# US 2020/0399588 A1 · Wnt compositions and methods of process from serum-free culturing conditions

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- Number: US 2020/0399588 A1
- Document ID: US20200399588A1
- Status: Published application
- Kind: A1
- Application number: 16/996,676
- Filed: 2020-08-18
- Published: 2020-12-24
- Earliest claimed: 2017-08-01
- Assignees: Ankasa Regenerative Therapeutics, Inc.
- Applicants: Ankasa Regenerative Therapeutics, Inc.
- Inventors: Ping Yuan, JR.; Jill Helms; Ying Zhu; Bo Liu; Stefanie Gaster
- Web page: https://patentgrep.com/patent/US20200399588A1

## CPC

- C07K 14/82: Translation products from oncogenes
- C12N 5/0031: {Serum-free culture media}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/127: Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
- A61K 47/26: Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
- A61K 47/6911: {the form being a liposome}
- C07K 1/18: Ion-exchange chromatography
- C07K 14/4705: {stimulating, promoting or activating activity}
- C12N 5/0602: {Vertebrate cells}
- A61K 35/32: Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
- 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)
- C12N 2501/415: Wnt; Frizzeled
- 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)
- 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)

## Abstract

Disclosed herein are methods and compositions for producing a Wnt polypeptide under a serum-free condition. Also disclosed herein are methods of purifying the Wnt polypeptide from a serum-free condition.

## Claims

### Claim 1

A method of preparing a functionally active Wnt polypeptide, comprising:
(a) co-expressing a Wnt polypeptide and a chaperone in a cell in a conditioned media to generate a plurality of Wnt polypeptide-chaperone complexes;
(b) harvesting the plurality of Wnt polypeptide-chaperone complexes from the conditioned media;
(c) incubating the plurality of Wnt polypeptide-chaperone complexes with a buffer comprising a sugar detergent to generate a mixture comprising a first Wnt composition comprising a functionally inactive Wnt polypeptide and a chaperone composition;
(d) separating the first Wnt composition from the mixture with a column immobilized with a sulfonated polyaromatic compound to generate a second Wnt composition comprising the functionally active Wnt polypeptide and the sugar detergent; and
(e) contacting the second Wnt composition with an aqueous solution of liposomes to generate a final Wnt composition comprising a functionally active Wnt polypeptide.

### Claim at position 2

2-21. (canceled)

### Claim 22 (depends on 1)

The method of claim 1, wherein the chaperone comprises a Frizzled protein.

### Claim 23 (depends on 1)

The method of claim 1, wherein the chaperone comprises a Frizzled-8 fusion protein.

### Claim 24 (depends on 23)

The method of claim 23, wherein the Frizzled-8 fusion protein comprises a truncated Frizzled-8 protein, and wherein the truncated Frizzled-8 protein comprises a cysteine-rich region (CRD) of Frizzled-8.

### Claim 25

(canceled)

### Claim 26

(canceled)

### Claim 27 (depends on 23)

The method of claim 23, wherein the Frizzled-8 fusion protein further comprises an IgG Fc portion.

### Claim at position 9

28-30. (canceled)

### Claim 31 (depends on 1)

The method of claim 1, wherein the Wnt polypeptide is a Wnt3A polypeptide.

### Claim 32 (depends on 31)

The method of claim 31, wherein the Wnt3A polypeptide comprises about 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 1.

### Claim 33 (depends on 31)

The method of claim 31, wherein the Wnt3A polypeptide comprises a truncation of about 1 to about 33 amino acids, optionally a C-terminal truncation.

### Claim 34

(canceled)

### Claim 35 (depends on 1)

The method of claim 1, wherein the Wnt polypeptide comprises a lipid modification at an amino acid position corresponding to amino acid residue 209 as set forth in SEQ ID NO: 1.

### Claim at position 15

36-100. (canceled)

### Claim 101

A mammalian cell comprising an exogenous Wnt polypeptide and an exogenous chaperone protein.

### Claim 102 (depends on 101)

The mammalian cell of claim 101, wherein the chaperone comprises a Frizzled protein.

### Claim 103 (depends on 102)

The mammalian cell of claim 102, wherein the chaperone comprises a Frizzled-8 fusion protein.

### Claim 104 (depends on 103)

The mammalian cell of claim 103, wherein the Frizzled-8 fusion protein comprises a truncated Frizzled-8 protein, wherein the truncated Frizzled-8 protein comprises a cysteine-rich region (CRD) of Frizzled-8.

### Claim 105 (depends on 101)

The mammalian cell of claim 101, wherein the Wnt polypeptide is a Wnt3A polypeptide.

### Claim 106 (depends on 105)

The mammalian cell of claim 105, wherein the Wnt3A polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1.

### Claim 107 (depends on 105)

The mammalian cell of claim 105, wherein the Wnt3A polypeptide comprises a truncation of about 1 to about 33 amino acids, optionally a C-terminal truncation.

### Claim 108

A pharmaceutical composition, wherein the pharmaceutical composition comprises a liposomal Wnt3A polypeptide, wherein the amount of active Wnt3A polypeptide present in the pharmaceutical composition provides for an EC50 of about equal to or less than 0.5 ng/uL when administered to a bone graft material.

### Claim 109 (depends on 108)

The pharmaceutical composition of claim 108, wherein the Wnt3A polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1.

### Claim 110 (depends on 108)

The pharmaceutical composition of claim 108, wherein the Wnt3A polypeptide comprises a truncation of about 1 to about 33 amino acids, optionally a C-terminal truncation.

### Claim 111 (depends on 108)

The pharmaceutical composition of claim 108, wherein the Wnt polypeptide comprises a lipid modification at an amino acid position corresponding to amino acid residue 209 as set forth in SEQ ID NO: 1.