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 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 linker, wherein the Fc fragment comprises the following sequence:
(SEQ ID NO: 22)
DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQI
SWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNK
ALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDI
DVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICA
VMHEALHNHYTQESLSHSPG
and wherein the insulin polypeptide comprises the following sequence:
(SEQ ID NO: 10)
FVNQHLCGSX 1 LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX 2 STCS
LDQLENYC
and wherein X 1 is not D and X 2 is not H.
2. The recombinant cell of claim 1 , wherein the cell is a HEK293 cell or a CHO cell.
3. A cDNA encoding a fusion protein comprising the following sequence:
(SEQ ID NO: 36)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLD
QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPK
DTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGT
YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYV
LPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLD
EDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG.
4. The cDNA of claim 3 , wherein the cDNA comprises the following nucleic acid sequence:
(SEQ ID NO: 35)
atggaatggagctgggtctactatatcctgtcagtaacgactggtgtccac
tcatcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcac
tcgtgtgcggcgagcggggcaccactacgggggtggcggaggaggactggt
ggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggac
cagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacag
ggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgct
cccgagatgctgggcggacccagcgtgacatcaccctcccaagcccaagga
cacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacct
ggatcccgaagaccccgaggtgcagatcagctggacgtggatggaaagcag
atgcagaccgccaagacccaaccccgggaagagcagactcaggcacctaca
gggtggtgagtgtgagcccatcggccaccaggactggctgaaggggaagca
attcacatgcaaggttaataacaaggccctgcccagccccatcgagaggac
catcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcc
cccatctagggaggaactgagcaagaacacagtcagccttacttgcctgat
caaggacttcacccaccggacatagacgtggagtggcagagtaacggccag
caggagcccgagagcaagtataggaccacaccgccccaactggacgaggac
ggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcag
cgaggcgacaccacatctgcgccgtgatgcacgaggctagcataaccacta
cacccaggagagcctgtcccacagccccggatag.
5. 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)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSL
DQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPK
PKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF
SGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQP
SVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTT
PPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHS
PG.
6. The recombinant cell of claim 5 , wherein the cDNA comprises the following nucleic acid sequence:
(SEQ ID NO: 35)
atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt
ccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctc
tggcactcgtgtgcggcgagcggggcttccactacgggggtggcggagga
ggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctg
ctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggag
gcggtggacagggtggaggtgggcagggaggaggcgggggagactgcccc
aagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccc
tcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacct
gcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctgg
ttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaaga
gcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccacc
aggactggctgaaggggaagcaattcacatgcaaggttaataacaaggcc
ctgcccagccccatcgagaggaccatcagcaaggccaggggccaggccca
ccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaaga
acacagtcagccttacttgcctgatcaaggacttcttcccaccggacata
gacgtggagtggcagagtaacggccagcaggagcccgagagcaagtatag
gaccacaccgccccaactggacgaggacggaagctacttcctctacagca
aattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgc
gccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtc
ccacagccccggatag.
7. The recombinant cell of claim 1 , wherein said recombinant cell is a stably transfected recombinant cell.
8. 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.
9. The method of claim 8 , 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.
10. The method of claim 9 , further comprising:
recovering said fusion protein from said cell culture supernatant.
11. The method of claim 10 , wherein said recovering step comprises one or more steps of centrifugation, filtration, or chromatography.
12. The method of claim 8 , wherein said transfecting step comprises stable transfection of said host cell.
13. The method of claim 8 , wherein said nucleic acid comprises cDNA encoding said fusion protein, said fusion protein comprising the following sequence:
(SEQ ID NO: 36)
FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSL
DQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPK
PKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF
SGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQP
SVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTT
PPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHS
PG.
14. The method of claim 13 , wherein the cDNA comprises the following nucleic acid sequence:
(SEQ ID NO: 35)
atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt
ccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctc
tggcactcgtgtgcggcgagcggggcttccactacgggggtggcggagga
ggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctg
ctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggag
gcggtggacagggtggaggtgggcagggaggaggcgggggagactgcccc
aagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccc
tcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacct
gcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctgg
ttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaaga
gcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccacc
aggactggctgaaggggaagcaattcacatgcaaggttaataacaaggcc
ctgcccagccccatcgagaggaccatcagcaaggccaggggccaggccca
ccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaaga
acacagtcagccttacttgcctgatcaaggacttcttcccaccggacata
gacgtggagtggcagagtaacggccagcaggagcccgagagcaagtatag
gaccacaccgccccaactggacgaggacggaagctacttcctctacagca
aattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgc
gccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtc
ccacagccccggatag.