# US 2021/0277090 A1 · Ulinastatin polypeptides

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- Number: US 2021/0277090 A1
- Document ID: US20210277090A1
- Status: Published application
- Kind: A1
- Application number: 17/193,481
- Filed: 2021-03-05
- Published: 2021-09-09
- Earliest claimed: 2020-03-05
- Assignees: DiaMedica USA Inc.
- Applicants: DiaMedica USA Inc.
- Inventors: Rick PAULS; Todd VERDOORN; Karl Frank JOHNSON; Gregory Thomas BLECK
- Web page: https://patentgrep.com/patent/US20210277090A1

## CPC

- C07K 14/81: Protease inhibitors
- A61K 38/57: from animals; from humans {(A61K38/553, A61K38/556 take precedence)}
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- C07K 14/76: Albumins
- C07K 14/8114: {Kunitz type inhibitors}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/02: containing a signal sequence
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Provided are ulinastatin glycoforms, ulinastatin fusion polypeptides, and related compositions, mixtures, and methods of use, including methods of recombinantly producing ulinastatin polypeptides and treating diseases.

## Claims

### Claim 1

An isolated, mature ulinastatin polypeptide, comprising (i) a modified O-linked glycosylation site at residues Glu-Gly-Ser-Gly (SEQ ID NO: 10) which reduces glycosylation at the O-linked glycosylation site, (ii) an N-linked glycan at residue at residue N45, and (iii) an O-linked glycan at residue T17, the residues being defined by SEQ ID NO: 2 or 4, wherein the ulinastatin polypeptide has at least one ulinastatin activity.

### Claim 2 (depends on 1)

The isolated, mature ulinastatin polypeptide of claim 1, which comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 or 4, wherein the ulinastatin polypeptide comprises or retains (i) the modified O-linked glycosylation site, (ii) the N-linked glycan at residue at residue N45, and (iii) the O-linked glycan at residue T17, wherein the ulinastatin polypeptide has at least one ulinastatin activity.

### Claim 3 (depends on 2)

The isolated, mature ulinastatin polypeptide of claim 2, which comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2, wherein the ulinastatin polypeptide comprises or retains (i) the S10A substitution of SEQ ID NO: 2, (ii) the N-linked glycan at residue at residue N45, and (iii) the O-linked glycan at residue T17, wherein the ulinastatin polypeptide has at least one ulinastatin activity.

### Claim 4 (depends on 2)

The isolated, mature ulinastatin polypeptide of claim 2, which comprises, consists, or consists essentially of SEQ ID NO: 2, and comprises the N-linked glycan at residue at residue N45 and the O-linked glycan at residue T17.

### Claim 5 (depends on 1)

The isolated, mature ulinastatin polypeptide of claim 1, wherein the at least one ulinastatin activity is selected from one or more of protease inhibitor activities, anti-inflammatory activities, and anti-metastatic activities.

### Claim 6 (depends on 1)

The isolated, mature ulinastatin polypeptide of claim 1, wherein the ulinastatin 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, wherein one unit (U) is an amount of the ulinastatin polypeptide that inhibits the activity of 2 trypsin by 50%.

### Claim 7 (depends on 1)

A therapeutic composition, comprising the isolated, mature ulinastatin polypeptide according to claim 1, and a pharmaceutically-acceptable carrier.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The therapeutic composition of claim 7, comprising a mixture of (a) the isolated, mature ulinastatin polypeptide according to any one of claims 1-6, and (b) a second, mature ulinastatin polypeptide that comprises an N-linked glycan at residue N45 and does not comprise an O-linked glycan at residue T17, wherein (a) and (b) have at least one ulinastatin activity.

### Claim 9 (depends on 8)

The therapeutic of claim 8, wherein (b) comprises a modified O-linked glycosylation site at residues Glu-Gly-Ser-Gly (SEQ ID NO: 10) which reduces glycosylation at the O-linked glycosylation site, and has at least one ulinastatin activity.

### Claim 10 (depends on 9)

The therapeutic composition of claim 9, wherein (b) comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 or 4, comprises or retains the modified O-linked glycosylation site and the N-linked glycan at residue N45, does not comprise the O-linked glycan at residue T17, and has at least one ulinastatin activity.

### Claim 11 (depends on 10)

The therapeutic composition of claim 10, wherein (b) comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2, comprises or retains the S10A substitution of SEQ ID NO: 2 and the N-linked glycan at residue N45, does not comprise the O-linked glycan at residue T17, and has at least one ulinastatin activity.

### Claim 12 (depends on 11)

The therapeutic composition of claim 11, wherein (b) comprises, consists, or consists essentially of SEQ ID NO: 2, comprises the N-linked glycan at residue N45, does not comprise the O-linked glycan at residue T17, and has at least one ulinastatin activity.

### Claim 13 (depends on 8)

The therapeutic composition of claim 8, wherein (a):(b) are present in the composition at 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.

### Claim 14 (depends on 7)

The therapeutic composition of claim 7, which is substantially free of other glycosylated isoforms (glycoforms) of ulinastatin.

### Claim 15 (depends on 7)

The therapeutic composition of claim 7, which has 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 is substantially aggregate-free.

### Claim 16

A ulinastatin fusion polypeptide, comprising, in an N-terminal to C-terminal orientation, a bovine alpha-lactalbumin signal peptide and a ulinastatin polypeptide, optionally wherein the ulinastatin polypeptide comprises a modified O-linked glycosylation site at residues Glu-Gly-Ser-Gly (SEQ ID NO: 10) which reduces glycosylation at the O-linked glycosylation site, wherein the ulinastatin polypeptide has at least one ulinastatin activity.

### Claim 17 (depends on 16)

The ulinastatin fusion polypeptide of claim 16, wherein the bovine alpha-lactalbumin signal peptide comprises, consists, or consists essentially an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 5.

### Claim 18 (depends on 16)

The ulinastatin fusion polypeptide of claim 16, wherein the ulinastatin polypeptide comprises, consists, 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: 1-4, wherein the ulinastatin polypeptide has at least one ulinastatin activity, and optionally wherein the ulinastatin polypeptide has or retains an S10A substitution as defined by the sequence of mature human ulinastatin.

### Claim 19 (depends on 16)

The ulinastatin fusion polypeptide of claim 16, wherein the ulinastatin fusion polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% to SEQ ID NO: 6 or 7, wherein the ulinastatin polypeptide has at least one ulinastatin activity, and optionally wherein the ulinastatin polypeptide has or retains an S10A substitution as defined by the sequence of mature human ulinastatin.

### Claim 20 (depends on 16)

The ulinastatin fusion polypeptide of claim 16, comprising a peptide linker between the bovine alpha-lactalbumin signal peptide and the ulinastatin 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.

### Claim 21 (depends on 20)

The ulinastatin fusion polypeptide of claim 20, wherein the peptide linker comprises a protease cleavage site.

### Claim 22 (depends on 16)

The ulinastatin fusion polypeptide of claim 16, comprising, consisting, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 6.

### Claim 23 (depends on 16)

The ulinastatin fusion polypeptide of claim 16, comprising, consisting, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 7.

### Claim 24 (depends on 16)

The ulinastatin fusion polypeptide of claim 16, wherein the at least one ulinastatin activity is selected from one or more of protease inhibitor activities, anti-inflammatory activities, and anti-metastatic activities.

### Claim 25 (depends on 16)

The ulinastatin fusion polypeptide of claim 16, wherein the ulinastatin 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, wherein one unit (U) is an amount of the ulinastatin polypeptide that inhibits the activity of 2 μg trypsin by 50%.

### Claim 26 (depends on 16)

A polynucleotide, encoding the ulinastatin fusion polypeptide of claim 16.

### Claim 27 (depends on 26)

The polynucleotide of claim 26, wherein the polynucleotide comprises, consists, or consists essentially of a nucleic acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% to SEQ ID NO: 8 or 9.

### Claim 28 (depends on 26)

An expression vector, comprising the polynucleotide of claim 26, which is operably linked to a promoter element.

### Claim 29 (depends on 28)

The expression vector of claim 28, wherein the expression vector is a retroviral vector that comprises, consists, or consist essentially of the following: in a 5′ to 3′ orientation, a 5′ long terminal repeat (LTR), a packaging region, a promoter region, the polynucleotide encoding the ulinastatin fusion polypeptide, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a 3′ LTR.

### Claim 30 (depends on 28)

A recombinant mammalian host cell, comprising expression vector of claim 28.

### Claim 31 (depends on 30)

The recombinant mammalian host cell of claim 30, selected from an HEK293 cell, and a chinese hamster ovary (CHO) cell, optionally a GPEx CHO (GCHO) cell.

### Claim 32 (depends on 31)

The recombinant mammalian host cell of claim 31, wherein the HEK293 cell constitutively expresses gag, pro, and pol proteins (optionally from murine leukemia virus (MLV)) and a separately-transfected env protein, and secretes replication incompetent retroviral particles that encode the ulinastatin fusion polypeptide.

### Claim 33 (depends on 31)

The mammalian host cell of claim 31, wherein the CHO cell expresses the ulinastatin fusion polypeptide, and expresses or over-expresses a furin polypeptide, optionally an exogenous furin polypeptide.

### Claim 34 (depends on 30)

A method for recombinantly-producing a ulinastatin polypeptide, comprising
(a) expressing the ulinastatin fusion polypeptide in the recombinant mammalian host cell of claim 30, optionally the CHO cell or GCHO; and
(b) isolating the ulinastatin polypeptide from the host cell or a medium containing the host cell,
thereby recombinantly-producing the ulinastatin fusion polypeptide.

### Claim 35 (depends on 34)

The method of claim 34, comprising cleaving the bovine alpha-lactalbumin signal peptide from the ulinastatin polypeptide, to produce a recombinant ulinastatin polypeptide that comprises, consists, 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: 1-4, wherein the ulinastatin polypeptide has at least one ulinastatin activity.

### Claim 36 (depends on 1, 2, 3, 4, 5, 6, 35)

The method of claim 35, wherein the ulinastatin polypeptide is the mature ulinastatin polypeptide according to any one of claims 1-6.

### Claim 37 (depends on 34)

The method of claim 34, comprising measuring at least one ulinastatin activity of the ulinastatin polypeptide under physiological conditions, optionally of temperature, salinity, and/or pH.

### Claim 38 (depends on 34)

The method of claim 34, comprising preparing a therapeutic composition that comprises the ulinastatin polypeptide, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or a weight-weight basis, and wherein the composition is substantially aggregate-free and substantially endotoxin-free.

### Claim 39

(canceled)

### Claim 40 (depends on 7)

A method of treating an inflammatory disease or condition in a subject in need thereof, comprising administering to the subject a therapeutic composition of claim 7, thereby treating the inflammatory disease or condition in the subject.

### Claim 41 (depends on 40)

The method of claim 40, wherein the inflammatory disease or condition is selected from one or more 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 related pro-inflammatory/secondary conditions (e.g., organ failure), systemic inflammatory response syndrome (SIRS), toxic epidermal necrolysis (TEN), Kawasaki disease, kidney disease (e.g., acute kidney failure, chronic kidney disease), ischemic conditions (e.g., ischemia-reperfusion injury in the liver, kidney, heart, lungs, brain), lung inflammation and inflammatory lung conditions (e.g., pulmonary infection, pneumonia, including infectious interstitial pneumonia associated with mixed connective tissue disease, 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 dysfunction, liver transplantation), lipopolysaccharide (LPS)-induced inflammation or tissue injury (e.g., lungs, liver, brain), inflammation or dysfunction secondary to diabetes (e.g., diabetes-induced cardiac dysfunction), burn injury, heat stroke, inflammatory or neuropathic pain, acute poisoning, hyperlipidemia-associated inflammation, autoimmunity-associated inflammation, allogeneic transplant or blood transfusion-associated inflammation, neuroinflammation, and cancer-associated inflammation.

### Claim 42 (depends on 40)

The method of claim 40, wherein administering the modified ulinastatin polypeptide reduces one or more of protease activity, endothelial activation/damage, proinflammatory cytokine and chemokine production/release (optionally, IL-1β, MIP-1α, MCP-1, and/or CXCL1), fibrinogen synthesis, neutrophil recruitment into organs, and/or organ injury in the subject.

### Claim 43 (depends on 7)

A method of treating, ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof, comprising administering to the subject a therapeutic composition of claim 7, thereby treating, ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof.

### Claim 44 (depends on 43)

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 myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, 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 cancers, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

### Claim 45 (depends on 43)

The method of claim 43, wherein the cancer is a metastatic cancer, optionally wherein administering the modified ulinastatin polypeptide reduces cancer cell invasion and/or angiogenesis.

### Claim 46 (depends on 45)

The method of claim 45, wherein the metastatic cancer is selected from one or more of:
(a) a bladder cancer which has metastasized to the bone, liver, and/or lungs;
(b) a breast cancer which has metastasized to the bone, brain, liver, and/or lungs;
(c) a colorectal cancer which has metastasized to the liver, lungs, and/or peritoneum;
(d) a kidney cancer which has metastasized to the adrenal glands, bone, brain, liver, and/or lungs;
(e) a lung cancer which has metastasized to the adrenal glands, bone, brain, liver, and/or other lung sites;
(f) a melanoma which has metastasized to the bone, brain, liver, lung, and/or skin/muscle;
(g) a ovarian cancer which has metastasized to the liver, lung, and/or peritoneum;
(h) a pancreatic cancer which has metastasized to the liver, lung, and/or peritoneum;
(i) a prostate cancer which has metastasized to the adrenal glands, bone, liver, and/or lungs;
(i) a stomach cancer which has metastasized to the liver, lung, and/or peritoneum;
(l) a thyroid cancer which has metastasized to the bone, liver, and/or lungs; and
(m) a uterine cancer which has metastasized to the bone, liver, lung, vagina, and/or peritoneum.