Ultra-long acting insulin-Fc fusion proteins
The present disclosure provides recombinantly manufactured ultra-long acting insulin-Fc fusion proteins for use in treating diabetes. The insulin-Fc fusion proteins comprise an insulin polypeptide linked via a peptide linker to an Fc-fragment of human origin. 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.
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 fusion protein comprises the following sequence:
(SEQ ID NO: 87)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSL
DQLENYCGGGGGAGGGGAGGGGAGGGGGDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPG.
2. 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 is of human origin and comprises the following sequence:
(SEQ ID NO: 77)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYX1STYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG
wherein X 1 is S, D, A, or R,
wherein the insulin polypeptide consists of an insulin B-chain analog linked to an insulin A-chain analog via a C-chain, wherein the 16th amino acid from the N-terminus of the insulin B-chain analog (i.e., B16) of the insulin polypeptide is alanine (i.e., B16A).
3. The fusion protein of claim 2 , wherein the insulin polypeptide comprises the following sequence:
FVNQHLCGSX 1 LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX 2 STCSLDQLENYC (SEQ ID NO: 9), wherein X 1 is not D, and X 2 is not H.
4. The fusion protein of claim 3 , wherein the insulin polypeptide comprises the following sequence:
(SEQ ID NO: 10)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLD
QLENYC.
5. The fusion protein of claim 2 , wherein the linker comprises the following sequence:
(SEQ ID NO: 67)
GGGGGAGGGGAGGGGAGGGGG.
6. The fusion protein of claim 2 , wherein the linker comprises the following sequence:
(SEQ ID NO: 13)
GGGGGQGGGGQGGGGQGGGGG.
7. The fusion protein of claim 6 , wherein the fusion protein comprises the following sequence:
(SEQ ID NO: 78)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLD
QLENYCGGGGGQGGGGQGGGGQGGGGGDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
8. The fusion protein of claim 6 , wherein the fusion protein comprises the following sequence:
(SEQ ID NO: 80)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLD
QLENYCGGGGGQGGGGQGGGGQGGGGGDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
9. The fusion protein of claim 6 , wherein the fusion protein comprises the following sequence:
(SEQ ID NO: 82)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLD
QLENYCGGGGGQGGGGQGGGGQGGGGGDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
10. The fusion protein of claim 6 , wherein the fusion protein comprises the following sequence:
(SEQ ID NO: 84)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLD
QLENYCGGGGGQGGGGQGGGGQGGGGGDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YRSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
11. The fusion protein of claim 2 , wherein the linker comprises the following sequence:
(SEQ ID NO: 11)
GGGGAGGGG.
12. The fusion protein of claim 11 , wherein the fusion protein comprises the following sequence:
(SEQ ID NO: 89)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLD
QLENYCGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
13. The fusion protein of claim 2 comprising domains in the following orientation from N- to C-terminus: (N-terminus)—insulin polypeptide—linker—Fc fragment—(C-terminus).
14. The fusion protein of claim 2 , wherein the fusion protein is a homodimer, and wherein the percentage homodimer of the fusion protein is greater than 90%.
15. The fusion protein of claim 2 , wherein the fusion protein is made using one of HEK293 cells or CHO cells, and the resulting homodimer titer after purification using Protein A beads or a Protein A column is greater than 150 mg/L.
16. The fusion protein of claim 2 , wherein the insulin receptor IC50 for the fusion protein is less than or equal to 5000 nM.
17. The fusion protein of claim 2 , wherein the human FcRn receptor EC50 for the fusion protein is less than or equal to 1000 ng/mL.
18. The fusion protein of claim 2 , wherein the human Fc(gamma)RI receptor assay OD450 Ratio at a biotinylated-Fc(gamma)RI concentration of 3000 ng/mL of the fusion protein is less than or equal to 0.50.
19. The fusion protein of claim 2 , wherein the human C1q assay OD450 Ratio at a biotinylated-C1q concentration of 1000 ng/mL is less than or equal to 0.35.
20. The fusion protein of claim 2 , wherein the fusion protein is formulated as a pharmaceutical composition, and wherein the fusion protein is present in the pharmaceutical composition at a concentration of about 3 mg/mL or greater.