Ultra-long acting insulin-Fc fusion proteins and methods of use
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.
1. A fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the Fc fragment comprises the following sequence:
(SEQ ID NO: 22)
DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEV
QISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCK
VNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKD
FFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ
RGDTFICAVMHEALHNHYTQESLSHSPG
wherein the insulin polypeptide and the Fc fragment are connected by a linker, and wherein the insulin polypeptide comprises a B-chain and an A-chain, said insulin polypeptide comprising an alanine substitution at B16.
2. 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.
3. 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).
4. The fusion protein of claim 1 , wherein the B-chain and the A-chain are linked by a C-chain comprising the following sequence:
(SEQ ID NO: 133)
GGGGGGSGGGG.
5. The fusion protein of claim 1 , wherein the fusion protein is a homodimer.
6. The fusion protein of claim 5 , wherein the percentage homodimer of the fusion protein is greater than 90%.
7. The fusion protein of claim 5 , 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.
8. The fusion protein of claim 1 , wherein the fusion protein is formulated as a pharmaceutical composition.
9. The fusion protein of claim 8 , wherein the fusion protein is present in the pharmaceutical composition at a concentration of about 3 mg/mL or greater.
10. The fusion protein of claim 8 , wherein the composition is suitable for subcutaneous administration.
11. 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.
12. The method of claim 11 in which the target animal is diagnosed with diabetes.
13. The method of claim 11 , wherein the fusion protein is administered subcutaneously.
14. The method of claim 13 , wherein the fusion protein is administered daily, twice weekly, or once weekly to the target animal.
15. The method of claim 13 , wherein the fusion protein is administered once weekly to the target animal at a dose between 0.025 and 0.5 mg/kg/week.