# US 10,308,924 B2 · Nucleic acid molecules encoding autoactivating type I pancreatic proelastase proteins

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- Number: US 10,308,924 B2
- Document ID: US10308924B2
- Status: Granted
- Kind: B2
- Application number: 15/919,814
- Filed: 2018-03-13
- Published: 2018-07-26
- Granted: 2019-06-04
- Earliest claimed: 2010-06-24
- Assignees: Proteon Therapeutics, Inc.
- Applicants: Proteon Therapeutics, Inc.
- Inventors: F. Nicholas Franano; Kimberly Bland; Marco D. Wong; Bee C. Ding
- Web page: https://patentgrep.com/patent/US10308924B2

## CPC

- C12N 9/6448: {Elastases, e.g. pancreatic elastase (3.4.21.36); leukocyte elastase (3.4.31.37)}
- A61K 9/19: lyophilised {, i.e. freeze-dried, solutions or dispersions (lyophilised products with subsequent particle size reduction A61K9/14; granules or pellets made by lyphilisation A61K9/1682; solid oral dosage forms made by lyophilisation A61K9/2095; lyophilisation additives A61K47/00)}
- A61K 38/486: {Elastase (3.4.21.36 or 3.4.21.37)}
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 1/14: Extraction; Separation; Purification
- C12N 9/50: Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)}
- C12N 9/64: derived from animal tissue
- C12N 15/815: {for yeasts other than Saccharomyces}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12Y 304/21036: Pancreatic elastase (3.4.21.36)
- 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 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/50: containing protease site
- C12Y 304/21037: Leukocyte elastase (3.4.21.37), i.e. neutrophil-elastase
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to methods for the manufacture, purification, formulation, and use of biologically active recombinant elastase proteins. Described are recombinant methods for producing therapeutically useful elastase proteins, as are pharmaceutical compositions comprising said elastase proteins. Novel recombinant elastase proteins and protein preparations are also disclosed. Methods are described for treating and preventing diseases of biological conduits using pharmaceutical compositions containing the elastase proteins of the invention.

## Claims

### Claim 1

A nucleic acid molecule encoding an autoactivating type I pancreatic proelastase protein, wherein the autoactivating type I pancreatic proelastase protein comprises an amino acid sequence which is at least 90% identical to a reference amino acid sequence consisting of SEQ ID NO:73 linked at its C-terminus to the N-terminus of SEQ ID NO:1, wherein the first thirteen amino acids of the reference amino acid sequence are P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-P1′-P2′-P3′; and wherein
(a) the amino acid residues at the P10-P5 and P1′-P3′ positions are each independently selected from any natural amino acid;
(b) the amino acid residue at the P4 position is any natural amino acid except glycine, lysine, phenylalanine, tyrosine, tryptophan, or arginine;
(c) the amino acid residue at the P3 position is any natural amino acid except proline or glycine;
(d) the amino acid residue at the P2 position is proline, alanine, leucine, isoleucine, glycine, valine, histidine, or threonine; and
(e) the amino acid residue at the P1 position is alanine, leucine, valine, isoleucine, or serine.

### Claim 2 (depends on 1)

The nucleic acid molecule of claim 1, wherein:
(a) the amino acid residue at the P5 position is glutamate, histidine, proline, glycine, asparagine, lysine, or alanine;
(b) the amino acid residue at the P4 position is threonine, alanine, proline, or histidine;
(c) the amino acid residue at the P3 position is alanine, leucine, isoleucine, methionine, lysine, asparagine, or valine;
(d) the amino acid residue at the P2 position is proline, alanine, leucine, isoleucine, glycine, valine, or threonine; and
(e) the amino acid residue at the P1 position is alanine, leucine, valine, isoleucine, or serine.

### Claim 3 (depends on 1)

The nucleic acid molecule of claim 1, wherein the amino acid residue at the P5 position is histidine.

### Claim 4 (depends on 1)

The nucleic acid molecule of claim 1, wherein the amino acid residue at the P4 position is threonine.

### Claim 5 (depends on 1)

The nucleic acid molecule of claim 1, wherein the amino acid residue at the P3 position is asparagine.

### Claim 6 (depends on 1)

The nucleic acid molecule of claim 1, wherein the amino acid residue at the P2 position is proline.

### Claim 7 (depends on 1)

The nucleic acid molecule of claim 1, wherein the amino acid residue at the P1 position is alanine.

### Claim 8 (depends on 1)

The nucleic acid molecule of claim 1, wherein the amino acid residues at the P3-P1 positions have the amino acid sequence of SEQ ID NO:20.

### Claim 9 (depends on 1)

The nucleic acid molecule of claim 1, wherein the amino acid residues at the P10-P1 positions have the amino acid sequence of SEQ ID NO:73.

### Claim 10 (depends on 1)

The nucleic acid molecule of claim 1, wherein the amino acid residues at the P10-P3′ positions have the amino acid sequence of SEQ ID NO:55.

### Claim 11 (depends on 1)

The nucleic acid molecule of claim 1, wherein the protein comprises an amino acid sequence which is at least 95% identical to the reference amino acid sequence.

### Claim 12 (depends on 1)

The nucleic acid molecule of claim 1, wherein the protein comprises an amino acid sequence which is at least 99% identical to the reference amino acid sequence.

### Claim 13 (depends on 1)

The nucleic acid molecule of claim 1, wherein the protein comprises an amino acid sequence which is identical to the reference amino acid sequence.

### Claim 14 (depends on 1)

The nucleic acid molecule of claim 1, wherein the protein further comprises a signal sequence.

### Claim 15 (depends on 1)

The nucleic acid molecule of claim 1, which is isolated.

### Claim 16 (depends on 1)

The nucleic acid molecule of claim 1, wherein the autoactivating type I pancreatic proelastase protein produces a mature type I pancreatic elastase having elastase activity following autoactivation.

### Claim 17 (depends on 1)

The nucleic acid molecule of claim 1, wherein the autoactivating type I pancreatic proelastase protein comprises a cleavage site that is cleaved by a mature type I pancreatic elastase having elastase activity.

### Claim 18 (depends on 1)

The nucleic acid molecule of claim 1, wherein autoactivation of the type I pancreatic proelastase protein produces a mature type I pancreatic elastase having elastase activity.

### Claim 19 (depends on 1)

The nucleic acid molecule of claim 1, wherein the protein lacks a signal sequence.

### Claim 20 (depends on 1)

A vector comprising the nucleic acid molecule of claim 1.

### Claim 21 (depends on 1)

A host cell genetically engineered to express the nucleic acid molecule of claim 1.

### Claim 22 (depends on 1)

A cell culture supernatant comprising the autoactivating type I pancreatic proelastase protein encoded by the nucleic acid molecule of claim 1.

### Claim 23 (depends on 21)

A method of producing an autoactivating type I pancreatic proelastase protein, comprising culturing the host cell of claim 21 under conditions in which the autoactivating type I pancreatic proelastase protein is produced.

### Claim 24 (depends on 23)

The method of claim 23, further comprising recovering the autoactivating type I pancreatic proelastase protein.