Polypeptide inhibitors of neutrophil elastase activity and uses thereof

Abstract

The present invention is characterized by a polypeptide comprising a plasminogen activator inhibitor 1 (PAI-1) variant, which has reduced ability to bind to brittle protein, reduced ability to interact with the PAI-1 clearance receptor LDL receptor-associated protein 1 (LRP1), and the ability to effectively inhibit neutrophil elastase in the presence of neutrophil extracellular traps (NETs). In some embodiments, the polypeptide of the present invention comprises a PAI-1 variant optionally fused to an Fc domain monomer or partially. The present invention is further characterized by pharmaceutical compositions and methods for treating diseases and conditions characterized by abnormal neutrophil elastase activity (e.g., idiopathic pulmonary fibrosis) using the polypeptide.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to U.S. Provisional Application No. 62 / 938,859, filed November 21, 2019, the entire contents of which are incorporated herein by reference. Invention Field

[0003] The present invention is characterized by polypeptides comprising plasminogen activator inhibitor 1 (PAI-1) variants, having reduced ability to bind to brittle protein, reduced ability to interact with the PAI-1 clearance receptor LDL receptor-associated protein 1 (LRP1), and the ability to effectively inhibit neutrophil elastase (NE) in the presence of neutrophil extracellular traps (NETs). In some embodiments, the polypeptides of the present invention comprise PAI-1 variants optionally fused to an Fc domain monomer or partially. The present invention is further characterized by pharmaceutical compositions and methods for treating diseases and conditions characterized by abnormal neutrophil elastase activity (e.g., idiopathic pulmonary fibrosis) using said polypeptides. Background Technology

[0004] Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic lung disease that leads to respiratory failure and death. IPF is the most common cause of death from progressive lung disease, affecting approximately 5 million people worldwide. The estimated median survival after diagnosis is only 3–5 years (see Chakraborty et al., (2014) Expert Opin Investig Drugs, 23:893-910; Spagnolo et al., (2015) Pharmacology & Therapeutics 152:18-27; Tzouvelekis et al., (2015) Therapeutics and Clinical Risk Management 11:359-370; Lederer DJ and Martinez FJ. The New England Journal of Medicine. 2018; 378:1811-1823). There are approximately 130,000 IPF patients in the United States, with an estimated 30,000 to 40,000 new cases diagnosed annually (see Ley, B., and Collard, HR 2013. Epidemiology of idiopathic pulmonary fibrosis. Clin. Epidemiol. 5:483-492; Lynch, JP, III, et al., 2016. Idiopathic Pulmonary Fibrosis: Epidemiology, Clinical Features, Prognosis, and Management. Semin. Respir. Crit Care Med. 37:331-357). The prevalence of IPF ranges from 14.0 to 42.7 cases per 100,000 people, and the annual incidence ranges from 6.8 to 16.3 cases per 100,000 people, depending on the stringency of the diagnostic criteria used (see Jones, MG, and Richeldi, L. Semin. Respir. Crit. CareMed. 2016; 37:477-484). The prevalence of IPF increases with age, with most patients diagnosed at age 60 or older. The disease is more common in men than in women (see Fernandez Perez ER et al., (2010) Chest 137(1):129-137), and most patients are current or former smokers.(See Jones, MG and Richeldi, L., Semin. Respir. Care Med. 2016, 37: 477-484).

[0005] The etiology of interstitial fibrosis (IPF) is unclear. However, potential factors such as smoking, dust exposure, and infectious agents have been associated with its development. IPF is characterized by progressive and irreversible distortion of lung structure due to apoptosis of epithelial and endothelial cells, fibroblast proliferation, and extracellular matrix remodeling (see Chakraborty et al., (2014) Expert Opin Investig Drugs, 23:893-910). As interstitial fibrosis progresses, accompanied by distortion of lung structure, the lungs become less compliant, increasing respiratory effort and leading to dyspnea. Lung function typically declines slowly over time, but some patients experience a rapid decline that can lead to hospitalization or death, especially in the later stages of the disease.

[0006] The development of drugs for treating IPF has been slow. The first two drugs for IPF, pirfenidone and nintedanib, were only approved at the end of 2014 (see King et al., (2014) N Engl J Med 370:2083-92; Richeldi et al., (2014) N Engl J Med 370:2071-82; Richeldi, L, et al., Am.J.Med.Sci.2019,357:370-373). However, these two drugs have limited efficacy and significant side effects, and require complex dosing regimens. Recent phase 3 clinical trials of pirfenidone, sildenafil, bosentan, etanercept, and interferon-γ-1b have failed to demonstrate efficacy at their primary endpoints. N-acetylcysteine ​​(NAC), corticosteroids, and immunosuppressants such as cyclophosphamide and azathioprine are commonly prescribed, but there is little evidence that their use improves patient outcomes or alters the natural course of the disease (see Collard HR et al., (2004) Chest 125(6):2169-2174; Walter N et al., (2006) Proc Am Thorac Soc 3(4):377-381). Lung transplantation is the only treatment to improve survival, but most IPF patients are not suitable for transplantation due to age or comorbidities. IPF patients are typically managed with supportive measures such as symptomatic treatment of cough and dyspnea, supplemental oxygen for hypoxemia, smoking cessation, pulmonary rehabilitation, and prevention and control of respiratory infections.

[0007] The treatment of IPF needs improvement.

[0008] This invention addresses this need. Summary of the Invention

[0009] Although it has been established that the plasminogen activator inhibitor 1 (PAI-1) complex with the target enzyme binds tightly to LDL receptor-associated protein 1 (LRP1), the molecular details of this interaction remain poorly defined. Furthermore, the nature of the interaction between free PAI-1 and LRP1 is subject to considerable debate in the literature. Experiments conducted during the development of embodiments of the present invention examined the binding of PAI-1 complexes with low molecular weight urokinase-type plasminogen activator (uPA) and the binding of free PAI-1 to LRP1. Data confirmed that the affinity of the uPA:PAI-1 complex for binding to LRP1 is ~100 times higher than that of PAI-1 alone. Chemical modifications of PAI-1 confirmed that lysine residues on PAI-1 are essential for the interaction of both PAI-1 and the uPA:PAI-1 complex with LRP1. Surface plasmon resonance measurements supported a bivalent binding model in which multiple sites on PAI-1 and the uPA:PAI-1 complex interact with complementary sites on LRP1. The ionic strength dependence of the binding indicates the involvement of two key charged residues for the interaction between PAI-1 and LRP1, and three charged residues for the interaction between the uPA:PAI-1 complex and LRP1. The enhanced affinity resulting from the interaction of the three regions of the uPA:PAI-1 complex with the LDLa repeats on LRP1 provides a molecular explanation for the increased affinity of the uPA:PAI-1 complex for LRP1. Mutation analysis revealed overlap between the binding sites of LRP1 and the small molecule inhibitor of PAI-1, CDE-096 (a specific PAI-1 inhibitor). K207 plays an important role in the interaction between PAI-1 and LRP1, and K207, K88, and K80 play important roles in the interaction between the uPA:PAI-1 complex and LRP1.

[0010] Experiments conducted during the development of embodiments of the present invention elucidated the relative binding affinity of PAI-1 and the protease:PAI-1 complex to LRP1. Furthermore, experiments were performed to determine whether the binding of the protease:PAI-1 complex to LRP1 is primarily attributable to determinants on PAI-1. Additionally, experiments were conducted to identify specific amino acid residues in the PAI-1 complex with the target protease (urokinase-type plasminogen activator (uPA)) and in free PAI-1 involved in its binding to LRP1. Indeed, mutation analysis revealed an overlap between the binding site of LRP1 and the binding site of the small molecule inhibitor CDE-096 of PAI-1, and that K207 plays a crucial role in the interaction between PAI-1 and LRP1, as well as the interactions of K207, K88, and K80 within the uPA:PAI-1 complex and LRP1.

[0011] IPF is characterized by the formation of interstitial scar tissue, which can significantly limit lung function. There are approximately 130,000 IPF patients in the United States, with an estimated 30,000 to 40,000 new cases diagnosed annually (see, Ley, B., and Collard, HR 2013. Epidemiology of idiopathic pulmonary fibrosis. Clin. Epidemiol. 5:483-492; Lynch, JP, III, et al., 2016. Idiopathic Pulmonary Fibrosis: Epidemiology, Clinical Features, Prognosis, and Management. Semin. Respir. Crit Care Med. 37:331-357). Life expectancy after an IPF diagnosis is typically three to five years (see, Lederer, DJ and Martinez, FJ, NEJM 2018, 378:1811-1823). There is no effective treatment other than lung transplantation. The lack of therapies to halt the progression of IPF presents a significant challenge and represents a major unmet medical need. The two currently approved drugs, pirfenidone and nintedanib, have been shown to slow disease progression, but they do not stop it or restore lost lung function. Therefore, new therapies for IPF treatment are essential for disease management.

[0012] This invention addresses this need by providing compositions comprising a mutant PA1-I polypeptide capable of inhibiting NE, and particularly inhibiting NE bound to NET. This document provides a PAI-1 variant capable of inhibiting NE activity while also exhibiting reduced binding ability to breccia and LRP1. Indeed, experiments conducted during the development of embodiments of this invention have demonstrated that such PAI-1 variants exhibit therapeutic efficacy for conditions associated with NE activity (e.g., IPF) by inhibiting the binding ability of PAI-1 to breccia via modification of the PAI-1 amino acid residues responsible for this breccia binding (e.g., R101A and Q123K).

[0013] In fact, experiments conducted during the development of embodiments of the present invention utilized serine protease inhibitor mapping technology and identified mutant forms of PA1-1 (e.g., those with one or more of the following mutations in the wild-type mature human PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V) as NE inhibitors capable of effectively inhibiting NE in the NET environment. This mutant form of PA1-I has been shown to irreversibly inhibit DNA-bound NE (a major component of NET), where the FDA-approved human plasma-derived A1AT trademark Aralast is invalid. In some embodiments, this mutant form of PA1-I is further associated with human IgG-Fc.

[0014] Therefore, the present invention is characterized by peptides comprising a PAI-1 variant capable of inhibiting NE activity, while exhibiting a weakened ability to bind to glassin by modifying the amino acid residues responsible for glassin binding (e.g., R101A and Q123K), and resulting in improved pharmacokinetics (PK) by modifying the amino acid residues responsible for LRP1 binding (e.g., K207, K88, and K80), thereby improving therapeutic efficacy for conditions associated with NE activity (e.g., IPF). In some embodiments, the peptides of the present invention comprise a PAI-1 variant fused to the N- or C-terminus of an Fc domain monomer or portion (e.g., for the purpose of improving PK). In some embodiments, the peptides of the present invention comprise a PAI-1 variant fused to the N- or C-terminus of an Fc domain monomer or portion. In some embodiments, the Fc domain monomer or portion enhances the stability of the peptide or improves the pharmacokinetics of the peptide.

[0015] Such moieties can be fused or linked via amino acids or other covalent bonds, which can improve the stability of the peptide. Peptides including PAI-1 variants fused to Fc domain monomers can also form dimers (e.g., homodimers or heterodimers) through interactions between the two Fc domain monomers. In some embodiments, the peptides described herein are linked to Fc domain monomers, which are fused to the peptide via linkers. In some embodiments, the linkers are amino acid spacers.

[0016] The peptides of the present invention can be used to inhibit NE activity and to inhibit NE activity bound to NET. Additionally, the peptides of the present invention can be used to treat subjects suffering from conditions characterized by abnormal NE activity (e.g., IPF). Furthermore, the peptides of the present invention can be used to prevent subjects from developing conditions characterized by abnormal NE activity (e.g., IPF). Moreover, the peptides of the present invention can also be used to influence NE activity in subjects at risk of developing or suffering from diseases or conditions involving abnormal NE activity.

[0017] In some embodiments, the invention is characterized by a polypeptide comprising a PAI-1 variant having one or more of the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V. In some embodiments, the PAI-1 variant comprises mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V, as shown in SEQ ID NO:5. In some embodiments, the PAI-1 variant includes the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): R101A and Q123K. In some embodiments, the PAI-1 variant includes the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, and R346V. In some embodiments, the PAI-1 variant includes the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, K207A, V343A, and R346V. The mutant should have an extended half-life, particularly in the Fc form, and possess complete activity against NE in NET.

[0018] In some embodiments, the present invention is characterized by comprising a peptide comprising a PAI-1 variant linked to an Fc domain monomer or portion thereof, said variant having one or more of the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V. In some embodiments, the PAI-1 variant linked to an Fc domain monomer or portion thereof comprises the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V. In some embodiments, the PAI-1 variant linked to the Fc domain monomer or partially includes the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): R101A and Q123K. In some embodiments, the PAI-1 variant linked to the Fc domain monomer or partially includes the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, and R346V (SEQ ID NO:7). In some embodiments, the PAI-1 variant linked to the Fc domain monomer or partially includes the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, K207A, V343A, and R346V.

[0019] In some embodiments, the peptides described herein are capable of inhibiting NE, and in particular, inhibiting NE bound to NET.

[0020] In some embodiments, the invention is characterized by a nucleic acid molecule encoding the polypeptide described herein (e.g., a polypeptide comprising a PAI-1 variant having one or more of the following mutations in the wild-type mature human PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V). In another aspect, the invention is further characterized by a vector comprising the nucleic acid molecule described herein.

[0021] In another aspect, the present invention is characterized by a host cell expressing the polypeptide described herein, wherein the host cell comprises the nucleic acid molecule or vector described in the preceding two aspects, wherein the nucleic acid molecule or vector is expressed in the host cell.

[0022] In another aspect, the present invention is characterized by a method for preparing the polypeptide described herein, wherein the method comprises: a) providing a host cell comprising the nucleic acid molecule or vector described herein, and b) expressing the nucleic acid molecule or vector in the host cell under conditions that allow the formation of the polypeptide.

[0023] In another aspect, the present invention is characterized by a pharmaceutical composition comprising the polypeptide, nucleic acid molecule, or carrier described herein, and one or more pharmaceutically acceptable loads or excipients. In some embodiments of the pharmaceutical composition, the polypeptide, nucleic acid molecule, or carrier is in a therapeutically effective amount.

[0024] In another aspect, the invention is characterized by comprising a construct (e.g., a homodimer) of two identical polypeptides, each polypeptide comprising a PAI-1 variant having one or more of the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V, wherein the variant is fused to the N- or C-terminus of an Fc domain monomer. The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0025] In another aspect, the invention is characterized by comprising a construct (e.g., a heterodimer) of two distinct polypeptides, each polypeptide comprising a PAI-1 variant having one or more different combinations of the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V, wherein the two variants are fused to the N- or C-terminus of an Fc domain monomer. The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0026] In another aspect, the present invention is characterized by a method for inhibiting NE activity in a subject with this need. In yet another aspect, the present invention is characterized by a method for inhibiting NE activity bound to NET in a subject with this need. The method comprises administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule, or carrier described herein, or a pharmaceutical composition described herein.

[0027] In some embodiments of the method for inhibiting NE activity in a subject or NE activity bound within a NET, the subject has IPF and / or a condition characterized by abnormal NE activity (e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), emphysema). In some embodiments of the method for inhibiting NE activity in a subject or NE activity bound within a NET, the subject has A1AT activity and / or expression defects.

[0028] In another aspect, the present invention is characterized by a method of treating a subject with IPF by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0029] In another aspect, the present invention is characterized by a method of treating a subject with cystic fibrosis by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0030] In another aspect, the present invention is characterized by a method of treating a subject with COPD by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0031] In another aspect, the present invention is characterized by a method of treating a subject with emphysema by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0032] In another aspect, the present invention is characterized by a method of treating a subject suffering from acute respiratory distress syndrome (ARDS) by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0033] In another aspect, the present invention is characterized by a method of treating a subject with ischemia-reperfusion injury by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0034] In another aspect, the present invention is characterized by a method of treating a subject suffering from ethanol-induced chronic pancreatitis by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0035] In another aspect, the present invention is characterized by a method for treating a subject with rheumatoid arthritis (RA) by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0036] In another aspect, the present invention is characterized by a method of treating a subject suffering from disseminated intravascular coagulation (DIC) by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0037] In another aspect, the present invention is characterized by a method of treating a subject with ulcerative colitis (UC) by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0038] In another aspect, the present invention is characterized by a method of treating a subject with Crohn's disease by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0039] In another aspect, the present invention is characterized by a method of treating a subject with a skin disease having neutrophil pathology by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0040] In another aspect, the present invention is characterized by a method of treating a subject with A1AT activity and / or expression defects by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0041] In another aspect, the present invention is characterized by a method of treating a subject suffering from any condition characterized by abnormal NE activity and / or expression by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0042] In another aspect, the present invention is characterized by a method of treating a subject suffering from any condition characterized by a lack of A1AT activity and / or expression by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0043] In any of the above embodiments, the subject has or is at risk of developing a condition characterized by abnormal NE activity (e.g., IPF, COPD, cystic fibrosis, emphysema, ARDS, ischemia-reperfusion, chronic pancreatitis, RA, DIC, UC, Crohn's disease, dermatitis).

[0044] In any of the above embodiments, the subject has or is at risk of developing a condition characterized by a lack of A1AT activity and / or expression. Attached Figure Description

[0045] Figure 1 The lysine residues on PAI-1 play a crucial role in the binding of PAI-1 and the LMWuPA:PAI-1 complex to LRP1. A. The binding of the LMWuPA:PAI-1 complex and free PAI-1 to LRP1 was analyzed by SPR, where Req values ​​were determined by equilibrium measurements. Three independent experiments were performed, and the mean ± SE was plotted. KD values ​​(0.9 ± 0.2 nM for LMWuPA:PAI-1 and 74 ± 13 nM for PAI-1) were determined by nonlinear regression analysis. B. PAI-1 (lane 1) and chemically modified PAI-1 (lane 2) formed complexes with LMWuPA (lanes 3 and 4, respectively). Lane 5, LMWuPA. C. 250 nM PAI-1 and 300 nM chemically modified PAI-1 with sulfonyl-NHS-acetate were injected into an SPR chip immobilized with LRP1. D. A complex formed by 9 nM LMWuPA:PAI-1 and 80 nM chemically modified PAI-1 was injected into an SPR chip immobilized with LRP1.

[0046] Figure 2 The binding of PAI-1 to LRP1 is ionic strength dependent. A. PAI-1 was injected into an LRP-1-coated SPR chip at increasingly higher concentrations in a buffer containing increasing concentrations of NaCl, and the Req values ​​were determined. For each NaCl concentration, the data were normalized relative to Rmax. NaCl concentrations from top to bottom of the curve: 150 mM, 250 mM, 500 mM, 750 mM, and 1000 mM. B. Debye-Hückel plot of PAI-1 binding to LRP1. KD values ​​were measured at each ionic strength (150, 250, 500, 750, and 1000 mM NaCl) by balanced SPR measurements. Three independent experiments were performed, and the plotted values ​​are mean ± SE. A slope of 1.5 ± 0.1 was determined by linear regression analysis. Similar slope values ​​were obtained by averaging the results of linear regression analyses of the individual experiments.

[0047] Figure 3The binding of PAI-1 to LRP1 was well described by a bivalent binding model. (A) Schematic diagram of the bivalent binding model showing the interaction between two different regions on PAI-1 and complementary sites on LRP1. (B) Increasing concentrations of PAI-1 (9.4, 37.5, 75, 300 nM) were injected into LRP1-coupled SPR chips. Dissociation at each concentration was measured from SPR data, with the initial values ​​at t=0 normalized to 100%. The data conformed to a double exponential decay (blue line). C. Increasing concentrations of PAI-1 (3.9, 7.8, 15.6, 31.2, 62.5, 125 nM) were injected into LRP1-coupled chips. The fit between the experimental data (black line) and the bivalent binding model is shown as a blue line. The data shown are representative from six independent experiments conducted.

[0048] Figure 4 The binding of PAI-1 to cluster IV from LRP1 fits the bivalent binding model well. (A) Schematic diagram of the domain organization of LRP1. Ligand clusters (red circles) binding repeat sequences are labeled I, II, III, and IV. (B) Increasing concentrations of PAI-1 (9.4, 15.6, 31, 62.5, 125 nM) were injected into LRP1 cluster IV-coupled SPR chips. Dissociation at each concentration was measured from SPR data, with the initial values ​​at t=0 normalized to 100%. The data conform to a double exponential decay (blue line). (B) Increasing concentrations of PAI-1 (3.9, 7.8, 15.6, 31.2, 62.5, 125 nM) were injected into LRP1 cluster IV-coupled chips. The fit of experimental data (black line) to the bivalent binding model is shown by the blue line. The data shown represent three independent experiments conducted.

[0049] Figure 5 CDE-096 inhibits the binding of the HMWuPA:PAI-1 complex to LRP1. A. 1 nM of the HMWuPA:PAI-1 complex was flowed through an LRP1-coupled SPR chip in the absence of CDE-096 (top curve) and in the presence of increased concentrations (15.6, 31.2, 62.5, 125, 250, 500 nM). B. Figure A is a graph of the initial slope of the binding period relative to the CDE-096 concentration. The IC50 of 70 ± 11 nM was determined by nonlinear regression analysis. Data represent two independent experiments.

[0050] Figure 6The binding of the LMWuPA:PAI-1 complex to LRP1 is ionic strength dependent. A. Increasing concentrations of the uPA:PAI-1 complex were passed through an LRP-1-coated SPR chip in the presence of increasing concentrations of NaCl, and Req values ​​were determined. For each NaCl concentration, the data were normalized relative to Rmax. NaCl concentrations from top to bottom of the curve: 150 mM, 250 mM, 500 mM, 750 mM, and 1000 mM. B. Debye-Hückel plot of LMWuPA:PAI-1 binding to LRP1. KD values ​​were measured at each ionic strength (150 mM, 250 mM, 500 mM, 750 mM, and 1000 mM NaCl) by equilibrium SPR measurements. Three independent experiments were performed, and the mean ± SE was plotted. A slope of 2.4 ± 0.4 was determined by linear regression analysis. The same value was obtained by averaging the results of the linear regression analysis of each experiment.

[0051] Figure 7 The LMWuPA:PAI-1 complex binds to LRP1 via a complex kinetic model. A) Models used to analyze the binding of the LMWuPA:PAI-1 complex to LRP1. In Scheme I, LMWuPA:PAI-1 binds via a bivalent model. At higher concentrations of LMWuPA:PAI-1, a monovalent binding model occurs (Scheme II). (B) Increasing concentrations of the LMWuPA:PAI-1 complex (3.12, 6.25, 12.5, 25, 50 nM) were injected into an LRP1 coupling chip. Dissociation at each concentration was measured from SPR data, and the initial value at t=0 was normalized to 100%. (B) Increasing concentrations of LMWuPA:PAI-1 (0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM) were injected into an LRP1 coupling chip. The experimental data (black line) is shown, along with the fit (blue line) to the models including schemes I and II. The data represent three independent experiments.

[0052] Figure 8 Kinetic analysis of the binding of the LMWuPA:PAI-1 complex to cluster IV of LRP1. A) Increasing concentrations of the uPA:PAI-1 complex (0.6, 1.2, 2.5, 5, 10, 20, and 40 nM) were injected into the LRP1 conjugate chip. Dissociation at each concentration was measured from SPR data, with the initial values ​​at t=0 normalized to 100%. B) Increasing concentrations of LMWuPA:PAI-1 (0.6, 1.2, 2.5, 5, 10, 20, and 40 nM) were injected into the LRP1 conjugate chip. Experimental data (black line) are fitted to models of schemes I and II (blue line). Data represent three independent experiments.

[0053] Figure 9 LRP1-mediated cellular uptake of LMWuPA:PAI-1 decreased when a complex was formed with PAI-1 containing a mutation at a lysine residue. In the absence or presence of excess RAP, WI-38 human fibroblasts were incubated with 5 nM 125I-labeled LMWuPA:PAI-1 complexes formed with I91L PAI-1 or designated mutant PAI-1 molecules at 37°C for 6 h at 37°C. The amount of internalized complex was quantified after incubation. Experiments were performed in triplicate.

[0054] Figure 10 The following are provided: wild-type PAI-1 nucleic acid sequence (SEQ ID NO:1); wild-type PAI-1 amino acid sequence (SEQ ID NO:2); and mature wild-type PAI-1 amino acid sequence (SEQ ID NO:3).

[0055] Figure 11 The study provides a mature variant PAI-1 nucleic acid sequence (SEQ ID NO:4) encoding a polypeptide with the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V; and a mature variant PAI-1 amino acid sequence (SEQ ID NO:4) with the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V.

[0056] Figure 12 The study provides a mature variant PAI-1 / Fc amino acid sequence (SEQ ID NO:6) encoding a polypeptide with the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, R346V; and a mature variant PAI-1 / Fc amino acid sequence (SEQ ID NO:7) with the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, R346V.

[0057] Figure 13 The results show that MDI-1001 targets the inflammation network better than Aralast.

[0058] Figure 14An in vitro comparison of Aralast, Avelestat, MDI-1002, MDI-1003, and MDI-1004 is shown.

[0059] Figure 15 This shows that MDI-1003 targets NETs in CF sputum.

[0060] Figure 16 The activity of elastase as a function of inhibitor concentration is shown.

[0061] Figure 17 MDI-1002 demonstrates protection against acute lung injury.

[0062] Figure 18 MDI-1002 demonstrates protection against pulmonary fibrosis.

[0063] Figure 19 The results showed that MDI-1002 did not improve recovery after bleomycin treatment.

[0064] Figure 20 The inhaled MDI-1003 is shown to protect against acute lung injury.

[0065] Figure 21 MDI-1003 demonstrates better protection against pulmonary fibrosis than MDI-1001.

[0066] Figure 22 The results show that MDI-1003 improved recovery after bleomycin treatment.

[0067] Figure 23 The Fc fusion constructs of MDI-1002 and MDI-1004 are shown.

[0068] Figure 24 The Fc fusion expression of MDI-1002 and MDI-1004 is shown.

[0069] Figure 25 This demonstrates how Fc fusion improves PK.

[0070] Figure 26 The results show that mutations in LRP1 binding residues do indeed affect the inhibition of neutrophil elastase in the presence of DNA NETs, ​​thus demonstrating reduced interaction with the clearance receptor LRP1 and preservation of elastase activity in NETs.

[0071] definition

[0072] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. An Fc domain is associated with at least C... H 2. Structural Domains and C HThe human Fc domain of the 3-domain complex has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100% sequence identity). The Fc domain monomer includes a second and a third antibody constant domain (C). H 2 and C H 3). In some embodiments, the Fc domain monomers further include a hinge domain. The Fc domain does not include any portion of the immunoglobulin that can serve as an antigen recognition region, such as a variable domain or complementarity-determining region (CDR). In the wild-type Fc domain, the two Fc domain monomers are connected by two C-terminals. H Dimerization occurs through interactions between the constant domains of the antibody and through one or more disulfide bonds formed between the hinge domains of the two dimerizing Fc domain monomers. In some embodiments, the Fc domain can be mutated to lack effector function, typically a "dead Fc domain". In some embodiments, each of the Fc domain monomers in the Fc domain includes a C H 2. Amino acid substitutions in the antibody constant domain reduce the interaction or binding between the Fc domain and the Fcγ receptor. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. The Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. Furthermore, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain can also be a non-naturally occurring Fc domain, such as a recombinant Fc domain.

[0073] As used herein, the terms “fusion” or “linkage” are used to describe the combination or linkage of two or more elements, components, or protein domains (e.g., peptides or polypeptides) through a combination of chemical conjugation, recombination, and chemical bonds (e.g., amide bonds). For example, two tandem peptides may be fused in any other way, such as through chemical conjugation, chemical bonds, peptide linkers, or covalent linkage, to form a continuous protein structure, such as a polypeptide.

[0074] As used herein, the term "peptide" describes a single polymer in which monomers are amino acid residues covalently linked together by amide bonds. The term peptide is intended to encompass any amino acid sequence, whether naturally occurring, recombinant, or synthetically produced.

[0075] As used herein, the term "homodimer" refers to a molecular construct formed from two identical macromolecules, such as proteins or nucleic acids. Two identical monomers can form a homodimer through covalent or non-covalent bonds. For example, if two Fc domain monomers contain the same sequence, the Fc domain can be a homodimer of the two Fc domain monomers. In another example, the peptide described herein comprising a PAI-1 variant fused with an Fc domain monomer can form a homodimer through the interaction of two Fc domain monomers, which form the Fc domain in the homodimer.

[0076] As used herein, the term "heterodimer" refers to a molecular construct formed from two distinct macromolecules, such as proteins or nucleic acids. Two monomers can form a heterodimer via covalent or non-covalent bonds. For example, the polypeptide described herein comprising a PAI-1 variant fused with an Fc domain monomer can form a heterodimer through the interaction of two Fc domain monomers, each Fc domain monomer fused with a different PAI-1 variant forming an Fc domain in the heterodimer.

[0077] As used herein, the term "host cell" refers to a vector that includes the necessary cellular components (e.g., organelles) required for protein expression from its corresponding nucleic acid. Nucleic acids are typically contained in nucleic acid vectors that can be introduced into host cells using conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). Host cells can be prokaryotic cells, such as bacterial cells, or eukaryotic cells, such as mammalian cells (e.g., CHO cells or HEK293 cells).

[0078] As used herein, the term "therapeuticly effective amount" refers to the amount of the polypeptide, nucleic acid, or carrier of the present invention, or a pharmaceutical composition comprising the polypeptide, nucleic acid, or carrier of the present invention, that effectively achieves the desired therapeutic effect in treating patients suffering from diseases such as any condition characterized by abnormal NE activity and / or lack of A1AT activity (e.g., IPF, COPD, cystic fibrosis, emphysema, ARDS, ischemia-reperfusion, chronic pancreatitis, RA, DIC, UC, Crohn's disease, dermatology)). The term "therapeuticly effective amount" also refers to the amount of the polypeptide, nucleic acid, or carrier of the present invention, or a pharmaceutical composition comprising the polypeptide, nucleic acid, or carrier of the present invention, that effectively achieves the desired therapeutic effect in treating patients suffering from such conditions. In particular, a therapeutically effective amount of the polypeptide, nucleic acid, or carrier avoids adverse side effects.

[0079] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical or pharmaceutical preparation comprising an active ingredient as well as excipients and diluents to suit a method of administration. The pharmaceutical compositions of the present invention comprise pharmaceutically acceptable components compatible with peptides, nucleic acids, or carriers. The pharmaceutical compositions may be in tablet or capsule form for oral administration or in an aqueous form for intravenous or subcutaneous administration.

[0080] As used herein, the term "pharmaceuticalally acceptable support or excipient" refers to an excipient or diluent in a pharmaceutical composition. A pharmaceutically acceptable support must be compatible with the other components of the formulation and harmless to the recipient. In this invention, a pharmaceutically acceptable support or excipient must provide sufficient pharmaceutical stability for a polypeptide including a PAI-1 variant, a nucleic acid molecule encoding that polypeptide, or a carrier containing one or more such nucleic acid molecules. The properties of the support or excipient vary depending on the route of administration. For example, for intravenous administration, aqueous solution supports are generally used; for oral administration, solid supports are preferred.

[0081] As used herein, the term "treatment and / or prevention" refers to the treatment and / or prevention of a disease, such as any condition characterized by abnormal NE activity and / or a lack of A1AT activity (e.g., IPF, COPD, cystic fibrosis, emphysema), using the methods and compositions of the present invention. Typically, treatment of such a disease occurs after the subject has developed the disease and / or has been diagnosed with it. Prevention of such a disease refers to steps or procedures taken when the subject is at risk of developing the disease. The subject may exhibit signs or mild symptoms that are judged by a physician as indications or risk factors for developing the disease, or have a family history or genetic predisposition to developing the disease, but has not yet developed the disease.

[0082] As used herein, the term "subject" refers to a mammal, preferably a human. Mammals include, but are not limited to, humans, as well as domesticated and farm animals such as monkeys, mice, dogs, cats, horses, and cattle. Detailed Implementation

[0083] IPF is characterized by the formation of interstitial scar tissue, which can significantly limit lung function. There are approximately 130,000 IPF patients in the United States, with an estimated 30,000 to 40,000 new cases diagnosed annually (see Ley, B., and Collard, HR 2013. Epidemiology of idiopathic pulmonary fibrosis. Clin. Epidemiol. 5:483-492; Lynch, JP, III, et al., 2016. Idiopathic Pulmonary Fibrosis: Epidemiology, Clinical Features, Prognosis, and Management. Semin. Respir. Crit Care Med. 37:331-357). Life expectancy after an IPF diagnosis is typically three to five years (see Lederer, DJ and Martinez, FJ, NEJM 2018, 378:1811-1823). There is no effective treatment other than lung transplantation. The lack of therapies to halt the progression of IPF presents a significant challenge and represents a major unmet medical need. The two currently approved drugs, pirfenidone and nintedanib, have been shown to slow disease progression, but they do not stop it or restore lost lung function. Therefore, new therapies for IPF treatment are essential for disease management.

[0084] The present invention addresses this need by providing a composition comprising a mutant PA1-I polypeptide capable of inhibiting NE, and in particular inhibiting NE bound to NET.

[0085] This invention addresses this need by providing compositions comprising a mutant PA1-I polypeptide capable of inhibiting NE, and particularly inhibiting NE bound to NET. This document provides a PAI-1 variant capable of inhibiting NE activity while also having reduced binding ability to breccia and / or LRP1. Indeed, experiments conducted during the development of embodiments of this invention have demonstrated that such PAI-1 variants exhibit therapeutic efficacy for conditions associated with NE activity (e.g., IPF) by inhibiting the binding ability of PAI-1 amino acid residues (e.g., R101A and Q123K) responsible for this breccia binding.

[0086] Experiments conducted during the development of embodiments of the present invention utilized serine protease inhibitor mapping technology and identified mutant forms of PA1-1 (e.g., those with one or more of the following mutations in the wild-type human mature PA1-I amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V) as NE inhibitors capable of effectively inhibiting NE in the NET environment. This mutant form of PA1-I has been shown to irreversibly inhibit DNA-bound NE (a major component of NET), where the FDA-approved human plasma-derived A1AT trademark Aralast is invalid. In some embodiments, this mutant form of PA1-I is further associated with human IgG-Fc.

[0087] As part of the innate immune response, neutrophils are the most abundant leukocytes in peripheral blood and are at the forefront of infection defense. Neutrophils effectively clear microbial infections through phagocytosis and through both oxygen-dependent and oxygen-independent mechanisms. Recently, a novel neutrophil antimicrobial mechanism has been described, involving the release of NETs, ​​composed of DNA, histones, and antimicrobial peptides. This mutant form of PAI-1 represents the first therapeutic agent specifically targeting NE, the cause of significant lung function loss in IPF (see Obayashi, Y., et al., 1997 Chest 112:1338-1343; Schaaf, B., et al., 2000 Respiration 67:52-59; Takemasa, A., et al., 2012 Eur. Respir. J 40:1475-1482; Kristensen, JH, et al., 2015 BMC. Pulm. Med. 15:53) and other devastating lung diseases (see Gregory, AD, et al., 2015 J Leukoc. Biol. 98:143-152) (e.g. cystic fibrosis and chronic obstructive pulmonary disease (COPD)). The prospect of more effective treatments and potential disease reversal is highly attractive to patients with this rare orphan disease.

[0088] Clinical indications for this variant of PAI-1 include idiopathic pulmonary fibrosis (because NE-NET is involved in the etiology of fibrosis, including the differentiation of lung fibroblasts), COPD (see Grabcanovic-Musija, F., et al., 2015 Respir. Res. 16: 59), cystic fibrosis (representing a lung disease in which NE-NET is elevated, and once these diseases develop, current treatment options are limited), emphysema, ARDS, ischemia-reperfusion, chronic pancreatitis, RA, DIC, UC, Crohn's disease, and skin diseases.

[0089] Therefore, the present invention is characterized by peptides comprising a PAI-1 variant capable of inhibiting NE activity while exhibiting weakened binding to glassin and LRP1 by modifying amino acid residues responsible for glassin binding (e.g., R101A and Q123K) and / or amino acid residues responsible for LRP1 binding (e.g., K207, K88, and K80), resulting in improved pharmacokinetics (PK) and improved therapeutic efficacy for conditions associated with NE activity (e.g., IPF). In some embodiments, the peptides of the present invention comprise a PAI-1 variant fused to the N- or C-terminus of an Fc domain monomer or portion (e.g., for the purpose of improving PK). In some embodiments, the peptides of the present invention comprise a PAI-1 variant fused to the N- or C-terminus of an Fc domain monomer or portion. In some embodiments, the Fc domain monomer or portion enhances the stability of the peptide or improves the pharmacokinetics of the peptide. Peptides including PAI-1 variants fused to Fc domain monomers can also form dimers (e.g., homodimers or heterodimers) through interactions between the two Fc domain monomers. The PAI-1 variants described herein are capable of inhibiting NE activity, wherein NE is bound within NETs. The invention also includes methods for treating diseases and conditions involving aberrant NE activity and / or a lack of A1AT activity in subjects by administering the peptides comprising the PAI-1 variants described herein.

[0090] Elastase is a serine protease released by activated neutrophils, macrophages, and monocytes. During the inflammatory response, neutrophils are activated and release elastase, which leads to tissue destruction through proteolysis. In the lungs, elastase degrades elastic tissue and contributes to emphysema. Elastase is also a contributing factor to cystic fibrosis (CF) and acute respiratory distress syndrome (ARDS) in both adults and infants. Elastase is also associated with TNF-mediated inflammation (see Massague, J. et al., Annu. Rev. Biochem. 62:515-541 (1993)) and HIV infection (Bristow, C. Letal., International Immunol. 7:239-249 (1995)).

[0091] Compared to plasminogen activators, elastases have a broader spectrum of reactivity, and each of them preferentially acts on a precursor substrate to activate it.

[0092] The natural defense against elastase is a protein called α1-antitrypsin (α1AT) or α1-protease inhibitor (α1PI). Patients lacking α1AT are prone to emphysema, especially smokers. Furthermore, smoking triggers inflammation. In this α1AT deficiency, the enzyme is present (CRM). + However, its function is impaired. Furthermore, even in individuals with normal enzyme levels, smoking directly inactivates α1AT. Therefore, there is a high expectation for improved elastase inhibitors to prevent emphysema in susceptible subjects or reverse the pathophysiological processes leading to this disease and other related conditions.

[0093] The major PAIs belong to the serine protease inhibitor (PRI) gene superfamily, which includes many protease inhibitors in the blood as well as other proteins with unrelated or unknown functions (see Gettins, PGW, and Olson, ST (2016) Inhibitory serpins. New insights into their folding, polymerization, regulation and clearance. Biochem. J. 473, 2273–2293). PAIs share a common tertiary structure and evolved from a common prototype. PAIs regulate many processes, including coagulation, fibrinolysis, complement activation, ovulation, angiogenesis, inflammation, tumor formation, viral disease progression, and allergic reactions.

[0094] Serine protease inhibitors, acting as suicide inhibitors, react only once with their target protease to form sodium dodecyl sulfate (SDS)-stable complexes. These complexes can dissociate to produce free active enzymes along with cleavage inhibitors similar to those seen in α1AT crystal structures (see Gettins, PGW, and Olson, ST (2016) Inhibitory serpins. New insights into their folding, polymerization, regulation and clearance. Biochem. J. 473, 2273–2293).

[0095] PAI-1 is considered one of the major regulators of the PA system. It is a single-chain glycoprotein with a molecular weight of 50 kDa (see Van Mourik JA et al., J Biol Chem (1984) 259:14914-14921) and is the most potent known inhibitor of both single-chain and double-chain forms of tPA and uPA (see Lawrence D et al., Eur J Biochem (1989) 186:523-533). PAI-1 also inhibits plasmin and trypsin (see Hekman CM et al., Biochemistry (1988) 27:2911-2918) and also inhibits thrombin and activated protein C, although with much lower efficiency.

[0096] PAI-1 cDNA encodes a 402-amino acid protein, including a typical secretion signal sequence (see Ny et al., ibid.; Ginsburg et al., 1986, ibid.). Mature human PAI-1 isolated from cell cultures consists of two variants of approximately equal proportions of 381 and 379 amino acids. Figure 10 The following are provided: human wild-type PAI-1 nucleic acid sequence (SEQ ID NO:1); human wild-type PAI-1 amino acid sequence (SEQ ID NO:2); and human mature wild-type PAI-1 amino acid sequence (SEQ ID NO:3).

[0097] PAI-1 is a glycoprotein containing 15% to 20% carbohydrates with three potential N-linked glycosylation sites (Van Mourik JA et al., ibid.). Mature PAI-1 does not contain cysteine ​​residues, which facilitates efficient expression and isolation of recombinant PAI-1 from *E. coli*. While unglycosylated, PAI-1 produced in *E. coli* is functionally very similar to native PAI-1. Recombinant PAI-1 can be isolated from *E. coli* in its intrinsically active form (see below), in contrast to PAI-1 purified from mammalian cell cultures (Lawrence et al., 1989, ibid.; Hekman et al., 1988, ibid.).

[0098] PAI-1 exists in its active form as it is produced by cells and secreted into the culture medium, and accumulates in the medium over time in an inactive or latent form (see Hekman CM et al, J Biol Chem (1985) 260:11581-11587, Levin EG et al, Blood (1987) 70:1090-1098). The active form spontaneously converts to a latent form with a half-life of approximately 1 hour at 37°C (see Lawrence et al., ibid., Hekman et al., ibid.; Levin EG et al, 1987, ibid.).

[0099] The latent form can be converted to the active form by treatment with denaturing agents, negatively charged phospholipids, or Vn (see Lampers et al., ibid.; Hekman et al., ibid.; Wun TC et al., J Biol Chem (1989) 264:7862-7868). The latent PAI-1 infused in rabbits was reactivated in vivo via an unknown mechanism. The reversible interconversion between the active and latent structures (likely due to conformational changes) is a unique feature of PAI-1 compared to other serine protein inhibitors. The latent form appears to be more energy-efficient.

[0100] The three-dimensional structure of the latent active form of PAI-1 has been identified. In this structure, the entire N-terminal side of the reactive central ring is inserted as a central chain into the β-sheet A (see, Mottonen et al., ibid.), which explains the increased stability (see, Lawrence, D.A et al., Biochemistry 33:3643-3648 (1994)) and the lack of inhibitory activity.

[0101] The activities of two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator, are regulated by plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor (serine protease inhibitor) that regulates fibrinolysis and wound healing and is associated with thrombotic and fibrotic diseases. Serine protease inhibitors exert their inhibitory function on serine proteases through a unique mechanism involving cleavage of the reaction center loop of the serine protease inhibitor, inducing a conformational change in the serine protease inhibitor, thereby leading to protease inhibition (see Gettins, PGW, and Olson, ST (2016) Biochem. J. 473, 2273–2293). Once the serine protease inhibitor complexes with the protease, the complex is rapidly removed from the hepatic circulation by binding to LDL receptor-associated protein 1 (LRP1) (see Kounnas, MZ, Church, FC, Argraves, WS, and Strickland, DK (1996) J. Biol. Chem. 271, 6523–6529).

[0102] LRP1 was initially identified as a liver receptor responsible for removing α2-macroglobulin protease complexes (see Ashcom, JD, et al., (1990) J. Cell Biol. 110, 1041–1048; Moestrup, SK, and Gliemann, J. (1989) J. Biol. Chem. 264, 15574–15577) and as a receptor for chylomicron residual lipoprotein particles (see Rohlmann, A., et al., (1998) J. Clin. Invest. 101, 689–695). In addition to its endocytic function, LRP1 also regulates various signaling pathways (see Gonias, SL (2018) Arter. Thromb Vasc Biol. 38, 2548–2549; Strickland, DK, et al., (2014) Thromb. Vasc. Biol. 34, 487–498). The extracellular domain of this large receptor is composed of modules consisting of clusters of LDLa repeats, EGF-like repeats, and β-propeller domains. Efficient delivery of newly synthesized LRP1 to the cell surface requires the participation of endoplasmic reticulum-resident molecular chaperones known as receptor-associated proteins (RAPs) (see Strickland, DK, et al., (1991) J. Biol. Chem. 266, 13364–13369; Willnow, TE, et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4537–41; Bu, G., et al., (1995) EMBO J. 14, 2269–80).

[0103] The fact that LRP1 recognizes many structurally unrelated ligands with relatively high affinity has raised questions about the nature of ligand / receptor interactions. Insights into how this might occur come from: the recognition that K256 and K270 are essential for the binding of LRP1 to the third domain of RAP (see, Migliorini, MM, et al., (2003) J. Biol. Chem. 278, 17986–17992) and the crystal structure of the third domain of RAP in a complex with two LDLa repeats from the LDL receptor (see, Fisher, C., et al., (2006) Mol. Cell. 22, 277–283). These studies suggest that the ε-amino groups of K256 and K270 on RAP form salt bridges with the carboxylates of aspartic residues within the LDLa repeats, thereby creating an acidic pocket on the receptor. To date, a variety of ligands, including α2-macroglobulin (α2M) (see Arandjelovic, S., Hall, BD, and Gonias, SL (2005) Arch. Biochem. Biophys. 438, 29–35) and coagulation factor VIII (see van den Biggelaar, et al., (2015) J. Biol. Chem. 290, 16463–76; Young, PA, et al., (2016) J. Biol. Chem. 291, 26035–26044), interact with LRP1 via interactions involving key lysine residues.

[0104] Although lysine residues appear to contribute to the interaction between PAI-1 and LRP1 (see, Horn, I., et al., (1998) Thromb. Haemost. 1, 20–22; Rodenburg, KW, et al., (1998) Biochem. J. 329, 55–63; Gettins, PGW, and Dolmer, K. (2016) J. Biol. Chem. 291, 800–812), studies investigating the interaction between PAI-1 and LRP1 have yielded conflicting data. First, there is a problem regarding the relative affinity of PAI-1 to proteases: the PAI-1 complex with LRP1 presents an issue. Most studies have shown that only PAI-1 complexed with proteases can bind to LRP1 with high affinity (see Horn, I., et al., (1998) Thromb. Haemost. 1, 20–22; Stefansson, S. (1998) J. Biol. Chem. 273, 6358–6366; Nykjaer, A., et al., (1992) J. Biol. Chem. 267, 14543–14546; Horn, IR, et al., (1997) J. Biol. Chem. 272, 13608–13613). In contrast, other studies (see Gettins, PGW, and Dolmer, K. (2016) J. Biol. Chem. 291, 800–812; Jensen, JK, et al. (2009) J. Biol. Chem. 284, 17989–17997) reported that PAI-1 alone binds to fragments from LRP1 with high affinity. Secondly, based on the observation that the protease:PAI-1 complex binds to LRP1 with a higher affinity than PAI-1 alone, some have proposed that the formation of the protease complex with PAI-1 exposes a hidden epitope on PAI-1 that is recognized by LRP1 (see Horn, I., et al. (1998) Thromb. Haemost. 1, 20–22; Stefansson, S. (1998) J. Biol. Chem. 273, 6358–6366). Conversely, others have suggested that the protease itself may interact with LRP1 and contribute to high-affinity interactions (see Skeldal, S., et al., (2006) FEBS J.273, 5143–5159).Finally, although many studies have reported changes in the affinity of PAI-1 for LRP1 when various basic residues are mutated to alanine (see, Horn, I., et al., (1998) Thromb. Haemost. 1, 20–22; Stefansson, S., (1998) J. Biol. Chem. 273, 6358–6366; Skeldal, S., et al., (2006) FEBS J. 273, 5143–5159), there seems to be little consensus on the binding site being lysine residues.

[0105] Experiments conducted during the development of embodiments of the present invention elucidated the relative binding affinity of PAI-1 and the protease:PAI-1 complex to LRP1. Furthermore, experiments were performed to determine whether the binding of the protease:PAI-1 complex to LRP1 is primarily attributable to determinants on PAI-1. Additionally, experiments were conducted to identify specific amino acid residues in the PAI-1 complex with the target protease (urokinase-type plasminogen activator (uPA)) and in free PAI-1 involved in its binding to LRP1. Indeed, mutation analysis revealed an overlap between the binding site of LRP1 and the binding site of the small molecule inhibitor CDE-096 of PAI-1, and that K207 plays a crucial role in the interaction between PAI-1 and LRP1, as well as the interactions of K207, K88, and K80 within the uPA:PAI-1 complex and LRP1.

[0106] Experiments conducted during the development of embodiments of the present invention utilized serine protease inhibitor mapping technology and identified mutant forms of PA1-1 (e.g., those with one or more of the following mutations in the wild-type human mature PA1-I amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V) as NE inhibitors capable of effectively inhibiting NE in the NET environment. This mutant form of PA1-I has been shown to irreversibly inhibit DNA-bound NE (a major component of NET), where the FDA-approved human plasma-derived A1AT trademark Aralast is invalid. In some embodiments, this mutant form of PA1-I is further associated with human IgG-Fc.

[0107] Therefore, the present invention is characterized by peptides comprising PAI-1 variants. In some embodiments, the peptides of the present invention comprise PAI-1 variants fused to the N- or C-terminus of an Fc domain monomer or portion. In some embodiments, the peptides of the present invention comprise PAI-1 variants fused to the N- or C-terminus of an Fc domain monomer or portion. In some embodiments, the Fc domain monomer or portion enhances the stability of the peptide or improves the pharmacokinetics of the peptide.

[0108] Such moieties can be fused or linked via amino acids or other covalent bonds, which can improve the stability of the peptide. Peptides including PAI-1 variants fused to Fc domain monomers can also form dimers (e.g., homodimers or heterodimers) through interactions between the two Fc domain monomers. In some embodiments, the peptides described herein are linked to Fc domain monomers, which are fused to the peptide via linkers. In some embodiments, the linkers are amino acid spacers.

[0109] The peptides of the present invention can be used to inhibit NE activity and to inhibit NE activity bound to NET. Additionally, the peptides of the present invention can be used to treat subjects suffering from conditions characterized by abnormal NE activity (e.g., IPF). Furthermore, the peptides of the present invention can be used to prevent subjects from developing conditions characterized by abnormal NE activity (e.g., IPF). Moreover, the peptides of the present invention can also be used to influence NE activity in subjects at risk of developing or suffering from diseases or conditions involving abnormal NE activity.

[0110] In some embodiments, the invention is characterized by a polypeptide comprising a PAI-1 variant having one or more of the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V. In some embodiments, the PAI-1 variant comprises mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V, as shown in SEQ ID NO:5. In some embodiments, the PAI-1 variant includes the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): R101A and Q123K. In some embodiments, the PAI-1 variant includes the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, and R346V.

[0111] In some embodiments, the present invention is characterized by comprising a peptide comprising a PAI-1 variant linked to an Fc domain monomer or portion thereof, the variant having one or more of the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V. In some embodiments, the PAI-1 variant linked to an Fc domain monomer or portion thereof comprises the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V. In some embodiments, the PAI-1 variant linked to the Fc domain monomer or part thereof includes the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): R101A and Q123K. In some embodiments, the PAI-1 variant linked to the Fc domain monomer or part thereof includes the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, R346V (SEQ ID NO:7).

[0112] In some embodiments, the peptides described herein are capable of inhibiting NE, and in particular, inhibiting NE bound to NET.

[0113] In some embodiments, the invention is characterized by a nucleic acid molecule encoding the polypeptide described herein (e.g., a polypeptide comprising a PAI-1 variant having one or more of the following mutations in the wild-type mature human PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V). In another aspect, the invention is further characterized by a vector comprising the nucleic acid molecule described herein.

[0114] In another aspect, the present invention is characterized by a host cell expressing the polypeptide described herein, wherein the host cell comprises the nucleic acid molecule or vector described in the preceding two aspects, wherein the nucleic acid molecule or vector is expressed in the host cell.

[0115] In another aspect, the present invention is characterized by a method for preparing the polypeptide described herein, wherein the method comprises: a) providing a host cell comprising the nucleic acid molecule or vector described herein, and b) expressing the nucleic acid molecule or vector in the host cell under conditions that allow for polypeptide formation.

[0116] In another aspect, the present invention is characterized by a pharmaceutical composition comprising the polypeptide, nucleic acid molecule, or carrier described herein, and one or more pharmaceutically acceptable loads or excipients. In some embodiments of the pharmaceutical composition, the polypeptide, nucleic acid molecule, or carrier is in a therapeutically effective amount.

[0117] Figure 11 The mature variant PAI-1 nucleic acid sequence (SEQ ID NO:4) is provided, which encodes a polypeptide having the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V; and the mature variant PAI-1 amino acid sequence (SEQ ID NO:5) is provided, which has the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V.

[0118] Figure 12 The mature variant PAI-1 / Fc amino acid sequence (SEQ ID NO:6) is provided, which encodes a polypeptide with the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, R346V; and the mature variant PAI-1 / Fc amino acid sequence (SEQ ID NO:7) is provided, which has the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, R346V.

[0119] In some embodiments, the peptides described herein may include a PAI-1 variant fused to an Fc domain monomer or a fragment of an immunoglobulin to increase the peptide's serum half-life. Peptides including a PAI-1 variant fused to an Fc domain monomer may also form a dimer (e.g., a homodimer or heterodimer) through an interaction between two Fc domain monomers, in which the two Fc domain monomers form the Fc domain. As is conventionally known in the art, the Fc domain is a protein structure found at the C-terminus of immunoglobulins. An Fc domain comprises two Fc domain monomers that interact through a C-terminus... H 3. Dimerization occurs due to interactions between the constant domains of the antibody. The wild-type Fc domain forms the minimal structure that binds to the Fc receptor, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV. In some embodiments, the Fc domain can be mutated to lack effector function, typically a "dead Fc domain." For example, the Fc domain may include specific amino acid substitutions known to minimize the interaction between the Fc domain and the Fcγ receptor.

[0120] The polypeptides of this invention can be produced by host cells. A host cell is a carrier containing the necessary cellular components (e.g., organelles) required for the expression of the polypeptides and fusion polypeptides described herein from their respective nucleic acids. Nucleic acids can be included in nucleic acid carriers that can be introduced into host cells using conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). The choice of nucleic acid carrier depends in part on the host cell to be used. Generally, preferred host cells are of eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.

[0121] Nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present invention can be prepared using a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding the polypeptides of the present invention can be obtained using standard techniques such as gene synthesis. Alternatively, nucleic acid molecules encoding wild-type PAI-1 can be obtained using standard techniques in the art such as QuikChange. TM Mutation is performed to induce mutations that include specific amino acid substitutions. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR technology.

[0122] The nucleic acid sequence encoding the polypeptide of the present invention can be inserted into a vector capable of replicating and expressing nucleic acid molecules in prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purposes of the present invention. Each vector may include various components that can be tuned and optimized to be compatible with a specific host cell. For example, vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding the target protein, and a transcription termination sequence.

[0123] In some embodiments, mammalian cells can be used as host cells of the present invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2 / O, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, *E. coli* cells can also be used as host cells of the present invention. Examples of *E. coli* strains include, but are not limited to, *E. coli* 294 (… 31,446), Escherichia coli λ1776 ( 31,537, Escherichia coli BL21(DE3)( BAA-1025) and Escherichia coli RV308 ( 31,608). Different host cells possess characteristics and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. Appropriate cell lines or host systems can be selected to ensure proper modification and processing of the expressed peptide. The aforementioned expression vectors can be introduced into suitable host cells using conventional techniques in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection). Once the vector is introduced into the host cells for protein production, the host cells are cultured in a conventional nutrient medium modified to induce promoters, select transformants, or amplify genes encoding desired sequences. Methods for expressing therapeutic proteins are known in the art, see, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2012.

[0124] The host cells used to produce the peptides of the present invention can be grown in culture media known in the art and suitable for culturing the selected host cells. Examples of suitable culture media for mammalian host cells include essential medium (MEM), Dulbecco modified Eagle medium (DMEM), and Expi 293. TM Expression medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable culture media for bacterial host cells include Luria broth (LB) with necessary supplements, such as selectants, for example, ampicillin. Host cells are cultured at suitable temperatures (e.g., from about 20°C to about 39°C, for example, from 25°C to about 37°C, preferably 37°C) and CO2 levels (e.g., 5% to 10%). The pH of the culture medium is typically from about 6.8 to 7.4, for example, 7.0, depending primarily on the host organism. If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for promoter activation.

[0125] In some embodiments, depending on the expression vector and host cell used, the expressed protein may be secreted from the host cell (e.g., mammalian host cell) into the cell culture medium. Protein recovery may involve filtering the cell culture medium to remove cell debris. The protein may be further purified. The peptides of the present invention can be purified by any method known in the field of protein purification, such as chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. For example, the protein can be separated and purified by appropriately selecting and combining affinity columns such as protein A columns (e.g., POROS protein A chromatography) with chromatographic columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting out, and dialysis procedures.

[0126] In another implementation, host cells can be disrupted (e.g., by osmotic shock, sonication, or lysis) to recover the expressed protein. Once the cells are disrupted, cell debris can be removed by centrifugation or filtration. In some cases, peptides can be conjugated with labeled sequences such as peptides to facilitate purification. An example of a labeled amino acid sequence is a six-histidine peptide (His-tag), which binds to a nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags that can be used for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from influenza hemagglutinin protein (see Wilson et al., Cell 37:767, 1984).

[0127] Alternatively, the polypeptides of the present invention can be produced by the cells of a subject (e.g., a human), for example, in the case of gene therapy, by administering a vector (such as a viral vector (e.g., a retroviral vector, adenovirus vector, poxvirus vector (e.g., vaccinia virus vector, such as modified Ankara vaccinia virus (MVA)), adeno-associated virus vector, and alphavirus vector)) containing a nucleic acid molecule encoding the polypeptide of the present invention. Once the vector enters the subject's cells (e.g., through transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), it will promote the expression of the polypeptide, which is then secreted from the cells. If the treatment of the disease or patient is the desired outcome, further action may not be necessary. If it is desired to collect the protein, blood can be collected from the subject and the protein can be purified from the blood by methods known in the art.

[0128] The present invention is characterized by pharmaceutical compositions comprising the polypeptides described herein (e.g., polypeptides comprising PAI-1 variants (e.g., PAI-1 variants having one or more of the following mutations within wild-type PAI-1 (SEQ ID NO: 1): K69A, K80A, K88A, I91L, R101A, K122A, Q123K, K176A, K207A, K263A, V343A, and R346V). In some embodiments, the pharmaceutical compositions of the present invention comprise a polypeptide containing a PAI-1 variant having a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) as a therapeutic protein. In some embodiments, the pharmaceutical compositions of the present invention... The pharmaceutical composition comprises a polypeptide containing a PAI-1 variant fused to a portion (e.g., an Fc domain monomer or dimer thereof, a wild-type Fc domain, or an Fc domain having an amino acid substitution (e.g., one or more substitutions that reduce dimerization)) as a therapeutic protein. In some embodiments, the pharmaceutical composition of the present invention comprises a polypeptide containing a PAI-1 variant fused to a first portion (e.g., an Fc domain monomer or dimer thereof, a wild-type Fc domain, or an Fc domain having an amino acid substitution (e.g., one or more substitutions that reduce dimerization)).

[0129] In some embodiments, the pharmaceutical compositions of the present invention comprising the polypeptides of the present invention may be used in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions in a therapy. In addition to a therapeutically effective amount of the polypeptide, the pharmaceutical composition may include one or more pharmaceutically acceptable loads or excipients, which may be formulated by methods known to those skilled in the art. In some embodiments, the pharmaceutical compositions of the present invention comprise a nucleic acid molecule (DNA or RNA, such as mRNA) encoding the polypeptide of the present invention, or a carrier comprising such a nucleic acid molecule.

[0130] The acceptable loading agents and excipients in the pharmaceutical composition are non-toxic to the recipient at the dosage and concentration used. Acceptable loading agents and excipients may include buffers such as phosphates, citrates, HEPES, and TAEs; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethyldiammonium chloride, octadecyl dimethyl benzyl ammonium chloride, resorcinol, and benzalkonium chloride; proteins such as human serum albumin, gelatin, dextran, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, histidine, and lysine; and carbohydrates such as glucose, mannose, sucrose, and sorbitol. The pharmaceutical compositions of the present invention can be administered parenterally in the form of injectable formulations. Sterile solutions or any pharmaceutically acceptable liquids can be used as carriers to formulate injectable pharmaceutical compositions. Pharmaceutically acceptable carriers include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco modified Eagle medium (DMEM), α-modified Eagle medium (α-MEM), F-12 medium). The preparation method is known in the art; see, for example, Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd ed.) Taylor & Francis Group, CRC Press (2015).

[0131] The pharmaceutical compositions of the present invention can be prepared into microcapsules, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules. The pharmaceutical compositions of the present invention can also be prepared in other drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. Such a technical description is found in Remington: The Science and Practice of Pharmacy 22. th In edition (2012), pharmaceutical compositions intended for internal administration must be sterile. This can be easily accomplished through filtration using a sterile filter membrane.

[0132] The pharmaceutical compositions of the present invention can also be prepared as sustained-release formulations. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers comprising the peptides of the present invention. Examples of sustained-release matrices include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and L-glutamic acid ethyl ester, non-degradable ethylene-vinyl acetate, and degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT. TMAnd poly-D-(-)-3-hydroxybutyric acid. Some sustained-release formulations are able to release molecules over several months (e.g., one to six months), while other formulations release the pharmaceutical compositions of the present invention over a shorter period of time (e.g., days to weeks).

[0133] As needed, the pharmaceutical composition can be formulated in unit dose form. The amount of the active ingredient (e.g., the polypeptide of the present invention) included in the pharmaceutical formulation enables the provision of a suitable dose within a specified range (e.g., a dose in the range of 0.01-100 mg / kg body weight).

[0134] Pharmaceutical compositions for gene therapy may be in an acceptable diluent or may include a sustained-release matrix incorporating a gene delivery vector. If hydrodynamic injection is used as the delivery method, the pharmaceutical composition comprising a nucleic acid molecule encoding the polypeptide described herein or a vector (e.g., a viral vector) containing such a nucleic acid molecule is rapidly delivered intravenously in a large fluid volume. Vectors that can be used as in vivo gene delivery vectors include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, such as modified Ankara vaccinia), adeno-associated virus vectors, and alphavirus vectors.

[0135] Pharmaceutical compositions comprising the polypeptides of the present invention as therapeutic proteins can be formulated for, for example, intravenous, parenteral, subcutaneous, intramuscular, intra-arterial, intrathecal, or intraperitoneal administration. The pharmaceutical compositions can also be formulated for or administered via oral, nasal, spray, aerosol, rectal, or vaginal administration. For injectable formulations, a variety of effective drug delivery systems are known in the art. See, for example, ASHP Handbook on Injectable Drugs, Toissel, 18th ed. (2014).

[0136] In some embodiments, pharmaceutical compositions comprising nucleic acid molecules encoding the polypeptides of the present invention or vectors containing such nucleic acid molecules can be administered via gene delivery. Methods of gene delivery are well known to those skilled in the art. Vectors that can be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenovirus vectors, poxvirus vectors (e.g., vaccinia virus vectors, such as modified Ankara vaccinia virus (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, mRNA molecules encoding the polypeptides of the present invention can be administered directly to a subject.

[0137] In some embodiments of the invention, nucleic acid molecules encoding the polypeptides described herein, or vectors containing such nucleic acid molecules, can be administered using a hydrodynamic injection platform. In hydrodynamic injection methods, nucleic acid molecules encoding the polypeptides described herein are placed under the control of a strong promoter in an engineered plasmid (e.g., a viral plasmid). The plasmid is typically delivered rapidly intravenously in a large fluid volume. Hydrodynamic injection uses controlled hydrodynamic pressure in the vein to enhance cell permeability, thereby rapidly injecting a large fluid volume and the resulting increased pressure causes fluid and plasmid to extravasate from the vein. Expression of the nucleic acid molecule is primarily driven by the liver. In mice, hydrodynamic injection is typically performed by injecting the plasmid into the tail vein. In some embodiments, mRNA molecules encoding the polypeptides described herein can be administered using hydrodynamic injection.

[0138] The dosage of the pharmaceutical composition of the present invention depends on factors including: route of administration, the disease to be treated, and the physical characteristics of the subject, such as age, weight, and general health status. The pharmaceutical composition of the present invention may include a dosage range of the polypeptide of the present invention from 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg), and in more specific embodiments, from about 0.1 to about 30 mg / kg, and in even more specific embodiments, from about 0.3 to about 30 mg / kg. The dosage may be adjusted by a physician based on conventional factors such as the severity of the disease and different parameters of the subject.

[0139] The pharmaceutical composition is administered in a dosage form compatible and therapeutically effective amount to achieve symptom improvement or relief. The pharmaceutical composition is administered in various dosage forms, such as intravenous, subcutaneous, and oral dosage forms (e.g., ingestible solutions, drug-release capsules). Typically, the dose of the therapeutic protein is 0.1-100 mg / kg, for example, 1-50 mg / kg. Pharmaceutical compositions comprising the polypeptides of the present invention can be administered to subjects in need, for example, once or multiple times (e.g., 1-10 times or more) daily, weekly, bi-weekly, monthly, bi-monthly, quarterly, semi-annually, annually, or as medically necessary. In some embodiments, pharmaceutical compositions comprising the polypeptides of the present invention can be administered to subjects in need weekly, bi-weekly, monthly, bi-monthly, or quarterly. Dosage can be provided in single-dose or multiple-dose regimens. The time between dosings can decrease as medical conditions improve or increase as the patient's health declines.

[0140] This invention is based on the discovery that replacing one or more specific amino acids in human PAI-1 enables it to inhibit NE activity and the NE activity in which NE binds to NET. These PAI-1 variant properties provide useful therapeutic agents that can be used to treat diseases characterized by abnormal NE activity and / or lack of A1AT activity.

[0141] In another aspect, the present invention is characterized by a method for inhibiting NE activity in a subject with this need. In yet another aspect, the present invention is characterized by a method for inhibiting NE activity bound to NET in a subject with this need. This method comprises administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule, or carrier described herein, or a pharmaceutical composition described herein.

[0142] In some embodiments of the method for inhibiting NE activity in a subject or NE activity bound within a NET, the subject has IPF and / or a condition characterized by abnormal NE activity (e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), emphysema). In some embodiments of the method for inhibiting NE activity in a subject or NE activity bound within a NET, the subject has A1AT activity and / or expression defects.

[0143] In another aspect, the present invention is characterized by a method of treating a subject with IPF by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0144] In another aspect, the present invention is characterized by a method of treating a subject with cystic fibrosis by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0145] In another aspect, the present invention is characterized by a method of treating a subject with COPD by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0146] In another aspect, the present invention is characterized by a method of treating a subject with emphysema by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0147] In another aspect, the present invention is characterized by a method of treating a subject suffering from acute respiratory distress syndrome (ARDS) by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0148] In another aspect, the present invention is characterized by a method of treating a subject with ischemia-reperfusion injury by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0149] In another aspect, the present invention is characterized by a method of treating a subject suffering from ethanol-induced chronic pancreatitis by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0150] In another aspect, the present invention is characterized by a method for treating a subject with rheumatoid arthritis (RA) by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0151] In another aspect, the present invention is characterized by a method of treating a subject suffering from disseminated intravascular coagulation (DIC) by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0152] In another aspect, the present invention is characterized by a method of treating a subject with ulcerative colitis (UC) by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0153] In another aspect, the present invention is characterized by a method of treating a subject with Crohn's disease by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0154] In another aspect, the present invention is characterized by a method of treating a subject with a skin disease having neutrophil pathology by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier or the pharmaceutical composition described herein.

[0155] In another aspect, the present invention is characterized by a method of treating a subject with A1AT activity and / or expression defects by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0156] In another aspect, the present invention is characterized by a method of treating a subject suffering from any condition characterized by abnormal NE activity and / or expression by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0157] In another aspect, the present invention is characterized by a method of treating a subject suffering from any condition characterized by a lack of A1AT activity and / or expression by administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or carrier described herein or the pharmaceutical composition described herein.

[0158] In any of the above embodiments, the subject has or is at risk of developing a condition characterized by abnormal NE activity (e.g., IPF, COPD, cystic fibrosis, emphysema).

[0159] In any of the above embodiments, the subject has or is at risk of developing a condition characterized by a lack of A1AT activity and / or expression.

[0160] Those skilled in the art will readily recognize that the foregoing represents only a detailed description of certain preferred embodiments of the invention. Various modifications and alterations to the above compositions and methods can be readily implemented using the expertise available in the art and are within the scope of the invention.

[0161] experiment

[0162] Example I.

[0163] This example demonstrates that the LMWuPA:PAI-1 complex binds to LRP1 with a higher affinity than PAI-1 alone.

[0164] To resolve the conflicting information in the literature regarding the relative affinity of PAI-1 and the protease:PAI-1 complex for LRP1, our initial experiments compared the binding of free PAI-1 and PAI-1 complexed with LMWuPA to LRP1. Notably, LMWuPA itself does not bind to LRP1. Because wild-type PAI-1 is relatively unstable and rapidly converts to its latent form, experiments in this study and all subsequent studies (unless otherwise stated) used the I91L mutant of PAI-1, which extended the stability of PAI-1 from a half-life of 2.0 h to 18.4 h (see Berkenpas, MB, Lawrence, DA, and Ginsburg, D. (1995) EMBO J. 14, 2969–77). Experiments were performed to form the I91L PAI-1 complex with LMWuPA, and its binding to LRP1 was compared using equilibrium SPR measurements. Data ( Figure 1 A) The LMWuPA:PAI-1 complex binds to LRP1 at a nearly two-order-of-magnitude tighter rate than PAI-1 alone (KL of LMWuPA:PAI-1). D=0.9±0.2nM, while K of PAI-1 D =74±13nM).

[0165] Example II.

[0166] This example demonstrates that the lysine residues on PAI-1 are crucial for the binding of both PAI-1 and the LMWuPA:PAI-1 complex to LRP1.

[0167] To determine the contribution of lysine residues to the interaction between PAI-1 and LRP1, these residues were chemically modified with sulfonyl-NHS-acetate, which forms a stable covalent amide bond with the primary amine of the lysine residue. This modification did not prevent PAI-1 from forming a stable complex with LMWuPA. Figure 1 B). However, it is worth noting that this modification prevents free PAI-1 (B). Figure 1 C) and the complex of LMWuPA and modified AI-1 both bind to LRP1 ( Figure 1 D). These results indicate that lysine residues on PAI-1 facilitate the binding of both PAI-1 and the LMWuPA:PA1-1 complex to LRP1.

[0168] Example III.

[0169] This example demonstrates that two charged residues are involved in the binding of PAI-1 to LRP1.

[0170] Next, experiments were conducted to examine the effect of ionic strength on the binding of PAI-1 to LRP1. The results of these experiments indicate that the binding of PAI-1 to LRP1 depends on the ionic strength ( Figure 2 A). Figure 2 B shows the data plotted in the form of a Debye-Hückel graph, where log 10 K D Plotting relative to ionic strength. These results indicate that two ionic interactions are involved in the binding of PAI-1 to LRP1, as shown by the slope (slope = 1.5 ± 0.1). This is consistent with the standard model of ligand binding to LDL receptor family members, in which two (or more) ε-amino groups of a specific lysine residue on the ligand form a salt bridge with the carboxylate of an aspartic residue within the LDL repeat (see Fisher, C., et al., (2006) Mol. Cell. 22, 277–283).

[0171] Example IV.

[0172] This example demonstrates that kinetic analysis supports a bivalent model combining PAI-1 and LRP1.

[0173] Figure 2 The data indicate that at least two charged residues are involved in the interaction between PAI-1 and LRP1, suggesting the possibility of a bivalent binding model, in which high-affinity binding is mediated by an affinity effect of the interaction between two regions on PAI-1 (each containing charged residues) and two repeating LDLa residues on LRP1. Figure 3 A). A similar model has been proposed for the binding of FVIII to LRP1 (16). To test this model, kinetic measurements were performed to examine the binding of I91L PAI-1 to LRP1 using surface plasmon resonance experiments. To gain a deeper understanding of the underlying mechanism, the kinetics of PAI-1 dissociation from LRP1 were initially compared at various ligand concentrations (A). Figure 3 B). These results indicate that the dissociation kinetics occur in two phases: a rapid phase followed by a much slower phase. Furthermore, as expected for the bivalent model, the dissociation kinetics are independent of ligand concentration. When both binding and dissociation kinetics are fitted to the global bivalent model simultaneously, the dissociation rate constant determined from the fitting of the experimental data is used as an initial estimate of the dissociation phase. This fitting shows that the bivalent binding model describes the experimental data well (B). Figure 3 C). The best-fit kinetic data (Table I) show that PAI-1 rapidly binds to LRP1 to form complex I, and then transforms into complex II with a half-life of 97 s. Importantly, the equilibrium binding constant K derived from the kinetic analysis... D The value (65±6 nM) is close to the K value of 56±4 nM determined by equilibrium analysis of SPR data. D To determine whether there are any differences between the binding of I91L PAI-1 and wild-type PAI-1 to LRP1, we also investigated the detailed binding kinetics of wild-type PAI-1 to LRP1. Kinetic and equilibrium binding data are summarized in Table I, and the kinetic constants and K0 of wild-type PAI-1 are revealed. D The values ​​are similar to those of the stable I91L mutant.

[0174] a Table I. Kinetics and equilibrium constants of the combination of WT PAI-1 and I91L PAI-1 with LRP1

[0175]

[0176]

[0177] a The kinetic constants are obtained by fitting the data to a bivalent model.

[0178] b Calculate the equilibrium binding constant K using the following equation.A :K A =(k a1 / k d1 )*(1+(k a2 / k d2 ), and K D Calculated as: K D =1 / K A

[0179] c The calculation is based on equilibrium SPR measurements, where Req is determined by fitting the combined data to a pseudo-first-order process.

[0180] d Three independent experiments were conducted, and the values ​​shown are the mean ± SE.

[0181] e Six independent experiments were conducted, and the values ​​shown are the mean ± SE.

[0182] The ligand-binding region of LRP1 is mainly located in repeating clusters of LDLa, referred to as clusters I, II, III, and IV. Figure 4 A). Among these clusters, most ligands bind to clusters II, III, or IV. Therefore, experiments were also conducted to examine the binding of PAI-1 to clusters II, III, and IV. Initial experiments showed that I91L PAI-1 interacts with clusters II and IV with similar affinities, but with a much weaker affinity for cluster III. Next, detailed experiments were conducted using cluster IV, the major ligand-binding region of LRP1. Figure 4 B confirmed that the dissociation of PAI-1 from cluster IV also occurs in two stages and is independent of PAI-1 concentration. Comparison with experimental data fitted to the model indicates that this binding conforms to the bivalent binding model. Figure 4 C). The best-fit parameters are summarized in Table I, which reveals the K values ​​derived from the kinetic data. D The value is 55±5 nM, close to the K value of 49±18 nM estimated by equilibrium analysis of SPR data. D Therefore, these results indicate that the binding of I91LPAI-1 to cluster IV is similar to its binding to full-length LRP1.

[0183] Example V.

[0184] This example demonstrates the crucial role of K207 in the combination of PAI-1 and LRP1.

[0185] Since chemical modifications of PAI-1 have revealed the crucial role of lysine residues in its interaction with LRP1, investigations were initiated to identify specific lysine residues that contribute to the binding of PAI-1 to LRP1. To further understand regions on PAI-1 that may be important for its interaction with LRP1, the potential of CDE-096 to block the binding of the HMWuPA:PAI-1 complex to LRP1 was investigated. CDE-096 is a small molecule inhibitor that reversibly binds to PAI-1 and inhibits the interaction of PAI-1 with the protease via an allosteric mechanism (see, Li, S.-H., et al., (2013) Proc. Natl. Acad. Sci. USA 110, E4941-9). CDE-096 also binds to the uPA:PAI-1 complex. When CDE-099 was added to the HMWuPA:PAI-1 complex, a dose-dependent inhibition of HMWuPA:PAI-1 binding to LRP1 was observed (…). Figure 5 A). By replotting the initial slope of the binding curve relative to the CDE-096 concentration ( Figure 5 B) The 70nm IC was determined. 50 .

[0186] Structural studies have shown that K207 and K263 facilitate the binding of CDE-096 to PAI-1 (see Li, S.-H., et al., (2013) Proc. Natl. Acad. Sci. USA 110, E4941-9). Therefore, experiments were conducted on these PAI-1 mutants included in the analysis, along with K69, K80, and K88, which had previously been identified as crucial for the binding of PAI-1 to LDLa repeats from cluster II (see Gettins, PGW, and Dolmer, K. (2016) J. Biol. Chem. 291, 800–812). In these studies, K207 and K263 were determined by SPR equilibrium measurements. D The values ​​and data are summarized in Table II. The data show that the K207 mutation to alanine has the greatest impact on the binding of PAI-1 to LRP1, reducing affinity by 1 / 19. Additionally, mutations to alanine in K69, K80, and K88 result in reductions in affinity to LRP1 of 1 / 7, 1 / 7, and 1 / 9, respectively. Interestingly, PAI-1 molecules with mutations in both K80 and K207, or mutants where K80, K207, and K88 are all converted to alanine, do not significantly reduce binding affinity compared to the K207A mutant alone, and instead lead to reductions in affinity for K69, K80, and K88. D Increased to 20 times and 21 times.

[0187] The data in Table II also show the surface integrals of specific side chains based on the three-dimensional structure of PAI-1. The accessible surface integral of a side chain group is the solvent-accessible area in the protein divided by the accessible surface area calculated for that residue and the extended Gly-Xaa-Gly tripeptide (see Willard, L., et al., (2003) Nucleic Acids Res. 31, 3316–3319), where values ​​close to 1 are fully accessible and values ​​close to 0 are hidden. The R76E PAI-1 mutant lacks LRP1 binding (see Stefansson, S. (1998) J. Biol. Chem. 273, 6358–6366), and it has been proposed that R76 is directly involved in the binding of PAI-1 to LRP1 (see Gettins, PGW, and Dolmer, K. (2016) J. Biol. Chem. 291, 800–812). However, the ASA value of 0.24 indicates that this residue is hidden and cannot interact directly with LRP1. Similarly, K263 and K122, which were involved in previous studies, are also partially hidden in the structure and therefore cannot interact directly with LRP1.

[0188] Table II. a Equilibrium binding constant of mutant PAI-1 to LRP1

[0189]

[0190] a The kinetic constants were obtained by fitting the data to a bivalent model. Three independent experiments were performed for each mutant, and the average values ​​are shown.

[0191] b The surface integral number was calculated using VADAR (Willard et al, Nucleic Acids Res 31,3316 (2003)) and 1dvm.pdb.

[0192] c The calculation is based on equilibrium measurements, where Req is determined by fitting the combined data to a pseudo-first-order process.

[0193] Example VI.

[0194] This example illustrates that the binding of the LMWuPA:PAI-1 complex to LRP1 occurs via a complex mechanism.

[0195] To characterize the binding of LMWuPA:PA1 to LRP1, experiments were conducted to examine the ionic strength dependence of the binding. Figure 6The results shown in Figure A demonstrate the significant dependence of binding on ionic strength. (Debye-Hückel plot) Figure 6 B) The slope of 2.4 ± 0.3 indicates that 2-3 ions interact to participate in the binding.

[0196] When examining the kinetics of the interaction, it was observed that the dissociation kinetics changed at higher concentrations of the LMWuPA:PAI-1 complex. Figure 7 B). From Figure 7 This is also evident in the data in C, where faster dissociation occurs at higher LMWuPA:PAI-1 concentrations, indicating the existence of multiple binding mechanisms. This has been previously observed in the binding of the RAP domain D1D2 to LRP1 (see Prasad, JM, et al., (2016) J. Biol. Chem. 291, 18430–18439). Therefore, the experiment also incorporated a second scheme into the model, in which the LMWuPA:PAI-1 complex can also bind to a second different site on LRP1 to form a monovalent complex (complex III). Figure 7 A, Scheme II). To simplify the model, assume that k in Scheme II... a1 and k d1 Compared to k in the first step of scheme I a1 and k d1 The same applies. When the experimental SPR data were fitted to a model that included both Scheme I and Scheme II, an excellent fit was obtained. Figure 7 C), and the kinetic parameters are summarized in Table III. The data confirm the high affinity binding of the LMWuPA:PAI-1 complex to LRP1, and its K0... D It is about 100 times higher than that of PAI-1 alone, mainly due to the slower dissociation rate of the LMWuPA:PAI-1 complex (compare the k values ​​of PAI-1 in Table 1). d1 and k d2 (Compare with those values ​​of the LMWuPA:PAI-1 complex in Table III).

[0197] a Table III. Kinetics and equilibrium binding constants of LMWuPA:I91L PAI-1 and mutants with cluster IV and LRP1.

[0198]

[0199] a The kinetic constants are obtained by fitting the data to a bivalent model. Figure 5 A).

[0200] bThree independent experiments were conducted, and the values ​​shown are mean ± SD.

[0201] The experiment also examined the binding of the LMWuPA:PAI-1 complex to cluster IV of LRP1 immobilized on an SPR chip. Similar to the binding of LMWuPA:PAI-1 to full-length LRP1, the dissociation kinetics were independent of ligand concentration. Figure 8 A), and therefore the experiment fits the data to Figure 7 The model described in A fits the data well. Figure 8 B) The parameters derived from these fits are summarized in Table III, and show that the binding of the LMWuPA:PAI-1 complex to cluster IV is slightly weaker than that to the full-length LRP1. These results suggest that the LMWuPA:PAI-1 complex may also interact with LRP1 regions outside of cluster IV.

[0202] Example VII.

[0203] This example demonstrates that the PA1-I mutant reveals additional residues involved in the interaction between the LMWuPA:PAI-1 complex and LRP1.

[0204] To determine whether similar lysine residues on PAI-1 are also involved in the interaction between the uPA:PAI-1 complex and LRP1, experiments were conducted to form complexes of LMWuPA and mutant PAI-1 molecules, and the binding of these complexes to LRP1 was measured. The results of these studies are summarized in Table II. Interestingly, unlike the binding of PAI-1 to LRP1, individual mutants of PAI-1 (K69A, K88A, and K207A) had the least impact on the binding of the LMWuPA:PAI-1 complex to LRP1, while K80A resulted in K... D The affinity was reduced to 1 / 5.8. PAI-1 molecules containing the K80A and K207A double mutants resulted in a 1 / 23 reduction in the affinity of the LMWuPA:PAI-1 complex to LRP1. Notably, the triple mutants of K80A, K207A, and K88A resulted in a 1 / 244 reduction in affinity (Table II), revealing the crucial role of these three residues in the LMWuPA:PAI-1 complex for binding to LRP1.

[0205] Subsequent experiments examined cellular uptake of PAI-1 complexes with LMWuPA formed from PAI-1 molecules containing double or triple mutations. Figure 9 The results showed that when combined with LMWuPA, the double and triple mutants of PAI-1 were not effectively taken up by cells expressing LRP1.

[0206] Example VIII.

[0207] This embodiment describes the materials and methods used in embodiments I-VII.

[0208] Reagents.

[0209] LMWuPA, HMWuPA, WT PAI-1HMWuPA:PAI-1 complex, and I91L PAI-1 were purchased from Molecular Innovations. Mutant PAI-1 protein was generated and purified as described. LRP1 was purified from human placenta as described (see Ashcom, JD, et al., (1990) J. Cell Biol. 110, 1041–1048). LRP1 ligands binding clusters II, III, and IV were purchased from RnD Systems. CDE096 was synthesized as described (see Li, S.-H., et al., (2013) Proc. Natl. Acad. Sci. USA 110, E4941-9). The LMWuPA:PAI-1 complex used in Biacore studies was formed by incubating PAI-1 with a 1.2-fold molar excess of LMWuPA in PBS at room temperature for 1 h. The formation of the complex was verified by analyzing the protein on a 4-20% Tris-gly gel (Novex) and staining with colloidal blue.

[0210] Chemical modification of I91LPAI-1.

[0211] I91L PAI-1 was chemically modified using sulfonyl-NHS-acetate (Thermo-Fisher Scientific) to block the primary amine in the lysine side chain. Sulfonyl-NHS-acetate was dissolved in PBS at 50 g / ml. 55 μg of I91LPAI1 was incubated in PBS with a 50-fold excess of sulfonyl-NHS-acetate relative to the total amino groups in I91L PAI-1 at 4°C for 3 h. NAP was used... TM -5 Sephadex G-25 column (GE Healthcare) desalted modified I91L PAI-1 protein into 0.01M HEPES, 0.15M NaCl pH 7.4 to remove excess sulfo-NHS-acetate.

[0212] Surface plasmon resonance and surface plasmon resonance.

[0213] Purified LRP1 was immobilized on the CM5 sensor chip surface using a working solution of 20 μg / ml LRP1 in 10 mM sodium acetate at pH 4 to achieve a response level of 10,000 units. Following the manufacturer's instructions (BIAcore AB), LRP1 ligands bound to cluster IV were immobilized on the CM5 sensor chip surface using a working solution of 20 μg / ml cluster IV in 10 mM sodium acetate at pH 4 to achieve a response level of 2,000 units. Another flow cell was activated and blocked with 1 M ethanolamine (protein-free) as a control surface. Unless otherwise specified, binding experiments were performed in HBS-P buffer (0.01 M HEPES, 0.15 M NaCl, 0.005% surfactant P, 1 mM CaCl2, pH 7.4). For ionic strength dependence, buffer solutions were prepared at pH 7.4 using 10 mM HEPES, 0.0005% surfactant P, 1 mM CaCl2, and various concentrations of NaCl (0.15 M, 0.25 M, 0.5 M, 0.75 M, and 1.0 M). All experiments were performed at 25 °C on an IAcore 3000 instrument using a flow rate of 20 μl / min. The sensor chip surface was regenerated by injecting 100 mM phosphate at a flow rate of 100 μl / min for 15 s.

[0214] SPR data analysis.

[0215] The dissociation rate was fitted to a 2-exponential decay using GraphPad Prism 7.04 software. The kinetic data were analyzed into a bivalent model (Scheme 1) using numerical integration algorithms available in BIAevaluationshotware.

[0216]

[0217] Option 1

[0218] Where A represents the ligand (PAI-1 or LMWuPA:PAI-1 complex), B represents LRP1, AB1 represents the ligand:LRP1 complex at site 1, and AB2 represents the ligand:LRP1 complex at site 2. To facilitate the fitting process, k is obtained by globally fitting the dissociation data to a double exponential decay model. d1 and k d2 The estimated values ​​are then used as initial estimates in the fitting process. In the case of the LMWuPA:PAI-1 complex, the data are fitted to the following scheme as previously described:

[0219]

[0220] Option 1

[0221] as well as

[0222]

[0223] Option 1I

[0224] Equilibrium binding data were determined by fitting the binding rate to a pseudo-first-order process to obtain Req. Req was then plotted against total ligand concentration and fitted to the binding isotherm using nonlinear regression analysis in GraphPad Prism 7.04 software.

[0225] y = Bmax * L / (KD + L)

[0226] Where Bmax is the Req value at saturation, L is the free ligand concentration, and KD is the equilibrium binding constant. Since the free ligand concentration is unknown in these experiments, this equation is used to assume that the total amount of added ligands is much greater than the amount of ligands bound to the LRP-1 coupled SPR chip.

[0227] Inhibition by CDE-096.

[0228] The HMWuPA-PAI-1 complex was diluted to 2 nM in a solution containing 0.01 M HEPES, 0.15 M NaCl, 1 mM CaCl2, 0.0005% surfactant P, and 0.1% DMSO at pH 7.8, with a concentration of 0 to 500 nM CDE-096. Binding to LRP1 was then performed on a Biacore as described above, but with a running buffer of 0.01 M HEPES, 0.15 M NaCl, 1 mM CaCl2, 0.0005% surfactant P, and 0.1% DMSO at pH 7.8.

[0229] Cellular uptake of the LMWuPA PAI-1 complex.

[0230] WI38 cells were plated in 12-well tissue culture plates pre-coated with poly-D-lysine hydrobromide (Sigma). Cells were incubated for 1 h in assay buffer (DMEM, 1% BSA, 20 mM HEPES) before treatment with the iodination complex. LMWuPA was iodinated with I-125 sodium iodide (PerkinElmer NEZ033) using Iodo-gen (Pierce) in PBS containing 1 mM 6-aminocaproic acid (Aldrich). The iodinated protein was desalted into PBS using a PD-10 column (GE Healthcare) to remove free iodine. Labeled complexes were formed by incubating I91L PAI-1 and its mutant (0.8 μM) with I-125 LMWuPA (0.4 μM) at room temperature for 1 h. The resulting complexes were diluted to 5 nM in a separate assay buffer or assay buffer containing 1 μM RAP and incubated on cells at 37°C for 6 h. Remove the culture medium, wash the cells with 2 ml of PBS, and treat with trypsin (Corning 25-0520) containing 50 μg / ml proteinase K. Centrifuge the cells at 4000 rpm for 4 min. Remove the supernatant and count the cell pellet to determine the number of moles internalized cells.

[0231] Example IX.

[0232] This example describes the purification in Escherichia coli of a PAI-1 variant with the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): R101A and Q123K (hereinafter referred to as "MDI-1003").

[0233] The supernatant obtained by centrifuging 200 mL of MDI-1003 fermenter lysate was dialyzed against 0.05 M sodium phosphate, 0.1 M sodium chloride, and 0.001 M EDTA at pH 6.6, followed by chromatographic separation on a Heparin-Sepharose 6B column (10 x 5.0 cm) at room temperature at a flow rate of approximately 0.5 mL / min. The Heparin-Sepharose 6B column was washed with 2 L of 0.05 M sodium phosphate, 0.1 M sodium chloride, and 0.001 M EDTA at pH 6.6, followed by elution to 1.0 M sodium chloride in a 600 mL gradient in the same buffer. Fractions containing PAI-1 were combined, and solid ammonium sulfate was added to 18% saturation. PAI-1 was separated chromatographically at room temperature at a flow rate of approximately 0.5 mL / min on a Phenyl-Sepharose Fast Flow (Low Sub) column (10 x 2.5 cm) pre-equilibrated in 0.05 M potassium phosphate, 0.1 M sodium chloride, 0.001 M EDTA, pH 6.6, and 30% saturated ammonium sulfate. The Phenyl-Sepharose Fast Flow column was washed with 500 mL of 0.05 M potassium phosphate, 0.1 M sodium chloride, 0.001 M EDTA, pH 6.6, and 30% saturated ammonium sulfate, followed by elution to 0% ammonium sulfate in the same buffer via a 400 mL gradient. Fractions containing PAI-1 were combined and precipitated by adding solid ammonium sulfate to 65% saturation. The precipitate was dissolved to 3.5 mg / mL in 0.05 M sodium phosphate, 0.1 M sodium chloride, 0.001 M EDTA, pH 6.6, followed by thorough dialyzing with the same buffer. The yield after the Heparin-Sepharose 6B column was 90 ml, containing 80 mg of protein (lane 1). The yield after the Phenyl-Sepharose Fast Flow column was 80 ml, containing 47 mg of protein (lane 2). The final yield was 12 ml, containing 38 mg of highly purified HPAI-AVI-AK protein.

[0234] Example X.

[0235] CF-sputum titration for elastase concentration.

[0236] Sputum from CF patients was extracted with 2 mL of cold PBS / 1 g sputum and then homogenized manually until smooth. The homogenate was centrifuged at 10,000 x g for 20 min (4 °C) and stored for elastase titration. Next, purified HNE was diluted to 40 nM and added to each well (black plate) with concentrations of 0, 2.5, 5, 7.5, 10, 12.5, 15, and 20 μL, respectively, and brought to a volume of 100 μL with 40 mM Hepes, 100 mM NaCl, pH 7.4, and 0.005% Tween-20. 100 μL of 500 μM MeOSuc-AAPV-AMC was added and the plate was dynamically read at ex 370 eM440 for 10 min. Note the slope and intercept.

[0237] CF-sputum was diluted and 0, 2.5, 5, 7.5, 10, 12.5, 15, and 20 μL were added to each well. The volume was brought to 100 μL with 40 mM Hepes, 100 mM NaCl, pH 7.4, and 0.005% Tween-20. Then, 100 μL of 500 μM MeOSuc-AAPV-AMC was added, and the volume was dynamically read for 10 min at ex370 and em440. The elastase concentration in CF-sputum was calculated using slope and intercept inversion of purified HNE.

[0238] IC 50 / dynamics.

[0239] Incubate 50 nM HNE or CF-sputum + / - salmon sperm DNA or heparin at room temperature for 30 min. Serially dilute the elastase inhibitor into the black plate (in 40 mM Hepes, 100 mM NaCl, pH 7.4, 0.005% Tween-20) to a final volume of 90 μL. Add 10 μL of HNE / DNA / heparin from the top to the 90 μL of inhibitor. Incubate for 30 seconds, 1 min, 2 min, or longer at room temperature if necessary. Add 100 μL of 500 μM MeOSucAAPV-AMC to each well and dynamically read at ex370, em440 for 10 min.

[0240] Dynamic calculations

[0241] K obs = Ln(nM residual elastase / nM initial elastase) / time (seconds)

[0242] K obs Plotting V relative to inhibitor concentration and determining V by nonlinearly fitting the Michaelis equation max and K m .

[0243] K i =V max / K m

[0244] PK Study of AVI and AVI-AK

[0245] The pharmacokinetic characteristics of AVI or AVI-AK were assessed in 8-week-old male C57BL / 6J mice after intravenous (IV) bolus administration of 20 mg / kg. Blood samples were collected at 0.5, 1, 2, 6, and 24 h to determine the amount of PAI-1 in plasma.

[0246] Acute pulmonary edema caused by LPS injection

[0247] Wild-type C57BL / 6J mice (male, 8 weeks old) were administered LPS (25 μL, 2 mg / mL) via intratracheal infusion, followed by intratracheal treatment with a carrier, AVI, AVI-AK, or Aralast (30 μL, 1.3 mg / mL). Eighteen hours later, the animals were perfused with PBS, and wet lung weight and total elastase were obtained.

[0248] Lung extraction and homogenization

[0249] Obtain the wet weight of the whole lung. Add 250 μL of 0.4 M Hepes, 0.1 M NaCl, pH 7.4, and 1% Tx-100, and homogenize at high speed for 1 min. Centrifuge at 10,000 x g for 10 min (4 °C). Transfer the supernatant to a new tube and centrifuge again at 10,000 x g for 10 min. Transfer the supernatant to a new test tube for determination.

[0250] Fibrosis determination

[0251] Fibrosis assays were performed essentially as described (Blood, 2011, 118:2313-2321). In brief, weight- and age-matched (6–8 weeks old, 18–22 g) WT mice were treated on day 0 with a single dose of intratracheal bleomycin (1.15 u / kg in 50 L sterile PBS) to induce pulmonary fibrosis. From day 1, mice were treated twice daily with MDI-1001, MDI-1002, MDI-1003, or saline (4 mg / kg IP) to treat the acute injury phase. On day 21, mice were sacrificed and pulmonary fibrosis was determined by hydroxyproline measurement as described (see Blood, 2011, 118:2313-2321).

[0252] Construction of HFc-AVI-AK expression vector and transfection of CHO cell line

[0253] The following mutations were introduced into the mature form of human PAI-1 cDNA using site-directed mutagenesis (QuickChange II Kit, Agilent, Santa Clara, CA): I91L, R101A, Q123K, V343A, and R346V. Mutations V343A, R346V, and I91L (AVI) transmit a stable, active PAI-1 phenotype, while mutations R101A and Q123K (AK) introduce a reduced brevisin-binding phenotype. The modified cDNA (named AVI-AK) was cloned into the pcDNA5 / FRT plasmid, which possesses an N-terminal fusion peptide sequence composed of a constant region of human immunoglobulin (IgG1) including domains 2 and 3, including a hinge region (HFc). The integrity of the construct was confirmed by restriction digestion screening and DNA sequencing. The validated HFc-AVI-AK fusion plasmid and pOG44 plasmid (containing the Flp recombinase gene) were co-transfected into CHO (Chinese hamster ovary) Flp-In cells (InVitrogen) at a 9:1 ratio using GeneJuice reagent (Novagen / Millipore) to promote single-copy integration of the fusion protein sequence. Transfected cells were incubated at 37°C and 6% CO2 for 48 hours, followed by the addition of 100 μg / ml hygromycin (Invivogen) to select cells carrying integrated AVI-AK cDNA. After selection and propagation of transfected cells in Ham's F12 medium (supplemented with 10% fetal bovine serum and hygromycin), cell cultures were adapted to grow in serum-free medium (CHOgro, MirusBio) to promote non-adherent growth, increase cell density, and simplify purification. Protein expression is regulated by a constitutive cytomegalovirus (CMV) promoter; therefore, cell supernatant containing HFc-AVI-AK was harvested approximately every 3–5 days for downstream processing.

[0254] Purification of HFc-AVI-AK

[0255] The conditioned medium was diluted 1:1 with phosphate-buffered saline (PBS, pH 7.0) and applied to a column containing 15–20 ml of protein A / protein G resin equilibrated with PBS, followed by thorough washing with PBS. The bound fusion protein was eluted with 0.1 M glycine, 0.1 M NaCl, pH 3.0 and collected in 0.5 M sodium acetate at pH 5.6 to stabilize the pH, yielding protein with >95% purity. The protein eluent was then immediately applied to heparinized agarose equilibrated with 0.05 M sodium phosphate, 0.1 M NaCl, pH 6.6, followed by washing and elution with 0.05 M sodium phosphate, 1 M NaCl, pH 6.6. The second step utilized the unique properties of PAI-1 to concentrate the protein to achieve >99% purity.

[0256] result

[0257] – MDI-1001 – contains the following stable mutation in the wild-type mature PAI-1 amino acid sequence (SEQ ID NO:3). PAI-1 variants: I91L and the mutants V343A and R346V capable of NE repression.

[0258] – MDI-1002 – Fc fusion protein with the MDI-1001 mutation

[0259] – MDI-1003– The MDI-1001 PAI-1 variant contains additional mutations, R101A and Q123K, within the wild-type mature human PAI-1 amino acid sequence (SEQ ID NO:3) that disable the brevisin-binding function of PAI-1.

[0260] –MDI-1004– An Fc fusion protein with the MDI-1003 mutation

[0261] Figure 13 The results show that MDI-1001 targets the inflammation network better than Aralast.

[0262] Figure 14 In vitro comparisons between Aralast, Avelestat, MDI-1002, MDI-1003, and MDI-1004 are shown.

[0263] Figure 15 This shows that MDI-1003 targets NETs in CF sputum.

[0264] Figure 16 The activity of elastase as a function of inhibitor concentration is shown.

[0265] Figure 17 MDI-1002 demonstrates protection against acute lung injury.

[0266] Figure 18 MDI-1002 demonstrates protection against pulmonary fibrosis.

[0267] Figure 19 The results showed that MDI-1002 did not improve recovery after bleomycin treatment.

[0268] Figure 20 The inhaled MDI-1003 is shown to protect against acute lung injury.

[0269] Figure 21 MDI-1003 demonstrates better protection against pulmonary fibrosis than MDI-1001.

[0270] Figure 22 The results show that MDI-1003 improved recovery after bleomycin treatment.

[0271] Figure 23The Fc fusion constructs of MDI-1002 and MDI-1004 are shown.

[0272] Figure 24 The Fc fusion expression of MDI-1002 and MDI-1004 is shown.

[0273] Figure 25 This demonstrates how Fc fusion improves PK.

[0274] Figure 26 Inhibition curves for neutrophil elastase with or without DNA NET are shown. These data indicate that the mutations providing optimal activity against neutrophil elastase can be combined with each of the mutations that reduce and clear receptor binding shown in Table II. The result is improved pharmacokinetics of the molecule. A total of seven variants were identified, six with a single receptor-binding mutation and one with two receptor-binding mutations. The latter double mutant demonstrates that mutations can be combined to have the potential to further reduce receptor binding. All of these also include the I91L, R101A, Q123K, V343A, and R346V mutations.

[0275] The MDI name for each variant is:

[0276] K69A is MDI-1005

[0277] K80A is MDI-1006

[0278] K88A is MDI-1007

[0279] K176A is MDI-1008

[0280] K207A is MDI-1009

[0281] K263A is MDI-1010

[0282] K69A-K207A is MDI-1011

[0283] These mutations indicate reduced binding to the clearance receptor while maintaining NET binding and inhibition of neutrophil elastase in the presence of DNA. The preferred mutation is K207A, as this reduces the clearance receptor binding of the free inhibitor to 1 / 19th, but only reduces the binding of the inhibited protease complex to 1 / 1.6th (see Table II in the application). This significantly increases the pharmacokinetics of the inhibitor with this mutation, while still allowing for the removal of the elastase complex after inhibition.

[0284] The present invention has now been fully described, and those skilled in the art will understand that it can also be carried out within a broad and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any of its embodiments. All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

[0285] By invoking and incorporating

[0286] For all purposes, the full disclosure of each patent document and scientific article mentioned herein is incorporated herein by reference, including but not limited to any references cited herein.

[0287] Equivalent form

[0288] This invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered illustrative in all respects and not to limit the invention described herein. Consequently, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes within the equivalent meaning and scope of the claims are intended to be included therein. sequence list <110> The Regents of the University of Michigan <120> Peptide inhibitors of neutrophil elastase activity and their uses <130> PPI22170938US <150> US 62 / 938,859 <151> 2019-11-21 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 4080 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 1 gaattcctgc agctcagcag ccgccgccag agcaggacga accgccaatc gcaaggcacc 60 cttaaggacg tcgagtcgtc ggcggcggtc tcgtcctgct tggcggttag cgttccgtgg 120 tctgagaact tcaggatgca gatgtctcca gccctcacct gcctagtcct gggcctggcc 180 agactcttga agtcctacgt ctacagaggt cgggagtgga cggatcagga cccggaccgg 240 cttgtctttg gtgaagggtc tgctgtgcac catcccccat cctacgtggc ccacctggcc 300 gaagaaac cacttcccag agcacaggtg gtagggggta ggatgcaccg ggtggaccgg 360 tcagacttcg gggtgagggt gtttcagcag gtggcgcagg cctccaagga ccgcaacgtg 420 agtctgaagc cccactccca caaagtcgtc caccgcgtcc ggaggttcct ggcgttgcac 480 gttttctcac cctatggggt ggcctcggtg ttggccatgc tccagctgac aacaggagga 540 caaaagagtg ggatacccca ccggagccac aaccggtacg aggtcgactg ttgtcctcct 600 gaaacccagc agcagattca agcagctatg ggattcaaga ttgatgacaa gggcatggcc 660 ctttgggtcg tcgtctaagt tcgtcgatac cctaagttct aactactgtt cccgtaccgg 720 cccgccctcc ggcatctgta caaggagctc atggggccat ggaacaagga tgagatcagc 780 gggcgggagg ccgtagacat gttcctcgag taccccggta ccttgttcct actctagtcg 840 accacagacg cgatcttcgt ccagcgggat ctgaagctgg tccagggctt catgccccac 900 tggtgtctgc gctagaagca ggtcgcccta gacttcgacc aggtcccgaa gtacggggtg 960 ttcttcaggc tgttccggag cacggtcaag caagtggact tttcagaggt ggagagagcc 1020 aagaagtccg acaaggcctc gtgccagttc gttcacctga aaagtctcca cctctctcgg 1080 agattcatca tcaatgactg ggtgaagaca cacacaaaag gtatgatcag caacttgctt 1140 tctaagtagt agttactgac ccacttctgt gtgtgttttc catactagtc gttgaacgaa 1200 gggaaaggag ccgtggacca gctgacacgg ctggtgctgg tgaatgccct ctacttcaac 1260 ccctttcctc ggcacctggt cgactgtgcc gaccacgacc acttacggga gatgaagttg 1320 ggccagtgga agactccctt ccccgactcc agcacccacc gccgcctctt ccacaaatca 1380 ccggtcacct tctgagggaa ggggctgagg tcgtgggtgg cggcggagaa ggtgtttagt 1440 gacggcagca ctgtctctgt gcccatgatg gctcagacca acaagttcaa ctatactgag 1500 ctgccgtcgt gacagagaca cgggtactac cgagtctggt tgttcaagtt gatatgactc 1560 ttcaccacgc ccgatggcca ttactacgac atcctggaac tgccctacca cggggacacc 1620 aagtggtgcg ggctaccggt aatgatgctg taggaccttg acgggatggt gcccctgtgg 1680 ctcagcatgt tcattgctgc cccttatgaa aaagaggtgc ctctctctgc cctcaccaac 1740 gagtcgtaca agtaacgacg gggaatactt tttctccacg gagagagacg ggagtggttg 1800 attctgagtg cccagctcat cagccactgg aaaggcaaca tgaccaggct gccccgcctc 1860 taagactcac gggtcgagta gtcggtgacc tttccgttgt actggtccga cggggcggag 1920 ctggttctgc ccaagttctc cctggagact gaagtcgacc tcaggaagcc cctagagaac 1980 gaccaagacg ggttcaagag ggacctctga cttcagctgg agtccttcgg ggatctcttg 2040 ctgggaatga ccgacatgtt cagacagttt caggctgact tcacgagtct ttcagaccaa 2100 gacccttact ggctgtacaa gtctgtcaaa gtccgactga agtgctcaga aagtctggtt 2160 gagcctctcc acgtcgcgca ggcgctgcag aaagtgaaga tcgaggtgaa cgagagtggc 2220 ctcggagagg tgcagcgcgt ccgcgacgtc tttcacttct agctccactt gctctcaccg 2280 acggtggcct cctcatccac agctgtcata gtctcagccc gcatggcccc cgaggagatc 2340 tgccaccgga ggagtaggtg tcgacagtat cagagtcggg cgtaccgggg gctcctctag 2400 atcatggaca gacccttcct ctttgtggtc cggcacaacc ccacaggaac agtccttttc 2460 tagtacctgt ctgggaagga gaaacaccag gccgtgttgg ggtgtccttg tcaggaaaag 2520 atgggccaag tgatggaacc ctgaccctgg ggaaagacgc cttcatctgg gacaaaactg 2580 tacccggttc actaccttgg gactgggacc cctttctgcg gaagtagacc ctgttttgac 2640 gagatgcatc gggaaagaag aaactccgaa gaaaagaatt ttagtgttaa tgactctttc 2700 ctctacgtag ccctttcttc tttgaggctt cttttcttaa aatcacaatt actgagaaag 2760 tgaaggaaga gaagacattt gccttttgtt aaaagatggt aaaccagatc tgtctccaag 2820 acttccttct cttctgtaaa cggaaaacaa ttttctacca tttggtctag acagaggttc 2880 accttggcct ctccttggag gacctttagg tcaaactccc tagtctccac ctgagaccct 2940 tggaaccgga gaggaacctc ctggaaatcc agtttgaggg atcagaggtg gactctggga 3000 gggagagag tttgaagcac aactccctta aggtctccaa accagacggt gacgcctgcg ccctctcttc aaacttcgtg ttgagggat tccagaggtt tggtctgcca ctgcggacgc 3120 ggaccatctg gggcacctgc ttccacccgt ctctctgccc actcgggtct gcagacctgg 3180 cctggtagac cccgtggacg aaggtgggca gagagacggg tgagcccaga cgtctggacc 3240 ttcccactga ggccctttgc aggatggac tacggggctt acaggagctt ttgtgtgcct 3300 aagggtgact ccgggaaacg tcctaccttg atgccccga tgtcctcga aacacacgga ggtagaaact atttctgttc cagtcacatt gccatcactc ttgtactgcc tgccaccgcg 3420 ccatctttga taaagacaag gtcagtgtaa cggtagtgag aacatgacgg acggtggcgc 3540. gtgacaggcc aaaggccagt ggaagaaca ccctttcatc tcagagtcca ctcctccgac cactgtccgg tttccggtca ccttctttgt gggaaagtag agtctcaggt 3600 ctgtggcact ggccacccct ccccagtaca ggggtgctgc aggtggcaga gtgaatgtcc 3660 gacaccgtga ccggtgggga ggggtcatgt ccccacgacg tccaccgtct cacttacagg 3720 cccatcatgt ggcccaactc tcctggcctg gccatctccc tccccagaaa cagtgtgcat 3780 gggtagtaca ccgggttgag aggaccggac cggtagaggg aggggtcttt gtcacacgta 3840 gggttatttt ggagtgtagg tgacttgttt actcattgaa gcagatttct gcttcctttt 3900 cccaataaaa cctcacatcc actgaacaaa tgagtaactt cgtctaaaga cgaaggaaaa 3960 atttttatag gaatagagga agaaatgtca gatgcgtgcc cagctcttca ccccccaatc 4020 taaaaatatc cttatctcct tctttacagt ctacgcacgg gtcgagaagt ggggggttag 4080 <210> 2 <211> 402 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 2 Met Gln Met Ser Pro Ala Leu Thr Cys Leu Val Leu Gly Leu Ala Leu 1 5 10 15 Val Phe Gly Glu Gly Ser Ala Val His His Pro Pro Ser Tyr Val Ala 20 25 30 His Leu Ala Ser Asp Phe Gly Val Arg Val Phe Gln Gln Val Ala Gln 35 40 45 Ala Ser Lys Asp Arg Asn Val Val Phe Ser Pro Tyr Gly Val Ala Ser 50 55 60 Val Leu Ala Met Leu Gln Leu Thr Thr Gly Gly Glu Thr Gln Gln Gln 65 70 75 80 Ile Gln Ala Ala Met Gly Phe Lys Ile Asp Asp Lys Gly Met Ala Pro 85 90 95 Ala Leu Arg His Leu Tyr Lys Glu Leu Met Gly Pro Trp Asn Lys Asp 100 105 110 Glu Ile Ser Thr Thr Asp Ala Ile Phe Val Gln Arg Asp Leu Lys Leu 115 120 125 Val Gln Gly Phe Met Pro His Phe Phe Arg Leu Phe Arg Ser Thr Val 130 135 140 Lys Gln Val Asp Phe Ser Glu Val Glu Arg Ala Arg Phe Ile Ile Asn 145 150 155 160 Asp Trp Val Lys Thr His Thr Lys Gly Met Ile Ser Asn Leu Leu Gly 165 170 175 Lys Gly Ala Val Asp Gln Leu Thr Arg Leu Val Leu Val Asn Ala Leu 180 185 190 Tyr Phe Asn Gly Gln Trp Lys Thr Pro Phe Pro Asp Ser Ser Thr His 195 200 205 Arg Arg Leu Phe His Lys Ser Asp Gly Ser Thr Val Ser Val Pro Met 210 215 220 Met Ala Gln Thr Asn Lys Phe Asn Tyr Thr Glu Phe Thr Thr Pro Asp 225 230 235 240 Gly His Tyr Tyr Asp Ile Leu Glu Leu Pro Tyr His Gly Asp Thr Leu 245 250 255 Ser Met Phe Ile Ala Ala Pro Tyr Glu Lys Glu Val Pro Leu Ser Ala 260 265 270 Leu Thr Asn Ile Leu Ser Ala Gln Leu Ile Ser His Trp Lys Gly Asn 275 280 285 Met Thr Arg Leu Pro Arg Leu Leu Val Leu Pro Lys Phe Ser Leu Glu 290 295 300 Thr Glu Val Asp Leu Arg Lys Pro Leu Glu Asn Leu Gly Met Thr Asp 305 310 315 320 Met Phe Arg Gln Phe Gln Ala Asp Phe Thr Ser Leu Ser Asp Gln Glu 325 330 335 Pro Leu His Val Ala Gln Ala Leu Gln Lys Val Lys Ile Glu Val Asn 340 345 350 Glu Ser Gly Thr Val Ala Ser Ser Ser Thr Ala Val Ile Val Ser Ala 355 360 365 Arg Met Ala Pro Glu Glu Ile Ile Met Asp Arg Pro Phe Leu Phe Val 370 375 380 Val Arg His Asn Pro Thr Gly Thr Val Leu Phe Met Gly Gln Val Met 385 390 395 400 Glu Pro <210> 3 <211> 379 <212> PRT <213> Artificial Sequence [[ID=十七]] <220> <223> Synthetic <400> 3 Val His His Pro Pro Ser Tyr Val Ala His Leu Ala Ser Asp Phe Gly 1 5 10 15 Val Arg Val Phe Gln Gln Val Ala Gln Ala Ser Lys Asp Arg Asn Val 20 25 30 Val Phe Ser Pro Tyr Gly Val Ala Ser Val Leu Ala Met Leu Gln Leu 35 40 45 Thr Thr Gly Gly Glu Thr Gln Gln Gln Ile Gln Ala Ala Met Gly Phe 50 55 60 Lys Ile Asp Asp Lys Gly Met Ala Pro Ala Leu Arg His Leu Tyr Lys<00009照抄03>65 70 75 80 Glu Leu Met Gly Pro Trp Asn Lys Asp Glu Ile Ser Thr Thr Asp Ala<00照抄00905>85 90 95 Ile Phe Val Gln Arg Asp Leu Lys Leu Val Gln Gly Phe Met Pro His 100 105 110 Phe Phe Arg Leu Phe Arg Ser Thr Val Lys Gln Val Asp Phe Ser Glu 115 120 125 Val Glu Arg Ala Arg Phe Ile Ile Asn Asp Trp Val Lys Thr His Thr 130 135 140 Lys Gly Met Ile Ser Asn Leu Leu Gly Lys Gly Ala Val Asp Gln Leu 145 150 155 160 Thr Arg Leu Val Leu Val Asn Ala Leu Tyr Phe Asn Gly Gln Trp Lys 165 170 175 Thr Pro Phe Pro Asp Ser Ser Thr His Arg Arg Leu Phe His Lys Ser 180 185 190 Asp Gly Ser Thr Val Ser Val Pro Met Met Ala Gln Thr Asn Lys Phe 195 200 205 Asn Tyr Thr Glu Phe Thr Thr Pro Asp Gly His Tyr Tyr Asp Ile Leu 210 215 220 Glu Leu Pro Tyr His Gly Asp Thr Leu Ser Met Phe Ile Ala Ala Pro 225 230 235 240 Tyr Glu Lys Glu Val Pro Leu Ser Ala Leu Thr Asn Ile Leu Ser Ala 245 250 255 Gln Leu Ile Ser His Trp Lys Gly Asn Met Thr Arg Leu Pro Arg Leu 260 265 270 Leu Val Leu Pro Lys Phe Ser Leu Glu Thr Glu Val Asp Leu Arg Lys 275 280 285 Pro Leu Glu Asn Leu Gly Met Thr Asp Met Phe Arg Gln Phe Gln Ala 290 295 300 Asp Phe Thr Ser Leu Ser Asp Gln Glu Pro Leu His Val Ala Gln Ala 305 310 315 320 Leu Gln Lys Val Lys Ile Glu Val Asn Glu Ser Gly Thr Val Ala Ser 325 330 335 Ser Ser Thr Ala Val Ile Val Ser Ala Arg Met Ala Pro Glu Glu Ile 340 345 350 Ile Met Asp Arg Pro Phe Leu Phe Val Val Arg His Asn Pro Thr Gly 355 360 365 Thr Val Leu Phe Met Gly Gln Val Met Glu Pro 370 375 <210> 4 <211> 2352 <212> Ms <213> artificial sequence <220> <223> synthesis <400> 4 gtgcaccatc ccccatccta cgtggcccac ctggcccacg tggtaggggg taggatgcac 60 cgggtggacc ggtcagactt cggggtgagg gtgtttcagc aggtggcgca ggcctccaag 120 gaccgcaacg tgagtctgaa gccccactcc cacaaagtcg tccaccgcgt ccggaggttc 180 ctggcgttgc acgttttctc accctatggg gtggcctcgg tgttggccat gctccagctg 240 acaacaggag gacaaaagag tgggataccc caccggagcc acaaccggta cgaggtcgac 300 tgttgtcctc ctgaaaccca gcagcagatt caagcagcta tgggattcaa gattgatgac 360 gcgggcatgg ccctttgggt cgtcgtctaa gttcgtcgat accctaagtt ctaactactg 420 cgcccgtacc ggcccgccct ccggcatctg taccgggagc tcatggggcc atggaacgcg 480 gatgagctca gcgggcggga ggccgtagac atggccctcg agtaccccgg taccttgcgc 540 ctactcgagt cgaccacaga cgcgatcttc gtccaggcgg atctgaagct ggtccagggc 600 ttcatgcccc actggtgtct gcgctagaag caggtccgcc tagacttcga ccaggtcccg 660 aagtacgggg tgttcttcag gctgttccgg agcacggtcg cgaaagtgga cttttcagag 720 gtggagagag ccaagaagtc cgacaaggcc tcgtgccagc gctttcacct gaaaagtctc 780 cacctctctc ggagattcat catcaatgac tgggtgaaga cacacacaaa aggtatgatc 840 agcaacttgc tttctaagta gtagttactg acccacttct gtgtgtgttt tccatactag 900 tcgttgaacg aagggaaagg agccgtggac cagctgacac ggctggtgct ggtgaatgcc 960 ctctacttca acccctttcc tcggcacctg gtcgactgtg ccgaccacga ccacttacgg 1020 gagatgaagt tgggccagtg ggcgactccc ttccccgact ccagcaccca ccgccgcctc 1080 ttccacaaat caccggtcac ccgctgaggg aaggggctga ggtcgtgggt ggcggcggag 1140 aaggtgttta gtgacggcag cactgtctct gtgcccatga tggctcagac caacgcgttc 1200 aactatactg agctgccgtc gtgacagaga cacgggtact accgagtctg gttgcgcaag 1260 ttgatatgac tcttcaccac gcccgatggc cattactacg acatcctgga actgccctac 1320 cacggggaca ccaagtggtg cgggctaccg gtaatgatgc tgtaggacct tgacgggatg 1380 gtgcccctgt ggctcagcat gttcattgct gccccttatg aaaaagaggt gcctctctct 1440 gccctcacca acgagtcgta caagtaacga cggggaatac tttttctcca cggagagaga 1500 cgggagtggt tgattctgag tgcccagctc atcagccact gggcaggcaa catgaccagg 1560 ctgccccgcc tctaagactc acgggtcgag tagtcggtga cccgtccgtt gtactggtcc 1620 gacggggcgg agctggttct gcccaagttc tccctggaga ctgaagtcga cctcaggaag 1680 cccctagaga acgaccaaga cgggttcaag agggacctct gacttcagct ggagtccttc 1740 ggggatctct tgctgggaat gaccgacatg ttcagacagt ttcaggctga cttcacgagt 1800 ctttcagacc aagaccctta ctggctgtac aagtctgtca aagtccgact gaagtgctca 1860 gaaagtctgg ttgagcctct ccacgtcgcg caggcgctgc agaaagtgaa gatcgaggtg 1920 aacgagagtg gcctcggaga ggtgcagcgc gtccgcgacg tctttcactt ctagctccac 1980 ttgctctcac cgacggtggc ctcctcatcc acagctgtca tagcctcagc cgtcatggcc 2040 cccgaggaga tctgccaccg gaggagtagg tgtcgacagt atcggagtcg gcagtaccgg 2100 gggctcctct agatcatgga cagacccttc ctctttgtgg tccggcacaa ccccacagga 2160 acagtccttt tctagtacct gtctgggaag gagaaacacc aggccgtgtt ggggtgtcct 2220 tgtcaggaaa agatgggcca agtgatggaa ccctgaccct ggggaaagac gccttcatct 2280 gggacaaaac tgtacccggt tcactacctt gggactggga cccctttctg cggaagtaga 2340 ccctgttttg ac 2352 <210> 5 <211> 379 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 5 Val His His Pro Pro Ser Tyr Val Ala His Leu Ala Ser Asp Phe Gly 1 5 10 15 Val Arg Val Phe Gln Gln Val Ala Gln Ala Ser Lys Asp Arg Asn Val 20 25 30 Val Phe Ser Pro Tyr Gly Val Ala Ser Val Leu Ala Met Leu Gln Leu 35 40 45 Thr Thr Gly Gly Glu Thr Gln Gln Gln Ile Gln Ala Ala Met Gly Phe 50 55 60 Lys Ile Asp Asp Ala Gly Met Ala Pro Ala Leu Arg His Leu Tyr Ala 65 70 75 80 Glu Leu Met Gly Pro Trp Asn Ala Asp Glu Leu Ser Thr Thr Asp Ala 85 90 95 Ile Phe Val Gln Ala Asp Leu Lys Leu Val Gln Gly Phe Met Pro His 100 105 110 Phe Phe Arg Leu Phe Arg Ser Thr Val Ala Lys Val Asp Phe Ser Glu 115 120 125 Val Glu Arg Ala Arg Phe Ile Ile Asn Asp Trp Val Lys Thr His Thr 130 135 140 Lys Gly Met Ile Ser Asn Leu Leu Gly Lys Gly Ala Val Asp Gln Leu 145 150 155 160 Thr Arg Leu Val Leu Val Asn Ala Leu Tyr Phe Asn Gly Gln Trp Ala 165 170 175 Thr Pro Phe Pro Asp Ser Ser Thr His Arg Arg Leu Phe His Lys Ser 180 185 190 Asp Gly Ser Thr Val Ser Val Pro Met Met Ala Gln Thr Asn Ala Phe 195 200 205 Asn Tyr Thr Glu Phe Thr Thr Pro Asp Gly His Tyr Tyr Asp Ile Leu 210 215 220 Glu Leu Pro Tyr His Gly Asp Thr Leu Ser Met Phe Ile Ala Ala Pro 225 230 235 240 Tyr Glu Lys Glu Val Pro Leu Ser Ala Leu Thr Asn Ile Leu Ser Ala 245 250 255 Gln Leu Ile Ser His Trp Ala Gly Asn Met Thr Arg Leu Pro Arg Leu 260 265 270 Leu Val Leu Pro Lys Phe Ser Leu Glu Thr Glu Val Asp Leu Arg Lys 275 280 285 Pro Leu Glu Asn Leu Gly Met Thr Asp Met Phe Arg Gln Phe Gln Ala 290 295 300 Asp Phe Thr Ser Leu Ser Asp Gln Glu Pro Leu His Val Ala Gln Ala 305 310 315 320 Leu Gln Lys Val Lys Ile Glu Val Asn Glu Ser Gly Thr Val Ala Ser 325 330 335 Ser Ser Thr Ala Val Ile Ala Ser Ala Val Met Ala Pro Glu Glu Ile 340 345 350 Ile Met Asp Arg Pro Phe Leu Phe Val Val Arg His Asn Pro Thr Gly 355 360 365 Thr Val Leu Phe Met Gly Gln Val Met Glu Pro 370 375 <210> 6 <211> 3834 <212> Ms <213> artificial sequence <220> <223> synthesis <400> 6 atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgc acttgtcacg 60 120 aattcggcac ctctcgagcc caaatctagt gacaaaactc acacatgccc accgtgccca 180 ttaagccgtg gagagctcgg gtttagatca ctgttttgag tgtgtacggg tggcacgggt 240 gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 300 cgtggacttg aggacccccc tggcagtcag aaggagaagg ggggttttgg gttcctgtgg 360 ctctacatca cccgggaacc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 420 gagatgtagt gggcccttgg actccagtgt acgcaccacc acctgcactc ggtgcttctg 480 540 ggactccagt tcaagttgac catgcacctg ccgcacctcc acgtattacg gttctgtttc 600 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 660 ggcgccctcc tcgtcatgtt gtcgtgcatg gcacaccagt cgcaggagtg gcaggacgtg 720 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 780 gtcctgaccg acttaccgtt cctcatgttc acgttccaga ggttgtttcg ggagggtcgg 840 cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 900 gggtagctct tttggtagag gtttcggttt cccgtcgggg ctcttggtgt ccacatgtgg 960 ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1020 gacgggggta gggccctcct ctactggttc ttggtccagt cggactggac ggaccagttt 1080 ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1140 ccgaagatag ggtcgctgta gcggcacctc accctctcgt tacccgtcgg cctcttgttg 1200 tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 1260 atgttctggt gcggagggca cgacctgagg ctgccgagga agaaggagat gtcgttcgag 1320 accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1380 tggcacctgt tctcgtccac cgtcgtcccc ttgcagaaga gtacgaggca ctacgtactc 1440 gctctgaagt tccactacac gcagaagagc ctctccctgt ctccgggtgc aactagtgtg 1500 cgagacttca aggtgatgtg cgtcttctcg gagagggaca gaggcccacg ttgatcacac 1560 caccatcccc catcctacgt ggcccacctg gcctcagact tcggggtgag ggtgtttcag 1620 gtggtagggg gtaggatgca ccgggtggac cggagtctga agccccactc ccacaaagtc 1680 caggtggcgc aggcctccaa ggaccgcaac gtggttttct caccctatgg ggtggcctcg 1740 gtccaccgcg tccggaggtt cctggcgttg caccaaaaga gtgggatacc ccaccggagc 1800 gtgttggcca tgctccagct gacaacagga ggagaaaccc agcagcagat tcaagcagct 1860 cacaaccggt acgaggtcga ctgttgtcct cctctttggg tcgtcgtcta agttcgtcga 1920 atgggattca agattgatga caagggcatg gcccccgccc tccggcatct gtacaaggag 1980 taccctaagt tctaactact gttcccgtac cgggggcggg aggccgtaga catgttcctc 2040 ctcatggggc catggaacaa ggatgagctc agcaccacag acgcgatctt cgtccaggcg 2100 gagtaccccg gtaccttgtt cctactcgag tcgtggtgtc tgcgctagaa gcaggtccgc 2160 gatctgaagc tggtccaggg cttcatgccc cacttcttca ggctgttccg gagcacggtc 2220 ctagacttcg accaggtccc gaagtacggg gtgaagaagt ccgacaaggc ctcgtgccag 2280 aagaaagtgg acttttcaga ggtggagaga gccagattca tcatcaatga ctgggtgaag 2340 ttctttcacc tgaaaagtct ccacctctct cggtctaagt agtagttact gacccacttc 2400 acacacacaa aaggtatgat cagcaacttg cttgggaaag gagccgtgga ccagctgaca 2460 tgtgtgtgtt ttccatacta gtcgttgaac gaaccctttc ctcggcacct ggtcgactgt 2520 cggctggtgc tggtgaatgc cctctacttc aacggccagt ggaagactcc cttccccgac 2580 gccgaccacg accacttacg ggagatgaag ttgccggtca ccttctgagg gaaggggctg 2640 tccagcaccc accgccgcct cttccacaaa tcagacggca gcactgtctc tgtgcccatg 2700 aggtcgtggg tggcggcgga gaaggtgttt agtctgccgt cgtgacagag acacgggtac 2760 atggctcaga ccaacaagtt caactatact gagttcacca cgcccgatgg ccattactac 2820 taccgagtct ggttgttcaa gttgatatga ctcaagtggt gcgggctacc ggtaatgatg 2880 gacatcctgg aactgcccta ccacggggac accctcagca tgttcattgc tgccccttat 2940 ctgtaggacc ttgacgggat ggtgcccctg tgggagtcgt acaagtaacg acggggaata 3000 gaaaaagagg tgcctctctc tgccctcacc aacattctga gtgcccagct catcagccac 3060 ctttttctcc acggagagag acgggagtgg ttgtaagact cacgggtcga gtagtcggtg 3120 tggaaaggca acatgaccag gctgccccgc ctcctggttc tgcccaagtt ctccctggag 3180 acctttccgt tgtactggtc cgacggggcg gaggaccaag acgggttcaa gagggacctc 3240 actgaagtcg acctcaggaa gcccctagag aacctgggaa tgaccgacat gttcagacag 3300 tgacttcagc tggagtcctt cggggatctc ttggaccctt actggctgta caagtctgtc 3360 tttcaggctg acttcacgag tctttcagac caagagcctc tccacgtcgc gcaggcgctg 3420 aaagtccgac tgaagtgctc agaaagtctg gttctcggag aggtgcagcg cgtccgcgac 3480 cagaaagtga agatcgaggt gaacgagagt ggcacggtgg cctcctcatc cacagctgtc 3540 gtctttcact tctagctcca cttgctctca ccgtgccacc ggaggagtag gtgtcgacag 3600 atagcctcag ccgtcatggc ccccgaggag atcatcatgg acagaccctt cctctttgtg 3660 tatcggagtc ggcagtaccg ggggctcctc tagtagtacc tgtctgggaa ggagaaacac 3720 gtccggcaca accccacagg aacagtcctt ttcatgggcc aagtgatgga accctgacag 3780 gccgtgttgg ggtgtccttg tcaggaaaag tacccggttc actaccttgg gact 3834 <210> 7 <211> 638 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Ala Leu Val Thr Asn Ser Ala Pro Leu Glu Pro Lys Ser Ser Asp Lys 20 25 30 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 35 40 45 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr 50 55 60 Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 65 70 75 80 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 85 90 95 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 100 105 110 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 115 120 125 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 130 135 140 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 145 150 155 160 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 165 170 175 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 180 185 190 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 195 200 205 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 210 215 220 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 225 230 235 240 Ala Leu Lys Phe His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 245 250 255 Ala Thr Ser Val His His Pro Pro Ser Tyr Val Ala His Leu Ala Ser 260 265 270 Asp Phe Gly Val Arg Val Phe Gln Gln Val Ala Gln Ala Ser Lys Asp 275 280 285 Arg Asn Val Val Phe Ser Pro Tyr Gly Val Ala Ser Val Leu Ala Met 290 295 300 Leu Gln Leu Thr Thr Gly Gly Glu Thr Gln Gln Gln Ile Gln Ala Ala 305 310 315 320 Met Gly Phe Lys Ile Asp Asp Lys Gly Met Ala Pro Ala Leu Arg His 325 330 335 Leu Tyr Lys Glu Leu Met Gly Pro Trp Asn Lys Asp Glu Leu Ser Thr 340 345 350 Thr Asp Ala Ile Phe Val Gln Ala Asp Leu Lys Leu Val Gln Gly Phe 355 360 365 Met Pro His Phe Phe Arg Leu Phe Arg Ser Thr Val Lys Lys Val Asp 370 375 380 Phe Ser Glu Val Glu Arg Ala Arg Phe Ile Ile Asn Asp Trp Val Lys 385 390 395 400 Thr His Thr Lys Gly Met Ile Ser Asn Leu Leu Gly Lys Gly Ala Val 405 410 415 Asp Gln Leu Thr Arg Leu Val Leu Val Asn Ala Leu Tyr Phe Asn Gly 420 425 430 Gln Trp Lys Thr Pro Phe Pro Asp Ser Ser Thr His Arg Arg Leu Phe 435 440 445 His Lys Ser Asp Gly Ser Thr Val Ser Val Pro Met Met Ala Gln Thr 450 455 460 Asn Lys Phe Asn Tyr Thr Glu Phe Thr Thr Pro Asp Gly His Tyr Tyr 465 470 475 480 Asp Ile Leu Glu Leu Pro Tyr His Gly Asp Thr Leu Ser Met Phe Ile 485 490 495 Ala Ala Pro Tyr Glu Lys Glu Val Pro Leu Ser Ala Leu Thr Asn Ile 500 505 510 Leu Ser Ala Gln Leu Ile Ser His Trp Lys Gly Asn Met Thr Arg Leu 515 520 525 Pro Arg Leu Leu Val Leu Pro Lys Phe Ser Leu Glu Thr Glu Val Asp 530 535 540 Leu Arg Lys Pro Leu Glu Asn Leu Gly Met Thr Asp Met Phe Arg Gln 545 550 555 560 Phe Gln Ala Asp Phe Thr Ser Leu Ser Asp Gln Glu Pro Leu His Val 565 570 575 Ala Gln Ala Leu Gln Lys Val Lys Ile Glu Val Asn Glu Ser Gly Thr 580 585 590 Val Ala Ser Ser Ser Thr Ala Val Ile Ala Ser Ala Val Met Ala Pro 595 600 605 Glu Glu Ile Ile Met Asp Arg Pro Phe Leu Phe Val Val Arg His Asn 610 615 620 Pro Thr Gly Thr Val Leu Phe Met Gly Gln Val Met Glu Pro 625 630 635

Claims

1. A polypeptide comprising a plasminogen activator inhibitor 1 (PAI-1) variant, wherein the PAI-1 variant contains mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3) of the following amino acids: I91L, R101A, Q123K, V343A, and R346V, wherein, The variant is connected to a monomer or part of the Fc domain.

2. The polypeptide according to claim 1, wherein, The PAI-1 variant also includes additional mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3), namely: 1, K69A; 2, K80A; 3, K88A; 4, K176A; 5, K207A; 6, K263A; 7, K69A and K207A.

3. The polypeptide according to claim 1, wherein, The variant is connected to the Fc structural domain or part thereof.

4. The polypeptide according to claim 1, wherein, The polypeptide has one or more of the following characteristics: a) it can inhibit neutrophil elastase (NE) activity, b) it has a weakened ability to bind to fibronectin, and c) it has a weakened ability to bind to LRP1.

5. The polypeptide according to claim 1, wherein, The polypeptide can inhibit neutrophil elastase activity, wherein the neutrophil elastase binds within the neutrophil extracellular trap (NET).

6. A nucleic acid molecule encoding the polypeptide according to claim 1.

7. A carrier comprising the nucleic acid molecule according to claim 6.

8. A host cell expressing the polypeptide according to claim 1, wherein, The host cell includes the nucleic acid molecule according to claim 6 or the vector according to claim 7, wherein the nucleic acid molecule or the vector is expressed in the host cell.

9. A method for preparing the polypeptide according to claim 1, wherein, The method includes: a) providing a host cell comprising a nucleic acid molecule according to claim 6 or a vector according to claim 7, and b) expressing the nucleic acid molecule or vector in the host cell under conditions that allow the formation of the polypeptide.

10. A pharmaceutical composition comprising a polypeptide according to claim 1, a nucleic acid molecule according to claim 6, or a carrier according to claim 7, and one or more pharmaceutically acceptable excipients.

11. The pharmaceutical composition according to claim 10, wherein, The polypeptide is at a therapeutically effective level.

12. Use of the polypeptide of claim 1, the nucleic acid molecule of claim 6, the carrier of claim 7, or the pharmaceutical composition of claim 10 in the preparation of a medicament for treating a subject with IPF.

13. Use of the polypeptide of claim 1, the nucleic acid molecule of claim 6, the carrier of claim 7, or the pharmaceutical composition of claim 10 in the preparation of a medicament for treating a subject suffering from cystic fibrosis.

14. Use of the polypeptide of claim 1, the nucleic acid molecule of claim 6, the carrier of claim 7, or the pharmaceutical composition of claim 10 in the preparation of a medicament for treating a subject suffering from ARDS.

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