# US 10,870,686 B2 · Ultra-long acting insulin-FC fusion proteins and methods of use

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,870,686 B2
- Document ID: US10870686B2
- Status: Granted
- Kind: B2
- Application number: 16/776,065
- Filed: 2020-01-29
- Published: 2020-05-21
- Granted: 2020-12-22
- Earliest claimed: 2018-06-29
- Assignees: Akston Biosciences Corporation
- Applicants: Akston Biosciences Corporation
- Inventors: Thomas M. Lancaster; Todd C. Zion
- Web page: https://patentgrep.com/patent/US10870686B2

## CPC

- C07K 14/62: Insulins
- 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 47/65: Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- C07H 21/04: with deoxyribosyl as saccharide radical
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 16/2869: {against hormone receptors (for antibodies against neuromediator receptors C07K16/286)}
- C12N 15/62: DNA sequences coding for fusion proteins
- 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 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- 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
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure provides recombinantly manufactured ultra-long acting insulin-Fc fusion proteins for use in treating canine and feline diabetes. The insulin-Fc fusion proteins comprise an insulin polypeptide linked via a peptide linker to an Fc-fragment of canine or feline origin. Based on the results obtained, creating a treatment that is amenable to low cost manufacturing, exhibits sufficient in vivo bioactivity, displays extended duration of bioactivity, does not induce anti-drug antibodies, and substantially retains is potency over multiple administrations, requires a non-obvious combination of insulin polypeptide, peptide linkers, and species-specific Fc fragment, in addition to selective mutations on one or more of these components. Exemplary ultra-long acting insulin-Fc fusion proteins, polynucleotides encoding these insulin-Fc fusion proteins, and pharmaceutical formulations of exemplary insulin-Fc fusion proteins are provided, in addition to methods of use and preparation.

## Claims

### Claim 1

A fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are connected by a linker, wherein the Fc fragment comprises the following sequence:
(SEQ ID NO: 22)
DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPE
VQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFT
CKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCL
IKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDK
SRWQRGDTFICAVMHEALHNHYTQESLSHSPG
and wherein the insulin polypeptide comprises the following sequence:
(SEQ ID NO: 10)
FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2S
TCSLDQLENYC
and wherein X1 is not D and X2 is not H.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein the insulin polypeptide comprises the following sequence:
(SEQ ID NO: 10)
FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2S
TCSLDQLENYC
wherein X1 is H and X2 is T.

### Claim 3 (depends on 1)

The fusion protein of claim 1 comprising domains in the following orientation from N- to C-terminus: (N-terminus)--insulin polypeptide--linker--Fc fragment--(C-terminus).

### Claim 4 (depends on 1)

The fusion protein of claim 1, wherein the insulin polypeptide and the Fc fragment are connected by a linker, comprising the following sequence:
(SEQ ID NO: 14)
GGGGGQGGGGQGGGGQGGGGG.

### Claim 5

A fusion protein comprising an insulin polypeptide linked to an Fc fragment, wherein the fusion protein comprises the following sequence:
(SEQ ID NO: 36)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTC
SLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFI
FPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQ
PREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISK
ARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQ
QEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEAL
HNHYTQESLSHSPG.

### Claim 6 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein is a homodimer.

### Claim 7 (depends on 6)

The fusion protein of claim 6, wherein the percentage homodimer of the fusion protein is greater than 90%.

### Claim 8 (depends on 6)

The fusion protein of claim 6, wherein the fusion protein is made using one of HEK293 or CHO cells, and the resulting homodimer titer after purification using Protein A beads or a Protein A column is greater than 50 mg/L.

### Claim 9 (depends on 1)

The fusion protein of claim 1, wherein the insulin receptor IC50 for the fusion protein is less than or equal to 5000 nM.

### Claim 10 (depends on 1)

The fusion protein of claim 1, wherein the serum half-life of the fusion protein in the blood or serum of a target animal upon administration is longer than about 3 days.

### Claim 11 (depends on 1)

The fusion protein of claim 1, wherein the time during which there is a statistically significant decrease in blood glucose level in a subject relative to a pre-dose level is longer than one of 2 hours, 6 hours, 9 hours, 12 hours, 18 hours, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer.

### Claim 12 (depends on 1)

The fusion protein of claim 1, wherein the NAOC after the first subcutaneous injection in a target animal is greater than 150% FBGL·days·kg/mg.

### Claim 13 (depends on 12)

The fusion protein of claim 12, wherein the ratio of the NAOC after the third weekly subcutaneous injection of the fusion protein in the target animal to the NAOC after the first subcutaneous injection of the fusion protein in the target animal is greater than 0.50.

### Claim 14 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein is formulated as a pharmaceutical composition.

### Claim 15 (depends on 14)

The pharmaceutical composition of claim 14, wherein the fusion protein is present in the pharmaceutical composition at a concentration of about 3 mg/mL or greater.

### Claim 16 (depends on 14)

The pharmaceutical composition of claim 14, wherein the composition is suitable for subcutaneous administration.

### Claim 17 (depends on 1)

A method for lowering the blood glucose of a target animal, the method comprising administering a physiologically effective amount of the fusion protein of claim 1 or a pharmaceutical composition thereof to the target animal, and wherein the target animal is a dog.

### Claim 18 (depends on 17)

The method of claim 17 in which the target animal is diagnosed with diabetes.

### Claim 19 (depends on 17)

The method of claim 17, wherein the fusion protein is administered subcutaneously.

### Claim 20 (depends on 17)

The method of claim 17, wherein the fusion protein is administered daily, twice weekly, or once weekly to the target animal.

### Claim 21 (depends on 17)

The method of claim 17, wherein the fusion protein is administered once weekly to the target animal at a dose between 0.025 and 0.5 mg/kg/week.