Ultra-long-acting insulin-Fc fusion protein and method of use

Abstract

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

Description

[0001] Priority and related applications

[0002] No. 62 / 774,682 filed on December 3, 2018, U.S. Provisional Patent Application Serial No. 62 / 743,358 filed on October 9, 2018, U.S. Provisional Patent Application Serial No. 62 / 774,682 filed on December 3, 2018, U.S. Provisional Patent Application Serial No. 62 / 774,682 filed on December 3, 2018, U.S. Provisional Patent Application Serial No. 62 / 774,682 filed on December 3, 2018, U.S. Provisional Patent Application Serial No. 62 / 774,682 filed on December 3, 2018, U.S. Provisional Patent Application Serial No. 62 / 774,682 filed on December 3, 2018, U.S. Provisional Patent Application Serial No. 62 / 774,682 filed on December 3, 2018, U.S. Provisional Patent Application Serial No. 62 / 774,682 filed on December 3, 2018, U.S. Provisional Patent Application Serial No. 62 / 774,682 filed on October ... No. 62 / 719,347 filed on August 17, 2018, U.S. Provisional Patent Application Serial No. 62 / 702,167 filed on July 23, 2018, U.S. Provisional Patent Application Serial No. 62 / 698,648 filed on July 16, 2018, U.S. Provisional Patent Application Serial No. 62 / 696,645 filed on July 11, 2018, U.S. Provisional Patent Application Serial No. 62 / 693,814 filed on July 3, 2018, U.S. Provisional Patent Application Serial No. 62 / 692,507 filed on June 29, 2018, and U.S. Patent Application Serial No. 62 / 692,498 filed on June 29, 2018. The contents of each of the foregoing patent applications are hereby incorporated by reference herein in their entirety. Technical Field

[0003] The present technology relates to compositions of insulin-Fc fusion proteins and their use in treating diabetes in companion animals such as dogs or cats. Background Art

[0004] The following description of the background of the present technology is provided only to help understand the present technology and is not considered to describe or constitute the prior art of the present technology.

[0005] Diabetes is a chronic condition characterized by a lack of insulin and / or ineffective use of insulin. Diabetics with an absolute lack of insulin are classified as having type 1 or insulin-dependent diabetes mellitus (IDDM). Type 1 diabetes is believed to be genetically predisposed, along with immune destruction of the insulin-producing beta cells of the pancreas. In contrast, diabetics who can still produce some insulin but are relatively deficient due to insulin resistance or other dysfunctions are classified as having type 2 or non-insulin-dependent diabetes mellitus (NIDDM). Type 2 diabetes is associated with genetic predisposition, obesity, and certain medications.

[0006] When a dog or cat does not produce insulin or cannot use insulin normally, blood sugar levels rise, leading to hyperglycemia. Dogs usually show atypical blood sugar phenotypes very similar to human type 1 diabetes. Dogs occasionally also show atypical blood sugars very similar to human type 2 diabetes. Female dogs can also produce temporary insulin resistance when they are in estrus or pregnant. In all cases, dogs are treated with chronic insulin injection therapy. Cats usually show atypical blood sugar phenotypes very similar to human type 2 diabetes (i.e. insulin resistance), but by the time the veterinarian diagnoses the disease, the disease has developed into a condition similar to type 1 diabetes (inflammatory disease in the pancreas, significant loss of β cell mass), and cats rely on exogenous insulin. Some diabetic cats can be controlled by dietary changes and oral medications, but most diabetic cats receive chronic insulin injection therapy to maintain adequate regulation. If not treated, diabetes in dogs and cats can lead to weight loss, loss of appetite, vomiting, dehydration, motor function problems, coma, and even death.

[0007] In the United States, approximately 0.24% of dogs and approximately 0.68% of cats have diabetes. Current treatments for diabetes in dogs and cats include the use of insulin, such as (Intervet Inc., dbaMERCK Animal Health, Summit, NJ), while the use of (Boehringer Ingelheim Vetmedica, Duluth, Georgia), is administered once or twice daily. The burden of frequent injections on owners often leads to lack of compliance with treatment regimens and underdosing, resulting in poor long-term health outcomes. In fact, the cost of insulin therapy and the practicality of dosing pets up to 14 times per week have led a large percentage of owners to choose euthanasia for their pets as an alternative to intensive management of diabetes. Therefore, there is a need for cost-effective and less burdensome treatment options for this disease. Summary of the invention

[0008] In one aspect, the present disclosure provides a fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are connected by a linker such as a peptide linker, wherein the Fc fragment is of non-human animal origin and comprises the following sequence:

[0009] DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 16). In some embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3 (SEQ ID NO: 6), wherein X1 is not D, X2 is not H, and X3 is absent or is N. In some embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3 (SEQ ID NO: 6), wherein X1 is H, X2 is T, and X3 is absent or is N. In an embodiment, the insulin polypeptide of the fusion protein and the Fc fragment are connected by a linker, such as a peptide linker, comprising the sequence GGGGQGGGGQGGGGGGGGG (SEQ ID NO: 14).

[0010] In embodiments, the fusion protein comprises the sequence FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO:32). In embodiments, the fusion protein comprises the sequence FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO:34).

[0011] In one aspect, the present disclosure provides a fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are connected by a linker, such as a peptide linker, wherein the Fc fragment comprises the sequence DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 22). In some embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is not D, and X2 is not H. In some embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is H, and X2 is T. In an embodiment, the insulin polypeptide and the Fc fragment are connected by a linker, such as a peptide linker, comprising the sequence GGGGGQGGGGQGGGGGGGGG (SEQ ID NO: 14).

[0012] In an embodiment, the fusion protein includes the sequence FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPR EEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:36).

[0013] In one aspect, the present disclosure provides a fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are connected by a linker such as a peptide linker, wherein the Fc fragment is of non-human animal origin and comprises the sequence DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 20). In an embodiment, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3 (SEQ ID NO: 6), wherein X1 is not D, X2 is not H, and X3 is absent. In an embodiment, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3 (SEQ ID NO: 6), wherein X1 is H, X2 is T, and X3 is absent. In an embodiment, the insulin polypeptide and the Fc fragment are connected by a linker such as a peptide linker, and the linker comprises the following sequence GGGGGQGGGGQGGGGGGGGG (SEQ ID NO: 14).

[0014] In an embodiment, the fusion protein includes the sequence FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPR EEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:38).

[0015] In one aspect, the present disclosure provides a fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are connected by a linker, such as a peptide linker, wherein the Fc fragment comprises the sequence DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 23). In embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is not D, and X2 is not H. In embodiments, the insulin polypeptide comprises the following sequence FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is H, and X2 is T. In embodiments, the insulin polypeptide and the Fc fragment are connected by a linker, such as a peptide linker, comprising the sequence GGGGGQGGGGQGGGGGGGGG (SEQ ID NO: 14).

[0016] In an embodiment, the fusion protein includes the sequence FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSP REEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:40).

[0017] In some aspects, the fusion protein described herein comprises homodimers. In an embodiment, the homodimer percentage of the fusion protein is greater than 90%. In an embodiment, the fusion protein described herein is prepared using HEK293 cells, and the homodimer titer obtained after purification using protein A beads or protein A columns is greater than 50 mg / L. In an embodiment, the insulin receptor IC50 of the fusion protein described herein is less than or equal to 5000 nM. In an embodiment, when administered, the serum half-life of the fusion protein described herein in the blood or serum of the target animal is longer than about 3 days. In an embodiment, for the fusion protein described herein, the time in which the blood glucose level in the subject is statistically significantly reduced relative to the pre-administration level is longer than 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 more. One of the longer time.

[0018] In some aspects, for the fusion proteins described herein, the NAOC after the first subcutaneous injection in the target animal is greater than 150% FBGL·day·kg / mg. In embodiments, for the fusion proteins described herein, the ratio of the NAOC after the third week of 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.

[0019] In some aspects, the fusion protein as described herein is formulated into a pharmaceutical composition. In an embodiment, in the pharmaceutical composition, the fusion protein is present at a concentration of about 3 mg / mL or higher. In an embodiment, the composition is suitable for subcutaneous administration.

[0020] In one aspect, a method for reducing the blood glucose level of a target animal is described, the method comprising administering to a patient a physiologically effective amount of a fusion protein as described herein or a pharmaceutical composition thereof. In an embodiment, the target animal is diagnosed with diabetes. In an embodiment, the target animal is a dog or a cat. In some embodiments, the fusion protein is administered subcutaneously. In some embodiments, the fusion protein is administered to the target animal every day, twice a week, or once a week. In an embodiment, the fusion protein is administered to the target animal once a week at a dosage of 0.025 to 0.5 mg / kg / week. In some aspects, a cell engineered to express a fusion protein as described herein is described. In an embodiment, a cell is transfected with a nucleic acid encoding the fusion protein. In an embodiment, the cell is a HEK293 cell or a CHO cell.

[0021] In one aspect, a cDNA encoding a fusion protein as described herein is described. In an embodiment, the cDNA comprises the nucleic acid sequence

[0022] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:31)。

[0023] In an embodiment, the cDNA comprises the nucleic acid sequence atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcaacggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:33).

[0024] In an embodiment, the cDNA comprises the nucleic acid sequence atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag (SEQ ID NO:35).

[0025] In an embodiment, the cDNA comprises the nucleic acid sequence atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcaacagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag(SEQ ID NO:37).

[0026] In an embodiment, the cDNA comprises the nucleic acid sequence atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcagcagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag(SEQ ID NO:39). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram of an exemplary insulin-Fc fusion protein homodimer is shown.

[0028] Figure 2 Shown are the mean fasting blood glucose levels in % from Day 0 to Day 3 for N=3 dogs dosed intravenously on Day 0 with 0.2 mg / kg of the homodimer of SEQ ID NO:42.

[0029] Figure 3 Shown is a side-by-side sequence comparison of SEQ ID NOs: 42, 44, 46, 48, and 50. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0030] Figure 4 Shown is a side-by-side sequence comparison of SEQ ID NOs: 42, 52, 54, and 56. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0031] Figure 5 Shown are the mean fasting blood glucose levels in % from Day 0 to Day 7 for N=3 dogs dosed intravenously on Day 0 with 0.2 mg / kg of the homodimer of SEQ ID NO:52.

[0032] Figure 6 Shown are the mean fasting blood glucose levels in % from Day 0 to Day 7 for N=6 dogs dosed subcutaneously on Day 0 with the homodimer of SEQ ID NO:52 at 0.33 mg / kg.

[0033] Figure 7 Shown are the average anti-drug antibody titers (μg / mL) for N=3 dogs dosed subcutaneously with the homodimer of SEQ ID NO:52 on Day 0 (0.30 mg / kg), Day 28 (0.33 mg / kg), Day 35 (0.33 mg / kg), Day 42 (0.50 mg / kg), Day 49 (1.00 mg / kg), and Day 56 (1.00 mg / kg).

[0034] Figure 8A side-by-side sequence comparison is shown for SEQ ID NOs: 58, 60, 62, and 64. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0035] Figure 9 Shown are mean anti-drug antibody titers (μg / mL) for N=1 dogs dosed subcutaneously with the homodimer of SEQ ID NO: 64 on Day 0 (0.33 mg / kg), Day 7 (0.50 mg / kg), Day 14 (0.50 mg / kg), and Day 21 (0.50 mg / kg).

[0036] Figure 10 Shown are mean anti-drug antibody titers (μg / mL) for N=1 dogs dosed subcutaneously with the homodimer of SEQ ID NO: 66 on Day 0 (0.33 mg / kg) and Day 14 (0.16 mg / kg).

[0037] Figure 11 Shown are the mean fasting blood glucose levels in % from Day 0 to Day 7 for N=2 dogs dosed subcutaneously on Day 0 with the homodimer of SEQ ID NO: 66 at 0.33 mg / kg.

[0038] Figure 12 Shown is a side-by-side sequence comparison of SEQ ID NOs: 66, 68, 70, 72, 74, and 76. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0039] Figure 13 Shown is a side-by-side sequence comparison of SEQ ID NOs: 66, 78, 80, 82, and 84. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0040] Figure 14 Shown is a side-by-side sequence comparison of SEQ ID NOs: 66, 76, and 86. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0041] Figure 15Shown is a side-by-side sequence comparison of SEQ ID NOs: 66, 82, 84, and 88. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0042] Figure 16 Shown is a side-by-side sequence comparison of SEQ ID NOs: 32, 34, 66, 90, 92, and 94. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0043] Figure 17 Shown are fasting blood glucose levels in % from Day 0 to Day 7 for N=1 dog dosed subcutaneously on Day 0 with the homodimer of SEQ ID NO: 34 at 0.16 mg / kg.

[0044] Figure 18 Shown are anti-drug antibody titers (μg / mL) for N=1 dogs dosed subcutaneously with the homodimer of SEQ ID NO: 34 on Day 0 (0.16 mg / kg), Day 14 (0.16 mg / kg), Day 28 (0.16 mg / kg), and Day 42 (0.16 mg / kg).

[0045] Figure 19 Shown are fasting blood glucose levels in % from Day 0 to Day 7 for N=1 dog dosed subcutaneously on Day 0 with the homodimer of SEQ ID NO: 32 at 0.33 mg / kg.

[0046] Figure 20 Shown are fasting blood glucose levels in % from Day 0 to Day 60 for N=1 dog dosed subcutaneously with the homodimer of SEQ ID NO: 32 on Day 0 (0.33 mg / kg), Day 15 (0.16 mg / kg), Day 31 (0.16 mg / kg), and Day 45 (0.15 mg / kg).

[0047] Figure 21 Shown are anti-drug antibody titers (μg / mL) for N=1 dogs dosed subcutaneously with the homodimer of SEQ ID NO: 32 on Day 0 (0.33 mg / kg), Day 15 (0.16 mg / kg), Day 31 (0.16 mg / kg), and Day 45 (0.15 mg / kg).

[0048] Figure 22Shown are fasting blood glucose levels in % from Day 0 to Day 7 for N=1 dog dosed subcutaneously on Day 0 with the homodimer of SEQ ID NO: 96 at 0.16 mg / kg.

[0049] Figure 23 Shown are fasting blood glucose levels in % from Day 0 to Day 7 for N=1 dog dosed subcutaneously on Day 0 with the homodimer of SEQ ID NO: 98 at 0.16 mg / kg.

[0050] Figure 24 Shown is a side-by-side sequence comparison of SEQ ID NOs: 102 and 104. "*" indicates complete homology across all sequences at a given sequence position, while ":", "." or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0051] Figure 25 Shown are the % fasting blood glucose levels from day 0 to day 7 for N=1 dog (the dog was administered 0.16 mg / kg subcutaneously of the homodimer of SEQ ID NO: 102 on day 0) and the % fasting blood glucose levels from day 0 to day 7 for N=1 dog (the dog was administered 0.16 mg / kg subcutaneously of the homodimer of SEQ ID NO: 104 on day 0).

[0052] Figure 26 Shown are fasting blood glucose levels in % from Day 0 to Day 7 for N=1 dog that was administered the homodimer of SEQ ID NO: 36 subcutaneously except on days when the dog was given food.

[0053] Figure 27 Shown are the mean fasting blood glucose levels in % from day 0 to day 7 for N=3 cats dosed subcutaneously on day 0 with the homodimer of SEQ ID NO: 106 at 0.8 mg / kg.

[0054] Figure 28 A side-by-side sequence comparison of SEQ ID Nos: 106, 108, 110 and 112 is shown. "*" indicates complete homology across all sequences at a given sequence position, while ":", "." or a space refers to a conservative amino acid mutation, a moderate amino acid mutation or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0055] Figure 29Shown are the average anti-drug antibody titers (μg / mL) for N=3 cats that were administered the homodimer of SEQ ID NO: 106 subcutaneously on Day 0 (0.8 mg / kg), Day 28 (0.6 mg / kg), Day 35 (0.6 mg / kg), Day 42 (0.6 mg / kg), and Day 48 (0.8 mg / kg).

[0056] Figure 30 A side-by-side sequence comparison of SEQ ID NOs: 108, 114, 116, and 118 is shown. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0057] Figure 31 A side-by-side sequence comparison of SEQ ID NOs: 106, 112, and 122 is shown. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space refers to a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequence at a given sequence position.

[0058] Figure 32 Shown are fasting blood glucose levels in % from day 0 to day 7 for N=1 cats dosed subcutaneously with the homodimer of SEQ ID NO: 122 on day 0 (0.16 mg / kg).

[0059] Figure 33 Shown are fasting blood glucose levels in % from day 0 to day 7 for N=1 cats that were dosed subcutaneously with the homodimer of SEQ ID NO: 38 on day 0 (0.16 mg / kg) except on days when the cats were given food.

[0060] Figure 34 Shown are anti-drug antibody titers (μg / mL) for N=1 cats dosed subcutaneously with the homodimer of SEQ ID NO: 38 on Day 0 (0.16 mg / kg), Day 14 (0.16 mg / kg), Day 28 (0.11 mg / kg), and Day 42 (0.09 mg / kg).

[0061] Figure 35 Shown are fasting blood glucose levels in % from day 0 to day 7 for N=1 cats dosed subcutaneously with the homodimer of SEQ ID NO: 124 on day 0 (0.16 mg / kg).

[0062] Figure 36Shown are the mean fasting blood glucose levels in % from day 0 to day 7 for N=3 cats dosed subcutaneously with the homodimer of SEQ ID NO: 40 on day 0 (0.10 mg / kg).

[0063] Figure 37 Shown are the mean fasting blood glucose levels in % from day 7 to day 14 for N=3 cats dosed subcutaneously with the homodimer of SEQ ID NO: 40 on day 7 (0.20 mg / kg).

[0064] Figure 38 The "full aa sequence" of the fusion protein (SEQ ID NO: 32) and its corresponding nucleic acid sequence (SEQ ID NO: 31) are shown.

[0065] Figure 39 The "full aa sequence" of the fusion protein (SEQ ID NO: 34) and its corresponding nucleic acid sequence (SEQ ID NO: 33) are shown.

[0066] Figure 40 The "full aa sequence" of the fusion protein (SEQ ID NO: 36) and its corresponding nucleic acid sequence (SEQ ID NO: 35) are shown.

[0067] Figure 41 The "full aa sequence" of the fusion protein (SEQ ID NO: 38) and its corresponding nucleic acid sequence (SEQ ID NO: 37) are shown.

[0068] Figure 42 The "full aa sequence" of the fusion protein (SEQ ID NO:40) and its corresponding nucleic acid sequence (SEQ ID NO:39) are shown. DETAILED DESCRIPTION

[0069] Insulin therapy that requires less frequent dosing (e.g., injections once a week) would be less burdensome to the owner, leading to better compliance, fewer euthanasias, and better outcomes for pets. For a given species (e.g., dog or cat), molecules suitable for ultra-long-acting treatment of diabetes should be manufactured in mammalian cells (e.g., human embryonic kidney (HEK, e.g., HEK293) cells) with acceptable titers of the desired homodimer product (e.g., greater than 50 mg / L homodimer titers from transiently transfected HEK cells, greater than 75 mg / L from transiently transfected HEK cells, greater than 100 mg / L from transiently transfected HEK cells, etc.). Candidates with only homodimer titers greater than 50 mg / L are considered useful in the present invention, because experience has shown that homodimer titers below this level are unlikely to result in commercial production of homodimer titers in Chinese hamster ovary (CHO) cells that meet the stringent low manufacturing cost requirements for veterinary products. In addition, the molecule must bind to the insulin receptor with considerable affinity (e.g., IC50 less than 5000nM, IC50 less than 4000nM, IC50 less than 3000nM, IC50 less than 2500nM, etc.), as measured in a 4°C IM-9 insulin receptor binding assay. As a rule of thumb, molecules that only exhibit an insulin receptor activity IC50 value of less than 5000nM are considered likely to exhibit the necessary biological activity in the target species. The molecule must also exhibit sustained biological activity in vivo (e.g., exhibiting glucose-lowering activity for greater than about 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) to justify less frequent dosing. The molecule must also exhibit an extended systemic residence time in the target animal (e.g., the serum half-life must be greater than 3 days, or longer). As described in Example 11, the bioactivity potential and duration of bioactivity can be quantitatively expressed by calculating the area on the curve normalized to the percentage of fasting blood glucose (%FBGL) for a given dose in mg / kg (NAOC), where the units are %FBGL·day·kg / mg. NAOC increases with greater decreases in %FBGL, which is the case where the molecule exhibits increased bioactivity, and when %FBGL takes longer to return to 100%, this is the case where the insulin-Fc fusion protein exhibits an increased duration of action. As described herein, in order to be useful, the molecule must exhibit a sufficiently high NAOC value (e.g., preferably a NAOC greater than 150%FBGL·day·kg / mg, more preferably a NAOC greater than 200%FBGL·day·kg / mg, and even more preferably a NAOC greater than 250%FBGL·day·kg / mg).As a rule of thumb, when the NAOC value is greater than 150% FBGL·day·kg / mg, the dosage requirements in the target species will be low enough to achieve an acceptable treatment cost. Finally, in order to be used to treat chronic diseases such as diabetes, the molecule must not induce the production of anti-drug antibodies (particularly antibodies that neutralize the biological activity of the molecule after repeated administration). Therefore, after multiple repeated administrations in the target animal, the molecule must exhibit similar duration and degree of biological activity (i.e., NAOC) (for example, the ratio of the NAOC after subcutaneous injection in the third week to the NAOC after subcutaneous injection of the molecule in the first week (i.e., the NAOC ratio (NAOCR) after the third dose) is preferably greater than 0.50, greater than 0.60, greater than 0.70, greater than 0.80 or greater than 0.90 or greater).

[0070] The recommended ultra-long-acting insulin therapy for clinical use in humans contains an insulin-Fc fusion protein that utilizes a human Fc fragment to prolong its in vivo action. Since the human Fc fragment is expected to be immunogenic and thus able to induce anti-drug antibodies in companion animals (e.g., dogs or cats), the human Fc fragment must be replaced with a species-specific (e.g., canine or feline) Fc fragment. However, it was unexpectedly found that a simple exchange between a human Fc fragment and a species-specific (e.g., canine or feline) Fc fragment did not produce a product with an acceptable homodimer titer (e.g., a homodimer titer greater than 50 mg / L) or a sufficiently high NAOC value (e.g., a NAOC value greater than 150% FBGL·day·kg / mg). For example, in some cases, only a specific isotype of the Fc fragment (e.g., canine IgGB or feline IgG1b) results in an insulin-Fc fusion protein with sufficiently high homodimer titers (e.g., homodimer titers greater than 50 mg / L) and an acceptably high NAOC value (e.g., a NAOC value greater than 150% FBGL·day·kg / mg). In other cases, specific amino acids of the insulin polypeptide are found to be immunogenic in the target species, thereby requiring site-directed mutagenesis to find a relatively small number of embodiments that are both non-immunogenic and biologically active in the target species, with an acceptably high NAOC value (e.g., a NAOC value greater than 150% FBGL·day·kg / mg) and a NAOCR value after subcutaneous administration in the third week of greater than 0.5. In other cases, when the Fc fragment was mutated to prevent glycosylation and thereby further reduce the immunogenicity of the insulin-Fc fusion protein, it was unexpectedly found that only specific amino acid mutations in the Fc fragment resulted in the desired homodimer titer (e.g., a homodimer titer greater than 50 mg / L) and NAOC value (e.g., a NAOC value greater than 150% FBGL·day·kg / mg). In addition, it was found that additional mutations were required in the insulin component to produce non-glycosylated insulin Fc-fusion proteins with these Fc mutations having the desired homodimer titer (e.g., a homodimer titer greater than 50 mg / L) and NAOC value (e.g., a NAOC value greater than 150% FBGL·day·kg / mg), while also obtaining a NAOCR value greater than 0.5 after subcutaneous administration in the third week. Therefore, the present invention provides a manufacturable, highly pure, long-acting, biologically active, non-immunogenic insulin-Fc fusion protein having an acceptably high homodimer titer (e.g., a homodimer titer greater than 50 mg / L), a NAOC value (e.g., a NAOC value greater than 150% FBGL·day·kg / mg), and a NAOCR value greater than 0.5 after subcutaneous administration in the third week, which is suitable for treating diabetes in companion animals (e.g., dogs or cats), each of which comprises an insulin polypeptide, an Fc fragment, and a linker between the insulin polypeptide and the Fc fragment.

[0071] definition

[0072] As used herein, the articles "a" and "an" refer to one or more than one (e.g., at least one) of the grammatical object of the article. When used in conjunction with the term "comprising" herein, the use of the words "a" and "an" can mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0073] As used herein, "about" and "approximately" generally refer to an acceptable degree of error for a measured quantity, given the nature or precision of a measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given range of values.

[0074] As used herein, an amount of a molecule, compound, conjugate, or substance effective to treat a disorder, such as a disorder described herein, a "therapeutically effective amount," or an "effective amount," refers to an amount of a molecule, compound, conjugate, or substance that, when administered in a single dose or multiple doses to a subject, treats a subject or cures, alleviates, relieves, or improves a subject suffering from a disorder, such as a disorder described herein, in excess of the amount that would be expected in the absence of such treatment.

[0075] As used herein, the term "analog" refers to a compound or conjugate that has a chemical structure that is similar to that of another compound or conjugate but differs therefrom in at least one aspect (e.g., a compound or conjugate as described herein, such as insulin).

[0076] As used herein, the term "antibody" or "antibody molecule" refers to an immunoglobulin molecule (Ig), an immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule containing an antigen binding site that specifically binds to an antigen (e.g., an immune response). As used herein, the term "antibody domain" refers to a variable region or constant region of an immunoglobulin. As used herein, the term "antibody domain" refers to a variable region or constant region of an immunoglobulin. It has been documented in the art that antibodies include several categories, such as IgA, IgM, or IgG in the case of mammals (e.g., humans and felines). The categories of immunoglobulins can be further divided into different isotypes, such as IgGA, IgGB, IgGC, and IgGD for canines, or IgG1a, IgG1b, and IgG2 for felines. Those skilled in the art will recognize that the immunoglobulin isotypes of a given immunoglobulin class will include different amino acid sequences, structures, and functional properties (e.g., different binding affinities to Fc (γ) receptors) from one another. "Specifically binds" or "immunoreactive with" means that the antibody reacts with one or more antigenic determinants of the desired antigen and has lower affinity for, eg, does not react with, other polypeptides.

[0077] As used herein, the term "area under the curve" or "AUC" refers to the integral area under the time curve of the experimenter's %FBGL after the insulin-Fc fusion protein of a given dose is used. As used herein, the term "area on the curve" or "AOC" is used as a measure of the biological effectiveness of insulin-Fc fusion protein, so that AOC equals the difference between the total possible area under the time curve and the AUC value of %FBGL. As used herein, "normalized area on the curve", "normalized AOC" or "NAOC" are the actual doses of the insulin-Fc fusion protein divided by the AOC value. As used herein, the term "standardized AOC ratio" or "NAOCR" is the ratio of the NAOC produced by the specific use of insulin-Fc fusion protein to the NAOC produced by the first use of insulin-Fc fusion protein in a series of uses. Therefore, NAOCR provides a measure of the biological activity changes of insulin-Fc fusion protein after repeated use.

[0078] As used herein, the terms "biological activity", "activity", "biological activity", "potency", "biological activity potency" or "biological potency" refer to the extent to which an insulin-Fc fusion protein activates the insulin receptor and / or achieves a reduction in blood glucose levels in a target subject. As used herein, "in vitro activity" or "insulin receptor activity" refers to the affinity with which an insulin-Fc fusion protein binds to an insulin receptor, and is typically measured by the concentration at which the insulin-Fc fusion protein displaces half of a reference standard of insulin from the insulin receptor in a competitive binding assay (i.e., IC50). As used herein, "in vivo activity" refers to the extent and duration of reduction in fasting blood glucose levels in a target subject following administration of an insulin-Fc fusion protein.

[0079] As used herein, the term "biosynthesis", "recombinant synthesis" or "recombinant preparation" refers to the process of expressing insulin-Fc fusion protein in host cells by transfecting cells with nucleic acid molecules (e.g., vectors) encoding insulin-Fc fusion protein (e.g., wherein the entire insulin-Fc fusion protein is encoded by a single nucleic acid molecule). Exemplary host cells include mammalian cells, such as HEK293 cells or CHO cells. Cells can be cultured using standard methods in the art, and the expressed insulin-Fc fusion protein can be harvested and purified from cell culture using standard methods in the art.

[0080] As used herein, the term "cell surface receptor" refers to a molecule, such as a protein, that is typically found on the outer surface of a cell membrane and interacts with a soluble molecule (e.g., a molecule that circulates in the blood supply). In some embodiments, a cell surface receptor may include a hormone receptor (e.g., an insulin hormone receptor or insulin receptor (IR)) or an Fc receptor that binds to an Fc fragment or Fc region of an antibody (e.g., an Fc (gamma) receptor, such as Fc (gamma) receptor I, or an Fc neonatal receptor, such as FcRn). As used herein, "in vitro activity" or "Fc(γ) receptor activity" or "Fc(γ) receptor binding" or "FcRn receptor activity" or "FcRn binding" refers to the affinity with which an insulin-Fc fusion protein binds to an Fc receptor (e.g., an Fc(γ) receptor or an FcRn receptor), and is typically measured by the concentration of the insulin-Fc fusion protein that gives the insulin-Fc fusion protein half of its maximal binding value (i.e., the EC50 value), as measured in an assay (e.g., an enzyme-linked immunosorbent assay (ELISA) assay) using an OD 450nm value measured on a microplate reader.

[0081] As used herein, the term "fasting blood glucose level" or "FBGL" refers to the average blood glucose level in a target subject at the end of a period during which no food is given and just before the time of administration of the insulin-Fc fusion protein. As used herein, the term "fasting blood glucose level percentage", "fasting blood glucose level %" or "%FBGL" refers to the ratio of a given blood glucose level to a fasting blood glucose level multiplied by 100.

[0082] As used herein, the term "immunogenic" or "immunogenicity" refers to the ability of a given molecule (e.g., an insulin-Fc fusion protein of the present invention) to stimulate the immune system of a target subject such that, after repeated administration of the molecule, the subject produces antibodies (i.e., anti-drug antibodies) that are able to specifically bind to the molecule. As used herein, the term "neutralizing," "neutralizing antibodies," or "neutralizing anti-drug antibodies" refers to the ability of an antibody to interfere with the biological activity of a compound in a target subject. As used herein, the term "immunogenic epitope," "immunogenic hotspot," or "hotspot" refers to a mutation or epitope of a given molecule (e.g., an insulin-Fc fusion protein of the present invention) that is responsible for moderate or strong binding of anti-drug antibodies.

[0083] As used herein, the term "insulin reference standard" is any of the following: (i) naturally occurring insulin from a mammal (e.g., human, dog, or cat); (ii) an insulin polypeptide that does not comprise an Fc fragment; or (iii) a standard of care insulin (e.g., commercially available insulin).

[0084] As used herein, the term "monomer" refers to a protein or fusion protein comprising a single polypeptide. In an embodiment, a "monomer" is a protein or fusion protein comprising an insulin polypeptide and an Fc fragment polypeptide, such as a single polypeptide, wherein the insulin and Fc fragment polypeptides are linked by a peptide bond to form a single polypeptide. In an embodiment, a monomer is encoded by a single nucleic acid molecule.

[0085] As used herein, "N-terminus" refers to the beginning of a protein or polypeptide initiated by an amino acid containing a free amine group, which is the α-amino group of an amino acid (e.g., a free amino group covalently linked to a carbon atom positioned adjacent to a second carbon atom, wherein the second carbon atom is part of the carbonyl group of the amino acid). As used herein, "C-terminus" refers to the end of a protein or polypeptide terminated by an amino acid containing a carboxylic acid group, wherein the carbon atom of the carboxylic acid group is positioned adjacent to the α-amino group of the amino acid.

[0086] As used herein, "pharmacodynamics" or "PD" generally refers to the biological effect of an insulin-Fc fusion protein in a subject. Specifically, PD herein refers to a measure of the decrease in fasting blood glucose levels over time in a subject after administration of an insulin-Fc fusion protein.

[0087] As used herein, "pharmacokinetics" or "PK" generally refers to the characteristic interactions of the insulin-Fc fusion protein and the subject's body in terms of its absorption, distribution, metabolism and excretion. Specifically, PK in this article refers to the concentration of the insulin-Fc fusion protein in the blood or serum of the subject at a given time after the administration of the insulin-Fc fusion protein. As used herein, "half-life" refers to the time taken for the concentration of the insulin-Fc fusion protein in the blood or serum of the subject to reach half of its original value, as calculated from a first-order exponential decay model for drug elimination. Insulin-Fc fusion proteins with greater "half-life" values ​​exhibit longer durations of action in target subjects.

[0088] As used herein, the terms "sequence identity", "sequence homology", "homology" or "identical" in an amino acid or nucleotide sequence describe the nucleotide or amino acid residues that are found identical in a variant and a reference sequence when a specified contiguous segment of the nucleotide sequence or amino acid sequence of the variant is aligned and compared to the nucleotide sequence or amino acid sequence of the reference sequence. Methods for sequence alignment and for determining identity between sequences are known in the art and include the use of Clustal Omega, which organizes, aligns and compares sequences for similarity, wherein the software highlights each sequence position and makes comparisons between all sequences at that position and assigns one of the following scores: "*" (asterisk) for sequence positions with a single completely conserved residue, ":" (colon) indicating conservation between groups of strong similarity with a score greater than 0.5 in the Gonnet PAM 250 matrix, and "." (period) indicating conservation between groups of weak similarity with a score less than or equal to 0.5 in the Gonnet PAM 250 matrix, "-" (dash) indicates a sequence gap, meaning that there is no local homology within a particular comparison set within a certain range of the sequences, and a space " " indicates that there is little or no sequence homology at that particular position in the compared sequences. See, e.g., Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978) in Atlas of Polypeptide Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). For the best comparison of two nucleotide sequences, the continuous section of the variant nucleotide sequence can have additional nucleotides or missing nucleotides relative to the reference nucleotide sequence. Similarly, for the best comparison of two amino acid sequences, the continuous section of the variant amino acid sequence can have additional amino acid residues or missing amino acid residues relative to the reference amino acid sequence. In certain embodiments, the continuous section for comparison with the reference nucleotide sequence or the reference amino acid sequence will include at least 6, 10, 15 or 20 continuous nucleotides or amino acid residues, and can be 30, 40, 50, 100 or more nucleotides or amino acid residues. The correction of the increased sequence identity associated with the nucleotide sequence or amino acid sequence of the variant can be performed by specifying a gap penalty. Methods of sequence alignment are known in the art.

[0089] In an embodiment, a mathematical algorithm is used to complete the determination of the identity percentage or "homology" between two sequences. For example, the Smith-Waterman homology search algorithm is used to determine the identity percentage of an amino acid sequence, and the algorithm uses an affine 6 gap search, wherein the gap opening penalty is 12, and the gap extension penalty is 2, BLOSUM matrix 62. The Smith-Waterman homology search algorithm is described in Smith and Waterman (1981) Adv.Appl.Math2:482-489 (which is incorporated herein by reference). In an embodiment, the Smith-Waterman homology search algorithm is used to determine the identity percentage of a nucleotide sequence, and the algorithm uses a gap opening penalty of 25 and a gap extension penalty of 5. Such determination of sequence identity can be performed using, for example, the DeCypher hardware accelerator from TimeLogic.

[0090] As used herein, the term "homology" is used to compare two or more proteins by locating common structural features and common spatial distributions of, for example, beta strands, spirals, and folds. Therefore, homologous protein structures are defined by spatial analysis. Measuring structural homology involves calculating geometric topological features of the space. A method for generating and analyzing three-dimensional (3D) protein structures is homology modeling (also referred to as comparative modeling or knowledge-based modeling), which works by finding similar sequences based on the fact that 3D similarities reflect 2D similarities. Homologous structures do not imply that sequence similarity is a necessary condition.

[0091] As used herein, the terms "subject" and "patient" are intended to include canines and felines. Exemplary canine and feline subjects include dogs and cats suffering from a disease or disorder (e.g., diabetes or another disease or disorder described herein), or normal subjects.

[0092] As used herein, the term "titer" or "yield" refers to the amount of fusion protein product (e.g., insulin-Fc fusion protein described herein) produced by biosynthesis (e.g., in mammalian cells, such as HEK293 cells or CHO cells) per volume of cell culture. The amount of the product can be determined at any step of the production process (e.g., before or after purification), but the yield or titer is always expressed per volume of the original cell culture. As used herein, the term "product yield" or "total protein yield" refers to the total amount of insulin-Fc fusion protein (e.g., protein A or protein G) expressed by cells and purified by at least one affinity chromatography step, and includes monomers of insulin-Fc fusion protein, homodimers of insulin-Fc fusion protein, and higher molecular aggregates of homodimers of insulin-Fc fusion protein. As used herein, the term "homodimer percentage" or "homodimer %" refers to the ratio of the fusion protein product (e.g., insulin-Fc fusion protein described herein) as the desired homodimer. As used herein, the term "homodimer titer" refers to the product of the % homodimer and the total protein yield after the Protein A purification step reported per volume of cell culture.

[0093] As used herein, the term "treat or treating" a subject with a disease or disorder refers to subjecting the subject to a treatment regimen, such as administering a fusion protein such as a fusion protein described herein, such that at least one symptom of the disease or disorder is cured, healed, alleviated, mitigated, altered, remedied, improved, or improved. Treatment includes administering an amount that effectively alleviates, mitigates, alters, remedies, improves, improves, or affects the disease or disorder or the symptoms of the disease or disorder. The treatment can inhibit the deterioration or aggravation of the symptoms of the disease or disorder.

[0094] Components and structure of insulin-Fc fusion protein

[0095] The present disclosure relates to a composition of a fusion protein (i.e., insulin-Fc fusion protein), which comprises an insulin polypeptide connected to a species-specific Fc fragment by a peptide linker, and the use of the same for treating diabetes in companion animals (e.g., dogs or cats). As used herein, the terms "fusion protein" and "insulin-Fc fusion protein" refer to a protein comprising more than one part, such as from different sources (different proteins, polypeptides, cells, etc.), which are covalently linked by peptide bonds. Insulin-Fc fusion protein is covalently linked by the following: (i) the gene encoding each part is linked to a single nucleic acid molecule, and (ii) the following protein encoded by the nucleic acid molecule is expressed in a host cell (e.g., HEK or CHO): (N-terminal)--insulin polypeptide--linker--Fc fragment--(C-terminal). Compared with the method in which insulin polypeptide and Fc fragment are synthesized separately and then chemically conjugated, a completely recombinant synthesis method is preferred. The chemical conjugation step and the subsequent purification process increase manufacturing complexity, reduce product yield, and increase cost.

[0096] As used herein, the term "dimer" refers to a protein or fusion protein comprising two covalently linked polypeptides. In an embodiment, two identical polypeptides are covalently linked (e.g., via a disulfide bond) to form a "homodimer" (in Figure 1 ). The disulfide bonds are shown schematically in Figure 1 In fact, the total number of disulfide bonds can be greater or less than Figure 1 In an embodiment, the homodimer is encoded by a single nucleic acid molecule, wherein the homodimer is produced recombinantly in a cell by first forming an insulin-Fc fusion protein monomer, and then assembling two identical insulin-Fc fusion protein monomers into the homodimer upon further processing in the cell.

[0097] As used herein, the term "multimer", "multimeric" or "multimeric state" refers to a non-covalent, associated form of Fc fusion protein dimers that can be in equilibrium with Fc fusion protein dimers or can serve as permanent aggregate forms of Fc fusion protein dimers (e.g., dimers of Fc fusion protein homodimers, trimers of Fc fusion protein homodimers, tetramers of Fc fusion protein homodimers, or higher order aggregates containing five or more Fc fusion protein homodimers). It can be expected that Fc fusion proteins in multimeric form may have different physical, stability or pharmacological activities from insulin-Fc fusion protein homodimers.

[0098] Insulin peptide

[0099] Insulin polypeptides can be, for example, insulin or insulin analogs produced by beta cells in the islets of Langerhans in the pancreas. Insulin works by regulating the absorption of glucose in the blood. After stimulation (such as increased protein and glucose levels), insulin is released from beta cells and binds to insulin receptors (IR), initiating signal cascades that affect many aspects of mammalian (e.g., human, canine or feline) metabolism. The interruption of this process is directly related to a variety of diseases, particularly diabetes, insulinoma, insulin resistance, metabolic syndrome and polycystic ovary syndrome. The insulin analogs disclosed herein can be related to the structure of insulin, but include one or more modifications. In some embodiments, insulin analogs include at least one amino acid substitution, deletion, addition or chemical modification relative to insulin, which can affect specific features or properties of insulin-Fc fusion proteins. For example, relative to a reference standard, the modifications or changes described herein may affect the structure, stability, pH sensitivity, biological activity or binding affinity of insulin-Fc fusion proteins to cell surface receptors (e.g., insulin hormone receptors).

[0100] The amino acid sequence of insulin is highly conserved throughout evolution, particularly among vertebrates. For example, natural canine insulin differs from human insulin by only one amino acid, and natural feline insulin differs from human insulin by only four amino acids. As used herein, the terms "B chain," "C peptide," or "C chain," and "A chain" refer to the amino acid sequence of insulin. Figure 1 1 and 2. Insulin is a 51 amino acid hormone containing two peptide chains (i.e., a B chain and an A chain) connected by a disulfide bond (e.g., a disulfide bond formed by one or more B chain cysteine ​​side chain thiols and one or more A chain cysteine ​​side chain thiols). The length of the A chain of insulin is 21 amino acids, and the length of the B chain of insulin is 30 amino acids. In the native form of insulin, the A chain contains an intrachain disulfide bond formed by two A chain cysteine ​​side chain thiols. For reference purposes, the sequences of the human insulin A chain of SEQ ID NO: 1 and the human insulin B chain of SEQ ID NO: 2 are shown below:

[0101] FVNQHLCGSHLVEALYLVCGERGFFYTPKT(SEQ ID NO:1)

[0102] GIVEQCCTSICSLYQLENYCN(SEQ ID NO:2)

[0103] As used herein, the term "insulin" or "insulin polypeptide" includes mature insulin, proinsulin, proinsulin and naturally occurring insulin or its analogs. In an embodiment, the insulin polypeptide can be a full-length insulin polypeptide or a fragment thereof. In an embodiment, the insulin polypeptide can include one or more fragments from mature insulin, proinsulin, proinsulin or naturally occurring insulin.

[0104] Insulin is usually constructed as an N-terminal-B chain: C chain: A chain-C-terminal polypeptide, wherein the C chain is cleaved to make it biologically active. For reference purposes, the sequence of the entire human insulin molecule (i.e., human proinsulin) including the C chain is shown below, wherein the C chain is underlined:

[0105] FVNQHLCGSHLVEALYLVCGERGFFYTPKT RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR GIVEQCCTSICSLYQLENYCN(SEQ ID NO:3)

[0106] Prior to glucose-stimulated insulin secretion, the conversion of single-chain insulin polypeptides to biologically active two-chain polypeptides is usually completed in the β cells of the islets of Langerhans by two endoproteases, i.e., type I endoproteases PC1 and PC3 that break the C-peptide-B chain connection and type II endoproteases PC2 that cleave the C-peptide-A chain bond just at the right site. However, cell systems (e.g., bacteria, yeast, and mammalian (e.g., HEK and CHO) cell systems) for biosynthesis of therapeutic molecules such as insulin do not have this pathway, and therefore the conversion must be performed after the single-chain polypeptide is expressed and harvested using chemical or enzymatic methods. All known techniques for cutting the C chain after expression and harvesting rely on first modifying the C chain so that it terminates at lysine just before the N-terminus of the A chain. Then, using an enzyme selected from the trypsin or Lys-C family (which specifically cleaves peptide bonds at the C-terminal end of lysine residues), the single-chain insulin polypeptide is cut at the C-terminal lysine of the C chain and at the C-terminal lysine at position 29 of the N-terminal end of the B chain. In some cases, the resulting biologically active two-chain insulin is used without reconnecting the sheared amino acid at the 30th position of the N-terminal of the B chain, and in some cases, an additional enzymatic method is used to add the sheared amino acid at the 30th position of the N-terminal of the B chain back into the molecule. Such a process is very effective for insulin because it contains only one lysine in its entire two-chain polypeptide form. However, this method cannot be used for the insulin-Fc fusion protein contained in this article because all known Fc fragments contain multiple lysine residues. Therefore, the enzymatic process digests the Fc fragment into a non-functional part, thereby eliminating the ability of the Fc fragment to prolong the effect of the insulin polypeptide in vivo. Therefore, the insulin-Fc fusion protein of the present invention must include an insulin polypeptide that does not need to cut the C chain, and therefore its single-chain form is biologically active.

[0107] A variety of biologically active single-chain insulin polypeptides have been described in the art. In all cases, the single-chain insulin polypeptide contains a C chain of a specific length and composition and an A chain and a B chain mutated at a specific amino acid site, so as to achieve electrostatic balance, prevent aggregation, enhance insulin receptor (IR) binding and / or downstream signaling, to achieve biological activity at a level comparable to that of natural two-chain insulin. In this article, the position of the mutation on the peptide segment is marked using the name of the segment (e.g., B chain, C chain, A chain) and the number of amino acids counted from the N-terminal of the fragment. For example, the symbol "B16" refers to the 16th amino acid at the N-terminal of the amino acid sequence of the B chain. The symbol "A8" refers to the 8th amino acid at the N-terminal of the A chain. In addition, if an amino acid mutates into a new amino acid from its native form at a specific position, the position is accompanied by a letter amino acid code for the new amino acid. For example, B16A refers to an alanine mutation at the 16th amino acid at the N-terminal of the amino acid sequence of the B chain, and A8H refers to a histidine mutation at the 8th amino acid at the N-terminal of the amino acid sequence of the A chain.

[0108] In one example, a single-chain insulin analog (having a C chain of the sequence GGGPRR and additional substitutions in the A chain and the B chain (SEQ ID NO: 4)) was developed by The Department of Biochemistry, Case Western Reserve University School of Medicine and the Department of Medicine, University of Chicago (see Hua, Q.-x, Nakagawa, SH, Jia, W., Huang, K., Phillips, NB, Hu, S.-q., Weiss, MA, (2008) J. Biol. Chem Vol. 283, No. 21 pp 14703-14716). In this example, at position 8 (i.e., A8) of the A chain, threonine is substituted with histidine; at position 10 (i.e., B10) of the B chain, histidine is substituted with aspartic acid; at position 28 (i.e., B28) of the B chain, proline is substituted with aspartic acid; and at position 29 (i.e., B29) of the B chain, lysine is substituted with proline. SEQ ID NO:4 is set forth below, with each unnatural amino acid underlined:

[0109] FVNQH D LVEALYLVCGERGFFYT DP T GGGPRR GIVEQCC HSICSLYQLENYCN (SEQ ID NO: 4)

[0110] In an embodiment, the tyrosine at position 16 (i.e., B16) from the N-terminus of the B chain in SEQ ID NO: 4 can be substituted with alanine to generate SEQ ID NO: 5, because alanine substitutions in this position are known to be less able to activate insulin-specific T cells (Alleva, DG, Gaur, A., Jin, L., Wegmann, D., Gottlieb, PA, Pahuja, A., Johnson, EB, Motheral, T., Putnam, A., Crowe, PD, Ling, N., Boehme, SA, Conlon, PJ, (2002) Diabetes Vol. 51, No. 7 pp 2126-2134). SEQ ID NO: 5 is listed below, with each unnatural amino acid underlined:

[0111] FVNQH D LVEAL A LVCGERGFFYT DP T GGGPRR GIVEQCC H SICSLYQLENYCN (SEQ ID NO: 5)

[0112] In some embodiments, it was unexpectedly found that specific amino acids in SEQ ID NO: 4 and SEQ ID NO: 5 resulted in the production of neutralizing anti-drug antibodies after repeated subcutaneous injections in target animals (e.g., dogs or cats). After multiple injections, the anti-drug antibodies resulted in an unacceptable decrease in NAOC (e.g., a NAOCR value of less than 0.5 after the third injection), thereby inactivating the associated insulin-Fc fusion protein. Specifically, it was found in the steps leading to the invention disclosed herein that the A8 mutation of histidine and the B10 mutation of aspartic acid accounted for the vast majority of the anti-drug antibody specificity, and therefore represented immunogenic "hot spots" (e.g., immunogenic epitopes) on the insulin-polypeptide. Therefore, in preferred embodiments, the insulin-polypeptide does not contain histidine at position A8, or does not contain aspartic acid at position B10 of the insulin polypeptide.

[0113] In one embodiment, it was demonstrated that simply keeping the A8 and B10 amino acids as their native threonine and histidine, respectively, did eliminate the anti-drug antibody response, but the resulting insulin-Fc fusion protein was not biologically active in the target species (e.g., NAOC was less than 150% FBGL·day·kg / mg). Therefore, it was necessary to test various A chain, B chain, and C chain variants to find a suitable solution. Most variants failed to achieve homodimer titers greater than 50 mg / L, and many variants that did meet these goals did not achieve acceptable levels of biological activity in the target species (e.g., acceptable NAOC values ​​greater than 150% FBGL·day·kg / mg). After screening more than 120 variants, the following insulin polypeptide of SEQ ID NO:6_NULL was considered suitable for achieving a homodimer titer greater than 50 mg / L, a NAOC value in the target species greater than 150% FBGL·day·kg / mg, minimal immunogenicity, and a NAOCR value greater than 0.5 after the third injection of the relevant insulin-Fc fusion protein in the target species (unnatural amino acids are underlined and missing natural amino acids are represented by underlined Z):

[0114] FVNQH X 1 LVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYCX 3 (SEQID NO:6_NULL)

[0115] Where X 1 Not D, X 2 Not H, and X 3 Does not exist or is N.

[0116] In certain embodiments, in SEQ ID NO:6_NULL, X 1 It is H, X 2 is T, and X 3 is absent or is N, resulting in the following SEQ ID NO:7_NULL (wherein the unnatural amino acids are underlined and the missing natural amino acid is represented by an underlined Z):

[0117] FVNQHLCGSHLVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYCX 3 (SEQID NO:7_NULL)

[0118] Where X 3 Does not exist or is N.

[0119] In a specific embodiment, in SEQ ID NO:7_NULL, X 3 is absent, resulting in the following SEQ ID NO:8_NULL (wherein the non-natural amino acids are underlined and the missing natural amino acids are represented by an underlined Z): FVNQHLCGSHLVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYCZ (SEQ ID NO:8_NULL)

[0120] In a specific embodiment, in SEQ ID NO:7_NULL, X 3 is N, resulting in the following SEQ ID NO:9_NULL (wherein the non-natural amino acids are underlined and the missing natural amino acids are represented by an underlined Z): FVNQHLCGSHLVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYCN (SEQ ID NO:9_NULL)

[0121] In some embodiments, the Fc fragment is mutated to prevent glycosylation during synthesis and potentially reduce the immunogenicity of the resulting insulin-Fc fusion protein in a target animal (e.g., dog or cat). Unexpectedly, it was found that there was an interaction between the insulin polypeptide and the mutated Fc fragment, so that another amino acid mutation was required on the insulin polypeptide so that the insulin-Fc fusion protein was sufficiently manufacturable (e.g., with a homodimer titer greater than 50 mg / L) and non-immunogenic, wherein the NAOC value in the target species was greater than 150% FBGL·day·kg / mg, and the NAOCR value after the third injection in the target species was greater than 0.5. Specifically, it was found that when the B16 amino acid was linked to a specific mutated non-glycosylated Fc fragment, the B16 amino acid needed to be mutated to an alanine on the insulin polypeptide, thereby producing the following insulin polypeptide SEQ ID NO: 10_NULL (wherein the non-natural amino acid was underlined and the missing natural amino acid was represented by an underlined Z):

[0122] FVNQH X 1 LVEALA LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYC Z (SEQID NO:10_NULL)

[0123] Where X 1 Not D, and X 2 Not H.

[0124] In a specific embodiment, in SEQ ID NO:10_NULL, X 1 is H, and X 2 is T, resulting in the following SEQ ID NO: 11_NULL (wherein the unnatural amino acids are underlined and the missing natural amino acids are represented by an underlined Z):

[0125] FVNQHLCGSHLVEAL A LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYC Z –SEQ ID NO:11_NULL

[0126] The following is a restatement of the above sequence, but with the missing amino acid having the symbol Z removed from the notation for the insulin polypeptide sequence. Again, in all cases, the unnatural amino acid is underlined. To avoid confusion, each original sequence containing the Z symbol is listed above the new sequence with the Z symbol removed. Despite having two separate notations, the paired sequences refer to exactly the same insulin polypeptide.

[0127] SEQ ID NO:6_NULL is restated as:

[0128] FVNQH X 1 LVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYC X 3 (SEQ ID NO:6)

[0129] Where X 1 Not D, X 2 Not H, and X 3 Does not exist or is N.

[0130] SEQ ID NO:7_NULL is restated as:

[0131] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYC X 3 (SEQ ID NO:7)

[0132] Where X 3 Does not exist or is N.

[0133] SEQ ID NO:8_NULL is restated as:

[0134] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYC (SEQ ID NO:8)

[0135] SEQ ID NO:9_NULL is restated as:

[0136] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYCN (SEQ ID NO:9)

[0137] SEQ ID NO:10_NULL is restated as:

[0138] FVNQH X 1 LVEAL A LVCGERGF H Y GGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYC (SEQ ID NO: 10)

[0139] Where X 1 No, and X 2 Not H.

[0140] SEQ ID NO:11_NULL is restated as:

[0141] FVNQHLCGSHLVEALA LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYC (SEQ ID NO: 11)

[0142] Connectors

[0143] The successful construction of the recombinantly prepared insulin-Fc fusion protein requires the insulin polypeptide to be connected to the linker of the Fc fragment. In an embodiment, the insulin-Fc fusion protein described herein comprises a peptide linker between the insulin polypeptide and the Fc fragment comprising amino acids (e.g., natural amino acids or non-natural amino acids). In an embodiment, the peptide linker can be encoded by a nucleic acid molecule, such as so that a single nucleic acid molecule can encode various peptides in the insulin polypeptide as well as peptide linkers and Fc fragments. The selection of peptide linkers (e.g., length, composition, hydrophobicity, and secondary structure) may affect the manufacturability (i.e., homodimer titer), chemical and enzymatic stability, biological activity (i.e., NAOC value) and immunogenicity of the insulin-Fc fusion protein (Chen, X., Zaro, J., Shen, WC, Adv Drug Deliv Rev. 2013 October 15; 65 (10): 1357-1369). Table 1 lists several linkers for designing insulin-Fc fusion proteins, with the purpose of improving homodimer titer and biological activity.

[0144] Table 1: Peptide linker between A chain and Fc fragment in insulin-Fc fusion protein GGGGAGGGG GGGGSGGGG GGGGGAGGGG GGGGSGGGGSGGGGSGGGG GGGGKGGGGKGGGGKGGGG GGGGGQGGGGQGGGGQGGGGG GGGGGAGGGGAGGGGAGGGGG SGGGGQGGGGQGGGGQGGGGG HGGGGQGGGGQGGGGQGGGGG PGGGGGQGGGGQGGGGQGGGGG

[0145] In an embodiment, the peptide linker comprises the sequence:

[0146] GGGGAGGGG (SEQ ID NO: 12).

[0147] In other embodiments, the peptide linker comprises the sequence:

[0148] GGGGSGGGG (SEQ ID NO: 13).

[0149] In a preferred embodiment, the peptide linker comprises the following sequence:

[0150] GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

[0151] When constructing a recombinantly prepared insulin-Fc fusion protein with a peptide linker (such as the linker in SEQ ID NO: 14), it is necessary to pay attention to the possibility of undesirable enzymatic cleavage between the C-terminus of the insulin A chain and the N-terminus of the peptide linker. Cutting the linker and Fc fragment from the insulin polypeptide will prevent the insulin-Fc fusion protein from providing an extended duration of biological activity. There are known enzymatic cleavage sites between asparagine-glycine bonds (Vlasak, J., Ionescu, R., (2011) MAbs Vol. 3, No. 3 pp 253-263). In the embodiment of multiple peptide linkers, including the preferred peptide linker of SEQ ID NO: 14, the N-terminal amino acid is glycine. In addition, the C-terminus of the insulin A chain (i.e., the 21st amino acid (i.e., A21) from the N-terminus of the A chain) is asparagine. Therefore, the A21 asparagine is omitted in the insulin polypeptides of SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11 to eliminate the enzymatically cleavable asparagine-glycine bond that may be formed between the A chain and the peptide linker. Unexpectedly, the insulin-Fc fusion protein constructed from the insulin polypeptide of SEQ ID NO: 9 (which retains the asparagine at the C-terminus of the A chain) exhibits manufacturability in mammalian cells, with acceptable homodimer titers (i.e., homodimer titers greater than 50 mg / L), acceptable in vivo bioactivity (i.e., NAOC greater than 150% FBGL·day·kg / mg in target animals) and sustained levels of bioactivity after multiple doses (i.e., NAOCR values ​​greater than 0.5 after the third injection in target animals). The results indicate that, contrary to expectations based on previous teachings, there is no risk of enzymatic cleavage or inactivation of insulin-Fc fusion proteins containing an asparagine-glycine linkage between the insulin polypeptide and the peptide linker, at least for insulin-Fc fusion proteins comprising the Fc fragment sequence disclosed herein.

[0152] Fc fragment

[0153] The terms "Fc fragment", "Fc region", "Fc domain" or "Fc polypeptide" are used herein to define the C-terminal region of an immunoglobulin heavy chain. The Fc fragment, region, domain or polypeptide may be a native sequence Fc region or a variant / mutant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, they generally include some or all of the hinge region of the heavy chain, the CH2 region of the heavy chain, and the CH3 region of the heavy chain. The hinge region of a canine or feline Fc fragment includes an amino acid sequence that connects the CH1 domain of the heavy chain to the CH2 region of the heavy chain, and includes one or more cysteines that form one or more inter-heavy chain disulfide bonds to form a homodimer of an Fc fusion protein from two identical but separate monomers of the Fc fusion protein. The hinge region may include all or part of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence.

[0154] Fc receptor (FcR) refers to a receptor that binds to the Fc region of an Fc fragment or antibody. In an embodiment, FcR is a native sequence of a canine or feline FcR. In an embodiment, FcR is an FcR that binds to the Fc fragment or Fc region of an IgG antibody (γ receptor), and includes but is not limited to receptors of Fc (γ) receptor I, Fc (γ) receptor IIa, Fc (γ) receptor IIb, and Fc (γ) receptor III subclasses, including allelic variants of these receptors and alternative splicing forms. "FcR" also includes neonatal receptor FcRn, which is responsible for transferring maternal IgG molecules to the fetus (Guyer et al., 1976 J. Immunol., 117: 587; and Kim et al., 1994, J. Immunol., 24: 249), and is also responsible for extending the in vivo elimination half-life of antibodies and Fc-fusion proteins in vivo. In an embodiment, a human-derived FcR is used in vitro (e.g., in an assay) to measure the binding of an insulin-Fc fusion protein containing a canine or feline Fc fragment to assess their FcR binding properties. It will be appreciated by those skilled in the art that a mammalian FcR from one species (e.g., a human-derived FcR) is sometimes able to bind in vitro to an Fc fragment from a second species (e.g., a canine or feline FcR). In an embodiment, a canine-derived FcR is used in vitro (e.g., in an assay) to measure the binding of an insulin-Fc fusion protein containing a canine or feline Fc fragment to assess their FcR binding properties. It will be appreciated by those skilled in the art that a mammalian FcR from one species (e.g., a canine-derived FcR) is able to bind in vitro to an insulin-Fc fusion protein containing an Fc fragment from the same species (e.g., canine-derived), and sometimes also to an insulin-Fc fusion protein containing an Fc fragment from another mammalian species (e.g., feline-derived).

[0155] In an embodiment, the Fc fragment comprises the Fc region (e.g., hinge region, CH2 domain, and CH3 domain) of a mammalian IgG, such as a canine IgGA Fc fragment (SEQ ID NO: 15), a canine IgGB Fc fragment (SEQ ID NO: 16), a canine IgGC Fc fragment (SEQ ID NO: 17), or a canine IgGD Fc fragment (SEQ ID NO: 18), or a feline IgG1a fragment (SEQ ID NO: 19), a feline IgG1b Fc fragment (SEQ ID NO: 20), or a feline IgG2 Fc fragment (SEQ ID NO: 21). In the embodiments, the C-terminal lysine (i.e., the lysine representing the last amino acid of the Fc fragment sequence) that is typically found in the amino acid sequence of a native canine or feline IgG isotype Fc fragment is omitted to prevent the accidental generation of unwanted amino acid sequence variants during production (e.g., Fc fragments containing the C-terminal lysine become mixed with Fc fragments in which the C-terminal lysine is omitted, which may occur during the production of a desired protein in a cell (Dick, LW., (2008) Biotechnol Bioeng. Aug 15; 100 (6) pp1132-43). Therefore, in the embodiments, the canine or feline Fc fragment sequence lacking the C-terminal lysine is:

[0156] RCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTIS KARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG(SEQ ID NO:15)

[0157] DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:16)

[0158] CNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG(SEQ ID NO:17)

[0159] CISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG(SEQ ID NO:18)

[0160] DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:19)

[0161] DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:20)

[0162] GEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:21)

[0163] Since the IgGA isotype lacks Fc (γ) effector function in dogs (very similar to the IgG2 isotype in humans), it is preferred to replace human Fc with canine IgGA to minimize any unwanted immunogenicity in dogs. However, in the embodiment containing the insulin polypeptide of SEQ ID NO: 5 and the peptide linker of SEQ ID NO: 12, it was unexpectedly found that the insulin-Fc fusion protein containing the canine IgGA fragment (SEQ ID NO: 15) was highly aggregated with low titers of the desired homodimer (i.e., the homodimer titer was less than 50 mg / L). In addition, the compound was not biologically active in dogs (i.e., NAOC values ​​were less than 150% FBGL·day·kg / mg), presumably due to its high aggregation level (e.g., low homodimer %). Despite mutations to the insulin polypeptide of SEQ ID NO: 5, the canine IgGA Fc fragment (SEQ ID NO: 15) and / or the linker, there is no embodiment based on the canine IgGA Fc fragment with sufficiently low aggregation and sufficiently high titer of the desired homodimer. In another aspect, the replacement of the canine IgGA Fc fragment (SEQ ID NO: 15) with the canine IgGB Fc fragment (SEQ ID NO: 16) resulted in a compound with much lower aggregation, which had a relatively high desired homodimer titer. In addition, the compound containing the insulin polypeptide of SEQ ID NO: 5 and the canine IgGB Fc fragment (SEQ ID NO: 16) was biologically active in dogs and exhibited glucose-lowering biological activity (i.e., NAOC values ​​greater than 150% FBGL·day·kg / mg) over multiple days.

[0164] In the examples containing the insulin polypeptide of SEQ ID NO: 8 and the peptide linker of SEQ ID NO: 14 (both of which are significantly different from the insulin polypeptide of SEQ ID NO: 5 and the peptide linker of SEQ ID NO: 12), it was demonstrated that the canine IgGB Fc fragment is superior to the canine IgGA Fc fragment. The insulin-Fc fusion protein containing the insulin polypeptide of SEQ ID NO: 8 and the peptide linker of SEQ ID NO: 14 was synthesized using the Fc fragment from the canine IgGA (SEQ ID NO: 15), canine IgGB (SEQ ID NO: 16), canine IgGC (SEQ ID NO: 17) or canine IgGD (SEQ ID NO: 18) immunoglobulin. Using conventional purification methods, only the compounds containing canine IgGA and canine IgGB showed any appreciable protein yield. However, as before, the canine IgGA version of the compound was highly aggregated and had low levels of bioactivity, while the canine IgGB version of the compound exhibited low aggregation (i.e., high homodimer %), high titers of the desired homodimer (i.e., homodimer titers greater than 50 mg / L), and appreciable levels of long-term glucose-lowering bioactivity in dogs (i.e., NAOC values ​​greater than 150% FBGL·day·kg / mg). Using an alternative purification method, a canine IgGC version of the compound was recovered with low aggregation, but it had the lowest bioactivity in dogs (i.e., NAOC values ​​less than 150% FBGL·day·kg / mg), presumably due to its low affinity for the FcRn receptor. Therefore, with respect to dog-specific products, canine IgGB (SEQ ID NO: 16) is the preferred Fc fragment of all insulin-Fc fusion proteins used in dogs, regardless of the insulin polypeptide selected.

[0165] Since the IgG2 isotype lacks Fc(γ) effector function in cats (very similar to the IgG2 isotype in humans), it is preferred to replace human Fc with feline IgG2 to minimize any unwanted immunogenicity in cats. Unlike the case of dogs, in the examples containing the insulin polypeptide of SEQ ID NO: 4, it was found that the insulin-Fc fusion protein comprising a feline IgG2 fragment (SEQ ID NO: 21) and a feline IgG1b fragment (SEQ ID NO: 20) was similarly highly produced, with low aggregation (i.e., homodimer titer greater than 50 mg / L) and appreciable insulin receptor affinity (i.e., insulin receptor IC50 value less than 5000 nM). However, unexpectedly, when the insulin polypeptide was changed to SEQ ID NO: 7, the insulin-Fc fusion protein containing the feline IgG2 fragment (SEQ ID NO: 21) had no biological activity in cats (i.e., NAOC was less than 150% FBGL·day·kg / mg), while the insulin-Fc fusion protein containing the feline IgG1b fragment (SEQ ID NO: 20) exhibited low aggregation (i.e., high homodimer %), high titer of the desired homodimer (i.e., homodimer titer greater than 50 mg / L), and long-term glucose-lowering biological activity at appreciable levels in cats (i.e., NAOC value greater than 150% FBGL·day·kg / mg). Therefore, for cat-specific products, when the insulin polypeptide sequence comprises SEQ ID NO: 7, the feline IgG1b fragment (SEQ ID NO: 20) is a preferred Fc fragment.

[0166] In view of the fact that canine IgGB and feline IgG1b isotypes interact with their respective species-specific Fc (γ) receptors with a higher affinity than their canine IgGA and feline IgG2 isotype counterparts, there may or may not be an unwanted immunogenicity risk after repeated injections. A method for reducing Fc (γ) interactions involves deglycosylation of Fc fragments during synthesis in host cells or preventing their glycosylation. Each IgG fragment contains a conserved asparagine (N)-glycosylation site in the CH2 domain of each heavy chain of the Fc region. In this article, the symbol used to refer to the conserved N-glycosylation site is "cNg". A method for removing attached glycans from synthetic insulin-Fc fusion proteins is to mutate cNg sites to prevent glycans from attaching together during production in host cells. In this article, the symbol used to describe cNg mutations is cNg-(substituted amino acid). For example, if the asparagine at the cNg site is mutated to serine, the mutation is named "cNg-S".

[0167] The absolute position of the cNg site from the N-terminus of the B chain of the insulin-Fc fusion protein varies according to the length of the insulin polypeptide, the length of the linker, and any omitted amino acids in the Fc fragment before the cNg site. In this article, the symbol used to represent the absolute position of the cNg site in a given insulin-Fc fusion protein sequence (as measured by counting from the N-terminus of the B chain of the insulin-Fc fusion protein) is "NB (number)". For example, if the cNg site is found at the 151st amino acid position from the N-terminus of the B chain, the absolute position of the site is referred to as cNg-NB151. As a further example, if the cNg site is found at the 151st amino acid position from the N-terminus of the B chain, and the asparagine at the site is mutated to serine, the mutation is referred to as "cNg-NB151-S".

[0168] In the embodiment containing the insulin polypeptide of SEQ ID NO: 5 and the canine IgGB Fc fragment with cNg-Q, cNg-S, cNg-D and cNg-K mutations, it was unexpectedly found that the compounds containing only the cNg-K and cNg-S mutations exhibited the necessary homodimer titer of greater than 50 mg / L and the lowest Fc(γ)RI binding affinity. In another aspect, in the embodiment containing the insulin polypeptide of SEQ ID NO: 8 and the canine IgGB Fc fragment with the cNg-S mutation, it was unexpectedly found that the resulting compounds were significantly less bioactive in dogs compared to the counterpart containing the native canine IgGB Fc (i.e., the NAOC value was significantly lower for the counterpart containing the native glycosylation site amino acid (e.g., cNg-N)). When the B16 amino acid was mutated to alanine, the biological activity was unexpectedly restored in the cNg-S mutant (i.e., the NAOC value was significantly increased), as described above for the insulin polypeptide SEQ ID NO: 11. In summary, there is an unexpected and significant interaction between the choice of cNg mutation and the composition of the insulin polypeptide, so that experiments are required to identify preferred embodiments. In a specific embodiment, a canine IgGB Fc mutant containing a cNg-S mutation is preferred, and the sequence of the cNg-S with underline is shown below:

[0169] DCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF SGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:22)

[0170] In a specific embodiment, a feline IgG1b Fc mutant containing a cNg-S mutation is preferred:

[0171] DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:23)

[0172] Insulin-Fc fusion protein

[0173] Provided herein is an insulin-Fc fusion protein comprising an insulin polypeptide, an Fc fragment, and a linker between the insulin polypeptide and the Fc fragment. In an embodiment, the insulin polypeptide comprises a domain in the following orientation from the N-terminus to the C-terminus: (N-terminus)--B chain--C chain--A chain--(C-terminus). In an embodiment, the insulin polypeptide is located at the N-terminal side of the Fc fragment. In an embodiment, as Figure 1 As shown, the fusion protein comprises domains in the following orientation from N-terminus to C-terminus: (N-terminus)—insulin polypeptide—linker—Fc fragment—(C-terminus) (e.g., (N-terminus)—B chain—C chain—A chain—linker—Fc fragment—(C-terminus)).

[0174] In a preferred embodiment, the preferred non-immunogenic, biologically active insulin polypeptide of SEQ ID NO: 6 is combined with the preferred canine IgGB Fc fragment of SEQ ID NO: 16 using the preferred linker of SEQ ID NO: 14 to produce a family of high homodimer titer-producing, non-aggregating, biologically active, non-immunogenic insulin-Fc fusion proteins of SEQ ID NO: 24, which exhibit homodimer titers greater than 50 mg / L, NAOC values ​​greater than 150% FBGL·day·kg / mg in dogs, and NAOCR values ​​greater than 0.5 after the third injection in a series of repeated injections in dogs. SEQ ID NO: 24 is shown below, with the unnatural amino acids underlined:

[0175] FVNQH X 1 LVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYC X 3 GGGGGQGGGGQGGGGQGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKA LPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:24)

[0176] Where X 1 Not D, X 2 Not H, and X 3 Does not exist or is N.

[0177] In a preferred embodiment comprising SEQ ID NO: 24, X 1 It is H, X 2 is T, and X 3Absent or N. The selection produces a high homodimer titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ ID NO: 25, which exhibits a homodimer titer greater than 50 mg / L, a NAOC value greater than 150% FBGL·day·kg / mg in dogs, and a NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. SEQ ID NO: 25 is shown below, wherein the non-natural amino acids are underlined:

[0178] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYC X 3 GGGGGQGGGGQGGGGQGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKA LPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:25)

[0179] X 3 Does not exist or is N.

[0180] In a preferred embodiment, in SEQ ID NO: 25, X 3 Absence, to produce a highly homodimer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ ID NO: 32, which exhibits a homodimer titer of greater than 50 mg / L, a NAOC value of greater than 150% FBGL·day·kg / mg in dogs, and a NAOCR value of greater than 0.5 after the third injection in a series of repeated injections in dogs. SEQ ID NO: 32 is shown below, wherein the unnatural amino acids are underlined:

[0181] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL DQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVN NKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:32)

[0182] In a preferred embodiment, in SEQ ID NO: 25, X 3 =N, to produce a high homodimer titer of SEQ ID NO: 34, a non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein, which exhibits a homodimer titer of greater than 50 mg / L, a NAOC value of greater than 150% FBGL·day·kg / mg in dogs, and a NAOCR value of greater than 0.5 after the third injection in a series of repeated injections in dogs. SEQ ID NO: 34 is shown below, wherein the non-natural amino acids are underlined:

[0183] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVN NKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:34)

[0184] In a preferred embodiment, the preferred non-glycosylated cNg-S mutant canine IgGB Fc fragment of SEQ ID NO:22 is combined with the preferred B16A mutant insulin polypeptide sequence of SEQ ID NO:10 using the preferred linker of SEQ ID NO:14 to produce a family of high homodimer titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion proteins of SEQ ID NO:26, which exhibit homodimer titers greater than 50 mg / L, NAOC values ​​greater than 150% FBGL·day·kg / mg in dogs, and NAOCR values ​​greater than 0.5 after the third injection of a series of repeated injections in dogs. SEQ ID NO:26 is shown below, wherein the non-natural amino acids are underlined:

[0185] FVNQH X 1 LVEAL A LVCGERGF H Y GGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:26)

[0186] Where X 1 Not D, and X 2 Not H.

[0187] In a preferred embodiment, in SEQ ID NO: 26, X 1 is H, and X 2is T, to produce a high homodimer titer of SEQ ID NO: 36, a non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein, which exhibits a homodimer titer of greater than 50 mg / L, a NAOC value of greater than 150% FBGL·day·kg / mg in dogs, and a NAOCR value of greater than 0.5 after the third injection in a series of repeated injections in dogs. SEQ ID NO: 36 is shown below, wherein the non-natural amino acids are underlined:

[0188] FVNQHLCGSHLVEAL A LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:36)

[0189] In a preferred embodiment, the preferred non-immunogenic, biologically active insulin polypeptide of SEQ ID NO: 6 (wherein X 3 10 (absent) is combined with the preferred feline IgG1b Fc fragment of SEQ ID NO: 20 to produce a family of high homodimer titer-producing, non-aggregating, biologically active, non-immunogenic insulin-Fc fusion proteins of SEQ ID NO: 27, which exhibit homodimer titers greater than 50 mg / L, NAOC values ​​greater than 150% FBGL·day·kg / mg in cats, and NAOCR values ​​greater than 0.5 after the third injection in a series of repeated injections in cats. SEQ ID NO: 27 is shown below, wherein the unnatural amino acids are underlined:

[0190] FVNQH X 1 LVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCX 2 S T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVN SKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:27)

[0191] Where X 1 Not D, and X 2 Not H.

[0192] In a preferred embodiment, in SEQ ID NO: 27, X1 is H and X2 is T to produce a high homodimer titer produced, non-aggregated, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ ID NO: 38, which exhibits a homodimer titer greater than 50 mg / L, a NAOC value greater than 150% FBGL·day·kg / mg in cats, and a NAOCR value greater than 0.5 after the third injection in a series of repeated injections in cats. SEQ ID NO: 38 is shown below, wherein the non-natural amino acids are underlined:

[0193] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVN SKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:38)

[0194] In a preferred embodiment, the preferred non-glycosylated cNg-S mutant feline IgG1b Fc fragment of SEQ ID NO:23 is combined with the preferred B16A mutant insulin polypeptide sequence of SEQ ID NO:10 using the preferred linker of SEQ ID NO:14 to produce a family of high homodimer titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion proteins of SEQ ID NO:28, which exhibit homodimer titers greater than 50 mg / L, NAOC values ​​greater than 150% FBGL·day·kg / mg in cats, and NAOCR values ​​greater than 0.5 after the third injection in a series of repeated injections in cats. SEQ ID NO:28 is shown below, wherein the non-natural amino acids are underlined:

[0195] FVNQH X 1 LVEAL A LVCGERGF H Y GGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:28)

[0196] Where X 1 Not D, and X 2 Not H.

[0197] In a preferred embodiment, in SEQ ID NO: 28, X 1 is H, and X 2is T, to produce a high homodimer titer of SEQ ID NO: 40, a non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein, which exhibits a homodimer titer of greater than 50 mg / L, a NAOC value of greater than 150% FBGL·day·kg / mg in cats, and a NAOCR value of greater than 0.5 after the third injection in a series of repeated injections in cats. SEQ ID NO: 40 is shown below, wherein the non-natural amino acids are underlined:

[0198] FVNQHLCGSHLVEAL A LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:40)

[0199] In some embodiments, the insulin-Fc fusion proteins described herein do not include a leader amino acid sequence at the N-terminus.

[0200] In other embodiments, the insulin-Fc fusion protein described herein includes a leader sequence, for example, at the N-terminus. An exemplary leader sequence includes the amino acid sequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 30). In some embodiments, the insulin-Fc fusion protein described herein is encoded by a nucleic acid molecule comprising a leader sequence, for example, for expression (e.g., recombinant expression) in a cell (e.g., a eukaryotic cell, e.g., a mammalian cell). In certain embodiments, for example, in cell culture, the leader sequence is cut off during expression. Exemplary nucleic acid sequences encoding the leader sequence include the following nucleic acid sequences:

[0201] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcc (SEQ ID NO: 29).

[0202] Also disclosed herein are nucleic acid sequences (eg, cDNAs) encoding the insulin-Fc fusion proteins of SEQ ID NOs: 032, 034, 036, 038, and 040.

[0203] In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO:32, the nucleic acid sequence (leader sequence is underlined) is:

[0204] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:31).

[0205] In the example of the insulin-Fc fusion protein comprising SEQ ID NO:34, the nucleic acid sequence (the leader sequence is underlined) is:

[0206] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcc ttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcaacggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:33)。

[0207] In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO:36, the nucleic acid sequence (leader sequence is underlined) is:

[0208] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:35).

[0209] In the example of the insulin-Fc fusion protein comprising SEQ ID NO:38, the nucleic acid sequence (leader sequence underlined) is:

[0210] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcc ttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcaacagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag(SEQ ID NO:37)。

[0211] In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO:40, the nucleic acid sequence (leader sequence is underlined) is:

[0212] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcagcagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag(SEQ ID NO:39).

[0213] Production of insulin-Fc fusion protein

[0214] In embodiments, the fusion protein can be expressed by a cell, as described in more detail in the Examples section.

[0215] Expression and purification

[0216] In an embodiment, insulin-Fc fusion protein can be, for example, recombinantly expressed in eukaryotic cells (e.g., mammalian cells or non-mammalian cells). Exemplary mammalian cells for expression include HEK cells (e.g., HEK293 cells) or CHO cells. CHO cells can be subdivided into various strains or subclasses (e.g., CHO DG44, CHO-M, and CHO-K1), and some of these cell strains can be genetically engineered to be optimally used for specific types of nucleic acid molecules (e.g., carriers comprising DNA) or specific cell growth medium compositions, as described in the example section. In an embodiment, cells are transfected with nucleic acid molecules (e.g., carriers) encoding insulin-Fc fusion proteins (e.g., wherein the entire insulin-Fc fusion protein is encoded by a single nucleic acid molecule). In an embodiment, HEK293 cells are transfected with a vector encoding insulin-Fc fusion protein, but only cause the insulin-Fc fusion protein to stop expressing a considerable level of insulin-Fc fusion protein (i.e. transient transfection) before the host cell stops expressing a temporary expression of a period of time (e.g., 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days or longer). HEK293 cells transiently transfected with a nucleic acid sequence encoding insulin-Fc fusion protein generally allow for more rapid production of recombinant proteins, which helps to prepare and screen a variety of insulin-Fc fusion protein candidates. In an embodiment, CHO cells are transfected with a vector permanently incorporated into the host cell DNA and causing the consistent and permanent expression (i.e., stable transfection) of insulin-Fc fusion protein, as long as the cells are appropriately cultured. CHO cells and CHO cell lines stably transfected with nucleic acids encoding insulin-Fc fusion protein generally require longer time to develop, but they generally produce higher protein yields, and are more suitable for manufacturing low-cost products (e.g., products for the veterinary drug market). Standard methods in the art can be used to culture cells and cell lines. In a preferred embodiment, HEK cells containing any one of the cDNA sequences of SEQ ID NO: 31, 33, 35, 37 and 39 are used to express insulin-Fc fusion protein. In a preferred embodiment, CHO cells containing any one of the cDNA sequences of SEQ ID NO: 31, 33, 35, 37 and 39 are used to express insulin-Fc fusion protein.

[0217] In certain embodiments, insulin-Fc fusion protein is purified or separated from cells (for example, by lysing cells). In other embodiments, insulin-Fc fusion protein is secreted by cells and purified or separated from the cell culture medium in which cells grow therein. The purification of insulin-Fc fusion protein can include using column chromatography (for example affinity chromatography) or using other separation methods based on the difference of size, charge and / or affinity to certain molecules. In an embodiment, the purification of insulin-Fc fusion protein relates to the protein containing Fc fragment selected or enriched, for example, by using protein A beads or protein A columns, which makes the protein containing Fc fragment become bound to the protein A covalently conjugated to the protein A beads with high affinity at neutral solution pH. Then, the combined insulin-Fc fusion protein can be eluted from the protein A beads by the change of solution variable (for example, the reduction of solution pH). Other separation methods can be used alternatively or additionally, such as ion exchange chromatography and / or gel filtration chromatography. In an embodiment, the purification of insulin-Fc fusion protein further comprises filtering or centrifugal protein preparations. In an embodiment, further purification of the insulin-Fc fusion protein comprises diafiltration, ultrafiltration and filtration through porous membranes of various sizes, and final formulation with excipients.

[0218] Purified insulin-Fc fusion proteins can be characterized using a variety of methods, such as purity, protein yield, structure and / or activity, such as 280nm absorbance (e.g., to determine protein yield), size exclusion or capillary electrophoresis (e.g., to determine molecular weight, aggregation percentage and / or purity), mass spectrometry (MS) and / or liquid chromatography (LC-MS) (e.g., to determine purity and / or glycosylation) and / or ELISA (e.g., to determine the degree of binding to anti-insulin antibodies, such as affinity). Exemplary characterization methods are also described in the Examples section.

[0219] In an embodiment, the protein yield of insulin-Fc fusion protein after production in transiently transfected HEK cells and protein A purification is greater than 5 mg / L, 10 mg / L or 20 mg / L. In a preferred embodiment, the protein yield of insulin-Fc fusion protein after production in transiently transfected HEK cells and protein A purification is greater than 50 mg / L (e.g., greater than 60 mg / L, greater than 70 mg / L, greater than 80 mg / L, greater than 90 mg / L, greater than 100 mg / L). In an embodiment, the homodimer % of insulin-Fc fusion protein after production in transiently transfected HEK cells and protein A purification is greater than 70% (e.g., greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%). In an embodiment, the homodimer titer of insulin-Fc fusion protein after production in transiently transfected HEK cells and protein A purification (which is calculated as the product between the yield of insulin-Fc fusion protein and the homodimer %) is greater than 50 mg / L (e.g., greater than 60 mg / L, greater than 70 mg / L, greater than 80 mg / L, greater than 90 mg / L, greater than 100 mg / L). Only candidates with homodimer titers greater than 50 mg / L are considered useful in the present invention, because experience has shown that homodimer titers below this level will be unlikely to produce commercial production titers in CHO cells that meet the stringent low manufacturing cost requirements for veterinary products.

[0220] In an embodiment, the protein yield of insulin-Fc fusion protein after production in stably transfected CHO cells (e.g., CHO cell line or CHO cell clone) and protein A purification is greater than 100 mg insulin-Fc fusion protein / L (e.g., mg / L culture medium). In a preferred embodiment, the protein yield of insulin-Fc fusion protein after production in stably transfected CHO cells (e.g., CHO cell line or CHO cell clone) and protein A purification is greater than 150 mg insulin-Fc fusion protein / L culture medium (e.g., greater than 200 mg / L, greater than 300 mg / L, greater than 400 mg / L, greater than 500 mg / L, greater than 600 mg / L or greater). In an embodiment, the homodimer % of insulin-Fc fusion protein after production in stably transfected CHO cells (e.g., CHO cell line or CHO cell clone) and protein A purification is greater than 70% (e.g., greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%). In an embodiment, the homodimer titer of insulin-Fc fusion protein after production in stably transfected CHO cells (e.g., CHO cell line or CHO cell clone) and protein A purification (calculated as the product between the insulin-Fc fusion protein yield and the homodimer %) is greater than 150 mg / L (e.g., greater than 200 mg / L, greater than 300 mg / L, greater than 400 mg / L, greater than 500 mg / L, greater than 600 mg / L or greater).

[0221] Functional characteristics of insulin-Fc fusion protein

[0222] Described herein are methods for interacting with an insulin receptor to lower blood glucose in a companion animal (e.g., a dog or cat), wherein the method comprises administering an insulin-Fc fusion protein, such as a fusion protein described herein, to a subject. In some embodiments, the subject has been diagnosed with diabetes (e.g., canine diabetes or feline diabetes).

[0223] In an embodiment, the insulin-Fc fusion protein described herein binds to the insulin receptor with considerable affinity, as measured by IC50 in the 4°C IM-9 insulin receptor binding assay described in Example 7 (e.g., IC50 is less than 5000nM, IC50 is less than 4000nM, IC50 is less than 3000nM, IC50 is less than 2500nM). According to experience, compounds that only exhibit an insulin receptor activity IC50 value of less than 5000nM are considered to be likely to exhibit biological activity in the target species. Generally, higher affinity insulin receptor binding (i.e., lower IC50 values) is preferred. However, it is well known that the clearance of insulin and insulin analogs (e.g., insulin polypeptides described herein) is mainly controlled by binding to the insulin receptor, followed by insulin receptor internalization and degradation in the cell. Therefore, insulin-Fc fusion proteins with too high insulin receptor binding affinity (i.e., too low IC50) may be cleared too quickly from the circulation, resulting in a duration less than the desired duration of glucose-lowering biological activity in the target animal.

[0224] In an embodiment, insulin-Fc fusion protein as described herein can reduce glucose level (for example, blood sugar level) after administration in a subject. In an embodiment, the glucose-lowering activity of insulin-Fc fusion protein is greater than the glucose-lowering activity of insulin reference standard. In certain embodiments, the activity duration of insulin-Fc fusion protein can be measured by fasting blood glucose relative to the reduction (for example, statistically significant reduction) of fasting blood glucose level before administration. In an embodiment, the activity duration of insulin-Fc fusion protein (for example, the time when fasting blood glucose level in the subject is statistically significantly reduced relative to the level before administration) is longer than about 2 hours. In an embodiment, the activity duration of insulin-Fc fusion protein (for example, the time when blood sugar level in the subject is statistically significantly reduced relative to the level before administration) is longer than about 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. In an embodiment, insulin-Fc fusion protein is long-acting (for example, having a long half-life in serum).

[0225] In an embodiment, the serum half-life of insulin-Fc fusion protein in the target animal (e.g., dog or cat) is longer than the serum half-life of insulin reference standard or control formulation. In an embodiment, the serum half-life of insulin-Fc fusion protein (e.g., in the blood of the subject when administered) in the target animal (e.g., dog or cat) is longer than about 2 hours. In an embodiment, the serum half-life of insulin-Fc fusion protein in the target animal (e.g., dog or cat) is about 0.5 days, 1 day, 2 days or 2.5 days. In a preferred embodiment, the serum half-life of insulin-Fc fusion protein in the target animal (e.g., dog or cat) is about 3 days or longer.

[0226] In an embodiment, the combination of potency and duration of biological activity can be quantified by calculating the area on the curve normalized to a given dose of fasting glucose percentage (%FBGL), in mg / kg (NAOC), where the unit is %FBGL·day·kg / mg. In an embodiment, the NAOC of the insulin-Fc fusion protein is greater than 150%FBGL·day·kg / mg (e.g., greater than 200%FBGL·day·kg / mg, greater than 250%FBGL·day·kg / mg or greater). Similarly, based on experience, when the NAOC value is greater than 150%FBGL·day·kg / mg, the dosage requirements in the target species will be low enough to achieve an acceptable treatment cost. In an embodiment, the NAOC of the insulin-Fc fusion protein must be maintained after repeated administration in the target species (i.e., the ratio of the NAOC after the third administration of the insulin-Fc fusion protein to the NAOC after the first administration is greater than 0.50 (e.g., greater than 0.60, greater than 0.70, greater than 0.80, greater than 0.90 or greater)).

[0227] In some embodiments, the insulin-Fc fusion proteins described herein bind to the Fc(γ) receptor with a lower affinity than a reference standard of insulin-Fc fusion proteins, as measured according to Example 8. In some embodiments, the ratio of the Fc(γ) receptor affinity of the insulin-Fc fusion protein to the Fc(γ) receptor affinity of the reference standard of insulin-Fc fusion proteins is less than 0.50 (e.g., less than 0.40, less than 0.30, less than 0.20).

[0228] Treatment methods and characteristics of subject selection

[0229] Described herein are methods for treating diabetes (eg, canine diabetes or feline diabetes) comprising administering to a subject an insulin-Fc fusion protein (eg, an insulin-Fc fusion protein described herein).

[0230] In embodiments, the reference standard used in any of the methods described herein comprises a reference treatment or a reference therapy. In some embodiments, the reference comprises a standard for a care agent for the treatment of diabetes (e.g., a standard for a care agent for canine diabetes or a standard for a care agent for feline diabetes). In some embodiments, the reference standard is commercially available insulin or insulin analogs. In some embodiments, the reference standard comprises long-acting insulin, intermediate-acting insulin, short-acting insulin, rapid-acting insulin, short-acting insulin, intermediate-acting insulin, long-acting insulin. In some embodiments, the reference standard comprises NPH insulin, insulin glargine or recombinant human insulin.

[0231] In embodiments, a reference standard used in any of the methods described herein comprises an outcome of a diabetes therapy (eg, a canine diabetes therapy or a feline diabetes therapy), such as an outcome described herein.

[0232] In an embodiment, the reference standard is the level of a marker (e.g., blood glucose or fructosamine) in a subject before starting therapy (e.g., insulin-Fc fusion protein therapy as described herein); wherein the subject suffers from diabetes. In an embodiment, before therapy begins, the blood glucose level in a companion animal (e.g., dog or cat) is greater than 200 mg / dL (e.g., greater than 250 mg / dL, 300 mg / dL, 350 mg / dL, 400 mg / dL or greater). In an embodiment, before therapy begins, the fructosamine level in a companion animal (e.g., dog or cat) is greater than 250 micromoles / liter, 350 micromoles / liter (e.g., greater than 400 micromoles / liter, 450 micromoles / liter, 500 micromoles / liter, 550 micromoles / liter, 600 micromoles / liter, 650 micromoles / liter, 700 micromoles / liter, 750 micromoles / liter or higher). In an embodiment, the reference standard is a measure of the presence or progression or severity of a disease. In embodiments, the reference standard is a measure of the presence or severity of disease symptoms prior to initiating therapy (eg, an insulin-Fc fusion protein therapy described herein, eg, where the subject has diabetes).

[0233] Pharmaceutical compositions and routes of administration

[0234] Provided herein is a pharmaceutical composition comprising insulin-Fc fusion protein as described herein, which can be used to reduce blood sugar in companion animals (e.g., dogs or cats). The amount and concentration of insulin-Fc fusion protein in the pharmaceutical composition and the amount of the pharmaceutical composition applied to the subject can be selected based on clinically relevant factors, such as the medically relevant features (e.g., age, weight, sex, other medical conditions, etc.) of the subject, the solubility of the compound in the pharmaceutical composition, the effectiveness and activity of the compound, and the mode of administration of the pharmaceutical composition. For further information on route of administration and dosage regimen, the reader may refer to Chapter 25.3 (Corwin Hansch; Chairman of Editorial Board) in Volume 5 of Comprehensive Medicinal Chemistry, Pergamon Press 1990.

[0235] Formulations of the present disclosure include those suitable for parenteral administration. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by intravenous or subcutaneous injection.

[0236] Examples of suitable aqueous carriers and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). For example, by using a coating material such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant such as a tween-like surfactant, appropriate fluidity can be maintained. In some embodiments, the pharmaceutical composition (e.g., as described herein) includes a tween-like surfactant, such as polysorbate-20, tween-20 or tween-80. In some embodiments, the pharmaceutical composition (e.g., as described herein) includes a tween-like surfactant, such as tween-80, at a concentration between about 0.001% and about 2%, or between about 0.005% and about 0.1%, or between about 0.01% and about 0.5%.

[0237] In some embodiments, the concentration of insulin-Fc fusion protein in the aqueous carrier is about 3 mg / mL. In some embodiments, the concentration of insulin-Fc fusion protein in the aqueous carrier is about 6 mg / mL. In some embodiments, the concentration of insulin-Fc fusion protein in the aqueous carrier is about 8 mg / mL, 9 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL or greater.

[0238] In certain embodiments, insulin-Fc fusion protein is administered in the form of pill, infusion or intravenous push. In certain embodiments, fusion protein is administered by syringe injection, pump, pen, needle or indwelling catheter. In certain embodiments, insulin-Fc fusion protein is administered by subcutaneous pill injection. The method of introduction can also be provided by rechargeable or biodegradable device. In recent years, a variety of sustained-release polymer devices have been developed and tested in vivo for controlled delivery of drugs (including protein biopharmaceuticals). A variety of biocompatible polymers (including hydrogels), including biodegradable and non-degradable polymers, can be used to form implants, for sustained release of compounds at specific target sites.

[0239] dose

[0240] The actual dosage level of insulin-Fc fusion protein can be varied so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular subject (e.g., dog or cat). The selected dosage level will depend on a variety of factors, including the activity of the particular fusion protein used or its ester, salt or amide, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular fusion protein used, the age, sex, weight, condition, general health and previous medical history of the subject being treated, and similar factors well known in the medical field.

[0241] Generally, the appropriate dose of insulin-Fc fusion protein will be the minimum dose that effectively produces the therapeutic effect.This effective dose will depend on the above factors generally.Generally, the intravenous and subcutaneous doses of insulin-Fc fusion protein for dogs or cats will be about 0.001 to about 1 mg per kilogram (e.g., mg / kg) body weight / day, for example, in the range of about 0.001 to 1 mg / kg / day, about 0.01 to 0.1 mg / kg / day, about 0.1 to 1 mg / kg / day or about 0.01 to 1 mg / kg / day.In other embodiments, the fusion protein is administered at a dosage of 0.025 to 4 mg / kg body weight / week, for example 0.025 to 0.5 mg / kg / week.

[0242] The disclosure contemplates the preparation of insulin-Fc fusion proteins in any of the aforementioned pharmaceutical compositions and preparations. In addition, the disclosure contemplates administration by any of the aforementioned routes of administration. Those skilled in the art can select suitable preparations and routes of administration according to the overall health, age and body type of the disease being treated and the patient being treated.

[0243] Examples

[0244] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way.

[0245] General methods, assays, and materials

[0246] Example 1: Method for synthesis and preparation of insulin-Fc fusion protein in HEK293 cells

[0247] Insulin-Fc fusion protein was synthesized as follows. The target gene sequence was constructed using proprietary software (LakePharma, Belmont, CA) and cloned into a high expression mammalian vector. HEK293 cells were seeded in shake flasks 24 hours before transfection and grown using serum-free chemically defined medium. A DNA expression construct encoding the target insulin-Fc fusion protein was transiently transfected into a suspension of HEK293 cells using the (LakePharma, Belmont, CA) standard operating procedure for transient transfection. After 20 hours, cells were counted to determine viability and viable cell counts and were expressed by (Pall ForteBio LLC, Fremont, CA) to measure titers. Additional readings were taken throughout the transient transfection production run. Cultures were harvested on or after day 5.

[0248] Example 2: Method for synthesis and preparation of insulin-Fc fusion protein in CHO cells

[0249] The CHO cell line was originally derived from CHO-K1 (LakePharma, Belmont, CA), and the endogenous glutamine synthetase (GS) gene was knocked out by recombinant technology using methods known in the art. Stable expression DNA vectors were designed and optimized for CHO expression and GS selection and integrated into a high expression mammalian vector (LakePharma, Belmont, CA). The sequence of each complete construct was confirmed before starting the scale-up experiment. The cells were cultured at 37°C in a humidified 5% CO 2 Suspension-adapted CHO cells were cultured in a chemically defined medium (CD OptiCHO; Invitrogen, Carlsbad, CA) in an incubator. No serum or other animal-derived products were used in the culture of CHO cells.

[0250] Approximately 80 million suspension-adapted CHO cells grown in CD OptiCHO medium in exponential growth phase were electroporated using Stable CHO cell lines were established for each insulin-Fc fusion protein by transfection with 80 μg of DNA using the MaxCyte system (MaxCyte, Inc., Gaithersburg, MD). After 24 hours, transfected cells were counted and placed under screening conditions for stable integration of the insulin-Fc fusion gene. Transfected cells were cultured in shake flasks at 0.5 × 10 6 The cells were seeded at a density of 10 cells / mL in CD OptiCHO selection medium containing 0-100 μM methionine sulfoxide (MSX) and incubated at 37°C in 5% CO. 2During the selection process, the cells were spun down and resuspended in fresh selection medium every 2-3 days until the CHO stable pool regained its growth rate and viability. The cell culture was monitored for growth and titer.

[0251] The cells were grown to 2.5 × 10 6 cells / mL. At the time of cell bank harvest, the viability was above 95%. The cells were then centrifuged and the cell pellet was resuspended in CD OptiCHO medium containing 7.5% dimethyl sulfoxide (DMSO) to a cell count of 15 × 10 6 The vials were cryopreserved for storage in liquid nitrogen.

[0252] CHO cells are used to carry out small-scale amplification production as follows.In the CD OptiCHO growth medium comprising 100 μM MSX, cells are scaled up at 37 ° C for production, and fed once every 2-4 days as required, wherein the CD OptiCHO growth medium is supplemented with glucose and other amino acids for about 14-21 days as required.The conditioned medium supernatant harvested from the stable pool production operation is clarified by centrifugal rotation.Albumen is run on a protein A (MabSelect, GE Healthcare, Little Chalfont, United Kingdom) post pre-balanced with binding buffer.Then wash buffer is passed through a chromatographic column until OD280 values ​​(NanoDrop, Thermo Scientific) are measured to be equal to or close to background levels.Low pH buffer is used to elute insulin-Fc fusion protein, elution fractions are collected, and the OD280 values ​​of each fraction are recorded.The fractions containing target insulin-Fc fusion protein are merged, and optionally 0.2 μM filter membranes are used to further filter.

[0253] The cell line is optionally further subcloned into monoclones using the limiting dilution method (a method known to those skilled in the art) and optionally further selected for high titer insulin-Fc-fusion protein expressing clones. After obtaining a high titer monoclonal insulin-Fc fusion protein expressing cell line, the production of insulin-Fc fusion protein is completed as described above in a growth medium without MSX or optionally in a growth medium containing MSX to obtain a cell culture supernatant containing recombinant CHO made insulin-Fc fusion protein. Over time, the MSX concentration is optionally increased to exert additional selectivity for clones capable of producing higher product titers.

[0254] Example 3: Purification of Insulin-Fc Fusion Protein

[0255] Purification of insulin-Fc fusion protein is carried out as follows.Harvest the conditioned medium supernatant containing secretory insulin-Fc fusion protein from the HEK production operation of transient or stable transfection, and clarify by centrifugation.The supernatant containing required insulin-Fc fusion protein is run on protein A or protein G column, and low pH gradient elution is used.Optionally, the recovery of insulin-Fc fusion protein can be improved by reloading the initial protein A or protein G column eluent onto the second protein A or protein G column.Then, the fractions of the elution containing the required protein are merged, and the buffer is exchanged into 200mM HEPES, 100mM NaCl, 50mM NaOAc, pH 7.0 buffer.Use 0.2 μm filter membrane to carry out final filtration step.Final protein concentration is calculated according to the solution optical density at 280nm.As required, further optional purification is carried out by ion exchange chromatography (for example, using anion exchange bead resin or cation exchange bead resin), gel filtration chromatography or other methods.

[0256] Example 4: Structural confirmation by non-reducing and reducing CE-SDS.

[0257] exist GXII (Perkin Elmer, Waltham, MA) was used to analyze the purified insulin-Fc fusion protein dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer by capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and the electrophoresis profile was plotted. Under non-reducing conditions, the samples were run against known molecular weight (MW) protein standards, and the elution peak represented the "apparent" MW of the insulin-Fc fusion protein homodimer.

[0258] As a possible correct way to determine the structural purity of the insulin-Fc fusion protein, the apparent MW of the resulting insulin-Fc fusion protein monomer is compared to half the molecular weight of the insulin-Fc fusion protein homodimer under reducing conditions (e.g., using β-mercaptoethanol to disrupt the disulfide bonds of the insulin-Fc fusion homodimer).

[0259] Example 5: Sequence Identification by Glycan-Removed LC-MS

[0260] In order to obtain an accurate estimate of the mass of insulin-Fc by mass spectrometry (MS), the sample is first processed to remove the naturally occurring polysaccharides that may interfere with MS analysis. First, the insulin-Fc fusion protein of 100 μL of 2.5 mg / mL is dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer using Zeba desalting columns (Pierce, ThermoFisher Scientific, Waltham, MA), and the buffer is exchanged into 0.1 M Tris, pH 8.0 buffers containing 5 mM EDTA. 1.67 μL of PNGase F enzyme (Prozyme N- polysaccharidase) is added to the solution to remove the polysaccharides (e.g., polysaccharides connected to the side chains of the asparagine located at the cNg-N sites) present in the fusion protein, and the mixture is incubated overnight at 37 ° C in an incubator. The samples were then analyzed by LC-MS (Nova Bioassays, Woburn, MA), yielding a molecular mass corresponding to the desired homodimer molecule without glycans. This mass was then further corrected because the enzymatic process used to cleave glycans from cNg-asparagine also deaminates the asparagine side chain to form aspartic acid, and in doing so, the enzymatically treated homodimer gains 2 Da overall, corresponding to a mass of 1 Da for each chain present in the homodimer. Thus, the actual molecular mass is the measured mass minus 2 Da to correct for enzymatic modifications of the insulin-Fc fusion protein structure in the analyzed sample.

[0261] Example 6: Homodimer % determined by size exclusion chromatography

[0262] Size exclusion chromatography (SEC-HPLC) of insulin-Fc fusion protein was performed at a wavelength of 280 nm using a Waters 2795HT HPLC (Waters Corporation, Milford, MA) connected to a 2998 photodiode array. 100 μL or less of a sample containing the target insulin-Fc fusion protein was injected into a MAbPac SEC-1, 5 μm, 4×300 mm column (ThermoFisher Scientific, Waltham, MA), which was operated at a flow rate of 0.2 mL / min and wherein the mobile phase contained 50 mM sodium phosphate, 300 mM NaCl, and 0.05% w / v sodium azide (pH 6.2). The MAbPac SEC-1 column works on the principle of molecular size separation. Therefore, larger soluble insulin-Fc aggregates (e.g., multimers of insulin-Fc fusion protein homodimers) elute at earlier retention times, and non-aggregated homodimers elute at later retention times. The purity of the insulin-Fc fusion protein solution (expressed as the percentage of non-aggregated homodimers) was determined when the homodimer mixture was separated from the aggregated multimeric homodimers by analytical SEC-HPLC.

[0263] Example 7: In vitro IM-9 insulin receptor binding of exemplary insulin-Fc fusion proteins at 4°C

[0264] Human IM-9 cells (ATTC#CCL-159) expressing human insulin receptor were cultured and maintained at 70-80% confluence in complete RPMI 5% FBS medium. The culture of IM-9 cells was centrifuged at 250×g (about 1000 rpm) for 10 min to pellet the cells. The cells were washed once with HBSS or PBS buffer and resuspended in cold FACS staining medium (HBSS / 2mMEDTA / 0.1% sodium azide + 4% horse serum) to a concentration of 8×10 6 cells / mL and stored on ice or at 4°C until test solutions were prepared. Insulin-Fc protein was diluted in 1:3 serial dilutions of FACS buffer at 2× concentration in 1.2 mL tubes (approximately 60 μL volume per dilution) and solutions were kept on ice until ready to pipette.

[0265] Biotinylated RHI was diluted to a concentration of 1.25 μg / mL in FACS staining medium. 40 μL of serially diluted test compounds and 8 μL of 1.25 μg / mL biotin-RHI were added to each well of a V-bottom microtiter plate, mixed by gentle vortexing, and placed on ice. 40 μL of IM-9 cell suspension (8×10 6Cells / mL), gently mixed again, and incubated on ice for 30min to allow competitive binding with insulin receptors on IM-9 cells. Then by centrifuging the V-shaped bottom plate at 3000rpm for 3min, and drawing the supernatant, wash the cells twice with 275μL of ice-cold FACS washing buffer (EDTA / 0.1% sodium azide+0.5% horse serum of HBSS / 2mM). Then resuspend the cells in 40μL of FACS staining medium containing 1:100 dilution of streptavidin-PE (Life Technologies) and place on ice for 20min. Then wash the cells once with 275μL of ice-cold FACS buffer, and finally fix them at room temperature for 10min with 3% paraformaldehyde. Then wash the cells once with 275μL of ice-cold FACS buffer, and resuspend them in 250μL of FACS buffer for analysis.

[0266] The V-shaped bottom plate containing cells is then analyzed on Guava 8-HT flow cytometer (Millipore).For the test compound of each concentration, the median fluorescence intensity (MFI) of cells on FACS FL-2 channel is quantitatively combined with the biotinylated RHI of insulin receptor.Control wells are only labeled with biotinylated RHI, and are used to calculate the inhibition percentage (%) produced by each test compound concentration.The inhibition % of the biotinylated RHI combination of the test compound on IM-9 cells has been drawn for the logarithmic concentration of the test compound, and the IC50 value of the gained of the test compound has been calculated using GraphPad Prism (GraphPad Software, LaJolla, CA).Therefore, the lower IC50 value of the test compound shows that the biotinylated RHI inhibition level is higher at lower concentrations, which shows that the stronger combination of insulin-Fc fusion protein and insulin receptor.Control compound (such as unlabeled recombinant human insulin (RHI)) is also used as an internal standard, to generate RHI IC50, from which the IC50 (IC50 (compound) / IC50 (RHI)) of a given compound can be inferred. Relative to RHI, a lower IC50 ratio has more similar binding to RHI (stronger binding to the insulin receptor), while a higher IC50 ratio has weaker binding to the insulin receptor.

[0267] Example 8: In vitro Fc(γ) receptor I binding affinity assay

[0268] At pH 7.4, the binding of insulin-Fc fusion protein to Fc (γ) receptor I was determined using ELISA as follows. Since canine and feline Fc (γ) receptor I are not commercially available, human Fc (γ) receptor I (i.e., rhFc (γ) receptor I) was used as an alternative mammalian receptor. The insulin-Fc compound was diluted to 10 μg / mL in sodium bicarbonate buffer at pH 9.6 and coated on Maxisorp (Nunc) microtiter plates overnight at 4°C, after which the microtiter plate strips were washed 5 times with PBST (PBS / 0.05% Tween-20) buffer and blocked with Superblock blocking agent (ThermoFisher). Serial dilutions of biotinylated rhFc(γ) receptor I (recombinant human Fc(γ) RI; R&D systems) were prepared in PBST / 10% Superblock buffer from 6000 ng / mL to 8.2 ng / mL and loaded at 100 μL / well onto microtiter strips coated with insulin-Fc fusion protein. The microtiter plate was incubated at room temperature for 1 hour, after which the microtiter strips were washed 5 times with PBST, and then 100 μL / well of 1:10000 diluted streptavidin-HRP was loaded in PBST / 10% Superblock buffer. After incubation for 45 min, the microtiter strips were washed 5 times with PBST again. TMB was added to reveal bound Fc(γ) receptor I protein and terminated with ELISA stop reagent (Boston Bioproducts). The plate was read in an ELISA plate reader at 450 nm and the OD values ​​(proportional to the binding of rhFc(γ) receptor I to the insulin-Fc protein) were plotted against the logarithmic concentration of rhFc(γ) receptor I added to each well using GraphPad Prism software to generate a binding curve.

[0269] Example 9: In vitro measurement of insulin-Fc fusion protein affinity for canine FcRn receptor

[0270] The in vitro binding affinity of insulin-Fc fusion proteins containing Fc fragments derived from canine or feline IgG to canine FcRn receptors was measured by ELISA technology performed at a solution pH of 5.5. Slightly acidic pH is a preferred binding environment for molecules containing Fc fragments to bind to FcRn receptors. In vivo, cells express FcRn on their surfaces and inside endosomes. When molecules containing Fc fragments enter cells through natural processes (such as endocytosis or endocytosis), the pH changes to a lower pH in the endosomes, where FcRn receptors bind to molecules containing Fc fragments, which would otherwise be degraded in the endosomal-lysosomal compartments, thereby recycling these molecules back to the cell surface where the pH is closer to neutral (e.g., pH 7.0-7.4). Neutral pH is not conducive to binding to FcRn receptors and allows molecules containing Fc fragments to be released back into the circulation. This is the main mechanism by which molecules containing Fc fragments present an extended circulating pharmacokinetic half-life in vivo.

[0271] Insulin-Fc fusion proteins containing Fc fragments of canine or feline origin were diluted to 10 μg / ml in sodium bicarbonate buffer at pH 9.6 and coated on Maxisorb ELISA plate strips in duplicate at room temperature for 1-2 hours. The strips were then washed 4 times with PBST (PBS / 0.1% Tween-20) buffer and blocked with Superblock blocking reagent (ThermoFisher). The strips were then washed twice again with pH 5.5 MES / NaCl / Tween (50mM MES / 150mM NaCl / 0.1% Tween-20) before adding the FcRn reagent (biotinylated canine FcRn; Immunitrack). Since no commercially available feline FcRn reagent was found, the binding of insulin-Fc fusion proteins containing canine Fc fragments or feline Fc fragments to canine FcRn was determined. In pH 5.5MES / NaCl / Tween / 10% Superblock buffer, serial dilutions of biotinylated FcRn reagents were prepared at concentrations of 1000ng / ml to 0.45ng / ml (1:3X dilutions), and the dilutions were loaded onto the strips coated with insulin-Fc fusion protein compounds at 100μL / well using a multichannel pipette. The assay plate was then incubated for 1 hour at room temperature. The FcRn binding strips were washed 4 times with pH 5.5 MES / NaCl / Tween buffer, and then loaded with 100μl / well streptavidin-HRP diluted 1:10000 in pH 5.5 MES / NaCl / 10% Superblock buffer. After incubation for 45 minutes, the strips were washed 4 times again with pH 5.5 MES / NaCl / Tween buffer. Finally, TMB was added to reveal the bound biotinylated canine FcRn reagent, and the color development was stopped with an ELISA stop reagent. Plates were read in an ELISA plate reader at a wavelength of 450 nm. OD values ​​(proportional to the binding of canine-FcRn to insulin-Fc fusion protein test compounds) were plotted relative to the logarithmic concentration graph of FcRn added to each well using GraphPad Prism software to generate binding curves. EC50 values ​​for each binding curve were calculated to compare the differences between different compounds.

[0272] Example 10: Determination of in vivo pharmacodynamics after a single administration of insulin Fc-fusion protein in dogs or cats (PD) general procedure.

[0273] The effect of insulin-Fc fusion protein on fasting blood glucose levels was evaluated as follows. N = 1, 2, 3 or more healthy, antibody-naive dogs weighing about 10-15 kg or cats weighing about 5 kg were used, one for each insulin-Fc fusion protein. The animals were also observed twice a day for signs of allergic reactions, lethargy, distress, pain, etc., and optionally for some compounds, treatment was continued for another three weekly subcutaneous injections or more to observe whether the glucose-lowering ability of the compound decreased over time, which is a key marker for the potential induction of neutralizing anti-drug antibodies. On day 0, animals receive a single injection of a pharmaceutical composition comprising an insulin Fc-fusion protein homodimer at a concentration between 1 and 10 mg / mL in a solution of 10-50 mM sodium hydrogen phosphate, 50-150 mM sodium chloride, 0.005-0.05% v / v Tween-80, and optionally a bacteriostatic agent (e.g., phenol, m-cresol, or methyl paraben) at a concentration of 0.02-1.00 mg / mL, by intravenous or subcutaneous administration at a solution pH of 7.0-8.0 at a dose of 0.08-0.80 mg insulin-Fc fusion protein / kg (or approximately equivalent to 1.2-12.3 nmol / kg or approximately equivalent to 0.4-4.0 U / kg insulin equivalent on a molar basis). On day 0, blood was collected from appropriate veins immediately before injection and at 15, 30, 45, 60, 120, 240, 360 and 480 minutes and on days 1, 2, 3, 4, 5, 6 and 7 after injection.

[0274] For each time point, collect at least 1 mL of whole blood. Glucose level readings were determined using an Aviva Plus glucometer, which requires approximately one drop of blood. The mean fasting blood glucose level % (%FBGL) from day 0 to day 7 was plotted to assess the bioactivity of a given insulin-Fc fusion protein.

[0275] Example 11: Repeated administration of insulin-Fc fusion protein in canines or felines to determine in vivo General Procedures for Pharmacodynamics (PD) .

[0276] The effect of insulin-Fc fusion protein on blood glucose levels during repeated injections was evaluated as follows. Healthy, antibody-naive dogs or cats weighing approximately 5 to 20 kg were used, and multiple doses of insulin-Fc fusion protein were administered to each animal. The animals were observed twice daily for signs of allergic reactions, lethargy, distress, pain, and other negative side effects, and treatment was optionally continued for some compounds, with up to 2 to 5 additional subcutaneous injections, to observe whether the glucose-lowering ability of the compound decreased over time, indicating the possible presence of neutralizing anti-drug antibodies in the body. On day 0, animals received a single subcutaneous injection of a pharmaceutical composition comprising insulin Fc-fusion protein in a solution of 10-50 mM sodium hydrogen phosphate, 50-150 mM sodium chloride, 0.005-0.05% v / v Tween-80 and optionally a bacteriostatic agent (e.g., phenol, m-cresol or methyl paraben) at a concentration of 0.02-1.00 mg / mL at a solution pH of 7.0-8.0 at a dose of 0.08-0.80 mg insulin-Fc fusion protein / kg (or approximately equal to 1.2-12.3 nmol / kg or approximately equal to 0.4-4.0 U / kg insulin equivalent on a molar basis). On day 0, blood was collected from an appropriate vein immediately before injection and at 15, 30, 45, 60, 120, 240, 360 and 480 minutes after injection, as well as on days 1, 2, 3, 4, 5, 6 and 7.

[0277] Subsequent subcutaneous injection frequency is no more than once a week, and in some cases, according to the pharmacodynamics of a given insulin-Fc fusion protein preparation, injection is given at different intervals. According to the pharmacodynamics of the insulin-Fc fusion protein demonstrated, the subsequent injection of each insulin-Fc fusion protein is adjusted to a higher dosage or a lower dosage. For example, if it is found that the dosage of the first injection on the 0th day is ineffective in reducing blood sugar, the dosage level of the insulin-Fc fusion protein injected subsequently is adjusted upward. In a similar manner, if it is found that the dosage of the first injection on the 0th day reduces glucose in a too strong manner, the dosage level of the insulin-Fc fusion protein injected subsequently is adjusted downward. It is also found that the mid-term dose or the final dose can be adjusted in a similar manner as needed. For each dosage, blood is collected from a suitable vein immediately before injection and 15, 30, 45, 60, 120, 240, 360 and 480 minutes and 1, 2, 3, 4, 5, 6, 7 days (and optionally 14 days) after injection. For each time point, at least 1mL of whole blood was collected. Immediately use a blood glucose meter ( Glucose level readings were measured using an Aviva Plus glucometer that requires approximately one drop of blood. The mean fasting blood glucose level % (%FBGL) over the entire study period was plotted versus time, which allowed the biological activity of the fusion protein to be determined.

[0278] To determine the bioactivity of each dose, an area on the curve (AOC) analysis was performed as follows. After constructing the data for %FBGL versus time, the data was then entered into the data analysis software (GraphPad Prism, GraphPad Software, San Diego CA). The software was first used to perform an area under the curve analysis (AUC) to integrate the area under the %FBGL versus time curve for each dose. To convert the AUC data to the desired AOC data, the following formula was used: AOC = TPA–AUC; where TPA is the total possible area obtained by multiplying each dose survival (e.g., 7 days, 14 days, etc.) by 100% (where 100% represents y = 100% of the %FBGL versus time curve). For example, given a 7-day dose survival and a calculated AUC of 500%FBGL·day, the following results for AOC are given: AOC = (100%FBGL x 7 days)–(500%FBGL·day) = 200%FBGL·day. Each injection dose in a series of injection doses can be analyzed to obtain the AOC value for injection 1, injection 2, injection 3, etc.

[0279] Since the dose of insulin-Fc fusion protein may vary as described above, it is usually more convenient to normalize all calculated AOC values ​​for a given insulin-Fc fusion protein to a specific dose of the insulin-Fc fusion protein. This allows for convenient comparison of the glucose-lowering efficacy of multiple injections of insulin-Fc fusion protein, even if the dose level changes between multiple injections in a given study. The normalized AOC (NAOC) for a given dose is calculated as follows: NAOC=AOC / D, where the unit is %FBGL·day·kg / mg; where D is the actual dose injected into the animal, in mg / kg. NAOC values ​​can be calculated for each injection in a series of injections for a given animal, and an average value can be taken for a group of animals receiving the same insulin-Fc fusion protein formulation.

[0280] For each injection in a series of injections for a given animal, the NAOC ratio (NAOCR) can also be calculated by taking the NAOC value for each injection (e.g., injections 1, 2, 3, ... N) and dividing each NAOC for a given injection by the NAOC value from injection 1, as shown below: NAOCR = (NAOC (Nth injection) / NAOC (injection 1)). By evaluating the NAOCR of a given insulin-Fc homodimer fusion protein preparation for the Nth injection in a series of injections, it can be determined whether the in vivo glucose-lowering activity of a given insulin-Fc fusion protein substantially maintains its biological activity over a series of N doses (e.g., the NAOCR for the Nth dose is greater than 0.5) or whether the in vivo glucose-lowering activity of a given insulin-Fc fusion protein loses most of its potency over the course of N doses (e.g., the NAOCR for the Nth dose is less than 0.5), indicating that neutralizing anti-drug antibodies may be formed in vivo. In a preferred embodiment, the ratio of the NAOC after the third subcutaneous injection to the NAOC after the first subcutaneous injection is greater than 0.5 (ie, the NAOCR of the third subcutaneous injection is greater than 0.5).

[0281] Example 12: General Procedure for Determining In Vivo Pharmacokinetics (PK) in Canine and Feline Serum

[0282] An assay was constructed as follows to measure the concentration of insulin-Fc fusion proteins containing Fc fragments of canine isotypes in canine serum. The assay comprises a sandwich ELISA format in which therapeutic compounds in serum samples are captured by anti-insulin / proinsulin monoclonal antibodies (mAbs) coated on ELISA plates and then detected by HRP-conjugated anti-canine IgG Fc-specific antibodies, followed by the use of a TMB substrate system for color development. Maxisorp ELISA plates (Nunc) were coated with anti-insulin mAb clone D6C4 (Biorad) in 5 μg / ml of coating buffer (pH=9.6 sodium carbonate-biocarbonate buffer) overnight at 4°C. The plates were then washed 5 times with PBST (PBS+0.05% Tween 20) and blocked with SuperBlock blocking solution (ThermoFisher) for at least 1 hour at room temperature (or overnight at 4°C). Test serum samples were diluted to 1:20 in PBST / SB / 20% HS sample dilution buffer (PBS + 0.1% Tween 20 + 10% SuperBlock + 20% horse serum). To make a standard curve, the target insulin-Fc fusion protein was diluted in a 1:2.5 serial dilution in sample dilution buffer (PBST / SB / 20% HS) + 5% pooled beagle serum (BioIVT) ranging from 200ng / ml to 0.82ng / ml. Standards and diluted serum samples were added to the blocked plate in duplicate at 100μl / well and incubated for 1 hour at room temperature. After incubation, samples and standards were washed 5 times with PBST. HRP-conjugated goat anti-canine IgG Fc (Sigma) detection antibody was diluted to approximately 1:15,000 in PBST / SB / 20% HS buffer, and 100μl was added to all wells and incubated for 45 minutes in the dark at room temperature. The plate was washed 5 times with PBST and washed once with deionized water, and developed for 8-10 minutes at room temperature by adding 100 μl / well TMB (Invitrogen). The color development was then stopped by adding 100 μl / well ELISA stop solution (Boston Bioproducts), and absorbance was read at 450nm using a SpectraMax plate reader (Molecular Devices) within 30 minutes. The concentration of insulin-Fc fusion protein compounds in the sample was calculated using SoftMaxPro software by interpolation on the 4-PL curve.

[0283] Similarly, a determination was constructed as follows for measuring the concentration of insulin-Fc fusion proteins containing Fc fragments of feline isotypes in feline serum. The determination comprises a sandwich ELISA format in which the therapeutic compound in the serum sample is captured by anti-insulin / proinsulin mAb coated on an ELISA plate, and then detected by a HRP-conjugated goat anti-feline IgG Fc-specific antibody, followed by a TMB substrate system for color development. Maxisorp ELISA plates (Nunc) were coated with anti-insulin mAb clone D6C4 (Biorad) in 5 μg / ml of coating buffer (pH=9.6 sodium carbonate-biological sodium carbonate buffer) overnight at 4°C. The plates were then washed 5 times with PBST (PBS+0.05% Tween 20) and blocked with SuperBlock blocking solution (ThermoFisher) for at least 1 hour at room temperature (or overnight at 4°C). The test serum samples were diluted to 1:20 in PBST / SB / 20% HS sample dilution buffer (PBS+0.1% Tween 20+10% SuperBlock+20% horse serum). To make a standard curve, the target insulin-Fc fusion protein compound was diluted in a 1:2.5 serial dilution in a sample dilution buffer (PBST / SB / 20% HS) + 5% normal cat serum (Jackson Immunoresearch) with a concentration range of 200ng / ml to 0.82ng / ml. Standards and diluted serum samples were added to the closed plate in duplicate at 100μl / well and incubated at room temperature for 1 hour. After incubation, the samples and standards were washed 5 times with PBST. The HRP-conjugated goat anti-feline IgG Fc (Bethyl Lab) detection antibody was diluted to approximately 1:20,000 in PBST / SB / 20% HS buffer, and 100μl was added to all wells and incubated in the dark at room temperature for 45 minutes. The plate was washed 5 times with PBST and washed once with deionized water, and developed for 8-10 minutes at room temperature by adding 100 μl / well TMB (Invitrogen). The color development was then stopped by adding 100 μl / well ELISA stop solution (Boston Bioproducts), and absorbance was read at 450nm using a SpectraMax plate reader (Molecular Devices) within 30 minutes. The concentration of insulin-Fc fusion protein compounds in the sample was calculated using SoftMaxPro software by interpolation on the 4-PL curve.

[0284] Example 13: Assay Protocol for Measuring Anti-Drug Antibodies in Canine Serum

[0285] Maxisorp ELISA plates (Nunc) were coated with target insulin-Fc fusion proteins diluted at 10 μg / mL in coating buffer (pH = 9.6 carbonate-biocarbonate buffer) overnight at 4°C for measurement of ADA against test compounds. To measure ADA against the insulin portion of the insulin-Fc fusion protein containing a canine IgG-derived Fc fragment, the plates were coated with 30 μg / mL of purified insulin in coating buffer. The plates were then washed 5 times with PBST (PBS + 0.05% Tween 20) and blocked with SuperBlock blocking solution (ThermoFisher, Waltham MA) for at least 1 hour (or overnight). To calculate ADA expressed in canine IgG units, the strips were directly coated overnight at 4°C with 1:2 serial dilutions of canine IgG in Carb-Biocarb coating buffer at pH = 9.6 at a concentration of 300-4.69 ng / ml and used to create a 7-point pseudo-standard curve. The standard strip plate was also washed and blocked with SuperBlock blocking solution for at least 1 hour (or overnight).

[0286] The test serum samples were diluted to greater than or equal to 1:100 (usually tested at 1:200) in PBST / SB / 20% HS sample dilution buffer (PBS+0.1% Tween 20+10% SuperBlock+20% horse serum) and added to the insulin-Fc fusion protein coated (or RHI coated) strips in duplicate at 100 μL / well. Repeat strips of the standard strips coated with canine IgG were also added to each plate and filled with PBST / SB (PBS+0.1% Tween 20+10% SuperBlock) buffer at 100 μL / well. The plates were incubated at room temperature for 1 hour and then washed 5 times with PBST. For detection of ADA, HRP-conjugated goat anti-feline IgG F(ab')2 (anti-feline IgG F(ab')2 reagent cross-reacts with canine antibodies; Jackson Immunoresearch Laboratories, West Grove PA) was diluted to 1:10,000 in PBST / SB and added to sample and standard wells at 100 μL / well and incubated at room temperature in the dark for 45 minutes. The plates were washed 5 times with PBST and then once with deionized water and then developed by adding 100 μL / well of TMB substrate (Invitrogen, ThermoFisher Scientific, Waltham MA) for 15-20 minutes at room temperature in the dark. The color development was then stopped by adding 100 μL / well of ELISA stop solution (Boston Bioproducts) and the absorbance was read at 450 nm using a SpectraMax plate reader within 30 minutes. Anti-drug antibody concentrations were determined by interpolation of OD values ​​from the 4-PL pseudo-standard curve using SoftMax Pro software (Molecular Devices, San Jose CA).

[0287] In order to determine the specificity of the detected ADA, an "inhibition" assay was performed. In the drug inhibition ADA assay, serum samples were diluted 1:100 in PBST / SB / 20% HS buffer and mixed with an equal volume of 300 μg / mL of relevant therapeutic compounds (the final sample dilution was 1:200, and the final inhibitory compound dilution was 150 μg / mL), and then incubated at room temperature for 30-40 minutes to allow anti-drug antibodies to bind to free inhibitors (i.e., therapeutic compounds). After pre-incubation, samples were added to insulin-Fc fusion protein-coated (or RHI-coated) strips in duplicate at 100 μL / well. Samples diluted 1:200 in PBST / SB / 20% HS buffer without inhibitory compounds were also tested in a sample plate together with duplicate strips of canine IgG-coated standards. The remaining steps of the assay procedure were performed as described above. The ADA measured in the drug-inhibited wells was matched with the non-inhibited ADA concentration to evaluate the specificity of ADA. If significant inhibition of ADA signal is observed in drug inhibited wells, this means that the ADA is specific for the therapeutic compound.

[0288] Example 14: Assay Protocol for Measuring Anti-Drug Antibodies in Feline Serum

[0289] Maxisorp ELISA plates (Nunc) were coated with target insulin-Fc fusion proteins diluted at 10 μg / mL in coating buffer (pH = 9.6 carbonate-biocarbonate buffer) overnight at 4°C for measuring ADA against insulin-Fc fusion proteins containing feline IgG-derived Fc fragments. To measure ADA against the insulin portion of the insulin-Fc fusion protein, the plates were coated with 30 μg / mL of purified insulin in coating buffer. The plates were then washed 5 times with PBST (PBS + 0.05% Tween 20) and blocked with SuperBlock blocking solution (ThermoFisher, Waltham MA) for at least 1 hour (or overnight). To calculate ADA expressed in feline IgG units, the strips were directly coated overnight at 4°C with 1:2 serial dilutions of canine IgG (Jackson Immunoresearch Laboratories, West Grove PA) at concentrations ranging from 300-4.69 ng / ml in sodium carbonate-bicarbonate coating buffer, pH = 9.6, and used to create a 7-point pseudo-standard curve. The standard strip plate was also washed and blocked with SuperBlock blocking solution for at least 1 hour (or overnight).

[0290] The test serum samples were diluted to greater than or equal to 1:100 (usually tested at 1:200) in PBST / SB / 20% HS sample dilution buffer (PBS+0.1% Tween 20+10% SuperBlock+20% horse serum) and added to the insulin-Fc fusion protein coated (or RHI coated) strips in duplicate at 100 μL / well. Repeat strips of the standard strips coated with feline IgG were also added to each plate and filled with PBST / SB (PBS+0.1% Tween 20+10% SuperBlock) buffer at 100 μL / well. The plate was incubated at room temperature for 1 hour and then washed 5 times with PBST. For the detection of ADA, HRP-conjugated goat anti-feline IgG F(ab')2 (Jackson Immunoresearch Laboratories, West Grove PA) was diluted in PBST / SB at a factor of 1:10000 and added to the sample wells and standard wells at 100 μL / well and incubated in the dark at room temperature for 45 minutes. The plate was washed 5 times with PBST and once with deionized water, and developed for 15-20 minutes in the dark by adding 100 μL / well of TMB substrate (Invitrogen). The color development was then stopped by adding 100 μL / well of ELISA stop solution (Boston Bioproducts, Ashland MA), and the absorbance was read at 450 nm using a SpectraMax plate reader within 30 minutes. Anti-drug antibody concentrations were determined by interpolating the OD values ​​in the 4-PL pseudo-standard curve using SoftMax Pro software (Molecular Devices, San Jose CA).

[0291] Example 15: Assay Procedure for Identification of Immunogenic Epitopes

[0292] Maxisorp ELISA microplates (Nunc) were coated with a library of insulin-Fc fusion protein homodimer compounds with known amino acid sequences, and the coated plates were blocked in a similar manner as described in Examples 13 and 14 for the anti-drug antibody ELISA assay, except that each compound in the library was coated on a separate strip of ELISA microplate wells. The compounds in the library comprised a range of insulin-Fc fusion proteins with different insulin polypeptide amino acid compositions, including various B-chain, C-chain and A-chain amino acid mutations, different linker compositions, and different Fc fragment compositions, including some compositions of human origin. In addition, as described in Examples 13 and 14, some of the strip wells were directly coated with 1:2 serial dilutions of canine or feline IgG (Jackson Immunoresearch Laboratories, West Grove PA) for calculation of anti-drug antibodies (ADA) in canine or feline IgG units, respectively.

[0293] Serum obtained from individual dogs or cats that received repeated administration of insulin-Fc fusion protein was first screened in an anti-drug antibody ELISA assay (Example 13 for dogs and Example 14 for cats). Serum samples that showed moderate or high positive results in the assay of Example 13 or Example 14 (e.g., moderate or high titers of antibodies) were serially diluted (1:200 to 1:8000) in PBST / SB / 20% HS sample dilution buffer (PBS+0.1% Tween 20+10% SuperBlock+20% horse serum) and added to a plate coated with a library of insulin-Fc fusion protein compounds and placed at room temperature for 1 hour. After incubation, the plate was washed 5 times with PBST. To detect canine or feline antibodies that can cross-react with the coated compound library, HRP-conjugated goat anti-feline IgG F(ab')2 (Jackson Immunoresearch Laboratories, West Grove PA), which is cross-reactive to both canine and feline IgG, was diluted to 1:10,000 in PBST / SB and added to the sample wells and standard wells at 100 μL / well and incubated in the dark at room temperature for 45 min. The plate was washed 5 times with PBST, washed once with deionized water, and developed by adding 100 μL / well of TMB substrate (Invitrogen, ThermoFisher Scientific, Waltham MA) at room temperature in the dark for 15-20 min. The color development was then stopped by adding 100 μL / well of ELISA stop solution (Boston Bioproducts, Ashland MA), and the absorbance was read at 450 nm using a SpectraMax plate reader within 30 min. The concentration of anti-compound cross-reactive antibodies present in serum samples was determined by interpolating the OD values ​​from the 4-PL pseudo-standard curve against directly coated canine or feline IgG antibody controls using SoftMax Pro Software (Molecular Devices, San Jose CA).

[0294] By correlating the resulting antibody concentrations from the assay with the known amino acid composition of the coated insulin-Fc fusion protein library, it can be determined whether a particular amino acid mutation or epitope results in no, some, most, or all of the total antibody signal on the assay, indicating no binding, weak binding, or strong binding to the various insulin-Fc fusion protein homodimers. Mutations or epitopes that are responsible for moderate or strong binding are referred to herein as immunogenic "hot spots."

[0295] Example 16: Acute and repeated testing for obtaining high homodimer titers and acceptable levels in target species Design process of insulin-Fc fusion protein for drug delivery

[0296] The process for meeting the design goal described in the specific embodiment of the present invention comprises the following steps. First, the insulin polypeptide of SEQ ID NO:4 or SEQ ID NO:5 is combined with the species-specific Fc fragment and joint of specific IgG isotype, so that the resulting insulin-Fc fusion protein is most likely to produce a long-acting biologically active product with minimal immunogenicity (for example, selecting the species-specific IgG isotype with minimal Fc (γ) receptor I binding). Prepare the DNA sequence encoding the required fusion protein, clone it into a carrier (LakePharma, San Carlos, CA), and then use the carrier to transiently transfect HEK cells according to the procedure described in Example 1. Then purify the insulin-Fc fusion protein according to Example 3, and measure the total protein yield and homodimer % according to Example 6. Only candidates with homodimer titers greater than 50mg / L are considered acceptable, because titers below this level are unlikely to produce commercial production titers that meet the strict low production cost requirements of veterinary products. Then as described in Example 7, the biological activity index of the selected insulin-Fc fusion protein is screened by in vitro insulin receptor binding studies. As a rule of thumb, only compounds that exhibit IR activity IC50 values ​​less than 5000 nM are considered likely to exhibit biological activity in the target species. Although the in vitro IR IC50 value is a useful qualitative screening tool, it utilizes human IM-9 cells expressing the human insulin receptor and may therefore not capture some small differences in affinity between canine or feline IR and human IR. In addition, factors other than insulin receptor binding may affect the in vivo biological activity of the compound (e.g., affinity for canine or feline FcRn to allow for extended in vivo pharmacokinetic elimination half-life). Therefore, the biological activity of the selected insulin-Fc fusion protein in the target animal (e.g., dog or cat) that is acceptable from the perspective of production and IR activity IC50 values ​​is further screened to screen out any material whose biological activity is less than the desired potency and / or duration (e.g., NAOC less than 150% FBGL·day·kg / mg). Similarly, as a rule of thumb, when the NAOC value is greater than 150% FBGL·day·kg / mg, the dosage requirements in the target species will be low enough to achieve acceptable treatment costs. Finally, an additional evaluation criterion was added that is rarely mentioned in the art. As discussed in more detail in the examples below, multiple insulin-Fc fusion protein embodiments that exhibited acceptable NAOC levels in target species after the first dose unexpectedly failed to maintain that level of bioactivity after repeated doses. In addition, in most cases, the reduction in bioactivity with repeated doses in the target species was associated with the production of neutralizing anti-drug antibodies. This tendency to produce anti-drug antibodies and the failure to maintain activity make such insulin-Fc fusion proteins unsuitable for the treatment of chronic diseases, such as canine diabetes or feline diabetes.Therefore, only insulin-Fc fusion proteins that exhibit acceptable levels of repeat dosing bioactivity (eg, NAOCR values ​​greater than 0.50 at the third dose relative to the first dose) and have minimal levels of anti-drug antibodies are considered useful in the present invention.

[0297] Results - Insulin-FC fusion protein containing canine Fc fragment

[0298] Example 17: Canine Insulin-Fc Fusion Protein Comprising Canine Fc IgGA Isotype

[0299] An attempt was made to produce an insulin-Fc fusion protein comprising the insulin polypeptide sequence of SEQ ID NO: 5 and an Fc fragment of the canine IgGA isotype (SEQ ID NO: 15) using the peptide linker of SEQ ID NO: 12. The complete amino acid sequence of the resulting insulin-Fc fusion protein is as follows:

[0300] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVS VLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG(SEQ ID NO:42).

[0301] The insulin-Fc fusion protein of SEQ ID NO:42 was synthesized in HEK cells according to Example 1 and purified according to Example 3. After the protein A purification step, the protein yield was 22 mg / L. The structure of the insulin-Fc fusion protein was confirmed by non-reducing and reducing CE-SDS according to Example 4, and the sequence was further identified by LC-MS removing polysaccharides according to Example 5. According to Example 6, the homodimer % was measured by size exclusion chromatography and was determined to be 24%, indicating a high degree of homodimer aggregates. Therefore, the homodimer titer obtained was only 5 mg / L. In short, the production of the insulin-Fc fusion protein of SEQ ID NO:42 in HEK cells produced a high level of aggregates and a low level of homodimer titer (5 mg / L), which did not meet the design goal of homodimer titer being greater than 50 mg / L.

[0302] However, the biological activity of the insulin-Fc fusion protein of SEQ ID NO: 42 was evaluated. First, the insulin receptor binding of the insulin-Fc fusion protein of SEQ ID NO: 42 was measured according to Example 7, and an IC50 value of 2,733 nM was obtained, indicating that the compound may have biological activity in vivo (i.e., IC50 is less than 5000 nM).

[0303] Next, according to Example 10, the in vivo pharmacodynamics (PD) of the insulin-Fc fusion protein of SEQ ID NO: 42 was measured after a single intravenous administration of the compound to N=3 canines. Figure 2 The percentage of fasting blood glucose level of SEQ NO:42 is shown as a function of time. According to the procedure of Example 11, the NAOC of SEQ ID NO:42 is calculated to be 105%FBGL·day·kg / mg. The in vivo half-life of SEQ ID NO:42 is calculated to be less than 1 day using the method of Example 12. The relatively low NAOC value may be the result of a large number of aggregates (i.e., low homodimer %) in the sample, but the soluble homodimer that remains in the circulation has only a pharmacodynamic elimination half-life of less than 1 day, which is considered unlikely to support once-weekly administration.

[0304] Example 18: Mutation of the Fc fragment region of an insulin-Fc fusion protein comprising a canine IgGA isotype

[0305] In an attempt to increase the homodimer content %, improve its biological activity and extend its half-life of the insulin-Fc fusion protein of SEQ ID NO: 42, mutations were inserted into the CH3 region of the Fc fragment in an attempt to prevent intermolecular association (e.g., Fc fragment-Fc fragment interactions between molecules) and promote stronger binding to the FcRn receptor (e.g., higher affinity for FcRn), thereby increasing recycling and systemic circulation time. The following insulin-Fc fusion protein was synthesized in HEK cells according to Example 1, purified according to Example 3, and tested according to Examples 4-7, as shown below. Figure 3 The sequence alignment of SEQ ID NOs: 44, 46, 48 and 50 relative to SEQ ID NO: 42 and the differences in amino acid sequences are shown in FIG.

[0306] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHEALHSHYTQKSLSLSPG(SEQ ID NO:44)

[0307] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHETLQSHYTDLSLSHSPG(SEQ ID NO:46)

[0308] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQSHYTDLSLSHSPG(SEQ ID NO:48)

[0309] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVS VLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHETLQNHYTDLSLSHSPG(SEQ ID NO:50)

[0310] Insulin-Fc fusion proteins based on canine IgGA variants and the corresponding protein yields, homodimer % and homodimer titers are listed in Table 2. The results showed that various mutations of the IgGA Fc fragment did not improve the homodimer % and homodimer titer, but instead produced highly aggregated proteins with very low homodimer titers below 5 mg / L. Therefore, the in vivo biological activity and pharmacokinetics of these compounds could not be evaluated.

[0311]

[0312] Example 19: Canine Insulin-Fc Fusion Proteins Using Other Canine Fc Fragment Isotypes

[0313] As described above, canine IgGA is considered to be the preferred isotype for the production of non-immunogenic insulin-Fc fusion proteins for dogs as Fc fragments because they lack Fc(γ)I effector functions in canines (very similar to the human IgG2 isotype in humans). However, insulin-Fc fusion proteins prepared with canine IgGA Fc fragments are highly aggregated, have unacceptably low homodimer titers, and unacceptably low levels of biological activity and duration of action. Therefore, Fc fragments from other canine IgG isotypes (canine IgGB of SEQ ID NO: 16, canine IgGC of SEQ ID NO: 17, and canine IgGD of SEQ ID NO: 18) were evaluated as alternatives to the canine IgGA Fc fragments of the insulin-Fc fusion of SEQ ID NO: 42. Using the insulin polypeptide of SEQ ID NO:5 identical to the insulin-Fc fusion protein of SEQ ID NO:42 and the peptide linker of SEQ ID NO:12, three insulin-Fc fusion proteins containing Fc fragments based on canine IgGB, IgGC and IgGD isotypes were synthesized. Protein was prepared in HEK293 cells according to Example 1. Then, the insulin-Fc fusion protein was purified using a protein A column according to Example 3. The structure of the insulin-Fc fusion protein was confirmed by non-reducing and reducing CE-SDS according to Example 4, and the sequence was further identified by LC-MS removing polysaccharides according to Example 5. According to Example 6, homodimer % was determined by size exclusion chromatography. Their sequences are shown below, and their sequence alignment comparison relative to SEQ ID NO:42 is shown in Figure 4 It shows:

[0314] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSV LPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:52)

[0315] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSV LPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG(SEQID NO:54)

[0316] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGCISPCPVPESLGGPSVFIFPPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVL PIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG(SEQID NO:56)

[0317] The resulting protein yields, homodimer % and homodimer titers are given in Table 3. Unexpectedly, only the insulin-Fc fusion protein of SEQ ID NO: 52 (comprising an Fc fragment based on the canine IgGB isotype) showed a homodimer titer that met the design criteria of greater than 50 mg / L. The insulin-Fc fusion protein of SEQ ID NO: 54 (comprising an Fc fragment based on the canine IgGC isotype) did not produce any compound at all, and the insulin-Fc fusion protein of SEQ ID NO: 56 (comprising an Fc fragment based on the canine IgGD isotype) showed a considerable protein yield, but with a high degree of aggregation and, therefore, an unacceptably low homodimer titer.

[0318] The insulin-Fc fusion proteins of SEQ ID NO:52 and SEQ ID NO:56 were tested for in vitro insulin receptor binding according to the procedure of Example 7. The insulin-Fc fusion protein of SEQ ID NO:56 showed an IC50 of greater than 5000 nM, indicating that the compound is highly unlikely to show in vivo biological activity. However, the insulin-Fc fusion protein of SEQ ID NO:52 showed an IC50 of 28 nM, indicating that the sequence may have in vivo biological activity.

[0319]

[0320] *DNM = Not Measured

[0321] Example 20: Insulin-containing insulin polypeptide of SEQ ID NO: 5 and canine IgGB isotype Fc fragment In vivo efficacy of Fc fusion proteins

[0322] In view of the promising homodimer titer and insulin receptor activity results in Example 19, the in vivo bioactivity of the insulin-Fc fusion protein of SEQ ID NO: 52 was tested according to Example 10 after intravenous injection into each of N = 3 healthy, antibody naive beagle dogs weighing approximately 10 kg. In a separate experiment, the compound was injected subcutaneously into N = 3 naive beagle dogs. Figure 5 shows % FBGL versus time for a single intravenous administration of the insulin-Fc fusion protein of SEQ ID NO: 52, and Figure 6 Shown are % FBGL versus time for a single subcutaneous administration of the insulin-Fc fusion protein of SEQ ID NO: 52, both of which demonstrate that the insulin-Fc fusion protein of SEQ ID NO: 52 has significant biological activity in dogs.

[0323] NAOC was calculated according to the procedure of Example 11 to determine the relative biological activity and duration of action of the insulin-Fc fusion protein. The NAOC of the insulin-Fc fusion protein of SEQ ID NO:52 injected intravenously was 399%FBGL·day·kg / mg, which was 3.8 times the NAOC of the insulin-Fc fusion protein of SEQ ID NO:42 injected intravenously, indicating that the biological activity of the insulin-Fc fusion protein containing the canine IgGB Fc fragment was significantly increased compared to the insulin-Fc fusion protein containing the canine IgGA Fc fragment. The NAOC of the insulin-Fc fusion protein of SEQ ID NO:52 injected subcutaneously was 366%FBGL·day·kg / mg, indicating that the level of biological activity obtained by subcutaneous administration was similar to that obtained by intravenous administration.

[0324] Example 21: Repeated subcutaneous administration of an insulin polypeptide comprising SEQ ID NO: 5 and a canine IgGB isotype In vivo immunogenicity screening of insulin-Fc fusion proteins after Fc fragmentation

[0325] Next, the subcutaneous bioactivity of the insulin-Fc fusion protein of SEQ ID NO:52 was tested in dogs according to the method described in Example 11. N = 3 animals were subcutaneously administered on days 0, 35 and 42, and the % FBGL in the 7-day window period after each administration was measured according to Example 11. For each repeated subcutaneous injection, NAOC and NAOCR were calculated according to the procedure of Example 11. As shown in Table 4, repeated subcutaneous administration in dogs unexpectedly showed a significant attenuation of bioactivity after the third administration, as measured by a significant decrease in NAOCR (i.e., the NAOC of the third injection was only 0.40 or 40% of the NAOC of the first injection).

[0326]

[0327] Without being bound by any particular explanation, it is assumed that the reason why the biological activity of the insulin-Fc fusion protein of SEQ ID NO: 52 was significantly reduced after the third repeated subcutaneous administration in dogs was due to the production of anti-drug antibodies that neutralize its biological activity. The anti-drug antibodies can be directed against the insulin polypeptide, linker or Fc fragment portion of the insulin-Fc fusion protein. The immunogenic response is manifested as an interaction between antigen presenting cells, T helper cells, B cells and their associated cytokines, which may lead to the production of endogenous antibodies (e.g., anti-drug antibodies) against the drug. Binding antibodies are all isotypes capable of binding to the insulin-Fc fusion protein, and these antibodies can be detected in an immunoassay as described in Example 13. Neutralizing antibodies that inhibit the functional activity of the insulin-Fc fusion protein are generally directed against epitopes required for biological activity. To evaluate whether this is the case, according to Example 13, sera collected before administration of each dose and at the end of the experiments described in Examples 11 and 12 were tested to quantify the levels of anti-drug antibodies. As Figure 7 As shown, the level of anti-drug antibodies did increase with multiple subcutaneous administrations of the compound, suggesting that the generation of neutralizing anti-drug antibodies after the third injection of the insulin Fc-fusion protein of SEQ ID NO: 52 was a possible reason for the decrease in NAOCR.

[0328] Example 22: Aglycosyltransferase comprising an insulin polypeptide of SEQ ID NO: 5 and a canine IgGB isotype Fc fragment Insulin-Fc fusion protein to reduce the potential risk of immunogenicity

[0329] As shown in Examples 19 and 20, the insulin-Fc fusion protein of SEQ ID NO: 52 showed acceptable homodimer content %, homodimer titer and biological activity in dogs; however, its use for chronic diseases such as diabetes was compromised by reduced biological activity (Example 21) and the production of anti-drug antibodies (Example 21) in the case of repeated subcutaneous administration. Without being bound by any particular theory, one possible reason for the production of anti-drug antibodies and reduced biological activity is the increased interaction of the canine IgGB Fc fragment with various receptors of the canine immune system (e.g., Fc (γ) receptors, such as Fc (γ) RI). However, the canine IgGB isotype is the only one of the four canine IgG isotypes that, when used for the Fc fragment, produces an insulin-Fc fusion protein that meets the design goals of manufacturability and single-dose biological activity (Example 16). As described in a specific embodiment of the present invention, a method for reducing Fc (γ) interactions involves mutating the Fc fragment cNg site to prevent glycosylation during synthesis in the host cell. Therefore, the cNg site mutation was performed on the Fc fragment region of SEQ ID NO:52 to reduce the binding affinity of the Fc fragment to the Fc(γ) receptor in vivo, as measured in the in vitro human Fc(γ)RI binding assay described in Example 8. Verification of the lack of glycans was performed using the LC-MS method of Example 5, but the PNGase F treatment step was omitted. The position of the cNg site in the insulin-Fc fusion protein of SEQ ID NO:52 is cNg-NB139. Mutations of SEQ ID NO:52 include SEQ ID NO:58 (comprising a mutation of cNg-NB139-Q); SEQ ID NO:60 (comprising a mutation of cNg-NB139-S); SEQ ID NO:62 (comprising a mutation of cNg-NB139-D); and SEQ ID NO:64 (comprising a mutation of cNg-NB139-K). The complete amino acid sequence of the cNg-mutated insulin-Fc fusion protein is listed below (with the NB139 position underlined) and is shown in Table 1. Figure 8 The resulting sequence alignment (Clustal Omega) is shown in:

[0330] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF QGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:58)

[0331] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:60)

[0332] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF D GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:62)

[0333] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF K GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:64)

[0334] Insulin-Fc fusion proteins were prepared in HEK293 cells according to Example 1 and purified using a protein A column according to Example 3. The structure of the insulin-Fc fusion protein was confirmed by non-reducing and reducing CE-SDS according to Example 4, and the sequence was further identified by LC-MS with glycan removal according to Example 5. Homodimer % was measured by size exclusion chromatography according to Example 6. As shown in Table 5, the homodimer titers of the insulin-Fc fusion proteins of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64 met the design target, while the insulin-Fc fusion protein of SEQ ID NO: 58 containing the cNg-NB139-Q mutation unexpectedly did not meet the design target for homodimer titer.

[0335]

[0336] To determine which of the remaining three compounds is most likely to exhibit reduced immunogenicity, Fc (γ) receptor binding was measured according to the procedure of Example 8. Low Fc (γ) receptor binding is most likely associated with the lowest immunogenicity. Table 6 compares the Fc (γ) receptor I binding of these insulin-Fc fusion proteins with the Fc (γ) receptor binding of the insulin-Fc fusion protein of SEQ ID NO: 52, and unexpectedly demonstrates that the Fc (γ) receptor binding activity exhibited by the insulin-Fc fusion protein of SEQ ID NO: 62 containing the cNg-D mutation is about twice that of the insulin-Fc fusion protein of SEQ ID NO: 60 containing the cNg-S mutation and the insulin-Fc fusion protein of SEQ ID NO: 64 containing the cNg-K mutation. Therefore, the insulin-Fc fusion protein containing only the latter two compounds containing the cNg-S mutation and the cNg-K mutation is considered suitable for repeated administration bioactivity testing in dogs.

[0337]

[0338] Example 23: SEQ ID NO: with non-glycosylated cNg-K and cNg-S canine IgGB isotype Fc fragments: Evaluation of in vivo bioactivity and immunogenicity of 5 insulin peptides

[0339] To determine whether the insulin-Fc fusion protein of SEQ ID NO:60 containing the cNg-S mutation improves the repeated administration bioactivity performance in dogs, the compound was subcutaneously administered to N=1 dogs on days 0, 7, 14 and 28 according to the procedure of Example 11. When the %FBGL of the dog dropped too low, food was provided to the dog to raise blood glucose to a safe level. The NAOC of the first injection was 191%FBGL·day·kg / mg, indicating that the insulin-Fc fusion protein of SEQ ID NO:60 has satisfactory bioactivity in vivo. The NAOC and NAOCR of each subsequent administration were also measured according to the general procedure of Example 11, calculated from the time of the dose administration until just before the next dose was administered. The NAOC and NAOCR shown in Table 7 show that the insulin-Fc fusion protein of SEQ ID NO:60 exhibits a significantly reduced NAOCR at doses 3 and 4 of the four-dose regimen. Therefore, although the Fc(γ)RI binding rate of the insulin-Fc fusion protein of SEQ ID NO:60 containing the cNg-S mutation is 4 times lower than that of the Fc(γ)RI binding rate of the insulin-Fc fusion protein of SEQ ID NO:52, the insulin-Fc fusion protein of SEQ ID NO:60 containing the cNg-S mutation cannot demonstrate repeated administration biological activity in dogs.

[0340]

[0341] In order to determine whether the insulin-Fc fusion protein of SEQ ID NO:64 containing cNg-K mutation improves the repeated administration bioactivity performance in dogs, the compound was subcutaneously administered to N=1 dogs on days 0, 7, 14 and 28 according to the procedure of Example 11. When the %FBGL of the dog dropped too low, food was provided to the dog to raise blood sugar to a safe level. The NAOC of the first injection was 449%FBGL·day·kg / mg, indicating that the insulin-Fc fusion protein of SEQ ID NO:64 has satisfactory bioactivity in vivo. The pharmacokinetic profile of the compound was also measured using ELISA by the method of Example 12, and a two-compartment model was fitted to the data to determine its elimination half-life (about 0.9 days). The NAOC and NAOCR of each subsequent administration were also measured according to the general procedure of Example 11, calculated from the time of the administration of the dose until just before the administration of the next dose. The NAOC and NAOCR shown in Table 8 indicate that the insulin-Fc fusion protein of SEQ ID NO: 64 maintained a NAOCR greater than 0.6 throughout the four doses. Thus, unexpectedly, the insulin-Fc fusion protein of SEQ ID NO: 64 containing the cNg-K mutation is the only non-glycosylated mutant of the insulin-Fc fusion protein of SEQ ID NO: 52, which significantly improves the repeated administration bioactivity in dogs.

[0342]

[0343] According to Example 13, the levels of anti-drug and anti-insulin antibodies were also measured throughout the treatment course (28 days) and for an additional two weeks. Fig. 9 It was demonstrated that the insulin-Fc fusion protein of SEQ ID NO: 64 still produced anti-drug antibodies under repeated subcutaneous administration in dogs, but the anti-drug antibody titers were much lower than those produced by the insulin-Fc fusion protein of SEQ ID NO: 52 (Example 19).

[0344] Example 24: Screening of canine sera for anti-drug antibodies and identification of the B10D and A8H sites of the insulin polypeptide Potential immunogenic epitopes

[0345] Mutating the cNg site of the canine IgGB Fc fragment to Lys (i.e., cNg-K) did improve the repeated administration biological activity of the insulin-fusion protein comprising the insulin polypeptide of SEQ ID NO: 5 and the peptide linker of SEQ ID NO: 12 (Example 23), but the resulting insulin-Fc fusion protein of SEQ ID NO: 64 still produced anti-drug antibodies (Example 23). Therefore, it is assumed that the insulin polypeptide of SEQ ID NO: 5 may unexpectedly contain a specific epitope (i.e., an immunogenic "hot spot") that is targeted by the dog's immune system. Therefore, the binding specificity of the antibodies present in the serum samples described in Example 13 was evaluated according to the general procedures of Example 15. Analysis of antibody-containing serum samples from repeated administration of insulin-Fc fusion proteins of SEQ ID NO: 52 (Example 19) to coated insulin-Fc fusion protein libraries showed that there were unexpectedly two major "hot spots" in the insulin polypeptide sequence of SEQ ID NO: 5: an aspartic acid mutation at position 10 from the N-terminus of the B chain (i.e., B10), and a histidine mutation at position 8 from the N-terminus of the A chain (i.e., A8). The results indicate that insulin-Fc fusion proteins containing insulin polypeptide amino acid compositions containing these two specific amino acid mutations may be immunogenic in dogs, and thus may generate anti-drug antibodies that neutralize biological activity after repeated injections. Therefore, it can be determined that insulin polypeptides that do not contain B10 aspartic acid and A8 histidine are preferably used for insulin-Fc fusion proteins that require long-term repeated administration in dogs (e.g., for the treatment of canine diabetes).

[0346] Example 25: Insulin polypeptide comprising SEQ ID NO: 5 and non-glycosylated canine IgGB isotype Fc fragment The insulin-Fc fusion protein of the present invention is a fragment of the insulin polypeptide, wherein the B10D and A8H mutations of the insulin polypeptide are restored to the natural composition to reduce the immune Potential risks of

[0347] To evaluate whether replacing the "hotspot" mutations would improve the immunogenicity and repeated-dosing bioactivity of an insulin-Fc fusion protein comprising an insulin polypeptide of SEQ ID NO:5 and a canine IgGB isotype fragment, an exemplary insulin-Fc fusion protein (SEQ ID NO:66) was synthesized in which the B10 and A8 amino acids of the insulin polypeptide were restored to their natural histidine and threonine composition, respectively (SEQ ID NO:125 listed below, in which the non-natural amino acids are underlined).

[0348] FVNQHLCGSHLVEAL A LVCGERGFFYT DP T GGGPRR GIVEQCCTSICSLYQLENYCN(SEQ ID NO:125)

[0349] In addition, considering the additional potential benefits of the non-glycosylated cNg mutant, the insulin-Fc fusion protein of SEQ ID NO: 66 contains a cNg-Q mutation. The complete amino acid sequence of the insulin-Fc fusion protein of SEQ ID NO: 66 is given below:

[0350] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFQGTYRVVSV LPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:66)

[0351] The insulin-Fc fusion protein of SEQ ID NO:66 was prepared in HEK293 cells according to Example 1 and purified using a protein A column according to Example 3. The resulting protein yield was only 21 mg / L. The structure was confirmed by non-reducing and reducing CE-SDS according to Example 4, and the sequence was further identified by LC-MS with glycans removed according to Example 5. The homodimer % measured by size exclusion chromatography according to Example 6 was 98.0%, indicating that the protein was relatively free of aggregates.

[0352] Despite the relatively low homodimer titer of 21 mg / L, the in vivo bioactivity and immunogenicity of the insulin-Fc fusion protein of SEQ ID NO: 66 were evaluated in dogs according to the procedures of Examples 11-13, respectively. Fig.10 It was shown that restoring the B10D and A8H mutations to their natural amino acids (ie, B10H and A8T) in the insulin-Fc fusion protein of SEQ ID NO: 66 did significantly reduce the immunogenicity of the parent compound (SEQ ID NO: 52).

[0353] However, if Fig.11 As shown, the insulin-Fc fusion protein of SEQ ID NO: 66 containing native B10 and A8 amino acids has no biological activity (ie, NAOC is essentially zero).

[0354] Example 26: Attempt to incorporate additional B chain and A chain mutations into the insulin polypeptide of SEQ ID NO: 125 to Improving the biological activity of related insulin-Fc fusion proteins containing canine IgGBFc fragments

[0355] The fact that the insulin-Fc fusion protein of SEQ ID NO: 66 did not generate anti-drug antibodies (Example 25) compared to the insulin-Fc fusion protein of SEQ ID NO: 52 (Example 20) provides strong evidence for the theory that the B10D and A8H mutations in the insulin polypeptide of SEQ ID NO: 5 may be immunogenic epitopes responsible for generating anti-drug antibodies. However, the lack of in vivo potency of the insulin-Fc fusion protein of SEQ ID NO: 66 compared to SEQ ID NO: 52 suggests that these two amino acid mutations also contribute to achieving acceptable levels of biological activity. The lack of in vivo potency of the insulin-Fc fusion protein of SEQ ID NO: 66, as measured by an insulin receptor binding assay according to the method of Example 7, correlates with its high IC50 (as shown in Table 9 below). Therefore, further efforts are needed to increase the biological activity of insulin-Fc fusion proteins (i.e., to reduce the insulin receptor binding assay IC50 value to less than 5000 nM, or more preferably less than 4000 nM, or even more preferably less than 3000 nM) while maintaining a low degree of immunogenicity by retaining the native B10 and A8 amino acids in the insulin polypeptide.

[0356] As is well known, each part of insulin B chain and A chain is necessary for strong binding to IR (Hubbard SR, "Structural biology: Insulin meets its receptor", Nature. 2013; 493 (7431): 171-172). Therefore, part of B chain or A chain is modified, while keeping B10 and A8 the same as native insulin and C chain and peptide linker constant. According to Example 1, several of these insulin-Fc fusion proteins are prepared in HEK293 cells, and according to Example 3, protein A column purification is used. According to Example 4, their structure is confirmed by non-reducing and reducing CE-SDS, and according to Example 5, the sequence is further identified by LC-MS removing polysaccharides. According to Example 6, their homodimer content % is measured by size exclusion chromatography, and their insulin receptor binding affinity is measured according to Example 7. Their sequences are shown below, and the resulting sequences are compared for SEQ ID NO: 66 in Fig.12 Shown in (Clustal Omega).

[0357] FVNQHLCGSHLVQALYLVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:68)

[0358] FVNQHLCGSELVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:70)

[0359] FVNQHLCGSHLVEAALALVCGEAGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:72)

[0360] FVNQHLCGSHLVEALALVCGERGFYYTDPTTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSV LPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:74)

[0361] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSV LPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:76)

[0362]

[0363] Compared to SEQ ID NO: 66, only in three cases (SEQ ID NO: 68, 70 and 74) did the proposed mutations improve IR binding (i.e., reduce IC50 values). However, none of these mutations produced compounds that met the production design goal of homodimer titers greater than 50 mg / L, and in some cases, these mutations resulted in significantly reduced manufacturability (e.g., homodimer titers less than 20 mg / L).

[0364] Example 27: Attempt to incorporate C-chain mutations into the insulin polypeptide of SEQ ID NO: 125 to improve the canine Biological activity of insulin-Fc fusion protein related to animal IgGB Fc fragment

[0365] The results obtained in Example 26 show that the attempts of all mutant SEQ ID NO:125 insulin polypeptide A chain and B chain result in unacceptable low HEK homodimer titers (i.e., homodimer titers less than or equal to 25 mg / L) of related insulin-Fc fusions. Therefore, it is necessary to conduct further experiments. In the present example, the C chain composition of the insulin polypeptide of SEQ ID NO:125 is mutated by making it longer or by increasing its flexibility. It has been shown that natural insulin (e.g., human insulin) undergoes significant conformational changes when combined with the insulin receptor, including the movement of B chain and A chain folding (e.g., such as Menting et al., Nature, 2013; 493 (7431): described in pp241–245). Different from the insulin polypeptide of the present invention, natural insulin can freely undergo this conformational change at the insulin receptor because it is a double-chain polypeptide in its native form, connected only by two disulfide bonds, without the mobility of C chain limiting A chain and B chain. Without being bound by any particular theory, it is hypothesized that the C chain contained in the insulin polypeptide of SEQ ID NO: 125 is too rigid (e.g., the amino acid composition and sequence does not allow easy movement between the B chain and the A chain) and / or too short (e.g., there are not enough amino acids between the C terminus of the B chain and the N terminus of the A chain), thereby preventing the insulin polypeptide from undergoing the necessary changes in molecular shape required for strong binding to the insulin receptor. Therefore, several insulin-Fc fusion proteins were synthesized based on the insulin-Fc fusion protein of SEQ ID NO: 66, wherein the changes in the insulin polypeptide C chain are shown below, wherein the resulting sequences are aligned against SEQ ID NO: 66 in Fig.13 Shown in (Clustal Omega).

[0366] FVNQHLCGSHLVQALYLVCGERGFFYTDPTQRGGGGGQRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSV LPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQID NO:78)

[0367] FVNQHLCGSHLVVEALALVCGERGFFYTDPTGGGGGGSGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVV SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPDIVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:80)

[0368] FVNQHLCGSHLVEAALALVCGERGFFYTDPGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:82)

[0369] FVNQHLCGSHLVEALALVCGERGFFYTPGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:84)

[0370]

[0371] Insulin-Fc fusion proteins were prepared in HEK293 cells according to Example 1, and purified using protein A columns according to Example 3. According to Example 4, their structures were confirmed by non-reducing and reducing CE-SDS, and according to Example 5, sequences were further identified by LC-MS removing polysaccharides. According to Example 6, their homodimer content % was measured by size exclusion chromatography, and according to Example 7, their insulin receptor binding affinity was measured. Only in one case (comprising the longest C chain (GGGGGGSGGGG) (SEQ ID NO:80)), compared with the insulin-Fc fusion protein of SEQ ID NO:66, the C chain mutation did significantly improve the insulin receptor binding affinity (IC50 was less than 3000nM). However, these insulin-Fc fusion proteins mutated by the C chain did not show a homodimer titer higher than the production design target of 50mg / L. In fact, in one case (SEQ ID NO:78), the C chain mutation unexpectedly resulted in a significantly lower homodimer titer.

[0372] Example 28: Attempting to incorporate peptide linker mutations into insulin polypeptides containing SEQ ID NO: 125 and canine IgGB Fc fragment in insulin-Fc fusion protein to improve biological activity

[0373] Without being bound by any particular theory, another possible reason for the poor insulin receptor binding of the insulin-Fc fusion protein of SEQ ID NO:66 is believed to involve steric hindrance between the insulin polypeptide and the insulin receptor, which is caused by the close proximity of the much larger Fc fragment molecule connected to the insulin polypeptide by a peptide linker. Shorter peptide linkers or more tightly folded peptide linkers are believed to exacerbate this problem, while longer peptide linkers or self-anti-folding peptide linkers (e.g., linkers with greater molecular rigidity) may alleviate this problem by creating more space between the insulin polypeptide and the Fc fragment. The increased space between the insulin polypeptide and the Fc fragment will also increase the distance between the insulin receptor and the Fc fragment, resulting in less interference during insulin receptor binding. The peptide linker (i.e., GGGGAGGGG) of SEQ ID NO:12 used to construct the insulin-Fc fusion protein of SEQ ID NO:66 is assumed to be too short and / or too flexible because the amino acids comprising the linker do not contain side chains (i.e., it only contains glycine and alanine amino acids). Therefore, to test this hypothesis, two additional insulin-Fc fusion protein variants of the insulin-Fc fusion protein of SEQ ID NO: 66 were synthesized. The insulin-Fc fusion protein of SEQ ID NO: 76 contains the same peptide linker as used to construct the insulin-Fc fusion protein of SEQ ID NO: 66, but with an insulin polypeptide in which the asparagine (i.e., A21) at position 21 from the N-terminus of the A chain is absent (i.e., des-A21). This particular mutation was incorporated to observe whether the connection between the A chain and the peptide linker affects protein yield and / or the biological activity of the molecule. The other insulin-Fc fusion protein of SEQ ID NO: 86 contains this des-A21N A chain mutation and a peptide linker that is more than twice the length of the insulin-Fc fusion protein used to construct SEQ ID NO: 66. In this longer peptide linker, alanine is not favored and is replaced by glutamine, which contains a polar amide side chain. The glutamine substitution is expected to increase the hydrophilicity of the peptide linker and potentially prevent the linker from folding onto itself. The sequence is shown below, wherein the resulting sequence alignment relative to SEQ ID NO: 66 is Fig.14 Shown in (Clustal Omega).

[0374] FVNQHLCGSHLVVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGGQGGGGQGGGGQGGGGDGCPKCPAPEMLGGPSVFIFPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPDIVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHPG(SEQ ID NO:86)

[0375] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:76)

[0376]

[0377]

[0378] According to example 1, two kinds of insulin-Fc fusion proteins were prepared in HEK293 cells, and protein A column purification was used according to example 3. According to example 4, their structures were confirmed by non-reducing and reducing CE-SDS, and according to example 5, the sequences were further identified by LC-MS removing polysaccharides. According to example 6, their homodimer content % was measured by size exclusion chromatography, and according to example 7, their insulin receptor binding affinity was measured. The longer peptide linker (GGGGGQGGGGQGGGGGGGGGG of SEQ ID NO:86 relative to GGGGAGGGG of SEQ ID NO:66) mixed with different compositions did improve insulin receptor binding, as measured by the significant reduction of IC50 values, which shows that longer linkers may be strategies for increasing the insulin receptor binding of other insulin-Fc fusion proteins. However, mixing longer linkers still did not increase homodimer titer to a production design target greater than 50mg / L.

[0379] Example 29: Attempting to delete a portion of the B chain of the insulin polypeptide of SEQ ID NO: 125, To increase the content of canine Homodimer titer of insulin-Fc fusion protein related to IgGBFc fragment

[0380] The result from Example 28 shows that the peptide linker can be modified to increase the insulin receptor binding affinity of the insulin-Fc fusion protein of SEQ ID NO:66, and the insulin-Fc fusion protein comprises natural B10 and A8 amino acids. However, the peptide linker mutation failed to increase the homodimer titer to be enough to meet the production design target. Since the homodimer titer is a function of several properties (including intracellular synthesis and intracellular processing), it is assumed that the insulin-Fc molecule may carry out intramolecular self-association (i.e. aggregation) between the two monomers of the homodimer during synthesis and after synthesis, or carry out intermolecular self-association between two or more independent homodimers. This aggregation will result in obtaining unacceptable low homodimer titer from the cell culture supernatant during the production process described in Examples 1, 3 and 6. This potential interaction between the insulin-Fc fusion protein molecules may be partly due to the well-known self-association of insulin and the tendency to form aggregates. A method for reducing the self-association tendency of insulin known in the art includes mutating the amino acids near the C-terminal of the B chain. For example, insulin lispro (B28K; B29P mutation) and insulin aspart (B28D mutation) are well-known commercial two-chain insulins with non-natural B chain mutations, which prevent association and aggregation, thereby producing the main monomer form of insulin in solution. Another method to prevent aggregation involves amino acid structural deletion. For example, the two-chain insulin (DPPI; see Brange J., Dodson GG, Edwards J., Holden PH, Whittingham J.L. 1997b. "A model of insulin fibrils derived from the x-ray crystal structure of a monomeric insulin (despentapeptide insulin)" Proteins 27 507–516) known as depentapeptide insulin is the same as natural two-chain human insulin, except that the five C-terminal amino acids of the B chain (YTPKT) are removed. Compared with natural two-chain human insulin, DPPI has a lower binding affinity to the insulin receptor, but is completely monomeric in solution, which means that there is no significant association or aggregation between DPPI molecules. Therefore, in an attempt to reduce the possibility of intramolecular and intermolecular self-association and to increase insulin-Fc fusion protein homodimer titer, several variants of the insulin-Fc fusion protein of SEQ ID NO: 66 were constructed using partial B chain amino acid truncation and B chain amino acid mutations, as described above for DPPI, lispro insulin and aspart insulin. The sequences are shown below, where the resulting sequence alignment relative to SEQ ID NO: 66 is Fig.15Shown in (Clustal Omega).

[0381] FVNQHLCGSHLVEALALVCGERGFFYTDPGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:82)

[0382] FVNQHLCGSHLVEALALVCGERGFFYTPGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:84)

[0383] FVNQHLCGSHLVEALALVCGERGFFYTQGGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSV LPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:88)

[0384]

[0385] Insulin-Fc fusion proteins were prepared in HEK293 cells according to Example 1 and purified using a protein A column according to Example 3. Their structures were confirmed by non-reducing and reducing CE-SDS according to Example 4, and the sequences were further identified by LC-MS with glycan removal according to Example 5. Their homodimer content % was measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinity was measured according to Example 7. Only in one case (SEQ ID NO: 82) was the homodimer titer of the resulting compound significantly increased, but surprisingly, for all mutated compounds, the insulin receptor affinity was improved (SEQ ID NO: 82, 88 and 84).

[0386] Example 30: Attempt to combine B chain, C chain and A chain mutations, B chain truncation and linker mutations with islet of SEQ ID NO: 66 Combined with TNF-α-Fc fusion protein to further improve homodimer titer and biological activity

[0387] As shown in Examples 26, 27, 28 and 29, no single strategy successfully incorporates insulin polypeptides containing non-immunogenic natural B10 and A8 amino acids with canine IgGB Fc fragments to form insulin-Fc fusion proteins with acceptable insulin receptor activity and homodimer titers. Therefore, the concepts of longer C chains, longer peptide linkers and C-terminal amino acid truncations of B chains are combined. In addition, in order to potentially further reduce the tendency of self-association and aggregation, lower hydrophobic amino acids (including amino acids with negatively charged or positively charged side groups at physiological pH) are used to introduce additional point mutations into the hydrophobic amino acid residue sites of natural insulin. Exemplary mutations include tyrosine to alanine, tyrosine to glutamic acid, isoleucine to threonine and phenylalanine to histidine. In addition, in order to simplify the analysis, in all cases, the cNg site of the canine IgGB Fc fragment is restored to its natural asparagine. The sequences of these insulin-Fc fusion protein variants are as follows, wherein the resulting sequence alignment relative to SEQ ID NO:66 is in Fig.16 Shown in (Clustal Omega).

[0388] FVNQHLCGSHLVEALELVCGERGFFYTPKTGGGSGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:90)

[0389] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNHGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:92)

[0390] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:34)

[0391] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:32)

[0392] FVNQHLCGSHLVEALELVCGERGFFYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:94)

[0393]

[0394] Insulin-Fc fusion proteins were prepared in HEK293 cells according to Example 1 and purified using a protein A column according to Example 3. According to Example 4, their structures were confirmed by non-reducing and reducing CE-SDS, and according to Example 5, the sequences were further identified by LC-MS with glycans removed. According to Example 6, their homodimer content % was measured by size exclusion chromatography, and according to Example 7, their insulin receptor binding affinity was measured. The results show that a combination of reducing the hydrophobicity of certain B-chain and A-chain amino acids, using longer and more flexible C-peptide sequences, truncating several C-terminal B-chain amino acids, and using longer peptide linkers leads to several useful insulin-Fc fusion proteins that meet the minimum homodimer titer and insulin receptor binding activity design criteria. SEQ ID NO: 92, 34, 32 and 94 (368d), (366d), (218d) and (375d) show more preferred insulin receptor IC50 values ​​(less than 3000 nM) and more preferred HEK homodimer titer values ​​(greater than 100 mg / L) compared to SEQ ID NO: 66 or SEQ ID NO: 90. Surprisingly, changing only a few amino acids resulted in a multiple-fold improvement in insulin receptor affinity, and in the case of the insulin-Fc fusion protein of SEQ ID NO: 32, the homodimer titer was significantly increased relative to the original insulin-Fc fusion protein of SEQ ID NO: 66.

[0395] Example 31: An insulin polypeptide of SEQ ID NO: 7, a peptide linker of SEQ ID NO: 14 and a peptide of SEQ ID NO: 16 In vivo bioactivity, repeated administration bioactivity and immunohistochemistry of insulin-Fc fusion protein constructed from canine IgGBFc fragment Immunogenicity

[0396] In view of the positive homodimer titer and insulin receptor binding activity from Example 30, two most promising insulin-Fc fusion proteins (SEQ ID No: 32 and 34) were tested in dogs to evaluate the biological activity and immunogenicity of repeated administration. Each compound contains a longer, more hydrophilic peptide linker of SEQ ID NO: 14 and a canine IgGB Fc fragment of SEQ ID NO: 16 that is easier to manufacture and less aggregated. Most importantly, both insulin-Fc fusion proteins contain insulin polypeptides with natural B10 and A8 amino acids with assumed lower immunogenicity (i.e., universal SEQ ID NO: 7). In the case of the insulin-Fc fusion protein of SEQ ID NO: 34, there is an asparagine at position A21 (i.e., the insulin polypeptide contains SEQ ID NO: 9). In the case of the insulin-Fc fusion protein of SEQ ID NO: 32, the asparagine at position A21 does not exist (i.e., the insulin polypeptide contains SEQ ID NO: 8).

[0397] According to the procedure of Example 10, the in vivo biological activity of the insulin-Fc fusion protein of SEQ ID NO: 34 was tested in N=1 dogs. Fig.17 The results of a single subcutaneous administration shown in , demonstrate that the insulin-Fc fusion protein of SEQ ID NO: 34 is indeed biologically active in vivo, wherein the NAOC calculated according to the procedure in Example 11 is 1076% FBGL·day·kg / mg. The pharmacokinetic profile of the insulin-Fc fusion protein of SEQ ID NO: 34 was measured by the method of Example 12 using an ELISA method, and a two-compartment model was fitted to the data to determine its elimination half-life (which was 3.5 days).

[0398] Then, according to the procedure of Example 8, the insulin-Fc fusion protein of SEQ ID NO:34 was continued to be administered subcutaneously to N=1 dog on the 14th day, the 28th day and the 42nd day after the initial injection to evaluate the repeated administration biological activity. When the %FBGL of the dog dropped too low, food was provided to the dog to raise blood sugar to a safe level. According to the general procedure of Example 11, the NAOC and NAOCR of each subsequent administration were measured, starting from the time of the administration of the dose until just before the next administration of the dose. The NAOC and NAOCR shown in Table 14 show that the insulin-Fc fusion protein of SEQ ID NO:34 maintained a NAOCR greater than 0.8 throughout the four administrations, thus meeting the design target of repeated administration biological activity.

[0399]

[0400] The immunogenicity of the insulin-Fc fusion protein of SEQ ID NO: 34 was tested according to the procedure of Example 13. Fig.18 This indicates that the insulin-Fc fusion protein of SEQ ID NO: 34 does not exhibit significant immunogenicity in vivo, which is consistent with maintaining in vivo biological activity throughout the repeated administration experiment.

[0401] The repeated administration biological activity performance of the insulin-Fc fusion protein of SEQ ID NO:32 (wherein the asparagine at A21 of the insulin polypeptide chain is deleted) in dogs was also evaluated. According to the procedure of Example 11, the compound was subcutaneously administered to N=1 dogs on days 0, 14, 28 and 42. When the %FBGL of the dog dropped too low, food was provided to the dog to raise blood sugar to a safe level. The NAOC of the first injection was an impressive 2278%FBGL·day·kg / mg, indicating that the insulin-Fc fusion protein of SEQ ID NO:32 had satisfactory in vivo biological activity, which was almost twice the potency of the insulin-Fc fusion protein of SEQ ID NO:34. Using ELISA, the pharmacokinetic profile of the insulin-Fc fusion protein was measured by the method of Example 12, and a two-compartment model was fitted to the data to determine its elimination half-life (which was 4.1±0.7 days). Fig.19 and 20 Single dose glycemic control and multiple dose, multi-week glycemic control of animals receiving homodimers of SEQ ID NO:32 are shown. The NAOC and NAOCR of each subsequent administration were also measured according to the general procedure of Example 11, calculated from the time of the administration of the dose until just before the next dose was administered. The NAOC and NAOCR shown in Table 15 show that the insulin-Fc fusion protein of SEQ ID NO:32 maintains a NAOCR greater than or equal to 1.0 during four administrations, thus meeting the repeated administration bioactivity design target described in Example 16.

[0402] The immunogenicity of the insulin-Fc fusion protein of SEQ ID NO: 32 was tested according to the procedure of Example 13. Fig.21 This indicates that the insulin-Fc fusion protein of SEQ ID NO: 32 does not exhibit significant immunogenicity in vivo, which is consistent with maintaining in vivo biological activity throughout the repeated administration experiment.

[0403]

[0404] As discussed in the specific embodiments of the present invention, known enzyme cleavage sites exist between asparagine-glycine bonds (Vlasak, J., Ionescu, R., (2011) MAbs Vol.3, No.3pp 253-263). The asparagine at the 21st amino acid (i.e., A21) of the A chain in the insulin polypeptide of SEQ ID NO:8 contained in the insulin-Fc fusion protein of SEQ ID NO:32 and the peptide linker of SEQ ID NO:14 eliminate the possibility of enzyme cleavage of the asparagine-glycine bond between the C-terminus of the A chain and the N-terminus of the peptide linker. However, the insulin-Fc fusion protein of SEQ ID NO:34 contains the peptide linker of SEQ ID NO:14 and the insulin polypeptide of SEQ ID NO:8, which retains asparagine at A21. Therefore, it is expected that the insulin-Fc fusion protein of SEQ ID NO:34 will be enzymatically digested during synthesis or after subcutaneous administration in vivo. However, unexpectedly, the insulin-Fc fusion protein of SEQ ID NO: 34 can be produced in HEK cells with acceptable homodimer titers and exhibits acceptable biological activity in vivo, with no evidence that enzymatic digestion impairs its biological activity.

[0405] Example 32: For a preferred insulin polypeptide comprising SEQ ID NO: 8 and a preferred peptide comprising SEQ ID NO: 14 Optimal Manufacturability and In Vivo Efficacy of Canine IgGB Isotype Fc Fragments with Linkers for Insulin-Fc Fusion Proteins recognize

[0406] As described in Examples 30 and 31, after discovering that novel insulin polypeptide and peptide linker combinations produce non-immunogenic, high-yield, high-purity and high-bioactive insulin-Fc fusion proteins, there remains a question as to whether canine IgGB Fc fragments are still the preferred isotype in terms of homodimer titer and bioactivity, as in the case of the insulin-Fc fusion proteins in Examples 19 and 20. Therefore, additional insulin-Fc fusion proteins were designed in which the insulin polypeptide (SEQ ID NO: 8) and peptide linker (SEQ ID NO: 14) of the insulin-Fc fusion protein of SEQ ID NO: 32 were kept constant, and the canine IgGB Fc fragment of SEQ ID NO: 16 was replaced by the canine IgGA Fc fragment of SEQ ID NO: 15, the canine IgGC Fc fragment of SEQ ID NO: 17, or the canine IgGD Fc fragment of SEQ ID NO: 18. The sequences of these resulting insulin-Fc fusion protein variants are shown below:

[0407] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:32)

[0408] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG(SEQ ID NO:96)

[0409] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG(SEQ ID NO:98)

[0410] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG(SEQ ID NO:100)

[0411] Insulin-Fc fusion proteins were prepared in HEK293 cells according to Example 1 and purified using protein A or protein G columns according to Example 3. According to Example 4, their structures were confirmed by non-reducing and reducing CE-SDS, and the sequences were further identified by LC-MS with glycan removal according to Example 5. According to Example 6, their homodimer content % was determined by size exclusion chromatography, and their insulin receptor binding affinity was measured according to Example 7. In addition, the affinity of insulin-Fc fusion proteins for canine FcRn receptors was measured according to Example 8. As shown in Table 16, the insulin-Fc fusion protein of SEQ ID NO: 32 containing canine IgGB Fc fragment showed the highest homodimer titer of these sequences. When purified using a protein A column, the insulin-Fc fusion protein of SEQ ID NO: 96 containing canine IgGA Fc fragment showed poor homodimer titer; however, when purified using a protein G column, the homodimer titer was significantly improved, exceeding the design target of greater than 50 mg / L. This was also true for the insulin-Fc fusion protein of SEQ ID NO: 98, which contains a canine IgGC Fc fragment. The insulin-Fc fusion protein of SEQ ID NO: 100, which contains a canine IgGD Fc fragment, did not produce any compound when purified with a protein A or protein G column. Thus, as demonstrated with the insulin-Fc fusion protein of SEQ ID NO: 52, which contains a different insulin polypeptide (SEQ ID NO: 5) and a peptide linker (SEQ ID NO: 12), canine IgGB is the preferred Fc fragment in terms of homodimer titer (see Example 19).

[0412]

[0413] DNM = not measured; # =Purified by Protein A; = Purified by Protein G.

[0414] The in vivo biological activity of the insulin-Fc fusion protein of SEQ ID NO: 96 comprising a canine IgGA Fc fragment purified via protein G was tested according to the procedure of Example 10. Fig. 22 The results shown indicate that the insulin-Fc fusion protein of SEQ ID NO: 96 has only a certain biological activity in vivo, wherein the NAOC calculated according to Example 11 is only 174% FBGL·day·kg / mg.

[0415] According to the procedure of Example 10, the in vivo biological activity of the insulin-Fc fusion protein of SEQ ID NO: 98 comprising a canine IgGC Fc fragment was purified by tested protein G. Fig.23The results shown indicate that the insulin-Fc fusion protein of SEQ ID NO: 98 has only a certain biological activity in vivo, wherein the NAOC calculated according to Example 11 is only 39% FBGL·day·kg / mg.

[0416] Thus, canine IgGB is the preferred Fc fragment in terms of biological activity as demonstrated with the insulin-Fc fusion protein of SEQ ID NO:52 containing a different insulin polypeptide (SEQ ID NO:5) and a peptide linker (SEQ ID NO:12) (see Examples 19 and 20 above and Table 16).

[0417] Example 33: Insulin polypeptide comprising SEQ ID NO: 8, a peptide linker of SEQ ID NO: 14, and canine IgGB Non-glycosylated Fc fragment of insulin-Fc fusion protein to reduce the potential risk of immunogenicity

[0418] Although the insulin-Fc fusion protein of SEQ ID NO: 32 meets all design goals (Example 16), there may or may not be a risk of immunogenicity over an extended treatment period (e.g., 6 months, 1 year, 2 years, or longer), which may compromise the use of the insulin-Fc fusion protein for the treatment of diabetes (if this occurs). As described in the specific embodiments of the present invention and in Examples 21 and 22, one possible reason for the reduction in bioactivity after repeated administration is the undesirable interaction of the canine IgGB Fc fragment with the dog's immune system, resulting in the production of neutralizing anti-drug antibodies. However, the results shown in Example 32 unexpectedly demonstrated that the canine IgGB isotype was the only option among the four canine IgG isotypes that produced the desired manufacturability and bioactivity. Therefore, further Fc mutations were explored to obtain a non-glycosylated insulin-Fc fusion protein with low Fc(γ)RI receptor binding, which could reduce the risk of long-term, chronic immunogenicity.

[0419] As described in a specific implementation of the present invention, a method for reducing Fc(γ)RI interactions includes mutating the cNg site of the Fc fragment to prevent glycosylation during synthesis in the host cell. Therefore, the cNg site mutation is performed on the Fc fragment region of SEQ ID NO:32 to reduce the binding affinity of the Fc fragment to the Fc(γ) receptor in vivo, as measured by the in vitro human Fc(γ)RI binding assay described in Example 8. The position of the cNg site in the insulin-Fc fusion protein of SEQ ID NO:32 is cNg-NB151. Mutations of SEQ ID NO:32 include SEQ ID NO:104 (comprising the cNg-NB151-S mutation) and SEQ ID NO:102 (comprising the same cNg-NB151-S mutation and the NB119-A mutation). In order to further try to reduce the interaction with Fc (γ) RI, NB119-A was incorporated, as described in Lo, M. et al. "Effector attenuating substitutions that maintain antibody stability and reduce toxicity in mice", J. Biol. Chem. (2017), pp. 1-20, which was used only in mouse antibodies. The complete amino acid sequence of the resulting insulin-Fc fusion protein and its sequence alignment (Clustal Omega) are listed below (NB119 and NB151 sites are underlined for clarity), as shown Fig.24 As shown:

[0420] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVV A LDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:102)

[0421] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:104)

[0422] Insulin-Fc fusion proteins were prepared in HEK293 cells according to Example 1 and purified using a protein A column according to Example 3. Their structures were confirmed by non-reducing and reducing CE-SDS according to Example 4, and the sequences were further identified by LC-MS with glycans removed according to Example 5. Their homodimer content % was measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinity was measured according to Example 7. As shown in Table 17, incorporation of the cNg-NB151-S mutation on the Fc fragment reduced the homodimer %, indicating that the aggregation level was unacceptably high (i.e., the homodimer % was reduced to slightly above 70%).

[0423]

[0424] According to the procedure of Example 10, the in vivo biological activities of insulin-Fc fusion proteins of SEQ ID NO: 102 and SEQ ID NO: 104 were tested in N=1 dogs, respectively. Fig.25 The results of a single subcutaneous administration shown in the Figures demonstrate that the biological activity of the two compounds in vivo is significantly lower than that of the insulin-Fc fusion protein of SEQ ID NO: 32 (NAOC of SEQ ID NO: 104 = 574% FBGL·day·kg / mg; NAOC of SEQ ID NO: 102 = 921% FBGL·day·kg / mg). The results indicate that incorporation of the cNg-NB151-S mutation on the Fc fragment to produce a non-glycosylated version of the insulin-Fc fusion protein of SEQ ID NO: 32 unexpectedly reduces the in vivo biological activity of the resulting compound.

[0425] In an attempt to reduce the aggregation and improve the biological activity of the insulin-Fc fusion protein of SEQ ID NO:104 containing the cNg-NB151-S site mutation, various insulin-polypeptide B chain variants were studied, with mutations in the region believed to be responsible for aggregation. The insulin-Fc fusion protein was prepared in HEK293 cells according to Example 1 and purified using a protein A column according to Example 3. According to Example 4, their structures were confirmed by non-reducing and reducing CE-SDS, and according to Example 5, the sequences were further identified by LC-MS with glycans removed. According to Example 6, their homodimer content % was measured by size exclusion chromatography. Among the tested B chain variants, it was unexpectedly found that an insulin Fc-fusion protein (SEQ ID NO:36) contained tyrosine to alanine substitution from the 16th amino acid at the N-terminal of the B chain (i.e., B16), and the insulin Fc-fusion protein had a high homodimer titer (105 mg / L) and low aggregation (99% homodimer), resulting in a homodimer titer of 104 mg / L. The insulin receptor binding measured according to Example 7 was acceptable, with an IC50 of 2040 nM. The FcRn receptor binding affinity EC50 value measured according to Example 9 was 1194 ng / mL. Using ELISA, the pharmacokinetic profile of the insulin-Fc fusion protein of SEQ ID NO:36 was measured by the method of Example 12, and a two-compartment model was fitted to the data to determine its elimination half-life (which was 4.1 ± 0.7 days). The sequence of SEQ ID NO:36 is shown below (the B16A and cNg-NB151-S mutations are underlined for clarity).

[0426] FVNQHLCGSHLVEAL A LVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:36)

[0427] The repeated administration bioactivity performance of the insulin-Fc fusion protein of SEQ ID NO:36 in dogs was then evaluated. According to the procedure of Example 11, the compound was subcutaneously administered to N=1 dogs on days 0, 7, 14, and 28. When the dog's %FBGL dropped too low, food was provided to the dog to raise blood sugar to a safe level. Unexpectedly, the NAOC of the insulin-Fc fusion protein of SEQ ID NO:36 containing the B16A mutation was significantly higher (1185% FBGL·day·kg / mg) for the first injection compared to the insulin-Fc fusion protein of SEQ ID NO:104. The in vivo bioactivity graph of the first administration is shown in FIG. Fig.26 14. The pharmacokinetic profile of the compound was measured by ELISA using the method of Example 12, and a two-compartment model was fitted to the data to determine its elimination half-life (which was 3.5 days). The NAOC and NAOCR of each subsequent administration were also measured according to the general procedure of Example 11, and were calculated from the time of the applied dosage until just before the next dose was applied. The NAOC and NAOCR shown in Table 18 show that the insulin-Fc fusion protein of SEQ ID NO:36 keeps a NAOCR greater than or equal to 0.6 throughout the four administrations, thus meeting the design target of repeated administration bioactivity. In a word, the result shows that it is necessary to mutate the insulin B chain sequence to obtain the suitable non-glycosylated cNg-S variant of SEQ ID NO:32. Therefore, for the non-glycosylated insulin-Fc fusion protein of the canine IgGB Fc fragment comprising cNg mutation, the insulin polypeptide of SEQ ID NO:11 is preferred.

[0428]

[0429] Finally, the possibility of the selected compounds interacting with the immune system was tested by measuring their Fc (γ) receptor binding activity according to the procedure of Example 8. Table 19 compares the Fc (γ) receptor I binding of these insulin-Fc fusion proteins with the Fc (γ) receptor binding of the insulin-Fc fusion protein of SEQ ID NO: 52. It can be seen that the non-glycosylated insulin-Fc fusion protein (achieved by cNg-S mutation) exhibits the lowest Fc (γ) receptor binding ratio with SEQ ID NO: 52.

[0430]

[0431] Example 34: Use of Fc fragments containing canine IgGB origin produced by stably transfected CHO cell lines Exemplary CHO-based production runs of preferred insulin-Fc fusion proteins

[0432] As described in Example 2, construct an isolated CHO cell line stably transfected with a vector encoding SEQ ID NO:32 or SEQ ID NO:36. The fed-batch shake flasks of the 14-day production run (0.5-2.0L culture medium scale) were inoculated with 500,000 cells / mL in an incubator-shaker set at 37°C and 5% carbon dioxide, and run as described in Example 2 above, except that CD OptiCHO was used instead of Dynamis as a growth medium (ThermoFisher) and efficient feed C (ThermoFisher) was used as feed. Feed was added at 3% v / v from the 3rd day of the production run, and on the 4th day, the shake flask temperature was adjusted to 32°C, and the incubator-shaker carbon dioxide concentration was reduced from 5% to 2%. During the run, cells increased to 8 million to 14 million cells / mL, and the production run was harvested on the 14th day to remove cells, and the culture supernatant was purified and tested to obtain the insulin-Fc fusion protein as described in Examples 3, 4, 5 and 6. Table 20 describes the production data obtained from production runs using stably transfected CHO cell lines.

[0433]

[0434]

[0435] Example 35: Use of Fc fragments containing canine IgGB origin produced by stably transfected CHO cell lines Exemplary CHO-based production runs of preferred insulin-Fc fusion proteins

[0436] As described in Example 2, construct a CHO cell line stably transfected with a vector encoding SEQ ID NO:34. The fed-batch shake flasks of 14-day production runs (0.5-2.0L culture medium scale) were inoculated with 500,000 cells / mL in an incubator-shake flask set at 37°C and 5% carbon dioxide, and run as described in Example 2, except that Dynamis was replaced with CD OptiCHO as a growth medium (ThermoFisher) and efficient feed C (ThermoFisher) was used as feed. Feed was added with 3% v / v from the 3rd day of production run, and on the 4th day, the shake flask temperature was adjusted to 32°C, and the incubator-shake flask carbon dioxide concentration was reduced to 2% from 5%. On the 14th day, harvest production runs to remove cells, and purification and test culture supernatants were used to obtain insulin-Fc fusion proteins as described in Examples 3, 4, 5 and 6. The resulting production run gave a protein yield of greater than 200 mg / L, greater than 95% homodimer, and a homodimer titer of SEQ ID NO:34 greater than 190 mg / L.

[0437] Results - Insulin-FC fusion protein containing feline FC fragment

[0438] Example 36: Insulin-Fc fusion protein comprising an Fc fragment of the feline IgG2 isotype

[0439] In order to develop a product suitable for cats, an attempt was made to produce an insulin-Fc fusion protein comprising the insulin polypeptide sequence of SEQ ID NO: 4 and an Fc fragment of the feline IgG2 isotype (SEQ ID NO: 21) using a peptide linker having the following amino acid sequence:

[0440] FVNQHLCGSDLVEALYLVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSV LPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQID NO:106)

[0441] The insulin-Fc fusion protein of SEQ ID NO:106 was synthesized in HEK cells according to Example 1 and purified according to Example 3. The structure of the insulin-Fc fusion protein was confirmed by non-reducing and reducing CE-SDS according to Example 4, and the sequence was further identified by LC-MS removing polysaccharides according to Example 5. The homodimer % of the obtained compound was measured by size exclusion chromatography according to Example 6 and was 88%. The homodimer titer obtained was only 20 mg / L, because HEK cells could not prepare the product in high yield (i.e., the protein yield after protein purification was only 23 mg / L). In short, the production of the insulin-Fc fusion protein of SEQ ID NO:106 in HEK cells resulted in a medium level of aggregates and a low homodimer titer of 20 mg / L, which did not meet the design target of a homodimer titer greater than 50 mg / L.

[0442] Nevertheless, the biological activity of the insulin-Fc fusion protein of SEQ ID NO: 106 was evaluated. First, the insulin receptor binding of the insulin-Fc fusion protein of SEQ ID NO: 106 was measured according to Example 7, and an IC50 value of 22 nM was obtained, indicating that the compound may have biological activity in vivo (i.e., IC50 is less than 5000 nM).

[0443] Next, according to Example 10, the in vivo pharmacodynamics (PD) of the insulin-Fc fusion protein of SEQ ID NO: 106 was measured after a single subcutaneous administration of the compound at a dose of 0.8 mg / kg to N=3 cats. Fig. 27 The percentage of fasting blood glucose level of the insulin-Fc fusion protein of SEQ NO:106 (161c) is shown as a function of time. According to the procedure of Example 11, the NAOC of the insulin-Fc fusion protein was calculated to be 215% FBGL·day·kg / mg. Surprisingly, unlike the similar insulin-Fc fusion protein of SEQ ID NO:42 for dogs comprising the insulin polypeptide of SEQ ID NO:5 and the peptide linker of SEQ ID NO:12, it was found that the insulin-Fc fusion protein of SEQ ID NO:106 for cats had much less aggregation in the target animal and significantly higher biological activity.

[0444] Since NAOC is acceptable and pharmacokinetic data support weekly administration, according to Example 11, cats were given additional subcutaneous doses on the 28th, 35th, 42nd and 49th days, and %FBGL was measured in the 7-day window period after each administration. For each repeated subcutaneous injection, NAOC and NAOCR were calculated according to the procedure of Example 11. As shown in Table 21, repeated subcutaneous administration in cats showed a significant attenuation of biological activity when the third administration, as measured by a significant reduction in NAOCR (i.e., the NAOC of the third injection was only 0.40 or 40% of the NAOC of the first injection, and the NAOC of the fourth injection was only 0.10 or 10% of the NAOC of the first injection). After repeated administration in cats, the significant decline in the biological activity of the insulin-Fc fusion protein of SEQ ID NO:106 was similar to the biological activity decline of the insulin-Fc fusion protein of SEQ ID NO:52 observed in dogs shown in Example 20.

[0445]

[0446] Example 37: Evaluation of Insulin Polypeptide Mutations and the Effects of Feline IgG1b or IgG2 Fc Fragments on Protein Yield, Purity and influence of insulin receptor activity on selection

[0447] In an attempt to increase the homodimer content % and protein yield of the insulin-Fc fusion protein of SEQ ID NO: 106, mutations were inserted into the sequence of the insulin polypeptide B chain (e.g., B16A mutation) and the peptide linker. In addition, in addition to the feline IgG2 Fc fragment (SEQ ID NO: 21) used to construct the insulin-Fc fusion protein of SEQ ID NO: 106, a feline IgG1b Fc fragment (SEQ ID NO: 20) was also evaluated. The resulting insulin-Fc fusion protein sequence is shown below, wherein the resulting sequence alignment relative to SEQ ID NO: 106 is Fig.28 Shown in (Clustal Omega).

[0448] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSV LPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ IDNO:108)

[0449] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSV LPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQID NO:110)

[0450] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSV LPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQID NO:112)

[0451] Insulin-Fc fusion proteins were prepared in HEK293 cells according to Example 1 and purified using a protein A column according to Example 3. According to Example 4, their structures were confirmed by non-reducing and reducing CE-SDS, and according to Example 5, the sequences were further identified by LC-MS removing polysaccharides. According to Example 6, their homodimer content % was measured by size exclusion chromatography, and according to Example 7, their insulin receptor binding affinity was measured. Insulin-Fc fusion protein variants and corresponding protein yields, homodimer % and homodimer titers are listed in Table 22. The results show that when combined with the insulin-Fc fusion protein of feline IgG1b isotype Fc fragment to produce SEQ ID NO:108, various mutations produce much higher protein yields, but the resulting protein is more aggregated (e.g., homodimer % lower than SEQ ID NO:106). This is surprising because feline IgG1b is more similar in function to canine IgGB Fc fragment isotypes, which are highly preferred Fc isotypes for producing canine insulin-Fc fusion proteins (Example 32). In feline compositions containing mutations of feline IgG2 isotypes, compositions comprising a B16A mutation of the insulin polypeptide B chain (i.e., SEQ ID NO: 110 and SEQ ID NO: 112) resulted in increased protein yield and homodimer titer. However, the mutated linker present in SEQ ID NO: 110 (i.e., GGGGAGGGG) appears to provide a further multiplication of protein yield and homodimer titer compared to SEQ ID NO: 112.

[0452]

[0453] Example 38: Repeated subcutaneous administration of an insulin polypeptide comprising SEQ ID NO: 4 and a feline IgG2 isotype In vivo immunogenicity screening of insulin-Fc fusion proteins after Fc fragmentation

[0454] Without being bound by any particular explanation, it is assumed that the reason why the biological activity of the insulin-Fc fusion protein of SEQ IDNO:106 was significantly reduced after the fourth repeated subcutaneous administration in cats (Example 36) is due to the production of anti-drug antibodies that neutralize its biological activity. Anti-drug antibodies can be directed against the insulin polypeptide, linker or Fc fragment portion of the insulin-Fc fusion protein. The immunogenic response is manifested as an interaction between antigen presenting cells, T helper cells, B cells and their associated cytokines, which may lead to the production of endogenous antibodies (e.g., anti-drug antibodies) against the drug. Binding antibodies are all isotypes capable of binding to insulin-Fc fusion proteins, and these antibodies can be detected in immunoassays such as those described in Example 14. Neutralizing antibodies that inhibit the functional activity of insulin-Fc fusion proteins are generally directed against biologically active sites. In order to evaluate whether this is the case, according to Example 14, serum collected before administration of each dose and at the end of the experiment described in Example 11 was tested to quantify the level of anti-drug antibodies. As Fig.29 As shown, the level of anti-drug antibodies did increase with multiple subcutaneous administrations of the compound, suggesting that the generation of neutralizing anti-drug antibodies after the fourth injection of the insulin Fc-fusion protein of SEQ ID NO: 106 was a possible reason for the decrease in NAOCR.

[0455] Example 39: Screening of feline sera for anti-drug antibodies and identification of the B10D and A8H sites of the insulin polypeptide Potentially immunogenic epitopes

[0456] As observed for SEQ ID NO: 52 in dogs (Example 20), repeated administration of the insulin-fusion protein of SEQ ID NO: 106 comprising the insulin polypeptide of SEQ ID NO: 4 and the peptide linker of SEQ ID NO: 13 still produced anti-drug antibodies (Example 38). Therefore, it was hypothesized that the insulin polypeptide of SEQ ID NO: 4 might unexpectedly contain a specific epitope (i.e., an immunogenic "hot spot") that the cat immune system is directed against. Therefore, the binding specificity of the antibodies present in the serum samples described in Example 38 was evaluated according to the general procedure of Example 15. Analysis of antibody-containing feline serum samples from repeated dosing of insulin-Fc fusion proteins of SEQ ID NO: 106 (Example 38) against coated insulin-Fc fusion protein libraries showed that there were unexpectedly two major "hot spots" in the insulin polypeptide sequence of SEQ ID NO: 4: the B10D site mutation (i.e., an aspartic acid mutation at the 10th position from the N-terminus of the B chain (i.e., B10)), and, respectively, the A8H site mutation (i.e., a histidine mutation at the 8th position from the N-terminus of the B chain (i.e., A8)). The results indicate that insulin-Fc fusion proteins comprising insulin polypeptide amino acid compositions containing these two specific amino acid mutations may be immunogenic in cats, and thus may generate anti-drug antibodies that neutralize biological activity after repeated injections. Therefore, it can be determined that insulin polypeptides that do not contain B10D and A8H are preferably used for insulin-Fc fusion proteins that require long-term repeated dosing in cats (e.g., for the treatment of feline diabetes).

[0457] Example 40: Insulin polypeptides comprising SEQ ID NO: 4 and glycosylated and non-glycosylated feline IgG1b and Insulin-Fc fusion protein of IgG2 isotype Fc fragment, wherein B10, A8 and other sites of insulin polypeptide are further Mutations to reduce potential risk of immunogenicity

[0458] To evaluate whether replacing the "hotspot" mutation will improve the immunogenicity and repeated administration biological activity of insulin-Fc fusion proteins, the insulin-Fc fusion proteins include the insulin polypeptide of SEQ ID NO:4 and the feline IgG2 isotype fragment, and the exemplary insulin-Fc fusion proteins of SEQ ID NO:114, 116 and 118 were synthesized, wherein the B10 and A8 amino acids of the insulin polypeptide were restored to their natural histidine and alanine composition, respectively, and the histidine at B16 was replaced by alanine (i.e., B16A), just as the case of the insulin polypeptide of SEQ ID NO:5 for various canine insulin-Fc fusion proteins. The A21N site of natural insulin is also missing. For this example, other insulin polypeptide amino acids were mutated to make their structure more similar to natural feline insulin (e.g., B30A, A8A, A10V and A18H). The sequence of the resulting insulin polypeptide (SEQ ID NO:120) is listed below, wherein the non-natural amino acids of feline insulin are underlined.

[0459] FVNQHLCGSHLVEAL A LVCGERGFFYT DP A GGGPRR GIVEQCCASVCSLYQLEHYC(SEQ ID NO:120)

[0460] In addition, given the additional potential benefits of the non-glycosylated cNg mutants discussed in Examples 22 and 33, two evaluated insulin-Fc fusion proteins (SEQ ID NOs: 116 and 118) contained the cNg-S mutation. The complete amino acid sequences of the insulin-Fc fusion proteins are shown below, with the resulting sequence alignment relative to SEQ ID NO: 108 being Fig.30 Shown in (Clustal Omega).

[0461] FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSP(SEQ IDNO:114)

[0462] FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ IDNO:116)

[0463] FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVALGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ IDNO:118)

[0464] Insulin-Fc fusion proteins were prepared in HEK293 cells according to Example 1 and purified using a protein A column according to Example 3. According to Example 4, their structures were confirmed by non-reducing and reducing CE-SDS, and according to Example 5, the sequences were further identified by LC-MS with glycans removed. According to Example 6, their homodimer content % was measured by size exclusion chromatography, and according to Example 7, their insulin receptor binding affinity was measured. Table 23 below shows the manufacturability and in vitro IR binding parameters of the resulting compounds.

[0465]

[0466] Unexpectedly, compared with the insulin-Fc fusion protein of SEQ ID NO:108, all three insulin-Fc fusion proteins give much lower protein yields. In fact, although it has sufficiently high insulin receptor binding affinity (IC50 is 707nM), the insulin-Fc fusion protein of SEQ ID NO:116 hardly produces protein yield. The insulin-Fc fusion protein of SEQ ID NO:118 gives unacceptable low protein yield and homodimer titer, and is considered unlikely to have in vivo biological activity due to its high IR binding IC50 value higher than 5000nM. Compared with the insulin-Fc fusion protein of SEQ ID NO:108, the albumen of SEQ ID NO:114 also produces unacceptable low protein yield and much lower insulin receptor binding affinity (higher IR IC50 value).

[0467] Example 41: An insulin polypeptide comprising SEQ ID NO: 8, a linker of SEQ ID NO: 14 and a feline IgG2 homologue Insulin-Fc fusion protein

[0468] In an attempt to obtain acceptable protein yields for an insulin-Fc fusion protein comprising an insulin polypeptide sequence without immunogenic "hotspot" mutations (i.e., B10D and A8H), learnings were obtained from the simultaneous and parallel development of a canine insulin-Fc fusion protein, which had shown that the use of an insulin polypeptide of SEQ ID NO: 8 and a peptide linker of SEQ ID NO: 14 on a canine IgGB isotype Fc fragment resulted in high protein and homodimer titers and acceptable IR binding affinity. Thus, a feline insulin-Fc fusion protein was constructed using an insulin polypeptide of SEQ ID NO: 8 and a peptide linker of SEQ ID NO: 14 on a feline IgG2 Fc fragment of SEQ ID NO: 21 to produce the following sequence:

[0469] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNS TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:122)

[0470] exist Fig.31 A sequence alignment of SEQ ID NO: 122 relative to Example 37 sequences SEQ ID NO: 106 and 112 is shown (Clustal Omega).

[0471]

[0472] The insulin-Fc fusion protein of SEQ ID NO: 122 was prepared in HEK293 cells according to Example 1 and purified using a protein A column according to Example 3. According to Example 4, their structures were confirmed by non-reducing and reducing CE-SDS, and according to Example 5, the sequences were further identified by LC-MS with polysaccharide removal. According to Example 6, their homodimer content% was measured by size exclusion chromatography, and according to Example 7, their insulin receptor binding affinity was measured. FcRn receptor binding affinity was measured according to Example 9. The protein yield was 146 mg / L, and the homodimer% was determined to be 99%, resulting in a homodimer titer of 145 mg / L that met the production design target. The IR binding affinity IC50 value was 2,536 nM, indicating that the compound may have in vivo biological activity. The FcRn receptor binding affinity EC50 value was 3114 ng / mL. Therefore, the insulin-Fc fusion protein of SEQ ID NO: 122 is a potential candidate for further in vivo testing.

[0473] Example 42: An insulin polypeptide of SEQ ID NO: 8, a peptide linker of SEQ ID NO: 14 and a peptide of SEQ ID NO: 21 In vivo biological activity of insulin-Fc fusion protein constructed from feline IgG2 Fc fragment

[0474] According to Example 10, the in vivo biological activity of the insulin-Fc fusion protein of SEQ ID NO: 122 was tested. Healthy, antibody-naive cats weighing about 5 kg were used. On day 0, the cats received a single injection of a pharmaceutical composition containing the insulin Fc-fusion protein of SEQ ID NO: 122. On day 0, blood was collected from a suitable vein before injection and at 15, 30, 45, 60, 120, 240, 360 and 480 minutes and 1, 2, 3, 4, 5, 6 and 7 days after injection. If the subject's blood sugar drops to a dangerous level, food and / or glucose injection are given to prevent symptomatic hypoglycemia.

[0475] Fig.32 The % FBGL for a single administration is shown, indicating that, unexpectedly, the insulin-Fc fusion protein of SEQ ID NO: 122 has only weak biological activity in vivo (NAOC is essentially 0% FBGL·day·kg / mg). This result is surprising, especially because the insulin-Fc fusion protein does not aggregate (i.e., has a high % homodimer content) and the molecule exhibits IR affinity in a similar range to canine insulin-Fc fusion proteins found to exhibit significant biological activity in dogs (Example 31). Due to the lack of biological activity on the first administration, no repeated administration was performed.

[0476] Example 43: Evaluation of the effect of feline IgG1b replacing feline IgG2 Fc fragment on pancreatic cancer comprising SEQ ID NO: 8 Yield, purity, biological activity and immunogenicity of insulin-Fc fusion protein containing insulin polypeptide and peptide linker of SEQ ID NO: 14 Sexual influence

[0477] Since the long-acting insulin research projects for dogs and cats were conducted in parallel, some of the knowledge from the canine insulin-Fc fusion protein research project was applied to the feline insulin-Fc protein research project. An important piece of knowledge gained from the canine insulin-Fc research project is how the selection of different IgG isotype Fc fragments (e.g., canine IgGA, canine IgGB, canine IgGC, and canine IgGD isotypes) can lead to significantly different production and in vivo efficacy performance. Therefore, the feline IgG2 Fc fragment of SEQ ID NO: 122 was replaced by the feline IgG1b Fc fragment of SEQ ID NO: 20, while retaining the insulin polypeptide of SEQ ID NO: 8 and the peptide linker of SEQ ID NO: 14, resulting in the following amino acid sequence:

[0478] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNST YRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:38)

[0479] The insulin-Fc fusion protein of SEQ ID NO:38 was synthesized in HEK293 cells according to the procedure of Example 1 and purified using a protein A column according to Example 3. According to Example 4, the structure was confirmed by non-reducing and reducing LC-MS, and according to Example 5, the sequence was further identified by LC-MS with polysaccharide removal. The protein yield at this stage was 158 mg / L. According to Example 6, the homodimer % of the sequence was measured by size exclusion chromatography and was determined to be 99.5%, resulting in a homodimer titer of 157 mg / L that met the production design target. The in vitro IM-9 insulin receptor binding IC50 value measured according to Example 7 was 2398 nM, which also met the design target. The FcRn receptor binding affinity EC50 value was measured according to Example 9 and was found to be 1552 ng / mL.

[0480] Then the in vivo biological activity of the insulin-Fc fusion protein of SEQ ID NO:38 was tested according to Example 10. Healthy, antibody-naive cats weighing about 5 kg received a single subcutaneous injection of a pharmaceutical composition containing the insulin Fc-fusion protein of SEQ ID NO:38, with a dose of 0.16 mg insulin Fc-fusion protein / kg. On day 0, blood was collected from a suitable vein before injection and at 15, 30, 45, 60, 120, 240, 360 and 480 minutes and 1, 2, 3, 4, 5, 6 and 7 days after injection. If the blood sugar of the experimenter drops to a dangerous level, food and / or glucose injection are given to prevent symptomatic hypoglycemia.

[0481] Fig.33The %FBGL after the first administration is shown. Animal food was given regularly to prevent symptomatic hypoglycemia, which shows that the insulin-Fc fusion protein of SEQ ID NO:38 has significant biological activity in vivo, with NAOC of 1838%FBGL·day·kg / mg. The pharmacokinetic profile of the compound was also measured using ELISA by the method of Example 12, and a two-compartment model was fitted to the data to determine its elimination half-life (which was 6.3±0.5). The differences in biological activity (in vitro and in vivo) between the insulin-Fc fusion protein of SEQ ID NO:38 and the insulin-Fc fusion protein of SEQ ID NO:122 unexpectedly indicate that when the insulin polypeptide sequence is modified as described in SEQ ID NO:8, the feline IgG1b isotype is more preferred than the feline IgG2 isotype for the Fc fragment.

[0482] Since NAOC is acceptable and pharmacokinetic data support weekly administration, additional subcutaneous administration of cats was given according to Example 11 on the 14th, 28th and 42nd days, and %FBGL was measured in the 7-day window period after each administration. For each repeated subcutaneous injection, NAOC and NAOCR were calculated according to the procedure of Example 11. As shown in Table 25, after multiple administrations, the insulin-Fc fusion protein of SEQ ID NO:38 showed acceptable biological activity in vivo.

[0483]

[0484] In addition, according to Example 14, serum was collected before each dose and once a week for two weeks after the end of the experiment to test for the presence and quantify the level of any anti-drug antibodies. Fig.34 As shown, there was no measurable increase above baseline in anti-drug antibodies after multiple administrations of the compound. Therefore, in order to obtain a feline insulin-Fc fusion protein candidate (e.g., SEQ ID NO: 38) that meets the design criteria of acceptable homodimer titer, in vivo bioactivity, and sustained bioactivity after repeated weekly injections in cats, it is necessary to replace the insulin polypeptide of SEQ ID NO: 4 with the insulin polypeptide of SEQ ID NO: 8 and to use the feline IgG1b Fc fragment of SEQ ID NO: 20 instead of the feline IgG2 Fc fragment of SEQ ID NO: 21.

[0485] Example 44: An insulin polypeptide comprising SEQ ID NO: 8, a peptide linker of SEQ ID NO: 14, and a feline Non-glycosylated insulin-Fc fusion protein of IgG1b Fc fragment to reduce the potential risk of immunogenicity

[0486] Although the insulin-Fc fusion protein of SEQ ID NO: 38 meets all design goals (Example 43), there may or may not be a risk of immunogenicity over an extended treatment period (e.g., 6 months, 1 year, 2 years, or longer), which may compromise the use of the insulin-Fc fusion protein for treating diabetes (if this occurs). As described in the specific embodiments of the invention, one possible reason for the reduction in biological activity after repeated administration is the undesirable interaction of the feline IgG1b Fc fragment with the cat's immune system, leading to the production of neutralizing anti-drug antibodies. However, the results shown in Example 43 unexpectedly demonstrate that the feline IgG1b isotype is superior to the less immunogenic feline IgG2 isotype in terms of in vivo biological activity. Therefore, further Fc mutations were explored to obtain a non-glycosylated insulin-Fc fusion protein with low Fc(γ)RI receptor binding, which can reduce the risk of long-term, chronic immunogenicity.

[0487] As described in a specific embodiment of the present invention, a method for reducing Fc (γ) RI interactions includes mutating the Fc fragment cNg site to prevent glycosylation during synthesis in the host cell. Therefore, the cNg site mutation is performed on the Fc fragment region of SEQ ID NO: 38 to reduce the binding affinity of the Fc fragment to the Fc (γ) receptor in vivo, as measured by binding in the in vitro human Fc (γ) RI assay described in Example 8. The position of the cNg site in the insulin-Fc fusion protein of SEQ ID NO: 38 is cNg-NB151. Again, using the knowledge obtained from the canine insulin-Fc fusion protein described in Example 33, the cNg-NB151-S mutation is introduced into the Fc fragment of SEQ ID NO: 38. The complete amino acid sequence of the resulting insulin-Fc fusion protein is shown below (cNg-NB151-S is underlined for clarity):

[0488] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF SSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:124)

[0489] The insulin-Fc fusion protein of SEQ ID NO:124 was synthesized in HEK293 cells according to the procedure of Example 1 and purified using a protein A column according to Example 3. According to Example 4, the structure of the insulin-Fc fusion protein was confirmed by non-reducing and reducing LC-MS, and according to Example 5, the sequence was further identified by LC-MS with polysaccharide removal. The protein yield at this stage was 202 mg / L. According to Example 6, the homodimer % of the sequence was measured by size exclusion chromatography and was determined to be 99%, resulting in a homodimer titer of 200 mg / L, which met the production design target. However, the in vitro IM-9 insulin receptor binding IC50 value measured according to Example 7 was greater than 5000 nM, which exceeded the design target of in vitro biological activity. The FcRn receptor binding affinity EC50 value was measured according to Example 9 and was 6922 ng / mL.

[0490] Although the insulin-Fc fusion protein of SEQ ID NO: 124 does not meet the insulin receptor binding design goal, its in vivo biological activity was tested according to Example 10. Healthy, antibody-naive cats weighing about 5 kg were used. On day 0, cats were given a single injection of a pharmaceutical composition containing the insulin Fc fusion protein of SEQ ID NO: 124 at a dose of 0.16 mg insulin-Fc fusion protein / kg. On day 0, blood was collected from a suitable vein before injection and at 15, 30, 45, 60, 120, 240, 360 and 480 minutes and 1, 2, 3, 4, 5, 6 and 7 days after injection. If the subject's blood sugar drops to a dangerous level, food and / or glucose injection are given to prevent symptomatic hypoglycemia.

[0491] Fig.35 The %FBGL of a single administration is shown, indicating that the insulin-Fc fusion protein of SEQ ID NO: 124 has only some biological activity in vivo, with a NAOC of 65%FBGL·day·kg / mg. Due to the lack of biological activity in the first administration, no repeated administration was performed.

[0492] Unexpectedly, as in the case of the canine insulin Fc-fusion protein of SEQ ID NO:36 in Example 33, it was found that mutating the insulin polypeptide sequence of SEQ ID NO:124 such that the 16th amino acid (B16) from the N-terminus of the B chain was mutated from tyrosine to alanine (i.e., B16A) made the resulting insulin Fc-fusion protein of SEQ ID NO:40 biologically active. The amino acid sequence of the resulting insulin-Fc fusion protein is shown below (for clarity, the B16A and cNg-NB151-S mutations are underlined):

[0493] FVNQHLCGSHLVEAL A LVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:40)

[0494] The insulin-Fc fusion protein of SEQ ID NO:40 was synthesized in HEK293 cells according to the procedure of Example 1 and purified using a protein A column according to Example 3. The structure of the insulin-Fc fusion protein was confirmed by non-reducing and reducing CE-SDS according to Example 4, and the sequence was further identified by LC-MS removing polysaccharides according to Example 5. The protein yield at this stage was 174 mg / L. According to Example 6, the homodimer % of the sequence was measured by size exclusion chromatography and was determined to be 98.9%, resulting in a homodimer titer of 172 mg / L, which met the production design criteria. The in vitro IM-9 insulin receptor binding IC50 value measured according to Example 7 was 4635 nM, which also met the design goals. Fc (γ) receptor activity was measured according to Example 8, and it was found that it was about 4 times lower than the activity obtained using the same procedure for the insulin-Fc fusion protein of SEQ ID NO:38, indicating that the insulin-Fc fusion protein may interact less adversely with the cat immune system. The FcRn receptor binding affinity EC50 value was measured according to Example 9 and was 8157 ng / mL.

[0495] Then the in vivo biological activity of the insulin-Fc fusion protein of SEQ ID NO:40 was tested according to Example 11. A healthy, antibody-naive cat weighing about 5 kg was used. On the 0th day, the 7th day and the 21st day, a single subcutaneous injection of a pharmaceutical composition of the insulin Fc fusion protein containing SEQ ID NO:40 was given to the cat at a dosage of 0.1 mg insulin-Fc fusion protein / kg. On the 0th day, blood was collected from a suitable vein before injection and at 15, 30, 45, 60, 120, 240, 360 and 480 minutes and 1, 2, 3, 4, 5, 6 and 7 days after injection. If the blood sugar of the experimenter drops to a dangerous level, food and / or glucose injection is given to prevent symptomatic hypoglycemia.

[0496] Fig.36 The % FBGL after the first administration is shown, indicating that the insulin-Fc fusion protein of SEQ ID NO:40 is biologically active in vivo, wherein for a subcutaneous dose of 0.1 mg insulin-Fc fusion protein / kg, the NAOC is 159% FBGL·day·kg / mg. A second higher subcutaneous dose of 0.2 mg insulin-Fc fusion protein / kg produced a much higher NAOC of 702% FBGL·day·kg / mg and Fig.37 16mg insulin-Fc fusion protein / kg) , the same compound containing tyrosine at B16 instead of alanine only has very weak biological activity. Therefore, for the non-glycosylated insulin-Fc fusion protein containing the feline IgG1b Fc fragment of cNg mutation, the insulin polypeptide of SEQ ID NO:11 is preferred.

[0497] In order to analyze the repeatable biological activity after multiple administration, at the 7th day, the 21st day and the 35th day, the insulin-Fc fusion protein of SEQ ID NO:40 of cat's additional dosage was given. When the %FBGL of cat decreased too low, food was provided to cat to raise blood sugar to a safe level. According to the general procedure of example 11, the NAOC and NAOCR of each subsequent administration were measured, and the time of the applied dosage was calculated until just before the next dosage was applied. NAOC and NAOCR shown in table 26 show that the insulin-Fc fusion protein of SEQ ID NO:40 has biological activity in vivo after multiple administration.

[0498]

[0499] In addition, according to Example 14, serum was collected before each dose and at the end of the experiment to test for the presence of any anti-drug antibodies and quantify their levels. After multiple administrations of the compound, there was no measurable increase in anti-drug antibodies above baseline. Therefore, in order to obtain a feline insulin-Fc fusion protein that meets the production and biological activity design standards and has significantly reduced Fc (γ) receptor activity, it is necessary not only to mutate cNg to serine, but also to mutate the insulin polypeptide B16 amino acid to alanine.

[0500] Example 45: Use of Fc fragments containing feline IgG1b origin produced by stably transfected CHO cell lines Exemplary CHO-based production runs of preferred insulin-Fc fusion proteins

[0501] As described in Example 2 above, a CHO cell line stably transfected with a vector encoding SEQ ID NO:38 was constructed. The fed batch shake flasks of the 14-day production run (0.5-2.0L culture medium scale) were inoculated with 500,000 cells / mL in an incubator-shake flask set at 37°C and 5% carbon dioxide, and operated as described in Example 2 above, except that Dynamis was replaced with CDOptiCHO as a growth medium (ThermoFisher) and efficient feed C (ThermoFisher) was used as feed. Feed was added with 3% v / v from the 3rd day of production run, and on the 4th day, the shake flask temperature was adjusted to 32°C, and the incubator-shake flask carbon dioxide concentration was reduced from 5% to 2%. During operation, cell density increased to 8 million-14 million cells / mL, and production runs were harvested on the 14th day to remove cells, and the culture supernatant was purified and characterized to obtain insulin-Fc fusion proteins as described in Examples 3, 4, 5 and 6. Table 27 describes the production data of the insulin-Fc fusion protein obtained by these stably transfected CHO cell line production runs.

[0502]

[0503] Example 46: Preferred pancreatic islets derived from feline IgG1b produced using a stably transfected CHO cell line Exemplary CHO-based production run of leukocyte antigen-Fc fusion protein

[0504] As described in Example 2 above, a CHO cell line stably transfected with a vector encoding SEQ ID NO:40 was constructed. The fed batch shake flasks of the 14-day production run (0.5-2.0L culture medium scale) were inoculated with 500,000 cells / mL in an incubator-shake flask set at 37°C and 5% carbon dioxide, and operated as described in Example 2 above, except that Dynamis was replaced with CDOptiCHO as a growth medium (ThermoFisher) and efficient feed C (ThermoFisher) was used as feed. Feed was added with 3% v / v from the 3rd day of production run, and on the 4th day, the shake flask temperature was adjusted to 32°C, and the incubator-shake flask carbon dioxide concentration was reduced to 2% from 5%. On the 14th day, production runs were harvested to remove cells, and the culture supernatant was purified and characterized to obtain the insulin-Fc fusion protein as described in Example 3, 4, 5 and 6. The resulting production run gave a protein yield of greater than 200 mg / L, greater than 95% homodimer, and a homodimer titer of greater than 190 mg / L for SEQ ID NO:40.

[0505] Example 47: Exemplary Insulin-Fc Fusion Protein Domains and Sequences

[0506] The exemplary insulin-Fc fusion protein amino acid sequences and corresponding DNA sequences used in the above examples are Fig.38 , 39 , 40, 41 and 42.

[0507] Equivalent

[0508] In the claims, articles such as "a," "an," and "the" may mean one or more than one, unless indicated to the contrary or obvious from the context. Claims or descriptions that include an "or" between one or more members of the group are deemed to comply if one, more than one, or all of the group members are present in, used in, or otherwise related to a given product or process, unless indicated to the contrary or obvious from the context. The present disclosure includes embodiments in which only one member of the group is present in, used in, or otherwise related to a given product or process. The present disclosure includes embodiments in which more than one or all of the group members are present in, used in, or otherwise related to a given product or process.

[0509] In addition, the present disclosure includes all variations, combinations and permutations, wherein one or more limitations, elements, clauses and descriptive terms from one or more listed claims are introduced into another claim. For example, any claim that is subordinate to another claim can be modified to include one or more limitations in any other claim that is subordinate to the same basic claim. In the case where the element exists in the form of a list (e.g., in the form of a Markush group), each subgroup of the element is also disclosed, and any element can be removed from the group. It should be understood that, in general, in the case where the present disclosure or aspects of the present disclosure are referred to as including specific elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure are composed of these elements and / or features or are substantially composed of these elements and / or features. For the purpose of simplicity, these embodiments are not specifically described herein. It should also be noted that the terms "comprise (s), comprising)" and "contain (s), containing" are intended to be open-ended, and their use allows the inclusion of additional elements or steps. In the case of a given range, the end point is included. In addition, unless otherwise stated or apparent from the context and understanding of one of ordinary skill in the art, in different embodiments of the present disclosure, values ​​expressed as ranges may take any specific value or sub-range within the range, up to the tenth of the unit of the lower limit of the range, unless the context clearly states otherwise. Sequence Listing <110> Akaston Biosciences <120> Ultra-long-acting insulin-FC fusion protein (Fusion Protein) and its use <130> ABC-3PC <160> 125 <170> PatentIn Version 3.5 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> hIns-B-chain <400> 1 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30 <210> 2 <211> twenty one <212> PRT <213> Artificial Sequence <220> <221> <222> <223> hIns-A-chain <400> 2 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu 1 5 10 15 Glu Asn Tyr Cys Asn 20 <210> 3 <211> 86 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Human Proinsulin <400> 3 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 4 <211> 57 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Ins Polypeptide <400> 4 Phe Val Asn Gln His Leu Cys Gly Ser Asp Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Pro Thr Gly Gly 20 25 30 Gly Pro Arg Arg Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 50 55 <210> 5 <211> 57 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Ins Polypeptide <400> 5 Phe Val Asn Gln His Leu Cys Gly Ser Asp Leu Val Glu Ala Leu Ala 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Pro Thr Gly Gly 20 25 30 Gly Pro Arg Arg Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser 35 40 45 Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 50 55 <210> 6 <211> 58 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Ins Polypeptide <220> <221> UNSURE <222> (10)..(10) <223> Xaa is not D <220> <221> UNSURE <222> (45)..(45) <223> Xaa is not H <220> <221> UNSURE <222> (58)..(58) <223> Xaa does not exist or is N <400> 6 Phe Val Asn Gln His Leu Cys Gly Ser Xaa Leu Val Glu Ala Leu Glu 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Gly Gly Gly Gly Gly Gly 20 25 30 Ser Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Xaa Ser Thr Cys 35 40 45 Ser Leu Asp Gln Leu Glu Asn Tyr Cys Xaa 50 55 <210> 7 <211> 58 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Ins Polypeptide <220> <221> UNSURE <222> (58)..(58) <223> Xaa does not exist or is N <400> 7 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Glu 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Gly Gly Gly Gly Gly Gly 20 25 30 Ser Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Thr Cys 35 40 45 Ser Leu Asp Gln Leu Glu Asn Tyr Cys Xaa 50 55 <210> 8 <211> 57 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Ins Polypeptide <400> 8 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Glu 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Gly Gly Gly Gly Gly Gly 20 25 30 Ser Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Thr Cys 35 40 45 Ser Leu Asp Gln Leu Glu Asn Tyr Cys 50 55 <210> 9 <211> 58 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Ins Polypeptide <400> 9 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Glu 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Gly Gly Gly Gly Gly Gly 20 25 30 Ser Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Thr Cys 35 40 45 Ser Leu Asp Gln Leu Glu Asn Tyr Cys Asn 50 55 <210> 10 <211> 57 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Ins Polypeptide <220> <221> UNSURE <222> (10)..(10) <223> Xaa is not D <220> <221> UNSURE <222> (45)..(45) <223> Xaa is not H <400> 10 Phe Val Asn Gln His Leu Cys Gly Ser Xaa Leu Val Glu Ala Leu Ala 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Gly Gly Gly Gly Gly Gly 20 25 30 Ser Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Xaa Ser Thr Cys 35 40 45 Ser Leu Asp Gln Leu Glu Asn Tyr Cys 50 55 <210> 11 <211> 57 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Ins Polypeptide <400> 11 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ala 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Gly Gly Gly Gly Gly Gly 20 25 30 Ser Gly Gly Gly Gly Gly Ile Val Glu Gln Cys Cys Thr Ser Thr Cys 35 40 45 Ser Leu Asp Gln Leu Glu Asn Tyr Cys 50 55 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Linker <400> 12 Gly Gly Gly Gly Ala Gly Gly Gly Gly 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Linker <400> 13 Gly Gly Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 14 <211> twenty one <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Linker <400> 14 Gly Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln Gly Gly Gly Gly Gln 1 5 10 15 Gly Gly Gly Gly Gly 20 <210> 15 <211> 228 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Canine IgGA Fc Fragment <400> 15 Arg Cys Thr Asp Thr Pro Pro Cys Pro Val Pro Glu Pro Leu Gly Gly 1 5 10 15 Pro Ser Val Leu Ile Phe Pro Pro Lys Pro Lys Asp Ile Leu Arg Ile 20 25 30 Thr Arg Thr Pro Glu Val Thr Cys Val Val Leu Asp Leu Gly Arg Glu 35 40 45 Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Glu Val His 50 55 60 Thr Ala Lys Thr Gln Ser Arg Glu Gln Gln Phe Asn Gly Thr Tyr Arg 65 70 75 80 Val Val Ser Val Leu Pro Ile Glu His Gln Asp Trp Leu Thr Gly Lys 85 90 95 Glu Phe Lys Cys Arg Val Asn His Ile Asp Leu Pro Ser Pro Ile Glu 100 105 110 Arg Thr Ile Ser Lys Ala Arg Gly Arg Ala His Lys Pro Ser Val Tyr 115 120 125 Val Leu Pro Pro Ser Pro Lys Glu Leu Ser Ser Ser Asp Thr Val Ser 130 135 140 Ile Thr Cys Leu Ile Lys Asp Phe Tyr Pro Pro Asp Ile Asp Val Glu 145 150 155 160 Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Arg Lys His Arg Met Thr 165 170 175 Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu 180 185 190 Ser Val Asp Lys Ser Arg Trp Gln Gln Gly Asp Pro Phe Thr Cys Ala 195 200 205 Val Met His Glu Thr Leu Gln Asn His Tyr Thr Asp Leu Ser Leu Ser 210 215 220 His Ser Pro Gly 225 <210> 16 <211> 224 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Canine IgGB Fc Fragment <400> 16 Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu Gly Gly Pro Ser Val 1 5 10 15 Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile Ala Arg Thr 20 25 30 Pro Glu Val Thr Cys Val Val Val Asp Leu Asp Pro Glu Asp Pro Glu 35 40 45 Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met Gln Thr Ala Lys 50 55 60 Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr Arg Val Val Ser 65 70 75 80 Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly Lys Gln Phe Thr 85 90 95 Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Arg Thr Ile 100 105 110 Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val Leu Pro 115 120 125 Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu Thr Cys Leu 130 135 140 Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn 145 150 155 160 Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro Pro Gln Leu 165 170 175 Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys 180 185 190 Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val Met His Glu 195 200 205 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser His Ser Pro Gly 210 215 220 <210> 17 <211> 225 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Canine IgGC Fc Fragment <400> 17 Cys Asn Asn Cys Pro Cys Pro Gly Cys Gly Leu Leu Gly Gly Pro Ser 1 5 10 15 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Ile Leu Val Thr Ala Arg 20 25 30 Thr Pro Thr Val Thr Cys Val Val Val Asp Leu Asp Pro Glu Asn Pro 35 40 45 Glu Val Gln Ile Ser Trp Phe Val Asp Ser Lys Gln Val Gln Thr Ala 50 55 60 Asn Thr Gln Pro Arg Glu Glu Gln Ser Asn Gly Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Ser Gly Lys Gln Phe 85 90 95 Lys Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Glu Ile 100 105 110 Ile Ser Lys Thr Pro Gly Gln Ala His Gln Pro Asn Val Tyr Val Leu 115 120 125 Pro Pro Ser Arg Asp Glu Met Ser Lys Asn Thr Val Thr Leu Thr Cys 130 135 140 Leu Val Lys Asp Phe Phe Pro Pro Glu Ile Asp Val Glu Trp Gln Ser 145 150 155 160 Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Met Thr Pro Pro Gln 165 170 175 Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp 180 185 190 Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val Met His 195 200 205 Glu Ala Leu His Asn His Tyr Thr Gln Ile Ser Leu Ser His Ser Pro 210 215 220 Gly 225 <210> 18 <211> 225 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Canine IgGD Fc Fragment <400> 18 Cys Ile Ser Pro Cys Pro Val Pro Glu Ser Leu Gly Gly Pro Ser Val 1 5 10 15 Phe Ile Phe Pro Pro Lys Pro Lys Asp Ile Leu Arg Ile Thr Arg Thr 20 25 30 Pro Glu Ile Thr Cys Val Val Leu Asp Leu Gly Arg Glu Asp Pro Glu 35 40 45 Val Gln Ile Ser Trp Phe Val Asp Gly Lys Glu Val His Thr Ala Lys 50 55 60 Thr Gln Pro Arg Glu Gln Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 65 70 75 80 Val Leu Pro Ile Glu His Gln Asp Trp Leu Thr Gly Lys Glu Phe Lys 85 90 95 Cys Arg Val Asn His Ile Gly Leu Pro Ser Pro Ile Glu Arg Thr Ile 100 105 110 Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val Leu Pro 115 120 125 Pro Ser Pro Lys Glu Leu Ser Ser Ser Asp Thr Val Thr Leu Thr Cys 130 135 140 Leu Ile Lys Asp Phe Phe Pro Pro Glu Ile Asp Val Glu Trp Gln Ser 145 150 155 160 Asn Gly Gln Pro Glu Pro Glu Ser Lys Tyr His Thr Thr Ala Pro Gln 165 170 175 Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp 180 185 190 Lys Ser Arg Trp Gln Gln Gly Asp Thr Phe Thr Cys Ala Val Met His 195 200 205 Glu Ala Leu Gln Asn His Tyr Thr Asp Leu Ser Leu Ser His Ser Pro 210 215 220 Gly 225 <210> 19 <211> 224 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Feline IgG1a Fc Fragment <400> 19 Asp Cys Pro Lys Cys Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile 1 5 10 15 Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser Arg Thr 20 25 30 Pro Glu Val Thr Cys Leu Val Val Asp Leu Gly Pro Asp Asp Ser Asp 35 40 45 Val Gln Ile Thr Trp Phe Val Asp Asn Thr Gln Val Tyr Thr Ala Lys 50 55 60 Thr Ser Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 65 70 75 80 Val Leu Pro Ile Leu His Gln Asp Trp Leu Lys Gly Lys Glu Phe Lys 85 90 95 Cys Lys Val Asn Ser Lys Ser Leu Pro Ser Pro Ile Glu Arg Thr Ile 100 105 110 Ser Lys Ala Lys Gly Gln Pro His Glu Pro Gln Val Tyr Val Leu Pro 115 120 125 Pro Ala Gln Glu Glu Leu Ser Arg Asn Lys Val Ser Val Thr Cys Leu 130 135 140 Ile Lys Ser Phe His Pro Pro Asp Ile Ala Val Glu Trp Glu Ile Thr 145 150 155 160 Gly Gln Pro Glu Pro Glu Asn Asn Tyr Arg Thr Thr Pro Pro Gln Leu 165 170 175 Asp Ser Asp Gly Thr Tyr Phe Val Tyr Ser Lys Leu Ser Val Asp Arg 180 185 190 Ser His Trp Gln Arg Gly Asn Thr Tyr Thr Cys Ser Val Ser His Glu 195 200 205 Ala Leu His Ser His His Thr Gln Lys Ser Leu Thr Gln Ser Pro Gly 210 215 220 <210> 20 <211> 224 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Feline IgG1b Fc Fragment <400> 20 Asp Cys Pro Lys Cys Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile 1 5 10 15 Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser Arg Thr 20 25 30 Pro Glu Val Thr Cys Leu Val Val Asp Leu Gly Pro Asp Asp Ser Asp 35 40 45 Val Gln Ile Thr Trp Phe Val Asp Asn Thr Gln Val Tyr Thr Ala Lys 50 55 60 Thr Ser Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 65 70 75 80 Val Leu Pro Ile Leu His Gln Asp Trp Leu Lys Gly Lys Glu Phe Lys 85 90 95 Cys Lys Val Asn Ser Lys Ser Leu Pro Ser Pro Ile Glu Arg Thr Ile 100 105 110 Ser Lys Asp Lys Gly Gln Pro His Glu Pro Gln Val Tyr Val Leu Pro 115 120 125 Pro Ala Gln Glu Glu Leu Ser Arg Asn Lys Val Ser Val Thr Cys Leu 130 135 140 Ile Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ile Thr 145 150 155 160 Gly Gln Pro Glu Pro Glu Asn Asn Tyr Arg Thr Thr Pro Pro Gln Leu 165 170 175 Asp Ser Asp Gly Thr Tyr Phe Leu Tyr Ser Arg Leu Ser Val Asp Arg 180 185 190 Ser Arg Trp Gln Arg Gly Asn Thr Tyr Thr Cys Ser Val Ser His Glu 195 200 205 Ala Leu His Ser His His Thr Gln Lys Ser Leu Thr Gln Ser Pro Gly 210 215 220 <210> 21 <211> 225 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Feline IgG2 Fc Fragment <400> 21 Gly Glu Gly Pro Lys Cys Pro Val Pro Glu Ile Pro Gly Ala Pro Ser 1 5 10 15 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Leu Val Val Asp Leu Gly Pro Asp Asp Ser 35 40 45 Asn Val Gln Ile Thr Trp Phe Val Asp Asn Thr Glu Met His Thr Ala 50 55 60 Lys Thr Arg Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Pro Ile Leu His Gln Asp Trp Leu Lys Gly Lys Glu Phe 85 90 95 Lys Cys Lys Val Asn Ser Lys Ser Leu Pro Ser Ala Met Glu Arg Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro His Glu Pro Gln Val Tyr Val Leu 115 120 125 Pro Pro Thr Gln Glu Glu Leu Ser Glu Asn Lys Val Ser Val Thr Cys 130 135 140 Leu Ile Lys Gly Phe His Pro Pro Asp Ile Ala Val Glu Trp Glu Ile 145 150 155 160 Thr Gly Gln Pro Glu Pro ...

Claims

1. A fusion protein consisting of the following sequence: FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 36). 2 . The fusion protein according to claim 1 , wherein the fusion protein is produced using one of HEK293 or CHO cells, and the homodimer titer obtained after purification using protein A beads or protein A columns is greater than 50 mg / L.

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

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

5. The fusion protein of claim 1, wherein the time for which a statistically significant reduction in blood glucose levels relative to pre-dose levels is achieved in a subject is greater 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 more. The fusion protein according to claim 1 , wherein the NAOC after the first subcutaneous injection in the target animal is greater than 150% FBGL·day·kg / mg.

7. The fusion protein of claim 6, wherein the ratio of 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.

8. A pharmaceutical composition comprising the fusion protein according to claim 1.

9. The pharmaceutical composition of claim 8, wherein the fusion protein is present in the pharmaceutical composition at a concentration of 3 mg / mL or higher.

10. The pharmaceutical composition according to claim 9, wherein the composition is suitable for subcutaneous administration.

11. Use of the fusion protein of claim 1 or the pharmaceutical composition of claim 8 for manufacturing a medicament for lowering blood glucose levels in a target animal, wherein the target animal is a dog.

12. The use according to claim 11, wherein the target animal is diagnosed with diabetes. 13 . The use according to claim 11 , wherein the fusion protein is administered to the target animal once a week at a dose of 0.025 to 0.5 mg / kg / week. The use according to claim 11 , wherein the fusion protein is administered subcutaneously.

15. The use according to claim 14, wherein the fusion protein is administered to the target animal daily, twice a week or once a week.

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