Polypeptide inhibitors of neutrophil elastase activity and uses thereof

Abstract

To provide an improved method for treating IdiopathicPulmonaryFibrosis (IPF).SOLUTION: The invention features a polypeptide comprising a variant of plasminogen-activator inhibitor-1 (PAI-1) having a reduced capacity to bind vitronectin, a reduced capacity to interact with the PAI-1 clearance receptor LDL-receptor-related-protein 1 (LRP1), and a capacity to efficiently inhibit neutrophil elastases in presence of neutrophil extracellular traps (NETs). In some embodiments, a polypeptide of the present invention comprises a PAI-1 variant optionally fused to an Fc domain monomer or moiety. The invention also features pharmaceutical compositions and methods of using the polypeptides to treat diseases and conditions characterized by aberrant neutrophil elastase activity (as an example, idiopathic pulmonary fibrosis).SELECTED DRAWING: Figure 1

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 938,859, filed November 21, 2019, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention features polypeptides comprising variants of plasminogen activator inhibitor 1 (PAI-1) that have a reduced ability to bind vitronectin, a reduced ability to interact with the PAI-1 clearance receptor LDL receptor-related protein 1 (LRP1), and the ability to efficiently inhibit neutrophil elastase (NE) in the presence of neutrophil extracellular traps (NETs). In some embodiments, the polypeptides of the invention comprise PAI-1 variants optionally fused to a monomer or portion of an Fc domain. The present invention also features pharmaceutical compositions and methods using the polypeptides for treating diseases and conditions characterized by aberrant neutrophil elastase activity (e.g., idiopathic pulmonary fibrosis).

[0003] [Introduction] Idiopathic pulmonary fibrosis (IPF) is a progressive, 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 patients with IPF in the United States, with an estimated 30,000 to 40,000 new cases diagnosed each year (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 rate 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. Care Med. 2016;37:477-484). The prevalence of IPF increases with age, and most patients with IPF are 60 years of age or older at the time of diagnosis.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).

[0004] The etiology of IPF remains unknown. However, potential factors such as smoking, dust exposure, and infectious agents have been associated with the development of IPF. IPF is characterized by progressive and irreversible distortion of lung architecture as a result of epithelial and endothelial cell apoptosis, fibroblast hyperplasia, and extracellular matrix remodeling (see Chakraborty et al., (2014) Expert Opin Investig Drugs, 23:893-910). As interstitial fibrosis progresses along with distortion of lung architecture, lung compliance decreases, increasing respiratory effort and leading to dyspnea. While lung function typically declines slowly over time, some patients experience a rapid decline, which can lead to hospitalization or death, especially in the later stages of the disease.

[0005] Development of therapeutic agents for IPF has been slow. The first two drugs for treating IPF, pirfenidone and nintedanib, were approved only 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, significant side effects, and require complex dosing regimens. Recent phase 3 clinical trials of pirfenidone, sildenafil, bosentan, etanercept, and interferon gamma-1b failed to demonstrate efficacy in their primary endpoints. N-acetylcysteine ​​(NAC), corticosteroids, and the immunosuppressants cyclophosphamide and azathioprine are commonly prescribed, but there is little evidence that the use of these medications improves patient outcomes or alters the natural history 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 that improves survival, but most patients with IPF are ineligible for transplant due to age or comorbid conditions. Patients with IPF are usually managed with supportive care, including symptomatic treatment of cough and dyspnea, supplemental oxygen for hypoxemia, smoking cessation, pulmonary rehabilitation, and prevention and control of respiratory tract infections.

[0006] Improved methods for treating IPF are needed.

[0007] The present invention addresses this need.

[0008] Summary of the Invention While it is well established that the complex of plasminogen activator inhibitor 1 (PAI-1) and its target enzyme binds tightly to LDL receptor-related protein 1 (LRP1), the molecular details of this interaction have not been fully elucidated. Furthermore, there is considerable debate in the literature regarding the nature of the interaction of free PAI-1 with LRP1. In experiments conducted in the course of developing embodiments of the present invention, the binding of free PAI-1 and a complex of PAI-1 and low-molecular-weight urokinase-type plasminogen activator (uPA) to LRP1 was tested. Data confirm that the uPA:PAI-1 complex binds to LRP1 with approximately 100-fold increased affinity over PAI-1 alone. Chemical modification of PAI-1 confirms that lysine residues in PAI-1 are essential and required for the interaction of both PAI-1 and the uPA:PAI-1 complex with LRP1. Surface plasmon resonance measurements support 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 binding suggests the involvement of two key charged residues for the interaction of PAI-1 with LRP1 and three charged residues for the interaction of the uPA:PAI-1 complex with LRP1. The enhanced affinity resulting from the interaction of three regions of the uPA:PAI-1 complex with the LDP1 repeats of LRP1 provides a molecular explanation for the increased affinity of the uPA:PAI-1 complex for LRP1. Mutational analysis reveals overlap between LRP1 binding and the binding site of CDE-096, a small molecule inhibitor of PAI-1 (specific PAI-1 inhibitor). It also reveals the critical roles of K207 in the interaction of PAI-1 with LRP1 and K207, K88, and K80 in the interaction of the uPA:PAI-1 complex with LRP1.

[0009] Experiments conducted during the development of embodiments of the present invention revealed the relative binding affinities of PAI-1 and the protease:PAI-1 complex with LRP1. Further experiments were conducted to determine whether binding of the protease:PAI-1 complex to LRP1 is primarily due to determinants on PAI-1. Furthermore, experiments were conducted to determine the specific amino acid residues involved in the binding of free PAI-1 and PAI-1 in complex with its target protease, urokinase-type plasminogen activator (uPA), to LRP1. Indeed, mutational analysis revealed overlap between LRP1 binding and the binding site of CDE-096, a small molecule inhibitor of PAI-1, and also revealed the critical roles of K207 in the interaction of PAI-1 with LRP1 and K207, K88, and K80 in the interaction of the uPA:PAI-1 complex with LRP1.

[0010] Idiopathic Pulmonary Fibrosis (IPF) is characterized by interstitial scar tissue formation, which can dramatically limit lung function. Approximately 130,000 people in the United States have IPF, with an estimated 30,000 to 40,000 new cases diagnosed each year (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 a diagnosis of IPF is typically 3 to 5 years (see Lederer, DJ and Martinez, FJ, NEJM 2018, 378:1811-1823). There is no effective curative treatment other than lung transplantation. The lack of treatments to halt the progression of IPF presents a significant challenge and represents a major unmet medical need. Two currently approved medications, pirfenidone and nintedanib, have been shown to slow disease progression but do not halt progression or restore lost lung function. Therefore, novel therapies for the treatment of IPF are needed for disease management.

[0011] The present invention addresses this need by providing compositions containing mutant PA1-I polypeptides capable of inhibiting NE, particularly NE bound to NETs. Provided herein are PAI-I mutants capable of inhibiting NE activity while reducing their ability to bind to vitronectin and LRP1. Indeed, experiments conducted during the course of developing embodiments of the present invention demonstrated that such PAI-I mutants have improved efficacy for treating conditions associated with NE activity (e.g., IPF) by inhibiting their ability to bind to vitronectin through modification of PAI-I amino acid residues involved in vitronectin binding (e.g., R101A and Q123K).

[0012] Indeed, experiments conducted during the development of embodiments of the present invention utilized serpin mapping techniques to identify mutant forms of PAI-1 (e.g., 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) as NE inhibitors capable of efficiently inhibiting NE within the NET environment. These mutant forms of PAI-1 were shown to irreversibly inhibit NE bound to DNA, a major component of NETs. The FDA-approved human plasma-derived A1AT, branded Aralast, was ineffective. In certain embodiments, these mutant forms of PAI-1 are further associated with human IgG-Fc.

[0013] Thus, the present invention features a polypeptide comprising a PAI-1 variant that can inhibit NE activity by modifying amino acid residues involved in vitronectin binding (e.g., R101A and Q123K), while reducing its ability to bind to vitronectin and thereby improving pharmacokinetics (PK) by modifying amino acid residues involved in LRP1 binding (e.g., K207, K88, and K80), thereby improving efficacy for treating conditions associated with NE activity (e.g., IPF). In some embodiments, the polypeptides 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 polypeptides 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 increases the stability or improves pharmacokinetics of the polypeptide.

[0014] Such moieties may be fused or attached by amino acid or other covalent bonds and may increase the stability of the polypeptide. A polypeptide comprising a PAI-1 variant fused to an Fc domain monomer may also form a dimer (e.g., a homodimer or heterodimer) through interaction between the two Fc domain monomers. In some embodiments, the polypeptide described herein to which an Fc domain monomer is attached is fused to the polypeptide by a linker. In some embodiments, the linker is an amino acid spacer.

[0015] The polypeptides of the present invention can be used to inhibit NE activity, and can be used to inhibit NE activity bound to NETs. Furthermore, the polypeptides of the present invention can be used to treat subjects with conditions characterized by abnormal NE activity (e.g., IPF). Furthermore, the polypeptides of the present invention can be used to prevent subjects from suffering from conditions characterized by abnormal NE activity (e.g., IPF). Furthermore, the polypeptides of the present invention can also be used to affect NE activity in subjects at risk of developing or having a disease or condition associated with abnormal NE activity.

[0016] In some embodiments, the invention provides a polypeptide comprising a PAI-1 variant, characterized in that the variant is a polypeptide 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 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, as set forth in SEQ ID NO: 5. In some embodiments, the PAI-1 variant comprises 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 comprises the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3): I91L, R101A, Q123K, V343A, R346V. In some embodiments, the PAI-1 variant comprises the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3): I91L, R101A, Q123K, K207A, V343A, R346V. This variant should have an extended half-life, particularly in the Fc form with full activity against NE in NETS.

[0017] In some embodiments, the invention features a polypeptide comprising a PAI-1 variant linked to a monomer or portion of an Fc domain, 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 a monomer or portion of an Fc domain 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 associated with a monomer or portion of an Fc domain comprises 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 associated with a monomer or portion of an Fc domain comprises 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). In some embodiments, the PAI-1 variant associated with a monomer or portion of an Fc domain comprises the following mutations in the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3): I91L, R101A, Q123K, K207A, V343A, R346V.

[0018] In some embodiments, the polypeptides described herein are capable of inhibiting NE, and in particular, inhibiting NE bound to NETs.

[0019] In some embodiments, the invention features a nucleic acid molecule encoding a polypeptide described herein (e.g., a polypeptide comprising a PAI-1 mutant 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 another aspect, the invention also features a vector comprising a nucleic acid molecule described herein.

[0020] In another aspect, the invention provides a host cell expressing a polypeptide as described herein, wherein the host cell comprises a nucleic acid molecule or a vector as described in the previous two aspects, and wherein the nucleic acid molecule or the vector is expressed in the host cell.

[0021] In another aspect, the invention features a method for preparing a polypeptide described herein, the method comprising the steps of: a) providing a host cell comprising a 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 the formation of the polypeptide.

[0022] In another aspect, the invention features a pharmaceutical composition comprising a polypeptide, nucleic acid molecule, or vector described herein and one or more pharmaceutically acceptable carriers or excipients. In some embodiments of the pharmaceutical composition, the polypeptide, nucleic acid molecule, or vector is in a therapeutically effective amount.

[0023] In another aspect, the invention also features a construct comprising two identical polypeptides (e.g., homodimers) each comprising a PAI-1 mutant 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 mutant is fused to the N-terminus or C-terminus of an Fc domain monomer, and wherein the two Fc domain monomers of the two polypeptides interact to form the Fc domain.

[0024] In another aspect, the present invention also features a construct comprising two different polypeptides (e.g., heterodimers) each containing a PAI-1 variant having a different combination of 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, wherein the two variants are fused to the N-terminus or C-terminus of an Fc domain monomer, and the two Fc domain monomers of the two polypeptides interact to form an Fc domain.

[0025] In another aspect, the invention features a method for inhibiting NE activity in a subject in need thereof. In another aspect, the invention features a method for inhibiting NE activity bound to NETs in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0026] In some embodiments of the methods for inhibiting NE activity or NE activity bound within NETs in a subject, 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 methods for inhibiting NE activity or NE activity bound within NETs in a subject, the subject has defective A1AT activity and / or expression.

[0027] In another aspect, the invention features a method of treating a subject with IPF by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0028] In another aspect, the invention features a method of treating a subject with cystic fibrosis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0029] In another aspect, the invention features a method of treating a subject with COPD by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0030] In another aspect, the invention features a method of treating a subject with emphysema by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0031] In another aspect, the invention features a method of treating a subject with acute respiratory distress syndrome (ARDS) by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0032] In another aspect, the invention features a method of treating a subject having ischemia-reperfusion injury by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0033] In another aspect, the invention features a method of treating a subject with ethanol-induced chronic pancreatitis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0034] In another aspect, the invention features a method of treating a subject with rheumatoid arthritis (RA) by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0035] In another aspect, the invention features a method of treating a subject having disseminated intravascular coagulation (DIC) by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0036] In another aspect, the invention features a method of treating a subject with ulcerative colitis (UC) by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0037] In another aspect, the invention features a method of treating a subject with Crohn's disease by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0038] In another aspect, the invention features a method of treating a subject having a skin disease associated with neutrophil pathology by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0039] In another aspect, the invention features a method for treating a subject having a deficiency in A1AT activity and / or expression by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0040] In another aspect, the invention features a method of treating a subject having any condition characterized by abnormal NE activity and / or expression by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0041] In another aspect, the invention features a method of treating a subject having any condition characterized by deficient A1AT activity and / or expression by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0042] In some embodiments of any of the above aspects, 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, skin diseases).

[0043] In some embodiments of any of the above aspects, the subject has or is at risk of developing a condition characterized by deficient A1AT activity and / or expression.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Essential role of lysine residues in PAI-1 for the binding of PAI-1 and the LMWuPA:PAI-1 complex to LRP1. A. Binding of the LMWuPA:PAI-1 complex and free PAI-1 to LRP1 was analyzed by SPR, and 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) form complexes with LMWuPA (lanes 3 and 4, respectively). Lane 5, LMWuPA. C. 250 nM PAI-1 and 300 nM PAI-1 chemically modified with sulfo-NHS-acetic acid were injected over an SPR chip with immobilized LRP1. D. 9 nM of the LMWuPA:PAI-1 complex and 80 nM of the complex formed with chemically modified PAI-1 were injected over an SPR chip on which LRP1 was immobilized.

[0045] Figure 2. Binding of PAI-1 to LRP1 depends on ionic strength. A. Increasing concentrations of PAI-1 in buffer containing increasing concentrations of NaCl were injected over an LRP-1-coated SPR chip, and Req values ​​were determined. Data are normalized to Rmax for each NaCl concentration. NaCl concentrations from the top curve down: 150 mM, 250 mM, 500 mM, 750 mM, and 1000 mM. B. Debye-Huckel plot of PAI-1 binding to LRP1. KD values ​​at each ionic strength (150, 250, 500, 750, and 1000 mM NaCl) were measured by equilibrium SPR measurements. Three independent experiments were performed, and plotted values ​​are the mean ± SE. A slope of 1.5 ± 0.1 was determined by linear regression analysis. Similar values ​​for slope were obtained by averaging the results of linear regression analyses from individual experiments.

[0046] Figure 3. Binding of PAI-1 to LRP1 is well described by a bivalent binding model. (A) Schematic of the bivalent binding model for the interaction of two different regions of PAI-1 with complementary sites on LRP1. (B) Increasing concentrations of PAI-1 (9.4, 37.5, 75, and 300 nM) were injected over an SPR chip bound to LRP1. The dissociation for each concentration was measured from the SPR data and normalized to 100% of the initial value at t = 0. The data were fitted to a biexponential decay (blue line). C. Increasing concentrations of PAI-1 (3.9, 7.8, 15.6, 31.2, 62.5, and 125 nM) were injected over an LRP1-bound chip. The fit of the experimental data (black line) to the bivalent binding model is shown in blue. The data shown is a representative experiment of six independent experiments performed.

[0047] Figure 4. PAI-1 binding to cluster IV of LRP1 fits well to a bivalent binding model. (A) Schematic diagram showing the domain organization of LRP1. Clusters of ligand-binding repeats (red circles) are labeled I, II, III, and IV. (B) Increasing concentrations of PAI-1 (9.4, 15.6, 31, 62.5, and 125 nM) were injected over an SPR chip bound to LRP1 cluster IV. Dissociation for each concentration was measured from the SPR data and normalized to 100% of the initial value at t = 0. The data were fitted to a biexponential decay (blue line). C. Increasing concentrations of PAI-1 (3.9, 7.8, 15.6, 31.2, 62.5, and 125 nM) were injected over a chip bound to LRP1 cluster IV. The fit of the experimental data (black line) to a bivalent binding model is shown in blue. Data shown are representative of three independent experiments performed.

[0048] Figure 5. CDE-096 inhibits the binding of HMWuPA:PAI-1 complexes to LRP1. A. 1 nM HMWuPA:PAI-1 complexes were flowed over an LRP1-bound SPR chip in the absence of CDE-096 (top curve) and in the presence of increasing concentrations of CDE-096 (15.6, 31.2, 62.5, 125, 250, 500 nM). B. Plot of the initial slope of the association phase from panel A versus CDE-096 concentration. An IC50 of 70 ± 11 nM was determined by nonlinear regression analysis. Data are representative of two independent experiments.

[0049] Figure 6. Binding of LMWuPA:PAI-1 complexes to LRP1 depends on ionic strength. A. Increasing concentrations of uPA:PAI-1 complexes were flowed over an LRP-1-coated SPR chip in the presence of increasing concentrations of NaCl, and Req values ​​were determined. Data are normalized to Rmax for each NaCl concentration. NaCl concentrations from the top curve down: 150 mM, 250 mM, 500 mM, 750 mM, and 1000 mM. B. Debye-Huckel plot of LMWuPA:PAI-1 binding to LRP1. KD values ​​at each ionic strength (150 mM, 250 mM, 500 mM, 750 mM, and 1000 mM NaCl) were measured by equilibrium SPR measurements. Three independent experiments were performed, and the mean ± SE is plotted. A slope of 2.4 ± 0.4 was determined by linear regression analysis. Identical values ​​were obtained by averaging the results of linear regression analysis of the individual experiments.

[0050] Figure 7. LMWuPA:PAI-1 complex binds to LRP1 via a complex kinetics model. A) Model used to analyze binding of 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 model of binding occurs (Scheme II). B) Increasing concentrations of LMWuPA:PAI-1 complex (3.12, 6.25, 12.5, 25, 50 nM) were injected over the chip with bound LRP1. The dissociation for each concentration was measured from the SPR data and normalized to 100% of the initial value at t = 0. B. Increasing concentrations of LMWuPA:PAI-1 (0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM) were injected over the chip with bound LRP1. Fitting of experimental data (black line) to the model comprising Schemes I and II is shown (blue line). Data are representative of three independent experiments.

[0051] Figure 8. Kinetic analysis of LMW uPA:PAI-1 complex binding to cluster IV of LRP1. A) Increasing concentrations of uPA:PAI-1 complex (0.6, 1.2, 2.5, 5, 10, 20, and 40 nM) were injected over the chip bound to LRP1. Dissociation for each concentration was measured from SPR data and normalized to 100% for the initial value at t = 0. B) Increasing concentrations of LMW uPA:PAI-1 (0.6, 1.2, 2.5, 5, 10, 20, and 40 nM) were injected over the LRP1-bound chip. Fitting of experimental data (black lines) to Scheme I and II models (blue lines). Data are representative of three independent experiments.

[0052] Figure 9. LRP1-mediated cellular uptake of LMWuPA:PAI-1 is reduced when complexed with PAI-1 containing lysine residue mutations. 5 nM 125I-labeled LMWuPA:PAI-1 complexes formed with I91L PAI-1 or the indicated mutant PAI-1 molecules were incubated with WI-38 human fibroblasts for 6 hours at 37°C in the absence or presence of excess RAP. After incubation, the amount of internalized complex was quantified. Experiments were performed in triplicate.

[0053] FIG. 10. The wild-type PAI-1 nucleic acid sequence (SEQ ID NO: 1); the wild-type PAI-1 amino acid sequence (SEQ ID NO: 2); and the mature wild-type PAI-1 amino acid sequence (SEQ ID NO: 3) are provided.

[0054] Figure 11. Provided is a mature variant PAI-1 nucleic acid sequence (SEQ ID NO:4) encoding a polypeptide having 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; and a mature variant PAI-1 amino acid sequence (SEQ ID NO:5) having 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.

[0055] Figure 12. Provided is a mature variant PAI-1 / Fc nucleic acid sequence (SEQ ID NO:6) encoding a polypeptide having the following mutations within 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) having the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, R346V.

[0056] FIG. 13 shows that MDI-1001 targets inflammatory nets better than Aralast.

[0057] FIG. 14 shows an in vitro comparison of Aralast, Avelestat, MDI-1002, MDI-1003, and MDI-1004.

[0058] FIG. 15 shows that MDI-1003 targets NETs in CF sputum.

[0059] FIG. 16 shows elastase activity as a function of inhibitor concentration.

[0060] FIG. 17 shows that MDI-1002 protects against acute lung injury.

[0061] FIG. 18 shows that MDI-1002 protects against pulmonary fibrosis.

[0062] FIG. 19 shows that MDI-1002 does not improve recovery after bleomycin.

[0063] FIG. 20 shows that inhaled MDI-1003 protects against acute lung injury.

[0064] FIG. 21 shows that MDI-1003 protects against pulmonary fibrosis better than MDI-1001.

[0065] FIG. 22 shows that MDI-1003 improves recovery after bleomycin.

[0066] FIG. 23 shows the constructs of Fc fusions of MDI-1002 and MDI-1004.

[0067] FIG. 24 shows the expression of Fc fusions of MDI-1002 and MDI-1004.

[0068] Figure 25 shows that Fc fusions improve PK.

[0069] Figure 26 shows that mutation of LRP1-binding residues affects the inhibition of neutrophil elastase in the presence of DNA NETs, ​​thereby demonstrating reduced interaction with the clearance receptor, LRP1, in NETs and retention of activity against elastase.

[0070] [Definition] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. An Fc domain comprises at least C H 2 domain and C HThe Fc domain monomer has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100% sequence identity) to a human Fc domain comprising three domains. The Fc domain monomer comprises a second and a third antibody constant domain (C H 2 and C H In some embodiments, the Fc domain monomer also includes a hinge domain. The Fc domain does not include any portion of an immunoglobulin that can act as an antigen recognition region (e.g., a variable domain or a complementarity determining region (CDR)). In a wild-type Fc domain, two Fc domain monomers are joined by two CDRs. H Dimerization occurs through interactions between the three antibody constant domains, as well as one or more disulfide bonds formed between the hinge domains of the two dimerizing Fc domain monomers. In some embodiments, the Fc domain may be mutated to lack the effector functions typical of a "dead Fc domain." In certain embodiments, each Fc domain monomer in the Fc domain may be mutated to reduce the interaction or binding between the Fc domain and the Fcγ receptor. H The Fc domain may comprise an amino acid substitution in the Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. The Fc domain may be of any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. Furthermore, the Fc domain may be of an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a non-naturally occurring Fc domain, for example, a recombinant Fc domain.

[0071] As used herein, the terms "fused" or "attached" are used to describe the combination or joining of two or more elements, components, or protein domains (e.g., peptides or polypeptides) by means including chemical conjugation, recombinant methods, and chemical bonds (e.g., amide bonds). For example, two single peptides in tandem can be fused to form one continuous protein structure (e.g., a polypeptide) via chemical conjugation, chemical bonds, peptide linkers, or any other covalent bonding means.

[0072] As used herein, the term "polypeptide" refers to a single polymer in which the monomers are amino acid residues covalently linked together through amide bonds. Polypeptide is intended to encompass any amino acid sequence, whether naturally occurring, recombinant, or synthetically produced.

[0073] As used herein, the term "homodimer" refers to a molecular construct formed by two identical macromolecules (e.g., proteins or nucleic acids). Two identical monomers can form a homodimer through covalent or non-covalent bonds. For example, an Fc domain can be a homodimer of two Fc domain monomers if the two Fc domain monomers contain the same sequence. In another example, a polypeptide described herein comprising a PAI-1 variant fused to an Fc domain monomer can form a homodimer through the interaction of the two Fc domain monomers that form the Fc domain in the homodimer.

[0074] As used herein, the term "heterodimer" refers to a molecular construct formed by two different macromolecules (e.g., proteins or nucleic acids). The two monomers may form a heterodimer through covalent or non-covalent bonds. For example, a polypeptide described herein comprising a PAI-1 variant fused to an Fc domain monomer may form a heterodimer through the interaction of the two Fc domain monomers, each fused to a different PAI-1 variant that forms the Fc domain in the heterodimer.

[0075] As used herein, the term "host cell" refers to a vehicle that contains the cellular components (e.g., organelles) necessary to express proteins from their corresponding nucleic acids. The nucleic acids are typically contained in a nucleic acid vector that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cell may be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a mammalian cell (e.g., a CHO cell or HEK293 cell)).

[0076] As used herein, the term "therapeutically effective amount" refers to an amount of a polypeptide, nucleic acid, or vector of the present invention, or a pharmaceutical composition comprising a polypeptide, nucleic acid, or vector of the present invention, effective to achieve a desired therapeutic effect in treating a patient with a disease such as any condition characterized by abnormal NE activity and / or deficient A1AT activity (e.g., IPF, COPD, cystic fibrosis, emphysema, ARDS, ischemia-reperfusion, chronic pancreatitis, RA, DIC, UC, Crohn's disease, skin diseases). The term "therapeutically effective amount" also refers to an amount of a polypeptide, nucleic acid, or vector of the present invention, or a pharmaceutical composition comprising a polypeptide, nucleic acid, or vector of the present invention, effective to achieve a desired therapeutic effect in treating a patient with such a condition. In particular, a therapeutically effective amount of a polypeptide, nucleic acid, or vector avoids adverse side effects.

[0077] As used herein, the term "pharmaceutical composition" refers to a medicinal or pharmaceutical preparation containing an active ingredient and an excipient and diluent to allow the active ingredient to be compatible with the administration method. The pharmaceutical composition of the present invention contains pharmaceutically acceptable ingredients that are compatible with the polypeptide, nucleic acid, or vector. The pharmaceutical composition can be in the form of a tablet or capsule for oral administration, or in an aqueous form for intravenous or subcutaneous administration.

[0078] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to an excipient or diluent in a pharmaceutical composition. A pharmaceutically acceptable carrier must be compatible with other ingredients of the formulation and not harmful to the patient. In the present invention, a pharmaceutically acceptable carrier or excipient must provide sufficient pharmaceutical stability for a polypeptide, including a PAI-1 variant, a nucleic acid molecule encoding the polypeptide, or a vector containing such a nucleic acid molecule. The nature of the carrier or excipient will vary depending on the mode of administration. For example, an aqueous carrier is generally used for intravenous administration, while a solid carrier is preferred for oral administration.

[0079] As used herein, the term "treating and / or preventing" refers to treating and / or preventing a disease (e.g., any condition characterized by having abnormal NE activity and / or deficient A1AT activity (e.g., IPF, COPD, cystic fibrosis, emphysema)) using the methods and compositions of the present invention. Generally, treating such a disease occurs after a subject has developed the disease and / or has already been diagnosed as having the disease. Preventing such a disease refers to a step or procedure performed when a subject is at risk of developing the disease. A subject may exhibit signs or mild symptoms that are determined by a physician to have symptoms or risk factors for developing the disease, or may have a family history or genetic predisposition to developing the disease but have not yet developed the disease.

[0080] As used herein, the term "subject" refers to a mammal (e.g., preferably a human). Mammals include, but are not limited to, humans, as well as domestic and farm animals such as monkeys, mice, dogs, cats, horses, and cattle.

[0081] Detailed Description of the Invention Idiopathic Pulmonary Fibrosis (IPF) is characterized by interstitial scar tissue formation, which can dramatically limit lung function. Approximately 130,000 people in the United States have IPF, 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 a diagnosis of IPF is typically 3 to 5 years (see Lederer, DJ, and Martinez, FJ, NEJM 2018; 378:1811-1823). There is no effective curative treatment other than lung transplantation. The lack of treatments to halt the progression of IPF presents a significant challenge and represents a major unmet medical need. Two currently approved medications, pirfenidone and nintedanib, have been shown to slow disease progression but do not halt progression or restore lost lung function. Therefore, novel therapies for the treatment of IPF are needed for disease management.

[0082] The present invention addresses this need by providing compositions comprising mutant PA1-I polypeptides that are capable of inhibiting NE, particularly NE bound to NETs.

[0083] The present invention addresses this need by providing compositions containing mutant PA1-I polypeptides capable of inhibiting NE, particularly NE bound to NETs. Provided herein are PAI-I mutants capable of inhibiting NE activity while reducing their ability to bind to vitronectin and / or LRP1. Indeed, experiments conducted during the course of developing embodiments of the present invention demonstrated that such PAI-I mutants have improved efficacy for treating conditions associated with NE activity (e.g., IPF) by inhibiting their ability to bind to vitronectin through modification of PAI-I amino acid residues involved in vitronectin binding (e.g., R101A and Q123K).

[0084] Experiments conducted during the development of embodiments of the present invention utilized serpin mapping techniques to identify mutant forms of PAI-I (e.g., having one or more of the following mutations in the wild-type human mature PAI-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 efficiently inhibiting NE in the NET environment. These mutant forms of PAI-I were shown to irreversibly inhibit NE bound to DNA, a major component of NETs. The FDA-approved human plasma-derived A1AT, branded Aralast, was ineffective. In certain embodiments, these mutant forms of PAI-I are further associated with human IgG-Fc.

[0085] As part of the innate immune response, neutrophils are the most abundant leukocytes in peripheral blood and are at the forefront of defense against infection. Neutrophils efficiently clear microbial infections by phagocytosis and by oxygen-dependent and oxygen-independent mechanisms. Recently, a novel neutrophil antibacterial mechanism has been described: the release of NETs, ​​which are composed of DNA, histones, and antimicrobial peptides. These mutant forms of PAI-1 represent the first therapeutic approach specifically targeting NE, which is responsible for a significant amount of 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)). This prospect of more effective treatment and possible disease reversal would be very appealing to patients suffering from this rare disease.

[0086] Clinical indications for these mutant forms of PAI-1 include idiopathic pulmonary fibrosis (as NE-NETs are involved in the pathogenesis of fibrosis, including the differentiation of lung fibroblasts), COPD (see Grabcanovic-Musija, F., et al., 2015 Respir. Res. 16:59), cystic fibrosis, where elevated NE-NETs represent pulmonary diseases that, when manifested, limit current treatment options, emphysema, ARDS, ischemia-reperfusion, chronic pancreatitis, RA, DIC, UC, Crohn's disease, and skin diseases.

[0087] Thus, the present invention features polypeptides comprising PAI-1 variants that can inhibit NE activity while reducing the ability to bind to vitronectin and LRP1 by modifying amino acid residues involved in vitronectin binding (e.g., R101A and Q123K) and / or amino acid residues involved in LRP1 binding (e.g., K207, K88, and K80), resulting in improved pharmacokinetics (PK) and efficacy for treating conditions associated with NE activity (e.g., IPF). In some embodiments, the polypeptides 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 polypeptides 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 increases the stability or improves pharmacokinetics of the polypeptide. Polypeptides comprising a PAI-1 variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interaction between the two Fc domain monomers. The PAI-1 variants described herein can inhibit NE activity, and can inhibit NE activity where NE is bound within NETs. The present invention also includes methods for treating diseases and conditions involving abnormal NE activity and / or deficient A1AT activity in a subject by administering to the subject a polypeptide comprising a PAI-1 variant described herein.

[0088] Elastase is a serine proteinase released by activated neutrophils, macrophages, and monocytes. During inflammatory responses, neutrophils are activated, releasing elastase and causing tissue destruction through proteolysis. In the lungs, elastase degrades elastic tissue, causing emphysema. Elastase is also an exacerbating factor in cystic fibrosis (CF) and acute respiratory distress syndrome (ARDS) in both adults and infants. Elastase is also involved in TNF-mediated inflammation (see Massague, J. et al., Annu. Rev. Biochem. 62:515-541 (1993)) and HIV infection (Bristow, CL et al., International Immunol. 7:239-249 (1995)).

[0089] Elastase has a broader spectrum of reactivity than plasminogen activators, each of which preferentially acts on and activates precursor substrates.

[0090] The natural defense against elastase is a protein called α1-antitrypsin (α1AT) or α1-proteinase inhibitor (α1PI). Patients with α1AT deficiency, especially smokers, are prone to emphysema. Furthermore, smoking induces inflammation. In such α1AT deficiency, the enzyme is present (CRM + ), but is functionally impaired. Furthermore, even in individuals with normal enzymes, smoking directly inactivates α1AT. Therefore, improved inhibitors of elastase would be highly desirable for the prevention of emphysema in susceptible subjects or for the reversal of the pathophysiological processes leading to this and other related diseases.

[0091] The major PAIs belong to the serine proteinase inhibitor (serpin) gene superfamily, which includes many proteinase inhibitors in the blood, as well as other proteins with unrelated or unknown function (see Gettins, PGW, and Olson, ST (2016) Inhibitory serpins. New insights into their folding, polymerization, regulation, and clearance. Biochem. J. 473, 2273-2293). Serpins share a common tertiary structure and have evolved from a common ancestor. Serpins regulate many processes, including coagulation, fibrinolysis, complement activation, ovulation, angiogenesis, inflammation, neoplasia, viral pathogenesis, and allergic responses.

[0092] Serpins act as suicide inhibitors, reacting only once with target proteinases to form sodium dodecyl sulfate (SDS)-stable complexes. These complexes can dissociate to produce free, active enzymes with cleaved inhibitors similar to those seen in the α1AT crystal structure (see Gettins, PGW, and Olson, ST (2016) Inhibitory serpins. New insights into their folding, polymerization, regulation, and clearance. Biochem. J. 473, 2273-2293).

[0093] 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 efficient inhibitor known of the single- and two-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 thrombin and activated protein C, although with much lower efficiency.

[0094] The PAI-1 cDNA encodes a 402-amino acid protein containing a typical secretory signal sequence (see Ny et al., supra; Ginsburg et al., 1986, supra). Mature human PAI-1 isolated from cell culture consists of two variants of approximately equal size, 381 and 379 amino acids. Figure 10 provides the human wild-type PAI-1 nucleic acid sequence (SEQ ID NO: 1); the human wild-type PAI-1 amino acid sequence (SEQ ID NO: 2); and the human mature wild-type PAI-1 amino acid sequence (SEQ ID NO: 3).

[0095] PAI-1 is a glycoprotein with three potential N-linked glycosylation sites containing 15–20% carbohydrate (Van Mourik JA et al., supra). Mature PAI-1 contains no cysteine ​​residues, facilitating efficient expression and isolation of recombinant PAI-1 from E. coli. PAI-1 produced in E. coli is unglycosylated but functionally very similar to native PAI-1. Recombinant PAI-1 can be isolated from E. coli in a constitutively active form (see below), in contrast to PAI-1 purified from mammalian cell culture (Lawrence et al., 1989, supra; Hekman et al., 1988, supra).

[0096] PAI-1 exists in an active form when produced by cells and secreted into the culture medium, and in an inactive or latent form that accumulates in the culture medium over time (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 the latent form with a half-life of approximately 1 hour at 37°C (see Lawrence et al., supra, Hekman et al., supra; Levin EG et al, 1987, supra).

[0097] The latent form can be converted to the active form by treatment with denaturing agents, negatively charged phospholipids or Vn (see Lambers et al., supra; Hekman et al., supra; Wun TC et al., J Biol Chem (1989) 264:7862-7868). Latent PAI-1 injected into rabbits was reactivated in vivo by an unknown mechanism. The reversible interconversion between active and latent structures, presumably by a conformational change, is a unique feature of PAI-1 compared with other serpins. The latent form appears to be more energetically favorable.

[0098] The three-dimensional structure of latent PAI-1 has been solved, in which the entire N-terminal portion of the reactive center loop is inserted as a central strand into β-sheet A (see Mottonen et al., supra), explaining its increased stability (see Lawrence, DA et al., Biochemistry 33: 3643-3648 (1994)) and lack of inhibitory activity.

[0099] The activity of two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (TPA), is regulated by plasminogen activator inhibitor 1 (PAI-1), a serine proteinase inhibitor (serpin) that regulates fibrinolysis and wound healing and is associated with thrombosis and fibrotic diseases. Serpins function to inhibit serine proteases by a unique mechanism following cleavage of the serpin's reactive center loop, which induces a conformational change in the serpin, resulting in protease inhibition (for a review, see (Gettins, PGW, and Olson, ST (2016) Biochem. J. 473, 2273-2293)). Once a serpin is complexed with a protease, the complex is rapidly removed from the circulation in the liver by binding to LDL receptor-related protein 1 (LRP1) (see Kounnas, MZ, Church, FC, Argraves, WS, and Strickland, DK (1996) J. Biol. Chem. 271, 6523-6529).

[0100] LRP1 was originally identified as a hepatic receptor involved in the clearance of the alpha2-macroglobulin protease complex (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 remnant lipoprotein particles (see Rohlmann, A., et al., (1998) J. Clin. Invest. 101, 689-695). In addition to its endocytic role, 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 modularly composed of LDLa repeats, EGF-like repeats, and clusters of β-propeller domains. Efficient delivery of newly synthesized LRP1 to the cell surface requires the participation of an endoplasmic reticulum-resident chaperone called receptor-associated protein (RAP) (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).

[0101] The fact that LRP1 recognizes multiple structurally unrelated ligands with relatively high affinity has raised questions regarding the nature of the ligand / receptor interaction. Insight into how this may occur comes from the recognition that K256 and K270 are essential for the third domain of RAP to bind to LRP1 (see Migliorini, MM, et al., (2003) J. Biol. Chem. 278, 17986-17992) and from the crystal structure of the third domain of RAP in complex with two LDLa repeats from the LDL receptor (see Fisher, C., et al., (2006) Mol. Cell. 22, 277-283). These studies revealed that the ε-amino groups of K256 and K270 of RAP form salt bridges with the carboxylate salts of aspartic acid residues within the LDLa repeats that form the acidic pocket on the receptor. To date, several ligands, including alpha2-macroglobulin (α2M) (see Arandjelovic, S., Hall, BD, and Gonias, SL (2005) Arch. Biochem. Biophys. 438, 29-35) and blood 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 through interactions involving key lysine residues.

[0102] Lysine residues appear to contribute to the interaction of PAI-1 with 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), but studies investigating the interaction of PAI-1 with LRP1 have yielded conflicting data. First, questions exist regarding the relative affinity of PAI-1 versus the protease:PAI-1 complex for LRP1. Most studies have demonstrated that only PAI-1 in complex with proteases binds 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) have reported that PAI-1 alone binds with high affinity to a fragment derived from LRP1.Second, based on the observation that the protease:PAI-1 complex binds to LRP1 with higher affinity than PAI-1 alone, some have proposed that the formation of a protease complex with PAI-1 exposes a cryptic 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). In contrast, others have argued that the protease itself may interact with LRP1 and contribute to the high-affinity interaction (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 appears to be little consensus that lysine residues constitute the binding site.

[0103] Experiments conducted during the development of embodiments of the present invention revealed the relative binding affinities of PAI-1 and protease:PAI-1 complexes with LRP1. Further experiments were conducted to determine whether binding of the protease:PAI-1 complex to LRP1 is primarily due to determinants on PAI-1. Furthermore, experiments were conducted to determine the specific amino acid residues in free PAI-1 and PAI-1 in complex with its target protease, urokinase-type plasminogen activator (uPA), that are involved in their binding to LRP1. Indeed, mutational analysis revealed an overlap between LRP1 binding and the binding site of CDE-096, a small molecule inhibitor of PAI-1, with K207 playing a key role in the interaction of PAI-1 with LRP1, and K207, K88, and K80 playing a key role in the interaction of the uPA:PAI-1 complex with LRP1.

[0104] In experiments conducted during the development of embodiments of the present invention, serpin mapping techniques were used to identify mutant forms of PAI-I (e.g., having one or more of the following mutations in the wild-type human mature PAI-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 efficiently inhibiting NE in the NET environment. These mutant forms of PAI-I were shown to irreversibly inhibit NE bound to DNA, a major component of NETs. The FDA-approved human plasma-derived A1AT, branded Aralast, was ineffective. In certain embodiments, these mutant forms of PAI-I further bind human IgG-Fc.

[0105] Thus, the present invention is characterized by a polypeptide comprising a PAI-1 variant. In some embodiments, the polypeptide of the present invention comprises a PAI-1 variant fused to the N-terminus or C-terminus of an Fc domain monomer or portion. In some embodiments, the polypeptide of the present invention comprises a PAI-1 variant fused to the N-terminus or C-terminus of an Fc domain monomer or portion. In some embodiments, the Fc domain monomer or portion increases the stability or improves the pharmacokinetics of the polypeptide.

[0106] Such moieties may be fused or attached by amino acid or other covalent bonds and may increase the stability of the polypeptide. A polypeptide comprising a PAI-1 variant fused to an Fc domain monomer may also form a dimer (e.g., a homodimer or heterodimer) through interaction between the two Fc domain monomers. In some embodiments, the polypeptide described herein to which an Fc domain monomer is attached is fused to the polypeptide by a linker. In some embodiments, the linker is an amino acid spacer.

[0107] The polypeptides of the present invention can be used to inhibit NE activity, and can be used to inhibit NE activity bound to NETs. Furthermore, the polypeptides of the present invention can be used to treat subjects with conditions characterized by abnormal NE activity (e.g., IPF). Furthermore, the polypeptides of the present invention can be used to prevent subjects from suffering from conditions characterized by abnormal NE activity (e.g., IPF). Furthermore, the polypeptides of the present invention can also be used to affect NE activity in subjects at risk of developing or having a disease or condition associated with abnormal NE activity.

[0108] In some embodiments, the invention provides a polypeptide comprising a PAI-1 variant, characterized in that the variant is a polypeptide 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 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, as set forth in SEQ ID NO: 5. In some embodiments, the PAI-1 variant comprises 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 comprises the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3): I91L, R101A, Q123K, V343A, R346V.

[0109] In some embodiments, the invention features a polypeptide comprising a PAI-1 variant linked to a monomer or portion of an Fc domain, 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 a monomer or portion of an Fc domain 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 associated with a monomer or portion of an Fc domain comprises 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 associated with a monomer or portion of an Fc domain comprises the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3): I91L, R101A, Q123K, V343A, R346V (SEQ ID NO: 7).

[0110] In some embodiments, the polypeptides described herein are capable of inhibiting NE, particularly inhibiting NE bound to NETs.

[0111] In some embodiments, the invention features a nucleic acid molecule encoding a polypeptide described herein (e.g., a polypeptide comprising a PAI-1 mutant 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 another aspect, the invention also features a vector comprising a nucleic acid molecule described herein.

[0112] In another aspect, the invention provides a host cell expressing a polypeptide as described herein, wherein the host cell comprises a nucleic acid molecule or a vector as described in the previous two aspects, and wherein the nucleic acid molecule or the vector is expressed in the host cell.

[0113] In another aspect, the invention features a method for preparing a polypeptide described herein, the method comprising the steps of: a) providing a host cell comprising a 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 the formation of the polypeptide.

[0114] In another aspect, the invention features a pharmaceutical composition comprising a polypeptide, nucleic acid molecule, or vector described herein and one or more pharmaceutically acceptable carriers or excipients. In some embodiments of the pharmaceutical composition, the polypeptide, nucleic acid molecule, or vector is in a therapeutically effective amount.

[0115] FIG. 11 provides the nucleic acid sequence (SEQ ID NO:4) of a mature mutant PAI-1 encoding a polypeptide having 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; and provides the mature mutant PAI-1 amino acid sequence (SEQ ID NO:5) having 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.

[0116] FIG. 12 provides a mature variant PAI-1 / Fc nucleic acid sequence (SEQ ID NO: 6) encoding a polypeptide having the following mutations within 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) having the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3): I91L, R101A, Q123K, V343A, R346V.

[0117] In some embodiments, the polypeptides described herein may comprise a PAI-1 variant fused to an Fc domain monomer or a fragment of an Fc domain of an immunoglobulin to increase the serum half-life of the polypeptide. A polypeptide comprising a PAI-1 variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a heterodimer) through the interaction between the two Fc domain monomers that form the Fc domain in the dimer. As conventionally known in the art, an Fc domain is a protein structure found at the C-terminus of an immunoglobulin. An Fc domain is a C H The Fc domain comprises two Fc domain monomers that are dimerized by interactions between the three antibody constant domains. Wild-type Fc domains form a minimal configuration that binds to Fc receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, FcγRIV). In some embodiments, the Fc domain can be mutated to lack effector functions typical of "dead" Fc domains. For example, the Fc domain can contain specific amino acid substitutions known to minimize interactions between the Fc domain and Fcγ receptors.

[0118] The polypeptides of the present invention can be produced from host cells. Host cells refer to vehicles containing the cellular components (e.g., organelles) necessary for expressing the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. The nucleic acids can be contained in nucleic acid vectors that can be introduced into host cells by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). The selection of nucleic acid vectors depends in part on the host cells used. Generally, preferred host cells are either eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.

[0119] Nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present invention can be prepared by various 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 conventional techniques (e.g., gene synthesis). Alternatively, nucleic acid molecules encoding wild-type PAI-1 can be mutated to contain specific amino acid substitutions using conventional techniques (e.g., QuikChange™ mutagenesis). Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR technology.

[0120] The nucleic acid sequence encoding the polypeptide of the present invention can be inserted into a vector capable of replicating and expressing the nucleic acid molecule 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 can contain various components that can be adjusted and optimized for compatibility with specific host cells.For example, vector components can include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding the target protein, and a transcription termination sequence.

[0121] In some embodiments, mammalian cells can be used as host cells for 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, NS0, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. coli cells can also be used as host cells for the present invention. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® 31,537), E. coli BL21(DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31,608). Different host cells have characteristic and specific mechanisms for the post-translational processing and modification (e.g., glycosylation) of protein products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed polypeptide. The above-described expression vectors can be introduced into suitable host cells using conventional techniques in the art, such as 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 conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the gene encoding the desired sequence.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.

[0122] Host cells used to produce the polypeptides of the present invention can be grown in media known in the art and appropriate for culturing the selected host cells. Examples of media suitable for mammalian host cells include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Expi293™ Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of media suitable for bacterial host cells include Luria broth (LB) supplemented with necessary supplements, such as a selection agent (e.g., ampicillin). Host cells are cultured at an appropriate temperature, such as about 20°C to about 39°C (e.g., 25°C to about 37°C, preferably 37°C), and at a CO2 level, such as 5-10%. The pH of the medium is generally about 6.8 to 7.4 (e.g., 7.0), depending primarily on the host organism. When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions appropriate for promoter activation.

[0123] In some embodiments, depending on the expression vector and host cells used, the expressed protein may be secreted from the host cells (e.g., mammalian host cells) into the cell culture medium. Protein recovery may include filtering the cell culture medium to remove cellular debris. The protein may be further purified. The polypeptides of the present invention may be purified by any method known in the art 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 purifying proteins. For example, proteins can be isolated and purified by appropriately selecting and combining affinity columns, such as Protein A columns (e.g., POROS Protein A chromatography) and chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting out, and dialysis.

[0124] In other embodiments, host cells can be disrupted by osmotic shock, sonication, or lysis to recover the expressed protein. Once the cells are disrupted, cellular debris can be removed by centrifugation or filtration. In some instances, the polypeptide can be conjugated to a marker sequence (e.g., a peptide) to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His tag), which binds to nickel-functionalized agarose affinity columns with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (see Wilson et al., Cell 37:767, 1984).

[0125] Alternatively, the polypeptides of the present invention can be produced by cells of a subject (e.g., a human) by administering a vector (such as a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxvirus vector (e.g., a vaccinia virus vector such as modified vaccinia Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector)) containing a nucleic acid molecule encoding a polypeptide of the present invention, for example, in the context of gene therapy. Once the vector is inside the subject's cells (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), it promotes expression of the polypeptide, which can then be secreted from the cells. If treatment of a disease or condition is the desired result, further treatment may not be required. If collection of protein is desired, blood can be collected from the subject and the protein can be purified from the blood by methods known in the art.

[0126] The present invention features pharmaceutical compositions comprising a polypeptide described herein (e.g., a polypeptide comprising a PAI-1 variant (e.g., a PAI-1 variant having one or more of the following mutations in 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, as a therapeutic protein, a polypeptide comprising a PAI-1 variant with a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids). In some embodiments, the pharmaceutical compositions of the present invention comprise, as a therapeutic protein, a polypeptide comprising a PAI-1 variant fused to a moiety (e.g., an Fc domain monomer or dimer thereof, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization)). In some embodiments, the pharmaceutical compositions of the invention comprise a polypeptide comprising a PAI-1 variant fused to a first moiety (e.g., an Fc domain monomer, or a dimer thereof, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization)).

[0127] In some embodiments, pharmaceutical compositions of the present invention comprising a polypeptide of the present invention may be used in therapy in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions. In addition to a therapeutically effective amount of the polypeptide, pharmaceutical compositions may contain one or more pharmaceutically acceptable carriers or excipients, which can be formulated by methods known to those skilled in the art. In some embodiments, pharmaceutical compositions of the present invention comprise a nucleic acid molecule (DNA or RNA (e.g., mRNA)) encoding a polypeptide of the present invention, or a vector comprising such a nucleic acid molecule.

[0128] Acceptable carriers and excipients in pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations used. Acceptable carriers and excipients may include buffers (such as phosphate, citrate, HEPES, and TAE), antioxidants (such as ascorbic acid and methionine), preservatives (such as hexamethonium chloride, octadecyldimethylbenzylammonium 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. Injectable pharmaceutical compositions can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, and cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagle Medium (α-MEM), F-12 medium). Formulation methods are 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).

[0129] The pharmaceutical compositions of the present invention can be prepared in microcapsules, such as hydroxymethylcellulose 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 techniques are described in Remington: The Science and Practice of Pharmacy 22 thPharmaceutical compositions to be used for in vivo administration must be sterile, which can be readily accomplished by filtration through sterile filtration membranes.

[0130] The pharmaceutical compositions of the present invention can also be prepared as sustained-release formulations. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptides of the present invention. Examples of sustained-release matrices include polyesters, hydrogels, polyactides, copolymers of L-glutamic acid and y-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™, and poly-D-(-)-3-hydroxybutyric acid. Some sustained-release formulations allow release of molecules over several months (e.g., 1 to 6 months), while other formulations release the pharmaceutical compositions of the present invention over shorter periods (e.g., days to weeks).

[0131] The pharmaceutical composition can be prepared in the form of a unit dose, if necessary. The amount of the active ingredient (e.g., the polypeptide of the present invention) contained in the pharmaceutical preparation is such that an appropriate dosage within a specified range is obtained (e.g., a dosage within the range of 0.01 to 100 mg / kg body weight).

[0132] The pharmaceutical composition for gene therapy can be in an acceptable diluent, or can comprise a slow-release matrix in which the gene delivery vehicle is embedded.When hydrodynamic injection is used as a delivery method, the pharmaceutical composition comprising the nucleic acid molecule encoding the polypeptide described herein or the vector (e.g., viral vector) comprising the nucleic acid molecule is rapidly delivered intravenously by large-volume infusion.Vector that can be used as in vivo gene delivery vehicle includes, but is not limited to, retrovirus vector, adenovirus vector, poxvirus vector (e.g., vaccinia virus vector such as modified vaccinia Ankara), adeno-associated virus vector, and alphavirus vector.

[0133] Pharmaceutical compositions containing the polypeptide of the present invention as a therapeutic protein can be formulated for, for example, intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intraarterial administration, intrathecal administration, or intraperitoneal administration. Pharmaceutical compositions can also be formulated for or administered via oral, nasal, spray, aerosol, rectal, or vaginal administration. For injectable formulations, various effective pharmaceutical carriers are known in the art. For example, see ASHP Handbook on Injectable Drugs, Toissel, 18th ed. (2014).

[0134] In some embodiments, pharmaceutical compositions containing nucleic acid molecules encoding the polypeptides of the present invention, or vectors containing such nucleic acid molecules, can be administered by 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, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors such as modified vaccinia Ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, mRNA molecules encoding the polypeptides of the present invention can be directly administered to subjects.

[0135] In some embodiments of the present 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, nucleic acid molecules encoding the polypeptides described herein are placed under the control of a strong promoter in a genetically engineered plasmid (e.g., a viral plasmid). The plasmid is often rapidly delivered intravenously via large volume infusion. Hydrodynamic injection uses controlled intravenous hydrodynamic pressure to enhance cell permeability, and the increased pressure from the rapid infusion of large volumes of fluid results in venous infusion and extravasation of the plasmid. Expression of the nucleic acid molecule is primarily driven by the liver. In mice, hydrodynamic injection is often performed by injecting the plasmid into the tail vein. In certain embodiments, mRNA molecules encoding the polypeptides described herein can be administered using hydrodynamic injection.

[0136] The dosage of the pharmaceutical compositions of the present invention depends on factors including the route of administration, the disease being treated, and the subject's physical characteristics (e.g., age, weight, general health). Pharmaceutical compositions of the present invention may contain a dosage of a polypeptide of the present invention ranging 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), or in more specific embodiments, from about 0.1 to about 30 mg / kg, and in more specific embodiments, from about 0.3 to about 30 mg / kg. The dosage can be adjusted by a physician according to conventional factors, such as the extent of the disease and various parameters of the subject.

[0137] Pharmaceutical compositions are administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective to improve or remediate symptoms. Pharmaceutical compositions are administered in various dosage forms, for example, intravenous, subcutaneous, and oral dosage forms (e.g., ingestible solutions, drug-release capsules). Generally, therapeutic proteins are administered at 0.1 to 100 mg / kg, for example, 1 to 50 mg / kg. Pharmaceutical compositions comprising the polypeptides of the present invention can be administered to a subject in need thereof, for example, one or more times (e.g., 1 to 10 or more times) daily, weekly, biweekly, monthly, bimonthly, quarterly, biennially, yearly, or as medically indicated. In some embodiments, pharmaceutical compositions comprising the polypeptides of the present invention can be administered to a subject in need thereof weekly, biweekly, monthly, bimonthly, or quarterly. Dosages can be given in either a single or multiple dose regimen. The timing between doses can be decreased as the condition improves or the patient's health increases.

[0138] The present invention is based on the discovery that substitution of one or more specific amino acids from human PAI-1 can inhibit NE activity and inhibit NE activity when NE is bound within NETs. The properties of these PAI-1 mutants make them useful therapeutic agents that can be used in the treatment of diseases characterized by abnormal NE activity and / or deficient A1AT activity.

[0139] In another aspect, the invention features a method of inhibiting NE activity in a subject in need thereof. In another aspect, the invention features a method of inhibiting NE activity bound to NETs in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0140] In some embodiments of the methods for inhibiting NE activity or the activity of NE bound in NETs in a subject, 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 methods for inhibiting NE activity or the activity of NE bound in NETs in a subject, the subject has defective A1AT activity and / or expression.

[0141] In another aspect, the invention features a method of treating a subject with IPF by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0142] In another aspect, the invention features a method of treating a subject with cystic fibrosis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0143] In another aspect, the invention features a method of treating a subject with COPD by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0144] In another aspect, the invention features a method of treating a subject with emphysema by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0145] In another aspect, the invention features a method of treating a subject with acute respiratory distress syndrome (ARDS) by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0146] In another aspect, the invention features a method of treating a subject having ischemia-reperfusion injury by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0147] In another aspect, the invention features a method of treating a subject with ethanol-induced chronic pancreatitis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0148] In another aspect, the invention features a method of treating a subject with rheumatoid arthritis (RA) by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0149] In another aspect, the invention features a method of treating a subject with disseminated intravascular coagulation (DIC) by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0150] In another aspect, the invention features a method of treating a subject with ulcerative colitis (UC) by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0151] In another aspect, the invention features a method of treating a subject with Crohn's disease by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0152] In another aspect, the invention features a method of treating a subject having a skin disease associated with neutrophil pathology by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0153] In another aspect, the invention features a method for treating a subject having a deficiency in A1AT activity and / or expression by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0154] In another aspect, the invention features a method of treating a subject having any condition characterized by abnormal NE activity and / or expression by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0155] In another aspect, the invention features a method of treating a subject having any condition characterized by deficient A1AT activity and / or expression by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0156] In some embodiments of any of the above aspects, the subject has or is at risk of developing a condition characterized by having aberrant NE activity (e.g., IPF, COPD, cystic fibrosis, emphysema).

[0157] In some embodiments of any of the above aspects, the subject has or is at risk of developing a condition characterized by having deficient A1AT activity and / or expression.

[0158] Those skilled in the art will readily recognize that the foregoing merely represents a detailed description of certain preferred embodiments of the present invention. Various modifications and variations of the compositions and methods described above can be readily accomplished using the expertise available in the art and are within the scope of the present invention.

[0159] 〔experiment〕 Example I In this example, we demonstrate that the LMWuPA:PAI-1 complex binds to LRP1 with higher affinity than PAI-1 alone.

[0160] To resolve the discrepancies in the literature regarding the relative affinities of PAI-1 and protease:PAI-1 complexes for LRP1, 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, the experiments in this example and all subsequent studies (unless otherwise specified) used the I91L mutant of PAI-1, which extends the stability of PAI-1 from a half-life of 2.0 hours to 18.4 hours (see Berkenpas, MB, Lawrence, DA, and Ginsburg, D. (1995) EMBO J. 14, 2969-77). Experiments were performed to form a complex between I91L PAI-1 and LMWuPA, and the binding of the complex to LRP1 was compared to that of free PAI-1 using equilibrium SPR measurements. From the data (Fig. 1A), it is clear that the LMWuPA:PAI-1 complex binds to LRP1 almost two orders of magnitude more strongly than PAI-1 alone (K of LMWuPA:PAI-1). D = 0.9 ± 0.2 nM, while KD of PAI-1 = 74 ± 13 nM).

[0161] Example II In this example, we demonstrate that lysine residues in PAI-1 are essential for binding of both PAI-1 and the LMWuPA:PAI-1 complex to LRP1.

[0162] To determine the contribution of lysine residues to the interaction of PAI-1 with LRP1, these residues were chemically modified with sulfo-NHS-acetate, which forms a stable covalent amide bond with the primary amine of the lysine residue. This modification does not prevent PAI-1 from forming a stable complex with LMWuPA (Figure 1B). Notably, however, this modification prevented the binding of both free PAI-1 (Figure 1C) and the complex of LMWuPA and modified PAI-1 (Figure 1D) to LRP1. These results clearly demonstrate that the lysine residues of PAI-1 contribute to the binding of both PAI-1 and the LMWuPA:PA1-1 complex to LRP1.

[0163] Example III In this example, we demonstrate that two charged residues are involved in the binding of PAI-1 to LRP1.

[0164] Next, we performed experiments to examine the effect of ionic strength on the binding of PAI-1 to LRP1. The results of these experiments clearly demonstrate that the binding of PAI-1 to LRP1 is dependent on ionic strength (Figure 2A). Figure 2B shows the Log 10 K D The data are plotted in the form of a Debye-Huckel plot, plotting σ against ionic strength. These results suggest the involvement of two ionic interactions in the binding of PAI-1 to LRP1, as indicated by the slope (slope = 1.5 ± 0.1). This is consistent with the standard model for ligand binding to LDL receptor family members, in which two (or more) ε-amino groups of specific lysine residues in the ligand form salt bridges with the carboxylate salts of aspartic acid residues within the LDL1 repeat (Fisher, C., et al., (2006) Mol. Cell. 22, 277-283).

[0165] Example IV In this example, we demonstrate that kinetic analysis supports a bivalent model for the binding of PAI-1 to LRP1.

[0166] The data in Figure 2 suggest the involvement of these two charged residues in the interaction of PAI-1 with LRP1, raising the possibility of a bivalent binding model in which high-affinity binding arises from an avidity effect mediated by the interaction of two regions of PAI-1, each containing a charged residue, with the two LDR repeats of LRP1 (Figure 3A). A similar model has been proposed for FVIII binding to LRP1 (16). To test this model, we performed kinetic measurements examining the binding of I91L PAI-1 to LRP1 using surface plasmon resonance experiments. To gain insight into the potential mechanism, we first compared the kinetics of PAI-1 dissociation from LRP1 at various concentrations of ligand (Figure 3B). These results clearly show that dissociation kinetics occurs in two phases: a fast phase followed by a much slower phase. Furthermore, as expected for a bivalent model, the dissociation kinetics is independent of ligand concentration. The dissociation rate constant determined from fitting the experimental data was used as an initial estimate for the dissociation phase when the association and dissociation kinetics were simultaneously fit to a global bivalent model. This fit revealed that the experimental data were well described by a bivalent binding model (Figure 3C). The kinetic data from the best fit (Table I) reveal rapid association of PAI-1 with LRP1 to form complex I, with conversion to complex II occurring with a half-life of 97 seconds. Importantly, the equilibrium binding constant K derived from the kinetic analysis (65 ± 6 nM) was 0.01. D A K value of 56 ± 4 nM was 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. The kinetic and equilibrium binding data are summarized in Table I, and the rate constants and K D The rate constants and K values ​​for wild-type PAI-1 are similar to those for D reveals the value.

[0167] [Table 1] The ligand-binding region of LRP1 is primarily localized to clusters of LDLa repeats, designated clusters I, II, III, and IV (Figure 4A). Among these clusters, most ligands bind to clusters II, III, or IV. Therefore, we performed experiments to examine PAI-1 binding to clusters II, III, and IV. Initial experiments revealed that I91L PAI-1 interacts with clusters II and IV with similar affinity, but with much weaker affinity to cluster III. Next, we performed detailed experiments using cluster IV, the major ligand-binding region of LRP1. Figure 4B confirms that dissociation of PAI-1 from cluster IV also occurs in two phases and is independent of PAI-1 concentration. Comparison of the fits with the experimental data reveals that binding is consistent with a bivalent binding model (Figure 4C). The best fitting parameters are summarized in Table I, with a K of 49 ± 18 nM estimated by equilibrium analysis of the SPR data. D A K of 55 ± 5 nM derived from kinetic data is close to the value D Thus, these results demonstrate that the binding of I91L PAI-1 to cluster IV is similar to its binding to full-length LRP1.

[0168] Example V In this example, we demonstrate the critical role of K207 in the binding of PAI-1 to LRP1.

[0169] Because chemical modification of PAI-1 revealed the critical role of lysine residues in its interaction with LRP1, we initiated studies to identify the specific lysine residues that contribute to PAI-1 binding to LRP1. To gain further insight into regions of PAI-1 that may be important for its interaction with LRP1, we tested the possibility that CDE-096 could block the binding of the HMWuPA:PAI-1 complex to LRP1. CDE-096 is a small-molecule inhibitor that reversibly binds to PAI-1 and inhibits its interaction with proteases 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-096 was added to the HMWuPA:PAI-1 complex, a dose-dependent inhibition of HMWuPA:PAI-1 binding to LRP1 was observed (Figure 5A). IC of 70 nM 50 was determined by replotting the initial slope of the association curve against CDE-096 concentration (Fig. 5B).

[0170] Structural studies have revealed that K207 and K263 contribute to 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 performed that included these mutants of PAI-1 in the analysis, along with K69, K80, and K88, which have previously been identified as important for PAI-1 binding to the LDLa repeats from cluster II (see Gettins, PGW, and Dolmer, K. (2016) J. Biol. Chem. 291, 800-812). In these studies, SPR equilibrium measurements revealed that K DK values ​​were determined and the data are summarized in Table II. The data demonstrate that mutation of K207 to alanine had the greatest effect on PAI-1 binding to LRP1, reducing affinity by 19-fold. Furthermore, mutation of K69, K80, and K88 to alanine resulted in a 7-fold, 7-fold, and 9-fold reduction in affinity for LRP1, respectively. Interestingly, PAI-1 molecules with mutations at both K80 and K207, or mutants in which K80, K207, and K88 were all replaced by alanine, did not substantially reduce the affinity of binding over the K207A mutant alone, resulting in a 20-fold and 21-fold reduction in K, respectively. D This resulted in an increase in

[0171] The data in Table II also show the specific surface area of ​​specific side chains based on the three-dimensional structure of PAI-1. The fraction of accessible surface area of ​​a side group is the solvent-accessible area of ​​the protein divided by the accessible surface area calculated for the residues in the extended Gly-Xaa-Gly tripeptide (see Willard, L., et al., (2003) Nucleic Acids Res. 31, 3316-3319), with values ​​near 1 being fully accessible and values ​​near zero being buried. The R76E PAI-1 mutant binds LRP1 poorly (see Stefansson, S. (1998) J. Biol. Chem. 273, 6358-6366). R76 has been proposed to be 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 reveals that this residue is buried and unavailable for direct interaction with LRP1. Similarly, K263 and K122, which have been implicated in previous studies, are also partially buried in the structure and therefore unavailable for direct interaction with LRP1.

[0172] [Table 2] Example VI In this example, we demonstrate that binding of the LMWuPA:PAI-1 complex to LRP1 occurs via a complex mechanism.

[0173] To characterize the binding of LMWuPA:PA1 to LRP1, experiments were performed to examine the ionic strength dependence of binding. The results, shown in Figure 6A, demonstrate a significant dependence of binding on ionic strength. The Debye-Huckel plot (Figure 6B) yielded a slope of 2.4 ± 0.3, suggesting the involvement of two to three ionic interactions in the binding.

[0174] When examining the interaction kinetics, we observed that the dissociation rate changed with higher concentrations of the LMWuPA:PAI-1 complex (Figure 7B). This is also evident from the data in Figure 7C, where faster dissociation was observed at higher LMWuPA:PAI-1 concentrations, suggesting multiple binding mechanisms. This was previously observed for the binding of RAP domains D1D2 to LRP1 (see Prasad, JM, et al., (2016) J. Biol. Chem. 291, 18430-18439). Therefore, we also incorporated a second scheme into the model in which the LMWuPA:PAI-1 complex can bind to a second, separate site on LRP1 to form a monovalent complex (Complex III, Figure 7A, Scheme II). To simplify the model, we used the k in Scheme II. a1 and k d1 is the first step in Scheme I a1 and k d1 and were assumed to be identical. Fitting the experimental SPR data to a model including both Scheme I and Scheme II gave an excellent fit (Figure 7C), and the kinetic parameters are summarized in Table III. The high affinity binding of the LMWuPA:PAI-1 complex to LRP1 was observed to be significantly higher than the K of PAI-1 alone. D K, which is about 100 times larger than D was confirmed, which is mainly due to the slow dissociation rate of the LMWuPA:PAI-1 complex (k for PAI-1 in Table I). d1and k d2 (Compare with those values ​​for the LMWuPA:PAI-1 complex in Table III).

[0175] [Table 3] In experiments, we 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 rate was not independent of ligand concentration (Figure 8A). Therefore, we fitted the data to the model described in Figure 7A. The data were well described by the fits (Figure 8B). The parameters obtained from these fits are summarized in Table III, revealing that the LMWuPA:PAI-1 complex binds slightly weaker to cluster IV than full-length LRP1. These results suggest that the LMWuPA:PAI-1 complex may also interact with regions of LRP1 outside of cluster IV.

[0176] Example VII In this example, we demonstrate that the PA1-I mutant reveals that additional residues are involved in the interaction of the LMWuPA:PAI-1 complex with LRP1.

[0177] To determine whether similar lysine residues in PAI-1 are also involved in the interaction of the uPA:PAI-1 complex with LRP1, experiments were performed in which LMWuPA was also complexed with 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, the individual mutants of PAI-1 (K69A, K88A, and K207A) had minimal effect on the binding of the LMWuPA:PAI-1 complex to LRP1, although K80A significantly reduced the K by 5.8-fold. DPAI-1 molecules containing the K80A and K207A double mutations reduced the binding of the LMWuPA:PAI-1 complex to LRP1 by 23-fold. Surprisingly, the triple mutation of K80A, K207A, and K88A resulted in a 244-fold decrease in affinity (Table II), revealing the critical role of these three residues in the LMWuPA:PAI-1 complex for binding to LRP1.

[0178] Next, we examined the cellular uptake of PAI-1 and LMWuPA complexes formed from PAI-1 molecules containing the double or triple mutation (Figure 9). The results revealed that the double and triple mutants of PAI-1 were not effectively taken up by LRP1-expressing cells when complexed with LMWuPA.

[0179] Example VIII In this example, the materials and methods utilized in Examples I-VII are described.

[0180] (reagent) LMWuPA, HMWuPA, WT PAI-1 HMWuPA:PAI-1 complex, and I91L PAI-1 were purchased from Molecular Innovations. Mutant PAI-1 proteins were produced 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 ligand-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 for 1 hour at room temperature. Complex formation was confirmed by analyzing the proteins on a 4–20% Tris-gly gel (Novex) and staining with colloidal blue stain.

[0181] (Chemical modification of I91LPAI-1) Chemical modification of I91L PAI-1 to block primary amines in lysine side chains was performed using sulfo-NHS-acetate (Thermo-Fisher Scientific). Sulfo-NHS-acetate was dissolved in PBS at 50 mg / ml. 55 μg of I91L PAI-1 was incubated with a 50-fold excess of sulfo-NHS-acetate relative to the total amino groups in I91L PAI-1 in PBS at 4°C for 3 hours. The modified I91L PAI-1 protein was desalted in 0.01 M HEPES, 0.15 M NaCl, pH 7.4 using a NAP™-5 Sephadex G-25 column (GE Healthcare) to remove excess sulfo-NHS-acetate.

[0182] (surface plasmon resonance) Purified LRP1 was immobilized onto a CM5 sensor chip surface to a level of 10,000 response units using a working solution of 20 μg / ml LRP1 in 10 mM sodium acetate, pH 4. LRP1 ligand-binding cluster IV was immobilized according to the manufacturer's instructions (BIAcore). Cluster IV was immobilized on a CM5 sensor chip surface to a level of 2,000 response units using a working solution of 20 μg / ml in 10 mM sodium acetate, pH 4, according to (AB). An additional flow cell was activated and blocked with protein-free 1 M ethanolamine to serve as a control surface. Unless otherwise noted, 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, a pH 7.4 buffer was made 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 on a BIAcore 3000 instrument at a flow rate of 20 μl / min at 25° C. The sensor chip surface was regenerated by a 15 s injection of 100 mM phosphoric acid at a flow rate of 100 μl / min.

[0183] (SPR data analysis) Dissociation rates were fitted to a 2-exponential decay using GraphPad Prism 7.04 software. Kinetic data were analyzed against a bivalent model (Scheme 1) using numerical integration algorithms available in BIAevaluation software:

[0184]

number

[0185]

number

[0186] (Inhibition by CDE-096) The HMWuPA-PAI-1 complex was diluted to 2 nM in 0.01 M HEPES, 0.15 M NaCl, 1 mM CaCl, 0.0005% surfactant P, 0.1% DMSO, and 0–500 nM CDE-096, pH 7.8. Binding to LRP1 on the Biacore was performed as described above, except that the running buffer was 0.01 M HEPES, 0.15 M NaCl, 1 mM CaCl, 0.0005% surfactant P, 0.1% DMSO, pH 7.8.

[0187] (Uptake of LMWuPA PAI-1 complex by cells) WI38 cells were plated in 12-well tissue culture plates precoated with poly-D-lysine hydrobromide (Sigma). Cells were incubated in assay buffer (DMEM, 1% BSA, 20 mM HEPES) for 1 hour before treatment with the iodinated conjugates. LMWuPA was iodinated with I-125 sodium iodide (Perkin Elmer NEZ033) using Iodo-gen (Pierce) in PBS containing 1 mM 6-aminocaproic acid (Aldrich). The iodinated proteins were desalted in PBS using a PD-10 column (GE Healthcare) to remove free iodine. Labeled conjugates were formed by incubating I91L PAI-1 and its mutants (0.8 μM) with I-125 LMWuPA (0.4 μM) at room temperature for 1 hour. The resulting complex was diluted to 5 nM in assay buffer alone or assay buffer containing 1 μM RAP and placed on cells for 6 hours at 37°C. The medium was removed, and cells were washed with 2 ml of PBS and treated with trypsin (Corning 25-0520) containing 50 μg / ml proteinase K. Cells were centrifuged at 4000 rpm for 4 minutes. The supernatant was removed, and the cell pellet was counted to determine the internalized moles.

[0188] Example IX In this example, we describe the purification in E. coli of a PAI-1 mutant (hereinafter "MDI-1003") having the following mutations R101A and Q123K within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3).

[0189] The supernatant was clarified by centrifugation, and 200 ml of MDI-1003 fermenter lysate was dialyzed against 0.05 M sodium phosphate, 0.1 M sodium chloride, 0.001 M EDTA, pH 6.6, and then chromatographed on a 10 x 5.0 cm heparin-Sepharose 6B column at a flow rate of approximately 0.5 ml / min at room temperature. The heparin-Sepharose 6B column was washed with 2 L of 0.05 M sodium phosphate, 0.1 M sodium chloride, 0.001 M EDTA, pH 6.6, followed by a 600 ml gradient elution to 1.0 M sodium chloride in the same buffer. PAI-1-containing fractions were pooled, and solid ammonium sulfate was added to 18% saturation. PAI-1 was chromatographed on a phenyl-Sepharose Fast Flow (Low Sub) column (10 × 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 at room temperature at a flow rate of approximately 0.5 ml / min. 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 a 400 ml gradient elution to 0% ammonium sulfate in the same buffer. PAI-1-containing fractions were pooled and precipitated by adding solid ammonium sulfate to 65% saturation. The precipitate was dissolved at 3.5 mg / ml in 0.05 M sodium phosphate, 0.1 M sodium chloride, 0.001 M EDTA, pH 6.6, and then extensively dialyzed against the same buffer. The yield after a heparin-Sepharose 6B column was 90 ml containing 80 mg of protein (lane 1). The yield after a 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.

[0190] Example X (CF sputum titration for elastase concentration) Sputum from CF patients was extracted in 2 mL of cold PBS per 1 g of sputum and then hand-homogenized until smooth. Centrifuge at 10,000 × g for 20 minutes (4°C) and save the supernatant for elastase titration. Purified HNE was then diluted to 40 nM and 0, 2.5, 5, 7.5, 10, 12.5, 15, and 20 μL was added to each well (black plate) and brought to 100 μL with 40 mM Hepes, 100 mM NaCl, pH 7.4, 0.005% Tween-20. 100 μL of 500 μM MeOSuc-AAPV-AMC was added and the kinetic reading was taken at 10 min (ex370 em440). Note the slope and intercept.

[0191] Dilute CF sputum and add 0, 2.5, 5, 7.5, 10, 12.5, 15, and 20 μL to each well. Bring the volume to 100 μL with 40 mM Hepes, 100 mM NaCL, pH 7.4, 0.005% Tween-20. Add 100 μL of 500 μM MeOSuc-AAPV-AMC. Measure the ex370 and em440 values ​​over 10 minutes using the slope and intercept of the purified HNE. Back-calculate the elastase concentration in CF sputum.

[0192] (I C 50 / speed) Incubate 50 nM HNE or CF sputum + / - salmon sperm DNA or heparin at room temperature for 30 minutes. Serially dilute elastase inhibitor in a black plate (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 to the 90 μL of inhibitor from above. Incubate at room temperature for 30 seconds, 1 minute, 2 minutes, or longer, as needed. Add 100 μL of 500 μM MeOSucAAPV-AMC to each well and read kinetically at 370°C and 440°C for 10 minutes.

[0193] (e speed calculation) K obs = Ln (nM elastase remaining / nM elastance onset) / time (sec) Inhibitor concentration and V max and K. m K plotted against obs is determined by a nonlinear Michaelis-Menten fit.

[0194] K i =V max / K m (PK study of AVI and AVI-AK) The pharmacokinetic properties of AVI or AVI-AK were evaluated in 8-week-old C57BL / 6J male mice after intravenous (IV) bolus administration of 20 mg / kg. Blood samples were collected at 0.5, 1, 2, 6, and 24 hours and plasma PAI-1 levels were measured.

[0195] (Acute pulmonary edema caused by LPS injection) Wild-type C57BL / 6J mice (8-week-old males) were intratracheally instilled with 25 microliters of LPS at 2 mg / mL, followed by intratracheal treatment with vehicle, AVI, AVI-AK, or Alast (30 microliters at 1.3 mg / mL). After 18 hours, animals were perfused with PBS, and wet lung weights and total elastase were obtained.

[0196] (Lung extraction and homogenization) Obtain a 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. Homogenize at high speed for 1 minute. Centrifuge at 10,000 × g (4 °C) for 10 minutes, transfer the supernatant to a new tube, and respin at 10,000 × g for 10 minutes. Transfer the supernatant to a new tube for assay.

[0197] Fibrosis assay Fibrosis assays were performed essentially as described (Blood, 2011, 118:2313-2321). Briefly, weight- and age-matched (6-8 weeks old, 18-22 g) WT mice were treated with a single dose of intratracheal bleomycin (1.15 μg / kg in 50 L of sterile PBS) on day 0 to induce pulmonary fibrosis. Starting on day 1, mice were treated with either MDI-1001, MDI-1002, MDI-1003, or saline (4 mg / kg IP) twice daily to treat the acute injury phase. Mice were sacrificed on day 21, and pulmonary fibrosis was determined from hydroxyproline measurements as described (Blood, 2011, 118:2313-2321).

[0198] (Construction of HFc-AVI-AK expression vector and transfected CHO cell line) The following mutations were introduced into the mature form of human PAI-1 cDNA by site-directed mutagenesis (QuickChange II Kit, Agilent, Santa Clara, CA): I91L, R101A, Q123K, V343A, and R346V. The V343A, R346V, and I91L mutation (AVI) conveys a stable, active PAI-1 phenotype, whereas the R101A, Q123K mutation (AK) induces a reduced vitronectin-binding phenotype. The modified cDNA (designated AVI-AK) was cloned into the pcDNA5 / FRT plasmid with an N-terminal fusion peptide sequence consisting of the human immunoglobulin (IgG1) constant region, consisting of domains 2 and 3, including the hinge region sequence (HFc). The integrity of the construct was confirmed by restriction digest screening and DNA sequencing. The validated HFc-AVI-AK fusion plasmid was co-transfected into Chinese hamster ovary (CHO) cells (InVitrogen) at a 9:1 ratio with the pOG44 plasmid (carrying the Flp recombinase gene) using GeneJuice reagent (Novagen / Millipore) to promote integration of a single copy of the fusion protein sequence. After incubating the transfected cells at 37°C and 6% CO for 48 hours, 100 μg / ml hygromycin (Invivogen) was added to select for cells with integrated AVI-AK cDNA. Following selection and growth of transfected cells in Ham's F12 medium (supplemented with 10% fetal bovine serum and hygromycin), the cell culture was adapted to growth in serum-free medium (CHOgro, Mirus Bio) to promote non-adherent growth, expand cell density, and simplify purification. Protein expression was regulated by a constitutive cytomegalovirus (CMV) promoter, and therefore, cell supernatant media containing HFc-AVI-AK was collected approximately every 3–5 days for downstream processing.

[0199] (Purification of HFc-AVI-AK) 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 in PBS, followed by extensive 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, pH 5.6, to stabilize the pH and yield >95% pure protein. The protein eluate was then immediately applied to heparin Sepharose equilibrated in 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. This second step exploits the unique properties of PAI-1 to concentrate the protein and achieve >99% purity.

[0200] (result) - MDI-1001 - A PAI-1 mutant with the following stabilizing mutations within the wild-type mature PAI-1 amino acid sequence (SEQ ID NO: 3): I91L, and mutations V343A and R346V that allow NE inhibition. -Fc fusion protein with MDI-1002-MDI-1001 variants - MDI-1003 - A PAI-1 variant of MDI-1001 with additional mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3): R101A and Q123K, which abolish the vitronectin binding function of PAI-1. -Fc fusion protein with MDI-1004-MDI-1003 variants FIG. 13 shows that MDI-1001 targets inflammatory nets better than Aralast.

[0201] FIG. 14 shows an in vitro comparison of Aralast, Avelestat, MDI-1002, MDI-1003, and MDI-1004.

[0202] FIG. 15 shows that MDI-1003 targets NETs in CF sputum.

[0203] FIG. 16 shows elastase activity as a function of inhibitor concentration.

[0204] FIG. 17 shows that MDI-1002 protects against acute lung injury.

[0205] FIG. 18 shows that MDI-1002 protects against pulmonary fibrosis.

[0206] FIG. 19 shows that MDI-1002 does not improve recovery after bleomycin.

[0207] FIG. 20 shows that inhaled MDI-1003 protects against acute lung injury.

[0208] FIG. 21 shows that MDI-1003 protects against pulmonary fibrosis better than MDI-1001.

[0209] FIG. 22 shows that MDI-1003 improves recovery after bleomycin.

[0210] FIG. 23 shows the constructs of Fc fusions of MDI-1002 and MDI-1004.

[0211] FIG. 24 shows the expression of Fc fusions of MDI-1002 and MDI-1004.

[0212] Figure 25 shows that Fc fusions improve PK.

[0213] Figure 26 shows the inhibition curves against neutrophil elastase plus or minus DNA NETs. This data indicates that the mutations that confer optimal activity against neutrophil elastase can be combined with each of the mutations shown in Table II that reduce binding to clearance receptors, resulting in improved pharmacokinetics of the molecule. There are seven mutants in total: six with single receptor-binding mutations and one with two receptor-binding mutations. The latter double mutant demonstrates that it is possible to combine mutations that potentially result in an even greater reduction in clearance receptor binding. These include the mutations I91L, R101A, Q123K, V343A, and R346V.

[0214] The MDI names for each variant are as follows: K69A is MDI-1005 K80A is MDI-1006 K88A is MDI-1007 K176A is MDI-1008 K207A is MDI-1009 K263A is MDI-1010 K69A-K207A is MDI-1011 These mutations demonstrate reduced binding to clearance receptors while retaining NET binding and neutrophil elastase inhibition in the presence of DNA. The preferred mutation is K207A, as it reduces clearance receptor binding of the free inhibitor by 19-fold but only reduces binding of the inhibited protease complex by 1.6-fold (see Table II herein). This significantly increases the pharmacokinetics of inhibitors with this mutation, while still allowing clearance of the elastase complex after inhibition.

[0215] Although the present invention has been fully described, it will be understood by those skilled in the art that the same can be practiced within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications, and publications mentioned herein are hereby fully incorporated by reference in their entirety.

[0216] [Incorporation by Reference] The entire disclosure of each of the patent documents and scientific articles referred to herein (including, but not limited to, the references set forth herein) is incorporated by reference for all purposes.

[0217] [Equivalent] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative rather than limiting on the invention described herein. The scope of the present invention is, therefore, indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. [Brief explanation of the drawings]

[0218] [Figure 1] The essential role of lysine residues in PAI-1 for 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, and 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) form complexes with LMWuPA (lanes 3 and 4, respectively). Lane 5, LMWuPA. C. 250 nM PAI-1 and 300 nM PAI-1 chemically modified with sulfo-NHS-acetic acid were injected over an SPR chip with immobilized LRP1. D. 9 nM of the LMWuPA:PAI-1 complex and 80 nM of the complex formed with chemically modified PAI-1 were injected over an SPR chip on which LRP1 was immobilized. [Figure 2]The binding of PAI-1 to LRP1 depends on ionic strength. A. Increasing concentrations of PAI-1 in buffer containing increasing concentrations of NaCl were injected over an LRP-1-coated SPR chip, and Req values ​​were determined. Data are normalized to Rmax for each NaCl concentration. NaCl concentrations from the top curve down: 150 mM, 250 mM, 500 mM, 750 mM, and 1000 mM. B. Debye-Huckel plot of PAI-1 binding to LRP1. KD values ​​at each ionic strength (150, 250, 500, 750, and 1000 mM NaCl) were measured by equilibrium SPR measurements. Three independent experiments were performed, and plotted values ​​are the mean ± SE. A slope of 1.5 ± 0.1 was determined by linear regression analysis. Similar values ​​for slope were obtained by averaging the results of linear regression analyses from individual experiments. [Figure 3] The binding of PAI-1 to LRP1 is well described by a bivalent binding model. (A) Schematic of the bivalent binding model for the interaction of two different regions of PAI-1 with complementary sites on LRP1. (B) Increasing concentrations of PAI-1 (9.4, 37.5, 75, and 300 nM) were injected over an SPR chip with bound LRP1. The dissociation for each concentration was measured from the SPR data and normalized to 100% of the initial value at t = 0. The data were fitted to a biexponential decay (blue line). C. Increasing concentrations of PAI-1 (3.9, 7.8, 15.6, 31.2, 62.5, and 125 nM) were injected over an LRP1-bound chip. The fit of the experimental data (black line) to the bivalent binding model is shown in blue. The data shown is a representative experiment of six independent experiments performed. [Figure 4]The binding of PAI-1 to cluster IV of LRP1 fits well to a bivalent binding model. (A) Schematic diagram showing the domain organization of LRP1. Clusters of ligand-binding repeats (red circles) are labeled I, II, III, and IV. (B) Increasing concentrations of PAI-1 (9.4, 15.6, 31, 62.5, and 125 nM) were injected over an SPR chip bound to LRP1 cluster IV. The dissociation for each concentration was measured from the SPR data and normalized to 100% of the initial value at t = 0. The data were fitted to a biexponential decay (blue line). C. Increasing concentrations of PAI-1 (3.9, 7.8, 15.6, 31.2, 62.5, and 125 nM) were injected over a chip bound to LRP1 cluster IV. The fit of the experimental data (black line) to a bivalent binding model is shown in blue. Data shown are representative of three independent experiments performed. [Figure 5] CDE-096 inhibits the binding of HMWuPA:PAI-1 complexes to LRP1. A. 1 nM HMWuPA:PAI-1 complexes were flowed over an LRP1-bound SPR chip in the absence of CDE-096 (top curve) and in the presence of increasing concentrations of CDE-096 (15.6, 31.2, 62.5, 125, 250, 500 nM). B. Plot of the initial slope of the association phase from panel A versus CDE-096 concentration. An IC50 of 70 ± 11 nM was determined by nonlinear regression analysis. Data are representative of two independent experiments. [Figure 6]Binding of LMWuPA:PAI-1 complexes to LRP1 depends on ionic strength. A. Increasing concentrations of uPA:PAI-1 complexes were flowed over an LRP-1-coated SPR chip in the presence of increasing concentrations of NaCl, and Req values ​​were determined. Data are normalized to Rmax for each NaCl concentration. NaCl concentrations from the top curve down: 150 mM, 250 mM, 500 mM, 750 mM, and 1000 mM. B. Debye-Huckel plot of LMWuPA:PAI-1 binding to LRP1. KD values ​​at each ionic strength (150 mM, 250 mM, 500 mM, 750 mM, and 1000 mM NaCl) were measured by equilibrium SPR measurements. Three independent experiments were performed, and the mean ± SE is plotted. A slope of 2.4 ± 0.4 was determined by linear regression analysis. Identical values ​​were obtained by averaging the results of linear regression analysis of the individual experiments. [Figure 7] The LMWuPA:PAI-1 complex binds to LRP1 via a complex kinetics model. A) Model 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 model of binding occurs (Scheme II). B) Increasing concentrations of the LMWuPA:PAI-1 complex (3.12, 6.25, 12.5, 25, 50 nM) were injected over the LRP1-bound chip. The dissociation for each concentration was measured from the SPR data and normalized to 100% of the initial value at t = 0. B. Increasing concentrations of LMWuPA:PAI-1 (0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM) were injected over the LRP1-bound chip. Fitting of experimental data (black line) to the model comprising Schemes I and II is shown (blue line). Data are representative of three independent experiments. [Figure 8]Kinetic analysis of LMW uPA:PAI-1 complex binding to cluster IV of LRP1. A) Increasing concentrations of uPA:PAI-1 complex (0.6, 1.2, 2.5, 5, 10, 20, and 40 nM) were injected over the chip bound to LRP1. Dissociation for each concentration was measured from SPR data and normalized to 100% for the initial value at t = 0. B) Increasing concentrations of LMW uPA:PAI-1 (0.6, 1.2, 2.5, 5, 10, 20, and 40 nM) were injected over the LRP1-bound chip. Fitting of experimental data (black lines) to the Scheme I and II model (blue lines). Data are representative of three independent experiments. [Figure 9] LRP1-mediated cellular uptake of LMWuPA:PAI-1 is reduced when complexed with PAI-1 containing lysine residue mutations. 5 nM 125I-labeled LMWuPA:PAI-1 complexes formed with I91L PAI-1 or the indicated mutant PAI-1 molecules were incubated with WI-38 human fibroblasts for 6 hours at 37°C in the absence or presence of excess RAP. After incubation, the amount of internalized complex was quantified. Experiments were performed in triplicate. [Figure 10-1] Provided are the wild-type PAI-1 nucleic acid sequence (SEQ ID NO:1); the wild-type PAI-1 amino acid sequence (SEQ ID NO:2); and the mature wild-type PAI-1 amino acid sequence (SEQ ID NO:3). [Figure 10-2] Provided are the wild-type PAI-1 nucleic acid sequence (SEQ ID NO:1); the wild-type PAI-1 amino acid sequence (SEQ ID NO:2); and the mature wild-type PAI-1 amino acid sequence (SEQ ID NO:3). [Figure 10-3] Provided are the wild-type PAI-1 nucleic acid sequence (SEQ ID NO:1); the wild-type PAI-1 amino acid sequence (SEQ ID NO:2); and the mature wild-type PAI-1 amino acid sequence (SEQ ID NO:3). [Figure 10-4] Provided are the wild-type PAI-1 nucleic acid sequence (SEQ ID NO:1); the wild-type PAI-1 amino acid sequence (SEQ ID NO:2); and the mature wild-type PAI-1 amino acid sequence (SEQ ID NO:3). [Figure 10-5]Provided are the wild-type PAI-1 nucleic acid sequence (SEQ ID NO:1); the wild-type PAI-1 amino acid sequence (SEQ ID NO:2); and the mature wild-type PAI-1 amino acid sequence (SEQ ID NO:3). [Figure 10-6] Provided are the wild-type PAI-1 nucleic acid sequence (SEQ ID NO:1); the wild-type PAI-1 amino acid sequence (SEQ ID NO:2); and the mature wild-type PAI-1 amino acid sequence (SEQ ID NO:3). [Figure 11] Provided are a mature variant PAI-1 nucleic acid sequence (SEQ ID NO:4) that encodes a polypeptide having 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; and a mature variant PAI-1 amino acid sequence (SEQ ID NO:5) that has 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. [Figure 12-1] Provided are a mature variant PAI-1 / Fc nucleic acid sequence (SEQ ID NO:6) encoding a polypeptide having the following mutations within 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) having the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, R346V. [Figure 12-2] Provided are a mature variant PAI-1 / Fc nucleic acid sequence (SEQ ID NO:6) encoding a polypeptide having the following mutations within 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) having the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, R346V. [Figure 12-3]Provided are a mature variant PAI-1 / Fc nucleic acid sequence (SEQ ID NO:6) encoding a polypeptide having the following mutations within 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) having the following mutations within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO:3): I91L, R101A, Q123K, V343A, R346V. [Figure 13] FIG. 13 shows that MDI-1001 targets inflammatory nets better than Aralast. [Figure 14] FIG. 14 shows an in vitro comparison of Aralast, Avelestat, MDI-1002, MDI-1003, and MDI-1004. [Figure 15] FIG. 15 shows that MDI-1003 targets NETs in CF sputum. [Figure 16] FIG. 16 shows elastase activity as a function of inhibitor concentration. [Figure 17] FIG. 17 shows that MDI-1002 protects against acute lung injury. [Figure 18] FIG. 18 shows that MDI-1002 protects against pulmonary fibrosis. [Figure 19] FIG. 19 shows that MDI-1002 does not improve recovery after bleomycin. [Figure 20] FIG. 20 shows that inhaled MDI-1003 protects against acute lung injury. [Figure 21] FIG. 21 shows that MDI-1003 protects against pulmonary fibrosis better than MDI-1001. [Figure 22] FIG. 22 shows that MDI-1003 improves recovery after bleomycin. [Figure 23] FIG. 23 shows the constructs of Fc fusions of MDI-1002 and MDI-1004. [Figure 24] FIG. 24 shows the expression of Fc fusions of MDI-1002 and MDI-1004. [Figure 25] Figure 25 shows that Fc fusions improve PK. [Figure 26-1] Figure 26 shows that mutation of LRP1-binding residues affects the inhibition of neutrophil elastase in the presence of DNA NETs, ​​thereby demonstrating reduced interaction with the clearance receptor, LRP1, in NETs and retention of activity against elastase. [Figure 26-2] Figure 26 shows that mutation of LRP1-binding residues affects the inhibition of neutrophil elastase in the presence of DNA NETs, ​​thereby demonstrating reduced interaction with the clearance receptor, LRP1, in NETs and retention of activity against elastase.

Claims

1. 1. A polypeptide comprising a plasminogen activator inhibitor 1 (PAI-1) mutant, wherein the PAI-1 mutant has the mutations R101A, Q123K, V343A, and R346V within the wild-type human mature PAI-1 amino acid sequence (SEQ ID NO: 3), and includes one or more of K69A, K80A, K88A, I91L, K122A, Q123K, K176A, K207A, K263A, and wherein the mutant is linked to a monomer or portion of an Fc domain.

2. 2. The polypeptide of claim 1, wherein the mutant comprises the substitutions I91L, R101A, Q123K, V343A, and R346V.

3. The polypeptide of claim 1 or 2, wherein the mutant further comprises at least one mutation selected from K207A, K80A, and K88A.

4. The polypeptide according to any one of claims 1 to 3, wherein the mutant is represented by the amino acid sequence set forth in SEQ ID NO:

7.

5. The polypeptide according to any one of claims 1 to 4, wherein the mutant has a reduced ability to bind to vitronectin compared to wild-type PAI-1.

6. The polypeptide of any one of claims 1 to 5, wherein the mutant has a reduced ability to bind to the PAI-1 clearance receptor low density lipoprotein receptor-related protein 1 (LRP1).

7. The polypeptide of any one of claims 1 to 6, wherein the mutant exhibits improved pharmacokinetic properties compared to wild-type PAI-1.

8. The polypeptide according to any one of claims 1 to 7, wherein the polypeptide is capable of inhibiting neutrophil elastase (NE) activity.

9. The polypeptide according to any one of claims 1 to 8, wherein the polypeptide is capable of inhibiting neutrophil elastase bound within neutrophil extracellular traps (NETs).

10. 10. The polypeptide of claim 1, wherein the Fc domain monomer or portion is derived from a human IgG isotype selected from IgG1, IgG2, IgG3, and IgG4.

11. The polypeptide of any one of claims 1 to 10, wherein the Fc domain monomer or portion comprises an amino acid substitution that reduces or eliminates an Fcγ receptor-mediated effector function.

12. The polypeptide of any one of claims 1 to 11, wherein two Fc domain monomers of the polypeptide form an Fc domain dimer.

13. The polypeptide of any one of claims 1 to 12, wherein the polypeptide is fused to the Fc domain monomer via an amino acid linker or spacer.

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