ggctctgtcc agtttgtggc atcctacaag 1020 gttgcctaca gtaatgacag tgcgaactgg actgagtacc aggaccccag gactggcagc 1080 agtaagatct tccctggcaa ctgggacaac cactcccaca agaagaactt gtttgagacg 1140 cccatcctgg ctcgctatgt gcgcatcctg cctgtagcct ggcacaaccg catcgccctg 1200 cgcctggagc tgctgggctg ttagtggcca cctgccaccc ccaggtcttc ctgctttcca 1260 tgggcccgct gcctcttggc ttctcagccc ctttaaatca ccatagggct ggggactggg 1320 gaaggggagg gtgttcagag gcagcaccac cacacagtca cccctccctc cctctttccc 1380 accctccacc tctcacgggc cctgccccag cccctaagcc ccgtccccta acccccagtc 1440 ctcactgtcc tgttttctta ggcactgagg gatctgagta ggtctgggat ggacaggaaa 1500 gggcaaagta gggcgtgtgg tttccctgcc cctgtccgga ccgccgatcc caggtgcgtg 1560 tgtctctgtc tctcctagcc cctctctcac acatcacatt cccatggtgg cctcaagaaa 1620 ggcccggaag cgccaggctg gagataacag cctcttgccc gtcggccctg cgtcggccct 1680 ggggtaccat gtggccacaa ctgctgtggc cccctgtccc caagacactt ccccttgtct 1740 ccctggttgc ctctcttgcc ccttgtcctg aagcccagcg acacagaagg gggtggggcg 1800 ​​​​​​​​​​​​​​​​​​​​​​​​His Asn Gly Gly Leu Cys Glu Glu Ile Ser Gln Glu Val Arg Gly Asp 35 40 45 Val Phe Pro Ser Tyr Thr Cys Thr Cys Leu Lys Gly Tyr Ala Gly Asn 50 55 60 His Cys Glu Thr Lys Cys Val Glu Pro Leu Gly Met Glu Asn Gly Asn 65 70 75 80 Ile Ala Asn Ser Gln Ile Ala Ala Ser Ser Val Arg Val Thr Phe Leu 85 90 95 Gly Leu Gln His Trp Val Pro Glu Leu Ala Arg Leu Asn Arg Ala Gly 100 105 110 Met Val Asn Ala Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro Trp Ile 115 120 125 Gln Val Asn Leu Leu Arg Arg Met Trp Val Thr Gly Val Val Thr Gln 130 135 140 Gly Ala Ser Arg Leu Ala Ser His Glu Tyr Leu Lys Ala Phe Lys Val 145 150 155 160 Ala Tyr Ser Leu Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn 165 170 175 Lys Lys His Lys Glu Phe Val Gly Asn Trp Asn Lys Asn Ala Val His 180 185 190 Val Asn Leu Phe Glu Thr Pro Val Glu Ala Gln Tyr Val Arg Leu Tyr 195 200 205 Pro Thr Ser Cys His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly 210 215 220 Cys Glu Leu Asn Gly Cys Ala Asn Pro Leu Gly Leu Lys Asn Asn Ser 225 230 235 240 Ile Pro Asp Lys Gln Ile Thr Ala Ser Ser Ser Tyr Lys Thr Trp Gly 245 250 255 Leu His Leu Phe Ser Trp Asn Pro Ser Tyr Ala Arg Leu Asp Lys Gln 260 265 270 Gly Asn Phe Asn Ala Trp Val Ala Gly Ser Tyr Gly Asn Asp Gln Trp 275 280 285 Leu Gln Val Asp Leu Gly Ser Ser Lys Glu Val Thr Gly Ile Ile Thr 290 295 300 Gln Gly Ala Arg Asn Phe Gly Ser Val Gln Phe Val Ala Ser Tyr Lys 305 310 315 320 Val Ala Tyr Ser Asn Asp Ser Ala Asn Trp Thr Glu Tyr Gln Asp Pro 325 330 335 Arg Thr Gly Ser Ser Lys Ile Phe Pro Gly Asn Trp Asp Asn His Ser 340 345 350 His Lys Lys Asn Leu Phe Glu Thr Pro Ile Leu Ala Arg Tyr Val Arg 355 360 365 Ile Leu Pro Val Ala Trp His Asn Arg Ile Ala Leu Arg Leu Glu Leu 370 375 380 Leu Gly Cys 385 <210> 13 <211> 2188 <212> DNA <213> Rattus norvegicus <400> 13 attcccctgt gagaggagcg gacgcaggaa ctctccggtc ccagcatcgg agcttgtgga 60 ccatttcccg cgtcccgcag catgcagttc tcccgtgtgc tggccgcgct gtgcggtgtg 120 ctgctctgcg cctccggcct cttcgctgcg tccggtgact tctgtgactc cagcctgtgc 180 ctgaatggtg ggacctgctt gatgggccaa gacaatgaca tctactgcct ctgccctgaa 240 ggcttcacag gccttgtgtg caacgagact gagaaaggac cgtgttcccc aaacccttgc 300 ttccacgatg ccaaatgcct ggtgactgag gacacacagc gaggggacat cttcactgag 360 tacatctgcc agtgccctgt gggctactcg ggcatccact gtgaactcga gaccacctcc 420 tacctggatg gagagtacct gtccagccca gccgtcccta ccacagccgt ccccaccaca 480 gccatcccca ccacagccgt ccccaccaca gccgtcccca ccacagccgt ccccaccccg 540 gcccccaacc ccgatctttc caaccaccta gcctcccgct gttccacaaa gctgggcttg 600 gaagggggcg ccattgccga ttcacagatt tctgcctcgt ctgtgtatat gggcttcatg 660 ggcttgcagc gctggggccc ggagctggct cgcctgtatc gcacagggat tgtcaatgca 720 tggacagcca gcagctatga tagcaagccc tggatccagg tggactttct gcggaagatg 780 cgggtatcag gtgtgatgac acagggtgcc agccgtgccg ggagggcgga atacctgaag 840 accttcaagg tggcttacag cctcgatgga cgcaggttcg agttcatcca ggatgaaagc 900 ggaaccggag acaaggagtt tatgggtaac caggacaaca acagcctgaa gattaacatg 960 ttcaacccca ctctggaggc acagtacata aggctgtacc ctgtctcgtg ccaccgcggc 1020 tgcaccctcc gcttcgagct cctgggctgc gagttgcatg gatgctctga gcccctgggc 1080 ctgaagaata acacgattcc tgacagccaa ataacagcct ccagcagcta caagacgtgg 1140 aacctgcgtg cctttggctg gtacccccac ttggggcggc tggacaatca gggcaagatc 1200 aatgcctgga cagctcagag caacagtgcc aaagtaatggc tgcaggttga cctgggcact 1260 cagaaaaaag tgacaggaat tatcacccag ggggcccgtg actttggcca catccagtat 1320 gtggcatcct ataaggtagc ccacagtgat gatggtgtgc agtggaccgt atataggaa 1380 caaggaacca gcaaggtctt ccagggcaac ttggacaaca actcccacaa gaagaacatc 1440 tttgagaaac ctttcatggc tcgctatgtg cgtgtccttc cactgtcctg gcataaccgt 1500 atcaccctgc gcctggagct gctgggctgt tagtgcccag tccttccagc ccaagtgacg 1560 aggacggcca gaggctgagg ggcctcctgg ccctgcctcc caggccctgc tgccttctgt 1620 ggctgacacc ttctcaatcc tccctcttga ttgcactggg actacaggca ggaagggcaa 1680 ggggggtttca gagttgcccc tcacccttcc cctcaccctg cagcccccac aggcctcctg 1740 ctagcccctt ctctcaggca ttctggggga gttggacagg tctgagatga your permission 1800 agagtgaagt tggggtatgt gggctgctgt accaaccacc ccaagtccta aactttctcc 1860 aggggttgac tcaagactaa agggaacctc tggttgccca cccgtctctg cacaccgcac 1920 atccctccat gttccattcc tggaaggaga ggcccacgtc cgcttgctgc cccttgggtc 1980 accagatcct gcctcttatc tcctgagacc cctcttgacc ctcgctctgg agcctcggtt 2040 gacaagagga ctgtcgggtc tggagagata gatgggctct gggtggttgg cgagctggct 2100 atgggacctc tgctggcttg ctacccaagc taacaagcag attccaaaat acatttgtgc 2160 tctccactgg aaaaaaaaaa aaaaaaaa 2188 <210> 14 <211> 483 <212> PRT <213> Rattus norvegicus <400> 14 Met Gln Phe Ser Arg Val Leu Ala Ala Leu Cys Gly Val Leu Leu Cys 1 5 10 15 Ala Ser Gly Leu Phe Ala Ala Ser Gly Asp Phe Cys Asp Ser Ser Leu 20 25 30 Cys Leu Asn Gly Gly Thr Cys Leu Met Gly Gln Asp Asn Asp Ile Tyr 35 40 45 Cys Leu Cys Pro Glu Gly Phe Thr Gly Leu Val Cys Asn Glu Thr Glu 50 55 60 Lys Gly Pro Cys Ser Pro Asn Pro Cys Phe His Asp Ala Lys Cys Leu 65 70 75 80 Val Thr Glu Asp Thr Gln Arg Gly Asp Ile Phe Thr Glu Tyr Ile Cys 85 90 95 Gln Cys Pro Val Gly Tyr Ser Gly Ile His Cys Glu Leu Glu Thr Thr 100 105 110 Ser Tyr Leu Asp Gly Glu Tyr Leu Ser Ser Pro Ala Val Pro Thr Thr 115 120 125 Ala Val Pro Thr Thr Ala Ile Pro Thr Thr Ala Val Pro Thr Thr Ala 130 135 140 Val Pro Thr Thr Ala Val Pro Thr Pro Ala Pro Asn Pro Asp Leu Ser 145 150 155 160 Asn His Leu Ala Ser Arg Cys Ser Thr Lys Leu Gly Leu Glu Gly Gly 165 170 175 Ala Ile Ala Asp Ser Gln Ile Ser Ala Ser Ser Val Tyr Met Gly Phe 180 185 190 Met Gly Leu Gln Arg Trp Gly Pro Glu Leu Ala Arg Leu Tyr Arg Thr 195 200 205 Gly Ile Val Asn Ala Trp Thr Ala Ser Ser Tyr Asp Ser Lys Pro Trp 210 215 220 Ile Gln Val Asp Phe Leu Arg Lys Met Arg Val Ser Gly Val Met Thr 225 230 235 240 Gln Gly Ala Ser Arg Ala Gly Arg Ala Glu Tyr Leu Lys Thr Phe Lys 245 250 255 Val Ala Tyr Ser Leu Asp Gly Arg Arg Phe Glu Phe Ile Gln Asp Glu 260 265 270 Ser Gly Thr Gly Asp Lys Glu Phe Met Gly Asn Gln Asp Asn Asn Ser 275 280 285 Leu Lys Ile Asn Met Phe Asn Pro Thr Leu Glu Ala Gln Tyr Ile Arg 290 295 300 Leu Tyr Pro Val Ser Cys His Arg Gly Cys Thr Leu Arg Phe Glu Leu 305 310 315 320 Leu Gly Cys Glu Leu His Gly Cys Ser Glu Pro Leu Gly Leu Lys Asn 325 330 335 Asn Thr Ile Pro Asp Ser Gln Ile Thr Ala Ser Ser Ser Tyr Lys Thr 340 345 350 Trp Asn Leu Arg Ala Phe Gly Trp Tyr Pro His Leu Gly Arg Leu Asp 355 360 365 Asn Gln Gly Lys Ile Asn Ala Trp Thr Ala Gln Ser Asn Ser Ala Lys 370 375 380 Glu Trp Leu Gln Val Asp Leu Gly Thr Gln Lys Lys Val Thr Gly Ile 385 390 395 400 Ile Thr Gln Gly Ala Arg Asp Phe Gly His Ile Gln Tyr Val Ala Ser 405 410 415 Tyr Lys Val Ala His Ser Asp Asp Gly Val Gln Trp Thr Val Tyr Glu 420 425 430 Glu Gln Gly Thr Ser Lys Val Phe Gln Gly Asn Leu Asp Asn Asn Ser 435 440 445 His Lys Lys Asn Ile Phe Glu Lys Pro Phe Met Ala Arg Tyr Val Arg 450 455 460 Val Leu Pro Leu Ser Trp His Asn Arg Ile Thr Leu Arg Leu Glu Leu 465 470 475 480 Leu Gly Cys <210> 15 <211> 2020 <212> DNA <213> Rattus norvegicus <400> 15 attcccctgt gagaggagcg gacgcaggaa ctctccggtc ccagcatcgg agcttgtgga 60 ccatttcccg cgtcccgcag catgcagttc tcccgtgtgc tggccgcgct gtgcggtgtg 120 ctgctctgcg cctccggcct cttcgctgcg tccggtgact tctgtgactc cagcctgtgc 180 ctgaatggtg ggacctgctt gatgggccaa gacaatgaca tctactgcct ctgccctgaa 240 ggcttcacag gccttgtgtg caacgagact gagaaaggac cgtgttcccc aaacccttgc 300 ttccacgatg ccaaatgcct ggtgactgag gacacacagc gaggggacat cttcactgag 360 tacatctgcc agtgccctgt gggctactcg ggcatccact gtgaactcgg ctgttccaca 420 aagctgggct tggaaggggg cgccattgcc gattcacaga tttctgcctc gtctgtgtat 480 atgggcttca tgggcttgca gcgctggggc ccggagctgg ctcgcctgta tcgcacaggg 540 attgtcaatg catggacagc cagcagctat gatagcaagc cctggatcca ggtggacttt 600 ctgcggaaga tgcgggtatc aggtgtgatg acacagggtg ccagccgtgc cgggagggcg 660 gaatacctga agaccttcaa ggtggcttac agcctcgatg gacgcaggtt cgagttcatc 720 caggatgaaa gcggaaccgg agacaaggag tttatgggta accaggacaa caacagcctg 780 aagattaaca tgttcaaccc cactctggag gcacagtaca taaggctgta ccctgtctcg 840 tgccaccgcg gctgcaccct ccgcttcgag ctcctgggct gcgagttgca tggatgctct 900 gagcccctgg gcctgaagaa taacacgatt cctgacagcc aaataacagc ctccagcagc 960 tacaagacgt ggaacctgcg tgcctttggc tggtaccccc acttggggcg gctggacaat 1020 cagggcaaga tcaatgcctg gacagctcag agcaacagtg ccaaggaatg gctgcaggtt 1080 gacctgggca ctcagaaaaa agtgacagga attatcaccc agggggcccg tgactttggc 1140 cacatccagt atgtggcatc ctataaggta gcccacagtg atgatggtgt gcagtggacc 1200 gtatatgagg aacaaggaac cagcaaggtc ttccagggca acttggacaa caactcccac 1260 aagaagaaca tctttgagaa acctttcatg gctcgctatg tgcgtgtcct tccactgtcc 1320 tggcataacc gtatcaccct gcgcctggag ctgctgggct gttagtgccc agtccttcca 1380 gcccaagtga cgaggacggc cagaggctga ggggcctcct ggccctgcct cccaggccct 1440 gctgccttct gtggctgaca ccttctcaat cctccctctt gattgcactg ggactacagg 1500 caggaagggc aagggggttt cagagttgcc cctcaccctt cccctcaccc tgcagccccc 1560 acaggcctcc tgctagcccc ttctctcagg cattctgggg gagttggaca ggtctgagat 1620 gaatagagaa gaagagtgaa gttggggtat gtgggctgct gtaccaacca ccccaagtcc 1680 taaactttct ccaggggttg actcaagact aaagggaacc tctggttgcc cacccgtctc 1740 tgcacaccgc acatccctcc atgttccatt cctggaagga gaggcccacg tccgcttgct 1800 gccccttggg tcaccagatc ctgcctctta tctcctgaga cccctcttga ccctcgctct 1860 ggagcctcgg ttgacaagag gactgtcggg tctggagaga tagatgggct ctgggtggtt 1920 ggcgagctgg ctatgggacc tctgctggct tgctacccaa gctaacaagc agattccaaa 1980 atacatttgt gctctccact ggaaaaaaaa aaaaaaaaaa 2020 <210> 16 <211> 427 <212> PRT <213> Rattus norvegicus <400> 16 Met Gln Phe Ser Arg Val Leu Ala Ala Leu Cys Gly Val Leu Leu Cys 1 5 10 15 ​​​​​​​​Cys Leu Cys Pro Glu Gly Phe Thr Gly Leu Val Cys Asn Glu Thr Glu 50 55 60 Lys Gly Pro Cys Ser Pro Asn Pro Cys Phe His Asp Ala Lys Cys Leu 65 70 75 80 Val Thr Glu Asp Thr Gln Arg Gly Asp Ile Phe Thr Glu Tyr Ile Cys 85 90 95 Gln Cys Pro Val Gly Tyr Ser Gly Ile His Cys Glu Leu Gly Cys Ser 100 105 110 Thr Lys Leu Gly Leu Glu Gly Gly Ala Ile Ala Asp Ser Gln Ile Ser 115 120 125 Ala Ser Ser Val Tyr Met Gly Phe Met Gly Leu Gln Arg Trp Gly Pro 130 135 140 Glu Leu Ala Arg Leu Tyr Arg Thr Gly Ile Val Asn Ala Trp Thr Ala 145 150 155 160 Ser Ser Tyr Asp Ser Lys Pro Trp Ile Gln Val Asp Phe Leu Arg Lys 165 170 175 Met Arg Val Ser Gly Val Met Thr Gln Gly Ala Ser Arg Ala Gly Arg 180 185 190 Ala Glu Tyr Leu Lys Thr Phe Lys Val Ala Tyr Ser Leu Asp Gly Arg 195 200 205 Arg Phe Glu Phe Ile Gln Asp Glu Ser Gly Thr Gly Asp Lys Glu Phe 210 215 220 Met Gly Asn Gln Asp Asn Asn Ser Leu Lys Ile Asn Met Phe Asn Pro 225 230 235 240 Thr Leu Glu Ala Gln Tyr Ile Arg Leu Tyr Pro Val Ser Cys His Arg 245 250 255 Gly Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys Glu Leu His Gly Cys 260 265 270 Ser Glu Pro Leu Gly Leu Lys Asn Asn Thr Ile Pro Asp Ser Gln Ile 275 280 285 Thr Ala Ser Ser Ser Tyr Lys Thr Trp Asn Leu Arg Ala Phe Gly Trp 290 295 300 Tyr Pro His Leu Gly Arg Leu Asp Asn Gln Gly Lys Ile Asn Ala Trp 305 310 315 320 Thr Ala Gln Ser Asn Ser Ala Lys Glu Trp Leu Gln Val Asp Leu Gly 325 330 335 Thr Gln Lys Lys Val Thr Gly Ile Ile Thr Gln Gly Ala Arg Asp Phe 340 345 350 Gly His Ile Gln Tyr Val Ala Ser Tyr Lys Val Ala His Ser Asp Asp 355 360 365 Gly Val Gln Trp Thr Val Tyr Glu Glu Gln Gly Thr Ser Lys Val Phe 370 375 380 Gln Gly Asn Leu Asp Asn Asn Ser His Lys Lys Asn Ile Phe Glu Lys 385,390,395,400 Pro Phe Met Ala Arg Tyr Val Arg Val Leu Pro Leu Ser Trp His Asn 405 410 415 Arg Ile Thr Leu Arg Leu Glu Leu Leu Gly Cys 420 425 <210> 17 <211> 2032 <212> DNA <213> Mus musculus <400> 17 ggcgcctgat ttattccgga gtgagaggag cggacgcagg aactctcgag tcccagcatc 60 agagcgcgtg gaccttttcc cgcgtcccgc agcatgcagg tctcccgtgt gctggccgcg 120 ctgtgcggca tgctactctg cgcctctggc ctcttcgccg cgtctggtga cttctgtgac 180 tccagcctgt gcctgaacgg tggcacctgc ttgacgggcc aagacaatga catctactgc 240 ctctgccctg aaggcttcac aggccttgtg tgcaatgaga ctgagagagg accatgctcc 300 ccaaaccctt gctacaatga tgccaaatgt ctggtgactt tggacacaca gcgtggggac 360 atcttcaccg aatacatctg ccagtgccct gtgggctact cgggcatcca ctgtgaaacc 420 ggttgttcta cacagctggg catggaaggg ggcgccattg ctgattcaca gatttccgcc 480 tcgtctgtgt atatgggttt catgggcttg cagcgctggg gcccggagct ggctcgtctg 540 taccgcacag ggatcgtcaa tgcctggaca gccagcaact atgatagcaa gccctggatc 600 caggtgaacc ttctgcggaa gatgcgggta tcaggtgtga tgacgcaggg tgccagccgt 660 gccgggagg cggagtacct gaagacctt aaggtggctt acagcctcga cggacgcaag 720 780 aacagcctga aggttaacat gttcaacccg actctggagg caagtacat aaagctgtac 840 cctgtttcgt gccaccgcgg ctgcaccctc cgcttcgagc tcctgggctg tgagttgcac 900 ggatgttctg agcccctggg cctgaagaat aacacaattc ctgacagcca gatgtcagcc 960 tccagcagct acaagacatg gaacctgcgt gcttttggct ggtaccccca cttgggaagg 1020 ctggataatc agggcaagat caatgcctgg acggctcaga gcaacagtgc caaggaatgg 1080 ctgcaggttg acctgggcac tcagaggcaa gtgacaggaa tcatcaccca gggggcccgt 1140 gactttggcc acatccagta tgtggcgtcc tacaaggtag cccacagtga tgatggtgtg 1200 cagtggactg tatatgagga gcaaggaagc agcaaggtct tccagggcaa cttggacaac 1260 aactcccaca agaagaacat cttcgagaaa cccttcatgg ctcgctacgt gcgtgtcctt 1320 ccagtgtcct ggcataaccg catcaccctg cgcctggagc tgctgggctg ttaatgctca 1380 gtcctgccag cccaaacgat gaggatggcc agaggctgag gggcctcctg gccctgcctc 1440 ccaggccctg ctgccttctg tggctgacga ccttcttggc cttcccttct gattgtactg 1500 gggctggagg caggaagggc caggggattt cagagttgcc cttcaccctt tccctcaccc 1560 tgcagccccc acaggcctcc tgctagcccc cttctctcag gcattctggg ggagttggac 1620 aggtctgaga tgaatagaga agaagagtga agttggggta tgtgggctat ctgtaccaac 1680 caccccaagt cctaaacttc ctgccagggc ttgactcagg actgaaggga gcccctgact 1740 gcccatccct ctctgcacac cacacattcc tccatgttcc attccgggaa ggagaggccc 1800 acgtccgctt gctgtccctt gggtcaccag gtcctgccctc ttatctcctg agacgccct 1860 tgacccttgc actggagcct cagttgacaa ggagactggc gggtctggag aggtcggtgg 1920 ctctgggtgg tgacaggtt ggctgtggga ccttgctgg cttgctaccc aagttaacaa 1980 gcagattcca aaatacattc gtgttctcca ctggaaaaa aaaaaaaaa aa 2032 <210> 18 <211> 426 <212> PRT <213> Muscles <400> 18 Met Gln Val Ser Arg Val Leu Ala Ala Leu Cys Gly Met Leu Leu Cys 1 5 10 15 Ala Ser Gly Leu Phe Ala Ala Ser Gly Asp Phe Cys Asp Ser Ser Leu 20 25 30 Cys Leu Asn Gly Gly Thr Cys Leu Thr Gly Gln Asp Asp Ile Tyr 35 40 45 Cys Leu Cys Pro Glu Gly Phe Thr Gly Leu Val Cys Asn Glu Thr Glu 50 55 60 Arg Gly Pro Cys Ser Pro Asn Pro Cys Tyr Asn Asp Ala Lys Cys Leu 65 70 75 80 Val Thr Leu Asp Thr Gln Arg Gly Asp Ile Phe Thr Glu Tyr Ile Cys 85 90 95 Gln Cys Pro Val Gly Tyr Ser Gly Ile His Cys Glu Thr Gly Cys Ser 100 105 110 Thr Gln Leu Gly Met Glu Gly Gly Ala Ile Ala Asp Ser Gln Ile Ser 115 120 125 Ala Ser Ser Val Tyr Met Gly Phe Met Gly Leu Gln Arg Trp Gly Pro 130 135 140 Glu Leu Ala Arg Leu Tyr Arg Thr Gly Ile Val Asn Ala Trp Thr Ala 145 150 155 160 Ser Asn Tyr Asp Ser Lys Pro Trp Ile Gln Val Asn Leu Leu Arg Lys 165 170 175 Met Arg Val Ser Gly Val Met Thr Gln Gly Ala Ser Arg Ala Gly Arg 180 185 190 Ala Glu Tyr Leu Lys Thr Phe Lys Val Ala Tyr Ser Leu Asp Gly Arg 195 200 205 Lys Phe Glu Phe Ile Gln Asp Glu Ser Gly Gly Asp Lys Glu Phe Leu 210 215 220 Gly Asn Leu Asp Asn Asn Ser Leu Lys Val Asn Met Phe Asn Pro Thr 225 230 235 240 Leu Glu Ala Gln Tyr Ile Lys Leu Tyr Pro Val Ser Cys His Arg Gly 245 250 255 Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys Glu Leu His Gly Cys Ser 260 265 270 Glu Pro Leu Gly Leu Lys Asn Asn Thr Ile Pro Asp Ser Gln Met Ser 275 280 285 Ala Ser Ser Ser Tyr Lys Thr Trp Asn Leu Arg Ala Phe Gly Trp Tyr 290 295 300 Pro His Leu Gly Arg Leu Asp Asn Gln Gly Lys Ile Asn Ala Trp Thr 305 310 315 320 Ala Gln Ser Asn Ser Ala Lys Glu Trp Leu Gln Val Asp Leu Gly Thr 325 330 335 Gln Arg Gln Val Thr Gly Ile Ile Thr Gln Gly Ala Arg Asp Phe Gly 340 345 350 His Ile Gln Tyr Val Ala Ser Tyr Lys Val Ala His Ser Asp Asp Gly 355 360 365 Val Gln Trp Thr Val Tyr Glu Glu Gln Gly Ser Ser Lys Val Phe Gln 370 375 380 Gly Asn Leu Asp Asn Asn Ser His Lys Lys Asn Ile Phe Glu Lys Pro 385 390 395 400 Phe Met Ala Arg Tyr Val Arg Val Leu Pro Val Ser Trp His Asn Arg 405 410 415 Ile Thr Leu Arg Leu Glu Leu Leu Gly Cys 420 425 <210> 19 <211> 2143 <212> DNA <213> Mus musculus <400> 19 ggcgcctgat ttattccgga gtgagaggag cggacgcagg aactctcgag tcccagcatc 60 agagcgcgtg gaccttttcc cgcgtcccgc agcatgcagg tctcccgtgt gctggccgcg 120 ctgtgcggca tgctactctg cgcctctggc ctcttcgccg cgtctggtga cttctgtgac 180 tccagcctgt gcctgaacgg tggcacctgc ttgacgggcc aagacaatga catctactgc 240 ctctgccctg aaggcttcac aggccttgtg tgcaatgaga ctgagagagg accatgctcc 300 ccaaaccctt gctacaatga tgccaaatgt ctggtgactt tggacacaca gcgtggggac 360 atcttcaccg aatacatctg ccagtgccct gtgggctact cgggcatcca ctgtgaaacc 420 gagaccaact actacaacct ggatggagaa tacatgttca ccacagccgt ccccaatact 480 gccgtcccca ccccggcccc cacccccgat ctttccaaca acctagcctc ccgttgttct 540 acacagctgg gcatggaagg gggcgccatt gctgattcac agatttccgc ctcgtctgtg 600 tatatgggtt tcatgggctt gcagcgctgg ggcccggagc tggctcgtct gtaccgcaca 660 gggatcgtca atgcctggac agccagcaac tatgatagca agccctggat ccaggtgaac 720 cttctgcgga agatgcgggt atcaggtgtg atgacgcagg gtgccagccg tgccgggagg 780 gcggagtacc tgaagacctt caaggtggct tacagcctcg acggacgcaa gtttgagttc 840 atccaggatg aaagcggtgg agacaaggag tttttgggta acctggacaa caacagcctg 900 aaggttaaca tgttcaaccc gactctggag gcacagtaca taaagctgta ccctgtttcg 960 tgccaccgcg gctgcaccct ccgcttcgag ctcctgggct gtgagttgca cggatgttct 1020 gagcccctgg gcctgaagaa taacacaatt cctgacagcc agatgtcagc ctccagcagc 1080 tacaagacat ggaacctgcg tgcttttggc tggtaccccc acttgggaag gctggataat 1140 cagggcaaga tcaatgcctg gacggctcag agcaacagtg ccaaggaatg gctgcaggtt 1200 gacctgggca ctcagaggca agtgacagga atcatcaccc agggggcccg tgactttggc 1260 cacatccagt atgtggcgtc ctacaaggta gcccacagtg atgatggtgt gcagtggact 1320 gtatatgagg agcaaggaag cagcaaggtc ttccagggca acttggacaa caactcccac 1380 aagaagaaca tcttcgagaa acccttcatg gctcgctacg tgcgtgtcct tccagtgtcc 1440 tggcataacc gcatcaccct gcgcctggag ctgctgggct gttaatgctc agtcctgcca 1500 gcccaaacga tgaggatggc cagaggctga ggggcctcct ggccctgcct cccaggccct 1560 gctgccttct gtggctgacg accttcttgg cttcccttc tgattgtact ggggctggag 1620 gcaggaaggg ccaggggatt tcagagttgc ccttcaccct ttccctcacc ctgcagcccc 1680 cacaggcctc ctgctagccc ccttctctca ggcattctgg gggagttgga caggtctgag 1740 atgaatagag aagaagagtg aagttggggt atgtgggcta tctgtaccaa ccaccccaag 1800 tcctaaactt cctgccaggg cttgactcag gactgaaggg agcccctgac tgcccatccc 1860 tctctgcaca ccacacattc ctccatgttc cattccggga aggagaggcc cacgtccgct 1920 tgctgtccct tgggtcacca ggtcctgcct ctttatctcct gagacgcctc ttgacccttg 1980 cactggagcc tcagttgaca aggagactgg cgggtctgga gaggtcggtg gctctgggtg 2040 gttgacaggt tggctgtggg acctctgctg gcttgctacc caagttaaca agcagattcc 2100 aaaatacatt cgtgttctcc actggaaaaa aaaaaaaaaa aaa 2143 <210> 20 <211> 463 <212> PRT <213> Mus musculus <400> 20 Met Gln Val Ser Arg Val Leu Ala Ala Leu Cys Gly Met Leu Leu Cys 1 5 10 15 Ala Ser Gly Leu Phe Ala Ala Ser Gly Asp Phe Cys Asp Ser Ser Leu 20 25 30 Cys Leu Asn Gly Gly Thr Cys Leu Thr Gly Gln Asp Asn Asp Ile Tyr 35 40 45 Cys Leu Cys Pro Glu Gly Phe Thr Gly Leu Val Cys Asn Glu Thr Glu 50 55 60 Arg Gly Pro Cys Ser Pro Asn Pro Cys Tyr Asn Asp Ala Lys Cys Leu 65 70 75 80 Val Thr Leu Asp Thr Gln Arg Gly Asp Ile Phe Thr Glu Tyr Ile Cys 85 90 95 Gln Cys Pro Val Gly Tyr Ser Gly Ile His Cys Glu Thr Glu Thr Asn 100 105 110 Tyr Tyr Asn Leu Asp Gly Glu Tyr Met Phe Thr Thr Ala Val Pro Asn 115 120 125 Thr Ala Val Pro Thr Pro Ala Pro Thr Pro Asp Leu Ser Asn Asn Leu 130 135 140 Ala Ser Arg Cys Ser Thr Gln Leu Gly Met Glu Gly Gly Ala Ile Ala 145 150 155 160 Asp Ser Gln Ile Ser Ala Ser Ser Val Tyr Met Gly Phe Met Gly Leu 165 170 175 Gln Arg Trp Gly Pro Glu Leu Ala Arg Leu Tyr Arg Thr Gly Ile Val 180 185 190 Asn Ala Trp Thr Ala Ser Asn Tyr Asp Ser Lys Pro Trp Ile Gln Val 195 200 205 Asn Leu Leu Arg Lys Met Arg Val Ser Gly Val Met Thr Gln Gly Ala 210 215 220 Ser Arg Ala Gly Arg Ala Glu Tyr Leu Lys Thr Phe Lys Val Ala Tyr 225 230 235 240 Ser Leu Asp Gly Arg Lys Phe Glu Phe Ile Gln Asp Glu Ser Gly Gly 245 250 255 Asp Lys Glu Phe Leu Gly Asn Leu Asp Asn Asn Ser Leu Lys Val Asn 260 265 270 Met Phe Asn Pro Thr Leu Glu Ala Gln Tyr Ile Lys Leu Tyr Pro Val 275 280 285 Ser Cys His Arg Gly Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys Glu 290 295 300 Leu His Gly Cys Ser Glu Pro Leu Gly Leu Lys Asn Asn Thr Ile Pro 305 310 315 320 Asp Ser Gln Met Ser Ala Ser Ser Ser Tyr Lys Thr Trp Asn Leu Arg 325 330 335 Ala Phe Gly Trp Tyr Pro His Leu Gly Arg Leu Asp Asn Gln Gly Lys 340 345 350 Ile Asn Ala Trp Thr Ala Gln Ser Asn Ser Ala Lys Glu Trp Leu Gln 355 360 365 Val Asp Leu Gly Thr Gln Arg Gln Val Thr Gly Ile Ile Thr Gln Gly 370 375 380 Ala Arg Asp Phe Gly His Ile Gln Tyr Val Ala Ser Tyr Lys Val Ala 385 390 395 400 His Ser Asp Asp Gly Val Gln Trp Thr Val Tyr Glu Glu Gln Gly Ser 405 410 415 Ser Lys Val Phe Gln Gly Asn Leu Asp Asn Asn Ser His Lys Lys Asn 420 425 430 Ile Phe Glu Lys Pro Phe Met Ala Arg Tyr Val Arg Val Leu Pro Val 435 440 445 Ser Trp His Asn Arg Ile Thr Leu Arg Leu Glu Leu Leu Gly Cys 450 455 460 <210> twenty one <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic 6xHis tag <400> twenty one His His His His His His 1 5 <210> twenty two <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> twenty two gttgtctcct gcgacttca 19 <210> twenty three <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> twenty three ggtggtccag ggtttctta 19 <210> twenty four <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> twenty four caatgcaatg aagaactgga ctgt 24 <210> 25 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 25 tcctacatct tctgagtttg gtga 24 <210> 26 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 26 gcagggttcc aacgatgttg 20 <210> 27 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 27 gcagccatcg actaggacag a 21 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 28 ctgacagagg caccactgaa 20 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 29 catctccaga gtccagcaca 20 <210> 30 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 30 tgacgtcact ggagttgtac gg 22 <210> 31 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 31 ggttcatgtc atggatggtg c 21 <210> 32 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 32 aactttgaga aggatggcaa gt 22 <210> 33 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 33 tgccacccat ggtaaacaa 19 <210> 34 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 34 ctggacagcc agacactaaa g 21 <210> 35 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 35 ctcgcggcaa gtcttcagag 20 <210> 36 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 36 gctctctgct cctcctgttc 20 <210> 37 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 37 ccatggtgtc tgagcgatgt 20 <210> 38 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 38 agctcccgga aaagattgat g 21 <210> 39 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 39 cagggtgctg gctgagtaga t 21 <210> 40 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 40 cacgcacgac gtcttcca 18 <210> 41 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic Primers <400> 41 aagcggtcct ggcagaaat 19

Claims

1. A pharmaceutical composition comprising a polypeptide, the polypeptide comprising: (i) Amino acid residues 1-225 of SEQ ID NO:10 or SEQ ID NO:12; or (ii) Amino acid residues 24-225 of SEQ ID NO:10 or SEQ ID NO:12, The polypeptide is missing amino acid residues 226-387 of SEQ ID NO:10 or SEQ ID NO:

12.

2. The pharmaceutical composition of claim 1, wherein the polypeptide comprises: (a) Amino acid residues 1-225 of SEQ ID NO:10 or SEQ ID NO:12; or (b) Amino acid residues 24-225 of SEQ ID NO:10 or SEQ ID NO:

12.

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

4. The pharmaceutical composition of claim 3, wherein the polypeptide can increase macrophage activity or inhibit fibrosis.

5. The pharmaceutical composition of claim 4, wherein the heterologous sequence comprises a FLAG tag, an HIS tag, and / or a GST.

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

7. The pharmaceutical composition of claim 6, wherein the heterologous sequence comprises the Fc portion of an immunoglobulin.

8. The pharmaceutical composition of claim 1, wherein the polypeptide is not glycosylated.

9. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers and / or diluents.

10. A nucleic acid molecule encoding an isolated polypeptide in a pharmaceutical composition according to claim 1.

11. The nucleic acid molecule of claim 10, wherein the nucleic acid molecule has 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 with any of the following: (i) nucleotides 61-735 of SEQ ID NO:9 or SEQ ID NO:11; and / or (ii) Nucleotides 130-735 of SEQ ID NO:9 or SEQ ID NO:

11.

12. A transport vector comprising the nucleic acid molecule as described in claim 10.

13. The carrier as claimed in claim 12, wherein the carrier is an expression carrier.

14. A host cell comprising the nucleic acid as described in claim 10.

15. The host cell of claim 14, wherein the host cell is Pichia pastoris.

16. A method for producing a polypeptide, the method comprising culturing a host cell as described in claim 14 in a culture medium to produce the polypeptide.

17. The use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for treating a disorder in a subject in need, wherein the disorder is cirrhosis.

18. The use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for treating a disorder in a subject in need, wherein the disorder is non-alcoholic steatohepatitis (NASH).

19. The use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for treating a disorder in a subject in need, wherein the disorder is idiopathic pulmonary fibrosis (IPF).

20. The use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for treating a disorder in a subject in need, wherein the disorder is myocardial infarction.

21. Use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for treating a disorder in a subject in need, wherein the disorder is Alzheimer's disease.

22. The application according to any one of claims 17-21, wherein the drug is administered via an intravenous, subcutaneous, intra-arterial, intraperitoneal, or intramuscular route.

23. The application as described in any one of claims 17-22, wherein the subject is a mammal.

24. The application as described in claim 23, wherein the mammal is a human, mouse, or rat.

25. The application as described in claim 24, wherein the mammal is a human.

26. A kit comprising the pharmaceutical composition according to any one of claims 1-9.

Images