Ultra-long acting insulin-Fc fusion protein and methods of use
The present disclosure relates to compositions of insulin-Fc fusion proteins and their use to treat feline diabetes.
1. A recombinant cell comprising a nucleic acid encoding a fusion protein, said fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are connected by a peptide linker, wherein the Fc fragment comprises the following sequence:
(SEQ ID NO: 21)
GEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNV
QITWFVDNTEMHTAKTRPREEQFKSTYRVVSVLPILHQDWLKGKEFKCKV
NSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFH
PPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGN
TYTCSVSHEALHSHHTQKSLTQSPG.
and wherein the insulin polypeptide comprises the following sequence:
(SEQ ID NO: 4)
FVNQHLCGSDLVEALYLVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLY
QLENYCN.
2. The recombinant cell of claim 1 , wherein the cell is a HEK293 cell or a CHO cell.
3. The recombinant cell of claim 1 , wherein said nucleic acid comprises cDNA encoding said fusion protein, said fusion protein comprising the following sequence:
(SEQ ID NO: 36)
FVNQHLCGSDLVEALYLVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLY
QLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTP
EVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFKSTYRVVSVLP
ILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEE
LSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYF
LYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG.
4. The recombinant cell of claim 3 , wherein the cDNA comprises the following nucleic acid sequence:
(SEQ ID NO: 37)
atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtg
tccactccttcgtgaaccagcacctgtgcggctccgacctggtggaagc
tctgtatctcgtgtgcggcgagcggggcttcttctacaccgatcccact
ggaggcggtccacgcagaggcatcgtggaacagtgctgccactccatct
gctccctgtaccagctggaaaactactgcaatggcggaggtggttcagg
aggcggtggaggtgagggccccaagtgccccgtgcccgagattcccggt
gcccccagcgtgttcatctttcccccaaaacccaaggacaccctgagca
tcagcaggacccccgaggtgacctgcctggtggtggacctgggacccga
cgacagcaacgtgcagatcacctggttcgtggacaacaccgagatgcac
accgccaagaccaggcctcgggaggagcagttcaagagcacctacaggg
tggtgagcgtgctgcccatcctgcaccaggactggctgaagggcaagga
gttcaagtgcaaggtgaacagcaagagcctgcccagcgccatggagagg
accatcagcaaggccaagggtcagccccacgagccccaagtgtacgtgc
ttcccccgacccaggaggagttgagcgagaacaaagtgagcgtgacctg
cctgatcaagggcttccaccctcccgacatcgccgtggagtgggagatc
accggccagcctgagccggaaaataactaccagaccacccctccacagc
tggacagcgacggcacctacttcctttacagcaggctgtctgtggaccg
aagccattggcaaaggggcaacacctacacctgcagcgtgagccacgag
gctctgcacagccaccacacccagaagtctctgacccagagccccggat
ag.
5. The recombinant cell of claim 1 , wherein said recombinant cell is a stably transfected recombinant cell.
6. A method of preparing a recombinant cell according to claim 1 , said method comprising:
transfecting a host cell with a nucleic acid encoding for said fusion protein comprising said insulin polypeptide and said Fc fragment connected by said linker, wherein said fusion protein is expressed in said recombinant cell after said transfecting step.
7. The method of claim 6 , further comprising:
culturing said recombinant cell in cell culture media; and harvesting cell culture supernatant from said cell culture media, said cell culture supernatant comprising said expressed fusion protein.
8. The method of claim 7 , further comprising:
recovering said fusion protein from said cell culture supernatant.
9. The method of claim 8 , wherein said recovering step comprises one or more steps of centrifugation, filtration, or chromatography.
10. The method of claim 6 , wherein said transfecting step comprises stable transfection of said host cell.
11. The method of claim 6 , wherein said nucleic acid comprises cDNA encoding said fusion protein, said fusion protein comprising the following sequence:
(SEQ ID NO: 36)
FVNQHLCGSDLVEALYLVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLY
QLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTP
EVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFKSTYRVVSVLP
ILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEE
LSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYF
LYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG.
12. The method of claim 11 , wherein the cDNA comprises the following nucleic acid sequence:
(SEQ ID NO: 37)
atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtg
tccactccttcgtgaaccagcacctgtgcggctccgacctggtggaagc
tctgtatctcgtgtgcggcgagcggggcttcttctacaccgatcccact
ggaggcggtccacgcagaggcatcgtggaacagtgctgccactccatct
gctccctgtaccagctggaaaactactgcaatggcggaggtggttcagg
aggcggtggaggtgagggccccaagtgccccgtgcccgagattcccggt
gcccccagcgtgttcatctttcccccaaaacccaaggacaccctgagca
tcagcaggacccccgaggtgacctgcctggtggtggacctgggacccga
cgacagcaacgtgcagatcacctggttcgtggacaacaccgagatgcac
accgccaagaccaggcctcgggaggagcagttcaagagcacctacaggg
tggtgagcgtgctgcccatcctgcaccaggactggctgaagggcaagga
gttcaagtgcaaggtgaacagcaagagcctgcccagcgccatggagagg
accatcagcaaggccaagggtcagccccacgagccccaagtgtacgtgc
ttcccccgacccaggaggagttgagcgagaacaaagtgagcgtgacctg
cctgatcaagggcttccaccctcccgacatcgccgtggagtgggagatc
accggccagcctgagccggaaaataactaccagaccacccctccacagc
tggacagcgacggcacctacttcctttacagcaggctgtctgtggaccg
aagccattggcaaaggggcaacacctacacctgcagcgtgagccacgag
gctctgcacagccaccacacccagaagtctctgacccagagccccggat
ag.
13. A cDNA encoding a fusion protein comprising the following sequence:
(SEQ ID NO: 36)
FVNQHLCGSDLVEALYLVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLY
QLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTP
EVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFKSTYRVVSVLP
ILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEE
LSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYF
LYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG.
14. The cDNA of claim 13 , wherein the cDNA comprises the following nucleic acid sequence:
(SEQ ID NO: 37)
atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtg
tccactccttcgtgaaccagcacctgtgcggctccgacctggtggaagc
tctgtatctcgtgtgcggcgagcggggcttcttctacaccgatcccact
ggaggcggtccacgcagaggcatcgtggaacagtgctgccactccatct
gctccctgtaccagctggaaaactactgcaatggcggaggtggttcagg
aggcggtggaggtgagggccccaagtgccccgtgcccgagattcccggt
gcccccagcgtgttcatctttcccccaaaacccaaggacaccctgagca
tcagcaggacccccgaggtgacctgcctggtggtggacctgggacccga
cgacagcaacgtgcagatcacctggttcgtggacaacaccgagatgcac
accgccaagaccaggcctcgggaggagcagttcaagagcacctacaggg
tggtgagcgtgctgcccatcctgcaccaggactggctgaagggcaagga
gttcaagtgcaaggtgaacagcaagagcctgcccagcgccatggagagg
accatcagcaaggccaagggtcagccccacgagccccaagtgtacgtgc
ttcccccgacccaggaggagttgagcgagaacaaagtgagcgtgacctg
cctgatcaagggcttccaccctcccgacatcgccgtggagtgggagatc
accggccagcctgagccggaaaataactaccagaccacccctccacagc
tggacagcgacggcacctacttcctttacagcaggctgtctgtggaccg
aagccattggcaaaggggcaacacctacacctgcagcgtgagccacgag
gctctgcacagccaccacacccagaagtctctgacccagagccccggat
ag.