# US 11,078,497 B2 · Compositions and methods to manufacture tumor suppressor fusions

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- Number: US 11,078,497 B2
- Document ID: US11078497B2
- Status: Granted
- Kind: B2
- Application number: 17/039,468
- Filed: 2020-09-30
- Published: 2021-03-18
- Granted: 2021-08-03
- Earliest claimed: 2019-04-12
- Assignees: TheraPten Biosciences Inc.
- Applicants: TheraPten Biosciences Inc.
- Inventors: Athanasios Alevizopoulos
- Web page: https://patentgrep.com/patent/US11078497B2

## CPC

- C12N 15/86: Viral vectors
- A61K 38/385: {Serum albumin}
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- A61P 35/00: Antineoplastic agents
- A61P 35/04: specific for metastasis
- C07K 14/765: Serum albumin, e.g. HSA
- C12N 9/16: acting on ester bonds (3.1)
- C12Y 301/03048: Protein-tyrosine-phosphatase (3.1.3.48)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- C12N 2710/16043: viral genome or elements thereof as genetic vector
- 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)
- C12Y: ENZYMES

## Abstract

Provided herein is a fusion protein comprising, or alternatively consisting essentially of, or yet further consisting of an optional signal peptide, a serum albumin, an optional linker, a Phosphatase and Tensin Homolog (PTEN), and an optional purification or detectable marker in any order. Relating polynucleotides, vectors, host cells, pharmaceutical compositions and kits are also disclosed. Further provided are methods for delivering a fusion protein to a subject, treating a cancer or tumor, and/or producing the fusion protein.

## Claims

### Claim 1

A fusion protein comprising human serum albumin (HSA) and human Phosphatase and Tensin Homolog (PTEN) tumor suppressor, wherein the HSA is upstream to the PTEN tumor suppressor.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein the HSA comprises the polypeptide of SEQ ID NO: 12 and the PTEN tumor suppressor comprises the polypeptide of SEQ ID NO: 2.

### Claim 3 (depends on 1)

A method of producing the fusion protein of claim 1, comprising
(i) culturing a host cell capable of expressing the fusion protein under conditions suitable for expression of the fusion protein; and
(ii) purifying or isolating the fusion protein optionally from the cell or cell culture medium,
whereby achieving one or more of: a high yield, a low aggregation, or a high stability compared to a produced wildtype PTEN protein, and optionally wherein after or during the culture, supernatant of host cells comprises more than about 5 μg/ml of secreted fusion protein.

### Claim 4 (depends on 3)

The method of claim 3, wherein the host cell comprises a polynucleotide encoding the fusion protein or a vector comprising the polynucleotide.

### Claim 5 (depends on 1)

A composition comprising the fusion protein of claim 1 and a carrier.

### Claim 6 (depends on 5)

The composition of claim 5, wherein the carrier is a pharmaceutically acceptable carrier.

### Claim 7 (depends on 1)

The fusion protein of claim 1, wherein the HSA comprises a polypeptide as set forth in any one of: SEQ ID NO: 12, SEQ ID NO: 163, SEQ ID NO: 13, amino acids 1-197 of SEQ ID NO:13, amino acids 1-395 of SEQ ID NO:13, amino acids 198-585 of SEQ ID NO:13, amino acids 35-385 of SEQ ID NO:13, or amino acids 63-585 of SEQ ID NO:13.

### Claim 8 (depends on 7)

The fusion protein of claim 7, wherein the HSA further comprises a mutation at a position aligned to amino acid 58 of SEQ ID NO: 12.

### Claim 9 (depends on 8)

The fusion protein of claim 8, wherein the PTEN tumor suppressor comprises the polypeptide as set forth in any one of SEQ ID NO: 1, 2, 3, 6, 7, 8, 88, 89, 162, 201, 202, or 203.

### Claim 10 (depends on 9)

The fusion protein of claim 9, wherein the PTEN tumor suppressor further comprises a mutation at a position aligned to any one of S65, S85, S89, S91, S94, S111, S115, S117, S129, S134, T140, S161, S163, S164 or S168 of SEQ ID NO:1 or S170, N292, S355, S360, 5366 or 5370 of SEQ ID NO:2.

### Claim 11 (depends on 7)

The fusion protein of claim 7, wherein the PTEN tumor suppressor comprises the polypeptide as set forth in any one of SEQ ID NO: 1, 2, 3, 6, 7, 8, 88, 89, 162, 201, 202, or 203.

### Claim 12 (depends on 11)

The fusion protein of claim 11, wherein the PTEN tumor suppressor further comprises a mutation at a position aligned to any one of S65, S85, S89, S91, S94, 5111, 5115, S117, S129, S134, T140, S161, S163, S164 or S168 of SEQ ID NO:1 or S170, N292, S355, S360, 5366 or 5370 of SEQ ID NO:2.

### Claim 13 (depends on 1)

The fusion protein of claim 1, wherein the PTEN tumor suppressor comprises the polypeptide as set forth in any one of SEQ ID NO: 1, 2, 3, 6, 7, 8, 88, 89, 162, 201, 202, or 203.

### Claim 14 (depends on 13)

The fusion protein of claim 13, wherein the PTEN tumor suppressor further comprises a mutation at a position aligned to any one of S65, S85, S89, S91, S94, 5111, 5115, S117, S129, S134, T140, S161, S163, S164 or S168 of SEQ ID NO:1 or S170, N292, S355, S360, S366 or 5370 of SEQ ID NO:2.

### Claim 15 (depends on 1)

The fusion protein of claim 1, wherein the HSA comprises a polypeptide as set forth in SEQ ID NO: 12 and the PTEN tumor suppressor comprises a polypeptide as set forth in SEQ ID NO: 2.

### Claim 16

A polynucleotide encoding a fusion protein comprising human serum albumin (HSA) and human Phosphatase and Tensin Homolog (PTEN) tumor suppressor, wherein the HSA is upstream to the PTEN tumor suppressor.

### Claim 17 (depends on 16)

The polynucleotide of claim 16, wherein the polynucleotide comprises SEQ ID NOs: 180, 176 and 172, or a polynucleotide at least 90% identical thereto and which encodes the same amino acid sequence as SEQ ID NOs: 180, 176 and 172, respectively.

### Claim 18 (depends on 16)

The polynucleotide of claim 16, further comprising a regulatory sequence for expression or replication of the polynucleotide in a host cell, optionally wherein the regulatory sequence comprises a promoter and optionally an enhancer.

### Claim 19 (depends on 16)

The polynucleotide of claim 16, consisting of polynucleotides as set forth in SEQ ID NOs: 180, 176 and 172.

### Claim 20 (depends on 16)

A vector comprising the polynucleotide of claim 16.

### Claim 21 (depends on 20)

The vector of claim 20, wherein the vector is a viral vector, optionally selected from the group of: rabies virus, flavivirus, lentivirus, baculovirus, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, alphavirus vectors, Seneca Valley virus, poliovirus, vaccinia virus, reovirus, Coxsackievirus, parvovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, or infectious tobacco mosaic virus (TMV).

### Claim 22 (depends on 20)

The vector of claim 20, wherein the vector is a non-viral vector, optionally selected from liposomes, cationic lipid, micelles biocompatible polymers, nanoparticles, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, metal particles, bacteria, bacteriophage, cosmid, plasmid, a yeast artificial chromosome, or fungal vectors.

### Claim 23 (depends on 20)

A composition comprising the vector of claim 20 and a carrier.

### Claim 24 (depends on 23)

The composition of claim 23, wherein the carrier is a pharmaceutically acceptable carrier.

### Claim 25 (depends on 16)

A composition comprising the polynucleotide of claim 16 and a carrier.

### Claim 26 (depends on 25)

The composition of claim 25, wherein the carrier is a pharmaceutically acceptable carrier.

### Claim 27

A host cell comprising a polynucleotide that encodes a fusion protein comprising human serum albumin (HSA) and human Phosphatase and Tensin Homolog (PTEN) tumor suppressor, wherein the HSA is upstream to the PTEN tumor suppressor.

### Claim 28 (depends on 27)

A method for producing the host cell of claim 27, comprising inserting a vector comprising the polynucleotide encoding the fusion protein into the host cell.

### Claim 29 (depends on 28)

The method of claim 28, further comprising generating the vector prior to inserting it into the host cell.