# US 2021/0330762 A1 · Medical application of mutant plasminogen and plasmin polypeptide therapeutics

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- Number: US 2021/0330762 A1
- Document ID: US20210330762A1
- Status: Published application
- Kind: A1
- Application number: 16/856,107
- Filed: 2020-04-23
- Published: 2021-10-28
- Earliest claimed: 2020-04-23
- Assignees: Shenzhen Bay Laboratory
- Applicants: Shenzhen Bay Laboratory
- Inventors: Xinli LIN; Jiali GAO
- Web page: https://patentgrep.com/patent/US20210330762A1

## CPC

- A61K 38/484: {Plasmin (3.4.21.7)}
- C12Y 304/21007: Plasmin (3.4.21.7), i.e. fibrinolysin
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- 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)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Provided are methods for producing biologically active mutant recombinant plasminogen polypeptides with desired pharmaceutical properties. The refolded polypeptides may be treated with a plasminogen activator, such as tPA, urokinase, or streptokinase to generate biologically active mutant plasmin polypeptide for pharmaceutical use. Methods are also provided to producing biologically active fusion recombinant mutant plasminogen/plasmin polypeptides that can cross the BBB through receptor mediated transcytosis. The fusion partner may carry the mutant plasminogen/plasmin polypeptides into the brain for increased therapeutic efficiency.

## Claims

### Claim 1

A method for treating and/or preventing diseases in patient caused by a pathogenic polypeptide, comprising:
administrating to the patient in need an effective dose of a mutant and/or fusion plasminogen or plasmin polypeptide therapeutics in a pharmaceutical formulation.

### Claim 2 (depends on 1)

The method of claim 1, wherein the diseases are thromboembolism related diseases including peripheral arterial occlusion, deep vein thrombosis, acute pulmonary embolism, and acute ischemic stroke.

### Claim 3 (depends on 1)

The method of claim 1, wherein the diseases are amyloid peptide caused diseases including Alzheimer's disease caused by a β-Amyloid peptide.

### Claim 4 (depends on 1)

The method of claim 1, wherein the diseases are caused by human tissue damages resulting from various causes including pulmonary fibrosis.

### Claim 5 (depends on 1)

The method of claim 1, wherein the diseases are caused by decreased in vivo plasminogen and plasmin polypeptide content or activity; wherein the diseases includes disseminate intravascular coagulation, sepsis, leukemia, hyaline membrane disease, cardiovascular diseases, Alzheimer's disease, pulmonary fibrosis, and liver diseases.

### Claim 6 (depends on 1)

The method of claim 1, wherein the mutant and/or fusion plasminogen and plasmin polypeptide is selected by the following methods:
(a) performing alanine scanning mutagenesis of the surface residues of the catalytic domain of plasmin (μPlasmin) that is involved in complex formation with α2-Antiplasmin; and selecting mutants that have similar catalytic activity as that of the wild type enzyme but also resist to α2-Antiplasmin inhibition; wherein the alanine scanning mutagenesis of changing all residues of μPlasmin individually into alanine and selecting mutants that have similar catalytic activity as that of the wild type enzyme but also resist to α2-Antiplasmin inhibition is used;
(b) performing saturation mutagenesis for the mutations selected from the alanine scanning mutagenesis of method (a); and selecting mutants that have similar catalytic activity as that of the wild type enzyme but also resist to α2-Antiplasmin inhibition;
(c) performing combinatorial mutagenesis of constructing combined mutations from mutants selected using method (a) or (b); and selecting mutants that have optimal catalytic activity toward a particular polypeptide pathogen but also resist to α2-Antiplasmin inhibition;
(d) performing Cys-PEGylation scanning mutagenesis of the surface residues of the catalytic domain of plasmin (μPlasmin) that is involved in complex formation with α2-Antiplasmin; and selecting mutants that have similar catalytic activity as that of the wild type enzyme but also resist to α2-Antiplasmin inhibition; wherein, the Cys-PEGylation scanning mutagenesis of changing all residues of μPlasmin individually into PEGylated-Cysteine and selecting mutants that have similar catalytic activity as that of the wild type enzyme but also resist to α2-Antiplasmin inhibition is used;
(e) performing albumination through albumin-fusion or fatty acid modification of plasmin (μPlasmin) residues that is involved in complex formation with α2-Antiplasmin; and selecting constructs that have similar catalytic activity as that of the wild type enzyme but also resist to α2-Antiplasmin inhibition;
(f) performing modification of a mutant plasmin (μPlasmin) that resulting in similar catalytic activity as that of the wild type enzyme but also resist to α2-Antiplasmin inhibition, including a better in vivo half-life and substrate specificity; wherein the substrate specificity comprises reduced catalytic efficiency toward fibrin or better catalytic efficiency toward small peptide substrates including β-amyloid peptides; wherein the modification includes PEGylation and albumination; and
(g) performing fusion construct of modified mutant plasmin (μPlasmin) that resulting in similar catalytic activity as that of the wild type enzyme but also resist to α2-Antiplasmin inhibition, having a better in vivo half-life, and is able to cross the blood brain barrier (BBB), for treating CNS diseases including Alzheimer's Disease.

### Claim 7 (depends on 1)

The method of claim 1, wherein the mutant plasmin polypeptide is derived from the corresponding mutant plasminogen polypeptide by treating with a plasminogen activator.

### Claim 8 (depends on 7)

The method of claim 7, wherein the mutant plasmin polypeptide is selected to be biologically active in cleaving and detoxifying pathogenic polypeptides and is also resisting α2-antiplasmin inhibition.

### Claim 9 (depends on 8)

The method of claim 8, wherein the mutant recombinant plasmin polypeptide contains >80% of the amino acid residues 542-791 of the SEQ ID NO. 1.

### Claim 10 (depends on 8)

The method of claim 8, wherein the mutant recombinant plasmin polypeptide contains >80% of the amino acid residues 440-791 of the SEQ ID NO. 1.

### Claim 11 (depends on 8)

The method of claim 8, wherein the mutant recombinant plasmin polypeptide contains >80% of the amino acid residues 1-791 of the SEQ ID NO. 1.

### Claim 12 (depends on 11)

The method of claim 11, wherein one or more kringle structures or other non-catalytic structures are deleted.

### Claim 13 (depends on 1)

The method of claim 1, wherein the mutant plasminogen polypeptide is expressed and purified from an E. coli expression system.

### Claim 14 (depends on 1)

The method of claim 1, wherein the mutant recombinant plasminogen polypeptide is expressed and purified from a yeast expression system.

### Claim 15 (depends on 1)

The method of claim 1, wherein the mutant recombinant plasminogen polypeptide is expressed and purified from an insect or a mammalian expression system.

### Claim 16 (depends on 8)

A composition comprising a mutant plasminogen and plasmin polypeptide produced by the method of claim 8.

### Claim 17 (depends on 16)

The composition of claim 16, further comprising a pharmaceutically acceptable excipient.

### Claim 18 (depends on 17)

The composition of claim 17, further comprising the delivery of the mutant plasminogen and plasmin polypeptide in a pharmaceutically acceptable excipient through intravenous, subcutaneous, submuscular, and aerosol routes; wherein the aerosol delivery comprises using inhale devices including Nebulizers, metered dose inhalers, and dry powder inhalers.