Urinastatin polypeptide

Abstract

To provide urinastatin polypeptides.SOLUTION: Urinastatin glycoforms, urinastatin fusion polypeptides, and related compositions, mixtures, and methods of use, including methods of recombinantly producing urinastatin polypeptides and methods of treating disease, are provided. Embodiments of the present disclosure include isolated mature urinastatin polypeptides comprising (i) an O-linked glycosylation site modified with the residue Glu-Gly-Ser-Gly (SEQ ID NO: 10) which reduces glycosylation at the O-linked glycosylation site, (ii) an N-linked glycan at residue N45, and (iii) an O-linked glycan at residue T17 which is a residue defined by SEQ ID NO: 2 or 4, wherein the urinastatin polypeptide has at least one urinastatin activity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 985,499, filed March 5, 2020, U.S. Provisional Patent Application No. 63 / 021,938, filed May 8, 2020, and U.S. Provisional Patent Application No. 63 / 108,773, filed November 2, 2020, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing Description 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 text file containing the Sequence Listing is named DIAM_039_03WO_ST25.txt. The text file is approximately 31 KB, was created on March 5, 2021, and has been submitted electronically via EFS-Web.

[0003] Embodiments of the present disclosure include urinastatin glycoforms, urinastatin fusion polypeptides, and related compositions, mixtures, and methods of use, including methods of recombinantly producing urinastatin polypeptides and methods of treating disease. [Background technology]

[0004] Urinastatin (also known as urinary trypsin inhibitor) is a glycoprotein protease inhibitor derived from human urine that inhibits the activity of trypsin, chymotrypsin, lactase, lipase, hyaluronidase, and various pancreatic enzymes. Highly purified urinastatin is used clinically to treat acute pancreatitis, chronic pancreatitis, Stevens-Johnson syndrome, burns, septic shock, toxic epidermal necrolysis (TEN), and other diseases.

[0005] Urinastatin has been approved for human use for a variety of conditions, including pancreatitis. However, because urinastatin is a potent protease inhibitor that irreversibly interacts with the active site of proteases and is therefore typically consumed in a 1:1 stoichiometry with its target, large amounts of urinastatin are required. This property, combined with the relatively low in vivo exposure achieved after systemic administration, creates challenges in generating therapeutics from the native urinastatin protein.

[0006] Thus, there is a need in the art for urinastatin polypeptides with improved characteristics related to their recombinant production and / or therapeutic utility, as well as related methods of producing recombinant urinastatin polypeptides. Summary of the Invention [Means for solving the problem]

[0007] Embodiments of the present disclosure include an isolated mature urinastatin polypeptide comprising: (i) an O-linked glycosylation site modified with residues Glu-Gly-Ser-Gly (SEQ ID NO: 10) 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, a residue defined by SEQ ID NO: 2 or 4, wherein the urinastatin polypeptide has at least one urinastatin activity.

[0008] In some embodiments, the isolated mature 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 or 4, wherein the urinastatin polypeptide comprises or retains (i) a modified O-linked glycosylation site, (ii) an N-linked glycan at residue N45, and (iii) an O-linked glycan at residue T17, and the urinastatin polypeptide has at least one urinastatin activity. In some embodiments, an isolated mature 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, wherein the urinastatin polypeptide comprises or retains (i) an S10A substitution of SEQ ID NO:2, (ii) an N-linked glycan at residue N45, and (iii) an O-linked glycan at residue T17, and the urinastatin polypeptide has at least one urinastatin activity. In certain embodiments, an isolated mature 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.

[0009] In some embodiments, the at least one urinastatin activity is selected from one or more of a protease inhibitory activity, an anti-inflammatory activity, and an anti-metastatic activity. In some embodiments, the urinastatin polypeptide has a specific activity of about or at least about 1000-3000 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 of trypsin by 50%.

[0010] Also included are therapeutic compositions comprising an isolated mature urinastatin polypeptide described herein and a pharmaceutically acceptable carrier. Some compositions comprise a mixture of (a) an isolated mature urinastatin polypeptide described herein and (b) a second mature urinastatin polypeptide comprising an N-linked glycan at residue N45 and no O-linked glycan at residue T17, wherein (a) and (b) have at least one urinastatin activity. In some embodiments, the mature urinastatin polypeptide (b) comprises an O-linked glycosylation site modified to residues Glu-Gly-Ser-Gly (SEQ ID NO: 10), which reduces glycosylation at the O-linked glycosylation site, and has at least one urinastatin activity. In some embodiments, the mature urinastatin polypeptide of (b) 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 or 4, includes or retains a modified O-linked glycosylation site and an N-linked glycan at residue N45, does not include an O-linked glycan at residue T17, and has at least one urinastatin activity. In some embodiments, the mature urinastatin polypeptide of (b) 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, includes or retains an S10A substitution and an N-linked glycan at residue N45, does not include an O-linked glycan at residue T17, and has at least one urinastatin activity. In certain embodiments, the mature urinastatin polypeptide of (b) comprises, consists of, or consists essentially of SEQ ID NO: 2, comprises an N-linked glycan at residue N45 and no O-linked glycan at residue T17, and has at least one urinastatin activity.

[0011] In some embodiments, the mature urinastatin polypeptides of (a):(b) are present in the composition at a ratio ranging from about 20:1 to about 1:20, optionally at 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.

[0012] Certain therapeutic compositions are substantially free of glycosylated isoforms (glycoforms) of urinastatin, have endotoxin levels of less than about 1 EU / mg protein, host cell protein of less than about 100 ng / mg total protein, host cell DNA of less than about 10 pg / mg total protein, and / or are substantially aggregate-free.

[0013] Certain embodiments include an urinastatin fusion polypeptide comprising, from N-terminal to C-terminal, a bovine alpha-lactalbumin signal peptide and an urinastatin polypeptide, e.g., the urinastatin polypeptide comprises an O-linked glycosylation site modified with residues Glu-Gly-Ser-Gly (SEQ ID NO: 10), which reduces glycosylation at the O-linked glycosylation site, and the urinastatin polypeptide has at least one urinastatin activity.

[0014] In certain embodiments, the bovine alpha-lactalbumin signal peptide 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: 5. In certain 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 NOs: 1-4, the urinastatin polypeptide has at least one urinastatin activity, and optionally, the urinastatin polypeptide has or retains an S10A substitution as defined by the sequence of mature human urinastatin.

[0015] In certain embodiments, the urinastatin fusion 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% of SEQ ID NO: 6 or 7, wherein the urinastatin polypeptide has at least one urinastatin activity, and optionally, the urinastatin polypeptide has or retains an S10A substitution as defined by the sequence of mature human urinastatin. Some embodiments include a peptide linker between the bovine alpha-lactalbumin signal peptide and the urinastatin polypeptide, and optionally the peptide linker is 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, 60, 70, 80, 90, or 100 amino acids in length. In certain embodiments, the peptide linker includes a protease cleavage site.

[0016] Some urinastatin fusion polypeptides comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 6. Some urinastatin fusion polypeptides comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the at least one urinastatin activity is selected from one or more of a protease inhibitor activity, an anti-inflammatory activity, and an anti-metastatic activity. In certain embodiments, the urinastatin polypeptide has a specific activity of about or at least about 1000-3000 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 of trypsin by 50%.

[0017] Also included are polynucleotides that encode the urinastatin fusion polypeptides described herein. In certain embodiments, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 8 or 9.

[0018] Also included are expression vectors comprising the polynucleotides described herein operably linked to a promoter element. In certain embodiments, the expression vector is a retroviral vector comprising, consisting of, or essentially consisting of, in the 5' to 3' direction: a 5' long terminal repeat (LTR), a packaging region, a promoter region, a polynucleotide encoding a urinastatin fusion polypeptide, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a 3' LTR.

[0019] Also included are recombinant mammalian host cells containing the polynucleotides or expression vectors described herein. Certain recombinant mammalian host cells are selected from HEK293 cells and Chinese hamster ovary (CHO) cells, such as GPEx CHO (GCHO) cells. In certain embodiments, HEK293 cells constitutively express gag, pro, and pol proteins (optionally derived from murine leukemia virus (MLV)) and a separately transfected env protein, and secrete replication-incompetent retroviral particles encoding a urinastatin fusion polypeptide. In certain embodiments, CHO cells express a urinastatin fusion polypeptide and express or overexpress a furin polypeptide, optionally an exogenous furin polypeptide.

[0020] Some embodiments provide a method for recombinantly producing a urinastatin polypeptide, comprising: (a) expressing a urinastatin fusion polypeptide in a recombinant mammalian host cell described herein, optionally a CHO cell or a GCHO cell; and (b) isolating the urinastatin polypeptide from the host cells or from a medium containing the host cells; This includes methods involving recombinantly producing urinastatin fusion polypeptides.

[0021] Certain embodiments include cleaving the bovine alpha-lactalbumin signal peptide from the urinastatin polypeptide to produce a recombinant urinastatin polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NOs: 1-4, wherein the urinastatin polypeptide has at least one urinastatin activity. In some cases, the urinastatin polypeptide has or retains an S10A substitution defined by the sequence of mature human urinastatin. In some cases, the urinastatin polypeptide is a mature urinastatin polypeptide described herein.

[0022] Some embodiments include measuring at least one urinastatin activity of the urinastatin polypeptide under physiological conditions, optionally under physiological conditions of temperature, salinity, and / or pH. Some embodiments include preparing a therapeutic composition comprising the urinastatin polypeptide, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or weight by weight basis, and the composition is substantially free of aggregates and substantially free of endotoxin.

[0023] As noted above, therapeutic compositions comprising the urinastatin polypeptides described herein, including mature urinastatin polypeptides described herein, mixtures thereof (e.g., mixtures comprising different urinastatin glycoforms), and therapeutic compositions prepared by the methods described herein are also included. Some compositions are for use in treating a disease in a subject in need thereof, for example, the disease is an inflammatory disease or cancer.

[0024] Also included are methods of treating an inflammatory disease or condition in a subject in need thereof, comprising administering to the subject a therapeutic composition described herein, thereby treating the inflammatory disease or condition in the subject. In some embodiments, the inflammatory disease or condition is selected from the group consisting of pancreatitis (e.g., acute pancreatitis, chronic pancreatitis, endoscopic retrograde cholangiopancreatography (ERCP)-induced pancreatitis), systemic inflammation, colitis, autoimmune encephalomyelitis, Stevens-Johnson syndrome, arthritis, renal failure, burns, sepsis / septic shock including severe sepsis and associated pro-inflammatory / secondary conditions (e.g., organ failure), systemic inflammatory response syndrome (SIRS), toxic epidermal necrolysis (TEN), Kawasaki disease, renal disease (e.g., acute renal failure, chronic renal disease), ischemic conditions (e.g., ischemia-reperfusion injury in the liver, kidney, heart, lung, brain), pulmonary inflammation and inflammatory pulmonary conditions (e.g., pulmonary inflammatory disease ... For example, the inflammation is selected from one or more of: pulmonary infection, infectious interstitial pneumonia associated with mixed connective tissue disease, pneumonia, pulmonary fibrosis, acute respiratory distress syndrome), liver inflammation including hepatitis, anaphylaxis, post-operative or post-surgical complications (e.g., renal function, cardiac surgery, lung surgery, cognitive impairment, liver transplant), lipopolysaccharide (LPS)-induced inflammation or tissue damage (e.g., lung, liver, brain), inflammation or dysfunction secondary to diabetes (e.g., diabetes-induced cardiac dysfunction), burns, heat stroke, inflammatory or neuropathic pain, acute poisoning, hyperlipidemia-associated inflammation, autoimmune-associated inflammation, allograft- or transfusion-associated inflammation, neuroinflammation, and cancer-associated inflammation.

[0025] In some embodiments, administration of a modified urinastatin polypeptide reduces one or more of protease activity, endothelial activation / damage, pro-inflammatory cytokine and chemokine production / release (optionally IL-1β, MIP-1α, MCP-1, and / or CXCL1), fibrinogen synthesis, neutrophil recruitment to an organ, and / or organ damage in a subject.

[0026] Also included are methods of treating cancer, ameliorating symptoms of cancer, or inhibiting the progression of cancer in a subject in need thereof, comprising administering to the subject a therapeutic composition described herein, thereby treating cancer, ameliorating symptoms of cancer, or inhibiting the progression of cancer in a subject in need thereof. In some embodiments, the cancer is chosen from one or more of melanoma (e.g., metastatic melanoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatocellular carcinoma (hepatocellular carcinoma), sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0027] In some embodiments, the cancer is a metastatic cancer, and optionally, administration of the modified urinastatin polypeptide reduces cancer cell invasion and / or angiogenesis. (a) bladder cancer that has metastasized to bone, liver, and / or lung; (b) breast cancer that has metastasized to the bone, brain, liver, and / or lungs; (c) colorectal cancer metastasizing to the liver, lung, and / or peritoneum; (d) kidney cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or lungs; (e) lung cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or other lung sites; (f) melanoma that has metastasized to bone, brain, liver, lung, and / or skin / muscle; (g) ovarian cancer that has metastasized to the liver, lung, and / or peritoneum; (h) pancreatic cancer that has metastasized to the liver, lung, and / or peritoneum; (i) prostate cancer that has metastasized to the adrenal glands, bone, liver, and / or lungs; (j) gastric cancer that has metastasized to the liver, lung, and / or peritoneum; (l) thyroid cancer that has metastasized to the bone, liver, and / or lungs; and (m) uterine cancer that has metastasized to the bone, liver, lung, vagina, and / or peritoneum. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows the vector map of pFCS-DM300FL-WPRE-SIN (novel ori), GDDDA01.0002, which encodes the full-length urinastatin fusion polypeptide; SEQ ID NO: 6 (polypeptide); and SEQ ID NO: 8 (nucleic acid). [Figure 2] FIG. 2 shows the vector map of pFCS-DM300DCS-WPRE-SIN (novel ori), GDDDA02.0002, which encodes the mature urinastatin fusion polypeptide; SEQ ID NO:7 (polypeptide); and SEQ ID NO:9 (nucleic acid). [Figure 3] Figure 3 shows a reducing SDS-PAGE gel analysis of the expressed urinastatin fusion polypeptides. Lane 4: Novex Sharp prestained standard. Lane 5: Urinary control urinastatin. Lane 6: Medium from day 4 of culture (full-length urinastatin fusion). Lane 7: Medium from day 8 of culture (full-length urinastatin fusion). Lane 8: Medium from day 12 of culture (full-length urinastatin fusion). Lane 9: Medium from day 4 of culture (mature urinastatin fusion). Lane 10: Medium from day 8 of culture (mature urinastatin fusion). Lane 11: Medium from day 12 of culture (mature urinastatin fusion). [Figure 4] Figure 4 shows a reducing SDS-PAGE gel analysis of the expressed urinastatin fusion polypeptide. Lane 1: Novex Sharp prestained standard. Lane 2: Blank. Lane 3: Urinary control urinastatin. Lane 4: Urinary control urinastatin (PNGase-treated). Lane 5: Blank. Lane 6: Full-length urinastatin. Lane 7: Full-length urinastatin (PNGase-treated). Lane 8: Blank. Lane 9: Mature urinastatin. Lane 10: Mature urinastatin (PNGase-treated). [Figure 5]Figure 5 shows that recombinant urinastatin polypeptide (T17) inhibits trypsin in vitro. Complete inhibition of trypsin is demonstrated at dilution factors (DF) of 200 and 4000. Trypsin inhibitory activity decreased as the dilution factor of urinastatin increased, demonstrating specific inhibitory activity against trypsin. [Figure 6A] 6A-6B show that a single dose of recombinant urinastatin polypeptide (T17) reduced mortality in a dose-dependent manner in a mouse model of sepsis. [Figure 6B] Same as above. [Figure 7] FIG. 7 shows the effect of recombinant urinastatin on serum α-amylase levels (32% reduction) in a mouse model of acute pancreatitis. [Figure 8] FIG. 8 shows the effect of recombinant urinastatin on serum lipase levels (25% reduction) in a mouse model of acute pancreatitis. [Figure 9] FIG. 9 shows the effect of recombinant urinastatin on gene markers of inflammation and oxidative stress in a mouse model of acute pancreatitis. DETAILED DESCRIPTION OF THE INVENTION

[0029] 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. Although any method, material, composition, reagent, cell similar to or equivalent to those described herein can be used in carrying out or testing the subject matter of this disclosure, preferred methods and materials are described. All publications and references cited herein, including but not limited to patents and patent applications, are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as if fully set forth. Any patent application to which this application claims priority is incorporated by reference in its entirety in the manner described above for publications and references.

[0030] 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, many of which are described below for illustrative purposes. Such techniques are explained fully in the literature, see, for example, 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,3 rded., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0031] Standard techniques for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection) may be used. 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 the various general and more specific references cited and discussed throughout the specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques of recombinant technology, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients may be used.

[0032] For purposes of this disclosure, the following terms are defined below.

[0033] 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" means one element or more than one element.

[0034] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0035] As used herein, the term "amino acid" is intended to refer to 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 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like, which are known to those skilled in the art. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications can include, for example, substitutions or replacements of chemical groups and moieties on amino acids or derivatization of amino acids. Amino acid mimetics include organic structures that exhibit functionally similar properties, such as the charge and charge spacing characteristics of the reference amino acids. For example, an organic structure that mimics arginine (Arg or R) has a positively charged moiety that is located in a similar molecular space and has the same degree of flexibility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures that maintain optimal spacing and charge interactions of amino acids or amino acid functional groups. Those skilled in the art will know or be able to determine which structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0036] "Biocompatible" generally refers to a material or compound that is not detrimental to biological functions and does not cause any degree of unacceptable toxicity, including allergenic and disease states.

[0037] By "coding sequence" is meant any nucleic acid sequence that is involved in encoding the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that is not directly involved in encoding the polypeptide product of a gene.

[0038] Throughout this disclosure, unless the context requires otherwise, the terms "comprises," "comprises," and "comprising" will be understood to mean the inclusion of the specified step or component or group of steps or components, but not the exclusion of any other step or component or group of steps or components.

[0039] "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed components are required or essential, and that other components may not be present. "Consisting essentially of" means including any components listed after the phrase, and is limited to other components that do not interfere with or participate in the activity or action specified in this disclosure for the listed components. Thus, the phrase "consisting essentially of" indicates that the listed components are required or essential, but that other components are optional and may or may not be present depending on whether they materially affect the activity or action of the listed components.

[0040] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to compositions, solvents, and / or vessels containing minimal amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects in a subject), preferably undetectable amounts. Endotoxins are toxins associated with certain microorganisms, such as bacteria, typically gram-negative bacteria, although endotoxins can also be found in gram-positive bacteria, such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), found in the outer membrane of various gram-negative bacteria and represent a central pathogenic property in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can produce fever, a drop in blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

[0041] Therefore, in pharmaceutical manufacturing, it is often desirable to remove most or all traces of endotoxin from pharmaceutical products and / or drug containers, since even small amounts can cause adverse reactions in humans. Because temperatures above 300°C are typically required to decompose most endotoxins, depyrogenating ovens may be used for this purpose. For example, based on primary packaging materials such as syringes or vials, a glass temperature of 250°C combined with a 30-minute holding time is often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods for removing endotoxin are contemplated, including, for example, chromatography and filtration methods described herein and known in the art.

[0042] Endotoxin can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte lysate assay, which utilizes blood from horseshoe crabs, is a highly sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable clotting of the Limulus lysate due to a powerful enzymatic 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, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to approximately 1 to 10 EU.

[0043] The "half-life" of a polypeptide can refer to the time it takes for the polypeptide to lose half of its pharmacological activity, physiological activity, or other activity, compared to such activity when administered to the serum or tissue of an organism, or compared to any other defined time point. "Half-life" can also refer to the time it takes for the amount or concentration of the polypeptide to be reduced to half of the starting amount administered to the serum or tissue of an organism, compared to such amount or concentration when administered to the serum or tissue of an organism, or compared to any defined time point. Half-life can be measured in serum and / or any one or more selected tissues.

[0044] The terms "modulating" and "altering" include "increasing," "enhancing," or "stimulating," as well as "decreasing," or "reducing," typically by a statistically significant or physiologically significant amount or degree 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, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and ranges in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the amount produced by no composition (e.g., absence of agent) or a control composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount and can include a 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% decrease (e.g., including all integers and ranges therebetween) in the amount produced by no composition (e.g., absence of agent) or a control composition. Examples of comparisons and "statistically significant" amounts are described herein.

[0045] 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 polypeptide or protein catalysts and, with respect to urinastatin, is used interchangeably with protein, polypeptide, or peptide. The terms include myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of signal sequences. The term "polypeptide" or "protein" refers to one or more chains of amino acids, each chain containing amino acids covalently linked by peptide bonds. Such polypeptides or proteins may include multiple chains non-covalently and / or covalently linked to each other by peptide bonds, having the sequence of a native protein, i.e., a protein produced by naturally occurring cells, particularly non-recombinant cells, or by genetic engineering or recombinant cells. These include molecules having the amino acid sequence of a native protein or molecules having deletions, additions, and / or substitutions of one or more amino acids from 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 produced by recombinant cells containing one or more recombinant DNA molecules, and are typically made from a heterologous polynucleotide sequence or combination of polynucleotide sequences not otherwise found in the cell.

[0046] As referred to herein, the term "isolated" polypeptide or protein means that the subject protein (1) is free from at least some other proteins with which it is typically found in nature; (2) is essentially free from other proteins from the same source, e.g., the same species; (3) is expressed by cells from a different species; (4) is separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) the "isolated protein" is not associated (by covalent or non-covalent interactions) with portions of proteins with which it is naturally associated; (6) is operably linked (by covalent or non-covalent interactions) to polypeptides with which it is not naturally associated; or (7) is not naturally occurring. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, or may be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, preventative, research, or otherwise).

[0047] In certain embodiments, the "purity" of any given agent (e.g., urinastatin polypeptide) in a composition may be specifically defined. For example, a particular composition may 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 fractions and ranges in between, 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.

[0048] The term "reference sequence" generally refers to a nucleic acid coding sequence or 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 in tables and sequence listings.

[0049] The term "sequence identity", or for example, as used herein, terms including "50% identical sequence", refer to the degree to which sequences are identical on a nucleotide-to-nucleotide basis or an amino acid-to-amino acid basis over a comparison window.Therefore, "sequence identity percentage" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical nucleic acid bases (for example, A, T, C, G, I) or identical amino acid residues (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occur in both sequences, resulting in 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 may be performed by computer 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 refinement and optimal alignment (i.e., resulting in the highest percentage of homology across the computer window) generated by any of a variety of selected methods. For example, see the BLAST family of programs disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0050] The term "solubility" refers to the ability of a drug provided herein (e.g., a urinastatin polypeptide) to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL) of solvent, mg / mL, etc.), molarity, molar concentration, molar fraction, or other similar descriptions of concentration. The maximum equilibrium amount of solute that can be dissolved per volume of solvent is the solubility of that solute in that solvent under specified conditions, including temperature, pressure, pH, and solvent properties. In certain embodiments, solubility is measured at physiological pH or other pHs, such as 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, such as PBS or NaCl (with or without NaP). In certain embodiments, solubility is measured at a relatively low pH (e.g., pH 6.0) and a 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, 21, 22, 23, 24, 25°C) or about body temperature (37°C). In certain embodiments, the agent has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml at room temperature or 37°C.

[0051] A "subject" or "subject in need thereof" or "patient" or "patient in need thereof" includes mammalian subjects, such as human subjects.

[0052] "Substantially" or "essentially" means nearly completely or entirely, for example, 95%, 96%, 97%, 98%, 99% or more of a given quantity.

[0053] " Statistically significant " means that the result is not considered to occur by chance.Statistical significance can be determined by any method known in the art.The commonly used significance measure includes p-value, which is the frequency or probability that the observed event occurs when the null hypothesis is true.If the obtained p-value is less than significance level, then the null hypothesis is rejected.In simple cases, significance level is defined as p-value of 0.05 or less.

[0054] "Therapeutic response" means an improvement in symptoms (whether sustained or not) following administration of one or more therapeutic agents.

[0055] 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 alter the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and may be administered prophylactically or following either the onset of a pathological phenomenon or contact with a pathogenic agent. Also included is "prophylactic" treatment, which may be directed toward 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 "prevention" does not necessarily indicate complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0056] 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 designated the "normal" or "wild-type" form of the gene.

[0057] Each embodiment herein should be applied mutatis mutandis to all other embodiments unless expressly stated otherwise.

[0058] Urinastatin glycoforms and fusion polypeptides Certain embodiments of the present disclosure generally relate to alternate glycoforms of human urinastatin, including mature human urinastatin polypeptides with an unexpected O-linked glycan at residue threonine 17 (T17), including active or functional variants and fragments thereof. Some embodiments relate to "urinastatin fusion polypeptides" comprising a "bovine alpha-lactalbumin signal peptide" and a "urinastatin polypeptide," including active or otherwise functional variants and fragments thereof.

[0059] "Urinatin" (also referred to as urinary trypsin inhibitor (UTI), HI-30, ASPI, or bikunin) is an acidic glycoprotein with a molecular weight of approximately 30 kDa by SDS-polyacrylamide gel electrophoresis. Wild-type, mature human urinastatin is a polyvalent Kunitz-type serine protease inhibitor found in human urine and blood, consisting of 147 amino acid residues but containing two Kunitz-type domains (see Table U1). It is produced by hepatocytes as a full-length precursor (see Table U1) in which urinastatin is linked to α1-microglobulin. In hepatocytes, different types of urinastatin-containing proteins are formed by the assembly of urinastatin with one or two of three evolutionarily related heavy chains (HC) 1, HC2, and HC3 via chondroitin sulfate chains. These proteins include members of the inter-α-inhibitor (IαI) family, including IαI, pre-α-inhibitor (PαI), inter-α-like inhibitor (IαLI), and free urinastatin. IαI, pαI, and IαLI consist of HC1 + HC2 + UTI, HC3 + UTI, and HC2 + UTI, respectively.

[0060] During inflammation, urinastatin is cleaved from IαI family proteins via proteolytic cleavage by neutrophil elastase in the peripheral circulation or at the site of inflammation, and plasma urinastatin levels and gene expression are altered in severe inflammatory conditions. Therefore, plasma urinastatin is considered to be part of the acute phase response. Furthermore, urinastatin is rapidly released into the urine upon infection and constitutes the majority of urinary antitrypsin activity, making it an excellent inflammatory marker. Urinastatin inhibits various serine proteases, including trypsin, chymotrypsin, kallikrein, plasmin, granulocyte elastase, cathepsin, thrombin, and factors IXa, Xa, XIa, and XlIa. Furthermore, urinastatin can suppress urokinase-type plasminogen activator (uPA) expression through inhibition of protein kinase C (PKC). Urinastatin appears to prevent organ damage by inhibiting the activity of these proteases.

[0061] Beyond inhibiting the inflammatory proteases mentioned above, urinastatin exhibits anti-inflammatory activity, suppressing neutrophil infiltration and their release of elastase and chemical mediators. Similarly, urinastatin inhibits tumor necrosis factor (TNF)-α and interleukin (IL)-1 production in LPS-stimulated human monocytes and IL-8 gene expression stimulated by LPS or neutrophil elastase in HL60 cells or bronchial epithelial cells in vitro. It has been shown to inhibit LPS-induced TNF-α and subsequent IL-1β and IL-6 induction by macrophages in vitro, at least in part through the expression of mitogen-activated protein kinase (MAPK) signaling pathways such as ERK1 / 2, JNK, and p38. Urinastatin also inhibits neutrophil-mediated endothelial cell damage in vitro, suggesting that it acts directly or indirectly on neutrophils to suppress the production and secretion of activated elastase. Furthermore, urinastatin down-regulates stimulated arachidonic acid metabolism in vitro, including thromboxane B2 production, which plays a role in the pathogenesis of sepsis.

[0062] In certain embodiments, the urinastatin polypeptide is a human urinastatin polypeptide, or a variant or fragment thereof. The amino acid sequences of exemplary human urinastatin polypeptides are provided in Table U1 below. [Table U1]

[0063] Thus, in some embodiments, a mature urinastatin polypeptide comprises, consists of, or consists essentially of an amino acid sequence at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 2 or 4 and having or retaining an O-linked glycan at residue T17 as defined by SEQ ID NO: 2 or 4, and the urinastatin polypeptide has at least one urinastatin activity. Also, in certain embodiments, the urinastatin polypeptide portion of a urinastatin fusion polypeptide comprises, consists of, or consists essentially of an amino acid sequence at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to a sequence selected from Table U1 (SEQ ID NOs: 1-4).

[0064] Certain urinastatin polypeptides have a modified O-linked glycosylation site, where serine 10 bears a chondroitin sulfate (CS) chain attached at the highly conserved Glu-Gly-Ser-Gly (SEQ ID NO: 10) O-linked glycosylation site. CS chains have 12-18 disaccharide repeats (GlcUA1,3-GalNAc1,4-) and a conventional linkage region (GlcUA1-3Gal1-3Gal1-4Xyl1)-O-Ser, and are relatively short (MWt approximately 8000). Approximately 30% of the GalNAc residues, typically those near the linkage region, are sulfated at the C-4 hydroxyl group. CS chains synthesized during inflammation are shortened due to reduced sulfation. Thus, in some cases, a urinastatin polypeptide contains at least one substitution and / or deletion at one or more of the Glu-Gly-Ser-Gly (SEQ ID NO: 10) residues of mature urinastatin, which reduces glycosylation at O-linked glycosylation sites. In certain embodiments, a urinastatin polypeptide contains a substitution or deletion at position S10, e.g., an S10A substitution, as defined by the mature urinastatin sequence. Also, in certain embodiments, a urinastatin polypeptide has a naturally occurring N-linked glycan at residue asparagine 45 (N45).

[0065] Accordingly, certain embodiments include an isolated mature urinastatin polypeptide comprising (i) an O-linked glycosylation site modified with residues Glu-Gly-Ser-Gly (SEQ ID NO: 10) 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, wherein the residues are defined by SEQ ID NO: 2 or 4, and the urinastatin polypeptide has at least one urinastatin activity. In some embodiments, the mature 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 or 4, the urinastatin polypeptide comprises or retains (i) a modified O-linked glycosylation site, (ii) an N-linked glycan at residue N45, and (iii) an O-linked glycan at residue T17, and the urinastatin polypeptide has at least one urinastatin activity. Specific examples of mature urinastatin polypeptides include, consist of, or consist essentially of an amino acid sequence at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:2, wherein the urinastatin polypeptide contains or retains (i) an S10A substitution of SEQ ID NO:2, (ii) an N-linked glycan at residue N45, and (iii) an O-linked glycan at residue T17, and wherein the urinastatin polypeptide has at least one urinastatin activity.

[0066] In some embodiments, the urinastatin polypeptide has at least one "urinastatin activity." The term "urinastatin activity" includes (a) protease inhibitory activity, including reducing the protease activity of one or more of trypsin, chymotrypsin, kallikrein, plasmin, granulocyte elastase, cathepsin, thrombin, and / or factor IXa, Xa, XIa, and X1Ia; (b) anti-inflammatory activity, including reducing inflammation and / or cytokine-dependent signaling pathways, e.g., to reduce organ damage following severe inflammation; and (c) reducing tumor invasion and metastasis, e.g., by reducing cathepsin B activity and / or reducing CD44 dimerization, at least the latter of which includes anti-metastatic activity, which inhibits the MAP kinase signaling cascade and reduces extracellular matrix (ECM) degradation, tumor cell invasion, and / or angiogenesis.

[0067] In certain embodiments, the urinastatin polypeptide has a ribonucleotide concentration of about or at least about 500-5000 U / mg, or about or at least about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300 It has a "specific activity" of 900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 U / mg of polypeptide, where 1 unit (U) is the amount of urinastatin polypeptide that inhibits the activity of 2 μg of trypsin by 50%.

[0068] As mentioned above, the urinastatin fusion polypeptides described herein contain a bovine alpha-lactalbumin signal peptide, or a variant or fragment thereof. Exemplary signal peptide sequences are provided in Table S1 below. [Table S1]

[0069] Thus, in certain embodiments, the bovine alpha-lactalbumin signal peptide portion of the urinastatin fusion 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:5.

[0070] The amino acid sequences of exemplary human urinastatin fusion polypeptides are provided below in Table U2. [Table U2]

[0071] Thus, in some embodiments, a urinastatin fusion polypeptide, or variant or fragment thereof, 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 a sequence selected from Table U2, and has at least one urinastatin activity. In certain embodiments, the urinastatin portion of a fusion polypeptide of Table U2 comprises or retains an O-linked glycosylation site modified with residues Glu-Gly-Ser-Gly (SEQ ID NO: 10), such as a substitution or deletion at position S10, e.g., an S10A substitution, which reduces glycosylation at the O-linked glycosylation site.

[0072] 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 retain the enzymatic or binding activity of the reference polypeptide. Such variants can result, for example, from genetic polymorphism and / or human manipulation.

[0073] In some cases, a variant contains one or more "conservative" changes or substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties, such that one skilled in the art of peptide chemistry would predict the secondary structure and hydropathic properties of the polypeptide to be substantially unchanged. As noted above, modifications may be made to the polynucleotide and polypeptide structures of the present disclosure to still obtain functional molecules encoding variant or derivative polypeptides with desired characteristics. When it is desired to alter 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 would typically change one or more of the codons in the encoding DNA sequence.

[0074] For example, certain amino acids can be substituted with other amino acids in a protein structure without significant loss of interaction binding ability with a structure, such as, for example, an antigen-binding region or binding site of an antibody on a substrate molecule. Because it is the interaction ability and properties of a protein that define its biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and of course, in its underlying DNA coding sequence, to obtain a protein with similar properties. Therefore, it is contemplated that various changes can be made in the peptide sequences of the disclosed compositions or the corresponding DNA sequences encoding the aforementioned peptides without significantly impairing their usefulness.

[0075] When making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the amino acid index, or hydropathic index, in conferring interactive biological function to a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic index characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the protein's interactions 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 with other amino acids having similar hydropathic indexes or scores, and still result in proteins with similar biological activity, i.e., obtain biologically functional equivalent proteins. In making such changes, substitution of amino acids exhibiting a hydropathic index within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0076] It is also understood in the art that substitutions of like amino acids can be usefully made 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 is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent protein, particularly an immunologically equivalent protein. In such changes, substitution of amino acids exhibiting hydrophilicity values ​​within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0077] As outlined above, amino acid substitutions are therefore generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration the various aforementioned characteristics 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.

[0078] Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups 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.

[0079] Variants may also, or alternatively, contain non-conservative changes. In some embodiments, variant polypeptides differ from the native or reference sequence by substitutions, deletions, or additions of no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or even a single amino acid. Variants may also (or alternatively) be modified by, for example, deletions or additions of amino acids that have minimal effect on the immunogenicity, secondary structure, enzymatic activity, and / or hydropathic properties of the polypeptide.

[0080] In certain embodiments, the polypeptide sequence is about, at least about, or up to 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, 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, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123, 124, 125, 126, 127, 12 4, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, or 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, The sequence may be 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 and may include all or a portion of a reference sequence (see, e.g., Table U1, Table S1, Table U2, Sequence Listing).

[0081] In some embodiments, the polypeptide sequence is about or 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, 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, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, , 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 more contiguous amino acids and may include all or a portion of a reference sequence (see, e.g., Table U1, Table S1, Table U2, Sequence Listing).

[0082] In certain embodiments, the polypeptide sequence is selected from the group consisting of about 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, 20-1000, 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 50-1000, 50-900, 50-800, 50-600, 50-800, 50-1000, 50-900, 50-800, 50-1000, 50-1000, 50-1000, 50-200, 50-300, 50-400, 50-300, 50-200, 50-1000, 50-500, 50-400, 50-300, 50-400, 50-500, 50-600, 50-600, 50-700, 50-800, 50-400, 50-300, 50-200, 50-1000, 50-500, 50-600, 50-600, 50-700, 50-80 0-700, 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 or a portion of a reference sequence (see, e.g., Table U1, Table S1, Table U2, and the Sequence Listing). In certain embodiments, the C-terminal or N-terminal region of any reference polypeptide may be truncated by 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 amino acids, or by about 10-50, 20-50, 50-100 or more amino acids, including all integers and ranges therebetween (e.g., 101, 102, 103, 104, 105), so long as the truncated polypeptide retains the binding properties and / or activity of the reference polypeptide (see, e.g., Table U1, Table S1, Table U2, Sequence Listing). Typically, biologically active fragments have about 1%, about 5%, about 10%, about 25%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% or more of the activity of the biologically active reference polypeptide from which they are derived.

[0083] 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, Table U2, Sequence Listing). Furthermore, approximately 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, 55, 60, 65, 70, 75, 80, 85 Sequences that differ from the native or parent sequence by the addition (e.g., C-terminal addition, N-terminal addition, both), deletion, truncation, insertion, or substitution (e.g., conservative substitution) of 90, 95, or 100 amino acids (including all integers and ranges therebetween) but retain the properties or activity of the parent or reference polypeptide sequence are contemplated (see, e.g., Table U1, Table S1, Table U2, Sequence Listing).

[0084] In some embodiments, a variant polypeptide differs from a reference sequence by at least one but less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3, or 2 amino acid residue(s). In certain embodiments, a variant polypeptide differs from a reference sequence by at least 1% but less than 20%, 15%, 10%, or 5% of the residues. (Where this comparison requires alignment, sequences should be aligned for maximum similarity. Sequences "out of" deletions or insertions, or mismatches, are considered differences.)

[0085] Calculation of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) 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 alignment (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may 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%, even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

[0086] The percent identity between 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, that need to be introduced for optimal alignment of the two sequences.

[0087] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. 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 incorporated into the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight 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 a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and those to be used unless otherwise specified) is the Blossum62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0088] 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 incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0089] The sequences described herein can be used as "query sequences" to search public databases, for example, to identify 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 using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 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). BLAST and Gapped BLAST programs can be used to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. When utilizing BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0090] In some embodiments, as described above, polynucleotides and / or polypeptides can be evaluated using BLAST alignment tools. Local alignments simply consist of paired sequence segments, with each one of the sequences being compared. A modification of the Smith-Waterman or Sellers algorithm will find all segment pairs, called high-scoring segment pairs (HSPs), whose scores cannot be improved by extension or trimming. The results of BLAST alignment include a statistical measure to indicate the likelihood that the BLAST score could be predicted by chance alone.

[0091] A raw score, S, is calculated from the number of gaps and substitutions associated with each aligned sequence, with higher similarity scores indicating more significant alignment. Substitution scores are given by lookup tables (see PAM, BLOSUM).

[0092] 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 will be G + Ln. The selection of gap costs, G and L, is empirical, but it is customary to choose a high value for G (10-15), e.g., 11, and a low value for L (1-2), e.g., 1.

[0093] The bit score S' results from the raw alignment score S, taking into account the statistical properties of the scoring system used. 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 indicates how good the alignment is; the higher the score, the better the alignment.

[0094] The E value, or expectation value, describes the likelihood that a sequence with a similar score will occur in a database by chance. It is a prediction 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 0.05 means that sequences with similar scores are very unlikely to occur simply by chance. In addition, the expected score for a random pairwise amino acid alignment must be negative; otherwise, long alignments tend to have high scores, regardless of whether the aligned segments are related. In addition, the BLAST algorithm uses an appropriate substitution matrix, nucleotide or amino acid, and gap alignments use 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.

[0095] In some embodiments, sequence similarity scores are reported from BLAST analyses conducted using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

[0096] For a particular Sporomus aeruginosa, the sequence identity / similarity scores provided herein refer to values ​​obtained using the following parameters: % identity and % similarity for nucleotide sequences using GAP version 10 (GCG, Accelrys, San Diego, Calif.) with a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89:10915-10919, 1992). GAP uses the algorithm of Needleman and Wunsch (J Mol Biol. 48:443-453, 1970) to find an alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.

[0097] In certain embodiments, the variant polypeptides are optimally aligned with a reference polypeptide sequence (see, e.g., Table U1, Table U2, Sequence Listing) and contain 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 , 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 greater BLAST bit scores or sequence similarity scores were obtained. BLAST alignments were performed using the BLOSUM62 matrix, a gap presence penalty of 11, and a gap extension penalty of 1.

[0098] As mentioned above, a reference polypeptide may be altered 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 a reference polypeptide can be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence changes 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," Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited therein. For guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest, see Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0099] The method of screening the gene product of the combinatorial library made by this modification, and the method of screening cDNA library for the gene product with selected properties are known in the art.This method can be adapted to the rapid screening of the gene library produced by combinatorial mutagenesis of reference polypeptide.As an example, recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in library, can be used in combination with screening assay to identify polypeptide variants (Arkin and Yourvan, PNAS USA 89:7811-7815,1992; Delgrave et al., Protein Engineering.6:327-331,1993).

[0100] In certain embodiments, a peptide linker sequence may be utilized to separate the bovine alpha-lactalbumin signal peptide(s) and the urinastatin polypeptide(s) by a distance sufficient to ensure that each polypeptide is folded into its desired secondary and tertiary structure and / or to facilitate cleavage of the signal peptide from the urinastatin polypeptide, if desired. Such peptide linker sequences can be incorporated into the fusion polypeptide using standard techniques well known in the art.

[0101] Certain peptide linker sequences may be selected based on the following exemplary factors: (1) their ability to adopt a flexibly extended conformation; (2) their ability to adopt a secondary structure that may interact with functional epitopes on the first and second polypeptides; (3) their physiological stability; and (4) the lack of hydrophobic or charged residues that may react with polypeptide functional epitopes, or other properties. See, e.g., George and See Heringa, J Protein Eng. 15:871-879, 2002.

[0102] Linker sequences may generally be from 1 to about 200 amino acids in length. Particular linkers may 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 amino acids. , 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, 60, 70, 80, 90, 100 or more amino acids.

[0103] Peptide linkers may utilize any one or more naturally occurring amino acids, non-naturally occurring amino acids(s), amino acid analogs, and / or amino acid mimetics as described elsewhere herein and known in the art. Certain amino acid sequences that can be usefully utilized as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA. 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. Certain peptide linker sequences contain Gly, Ser, and / or Asn residues. Other near-neutral amino acids, such as Thr and Ala, may also be utilized in peptide linker sequences if desired.

[0104] Certain exemplary linkers are: [G] x , [S] x , [N] x , [GS] x , [GGS] x , [GSS] x , [GSGS] x (SEQ ID NO: 11), [GGSG] x (SEQ ID NO: 10), [GGGS] x (SEQ ID NO: 12), [GGGGS] x (SEQ ID NO: 13), [GN] x , [GGN] x , [GNN] x , [GNGN] x (SEQ ID NO: 14), [GGNG] x (SEQ ID NO: 15), [GGGN] x (SEQ ID NO: 16), [GGGGN] x (SEQ ID NO: 17) LINKER (wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more). Other combinations of these and related amino acids will be apparent to one of skill in the art.

[0105] Further examples of linker peptides include those with the following amino acid sequences: Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 18); Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 19); Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly- Examples of amino acids that may be used include, but are not limited to, Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 20); Asp-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-Ala-Ala-Ala-Ala-Arg-Asp-Ala-Ala-Ala-Lys-(SEQ ID NO: 21); and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg-(SEQ ID NO: 22).

[0106] Further, non-limiting examples of linker peptides include DGGGS (SEQ ID NO: 23); TGEKP (SEQ ID NO: 24) (see, e.g., Liu et al., PNAS. 94:5525-5530, 1997); GGRR (SEQ ID NO: 25) (Pomerantz et al.1995);(GGGGS) n (SEQ ID NO: 13) (Kim et al., PNAS. 93: 1156-1160, 1996); EGKSSGSGSESKVD (SEQ ID NO: 26) (Chaudhary et al., PNAS. 87: 1066-1070, 1990); KESGSVSSEQLAQFRSLD (SEQ ID NO: 27) (Bird et al., Science. 242:423-426, 1988), GGRRGGGS (SEQ ID NO: 28); LQRDGERP (SEQ ID NO: 29); LRQKDGGGSERP (SEQ ID NO: 30); LRQKd(GGGS)2ERP (SEQ ID NO: 31). In certain embodiments, the linker sequence comprises a Gly3 linker sequence containing three glycine residues. In certain embodiments, flexible linkers can be readily designed using computer programs capable of modeling both DNA binding sites and the peptides themselves (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and PNAS. 91:11099-11103, 1994) or by phage display methods.

[0107] In certain embodiments, the linker peptide comprises an autocatalytic or autocleaving peptide cleavage site. In certain embodiments, the autocleaving peptide comprises the potyvirus and cardiovirus 2A peptide, its polypeptide sequence obtained from FMDV (foot-and-mouth disease virus), equine rhinitis A virus, sosachinavirus, and porcine teschovirus. In certain embodiments, the autocleaving polypeptide site comprises a 2A or 2A-like site, sequence, or domain (Donnelly et al., J. Gen. Virol. 82:1027-1041, 2001). Exemplary 2A sites include the following sequences: LLNFDLLKLAGDVESNPGP (SEQ ID NO: 32); TLNFDLLKLAGDVESNPGP (SEQ ID NO: 33); LLKLAGDVESNPGP (SEQ ID NO: 34); NFDLLKLAGDVESNPGP (SEQ ID NO: 35); QLLNFDLLKLAGDVESNPGP (SEQ ID NO: 36); APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 37); VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT (SEQ ID NO: 38); LNFDLLKLAGDVESNPGP (SEQ ID NO: 39); LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 40); and EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 41). In some embodiments, the autocatalytic peptide cleavage site comprises a translational 2A signal sequence, such as the 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein, which is an 18-amino acid sequence. Further examples of 2A-like sequences that can be used include insect virus polyproteins in the genus Trypanosoma, as described, for example, in Donnelly et al., Journal of General Virology. 82:1027-1041, 2001, the NS34 protein of type C rotavirus, and repeat sequences.

[0108] Suitable protease cleavage sites and self-cleaving peptides are known to those of skill in the art (see, for example, Ryan et al., J. Gener. Virol. 78:699-722, 1997; and Scymczak et al., Nature Biotech. 5:589-594, 2004). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, biovirus NIa protease, biovirus RNA-2 encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Rice tungrospheroidal virus) 3C-like protease, PYVF (Parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to their high cleavage stringency, TEV (Tobacco Etch Virus) protease cleavage sites are included in some embodiments, e.g., EXXYXQ(G / S) (SEQ ID NO: 42), e.g., ENLYFQG (SEQ ID NO: 43) and ENLYFQS (SEQ ID NO: 44), where X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).

[0109] Additionally, examples of enzymatically degradable linkers suitable for use in certain embodiments include, but are not limited to, amino acid sequences that are cleaved by serine proteases, such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or subtilisin. Illustrative examples of thrombin-cleavable amino acid sequences include, but are not limited to, -Gly-Arg-Gly-Asp-(SEQ ID NO:45), -Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro-(SEQ ID NO:46), -Gly-Arg-Gly-Asp-Ser-(SEQ ID NO:47), -Gly-Arg-Gly-Asp-Ser-Pro-Lys-(SEQ ID NO:48), -Gly-Pro-Arg-, -Val-Pro-Arg-, and -Phe-Val-Arg-. Illustrative examples of elastase-cleavable amino acid sequences include, but are not limited to, -Ala-Ala-Ala-, -Ala-Ala-Pro-Val- (SEQ ID NO: 49), -Ala-Ala-Pro-Leu- (SEQ ID NO: 50), -Ala-Ala-Pro-Phe- (SEQ ID NO: 51), -Ala-Ala-Pro-Ala- (SEQ ID NO: 52), and -Ala-Tyr-Leu-Val- (SEQ ID NO: 53).

[0110] Enzymatically degradable linkers also include amino acid sequences that can be cleaved by matrix metalloproteinases, such as collagenase, stromelysin, and gelatinase. Illustrative examples of amino acid sequences cleavable by matrix metalloproteinases include, but are not limited to, -Gly-Pro-Y-Gly-Pro-Z- (SEQ ID NO: 54), -Gly-Pro-, Leu-Gly-Pro-Z- (SEQ ID NO: 55), -Gly-Pro-Ile-Gly-Pro-Z- (SEQ ID NO: 56), and -Ala-Pro-Gly-Leu-Z- (SEQ ID NO: 57), where Y and Z are amino acids. Illustrative examples of collagenase-cleavable amino acid sequences include, but are not limited to, -Pro-Leu-Gly-Pro-D-Arg-Z- (SEQ ID NO: 58), -Pro-Leu-Gly-Leu-Leu-Gly-Z- (SEQ ID NO: 59), -Pro-Gln-Gly-Ile-Ala-Gly-Trp- (SEQ ID NO: 60), -Pro-Leu-Gly-Cys(Me)-His- (SEQ ID NO: 61), -Pro-Leu-Gly-Leu-Tyr-Ala- (SEQ ID NO: 62), -Pro-Leu-Ala-Leu-Trp-Ala-Arg- (SEQ ID NO: 63), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg- (SEQ ID NO: 64), where Z is an amino acid. An illustrative example of an amino acid sequence cleavable by stromelysin is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg- (SEQ ID NO: 65), and an example of an amino acid sequence cleavable by gelatinase is -Pro-Leu-Gly-Met-Tyr-Ser-Arg- (SEQ ID NO: 66).

[0111] Enzymatically degradable linkers suitable for use in certain embodiments include amino acid sequences that can be cleaved by angiotensin-converting enzyme, such as, for example, -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro- (SEQ ID NO: 67), and -Gly-Ser-Asp-Lys-Pro- (SEQ ID NO: 68).

[0112] Enzymatically degradable linkers suitable for use in certain embodiments include amino acid sequences that can be degraded by cathepsin B, such as, for example, Val-Cit, Ala-Leu-Ala-Leu- (SEQ ID NO: 69), Gly-Phe-Leu-Gly- (SEQ ID NO: 70), and Phe-Lys.

[0113] However, in certain embodiments, any one or more peptide linkers are optional, e.g., linker sequences are not required when the first and second polypeptides have non-essential N- and / or C-terminal amino acid regions that can be used to separate functional domains and prevent biological interference.

[0114] The urinastatin fusion polypeptides can be used in any of the compositions, methods, and / or kits described herein.

[0115] Polynucleotides, expression vectors, and host cells Certain embodiments relate to polynucleotides encoding the urinastatin fusion polypeptides described herein. Accordingly, certain embodiments include polynucleotides encoding any one or more of the individual urinastatin fusion polypeptides in Table U1 or Table U2, including variants and / or fragments thereof. For example, certain polynucleotides encode urinastatin fusion polypeptides comprising, consisting of, or consisting essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a reference amino acid sequence selected from Table U1 or Table U2.

[0116] Exemplary nucleic acid coding sequences are provided below in Table U3. [Table U3-1] [Table U3-2]

[0117] Accordingly, certain embodiments include polynucleotides, e.g., isolated polypeptides, that encode urinastatin fusion polypeptides, where the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a nucleic acid sequence of Table U3 (e.g., SEQ ID NO: 8 or 9).

[0118] Among other uses, these and related embodiments may be utilized to recombinantly produce urinastatin polypeptides in host cells. Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides may bear minimal homology to the nucleotide sequence of any native gene. Nevertheless, due to differences in codon usage, polynucleotides that vary, such as polynucleotides optimized for human, yeast, or bacterial codon preferences, are specifically contemplated.

[0119] As will be appreciated by those skilled in the art, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. Polynucleotides may comprise native sequences or may comprise variants or biologically functional equivalents of such sequences. Polynucleotide variants may contain one or more substitutions, additions, deletions, and / or insertions described herein, preferably such that the activity of the variant polypeptide is not substantially diminished compared to the unmodified polypeptide.

[0120] Additional coding or non-coding sequences can be, but are not required to be, present within a polynucleotide, and polynucleotides can be, but are not required to be, linked to other molecules and / or supporting materials. Thus, polynucleotides can be combined with other DNA or RNA sequences, such as promoters, enhancements, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably, regardless of the length of the coding sequence itself.

[0121] Polynucleotide sequences may also be of mixed genomic, cDNA, RNA, and synthetic origin. For example, a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding a polypeptide, and the DNA or RNA sequence may then be modified at that site by inserting a synthetic oligonucleotide encoding the desired amino acid sequence for homologous recombination according to well-known techniques, or, preferably, by PCR using appropriate oligonucleotides to generate the desired sequence. In some embodiments, a signal sequence may be included before the coding sequence. This sequence encodes a signal peptide at the N-terminus of the coding sequence that signals the host cell to target the polypeptide to the cell surface or secrete the polypeptide into the medium. Typically, the signal peptide is cleaved off by the host cell before the protein leaves the cell. Signal peptides can be found in a variety of proteins in prokaryotes and eukaryotes.

[0122] One or more polynucleotides can encode the urinastatin polypeptides described herein. Furthermore, the polynucleotide sequence can be manipulated for a variety of reasons, including, but not limited to, the incorporation of preferred codons to enhance expression of the polynucleotide in various organisms (see generally Nakamura et al., Nuc. Acid. Res. 28:292, 2000).

[0123] Also included are expression vectors containing polynucleotides, and host cells containing polynucleotides and / or expression vectors.Urinastatin polypeptides can be produced by expressing DNA or RNA sequences encoding the polypeptides in suitable host cells using well-known techniques.The term "host cell" refers to a cell into which a nucleic acid sequence encoding one or more of the polypeptides described herein has been introduced, or into which a nucleic acid sequence encoding one or more of the polypeptides described herein can be introduced, and to a cell that further expresses or can express a polypeptide of interest, such as a polynucleotide encoding any of the polypeptides described herein.This term also includes the progeny of a parent cell, regardless of whether the progeny is identical in morphology or genetic makeup to the original parent, as long as the selected gene is present.

[0124] In some cases, polynucleotides or expression vectors contain additional non-coding sequences. For example, the "regulatory elements" or "control sequences" present in expression vectors are untranslated regions of the vector, including enhancers, promoters, 5' and 3' untranslated regions, which interact with host cell proteins to transcribe and translate. Such elements can vary in their strength and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements, including constitutive promoters and inducible promoters, may be used.

[0125] A variety of expression vector / host systems are known and may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with expression vectors, e.g., recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems, including mammalian cells transformed with viral, plasmid, episomal, or integrative expression vectors, and more specifically, human cell systems. Accordingly, certain embodiments include expression vectors containing polynucleotide sequences encoding the polypeptides described herein, e.g., urinastatin fusion polypeptides. In certain embodiments, the expression vector is a retroviral vector (or retrovector), e.g., as illustrated in Figures 2-3 and Tables E1-E2.

[0126] Host cells containing the polynucleotide and / or expression vector are also included. Numerous expression systems for mammalian host cells are well known in the art and commercially available. Exemplary mammalian host cells and systems include, for example, HEK293 cells, CHO cells, including the GPEx® Chinese Hamster Ovary (GCHO) cell line, HeLa cells, and the like. Mammalian expression systems can utilize adherent cell lines in, for example, T-flasks, roller bottles, shaker flasks (e.g., 2.8 L), and / or cell factories, or suspension cultures, e.g., 1 L and 5 L spinners, 5 L, 14 L, 40 L, 100 L, and 200 L stirred-tank reactors, or 20 / 50 L and 100 / 200 L WAVE reactors, among others known in the art. Thus, certain embodiments include recombinant host cells, e.g., mammalian host cells, containing a polynucleotide encoding the urinastatin fusion polypeptide described herein. In certain embodiments, the host cell expresses or overexpresses a protease, such as a furin polypeptide, that cleaves full-length urinastatin to produce mature urinastatin. Certain host cells (e.g., HEK293 cells) can be used to produce high-titer replication-incompetent retrovector particles, and certain host cells (CHO, GCHO) can be transduced with the retrovector particles to produce urinastatin-expressing cells.

[0127] Also included are methods for recombinantly producing the urinastatin polypeptides described herein. In some embodiments, a polynucleotide or expression vector encoding a urinastatin fusion polypeptide is directly introduced into a host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded protein(s). Accordingly, certain embodiments relate to a method for recombinantly producing a urinastatin polypeptide, comprising: (a) expressing a urinastatin fusion polypeptide in a recombinant mammalian host cell described herein, optionally a CHO cell or GCHO cell; and (b) isolating the urinastatin polypeptide from the host cell or from a medium containing the host cell, thereby recombinantly producing the urinastatin polypeptide. Expression of the urinastatin polypeptide in a host cell can be achieved by culturing a recombinant host cell containing the polynucleotide under appropriate conditions (see, e.g., Example 1). After production by expression, the urinastatin polypeptide may be isolated and / or purified using any suitable technique and then used as desired. Additionally, after expression of the mature urinastatin fusion polypeptide, certain embodiments include a step of cleaving the signal peptide, for example, with a protease.

[0128] The urinastatin polypeptide produced by recombinant host cells can be purified and characterized according to various techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems) and high performance liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary purification chemistries include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reversed-phase, ceramic HYPERD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. See also the Examples.

[0129] Also included are methods for assessing or measuring the activity of a purified urinastatin polypeptide produced under physiological conditions, optionally at physiological temperature and pH, wherein the urinastatin polypeptide has activity under physiological conditions. In some embodiments, the urinastatin polypeptide has at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more activity compared to the urinastatin polypeptide of SEQ ID NO: 2 (mature human urinastatin) under equivalent physiological conditions.

[0130] Certain embodiments include preparing a composition comprising a urinastatin polypeptide, e.g., the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or weight by weight basis, the composition is substantially free of aggregates, and is substantially free of endotoxin.

[0131] Compositions and Methods of Use Certain embodiments include therapeutic compositions comprising urinastatin polypeptides, for example, urinastatin polypeptides produced according to the methods described herein and methods of using same for the treatment of various diseases.

[0132] Some embodiments include compositions, e.g., therapeutic or pharmaceutical compositions, comprising a mature urinastatin polypeptide described herein, including an urinastatin polypeptide prepared or produced according to the methods described herein, and a pharmaceutically acceptable carrier. For example, certain compositions include a mature urinastatin polypeptide having a T17-O-linked glycan, including (i) a modified O-linked glycosylation site at residues Glu-Gly-Ser-Gly (SEQ ID NO: 10) that reduces glycosylation at the O-linked glycosylation site, (ii) an N-linked glycan at residue N45, and (iii) an O-linked glycan at residue T17, wherein the residues are defined by SEQ ID NO: 2 or 4, as described herein. Specific examples of mature urinastatin polypeptides and active variants and fragments thereof are described herein (see, e.g., Table U1 and related disclosures).

[0133] Certain compositions comprise a mixture of urinastatin glycoforms. For example, certain compositions comprise (a) a first mature urinastatin polypeptide comprising a T17-O-linked glycan described herein, and (b) a second mature urinastatin polypeptide comprising an N-linked glycan at residue N45 and no O-linked glycan at residue T17. In some embodiments, the second mature urinastatin polypeptide has a modified O-linked glycosylation site, e.g., an S10A substitution at residues Glu-Gly-Ser-Gly (SEQ ID NO: 10), described herein, which reduces glycosylation at the O-linked glycosylation site. In some embodiments, the second mature urinastatin polypeptide of (b) 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 or 4, includes or retains a modified O-linked glycosylation site (e.g., an S10A substitution) and an N-linked glycan at residue N45, does not include an O-linked glycan at residue T17, and has at least one urinastatin activity.

[0134] In some embodiments, the mature urinastatin polypeptides of (a):(b) are present in the composition at a ratio ranging from about 20:1 to about 1:20, optionally at 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.

[0135] 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-to-weight basis, and are substantially free of aggregates, for example, less than about 10%, 9%, 8%, 7%, 6%, or 5% aggregates.Certain compositions are substantially free of endotoxins as described herein.

[0136] Compositions may be prepared by methodologies well known in the pharmaceutical arts. For example, compositions intended to be administered by injection can be prepared by combining a composition containing the urinastatin polypeptide described herein and, optionally, one or more buffers or excipients, with distilled water to form a solution. Certain compositions contain 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 can be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that do not covalently interact with the urinastatin polypeptide in the composition to facilitate dissolution or uniform suspension of the polypeptide in an aqueous delivery system.

[0137] Certain compositions have a pharmaceutically acceptable pH. For example, in certain embodiments, the pharmaceutically 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.

[0138] In certain embodiments, the urinastatin polypeptide has at least one urinastatin activity at a pH that approximates 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.

[0139] In certain embodiments, the composition has one or more of 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 is substantially free of aggregates.

[0140] Also included are methods of treating a disease, ameliorating symptoms of a disease, or inhibiting the progression of a disease in a subject in need thereof, comprising administering to the subject a composition comprising at least one urinastatin polypeptide produced according to the methods described herein.

[0141] The methods and compositions described herein can be used in the treatment of various diseases or conditions. For example, certain embodiments include a method of treating an inflammatory disease or condition in a subject in need thereof, comprising administering to the subject a therapeutic composition described herein.

[0142] Exemplary inflammatory diseases or conditions include pancreatitis (e.g., acute pancreatitis, chronic pancreatitis, endoscopic retrograde cholangiopancreatography (ERCP)-induced pancreatitis), systemic inflammation, colitis, autoimmune encephalomyelitis, Stevens-Johnson syndrome, arthritis, renal failure, burns, severe sepsis and related pro-inflammatory / secondary conditions (e.g., organ failure) including sepsis / septic shock, systemic inflammatory response syndrome (SIRS), toxic epidermal necrolysis (TEN), Kawasaki disease, renal disease (e.g., acute renal failure, chronic renal disease), ischemic conditions (e.g., ischemia-reperfusion injury in the liver, kidney, heart, lung, brain), pulmonary inflammation, and inflammatory Pulmonary conditions (e.g., pulmonary infections, including infectious interstitial pneumonia associated with mixed connective tissue disease, pneumonia, pulmonary fibrosis, acute respiratory distress syndrome), liver inflammation including hepatitis, anaphylaxis, post-operative or post-surgical complications (e.g., renal function, cardiac surgery, pulmonary surgery, cognitive impairment, liver transplant), lipopolysaccharide (LPS)-induced inflammation or tissue damage (e.g., lung, liver, brain), inflammation or dysfunction secondary to diabetes (e.g., diabetes-induced cardiac dysfunction), burns, heat stroke, inflammatory or neuropathic pain, acute intoxication, hyperlipidemia-associated inflammation, autoimmune-associated inflammation, allograft- or transfusion-associated inflammation, and neuroinflammation.

[0143] Also included are methods of treating cancer, ameliorating the symptoms of cancer, or inhibiting the progression of cancer in a subject in need thereof, comprising administering to the subject a therapeutic composition described herein. The methods and therapeutic compositions described herein can be used in the treatment of any of a variety of cancers or tumors. In some embodiments, the cancer is a primary cancer, i.e., a cancer that arises at an anatomical site where tumor progression begins and produces a cancerous mass. In some embodiments, the cancer is a secondary or metastatic cancer, i.e., a cancer that has spread from the original primary site or tissue to one or more different sites or tissues. In some embodiments, the subject has a cancer chosen from one or more of melanoma (e.g., metastatic melanoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatocellular carcinoma (hepatocellular carcinoma), sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0144] In some embodiments, the cancer or tumor is a metastatic cancer, as described above. In addition to the cancers mentioned above, exemplary metastatic cancers include, but are not limited to, bladder cancer that has metastasized to the bone, liver, and / or lungs; breast cancer that has metastasized to the bone, brain, liver, and / or lungs; colorectal cancer that has metastasized to the liver, lungs, and / or peritoneum; kidney cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or lungs; lung cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or other pulmonary sites; melanoma that has metastasized to the bone, brain, liver, lungs, and / or skin / muscle; ovarian cancer that has metastasized to the liver, lungs, and / or peritoneum; pancreatic cancer that has metastasized to the liver, lungs, and / or peritoneum; prostate cancer that has metastasized to the adrenal glands, bone, liver, and / or lungs; gastric cancer that has metastasized to the liver, lungs, and / or peritoneum; thyroid cancer that has metastasized to the bone, liver, and / or lungs; and uterine cancer that has metastasized to the bone, liver, lungs, peritoneum, and / or vagina, among others.

[0145] In some cases, administration of the therapeutic composition reduces inflammation or one or more inflammatory responses in the subject. For example, in some cases, administration of the therapeutic composition reduces one or more of endothelial activation / damage, pro-inflammatory cytokine and chemokine production / release (e.g., IL-1β, MIP-1α, MCP-1, and CXCL1), fibrinogen synthesis, neutrophil recruitment to an organ, and / or organ damage in the subject.

[0146] In some embodiments, the methods or compositions described herein increase patient median survival by 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 weeks or more. In certain embodiments, the methods or compositions described herein increase patient median survival by 1, 2, 3 years or more.

[0147] In certain embodiments, for example, in the treatment of cancer, the administered composition is sufficient to cause tumor regression, as indicated by a statistically significant reduction in the amount of viable tumor, for example, by at least 10%, 20%, 30%, 40%, 50% or more reduction in tumor mass, or by a change (e.g., a statistically significant reduction) in scan dimension.In certain embodiments, the administered composition is sufficient to cause stable disease.In certain embodiments, the administered composition is sufficient to cause stabilization or clinically correlated reduction of the symptoms of specific disease indications known to those skilled in the art.

[0148] Methods for identifying a subject having one or more of the diseases or conditions described herein are known in the art.

[0149] Administration can be achieved by a variety of different routes. The administration method 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, intrainterstitially, intracatheterically, intraarterially, subcutaneously, intraocularly, intrasynovially, intraepithelially, and / or transdermally. Certain embodiments include administration by IV infusion.

[0150] The exact dosage and duration of treatment are a function of the disease being treated and may be empirically determined using known testing protocols or by testing the composition in model systems known in the art and extrapolating from them.Controlled clinical trials may also be conducted.Dosage may 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 may be administered once or divided into multiple smaller doses to be administered at time intervals.For any particular subject, the specific dosage regimen may be adjusted over time according to individual needs.

[0151] In some embodiments, a therapeutically effective amount or therapeutic dosage of a composition described herein is an amount that is effective to reduce inflammation or an inflammatory response in a subject. In some cases, treatment is initiated with a low dosage that can be increased in small increments until the optimum effect under the circumstances is achieved.

[0152] In some embodiments, the dosage is administered from about once per day to about once every two or three weeks. For example, in certain embodiments, the dosage is administered about once every 1, 2, 3, 4, 5, 6, or 7 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.

[0153] Certain embodiments include about 1×10, including all ranges and integers therebetween. 4 U ~ approx. 1 × 10 5 U ~ approx. 100 x 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 x 10 4 U, 9 x 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 x 10 5 U, 9 x 10 5 U, 10 x 10 5 U, 11 x 10 5 U, 12 x 10 5 U, 15 x 10 5 U, 20 x 10 5 U, 30 x 10 5 U, 40 x 10 5 U, 50 x 10 5 U, 60 x 10 5 U, 70 x 10 5 U, 80 x 10 5U, or 100 x 10 5 U / kg. Certain embodiments include administering a urinastatin polypeptide at a dosage (e.g., daily dose) of about, at least about, or less than about 50,000 U / kg, 125,000 U / kg, 250,000 U / kg, 500,000 U / kg, 750,000 U / kg, or 1,000,000 U / kg; or about 50,000-1,000,000 U / kg, about 125,000-1,000,000 U / kg, about 250,000-1,000,000 U / kg, about 500,000-1,000,000 U / kg, about 750,000-1,000,000 U / kg, about 50,000-750,000 U / kg. U / kg, about 125,000-750,000 U / kg, about 250,000-750,000 U / kg, about 500,000-750,000 U / kg, about 50,000-500,000 U / kg, about 125,000-500,000 U / kg, about 250,000-500,000 U / kg, about 50,000-250,000 U / kg, about 125,000-250,000 U / kg, or about 50,000-125,000 U / kg.

[0154] Certain embodiments include administering a subcutaneous dosage. Some embodiments include administering an intravenous dosage, for example, by intravenous infusion of the daily dose over about 1, 2, or 3 hours. Certain embodiments include infusing the daily dose over about 1, 2, or 3 hours, optionally about 1, 2, or 3 times a day, and optionally for about 2, 3, 4, 5, 6, or 7 or more consecutive days.

[0155] Also included are patient care kits containing one or more compositions or urinastatin polypeptides produced according to the methods described herein. Certain kits also contain one or more pharmaceutically acceptable diluents or solvents, such as water (e.g., sterile water) or saline. In some embodiments, the compositions or urinastatin polypeptides are stored in vials, cartridges, dual-chamber syringes, and / or pre-filled mixing systems.

[0156] The kits herein may include one or more additional therapeutic agents or other components appropriate or desirable for the indication being treated or the desired diagnostic application. The kits herein may also include one or more syringes or other components necessary or desirable to facilitate the intended mode of delivery (e.g., stent, implantable depot, etc.).

[0157] 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.

[0158] 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 those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are offered merely by way of illustration and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results. [Example]

[0159] Example 1 Preparation and Expression of Urinastatin Fusion Polypeptides Two fusion constructs were prepared to obtain optimal urinastatin expression in Chinese hamster ovary (CHO) cells. The bovine alpha-lactalbumin signal peptide was fused to the N-terminus of full-length human urinastatin and to the mature form of human urinastatin. Each of the urinastatin sequences was modified to incorporate a serine-to-alanine mutation at residue 10 (S10A) of the mature urinastatin sequence to prevent glycosaminoglycan attachment at that residue. The full-length urinastatin fusion polypeptide requires enzymatic cleavage (furin) in CHO cells to produce mature urinastatin. The mature urinastatin fusion polypeptide requires only cleavage of the bovine alpha-lactalbumin signal peptide to produce mature urinastatin. The amino acid sequences of the fusion polypeptides are shown in Table U2, and the nucleic acid coding sequences are shown in Table U3. The DNA sequences were confirmed by DNA sequencing.

[0160] Retroviral vector production. The fusion protein constructs outlined above were introduced into retroviral vectors as illustrated in Figure 2 (FL urinastatin) and Figure 3 (mature urinastatin). Details of the vector components are provided below in Tables E1 and E2. [Table E1] [Table E2]

[0161] The retrovectors were transfected into a HEK293 cell line that constitutively produces MLV gag, pro, and pol proteins. An envelope-containing expression plasmid was also cotransfected with each of the two retrovector constructs. Cotransfection resulted in the production of high-titer replication-incompetent retrovectors for each of the two gene constructs, which were concentrated by ultracentrifugation and used for cell transduction (Bleck, An alternative method for the rapid generation of stable, high-expressing retrovectors). See, e.g., "Mammalian Cell Lines," Bioprocessing J. Sept / Oct. pp 1-7, 2005; and "Bleck, GPEx® A Flexible Method for the Rapid Generation of Stable, High Expressing, Antibody Producing Mammalian Cell Lines," Chapter 4 In: Current Trends in Monoclonal Antibody Development and Manufacturing, Biotechnology: Pharmaceutical Aspects, Edited by: S.J. Shire et al.® 2010 American Association of Pharmaceutical Scientists, DOI 10.1007 / 978-0-387-76643-0_4.

[0162] Pooled cell lines for each retroviral construct were generated by three cycles of cell transduction of the GPEx® Chinese Hamster Ovary (GCHO) parent cell line with replication-incompetent, high-titer retrovectors. The full-length urinastatin retrovector was transduced into a GCHO cell line overexpressing the furin enzyme, which digests the full-length molecule to produce mature urinastatin. The mature urinastatin retrovector was transduced into normal GCHO cells.

[0163] After transduction, pooled cell lines for each of the two constructs were scaled up for production in fed-batch studies in 2.8 L spinner flasks. Each spinner flask was seeded with 300,000 viable cells per mL in G12.1 medium (Irvine Scientific) and incubated at 5% CO2 and 37°C in a humidified (70-80%) shaking incubator at 80 rpm. Cultures were challenged seven times during the production run using two different nutrient supplements. The culture temperature was lowered from 37°C to 34°C on day 5 of culture. Cultures were terminated when viability was ≤80%.

[0164] Verification of protein production was determined by reducing SDS-PAGE gel analysis, as shown in Figure 3. The urinary-derived urinastatin control contains both N-linked glycosylation and glycosaminoglycan (GAG) molecules. Because the attachment site for GAG molecules was mutated, the full-length and mature urinastatin fusion constructs should have only N-linked glycosylation. The full-length urinastatin sample appears to be completely cleaved by the added furin in the cell line. The gel shows both the large fragment of the cleaved protein (186 amino acids and two N-linked glycosylation sites (upper band)) and the mature urinastatin fragment (147 amino acids and one N-linked glycosylation site (lower band)).

[0165] The mature urinastatin sample contains only the smaller-sized mature urinastatin fragment. The smaller molecular weight mature urinastatin product appears to consist of two bands when run on a reducing SDS-PAGE gel. This doublet was not observed in the full-length sample (compare lane 6 with lane 9, see Figure 3).

[0166] To examine the two bands observed in the mature urinastatin sample in lane 9 of Figure 3, the material was first examined to determine whether the second band was due to N-linked glycosylation. Therefore, urinary urinastatin, full-length urinastatin, and mature urinastatin samples were digested with PNGase to remove any possible N-linked glycans and then electrophoresed on a gel. If the duplex were caused by N-linked glycosylation, PNGase digestion would be expected to result in a single band on a reducing SDS-PAGE gel. However, as shown in Figure 4, PNGase digestion did not alter the duplex observed in the mature urinastatin sample (see lane 10). Instead, removal of the N-linked glycans, which resulted in a molecular weight shift (compare lanes 9 and 10 in Figure 4), maintained the duplex, indicating that additional N-linked glycosylation did not result in the second band. Additionally, PNGase treatment of full-length urinastatin confirms that the dual modification occurs only in the mature urinastatin sample, but not in the full-length urinastatin sample.

[0167] The mature urinastatin sample was peptide mapped to determine whether the duplication was due to a variant protein sequence or another modification to the protein structure. The analysis not only confirmed the absence of modifications to the mature urinastatin sequence, but also surprisingly demonstrated that the upper band in the mature urinastatin sample was caused by an otherwise unnatural O-linked glycosylation at threonine 17 (T17). This O-linked glycosylation is absent from full-length urinastatin / bovine alpha-lactalbumin signal peptide fusion protein preparations, even after in situ enzymatic (furin) cleavage to produce the mature form of urinastatin. It occurs only in mature urinastatin / bovine alpha-lactalbumin signal peptide fusion protein preparations. Materials containing mature urinastatin with N-linked glycosylation and the unexpected O-linked glycosylation at T17 were tested and found to inhibit trypsin in vitro (see Figure 5). Here, trypsin inhibitory activity was analyzed using a BioVision™ trypsin activity kit. Dilutions were performed using samples containing approximately 5000 U / mg of recombinant urinastatin (T17) at dilution factors (DF) of DF2000, DF4000, DF8000, DF16000, and DF32000.

[0168] Example 2 Activity of recombinant urinastatin in a mouse model of sepsis A study was conducted to evaluate the efficacy of recombinant urinastatin in a mouse model of sepsis (see Example 1; O-linked glycosylation at T17). Male C57BL|6 mice underwent colonic puncture (CLP) surgery to induce mild to moderate grade sepsis. The study schedule is shown in Table E3. [Table E3]

[0169] Thirty (30) minutes after surgery, mice were administered the urinastatin test article, a negative control (phosphate buffered saline, PBS) via intravenous (IV) injection once daily on days 0 through 4, or a positive control, imipenem, via subcutaneous (SC) injection twice daily on days 0 through 4. Six days after CLP surgery, animal survival was followed and the efficacy of the test article was determined by comparing survival between the test article and the negative control.

[0170] The data are shown in Figures 6A-6B. Together, the data indicate that intestinal perforation using a 22-gauge needle resulted in septic infection in male C57BL|6 mice. The PBS vehicle CLP group developed mild to moderate-grade sepsis with a 40% mortality rate, while mice treated with imipenem had 0% mortality, which is within the range observed in previous studies. There was a dose response observed in the recombinant urinastatin-treated group, with 50% mortality at the lowest concentration (50,000 U / kg) and only 20% mortality at the higher concentrations tested (125,000 and 250,000 U / kg).

[0171] Example 3 Activity of recombinant urinastatin in a mouse model of acute pancreatitis Experiments were conducted to evaluate the activity of recombinant urinastatin (T17) at three dose levels in a caerulein-induced acute pancreatitis model in mice.

[0172] Acute pancreatitis was induced in male ICR mice by intraperitoneal (IP) injection of cerulein (100 μg / kg) three times at two-hour intervals. The test article or vehicle (sterile PBS) was administered intravenously (IV) 1 hour after each cerulein challenge (a total of three doses). The reference compound, devazepide, was administered orally (PO) 1 hour after each cerulein challenge. Mice were sacrificed 9 hours after the first cerulein challenge. Blood was collected, and serum α-amylase and lipase were detected using an automatic analyzer (TBA-120FR, Toshiba, Japan).

[0173] The results are summarized in Figures 7-8 and Table E4 below. Table E4

[0174] Serum α-amylase and lipase, indicators of acute pancreatitis, were significantly (p<0.05) increased by repeated cerulein challenge compared with the normal control group, indicating successful induction of acute pancreatitis in the vehicle group 9 hours after the first cerulein injection. Oral administration of devazepide, a positive control, at 0.1 mg / kg × 3 significantly (p<0.05) reduced serum α-amylase and lipase levels 9 hours after the first cerulein injection compared with the vehicle group.

[0175] The test article (recombinant urinastatin; T17) given by oral gavage at 16.5 mg / kg x 3 (83,333 U / kg x 3 = 250,000 U / kg total) and 33 mg / kg x 3 (166,667 U / kg x 3 = 500,000 U / kg total) showed a significant (p<0.05) decrease in serum α-amylase levels 9 hours after the first caerulein injection when compared to the vehicle group. There was a slight effect on serum lipase at all three dose levels of recombinant urinastatin compared to the vehicle group.

[0176] At the end of the study, pancreatic samples from all animals were collected in RNAlater. Quantitative real-time PCR biomarker analysis was performed on the pancreatic samples. Each assay was performed in triplicate on an Applied Biosystems 7900HT real-time PCR system, and expression fold changes were determined using the comparative CT method, with GAPDH as the endogenous control and sample "1-1" as the standard. The final results were expressed as n-fold differences in gene expression or relative quantities (RQ), with the RQ of the control sample always equal to 1.

[0177] As shown in Figure 9, mRNA levels of gene markers of inflammation (IL-6) and oxidative stress (HMOX1) were significantly (p<0.05) increased by repeated caerulein challenge compared with the normal control group. Oral administration of devazepide at 0.1 mg / kg × 3 moderately attenuated IL-6 mRNA expression and significantly (p<0.05) attenuated HMOX1 mRNA expression compared with the vehicle group. Recombinant urinastatin (T17) given at 16.5 mg / kg × 3 PO (83,333 U / kg × 3 = 250,000 U / kg total) and 33 mg / kg × 3 PO (166,667 U / kg × 3 = 500,000 U / kg total) attenuated IL-6 mRNA expression compared with the vehicle group. Recombinant urinastatin given at 3.3 mg / kg × 3PO (16,667 U / kg × 3 = 50,000 U / kg total), 16.5 mg / kg × 3PO (83,333 U / kg × 3 = 250,000 U / kg total), and 33 mg / kg × 3PO (166,667 U / kg × 3 = 500,000 U / kg total) showed an inverse dose-response of HMOX1 mRNA expression compared with vehicle control. Collagen I levels were also restored to normal control levels by treatment with recombinant urinastatin.

[0178] Overall, recombinant urinastatin given at 16.5 mg / kg × 3PO (83,333 U / kg × 3 = 250,000 U / kg total) and 33 mg / kg × 3PO (166,667 U / kg × 3 = 500,000 U / kg total) significantly reduced serum α-amylase levels, had significant effects on serum lipase levels, IL-6 mRNA expression, and restored collagen I levels 9 hours after the first caerulein injection in a mouse acute pancreatitis model. The present invention provides, for example, the following items. (Item 1) An isolated mature urinastatin polypeptide comprising: (i) a modified O-linked glycosylation site at residues Glu-Gly-Ser-Gly (SEQ ID NO: 10) that reduces glycosylation at the O-linked glycosylation site; (ii) an N-linked glycan at residue N45; and (iii) an O-linked glycan at residue T17, wherein the residues are defined by SEQ ID NO: 2 or 4, and the urinastatin polypeptide has at least one urinastatin activity. (Item 2) 2. The isolated mature urinastatin polypeptide of claim 1, comprising, consisting of, or consisting essentially of an amino acid sequence at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 or 4, wherein the urinastatin polypeptide comprises or retains (i) the modified O-linked glycosylation site, (ii) the N-linked glycan at residue N45, and (iii) the O-linked glycan at residue T17, and wherein the urinastatin polypeptide has at least one urinastatin activity. (Item 3) 3. The isolated mature urinastatin polypeptide of claim 2, comprising, consisting of, or consisting essentially of an amino acid sequence at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:2, wherein the urinastatin polypeptide comprises or retains (i) an S10A substitution of SEQ ID NO:2, (ii) the N-linked glycan at residue N45, and (iii) the O-linked glycan at residue T17, and wherein the urinastatin polypeptide has at least one urinastatin activity. (Item 4) 3. The isolated mature urinastatin polypeptide of claim 2, comprising, consisting of, or consisting essentially of SEQ ID NO: 2, and comprising the N-linked glycan at residue N45 and the O-linked glycan at residue T17. (Item 5) 5. The isolated mature urinastatin polypeptide of any one of items 1 to 4, wherein the at least one urinastatin activity is selected from one or more of a protease inhibitory activity, an anti-inflammatory activity, and an anti-metastatic activity. (Item 6) 5. The isolated mature urinastatin polypeptide of any one of Items 1 to 4, wherein 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, wherein 1 unit (U) is the amount of the urinastatin polypeptide that inhibits the activity of 2 μg of trypsin by 50%. (Item 7) A therapeutic composition comprising the isolated mature urinastatin polypeptide according to any one of items 1 to 6 and a pharmaceutically acceptable carrier. (Item 8) 8. The therapeutic composition of claim 7, comprising a mixture of (a) an isolated mature urinastatin polypeptide of any one of claims 1 to 6 and (b) a second mature urinastatin polypeptide comprising an N-linked glycan at residue N45 and no O-linked glycan at residue T17, wherein (a) and (b) have at least one urinastatin activity. (Item 9) 9. The therapeutic use of item 8, wherein (b) comprises an O-linked glycosylation site modified with residues Glu-Gly-Ser-Gly (SEQ ID NO: 10), which reduces glycosylation at the O-linked glycosylation site, and has at least one urinastatin activity. (Item 10) 10. The therapeutic composition of item 9, wherein (b) 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 or 4, and includes or retains the modified O-linked glycosylation site and the N-linked glycan at residue N45 and does not include the O-linked glycan at residue T17, and has at least one urinastatin activity. (Item 11) 11. The therapeutic composition of item 10, wherein (b) 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, and includes or retains the S10A substitution and the N-linked glycan at residue N45 and does not include the O-linked glycan at residue T17 of SEQ ID NO:2, and has at least one urinastatin activity. (Item 12) 12. The therapeutic composition of claim 11, wherein (b) comprises, consists of, or consists essentially of SEQ ID NO:2, comprises the N-linked glycan at residue N45 and does not comprise the O-linked glycan at residue T17, and has at least one urinastatin activity. (Item 13) 13. The therapeutic composition of any one of items 7-12, wherein (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. (Item 14) 14. The therapeutic composition according to any one of items 7 to 13, which is substantially free of other glycosylated isoforms (glycoforms) of urinastatin. (Item 15) 15. The therapeutic composition of any one of items 7 to 14, having an endotoxin level of less than about 1 EU / mg protein, host cell protein of less than about 100 ng / mg total protein, host cell DNA of less than about 10 pg / mg total protein, and / or being substantially free of aggregates. (Item 16) An urinastatin fusion polypeptide comprising, in an N-terminal to C-terminal direction, a bovine alpha-lactalbumin signal peptide and an urinastatin polypeptide, optionally wherein the urinastatin polypeptide comprises an O-linked glycosylation site modified with residues Glu-Gly-Ser-Gly (SEQ ID NO: 10), which reduces glycosylation at the O-linked glycosylation site, and wherein the urinastatin polypeptide has at least one urinastatin activity. (Item 17) 17. The urinastatin fusion polypeptide of item 16, wherein the bovine alpha-lactalbumin signal peptide 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:5. (Item 18) 18. The urinastatin fusion polypeptide of claim 16 or 17, wherein 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 NOs: 1-4, wherein the urinastatin polypeptide has at least one urinastatin activity, and optionally the urinastatin polypeptide has or retains an S10A substitution as defined by the sequence of mature human urinastatin. (Item 19) 19. The urinastatin fusion polypeptide of any one of Paragraphs 16 to 18, wherein the urinastatin fusion polypeptide comprises, consists of, or consists essentially of an amino acid sequence which is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 6 or 7, wherein the urinastatin polypeptide has at least one urinastatin activity, and optionally the urinastatin polypeptide has or retains an S10A substitution as defined by the sequence of mature human urinastatin. (Item 20) 20. The urinastatin fusion polypeptide of any one of items 16 to 19, comprising a peptide linker between the bovine alpha-lactalbumin signal peptide and the urinastatin polypeptide, optionally wherein the peptide linker is 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, 60, 70, 80, 90, 100 amino acids in length. (Item 21) 21. The urinastatin fusion polypeptide of claim 20, wherein the peptide linker comprises a protease cleavage site. (Item 22) 22. The urinastatin fusion polypeptide according to any one of items 16 to 21, comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:6. (Item 23) 23. The urinastatin fusion polypeptide according to any one of items 16 to 22, comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:7. (Item 24) 24. The urinastatin fusion polypeptide according to any one of Items 16 to 23, wherein the at least one urinastatin activity is selected from one or more of a protease inhibitory activity, an anti-inflammatory activity, and an anti-metastatic activity. (Item 25) 25. The urinastatin fusion polypeptide of any one of Items 16 to 24, wherein 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, wherein 1 unit (U) is the amount of the urinastatin polypeptide that inhibits the activity of 2 μg of trypsin by 50%. (Item 26) A polynucleotide encoding the urinastatin fusion polypeptide according to any one of Items 16 to 25. (Item 27) 27. The polynucleotide of item 26, wherein the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 8 or 9. (Item 28) 28. An expression vector comprising the polynucleotide of item 26 or 27 operably linked to a promoter element. (Item 29) 29. The expression vector of item 28, wherein the expression vector is a retroviral vector comprising, consisting of, or consisting essentially of, in 5' to 3' orientation: a 5' long terminal repeat (LTR), a packaging region, a promoter region, the polynucleotide encoding the urinastatin fusion polypeptide, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a 3' LTR. (Item 30) 29. A recombinant mammalian host cell comprising the polynucleotide of item 26 or 27, or the expression vector of item 28 or 29. (Item 31) 31. The recombinant mammalian host cell of item 30, selected from HEK293 cells and Chinese hamster ovary (CHO) cells, optionally GPEx CHO (GCHO) cells. (Item 32) 32. The recombinant mammalian host cell of Item 31, wherein the HEK293 cells constitutively express gag, pro, and pol proteins (optionally derived from murine leukemia virus (MLV)) and a separately introduced env protein, and secrete replication-incompetent retroviral particles encoding the urinastatin fusion polypeptide. (Item 33) 32. The mammalian host cell of claim 31, wherein the CHO cell expresses the urinastatin fusion polypeptide and expresses or overexpresses a furin polypeptide, optionally an exogenous furin polypeptide. (Item 34) 1. A method for recombinantly producing a urinastatin polypeptide, comprising: (a) expressing the urinastatin fusion polypeptide in a recombinant mammalian host cell according to any one of items 30 to 33, optionally a CHO cell or a GCHO cell; and (b) isolating the urinastatin polypeptide from the host cells or from a medium containing the host cells; Thus, the method comprises recombinantly producing the urinastatin fusion polypeptide. (Item 35) 35. The method of claim 34, comprising cleaving the bovine alpha-lactalbumin signal peptide from the urinastatin polypeptide to produce a recombinant urinastatin polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NOs: 1-4, wherein the urinastatin polypeptide has at least one urinastatin activity. (Item 36) Item 36. The method according to Item 35, wherein the urinastatin polypeptide is the mature urinastatin polypeptide according to any one of Items 1 to 6. (Item 37) 37. The method of any one of Items 34 to 36, comprising measuring at least one urinastatin activity of the urinastatin polypeptide under physiological conditions, optionally under physiological conditions of temperature, salinity, and / or pH. (Item 38) 38. The method of any one of items 34 to 37, comprising preparing a therapeutic composition comprising the urinastatin polypeptide, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or weight by weight basis, and wherein the composition is substantially free of aggregates and substantially free of endotoxin. (Item 39) A therapeutic composition according to any one of items 7 to 15, or a therapeutic composition prepared according to the method of item 38, for use in treating a disease in a subject in need thereof, wherein optionally the disease is an inflammatory disease or cancer. thing. (Item 40) A method for treating an inflammatory disease or condition in a subject in need thereof, comprising administering to the subject the therapeutic composition according to any one of Items 7 to 15, or the therapeutic composition prepared according to the method according to Item 38, thereby treating the inflammatory disease or condition in the subject. (Item 41) The inflammatory disease or condition may be pancreatitis (e.g., acute pancreatitis, chronic pancreatitis, endoscopic retrograde cholangiopancreatography (ERCP)-induced pancreatitis), systemic inflammation, colitis, autoimmune encephalomyelitis, Stevens-Johnson syndrome, arthritis, renal failure, burns, severe sepsis and related pro-inflammatory / secondary conditions (e.g., organ failure) including sepsis / septic shock, systemic inflammatory response syndrome (SIRS), toxic epidermal necrolysis (TEN), Kawasaki disease, renal disease (e.g., acute renal failure, chronic renal disease), ischemic conditions (e.g., ischemia-reperfusion injury in the liver, kidney, heart, lung, brain), pulmonary inflammation and inflammatory lung conditions (e.g., pulmonary infection, mixed 41. The method of claim 40, wherein the inflammation is selected from one or more of: pneumonia, including infectious interstitial pneumonia associated with chronic connective tissue diseases, pulmonary fibrosis, acute respiratory distress syndrome, liver inflammation including hepatitis, anaphylaxis, post-operative or post-surgical complications (e.g., renal function, cardiac surgery, lung surgery, cognitive impairment, liver transplant), lipopolysaccharide (LPS)-induced inflammation or tissue damage (e.g., lung, liver, brain), inflammation or dysfunction secondary to diabetes (e.g., diabetes-induced cardiac dysfunction), burns, heat stroke, inflammatory or neuropathic pain, acute poisoning, hyperlipidemia-associated inflammation, autoimmune-associated inflammation, allograft- or transfusion-associated inflammation, neuroinflammation, and cancer-associated inflammation. (Item 42) 42. The method of claim 40 or 41, wherein administering the modified ulinastatin polypeptide reduces one or more of protease activity, endothelial activation / damage, pro-inflammatory cytokine and chemokine production / release (optionally IL-1β, MIP-1α, MCP-1, and / or CXCL1), fibrinogen synthesis, neutrophil recruitment to organs, and / or organ damage in the subject. (Item 43) A method for treating cancer, ameliorating symptoms of cancer, or inhibiting the progression of cancer in a subject in need thereof, comprising administering to the subject the therapeutic composition according to any one of Items 7 to 15, or the therapeutic composition prepared according to the method according to Item 38, thereby treating cancer, ameliorating symptoms of cancer, or inhibiting the progression of cancer in the subject in need thereof. (Item 44) 44. The method of claim 43, wherein the cancer is selected from one or more of melanoma (e.g., metastatic melanoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatocellular carcinoma (hepatocellular carcinoma), sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer. (Item 45) 45. The method of item 43 or 44, wherein the cancer is a metastatic cancer, and optionally, administration of the modified urinastatin polypeptide reduces cancer cell invasion and / or angiogenesis. (Item 46) the metastatic cancer is (a) bladder cancer that has metastasized to bone, liver, and / or lung; (b) breast cancer that has metastasized to the bone, brain, liver, and / or lungs; (c) colorectal cancer metastasizing to the liver, lung, and / or peritoneum; (d) kidney cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or lungs; (e) lung cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or other lung sites; (f) melanoma that has metastasized to bone, brain, liver, lung, and / or skin / muscle; (g) ovarian cancer that has metastasized to the liver, lung, and / or peritoneum; (h) pancreatic cancer that has metastasized to the liver, lung, and / or peritoneum; (i) prostate cancer that has metastasized to the adrenal glands, bone, liver, and / or lungs; (j) gastric cancer that has metastasized to the liver, lung, and / or peritoneum; (l) thyroid cancer that has metastasized to the bone, liver, and / or lungs; and (m) uterine cancer that has metastasized to the bone, liver, lung, vagina, and / or peritoneum.

Claims

[Claim 1] The invention described in the present specification.

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