Urinastatin Polypeptides for Treating Diseases - Patent application

Abstract

Methods and compositions are provided that include an urinastatin polypeptide for treating diseases associated with elevated neutrophil elastase (NE), including NE-associated lung and skin diseases. The urinastatin polypeptide can be a recombinant urinastatin polypeptide or a urinary-derived urinastatin polypeptide. The urinastatin polypeptide can comprise, consist of, or consist essentially of SEQ ID NO:2, and can include an N-linked glycan at residue N45 and an O-linked glycan at residue T17.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 143,403, filed January 29, 2021, which is incorporated by reference in its entirety.

[0002] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing said sequence listing is DIAM_041_01WO_ST25.txt. The text file is approximately 29KB, has a creation date of January 24, 2022, and has been submitted electronically via EFS-Web.

[0003] background Technical Field Embodiments of the present disclosure include methods and compositions comprising urinastatin polypeptides for treating diseases associated with elevated neutrophil elastase (NE), including NE-associated lung and skin diseases. [Background technology]

[0004] 2. Description of Related Art Urinastatin (also known as urinary trypsin inhibitor) is a glycoprotein proteinase inhibitor derived from human urine that inhibits the activity of trypsin, chymotrypsin, lactate, lipase, hyaluronidase, and various pancreatic enzymes.

[0005] Highly purified urinastatin is used clinically for the treatment of acute pancreatitis, chronic pancreatitis, Stevens-Johnson syndrome, burns, septic shock, toxic epidermal necrolysis (TEN), and other diseases. However, large amounts of urinastatin are required because it is a serpin, a potent protease inhibitor that reacts irreversibly with the active sites of proteases and is therefore typically consumed in a 1:1 stoichiometry with its target. This property, combined with the relatively low in vivo exposure achieved after systemic administration, creates a challenge in generating therapeutic agents from the native urinastatin protein.

[0006] Neutrophil elastase (NE) is a serine proteinase secreted by neutrophils and macrophages during inflammation. Excessive activity of neutrophil elastase and similar proteases has been reported to cause tissue damage and alter remodeling processes in many pulmonary conditions such as pneumonia, respiratory distress, and acute lung injury (Polverino et al., Chest. 152(2):249-262, 2017), as well as skin conditions such as pemphigoid (Liu et al., J Clin Invest. 105(1):113-123, 2000). There is also a need in the art for recombinant production of urinastatin polypeptides and / or urinastatin polypeptides having improved properties related to their therapeutic utility, as well as for extending the clinical utility of urinastatin polypeptides to other diseases. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Polverino et al., Chest. 152(2):249-262, 2017 [Non-Patent Document 2] Liu et al., J Clin Invest. 105(1):113-123, 2000 Summary of the Invention [Means for solving the problem]

[0008] A brief summary Embodiments of the present disclosure include a method of treating a neutrophil elastase (NE)-associated disease, ameliorating symptoms of the disease, or inhibiting progression of the disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a urinastatin polypeptide.

[0009] In certain embodiments, the NE-related disease comprises pulmonary disease.In some embodiments, the pulmonary disease is selected from one or more of alpha-1 antitrypsin (A1AT) deficiency (including its complications), cystic fibrosis, pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), bronchiectasis, and optionally virus-induced lung injury (including combinations thereof) associated with coronavirus (e.g., Covid-19) or influenza infection.In some embodiments, the NE-related disease is alpha-1 antitrypsin (A1AT) deficiency (including its complications).

[0010] In certain embodiments, the subject has a reduced level of serum A1AT, optionally about 80% or less than about 80% of the normal serum level of A1AT, about 60% or less than about 60% of the normal serum level of A1AT, about 40% or less than about 40% of the normal serum level of A1AT, or about 10-15% of the normal serum level of A1AT.

[0011] In some embodiments, the subject optionally has an A1AT phenotype / genotype selected from PiMS, PiSS, PiMZ, PiSZ and PiZZ.In certain embodiments, the subject optionally has an A1AT genotype selected from a deficiency A1AT allele and a null A1AT allele.In certain embodiments, the A1AT deficiency allele is an E342K allele (Z allele) or an E264V allele (S allele).

[0012] Some embodiments include (a) determining serum A1AT levels and / or A1AT phenotype / genotype in the subject; and (b) administering the pharmaceutical composition to the subject if the subject has an A1AT phenotype / genotype and / or a reduced serum level of A1AT compared to normal; Includes:

[0013] In certain embodiments, the A1AT phenotype / genotype is selected from PiMS, PiSS, PiMZ, PiSZ and PiZZ; the A1AT genotype comprises a deficiency A1AT allele or a null A1AT allele; and / or the serum A1AT level is about 80%, 60%, 40%, 20%, 15%, or 10% or less than about 80%, 60%, 40%, 20%, 15%, or 10% of the normal serum level of A1AT. In some embodiments, the A1AT deficiency allele is E342K allele (Z allele) or E264V allele (S allele).

[0014] In certain embodiments, the A1AT-deficiency complications include one or more of asthma, bronchiectasis, liver cirrhosis, COPD, respiratory failure, and vasculitis.In certain embodiments, the subject has one or more of the following shortness of breath (on exertion and then at rest as needed), wheezing, sputum production, or recurrent respiratory infections.

[0015] Certain embodiments include administering a pharmaceutical composition comprising the urinastatin polypeptide described above in combination with an additional therapeutic agent for treating A1AT-deficiency, optionally an RNA interference (RNAi) agent directed against a disease-associated A1AT mutant, an A1AT augmentation therapy, a bronchodilator, an inhaled steroid, an antibiotic, or an antiviral agent.

[0016] In certain embodiments, the NE-associated disease is cystic fibrosis and the subject has a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some embodiments, the NE-associated disease comprises a disease of the skin or mucous membranes. In certain embodiments, the disease of the skin or mucous membranes is Behcet's disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, and Kawasaki disease.

[0017] In some embodiments, the NE-associated disease is an autoimmune disease, e.g., an autoimmune disease selected from myasthenia gravis, warm autoimmune hemolytic anemia, thyroid eye disease, idiopathic thrombocytopenic purpura, chronic inflammatory demyelinating polyneuropathy, neuromyelitis optica, Guillain-Barré syndrome, and PLA2R+ membranous nephropathy.

[0018] In certain embodiments, the subject has increased NE levels or activity compared to a reference standard or healthy control.

[0019] Some embodiments include (a) determining NE level or activity in a sample from the subject; and (b) if the sample has an increased NE level or activity compared to a reference standard or a healthy control, administering the pharmaceutical composition to the subject; Includes:

[0020] In some embodiments, the sample is a sputum sample (optionally including mucus from the lower respiratory tract), a blood or serum sample, or a skin or mucosal sample. In some embodiments, the sputum sample contains airway neutrophils, and the method comprises determining the level or activity of surface-bound NE in the sample. In some embodiments, the method comprises determining the level or activity of free NE in the sample.

[0021] In some embodiments, the urinastatin polypeptide is a recombinant urinastatin polypeptide or a urinary-derived urinastatin polypeptide. In some embodiments, the urinastatin polypeptide comprises, consists of, or consists essentially of SEQ ID NO:2, and comprises an N-linked glycan at residue N45 and an O-linked glycan at residue T17. In some embodiments, the urinastatin polypeptide comprises, consists of, or consists essentially of an amino acid sequence selected from Table U1, or an active variant or fragment thereof having at least one urinastatin activity.

[0022] In some embodiments, the urinastatin polypeptide is (a) SEQ ID NO:1, or a variant of SEQ ID NO:1 having at least one modification to O-linked glycosylation and at least one urinastatin activity; (b) a fragment of SEQ ID NO:1 or (a), having at least one urinastatin activity; or (c) a variant of (a) or (b) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to (a) or (b) and has at least one urinastatin activity; Comprising, consisting of, or consisting essentially of.

[0023] In some embodiments, (a) comprises a substitution or deletion at the O-linked glycosylation site of residues 8-11 of SEQ ID NO:1, optionally a substitution or deletion at residue S10 of SEQ ID NO:1, optionally an S10A substitution, or (b) comprises, consists of, or consists essentially of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 contiguous amino acids of SEQ ID NO:1 or 2.

[0024] In some embodiments, (b) is about 20 to 130, 20 to 120, 20 to 110, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 30 to 130, 30 to 120, 30 to 110, or 30 to 150 amino acids of SEQ ID NO: 1 or 2. pieces, 30~100 pieces, 30~90 pieces, 30~80 pieces, 30~70 pieces, 30~60 pieces, 30~50 pieces, 30~40 pieces, 40~130 pieces, 40~120 pieces, 40~110 pieces, 40~100 pieces, 40~90 pieces, 40~80 pieces, 40~70 pieces, 40~60 pieces, 40~50 pieces, 50~130 pieces, 50~120 pieces, 5 0~110 pieces, 50~100 pieces, 50~90 pieces, 50~80 pieces, 50~70 pieces, 50~60 pieces, 60~130 pieces, 60~120 pieces, 60~110 pieces, 6 0~100 pieces, 60~90 pieces, 60~80 pieces, 60~70 pieces, 70~130 pieces, 70~120 pieces, 70~110 pieces, 70~100 pieces, 70~90 pieces, 7 comprising, consisting of, or consisting essentially of 0 to 80, 80 to 130, 80 to 120, 80 to 110, 80 to 100, 80 to 90, 90 to 130, 90 to 120, 90 to 110, 90 to 100, 100 to 130, 100 to 120, or 100 to 110 consecutive amino acids.

[0025] In some embodiments, (b) comprises, consists of, or consists essentially of domain 1 of SEQ ID NO: 1 or 2. In some embodiments, (b) comprises, consists of, or consists essentially of domain 2 of SEQ ID NO: 1 or 2.

[0026] In some embodiments, the urinastatin polypeptide comprises, consists of, or consists essentially of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2 (UTIΔCS) and retains a substitution at position S10, optionally an S10A substitution, of SEQ ID NO:2. In some embodiments, the urinastatin polypeptide has an N-linked glycan at residue N45 and an O-linked glycan at residue T17, wherein the residues are defined by SEQ ID NO:1 or 2.

[0027] In some embodiments, the urinastatin polypeptide is fused to an Fc region to form an urinastatin-Fc fusion polypeptide, wherein the urinastatin-Fc fusion polypeptide has at least one urinastatin activity. In some embodiments, the Fc region comprises, consists of, or consists essentially of the CH2 region, CH3 region, CH4 region, and / or hinge region of an IgA, IgD, IgE, IgG, or IgM immunoglobulin heavy chain. In some embodiments, the Fc region comprises, consists of, or consists essentially of one or more of the human Fc region amino acid sequences of Table F1 (including variants, fragments, homologs, orthologs, paralogs, and combinations thereof). In some embodiments, the urinastatin-Fc fusion polypeptide comprises a modified Fc region having at least one altered effector function and / or pharmacokinetic (PK) property compared to a wild-type Fc region.

[0028] In some embodiments, the at least one urinastatin activity comprises a protease inhibitor activity, optionally a neutrophil elastase (NE) inhibitor activity or a trypsin inhibitor activity, and optionally an anti-inflammatory activity. In some embodiments, the at least one urinastatin activity is a trypsin inhibitor activity and the urinastatin polypeptide has a specific activity as a trypsin inhibitor of about or at least about 1000 to 3000 U / mg, or about or at least about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 U / mg, where 1 unit (U) is the amount of urinastatin polypeptide that inhibits the activity of 2 μg trypsin by about 50%. In some embodiments, the at least one urinastatin activity is a NE inhibitor activity, and the urinastatin polypeptide has an IC of about 10 μg / ml or less in an in vitro assay of neutrophil elastase activity. 50 , an IC of about 10 μg / ml, 9 μg / ml, 8 μg / ml, 7 μg / ml, 6 μg / ml, 5 μg / ml, 4 μg / ml, 3 μg / ml, 2 μg / ml, or 1 μg / ml or lower, as appropriate (including all ranges and integers therebetween). 50 , as necessary, about 1~10μg / ml, 1~9μg / ml, 1~8μg / ml, 1~7μg / ml, 1~6μg / ml, 1~5μg / ml, 1~4μg / ml, 1~3μg / ml, 1~2μg / ml, 2~10μg / ml, 2~9μg / ml, 2 ~8μg / ml, 2~7μg / ml, 2~6μg / ml, 2~5μg / ml, 2~4μg / ml, 2~3μg / ml, 3~10μg / ml, 3~9μg / ml, 3~9μg / ml, 3~7μg / ml, 3~6μg / ml, 3~5μg / ml, 3~4 IC of 10μg / ml 50 In some embodiments, the urinastatin polypeptide has an IC of about 100 nM or less in an in vitro assay of neutrophil elastase activity. 50, or an IC of about 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM or lower, as appropriate (including all ranges and integers therebetween). 50 , as needed, about 10-100nM, 10-90nM, 10-80nM, 10-70nM, 10-60nM, 10-50nM, 10-40nM, 10-30nM, 10-20nM, 20-100nM, 20-90nM, 20-80nM, 20-70nM, 20-60nM, 20-50nM IC of 20-40nM, 20-30nM, 30-100nM, 30-90nM, 30-80nM, 30-70nM, 30-60nM, 30-50nM, 30-40nM, 40-100nM, 40-90nM, 40-80nM, 40-70nM, 40-60nM, or 40-50nM 50 has.

[0029] In some embodiments, the pharmaceutical composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein or weight-by-weight basis, is substantially free of aggregation, optionally less than about 10%, 9%, 8%, 7%, 6%, or 5% aggregated, and the composition is substantially endotoxin-free. In some embodiments, the pharmaceutical composition has less than about 1 EU endotoxin / mg protein, less than about 100 ng host cell protein / mg protein, less than about 10 pg host cell DNA / mg protein, and / or a single peak purity of greater than about 95% by SEC-HPLC.

[0030] Certain embodiments include administering the pharmaceutical composition to the subject intravenously (IV), subcutaneously (SC), by inhalation, topically, or orally. Certain embodiments include administering the pharmaceutical composition to the subject with pulmonary disease by inhalation, optionally with a nebulizer, metered dose inhaler, or dry powder inhaler. Certain embodiments include administering the pharmaceutical composition to the subject with skin or mucous membrane disease by topical or oral administration, optionally as a mouthwash.

[0031] In some embodiments, the pharmaceutical composition comprises the urinastatin polypeptide in a dosage of about 50,000 U / kg to about 1,000,000 U / kg, including all integers and ranges therebetween.

[0032] In some embodiments, administration of the pharmaceutical composition improves NE levels or activity in the subject, as appropriate, when measured over a period of about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months or longer, as appropriate, compared to a control or reference. or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. In some embodiments, administration of the pharmaceutical composition extends the life expectancy of the subject in need thereof by about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 ​​years, 49 years, 50 years, 51 years, 52 years, 53 years, 54 years, 55 years, 56 years, 57 years, 58 years, 59 years, 60 years, 61 years, 62 years, 63 years, 64 years, 65 years, 66 years, 67 years, 68 years, 69 years, 70 years, 71 years, 72 years, 73 years, 74 years, 75 years, 76 years, 77 years, 78 years, 79 years, 80 years, 81 years, 82 years, 83 years, 84 years, 85 years, 86 years, 87 years, 88 years, 89 years, 90 years, 91 years, 92 years, 93 years, 94 years, 95 years, 96 years, 97 years, 98 years, 99 years, 10 4 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 Year, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60 or more years, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 years , 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 ​​years, 49 years, 50 years, 51 years, 52 years, 53 years, 54 years, 55 years, 56 years, 57 years, 58 years, 59 years, 60 years or more.

[0033] In some embodiments, the subject has a pulmonary disease and administration of the pharmaceutical composition improves lung function by about 10%, 20%, 30% or more compared to a control or reference when measured over a period of about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months or more, as appropriate. , 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. In some embodiments, the improved respiratory function is selected from one or more of increased expiratory time, increased inspiratory time, decreased maximum expiratory flow, decreased maximum airflow, decreased minute ventilation (RMV), and decreased respiratory rate.

[0034] In some embodiments, the subject has a skin or mucosal disorder and administration of the pharmaceutical composition results in an improvement in disease progression of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, or more compared to a control or reference when measured over a period of about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months or more, as appropriate. or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 shows that recombinant urinastatin (DM300) has significant inhibitory activity in an in vitro assay of neutrophil elastase activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used in carrying out or testing the subject matter of this disclosure, but preferred methods and materials are described.All publications and references cited herein (including but not limited to patents and patent applications) are incorporated herein by reference in their entirety as if each individual publication and reference was specifically and individually indicated to be incorporated herein by reference as if fully indicated.Any patent application to which this application claims priority is also incorporated herein by reference in its entirety in the manner described above for publications and references.

[0037] The practice of the present disclosure will employ, unless specifically indicated to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the skill of the art, many of which are described below for illustrative purposes, and such techniques are fully explained in the literature. See, e.g., Current Protocols in Protein Science, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (ed. D. Glover); Oligonucleotide Synthesis (ed. N. Gait, 1984); Nucleic Acid Hybridization (eds. B. Hames & S. Higgins, 1985); Transcription and Translation (eds. B. Hames & S. Higgins, 1984); Animal Cell Culture (ed. R. Freshney, 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar publications.

[0038] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may generally be performed according to conventional methods well known in the art and as described in various general and more detailed references cited and discussed throughout this specification. Unless specific definitions are provided, the nomenclature utilized in connection with molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and drug discovery chemistry described herein, as well as the experimental procedures and techniques thereof, are well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0039] For purposes of this disclosure, the following terms are defined below.

[0040] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0041] "About" means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0042] As used herein, the term "amino acid" is intended to mean both naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis, as well as others, such as, for example, 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, the (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like, which are known to those of skill in the art. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications may include, for example, by substitution or replacement of chemical groups and moieties on the amino acid, or by derivatization of the amino acid. Amino acid mimetics include organic structures that exhibit functionally similar properties, such as, for example, charge and charge spacing characteristic of the reference amino acid. For example, an organic structure that mimics arginine (Arg or R) has a positively charged portion that is located in a similar molecular space and has the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include structures that are constrained to maintain optimal placement and charge interactions of amino acids or amino acid functional groups. Those skilled in the art will know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0043] "Biocompatible" refers to a substance or compound that is generally not detrimental to biological functions and does not cause any degree of unacceptable toxicity (including allergenic and disease states).

[0044] "Coding sequence" refers to any nucleic acid sequence that contributes to the coding of the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the coding of the polypeptide product of a gene.

[0045] Throughout this disclosure, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements.

[0046] "Consisting of" means inclusive and limited to everything following the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that no other elements may be present. "Consisting essentially of" means including any elements recited after the phrase, but limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they essentially affect the activity or action of the recited elements.

[0047] The terms "endotoxin free" or "substantially endotoxin free" generally refer to compositions, solvents, and / or containers that contain at most trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects on a subject), and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms (e.g., bacteria, typically gram-negative bacteria), although endotoxins can also be found in gram-positive bacteria (e.g., Listeria monocytogenes). The most widely recognized endotoxins are lipopolysaccharides (LPS) or lipo-oligo-saccharides (LOS), which are found in the outer membrane of various gram-negative bacteria and represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can cause fever, a drop in blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

[0048] Thus, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxin from the drug product and / or drug container, since even small amounts can cause adverse effects in humans. Depyrogenation ovens can be used for this purpose, since temperatures above 300°C are typically required to break down most endotoxins. For example, based on the primary packaging material, such as a syringe or vial, a combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels. Other methods of removing endotoxin are contemplated, including, for example, chromatography and filtration methods, as described herein and known in the art.

[0049] Endotoxin can be detected by using conventional techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes horseshoe crab blood, is a very sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of horseshoe crab lysate due to a powerful enzyme cascade that amplifies this reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, endotoxin levels can be less than about 0.001 EU / mg, less than 0.005 EU / mg, less than 0.01 EU / mg, less than 0.02 EU / mg, less than 0.03 EU / mg, less than 0.04 EU / mg, less than 0.05 EU / mg, less than 0.06 EU / mg, less than 0.08 EU / mg, less than 0.09 EU / mg, less than 0.1 EU / mg, less than 0.5 EU / mg, less than 1.0 EU / mg, less than 1.5 EU / mg, less than 2 EU / mg, less than 2.5 EU / mg, less than 3 EU / mg, less than 4 EU / mg, less than 5 EU / mg, less than 6 EU / mg, less than 7 EU / mg, less than 8 EU / mg, less than 9 EU / mg, or less than 10 EU / mg of active compound. Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

[0050] The "half-life" of a polypeptide may refer to the time it takes for the polypeptide to lose half of its pharmacological, physiological, or other activity, as compared to such activity upon administration to the serum or tissue of an organism, or as compared to any other defined time point. "Half-life" may also refer to the time it takes for the amount or concentration of the polypeptide to be reduced by half of the starting amount administered to the serum or tissue of an organism, as compared to such amount or concentration upon administration to the serum or tissue of an organism, or as compared to any other defined time point. Half-life may be measured in serum and / or in any one or more selected tissues.

[0051] The terms "modulating" and "altering" include "increasing," "enhancing," or "stimulating," as well as "decreasing" or "reducing," typically in a statistically significant or physiologically significant amount or degree as compared to a control. An "increased," "stimulated," or "enhanced" amount is typically a "statistically significant" amount and can include an increase that is 1.1-fold, 1.2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or greater (e.g., 500-fold, 1000-fold) (including all integers and ranges therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the amount produced without the composition (e.g., without agent) or with a control composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount and can include a reduction of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all integers and ranges therebetween) of the amount produced without the composition (e.g., without the agent) or with a control composition. Examples of comparisons and "statistically significant" amounts are described herein.

[0052] The terms "polypeptide", "protein" and "peptide" are used interchangeably and refer to a polymer of amino acids not limited to any particular length. The term "enzyme" includes a polypeptide or protein catalyst and, in the context of urinastatin, is used interchangeably with protein, polypeptide, or peptide. The terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and addition or deletion of a signal sequence. The term "polypeptide" or "protein" refers to one or more chains of amino acids, where each chain includes amino acids covalently linked by peptide bonds, and the polypeptide or protein can include multiple chains having the sequence of a native protein, i.e., a protein that occurs in nature, specifically produced by non-recombinant cells, or genetically engineered or recombinant cells, and includes molecules having the amino acid sequence of a native protein, or molecules having deletions, additions, and / or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass the urinastatin proteins described herein, or sequences having one or more amino acid deletions, additions, and / or substitutions of the urinastatin proteins. In certain embodiments, the polypeptides are "recombinant" polypeptides, which are produced by recombinant cells that contain one or more recombinant DNA molecules, typically made from heterologous polynucleotide sequences or combinations of polynucleotide sequences not found elsewhere in the cell.

[0053] The term "isolated" polypeptide or protein, as referred to herein, means that the protein is (1) free from at least some other proteins with which it is typically found in nature, (2) essentially free from other proteins from the same source, e.g., from the same species, (3) expressed by cells from a different species, (4) separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) not associated (by covalent or non-covalent interactions) with portions of proteins with which it is naturally associated, (6) operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (7) not occurring in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA or other RNA, may be of synthetic origin, or any combination of these. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants that are found in its natural environment and would interfere with its use (therapeutic, diagnostic, prophylactic, research or other).

[0054] In certain embodiments, the "purity" of any given agent (e.g., urinastatin polypeptide) in a composition can be specifically defined. For example, a certain composition can contain an agent that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure (e.g., on a protein basis) (including all decimal points and ranges therebetween) as measured, for example, by high performance liquid chromatography (HPLC), a well-known form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0055] The term "reference sequence" generally refers to a nucleic acid coding sequence, or an amino acid sequence, to which another sequence is compared. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including those described by name and those described in the Tables and Sequence Listing.

[0056] The term "sequence identity", or, for example, including "sequence 50% identical to", as used herein, refers to the degree to which sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis over a comparison window.Thus, "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for aligning a comparison window can be performed by computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection of the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by any of the various methods selected. Reference can also be made to the BLAST family of programs, as disclosed, for example, by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0057] The term "solubility" refers to the property of an agent provided herein (e.g., an urinastatin polypeptide) to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as concentration (either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g / dL (100 mL), mg / ml, etc.)), molar concentration, critical molar concentration, molar fraction, or other similar description of concentration. The maximum equilibrium amount of solute that can be dissolved per amount of solvent is the solubility of that solute in that solvent under specified conditions (including temperature, pressure, pH, and properties of the solvent). In certain embodiments, solubility is measured at physiological pH, or other pHs (e.g., pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8)). In certain embodiments, solubility is measured in water or a physiological buffer (e.g., PBS or NaCl, with or without NaP). In specific embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt (e.g., 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C) or about body temperature (37°C).In certain embodiments, the agent has a concentration of at least about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, 40 mg / ml, 41 mg / ml, 42 mg / ml, 43 mg / ml, 44 mg / ml, 45 mg / ml, 46 mg / ml, 47 mg / ml, 48 mg / ml, 49 mg / ml, 50 mg / ml, 51 mg / ml, 52 mg / ml, 53 mg / ml, 54 mg / ml, 55 mg / ml, 56 mg / ml, 57 mg / ml, 58 mg / ml, 59 mg / ml, 60 mg / ml, 61 mg / ml, 62 mg / ml, 63 mg / ml, 64 mg / ml, 65 mg / ml, 66 mg / ml, 50mg / ml, 60mg / ml, 70mg / ml, 80mg / ml, 90mg / ml or 100mg / ml.

[0058] A "subject" or "subject in need thereof" or "patient" or "patient in need thereof" includes mammalian subjects such as human subjects.

[0059] "Substantially" or "essentially" means nearly completely or entirely of some given amount, e.g., 95%, 96%, 97%, 98%, 99% or greater.

[0060] By "statistically significant" we mean that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. A commonly used measure of significance includes the p-value, which is the frequency or probability that an observed event would occur if the null hypothesis were true. If the resulting p-value is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.

[0061] "Therapeutic response" refers to an improvement in symptoms, whether sustained or not, following administration of one or more therapeutic agents.

[0062] As used herein, "treatment" of a subject (e.g., a mammal such as a human) or cell is any type of intervention used in an attempt to change the natural course of the individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathological event or contact with a pathogenic agent. Also included are "prophylactic" treatments, which may relate to reducing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prophylaxis" does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or its associated symptoms.

[0063] The term "wild-type" refers to a gene or gene product (e.g., a polypeptide) that is most frequently observed in a population and is thus arbitrarily designed to be the "normal" or "wild-type" form of that gene.

[0064] Each embodiment herein should be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0065] The present disclosure relates to a method of treating, improving symptoms, or inhibiting the progression of a neutrophil elastase (NE)-associated disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a urinastatin polypeptide. Neutrophil elastase (NE; UniProt: P08246) is a serine protease that hydrolyzes proteins in neutrophil lysosomes (azurophilic granules) and extracellular matrix (ECM) proteins upon release from activated neutrophils. It has been shown to play a role in degenerative and inflammatory diseases by hydrolyzing ECM type IV collagen and elastin, among other mechanisms. Thus, a "NE-associated disease" includes a disease or condition associated with abnormal or elevated expression or activity of neutrophil elastase compared to a reference standard or healthy control. Human neutrophil elastase is encoded by the ELANE gene on chromosome 19.

[0066] In certain embodiments, the NE-associated disease comprises a pulmonary disease (see, e.g., Sandhaus and Turino, COPD. 10 Suppl 1:60-3, 2013; and Polverino et al., Chest. 152(2):249-262, 2017). Examples of NE-associated pulmonary diseases include alpha-1 antitrypsin (A1AT) deficiency and its complications, cystic fibrosis, pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), bronchiectasis, and virus-induced lung injury. In specific embodiments, the virus-induced lung injury is associated with coronavirus infection, e.g., COVID-19, associated with SARS-Cov2 infection. In some embodiments, the virus-induced lung injury is associated with influenza virus infection.

[0067] In a specific embodiment, the NE-related disease is alpha-1 antitrypsin (A1AT) deficiency, or A1AD, a genetic disorder that often occurs in lung or liver disease. A1AD is caused by a mutation in the SERPINA1 gene, which encodes the A1AT protein, a protease inhibitor that belongs to the serpin superfamily. A1AT is produced in the liver, and one of its functions is to protect the lung from neutrophil elastase, an enzyme that can destroy connective tissue.

[0068] Normal blood or serum levels of A1AT vary depending on the analytical method, but are typically approximately 1.0-2.7 g / l or about 1.26-2.26 g / L (SI Units) or about 126-226 mg / dL (conventional units). Mutations in the SERPINA1 gene result in reduced levels of blood or serum A1AT. Thus, in certain embodiments, the subject has reduced levels of blood or serum A1AT, e.g., about 80% or less than about 80% of the normal serum level of A1AT, about 60% or less than about 60% of the normal serum level of A1AT, about 40% or less than about 40% of the normal serum level of A1AT, or about 10-15% or less than about 10-15% of the normal serum level of A1AT.

[0069] In certain embodiments, the subject has an A1AT phenotype / genotype, for example, an A1AT phenotype / genotype selected from PiMS, PiSS, PiMZ, PiSZ, and PiZZ. In some embodiments, the subject has a PiMS phenotype / genotype and about 80% or less than about 80% of the normal serum level of A1AT. In some embodiments, the subject has a PiSS phenotype / genotype and about 60% or less than about 60% of the normal serum level of A1AT. In some embodiments, the subject has a PiMZ phenotype / genotype and about 60% or less than about 60% of the normal serum level of A1AT. In certain embodiments, the subject has a PiSZ phenotype / genotype and about 40% or less than about 40% of the normal serum level of A1AT. In certain embodiments, the subject has a PiZZ phenotype / genotype and about 10-15% or less than about 10-15% of the normal serum level of A1AT. In some embodiments, the subject has an A1AT genotype selected from a deficiency A1AT allele and a null A1AT allele (e.g., an RNA null that does not produce an A1AT RNA transcript, or a protein null that does not produce an A1AT protein). Many deficiency A1AT alleles are known in the art, the main examples of which include the E342K allele (or Z allele) and the E264V allele (or S allele).

[0070] In certain embodiments, the subject has one or more complications and / or symptoms of A1AT deficiency or other NE-related lung diseases.Examples of complications of A1AT deficiency include asthma, bronchiectasis, liver cirrhosis, COPD, respiratory failure and vasculitis.Exemplary symptoms of A1AT deficiency or other lung diseases include shortness of breath (e.g., during exertion and then at rest), wheezing, sputum production and recurrent respiratory infections.

[0071] Certain embodiments include determining the serum A1AT level and / or A1AT phenotype / genotype in the subject, and administering a pharmaceutical composition (comprising an urinastatin polypeptide) to the subject when the subject has an A1AT phenotype / genotype and / or a serum level of A1AT level that is reduced compared to normal.Methods for determining serum A1AT level and / or A1AT phenotype / genotype are known in the art (see, for example, Silverman and Sandhaus, N Engl J Med. 360(26):2749-57, 2009), and include protein electrophoresis assay, nephelometric assay or immunonephelometric assay, enzyme-linked immunosorbent assay (ELISA), and radial immunodiffusion. Certain embodiments include administering the pharmaceutical composition when the subject is determined to have an A1AT phenotype / genotype selected from PiMS, PiSS, PiMZ, PiSZ, and PiZZ; when the subject is determined to have a deficient A1AT allele (e.g., Z allele, S allele) or a null A1AT allele; and / or when the subject is determined to have a serum A1AT level that is about 80%, 60%, 40%, 20%, 15%, or 10% of the normal serum level of A1AT, or less than about 80%, 60%, 40%, 20%, 15%, or 10%.

[0072] Certain embodiments include combination therapy for treating A1AT deficiency.For example, certain embodiments include administering the pharmaceutical composition comprising the above-mentioned urinastatin polypeptide in combination with additional therapeutic agent for treating A1AT-deficiency (for example, RNA interference (RNAi) agent directed against disease-related A1AT mutant (for example, ARO-ATT), A1AT enhancement therapy, bronchodilator, inhaled steroid, antibiotic, or antiviral agent).

[0073] In certain embodiments, the NE-associated lung disease is cystic fibrosis, an autosomal recessive disorder associated with a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Neutrophil elastase is an important risk factor for the severity of cystic fibrosis lung disease (see, e.g., Dittrich et al., Eur Respir J. 51(3):1701910, 2018). Thus, in certain embodiments, the subject has cystic fibrosis and a mutation in the CFTR gene.

[0074] In certain embodiments, the NE-related disease comprises a skin or mucosal disease (see, e.g., Liu et al., J Clin Invest. 105(1):113-123, 2000). Examples of skin or mucosal diseases include Behcet's disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, and Kawasaki disease.

[0075] In certain embodiments, the NE-related disease comprises an autoimmune disease, examples of which include myasthenia gravis, warm autoimmune hemolytic anemia, thyroid eye disease, idiopathic thrombocytopenic purpura, chronic inflammatory demyelinating polyneuropathy, neuromyelitis optica, Guillain-Barré syndrome, and PLA2R+ membranous nephropathy.

[0076] In certain embodiments, the subject has increased neutrophil elastase (NE) levels and / or activity compared to a reference standard or healthy control. In some embodiments, the subject has increased NE levels in blood / serum, increased NE levels in sputum, and / or increased NE levels in skin or mucous membranes. For example, in some embodiments, the NE level in the subject is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, compared to a reference standard or a healthy control. Certain embodiments include determining NE level and / or activity in a sample from the subject, and administering the pharmaceutical composition (comprising the urinastatin polypeptide) to the subject if the sample has increased NE level and / or activity compared to a reference standard or a healthy control. In some embodiments, the sample is a sputum sample (e.g., containing mucus from the lower respiratory tract), a blood or serum sample, or a skin or mucosal sample. In a specific embodiment, the sputum sample contains airway neutrophils, and the method comprises determining the level or activity of surface-bound NE in the sample; in a particular embodiment, the method comprises determining the level or activity of free NE in the sample. Methods for determining NE levels and / or activity are known in the art (see, e.g., Shoemark et al., Eur Respir J. 53(6):1900303, 2019).

[0077] The methods provided herein include administering a pharmaceutical composition comprising a "ulinastatin polypeptide." "Ulinastatin" (also referred to as urinary trypsin inhibitor (UTI), HI-30, ASPI, or bikunin) is an acidic glycoprotein with a molecular weight of about 30 kDa by SDS polypeptide gel electrophoresis. Wild-type human urinastatin is found in human urine and blood and is a multivalent Kunitz-type serine protease inhibitor that contains two Kunitz-type domains. It is expressed as a full-length protein and processed into the mature or active form of urinastatin. In certain embodiments, the urinastatin polypeptide is a recombinant urinastatin polypeptide or a urinary-derived urinastatin polypeptide. In specific embodiments, the urinastatin polypeptide is the mature form of urinastatin, or a variant or fragment or domain thereof.

[0078] Exemplary polypeptide sequences of mature urinastatin and its domains are provided in Table U1. [Table U1]

[0079] Thus, in some embodiments, the urinastatin polypeptide comprises, consists of, or consists essentially of an amino acid sequence selected from Table U1, or an active variant or fragment thereof having at least one urinastatin activity. (a) SEQ ID NO:1, or a variant of SEQ ID NO:1 having at least one modification to O-linked glycosylation and at least one urinastatin activity; (b) a fragment of SEQ ID NO:1 or (a), having at least one urinastatin activity; or (c) a variant of (a) or (b) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to (a) or (b) and has at least one urinastatin activity; Comprising, consisting of, or consisting essentially of.

[0080] Certain urinastatin polypeptides have an altered O-linked glycosylation site, where serine 10 has a chondroitin sulfate (CS) chain attached at the well-conserved Glu-Gly-Ser-Gly (SEQ ID NO: 5) O-linked glycosylation site. The CS chain is relatively short (Mwt approx. 8000), with 12-18 disaccharide repeats (GlcUA 1,3-GalNac 1,4-) and a conventional linkage region (GlcUA 1-3Gal 1-3Gal 1- 4Xyl 1)-O-Ser. Approximately 30% of GalNAcs (usually those near the linkage region) are sulfated at the C-4 hydroxyl group. CS chains synthesized during inflammation are shorter and have reduced sulfation. Thus, in some cases, the urinastatin polypeptide comprises at least one substitution and / or deletion at one or more of the Glu-Gly-Ser-Gly (SEQ ID NO:5) residues of SEQ ID NO:1 that reduces glycosylation at O-linked glycosylation sites. In a specific embodiment, the urinastatin polypeptide comprises a substitution or deletion at position S10 of SEQ ID NO:1, e.g., an S10A substitution (see SEQ ID NO:2) or other conservative substitution. In some embodiments, the urinastatin polypeptide comprises a substitution or deletion at the O-linked glycosylation site at residues 8-11 of SEQ ID NO:1, optionally a substitution or deletion at residue S10 of SEQ ID NO:1, optionally an S10A substitution; or (b) comprises, or consists essentially of, about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 contiguous amino acids of SEQ ID NO:1 or 2.

[0081] In some cases, the urinastatin polypeptide comprises, consists of, or consists essentially of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 consecutive amino acids of SEQ ID NO:1 or 2. In some cases, the urinastatin polypeptide may be about 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-130, 30-120, 30-140, 30-200, 30-220, 30-240, 30-360, 30-400, 30-420, 30-500, 30-600, 30-700, 30-800, 30-900, 30-1000, 30-1400, 30-2400, 30-3600, 30-400, 30-500, 30-600, 30-700, 30-800, 30-900, 30-1400, 30-1600, 30-1800, 30-2400, 30-2400, 30-3600, 30-4000, 30-5000, 30-6000, 30-7000, 30-8000, 30-9000, 30-1800, 30-1900, 30-2400, 30-2400, 30-3600, 30-4000, 30-5000, 30-6000, 30-7000, 30-8000, 30-9000, 30-1900, 30- 10 pieces, 30~100 pieces, 30~90 pieces, 30~80 pieces, 30~70 pieces, 30~60 pieces, 30~50 pieces, 30~40 pieces, 40~130 pieces, 40~120 pieces pieces, 40~110 pieces, 40~100 pieces, 40~90 pieces, 40~80 pieces, 40~70 pieces, 40~60 pieces, 40~50 pieces, 50~130 pieces, 50~120 pieces , 50~110 pieces, 50~100 pieces, 50~90 pieces, 50~80 pieces, 50~70 pieces, 50~60 pieces, 60~130 pieces, 60~120 pieces, 60~110 pieces , 60~100 pieces, 60~90 pieces, 60~80 pieces, 60~70 pieces, 70~130 pieces, 70~120 pieces, 70~110 pieces, 70~100 pieces, 70~90 pieces , 70-80, 80-130, 80-120, 80-110, 80-100, 80-90, 90-130, 90-120, 90-110, 90-100, 100-130, 100-120, or 100-110 consecutive amino acids.

[0082] In some embodiments, the urinastatin polypeptide comprises, consists of, or consists essentially of Domain 1 of SEQ ID NO: 1 or 2, or an active variant thereof. In certain embodiments, the urinastatin polypeptide comprises, consists of, or consists essentially of Domain 2 of SEQ ID NO: 1 or 2, or an active variant thereof.

[0083] In certain embodiments, the urinastatin polypeptide has an N-linked glycan at residue N45 and / or a non-natural O-linked glycan at residue T17, wherein said residues are defined by SEQ ID NO:1 or SEQ ID NO:2.

[0084] In some embodiments, the urinastatin polypeptide comprises, consists of, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:2 and retains a substitution at position S10, optionally an S10A substitution, of SEQ ID NO:2. In specific embodiments, the urinastatin polypeptide comprises, consists of, or consists essentially of SEQ ID NO:2 and comprises an N-linked glycan at residue N45 and / or an O-linked glycan at residue T17.

[0085] In certain embodiments, the urinastatin polypeptide is fused to an Fc region to form an urinastatin-Fc fusion polypeptide, and the urinastatin-Fc fusion polypeptide has at least one urinastatin activity. In certain embodiments, the Fc region comprises, consists of, or consists essentially of the CH2 region, CH3 region, CH4 region, and / or hinge region of an IgA, IgD, IgE, IgG, or IgM immunoglobulin heavy chain. In some cases, the Fc region is a human Fc region. In some embodiments, the Fc region has high effector function in humans (e.g., IgG1 Fc region or IgG3 Fc region). In some embodiments, the Fc region has low effector function in humans (e.g., IgG2 Fc region or IgG4 Fc region).

[0086] The amino acids of the CH2, CH3, CH4, and hinge regions of exemplary wild-type human IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM immunoglobulins are shown in Table F1 below. [Table F1-1] [Table F1-2] [Table F1-3]

[0087] Thus, in some embodiments, the Fc region in the urinastatin-Fc fusion polypeptide comprises, consists of, or consists essentially of one or more of the human Fc region amino acid sequences of Table F1 (including variants, fragments, homologs, orthologs, paralogs, and combinations thereof). Certain exemplary embodiments include an Fc region ranging in size from about 20-50, 20-100, 20-150, 20-200, 20-250, 20-300, 20-400, 50-100, 50-150, 50-200, 50-250, 50-300, 50-400, 100-150, 100-200, 100-250, 100-300, 100-350, 100-400, 200-250, 200-300, 200-350, or 200-400 amino acids in length, and optionally comprising, consisting of, or consisting essentially of, any one or more of the sequences in Table F1. Certain embodiments comprise an Fc region of up to about 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 110, up to 120, up to 130, up to 140, up to 150, up to 160, up to 170, up to 180, up to 190, up to 200, up to 210, up to 220, up to 230, up to 240, up to 250, up to 300, up to 350, up to 400 or more amino acids, optionally comprising, consisting of, or consisting essentially of any one or more of the amino acid sequences in Table F1.

[0088] Certain urinastatin Fc fusion polypeptides comprise modified Fc regions, including Fc regions having altered properties or biological activities compared to the wild-type Fc region. In certain embodiments, the modified Fc region has at least one altered effector function and / or pharmacokinetics (PK) characteristic compared to the wild-type Fc region. Thus, in certain embodiments, the fusion polypeptide has a modified Fc region with an altered functional characteristic (e.g., reduced or enhanced CDC, ADCC, or ADCP activity, reduced or enhanced binding affinity to a particular FcγR, or increased serum half-life). Specific embodiments include modified Fc regions that have altered (e.g., increased, decreased) binding to FcR (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, and / or FcRn); pharmacokinetic properties (e.g., stability or half-life, bioavailability, tissue distribution, volume of distribution, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, Cmax, tmax, Cmin, and / or variability); immunogenicity; complement fixation or activation; and / or CDC / ADCC / ADCP-related activity compared to the corresponding wild-type Fc region. Modified Fc regions of human and / or murine origin are included.

[0089] In certain embodiments, a peptide linker sequence may be used to separate the urinastatin polypeptide and the Fc region by a distance sufficient to ensure that each polypeptide folds into its desired secondary and tertiary structure and / or, if desired, to facilitate cleavage of the signal peptide from the urinastatin polypeptide. Such peptide linker sequences may be incorporated into the fusion polypeptide using standard techniques well known in the art.

[0090] Certain peptide linker sequences can be selected based on the following exemplary factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt secondary structures that may interact with functional epitopes on the first and second polypeptides; (3) their physiological stability; and (4) the absence of hydrophobic or charged residues that may react with functional epitopes or other features of the polypeptides. See, e.g., George and Heringa, J Protein Eng. 15:871-879, 2002.

[0091] Linker sequences can generally be from 1 to about 200 amino acids in length. Particular linkers can be from about 1 to 200 amino acids, 1 to 150 amino acids, 1 to 100 amino acids, 1 to 90 amino acids, 1 to 80 amino acids, 1 to 70 amino acids, 1 to 60 amino acids, 1 to 50 amino acids, 1 to 40 amino acids, 1 to 30 amino acids, 1 to 20 amino acids, 1 to 10 amino acids, 1 to 5 amino acids, 1 to 4 amino acids, 1 to 3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 It may have an overall amino acid length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more amino acids.

[0092] In certain embodiments, the urinastatin polypeptide (including variants, fragments, and fusion polypeptides) has at least one urinastatin activity (including protease inhibitor activity and anti-inflammatory activity). In some embodiments, the urinastatin polypeptide has a specific activity of about or at least about 1000 to 3000 U / mg, or about or at least about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 U / mg, where 1 unit (U) is the amount of urinastatin polypeptide that inhibits the activity of 2 μg trypsin by about 50%. In some embodiments, the at least one urinastatin activity has neutrophil elastase (NE) inhibitor activity. In certain embodiments, the urinastatin polypeptide has an NE inhibitor activity of about 10 μg / ml or less, e.g., about 10 μg / ml, 9 μg / ml, 8 μg / ml, 7 μg / ml, 6 μg / ml, 5 μg / ml, 4 μg / ml, 3 μg / ml, 2 μg / ml, or 1 μg / ml or lower (and all ranges and integers therebetween, e.g., about 1-10 μg / ml) in an in vitro assay of neutrophil elastase activity (see Example 1). ml, 1~9μg / ml, 1~8μg / ml, 1~7μg / ml, 1~6μg / ml, 1~5μg / ml, 1~4μg / ml, 1~3μg / ml, 1~2μg / ml, 2~10μg / ml, 2~9μg / ml, 2~8μg / ml, 2~7μg / ml, 2~6μg / ml, 2~5μg / ml, 2~4μg / ml, 2~3μg / ml, 3~10μg / ml, 3~9μg / ml, 3~9μg / ml, 3~7μg / ml, 3~6μg / ml, 3~5μg / ml, 3~4 IC of 10μg / ml) 50 In some embodiments, the urinastatin polypeptide has an IC of about 100 nM or less in an in vitro assay of neutrophil elastase activity. 50, or an IC of about 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM or lower, as appropriate (including all ranges and integers therebetween). 50 , as needed, about 10-100nM, 10-90nM, 10-80nM, 10-70nM, 10-60nM, 10-50nM, 10-40nM, 10-30nM, 10-20nM, 20-100nM, 20-90nM, 20-80nM, 20-70nM, 20-60nM, 20-50nM IC of 20-40nM, 20-30nM, 30-100nM, 30-90nM, 30-80nM, 30-70nM, 30-60nM, 30-50nM, 30-40nM, 40-100nM, 40-90nM, 40-80nM, 40-70nM, 40-60nM, or 40-50nM 50 In certain embodiments, the at least one urinastatin activity is measured or characterized under physiological conditions (e.g., temperature, salinity, and / or pH).

[0093] As noted above, certain embodiments include "variants" of the urinastatin polypeptides or fusion polypeptides described herein. A "variant" sequence refers to a polypeptide or polynucleotide sequence that differs from a reference sequence by one or more substitutions, deletions (e.g., truncations), additions, and / or insertions. Thus, certain variants include fragments of the reference sequences described herein. Variant polypeptides are biologically active, i.e., they continue to have the enzymatic or binding activity of the reference polypeptide. Such variants may result, for example, from genetic polymorphism and / or human manipulation.

[0094] In some cases, the variants contain one or more "conservative changes or substitutions. A "conservative substitution" is the substitution of one amino acid for another having similar properties, such that one skilled in the art of peptide chemistry would predict that the secondary structure and hydropathic properties of the polypeptide will not be substantially altered. As noted above, modifications can be made in the structures of the polynucleotides and polypeptides of the present disclosure and still obtain functional molecules that encode variant or derivative polypeptides having desirable properties. When it is desired to change the amino acid sequence of a polypeptide to produce an equivalent or even improved variant or portion of the polypeptides described herein, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence.

[0095] For example, certain amino acids can be substituted for other amino acids in protein structures without obvious loss of interactive binding ability with structures such as, for example, the antigen-binding region of an antibody or the binding site on a substrate molecule. Because it is the interactive ability and properties of a protein that define the biological functional activity of the protein, certain amino acid sequence substitutions can be made in the protein sequence, and of course, in the underlying DNA coding sequence, to nevertheless obtain a protein with similar properties. Thus, it is contemplated that various changes can be made in the peptide sequences of the compositions of the present disclosure, or in the corresponding DNA sequences encoding said peptides, without obvious loss of their usefulness.

[0096] In making such changes, the hydropathic index of amino acids may be considered. The importance of the amino acid hydropathic index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). The relative hydropathic characteristics of amino acids are allowed to contribute to the secondary structure of a resulting protein, which in turn defines the interactions of that protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982). These values ​​are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is known in the art that certain amino acids can be substituted by other amino acids having a similar hydropathic index or score and still result in a protein having similar biological activity, i.e., a biologically functional equivalent protein can be obtained. In making such changes, substitutions of amino acids having hydropathic indices within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0097] It is also understood in the art that substitutions of similar amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101 (specifically incorporated herein by reference in its entirety) states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It will be understood that an amino acid may be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent, protein. In such alterations, substitutions of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0098] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account various of the foregoing properties are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0099] Amino acid substitutions can also be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residue.For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; uncharged polar head group amino acids with similar hydrophilicity values ​​include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0100] Variants may also or alternatively include non-conservative changes. In some embodiments, variant polypeptides differ from the native or reference sequence by substitution, deletion or addition of about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or less than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid. Variants may also (or alternatively) be modified by, for example, deletion or addition of amino acids that may have minimal effect on the immunogenicity, secondary structure, enzymatic activity, and / or hydropathic properties of the polypeptide.

[0101] In certain embodiments, the polypeptide sequence is about, at least about, or at most about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380 pieces, 390 pieces, 400 pieces, 410 pieces, 420 pieces, 430 pieces, 440 pieces, 450 pieces, 460 pieces, 470 pieces, 480 pieces, 490 pieces, 500 pieces, 510 pieces, 520 pieces, 530 pieces, 540 pieces, 550 pieces, 560 pieces, 570 pieces, 580 pieces, 590 pieces, 600 pieces, 610 pieces, 620 pieces, 630 pieces, 640 pieces, 650 pieces, 660 pieces, 670 pieces, 680 pieces, 690 pieces, 700 pieces, 710 pieces, 720 pieces, 730 pieces, 740 pieces, 750 pieces, 760 pieces, 770 pieces, 780, 790, 800, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids in length (including all integers in between, and may include all or a portion of a reference sequence (see, e.g., Table U1, Sequence Listing)).

[0102] In some embodiments, the polypeptide sequence is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, or about 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less, 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less, 30 or less, 31 or less, 32 or less, 33 or less, 34 or less, 35 or less, 36 or less, 37 or less, 38 or less, 39 or less, 40 or less, 41 or less, 42 or less, 43 or less, 44 or less, 45 or less, 46 or less, 47 or less, 48 or less, 49 or less, 50 or less, 55 or less, 60 or less, 65 or less,70 or less, 75 or less, 80 or less, 85 or less, 90 or less, 95 or less, 100 or less, 110 or less, 120 or less, 130 or less, 140 or less, 150 or less, 160 or less, 170 or less, 180 Less than 190 pieces, 200 pieces or less, 210 pieces or less, 220 pieces or less, 230 pieces or less, 240 pieces or less, 250 pieces or less, 260 pieces or less, 270 pieces or less, 280 pieces or less, 290 pieces or less, 300 pieces or less, 310 pieces or less, 320 or less, 330 or less, 340 or less, 350 or less, 360 or less, 370 or less, 380 or less, 390 or less, 400 or less, 410 or less, 420 or less, 430 or less, 440 or less, 450 Below, 460 or less, 470 or less, 480 or less, 490 or less, 500 or less, 510 or less, 520 or less, 530 or less, 540 or less, 550 or less, 560 or less, 570 or less, 580 or less, 59 0 or less, 600 or less, 610 or less, 620 or less, 630 or less, 640 or less, 650 or less, 660 or less, 670 or less, 680 or less, 690 or less, 700 or less, 710 or less, 720 or less , 730 or less, 740 or less, 750 or less, 760 or less, 770 or less, 780 or less, 790 or less, 800 or less, 800 or less, 810 or less, 820 or less, 830 or less, 840 or less, 850 50 or less, 860 or less, 870 or less, 880 or less, 890 or less, 900 or less, 900 or less, 910 or less, 920 or less, 930 or less, 940 or less, 950 or less, 960 or less, 970 or less, 980 or less, 990 or less, 1000 or less, or more contiguous amino acids (including all integers in between, and may include all or a portion of a reference sequence (see, e.g., Table U1, Sequence Listing)).

[0103] In certain embodiments, the polypeptide sequence is about 10 to 1000, 10 to 900, 10 to 800, 10 to 700, 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 10 to 40, 10 to 30, 10 to 20 , 20~1000 pieces, 20~900 pieces, 20~800 pieces, 20~700 pieces, 20~600 pieces, 20~500 pieces, 20~400 pieces, 20~300 pieces, 2 0~200 pieces, 20~100 pieces, 20~50 pieces, 20~40 pieces, 20~30 pieces, 50~1000 pieces, 50~900 pieces, 50~800 pieces, 50~700 pieces , 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, or 200-300 consecutive amino acids (including all integers therebetween, and including all or a portion of a reference sequence (see, e.g., Table U1, Sequence Listing)). In certain embodiments, the C-terminal or N-terminal regions of any reference polypeptide can be truncated to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more of the above, so long as the truncated polypeptide retains the binding properties and / or activity of the reference polypeptide (see, e.g., Table U1, Sequence Listing). A biologically active fragment may be truncated by about 10 to 50, 20 to 50, 50 to 100 or more amino acids, including all integers and ranges therebetween (e.g., 101, 102, 103, 104, 105). Typically, a biologically active fragment has about 1%, about 5%, about 10%, about 25%, or about 50% or more of the activity of the biologically active reference polypeptide from which the fragment is derived.

[0104] Generally, variants will exhibit at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identity or sequence homology to the reference polypeptide sequence (see, e.g., Table U1, Sequence Listing). In addition, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 Contemplated are sequences that differ from a native or parent sequence by the addition (e.g., C-terminal additions, N-terminal additions, both), deletions, truncations, insertions, or substitutions (e.g., conservative substitutions) of 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids (including all integers and ranges therebetween), but which retain a property or activity of the parent or reference polypeptide sequence (see, e.g., Table U1, Sequence Listing).

[0105] In some embodiments, a variant polypeptide differs from a reference sequence by at least about 1, but by fewer than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3, or 2 amino acid residues. In certain embodiments, a variant polypeptide differs from a reference sequence by at least about 1%, but by about 20%, 15%, 10%, or 5% of its residues (if this comparison requires alignment, the sequences should be aligned for maximum similarity. Deletions or insertions, or sequences "looped" out from mismatches, are considered differences).

[0106] Calculation of sequence similarity or sequence identity (these terms are used interchangeably herein) between sequences is performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In certain embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0107] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

[0108] Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. 48: 444-453, 1970) algorithm, which is incorporated into the GAP program in the GCG software package, using either the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix, and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the set that should be used unless otherwise specified) is a Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0109] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4:11-17, 1989) as implemented in the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0110] The sequences described herein can be used as "query sequences" to perform searches against public databases to identify, for example, other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0111] In some embodiments, as noted above, polynucleotides and / or polypeptides can be evaluated using the BLAST alignment tool. Local alignment simply consists of pairs of sequence segments, one from each of the sequences being compared. A modification of the Smith-Waterman or Sellers algorithm finds all segment pairs whose scores cannot be improved by extension or trimming (referred to as high-scoring segment pairs (HSPs)). The results of BLAST alignment include a statistical measure that indicates the likelihood that the BLAST score could be predicted by chance alone.

[0112] A raw score, S, is calculated from the number of gaps and substitutions associated with each aligned sequence, where a higher similarity score indicates a more significant alignment. Substitution scores are given by lookup tables (see PAM, BLOSUM).

[0113] A gap score is typically calculated as the sum of G, the gap opening penalty, and L, the gap extension penalty. For a gap of length n, the gap cost is G+Ln. The selection of gap costs, G and L, is empirical, but it is conventional to select a high value of G (10-15), e.g., 11, and a low value of L (1-2), e.g., 1.

[0114] The bit scores, S', are derived from the raw alignment scores S, where the statistical properties of the scoring system used are taken into account. The bit scores are normalized with respect to the scoring system, so that they can be used to compare alignment scores from different searches. The terms "bit score" and "similarity score" are used interchangeably. The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment.

[0115] The E value, or expectation value, describes the likelihood that sequences with similar scores will occur in a database by chance. It is an estimate of the number of different alignments with a score equal to or better than S that would be expected to occur by chance in a database search. The smaller the E value, the more significant the alignment. For example, e -117An alignment with an E-value of 0.01 means that sequences with similar scores are highly unlikely to occur simply by chance. Furthermore, the expected score for aligning random pairs of amino acids is required to be negative, otherwise long alignments tend to have high scores, regardless of whether the aligned segments are related or not. Furthermore, the BLAST algorithm uses an appropriate substitution matrix, nucleotide or amino acid, and for gapped alignments, gap creation and extension penalties. For example, BLAST alignments and comparisons of polypeptide sequences are typically performed using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

[0116] In some embodiments, sequence similarity scores are reported from BLAST analyses performed using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

[0117] In certain embodiments, the sequence identity / similarity scores provided herein refer to values ​​obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using the following parameters: % identity and % similarity for nucleotide sequences using GAP weight 50 and length weight 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using GAP weight 8 and length weight 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89:10915-10919, 1992). GAP uses the Needleman and Wunsch algorithm (J Mol Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.

[0118] In certain embodiments, the variant polypeptide has at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620 , 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, or higher (including all integers and ranges therebetween), where the BLAST alignment used a BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

[0119] As noted above, the reference polypeptide can be modified in various ways, including amino acid substitution, deletion, truncation, addition, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the reference polypeptide can be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are well known in the art. See, for example, Kunkel (PNAS USA. 82: 488-492, 1985); Kunkel et al. (Methods in Enzymol. 154: 367-382, 1987), U.S. Patent No. 4,873,192, Watson, JD et al. ("Molecular Biology of the Gene", 4th ed., Benjamin / Cummings, Menlo Park, Calif., 1987) and references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0120] The method of screening the gene products of the combinatorial library made by such modification and screening the cDNA library for gene products with selected properties is known in the art. Such method is adaptable for rapid screening of the gene library generated by combinatorial mutagenesis of reference polypeptide. As an example, recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in a library, can be used in combination with screening assays to identify polypeptide variants (Arkin and Yourvan, PNAS USA 89: 7811-7815, 1992; Delgrave et al., Protein Engineering. 6: 327-331, 1993).

[0121] For in vivo use, the urinastatin polypeptides and fusion polypeptides described herein are typically formulated as part of a pharmaceutical or therapeutic composition. Certain embodiments thus use a composition (e.g., a therapeutic or pharmaceutical composition) that includes the urinastatin polypeptides described herein and a pharma- ceutically acceptable carrier. A "carrier" can include, for example, a pharma- ceutically or physiologically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to the carrier at the dosages and concentrations used. Often, the physiologically acceptable carrier is an aqueous pH-buffered solution. Examples of physiologically acceptable carriers include: buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents (e.g., EDTA); sugar alcohols (e.g., mannitol or sorbitol); salt-forming counterions (e.g., sodium); and / or non-ionic surfactants (e.g., polysorbate 20 (TWEEN TM ) Polyethylene glycol (PEG) and poloxamer (PLURONICS TM ) etc.

[0122] The composition may be prepared by methodologies well known in the pharmaceutical arts. For example, the composition may be prepared by combining a composition comprising an urinastatin polypeptide (as described herein) and one or more of a buffer or excipient as needed, with sterile distilled water to form a solution as needed. Certain compositions include saline solution (e.g., 0.9% normal saline) or dextrose (e.g., about 1-10% dextrose, or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% dextrose). Surfactants may be added to facilitate the formation of a homogenous solution or suspension. Surfactants are compounds that covalently interact with the urinastatin polypeptide in a composition to facilitate dissolution or homogenous suspension of the polypeptide in an aqueous delivery system.

[0123] Pharmaceutical carriers can be liquid, semi-liquid, or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application can include, for example, sterile diluents (e.g., water), saline solution (e.g., phosphate buffered saline; PBS), non-volatile oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antimicrobial agents (e.g., benzyl alcohol and methylparaben); antioxidants (e.g., ascorbic acid and sodium bisulfite) and chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); buffers (e.g., acetate, citrate, and phosphate). When administered intravenously (e.g., by IV infusion), suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickeners and solubilizers (e.g., glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof).

[0124] An exemplary composition comprises a mature urinastatin polypeptide having a T17-O-linked glycan, including (i) an O-linked glycosylation site modified at residues Glu-Gly-Ser-Gly (SEQ ID NO:5) to reduce glycosylation at the O-linked glycosylation site, (ii) an N-linked glycan at residue N45, and (iii) an O-linked glycan at residue T17 (said residues are defined by SEQ ID NO:1 or 2, as described herein), and a pharma- ceutically acceptable carrier. Further examples of mature urinastatin polypeptides, and active variants and fragments thereof, are described herein (see, e.g., Table U1 and related disclosures).

[0125] Certain compositions include a mixture of urinastatin glycoforms. For example, certain compositions include (a) a first mature urinastatin polypeptide (as described herein) that includes a T17-O-linked glycan, and (b) a second mature urinastatin polypeptide that includes an N-linked glycan at residue N45 and does not include an O-linked glycan at residue T17. In some embodiments, the second mature urinastatin polypeptide has an altered O-linked glycosylation site (as described herein) at residue Glu-Gly-Ser-Gly (SEQ ID NO: 5), for example, an S10A substitution that reduces glycosylation at the O-linked glycosylation site. In some embodiments, the second mature urinastatin polypeptide of (b) above comprises, consists of, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2 or 4, comprises or retains the modified O-linked glycosylation site (e.g., the S10A substitution described above) and an N-linked glycan at residue N45, does not comprise an O-linked glycan at residue T17, and has at least one urinastatin activity. In some embodiments, the mature urinastatin polypeptides of (a):(b) are present in the composition in a ratio ranging from about 20:1 to about 1:20, optionally about 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, or 1:20.

[0126] Certain compositions are substantially pure on a protein or weight basis. For example, as described above, certain compositions have a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein or weight-weight basis, and are substantially free of aggregates, for example, less than about 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% aggregated. Certain compositions are substantially free of endotoxins, as described herein. In specific embodiments, the compositions have one or more of the following determinations of purity: less than about 1 EU endotoxin / mg protein, less than about 100 ng host cell protein / mg protein, less than about 10 pg host cell DNA / mg protein, and / or are substantially free of aggregates.

[0127] Certain compositions are at a pharma- ceutically acceptable pH. For example, in certain embodiments, the pharma-ceutically acceptable pH is about 5.0 to about 8.0 (±0.01 to ±0.1), or about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 (±0.01 to ±0.1), including all integers and ranges therebetween.

[0128] In certain embodiments, the urinastatin polypeptide has at least one urinastatin activity at a pH that is close to the physiological pH of human blood. Thus, in some embodiments, the urinastatin polypeptide has at least one urinastatin activity at a pH of about 4 to about 10.8, or about 6 to about 8, or about 6.5 to about 7.5. In certain embodiments, the urinastatin polypeptide has effective urinastatin activity at about pH 7.4.

[0129] Administration can be accomplished by a variety of different routes. The mode of administration depends on the nature of the condition to be treated or prevented. For example, the composition can be administered orally, intranasally, intraperitoneally, parenterally, intravenously, intralymphatically, intratumorally, intramuscularly, interstitially, intraintestinally, intraarterially, subcutaneously, intraocularly, intrasynovially, transepithelially, and / or transdermally. Particular embodiments include by intravenous (IV), subcutaneous (SC), inhalation, topical, or oral administration.

[0130] Certain embodiments include administering the pharmaceutical composition to the subject with pulmonary disease by inhalation administration, for example, certain embodiments include administration with a nebulizer, a metered dose inhaler, or a dry powder inhaler.

[0131] Specific embodiments include administering the pharmaceutical composition to the subject with skin or mucous membrane disease by topical or oral administration, for example, as a mouthwash. For topical administration, the carrier may comprise a solution, emulsion, ointment, or gel base. The base may comprise, for example, one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents (e.g., water and alcohol), and emulsifiers and stabilizers. Thickeners may be present in therapeutic or pharmaceutical compositions for topical administration. When intended for transdermal administration, the composition may comprise a transdermal patch or iontophoresis device.

[0132] The exact dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known test protocols or by testing the composition in model systems known in the art and extrapolating therefrom. Controlled clinical trials can also be performed. Dosage can also vary with the severity of the condition to be alleviated. Pharmaceutical compositions are generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition can be administered at once or divided into many smaller doses to be administered at intervals of time. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs.

[0133] In some embodiments, dosages can be administered from about once per day to about once every two or three weeks. For example, in certain embodiments, dosages are administered about once per day, two days, three days, four days, five days, six days, or seven days, or about once per week, or about twice per week, or about three times per week, or about once per two or three weeks.

[0134] In one particular embodiment, the 4 U ~ approx. 1 x 10 5 U~approx. 100×10 5 U, or approximately 1 x 10 4 U, 2×10 4 U, 3×10 4 U, 4×10 4 U, 5×10 4 U, 6×10 4 U, 7×10 4 U, 8×10 4 U, 9×10 4 U, 1×10 5 U, 2×10 5 U, 3×10 5 U, 4×10 5 U, 5×10 5 U, 6×10 5 U, 7×10 5 U, 8×10 5 U, 9×10 5 U, 10×105 U, 11×10 5 U, 12×10 5 U, 15×10 5 U, 20×10 5 U、30×10 5 U, 40×10 5 U、50×10 5 U、60×10 5 U、70×10 5 U、80×10 5 U、or 100×10 5In certain embodiments, the urinastatin polypeptide is administered in a dosage (e.g., daily dosage) of about 50,000 U / kg to about 1,000,000 U / kg (including all ranges and integers therebetween). Dosages (e.g., daily dosages) in U / kg (including all integers and ranges therebetween) (e.g., about 50,000 U / kg, 55,000 U / kg, 60,000 U / kg, 65,000 U / kg, 70,000 U / kg, 75,000 U / kg, 80,000 U / kg, 85,000 U / kg, 90,000 U / kg, 95,000 U / kg, 100,000 U / kg, 115,000 U / kg, 120,000 U / kg, 125,000 U / kg, 130,000 U / kg, 135,000 U / kg, / kg, 140,000U / kg, 145,000U / kg, 150,000U / kg, 155,000U / kg, 160,000U / kg, 165,000U / kg, 170,000U / kg, 175,000U / kg, 180,000U / kg , 185,000U / kg, 190,000U / kg, 195,000U / kg, 200,000U / kg, 200,000U / kg, 215,000U / kg, 220,000U / kg, 225,000U / kg, 230,000U / kg, 235 ,000U / kg,240,000U / kg,245,000U / kg,250,000U / kg,255,000U / kg,260,000U / kg,265,000U / kg,270,000U / kg,275,000U / kg,280,00 0U / kg, 285,000U / kg, 290,000U / kg, 295,000U / kg, 300,000U / kg, 300,000U / kg, 315,000U / kg, 320,000U / kg, 325,000U / kg, 330,000U / kg kg, 335,000U / kg, 340,000U / kg, 345,000U / kg, 350,000U / kg, 355,000U / kg, 360,000U / kg, 365,000U / kg, 370,000U / kg, 375,000U / kg, 380,000U / kg, 385,000U / kg, 390,000U / kg, 395,000U / kg, 400,000U / kg, 400,000U / kg, 415,000U / kg, 420,000U / kg, 425,000U / kg, 430,000U / kg、435,000U / kg、440,000U / kg、445,000U / kg、450,000U / kg、455,000U / kg、460,000U / kg、465,000U / kg、470,000U / kg、475,000U / kg、480,000U / kg、485,000U / kg、490,000U / kg、495,000U / kg、500,000U / kg、500,000U / kg、515,000U / kg、520,000U / kg、525,000U / kg、530,000U / kg、535,000U / kg、540,000U / kg、545,000U / kg、550,000U / kg、555,000U / kg、560,000U / kg、565,000U / kg、570,000U / kg、575,000U / kg、580,000U / kg、585,000U / kg、590,000U / kg、595,000U / kg、600,000U / kg、600,000U / kg、615,000U / kg、620,000U / kg、625,000U / kg、630,000U / kg、635,000U / kg、640,000U / kg、645,000U / kg、650,000U / kg、655,000U / kg、660,000U / kg、665,000U / kg、670,000U / kg、675,000U / kg、680,000U / kg、685,000U / kg、690,000U / kg、695,000U / kg、700,000U / kg、700,000U / kg、715,000U / kg、720,000U / kg、725,000U / kg、730,000U / kg、735,000U / kg、740,000U / kg、745,000U / kg、750,000U / kg、755,000U / kg、760,000U / kg、765,000U / kg、770,000U / kg、775,000U / kg、780,000U / kg、785,000U / kg、790,000U / kg、795,000U / kg、800,000U / kg、800,000U / kg、815,000U / kg、820,000U / kg、825,000U / kg、830,000U / kg、835,000U / kg、840,000U / kg、845,000U / kg、850,000U / kg、855,000U / kg、860,000U / kg、865,000U / kg, 870,000U / kg, 875,000U / kg, 880,000U / kg, 885,000U / kg, 890,000U / kg, 895,000U / kg, 900,000U / kg, 900,000U / kg, 915,000U / kg, 920,000U / kg, 925,000U / kg, 930,000U / kg, 935,000U / kg, 940,000U / kg, 945,000U / In certain cases, treatment may be initiated with small dosages, which may be increased by small increments until the optimum effect under the circumstances is reached.

[0135] In some embodiments, a therapeutically effective amount or therapeutic dose of a composition described herein is an amount that is effective to reduce NE levels or activity in a subject and / or improve one or more clinical characteristics of a disease. For example, in some embodiments, administration of a pharmaceutical composition reduces NE levels or activity in the subject by about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months or longer, as compared to a control or reference. 0%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more.

[0136] In certain embodiments, administration of the pharmaceutical composition extends the life expectancy of the subject in need thereof by, for example, about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 5 9 years, 60 years or more, or at least about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, Increase by 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 ​​years, 49 years, 50 years, 51 years, 52 years, 53 years, 54 years, 55 years, 56 years, 57 years, 58 years, 59 years, 60 years or more.

[0137] In some embodiments, the subject has a pulmonary disease and administration of the pharmaceutical composition improves or reduces lung disease by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, or more, as compared to a control or reference, e.g., when measured over a period of about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months or longer. Improve respiratory function in the subject by 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. In some cases, the improved respiratory function is selected from one or more of: increased expiratory time, increased inspiratory time, decreased maximum expiratory flow, decreased maximum airflow, decreased minute ventilation (RMV), and decreased respiratory rate. In certain embodiments, the subject has a skin or mucosal disorder, and administration of the pharmaceutical composition results in an improvement in disease progression, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, or more, compared to a control or reference, when measured over a period of, e.g., about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months or longer. or greater, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or greater.Methods and assays for measuring such symptoms are known in the art.

[0138] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0139] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made to the appended claims without departing from the spirit or scope thereof. The following examples are offered by way of illustration and not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to obtain essentially similar results. EXAMPLES

[0140] Example 1 Inhibition of neutrophil elastase In vitro studies were performed to test the ability of urinastatin to inhibit neutrophil elastase (NE). The recombinant urinastatin polypeptide (DM300) in this experiment was a UTIΔCS variant (SEQ ID NO:2), which has an N-linked glycan at residue N45 and an unnatural O-linked glycan at residue T17 after recombinant preparation according to the methods described in U.S. Provisional Patent Applications Nos. 63 / 108,733 and 63 / 021,938, which are incorporated herein by reference.

[0141] Recombinant urinastatin (DM300) was tested for inhibition of neutrophil elastase at eight concentrations of DM300, with each compound concentration measured in triplicate. Neutrophil elastase activity was measured using the Neutrophil Elastase Colorimetric Drug Discovery Assay (Enzo Life Sciences) according to the manufacturer's protocol. 65 μl of assay buffer was placed into the required number of wells of a 96-well clear half-area plate and equilibrated at 37°C. Inhibitors were added by adding 20 μl of 5× concentration of DM300 diluted in assay buffer followed by the addition of 10 μl of diluted neutrophil elastase to give final reaction concentrations of 100 μg / mL, 31.6 μg / mL, 10 μg / mL, 3.16 μg / mL, 1 μg / mL, 0.316 μg / mL, 0.1 μg / mL, 0.0316 μg / mL, or 0 μg / mL DM300. 100 μM elastatinal was also included as a control. After 15 minutes at 37° C., 5 μl of diluted substrate was added and the plate was read on a SpectraMax m5 Microplate Reader equilibrated to 37° C. The linear portion of the data was selected and the resulting data was analyzed using GraphPad Prism V.8.2.1.

[0142] The results are shown in FIG. 1 and Table E1 below. [Table E1]

[0143] Under the conditions tested, the IC 50 was about 3.153 μg / ml or about 43.79 nM (assuming 1 Da=1 g / mol for an approximately 17.2 kDa protein). Recombinant urinastatin (e.g., DM300) thus has potential therapeutic utility as an inhibitor of neutrophil elastase activity, for example, in the treatment of neutrophil elastase (NE)-related diseases (e.g., alpha-1 antitrypsin (A1AT) deficiency).

Claims

1. A pharmaceutical composition for use in treating, ameliorating the symptoms of, or inhibiting the progression of a neutrophil elastase (NE)-associated disease in a subject in need thereof, wherein the NE-associated disease is alpha-1 antitrypsin (A1AT) deficiency including its complications, said composition comprising an urinastatin polypeptide, said urinastatin polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-4, or an active variant thereof that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-4 and has at least one urinastatin activity.

2. 2. The pharmaceutical composition for use according to claim 1, wherein the subject has a reduced level of serum A1AT, optionally about 80% or less than about 80% of the normal serum level of A1AT, about 60% or less than about 60% of the normal serum level of A1AT, about 40% or less than about 40% of the normal serum level of A1AT, or about 10-15% of the normal serum level of A1AT.

3. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the subject has an A1AT phenotype / genotype optionally selected from PiMS, PiSS, PiMZ, PiSZ, and PiZZ.

4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the subject has an A1AT genotype selected from a deficiency A1AT allele and a null A1AT allele.

5. The pharmaceutical composition for use according to claim 4, wherein the A1AT deficiency allele is an E342K allele (Z allele) or an E264V allele (S allele).

6. The use, (a) determining serum A1AT level and / or A1AT phenotype / genotype in said subject; and (b) administering the pharmaceutical composition to the subject if the subject has an A1AT phenotype / genotype and / or a reduced serum level of A1AT compared to normal; A pharmaceutical composition for use according to any one of claims 1 to 5, comprising:

7. The pharmaceutical composition for use according to claim 6, wherein the A1AT phenotype / genotype is selected from PiMS, PiSS, PiMZ, PiSZ, and PiZZ; the A1AT genotype comprises a deficient A1AT allele or a null A1AT allele; and / or the serum A1AT level is about 80%, 60%, 40%, 20%, 15%, or 10%, or less than about 80%, 60%, 40%, 20%, 15%, or 10% of the normal serum level of A1AT.

8. The pharmaceutical composition for use according to claim 7, wherein the A1AT deficiency allele is an E342K allele (Z allele) or an E264V allele (S allele).

9. 9. The pharmaceutical composition for use according to any one of claims 1 to 8, wherein the complications of A1AT deficiency include one or more of asthma, bronchiectasis, cirrhosis, COPD, respiratory failure, and vasculitis.

10. 10. The pharmaceutical composition for use according to any one of claims 1 to 9, wherein the subject has any one or more of the following: shortness of breath (on exertion and then at rest if necessary), wheezing, sputum production, or recurrent respiratory infections.

11. The pharmaceutical composition for use according to any one of claims 1 to 10, characterized in that the pharmaceutical composition comprising the urinastatin polypeptide is administered in combination with a further therapeutic agent for treating A1AT deficiency, optionally an RNA interference (RNAi) agent directed against a disease-associated A1AT variant, an A1AT potentiation therapy, a bronchodilator, an inhaled steroid, an antibiotic, or an antiviral agent.

12. The pharmaceutical composition for use according to any one of claims 1 to 11, wherein the subject has increased NE levels or activity compared to a reference standard or healthy control.

13. The use, (a) determining NE level or activity in a sample from the subject; and (b) if the sample has an increased NE level or activity compared to a reference standard or a healthy control, administering the pharmaceutical composition to the subject; A pharmaceutical composition for use according to any one of claims 1 to 12, comprising:

14. 14. The pharmaceutical composition for use according to claim 13, wherein the sample is a sputum sample (optionally including mucus from the lower respiratory tract), a blood or serum sample, or a skin or mucous membrane sample.

15. 15. The pharmaceutical composition for use according to claim 14, wherein the sputum sample contains airway neutrophils and the use comprises determining the level or activity of surface-bound NE in the sample.

16. 16. The pharmaceutical composition for use according to claim 14 or 15, wherein said use comprises determining the level or activity of free NE in said sample.

17. The pharmaceutical composition for use according to any one of claims 1 to 16, wherein the urinastatin polypeptide is a recombinant urinastatin polypeptide or a urinary-derived urinastatin polypeptide.

18. The pharmaceutical composition for use according to any one of claims 1 to 17, wherein the urinastatin polypeptide comprises SEQ ID NO:2 and comprises an N-linked glycan at residue N45 and an O-linked glycan at residue T17.

19. The pharmaceutical composition for use according to any one of claims 1 to 17, wherein the urinastatin polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 (UTIΔCS), bearing a substitution at position S10 of SEQ ID NO:

2.

20. 20. The pharmaceutical composition for use according to claim 19, wherein the urinastatin polypeptide has an S10A substitution, an N-linked glycan at residue N45, and an O-linked glycan at residue T17, said residues being defined by SEQ ID NO:

2.

21. The pharmaceutical composition for use according to any one of claims 1 to 20, wherein the urinastatin polypeptide is fused to an Fc region to form an urinastatin-Fc fusion polypeptide, wherein the urinastatin-Fc fusion polypeptide has at least one urinastatin activity.

22. 22. The pharmaceutical composition for use according to any one of claims 1 to 21, wherein the at least one urinastatin activity comprises a protease inhibitor activity, optionally a neutrophil elastase (NE) inhibitor activity or a trypsin inhibitor activity, and optionally an anti-inflammatory activity.

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