Super long acting insulin-FC fusion proteins and methods of use

Abstract

The present disclosure provides recombinant manufactured ultra long acting insulin-Fc fusion proteins for use in the treatment of canine and feline diabetes mellitus. The insulin-Fc fusion protein comprises an insulin polypeptide linked by a peptide linker to an Fc fragment of canine or feline origin. Based on the results obtained, a therapy is created which is suitable for low cost manufacture, exhibits sufficient in vivo biological activity, exhibits extended biological activity duration, does not induce anti-drug antibodies, and remains substantially potency during multiple administration, in addition to selective mutations on one or more of these components, and is not susceptible to the occurrence of a disruption in the body of the patient. A non-noticeable combination of an insulin polypeptide, a peptide linker, and a species-specific Fc fragment is also required. 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 also provided in addition to methods of use and preparation.

Description

[0001] This invention is a divisional application of a case with an application date of June 28, 2019, application number 201980056947.5, and invention name “Ultra-long-acting insulin-FC fusion protein and method of use”.

[0002] Priority and related applications

[0003] No. 62 / 781,378 filed on December 18, 2018, U.S. Provisional Patent Application Serial No. 62 / 781,368 filed on December 18, 2018, U.S. Provisional Patent Application Serial 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 / 743,358 filed on October 3, 2018, 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. Provisional 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

[0004] 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

[0005] 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 prior art for the present technology.

[0006] Diabetes is a chronic condition characterized by insulin deficiency and / or ineffective use of insulin. Diabetic patients with an absolute deficiency of insulin are classified as having type 1 or insulin-dependent diabetes mellitus (IDDM). Type 1 diabetes is believed to be genetically predisposed and is accompanied by immune destruction of the insulin-producing beta cells of the pancreas. In contrast, diabetic patients 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 related to genetic predisposition, obesity, and certain medications.

[0007] When a dog or cat does not produce insulin or cannot use insulin normally, blood sugar levels rise, leading to hyperglycemia. Dogs typically exhibit an atypical blood sugar phenotype very similar to human type 1 diabetes. Dogs occasionally also exhibit atypical blood sugars very similar to human type 2 diabetes. Female dogs can also develop temporary insulin resistance when they are in estrus or pregnant. In all cases, dogs are treated with chronic insulin injection therapy. Cats typically exhibit an atypical blood sugar phenotype very similar to human type 2 diabetes (i.e., insulin resistance), but by the time a veterinarian diagnoses the disease, the disease has developed into a condition similar to type 1 diabetes (inflammatory disease in the pancreas, significant loss of beta cell mass), and the cats are dependent on exogenous insulin. Some diabetic cats can be controlled with dietary changes and oral medications, but most diabetic cats receive chronic insulin injection therapy to maintain adequate regulation. If left untreated, diabetes in dogs and cats can lead to weight loss, loss of appetite, vomiting, dehydration, motor function problems, coma, and even death.

[0008] In the United States, approximately 0.24% of dogs and 0.68% of cats have diabetes. Current diabetes treatments for dogs and cats include the use of insulin, such as (Intervet Inc., dbaMERCK Animal Health, Summit, NJ), and the use of (Boehringer Ingelheim Vetmedica, Duluth, Georgia), administered once or twice daily. The burden of frequent injections on owners often leads to a lack of adherence to treatment regimens and inadequate dosing, which can lead to 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 proportion 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

[0009] 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:

[0010] 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 GGGGQGGGGQGGGGGGG (SEQ ID NO: 14).

[0011] In an embodiment, the fusion protein comprises the sequence FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO:32). In an embodiment, the fusion protein comprises the sequence FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO:34).

[0012] 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 FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (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 FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (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 GGGGGQGGGGQGGGGGGG (SEQ ID NO: 14).

[0013] In an embodiment, the fusion protein includes the sequence FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPR EEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:36).

[0014] 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 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. In 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. In embodiments, the insulin polypeptide and the Fc fragment are connected by a linker, such as a peptide linker, comprising the following sequence GGGGGQGGGGQGGGGGGG (SEQ ID NO: 14).

[0015] In an embodiment, the fusion protein includes the sequence FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPR EEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:38).

[0016] 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 FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is not D, and X2 is not H. In embodiments, the insulin polypeptide comprises the sequence FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (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 GGGGGQGGGGQGGGGGGG (SEQ ID NO: 14).

[0017] In an embodiment, the fusion protein includes the sequence FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSP REEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:40).

[0018] In some aspects, fusion protein as described herein comprises homodimer (homodimer).In an embodiment, the homodimer percentage of fusion protein is greater than 90%.In an embodiment, fusion protein as described herein is prepared using HEK293 cells, and the homodimer titer obtained after purification using protein A beads or protein A column is greater than 50mg / L.In an embodiment, the insulin receptor IC50 of fusion protein as described herein is less than or equal to 5000nM.In an embodiment, when administered, the serum half-life of fusion protein as described herein in the blood or serum of target animal is longer than about 3 days.In an embodiment, for fusion protein as described herein, wherein in subject, blood glucose level has statistically significant reduction time relative to pre-administration level 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 one of longer time.

[0019] 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 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.

[0020] In some aspects, the fusion protein as described herein is formulated into a pharmaceutical composition. In an embodiment, the fusion protein is present in a pharmaceutical composition at a concentration of about 3 mg / mL or higher. In an embodiment, the composition is suitable for subcutaneous administration.

[0021] In one aspect, a method for lowering the blood glucose level of a target animal is described, comprising administering to the patient a physiologically effective amount of a fusion protein as described herein or a pharmaceutical composition thereof. In embodiments, the target animal is diagnosed with diabetes. In embodiments, 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 daily, twice a week, or once a week. In embodiments, the fusion protein is administered to the target animal once a week at a dose 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 embodiments, the cell is transfected with a nucleic acid encoding the fusion protein. In embodiments, the cell is a HEK293 cell or a CHO cell.

[0022] In one aspect, a cDNA encoding a fusion protein as described herein is described. In an embodiment, the cDNA comprises the nucleic acid sequence

[0023] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:31)。

[0024] In an embodiment, the cDNA contains the nucleic acid sequence atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcaacggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:33).

[0025] In an embodiment, the cDNA comprises the nucleic acid sequence atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:35).

[0026] In an embodiment, the cDNA comprises the nucleic acid sequence atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcaacagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag (SEQ ID NO:37).

[0027] In an embodiment, the cDNA comprises the nucleic acid sequence atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcagcagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag (SEQ ID NO:39). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A schematic diagram of an exemplary insulin-Fc fusion protein homodimer is shown.

[0029] Figure 2 Shown are the mean % fasting blood glucose levels 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.

[0030] Figure 3 Shown is a side-by-side sequence comparison of SEQ ID NOs: 42, 44, 46, 48, and 50. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates 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 4 Shown is a side-by-side sequence comparison of SEQ ID NOs: 42, 52, 54, and 56. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequences at a given sequence position.

[0032] Figure 5 Shown are the mean % fasting blood glucose levels 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.

[0033] Figure 6 Shown are the mean % fasting blood glucose levels from Day 0 to Day 7 for N=6 dogs dosed subcutaneously on Day 0 with 0.33 mg / kg of the homodimer of SEQ ID NO: 52.

[0034] Figure 7 Shown are the mean 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).

[0035] Figure 8Shown is a side-by-side sequence comparison of SEQ ID NOs: 58, 60, 62, and 64. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequences at a given sequence position.

[0036] 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).

[0037] 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).

[0038] Figure 11 Shown are the mean % fasting blood glucose levels from Day 0 to Day 7 for N=2 dogs dosed subcutaneously on Day 0 with 0.33 mg / kg of the homodimer of SEQ ID NO: 66.

[0039] Figure 12 Shown is a side-by-side sequence comparison of SEQ ID NOs: 66, 68, 70, 72, 74, and 76. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates 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 13 Shown is a side-by-side sequence comparison of SEQ ID NOs: 66, 78, 80, 82, and 84. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates 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 14 Shown is a side-by-side sequence comparison of SEQ ID NOs: 66, 76, and 86. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequences at a given sequence position.

[0042] Figure 15Shown is a side-by-side sequence comparison of SEQ ID NOs: 66, 82, 84, and 88. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates 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 16 Shown is a side-by-side sequence comparison of SEQ ID NOs: 32, 34, 66, 90, 92, and 94. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates 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.

[0044] Figure 17 Shown are the % fasting blood glucose levels from Day 0 to Day 7 for N=1 dog dosed subcutaneously with 0.16 mg / kg of the homodimer of SEQ ID NO: 34 on Day 0.

[0045] 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).

[0046] Figure 19 Shown are the % fasting blood glucose levels from Day 0 to Day 7 for N=1 dog dosed subcutaneously with 0.33 mg / kg of the homodimer of SEQ ID NO: 32 on Day 0.

[0047] Figure 20 Shown are % fasting blood glucose levels 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).

[0048] 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).

[0049] Figure 22Shown are the % fasting blood glucose levels from Day 0 to Day 7 for N=1 dogs dosed subcutaneously with 0.16 mg / kg of the homodimer of SEQ ID NO: 96 on Day 0.

[0050] Figure 23 Shown are the fasting blood glucose levels in % from day 0 to day 7 for N=1 dog dosed subcutaneously with 0.16 mg / kg of the homodimer of SEQ ID NO: 98 on day 0.

[0051] Figure 24 Shown is a side-by-side sequence comparison of SEQ ID NOs: 102 and 104. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequences at a given sequence position.

[0052] Figure 25 Shown are the % fasting blood glucose levels from day 0 to day 7 for N=1 dog 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 administered 0.16 mg / kg subcutaneously of the homodimer of SEQ ID NO: 104 on day 0.

[0053] Figure 26 Shown are the 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.

[0054] Figure 27 Shown are the mean % fasting blood glucose levels from day 0 to day 7 for N=3 cats dosed subcutaneously on day 0 with 0.8 mg / kg of the homodimer of SEQ ID NO: 106.

[0055] Figure 28 A side-by-side sequence comparison is shown for SEQ ID Nos: 106, 108, 110, and 112. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates 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.

[0056] Figure 29Shown are the mean anti-drug antibody titers (μg / mL) for N=3 cats administered subcutaneously a homodimer of SEQ ID NO: 106 on Days 0 (0.8 mg / kg), 28 (0.6 mg / kg), 35 (0.6 mg / kg), 42 (0.6 mg / kg), and 48 (0.8 mg / kg).

[0057] Figure 30 Shown is a side-by-side sequence comparison of SEQ ID NOs: 108, 114, 116, and 118. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates 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 31 Shown is a side-by-side sequence comparison of SEQ ID NOs: 106, 112, and 122. "*" indicates perfect homology across all sequences at a given sequence position, while ":," ".", or a space indicates a conservative amino acid mutation, a moderate amino acid mutation, or a very different amino acid mutation, respectively, across the sequences at a given sequence position.

[0059] Figure 32 Shown are the 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).

[0060] Figure 33 Shown are the 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.

[0061] 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 days 0 (0.16 mg / kg), 14 (0.16 mg / kg), 28 (0.11 mg / kg), and 42 (0.09 mg / kg).

[0062] Figure 35 Shown are the 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).

[0063] Figure 36Shown are the mean % fasting blood glucose levels from day 0 to day 7 for N=3 cats that were dosed subcutaneously with the homodimer of SEQ ID NO: 40 on day 0 (0.10 mg / kg).

[0064] Figure 37 Shown are the mean % fasting blood glucose levels from day 7 to day 14 for N=3 cats that were dosed subcutaneously with the homodimer of SEQ ID NO: 40 on day 7 (0.20 mg / kg).

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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

[0070] Insulin therapy that requires less frequent dosing (e.g., once a week) would be less burdensome on 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 titer 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.). Only candidates with a homodimer titer greater than 50 mg / L are considered useful in the present invention, as experience has shown that homodimer titers below this level are unlikely to result in commercial production 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 5000 nM, IC50 less than 4000 nM, IC50 less than 3000 nM, IC50 less than 2500 nM, etc.), as measured in a 4°C IM-9 insulin receptor binding assay. As a rule of thumb, molecules that only exhibit an IC50 value of insulin receptor activity less than 5000 nM 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 quantified by calculating the area under the curve normalized to a percentage of fasting blood glucose (%FBGL) for a given dose in mg / kg (NAOC), where the units are %FBGL·day·kg / mg. An increase in NAOC with a greater decrease in %FBGL is a condition in which the molecule exhibits increased bioactivity, and an increase in %FBGL with a longer recovery time to 100% is a condition in which the insulin-Fc fusion protein exhibits an increased duration of action. As described herein, to be useful, a 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 after the third dose (NAOCR)) 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).

[0071] The recommended ultra-long-acting insulin therapy for clinical use in humans comprises 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 therefore 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 discovered 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., an 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, requiring site-directed mutagenesis to discover a relatively small number of examples that are both non-immunogenic and biologically active in the target species, with acceptably high NAOC values ​​(e.g., a NAOC value greater than 150% FBGL·day·kg / mg) and a NAOCR value after subcutaneous administration of greater than 0.5 in the third week. In another case, 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., homodimer titer greater than 50 mg / L) and NAOC value (e.g., NAOC value greater than 150% FBGL·day·kg / mg). In addition, it was found that additional mutations in the insulin component were required to produce non-glycosylated insulin Fc-fusion proteins with these Fc mutations having the desired homodimer titer (e.g., homodimer titer greater than 50 mg / L) and NAOC value (e.g., 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. Thus, provided herein are manufacturable, highly pure, long-acting, bioactive, non-immunogenic insulin-Fc fusion proteins 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, 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.

[0072] definition

[0073] 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."

[0074] As used herein, "about" and "approximately" generally refer to an acceptable degree of error for a measured quantity, given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given range of values.

[0075] 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 the subject or cures, alleviates, relieves or improves the condition of 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.

[0076] 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 respect (e.g., a compound or conjugate as described herein, such as insulin).

[0077] As used herein, the term "antibody" or "antibody molecule" refers to an immunoglobulin molecule (Ig), the 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 the variable or constant region of an immunoglobulin. As used herein, the term "antibody domain" refers to the variable or constant region of an immunoglobulin. It has been documented in the art that antibodies comprise several classes, such as IgA, IgM, or IgG in the case of mammals (e.g., humans and felines). The class 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 comprise amino acid sequences, structures, and functional properties (e.g., different binding affinities to Fc (γ) receptors) that are different from each other. "Specifically binds to" 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.

[0078] 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 administering a given dose of insulin-Fc fusion protein. 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, such that AOC equals the difference between the total possible area under the time curve and the AUC value for %FBGL. As used herein, "normalized area on the curve," "normalized AOC," or "NAOC" are the AOC values ​​divided by the actual dosage of the insulin-Fc fusion protein administered. As used herein, the term "standardized AOC ratio" or "NAOCR" is the ratio of the NAOC produced by the specific administration of insulin-Fc fusion protein to the NAOC produced by the first administration of insulin-Fc fusion protein in a series of administrations. Therefore, NAOCR provides a measure of the biological activity changes of insulin-Fc fusion protein after repeated administrations.

[0079] As used herein, the terms "biological activity," "activity," "biological activity," "potency," "biological activity potency," or "biological efficacy" 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 the 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.

[0080] As used herein, the terms "biosynthesis," "recombinant synthesis," or "recombinant preparation" refer to the process of expressing an insulin-Fc fusion protein in a host cell by transfecting the cell with a nucleic acid molecule (e.g., a vector) encoding the 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. The cells can be cultured using standard methods in the art, and the expressed insulin-Fc fusion protein can be harvested and purified from the cell culture using standard methods in the art.

[0081] As used herein, the term "cell surface receptor" refers to a molecule, such as a protein, typically found on the outer surface of a cell membrane and that interacts with a soluble molecule (e.g., a molecule circulating in the blood supply). In some embodiments, a cell surface receptor can include a hormone receptor (e.g., the insulin hormone receptor or insulin receptor (IR)) or an Fc receptor that binds to the 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 OD 450nm values ​​measured on a microplate reader.

[0082] As used herein, the term "fasting blood glucose level" or "FBGL" refers to the average blood glucose level in a subject at the end of a period during which no food is given and immediately before the time of administration of the insulin-Fc fusion protein. As used herein, the term "percent fasting blood glucose level," "% fasting blood glucose level," or "%FBGL" refers to the ratio of a given blood glucose level to the fasting blood glucose level multiplied by 100.

[0083] 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 can specifically bind to the molecule. As used herein, the term "neutralizing," "neutralizing antibody," or "neutralizing anti-drug antibody" 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.

[0084] 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).

[0085] As used herein, the term "monomer" refers to a protein or fusion protein comprising a single polypeptide. In embodiments, a "monomer" is a protein or fusion protein comprising an insulin polypeptide and an Fc fragment polypeptide, e.g., a single polypeptide, wherein the insulin and Fc fragment polypeptides are linked by a peptide bond to form a single polypeptide. In embodiments, a monomer is encoded by a single nucleic acid molecule.

[0086] As used herein, the "N-terminus" refers to the beginning of a protein or polypeptide that is initiated by an amino acid containing a free amine group, which is the α-amino group of the 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, the "C-terminus" refers to the end of a protein or polypeptide that is 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.

[0087] 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 reduction in fasting blood glucose levels over time in a subject after administration of an insulin-Fc fusion protein.

[0088] As used herein, "pharmacokinetics" or "PK" generally refers to the characteristic interactions between an insulin-Fc fusion protein and a subject's body in terms of its absorption, distribution, metabolism, and excretion. Specifically, in this article, PK refers to the concentration of the insulin-Fc fusion protein in a subject's blood or serum at a given time after administration of the insulin-Fc fusion protein. As used herein, "half-life" refers to the time it takes for the concentration of the insulin-Fc fusion protein in a subject's blood or serum 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 the target subject.

[0089] As used herein, the terms "sequence identity," "sequence homology," "homology," or "identical" in amino acid or nucleotide sequences describe the nucleotide or amino acid residues that are found to be identical in a variant and a reference sequence when a specified contiguous segment of the nucleotide or amino acid sequence of the variant is aligned and compared to the nucleotide or amino acid sequence of the reference sequence. Methods for aligning sequences 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 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). With respect to the optimal comparison of two nucleotide sequences, the continuous section of the variant nucleotide sequence can have additional nucleotides or deleted nucleotides relative to the reference nucleotide sequence. Similarly, for the optimal comparison of two amino acid sequences, the continuous section of the variant amino acid sequence can have additional amino acid residues or deleted amino acid residues relative to the reference amino acid sequence. In certain embodiments, the continuous section for comparison with the reference nucleotide sequence or reference amino acid sequence will comprise 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 carried out by specifying a gap penalty. Methods of sequence alignment are known in the art.

[0090] In an embodiment, mathematical algorithms are used to complete the determination of identity percentage or " homology " between two sequences.For example, the Smith-Waterman homology search algorithm is used to determine the identity percentage of amino acid sequence, and the algorithm uses affine 6 gap searches, wherein the gap open 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.Math 2:482-489 (which is incorporated herein by reference).In an embodiment, the Smith-Waterman homology search algorithm is used to determine the identity percentage of nucleotide sequence, and the algorithm uses a gap open penalty of 25 and a gap extension penalty of 5.This type of determination of sequence identity can be carried out using, for example, the DeCypher hardware accelerator from TimeLogic.

[0091] As used herein, the term "homology" is used to compare two or more proteins by locating common structural features and common spatial distribution of, for example, beta strands, helices, and folds. Therefore, homologous protein structures are defined by spatial analysis. Measuring structural homology involves calculating the geometric topological characteristics 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 prerequisite.

[0092] 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.

[0093] As used herein, the term "titer" or "yield" refers to the amount of fusion protein product (e.g., insulin-Fc fusion protein as 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 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 proportion of fusion protein product (e.g., insulin-Fc fusion protein as described herein) that is the desired homodimer. As used herein, the term "homodimer titer" refers to the product of the % homodimer and the total protein yield reported per volume of cell culture after the Protein A purification step.

[0094] 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, relieved, altered, remedied, improved, or improved. Treatment includes administering an amount that effectively alleviates, alleviates, alters, remedies, improves, improves, or affects the disease or disorder or the symptoms of the disease or disorder. The treatment can inhibit the worsening or aggravation of the symptoms of the disease or disorder.

[0095] Components and structure of insulin-Fc fusion protein

[0096] 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 purposes of 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 to 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.

[0097] 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" (e.g., Figure 1 ). Disulfide bonds such as 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 during further processing in the cell.

[0098] As used herein, the terms "multimer," "multimeric," or "multimeric state" refer to non-covalent, associated forms of Fc-fusion protein dimers that can be in equilibrium with Fc-fusion protein dimers or can serve as permanent aggregates 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 is anticipated that Fc-fusion proteins in multimeric form may have different physical, stability, or pharmacological activities than insulin-Fc fusion protein homodimers.

[0099] Insulin peptide

[0100] 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 the insulin receptor (IR), initiating a signaling cascade that affects many aspects of mammalian (e.g., human, canine or feline) metabolism. 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 may be related to the structure of insulin, but contain one or more modifications. In some embodiments, the insulin analogs contain at least one amino acid substitution, deletion, addition or chemical modification relative to insulin, which may affect the specific features or properties of the insulin-Fc fusion protein. 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 the insulin-Fc fusion protein to cell surface receptors (e.g., insulin hormone receptors).

[0101] 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:

[0102] FVNQHLCGSHLVEALYLVCGERGFFYTPKT(SEQ ID NO:1)

[0103] GIVEQCCTSICSLYQLENYCN(SEQ ID NO:2)

[0104] 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 comprise one or more fragments from mature insulin, proinsulin, proinsulin, or naturally occurring insulin.

[0105] Insulin is generally constructed as an N-terminal-B chain:C chain:A chain-C-terminal polypeptide, wherein the C chain is cleaved to render 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, with the C chain underlined:

[0106] FVNQHLCGSHLVEALYLVCGERGFFYTPKT RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR GIVEQCCTSICSLYQLENYCN(SEQ ID NO:3)

[0107] Prior to glucose-stimulated insulin secretion, the conversion of single-chain insulin polypeptides to biologically active two-chain polypeptides is typically accomplished in the β cells of the islets of Langerhans by two endoproteases: type I endoproteases PC1 and PC3, which break the C-peptide-B chain connection, and type II endoproteases PC2, which cleave the C-peptide-A chain bond at the correct site. However, cell systems used to biosynthesize therapeutic molecules such as insulin (e.g., bacteria, yeast, and mammalian (e.g., HEK and CHO) cell systems) do not have this pathway, and therefore conversion must be performed after expressing and harvesting the single-chain polypeptide using chemical or enzymatic methods. All known techniques for cleaving the C-chain after expression and harvesting rely on first modifying the C-chain so that it terminates at a 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 cleaved at the C-terminal lysine of the C-chain and at the C-terminal lysine at position 29 of the N-terminus of the B-chain. In some cases, the resulting biologically active two-chain insulin is used without reconnecting the sheared amino acid at position 30 of the N-terminal B chain, and in some cases, an additional enzymatic method is used to add the sheared amino acid at position 30 of the N-terminal B chain back into the molecule. This type of process works well 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 herein because all known Fc fragments contain multiple lysine residues. Therefore, the enzymatic cleavage process digests the Fc fragment into non-functional parts, thereby eliminating the ability of the Fc fragment to prolong the action of the insulin polypeptide in vivo. Therefore, the insulin-Fc fusion protein of the present invention must contain an insulin polypeptide that does not require cleavage of the C chain, and therefore its single-chain form is biologically active.

[0108] 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 to achieve electrostatic equilibrium, 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 from the N-terminal of the amino acid sequence of the B chain. The symbol "A8" refers to the 8th amino acid from the N-terminal of the A chain. In addition, if an amino acid mutates to a new amino acid from its native form at a specific position, the position is accompanied by a one-letter amino acid code for the new amino acid. For example, B16A refers to an alanine mutation at the 16th amino acid from the N-terminal of the amino acid sequence of the B chain, and A8H refers to a histidine mutation at the 8th amino acid from the N-terminal of the amino acid sequence of the A chain.

[0109] In one example, a single-chain insulin analog having a C chain with the sequence GGGPRR and additional substitutions in the A and B chains (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 was substituted with histidine; at position 10 (i.e., B10) of the B chain, histidine was substituted with aspartic acid; at position 28 (i.e., B28) of the B chain, proline was substituted with aspartic acid; and at position 29 (i.e., B29) of the B chain, lysine was substituted with proline. SEQ ID NO:4 is set forth below, with each unnatural amino acid underlined:

[0110] FVNQHLCGS D LVEALYLVCGERGFFYT DP T GGGPRR GIVEQCC HSICSLYQLENYCN (SEQ ID NO: 4)

[0111] In an embodiment, the tyrosine at position 16 (i.e., B16) of the N-terminal end of the B chain in SEQ ID NO: 4 can be substituted with alanine to generate SEQ ID NO: 5, because alanine substitutions at 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:

[0112] FVNQHLCGS D LVEAL A LVCGERGFFYT DP T GGGPRR GIVEQCC H SICSLYQLENYCN (SEQ ID NO: 5)

[0113] In some embodiments, it was unexpectedly discovered 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 injection in a target animal (e.g., a dog or cat). 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, in the steps leading to the invention disclosed herein, it was discovered that mutations at A8 to histidine and mutations at B10 to aspartic acid accounted for the vast majority of anti-drug antibody specificity and therefore represent immunogenic "hot spots" (e.g., immunogenic epitopes) on insulin polypeptides. Therefore, in preferred embodiments, the insulin polypeptide does not contain histidine at position A8 or aspartic acid at position B10 of the insulin polypeptide.

[0114] In one embodiment, it was demonstrated that simply maintaining 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 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 bioactivity 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 found to be suitable for achieving a homodimer titer greater than 50 mg / L, an NAOC value in the target species greater than 150% FBGL·day·kg / mg, minimal immunogenicity, and an 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 an underlined Z):

[0115] FVNQHLCGS X1 LVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCC X2 S T CSL D QLENYC X3 (SEQID NO:6_NULL)

[0116] Where X1 is not D, X2 is not H, and X3 does not exist or is N.

[0117] In a specific embodiment, in SEQ ID NO:6_NULL, X1 is H, X2 is T, and X3 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):

[0118] FVNQHLCGSHLVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYC X3 (SEQID NO:7_NULL)

[0119] Where X3 does not exist or is N.

[0120] In a specific embodiment, in SEQ ID NO:7_NULL, X3 is absent, resulting in the following SEQ ID NO:8_NULL (wherein the unnatural amino acids are underlined and the missing natural amino acid is represented by an underlined Z):

[0121] FVNQHLCGSHLVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYC Z (SEQ ID NO:8_NULL)

[0122] In a specific embodiment, in SEQ ID NO:7_NULL, X3 is N, resulting in the following SEQ ID NO:9_NULL (wherein the unnatural amino acids are underlined and the missing natural amino acid is represented by an underlined Z): FVNQHLCGSHLVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYCN (SEQ ID NO:9_NULL)

[0123] 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 the 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, necessitating another amino acid mutation on the insulin polypeptide in order to render the insulin-Fc fusion protein sufficiently manufacturable (e.g., having a homodimer titer greater than 50 mg / L) and non-immunogenic, with an NAOC value in the target species greater than 150% FBGL·day·kg / mg, and an NAOCR value after the third injection in the target species 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, resulting in the following insulin polypeptide SEQ ID NO: 10_NULL (wherein the non-natural amino acid is underlined and the missing natural amino acid is represented by an underlined Z):

[0124] FVNQHLCGS X1 LVEAL A LVCGERGF H Y ​ GIVEQCC X2 S TCSL D QLENYC Z (SEQID NO:10_NULL)

[0125] Where X1 is not D, and X2 is not H.

[0126] In a specific embodiment, in SEQ ID NO: 10_NULL, X1 is H, and X2 is T, resulting in the following SEQ ID NO: 11_NULL (wherein the unnatural amino acids are underlined and the missing natural amino acid is represented by an underlined Z):

[0127] FVNQHLCGSHLVEAL A LVCGERGF H Y ​ GIVEQCCTS T CSL D QLENYC Z –SEQ ID NO:11_NULL

[0128] The following is a restatement of the above sequence, but with the missing amino acid symbol Z removed from the insulin polypeptide sequence notation. 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 symbols, the paired sequences refer to the exact same insulin polypeptide.

[0129] SEQ ID NO:6_NULL is restated as:

[0130] FVNQHLCGS X1 LVEAL E LVCGERGF H Y ​ GIVEQCC X2 S T CSL D QLENYC X3 (SEQ ID NO: 6)

[0131] Where X1 is not D, X2 is not H, and X3 does not exist or is N.

[0132] SEQ ID NO:7_NULL is restated as:

[0133] FVNQHLCGSHLVEAL E LVCGERGF H Y ​ GIVEQCCTS T CSL D QLENYCX3 (SEQ ID NO:7)

[0134] Where X3 does not exist or is N.

[0135] SEQ ID NO:8_NULL is restated as:

[0136] FVNQHLCGSHLVEAL E LVCGERGF H Y ​ GIVEQCCTS T CSL D QLENYC (SEQ ID NO: 8)

[0137] SEQ ID NO:9_NULL is restated as:

[0138] FVNQHLCGSHLVEAL E LVCGERGF H Y ​ GIVEQCCTS T CSL D QLENYCN (SEQ ID NO: 9)

[0139] SEQ ID NO:10_NULL is restated as:

[0140] FVNQHLCGS X1 LVEAL A LVCGERGF H Y ​ GIVEQCC X2 S T CSL D QLENYC (SEQ ID NO: 10)

[0141] Where X1 is not , and X2 is not H.

[0142] SEQ ID NO: 11_NULL is restated as:

[0143] FVNQHLCGSHLVEAL A LVCGERGF H Y ​ GIVEQCCTS T CSL D QLENYC (SEQ ID NO: 11)

[0144] connector

[0145] The successful construction of recombinantly prepared insulin-Fc fusion proteins requires a linker to connect the insulin polypeptide to 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, for example, so that a single nucleic acid molecule can encode various peptides within the insulin polypeptide as well as a peptide linker and an Fc fragment. 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 used to design insulin-Fc fusion proteins with the aim of improving homodimer titer and biological activity.

[0146] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0147] In an embodiment, the peptide linker comprises the sequence:

[0148] GGGGAGGGG (SEQ ID NO: 12).

[0149] In other embodiments, the peptide linker comprises the sequence:

[0150] GGGGSGGGG (SEQ ID NO: 13).

[0151] In a preferred embodiment, the peptide linker comprises the following sequence:

[0152] GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

[0153] When constructing a recombinantly prepared insulin-Fc fusion protein with a peptide linker (such as the linker in SEQ ID NO: 14), it is important to note 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 extended biological activity duration. There is a known enzymatic cleavage site between the asparagine-glycine bond (Vlasak, J., Ionescu, R., (2011) MAbs Vol. 3, No. 3 pp 253-263). In embodiments 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 was omitted from 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 could form 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) exhibited 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.

[0154] Fc fragment

[0155] 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 comprise 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 comprises an amino acid sequence that connects the CH1 domain of the heavy chain to the CH2 region of the heavy chain, and comprises one or more cysteines that form one or more inter-heavy chain disulfide bonds to form a homodimer of the Fc fusion protein from two identical but separate monomers of the Fc fusion protein. The hinge region may comprise all or part of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence.

[0156] 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 Fc fragment or Fc region that binds to an IgG antibody (γ receptor), and includes but is not limited to receptors of the Fc (γ) receptor I, Fc (γ) receptor IIa, Fc (γ) receptor IIb, and Fc (γ) receptor III subclasses, including allelic variants and alternative splicing forms of these receptors. "FcR" also includes the 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 comprising a canine or feline Fc fragment in order to assess their FcR binding properties. One skilled in the art will appreciate that a mammalian FcR from one species (e.g., a human-derived FcR) is sometimes capable of binding in vitro to an Fc fragment from a second species (e.g., a canine or feline-derived 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 comprising a canine or feline-derived Fc fragment in order to assess their FcR binding properties. One skilled in the art will appreciate that a mammalian FcR from one species (e.g., a canine-derived FcR) is capable of binding in vitro to an insulin-Fc fusion protein comprising an Fc fragment from the same species (e.g., canine-derived), and sometimes also to an insulin-Fc fusion protein comprising an Fc fragment from another mammalian species (e.g., feline-derived).

[0157] In embodiments, the Fc fragment comprises the Fc region (e.g., hinge region, CH2 domain, and CH3 domain) of a mammalian IgG, such as a canine IgGAFc fragment (SEQ ID NO: 15), a canine IgGBFc fragment (SEQ ID NO: 16), a canine IgGCFc fragment (SEQ ID NO: 17), or a canine IgGDFc fragment (SEQ ID NO: 18), or a feline IgG1a fragment (SEQ ID NO: 19), a feline IgG1bFc fragment (SEQ ID NO: 20), or a feline IgG2Fc fragment (SEQ ID NO: 21). In the Examples, the C-terminal lysine (i.e., the lysine representing the last amino acid of the Fc fragment sequence) 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 production of the desired protein in a cell (Dick, LW., (2008) Biotechnol Bioeng. Aug 15; 100(6) pp1132-43). Thus, in the Examples, the sequence of the canine or feline Fc fragment lacking the C-terminal lysine is:

[0158] RCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTIS KARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG(SEQ ID NO:15)

[0159] DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:16)

[0160] CNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG(SEQ ID NO:17)

[0161] CISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTY

[0162] RVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPP

[0163] EIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG(SEQ ID NO:18)

[0164] DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNST

[0165] YRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHP

[0166] PDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:19)

[0167] DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNST

[0168] YRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYP

[0169] SDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:20)

[0170] GEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNST

[0171] YRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPP

[0172] DIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:21)

[0173] Since the IgGA isotype lacks Fc (γ) effector function in dogs (very similar to the IgG2 isotype in humans), it is preferred to replace the human Fc with canine IgGA to minimize any unwanted immunogenicity in dogs. However, in the example 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., homodimer titer less than 50 mg / L). In addition, the compound was not biologically active in dogs (i.e., NAOC value 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 are no examples based on canine IgGA Fc fragments that exhibit sufficiently low aggregation and sufficiently high titers of the desired homodimers. In another aspect, replacing the canine IgGA Fc fragment (SEQ ID NO:15) with a canine IgGB Fc fragment (SEQ ID NO:16) resulted in a compound with much lower aggregation and a relatively high desired homodimer titer. Furthermore, compounds containing the insulin polypeptide of SEQ ID NO:5 and the canine IgGB Fc fragment (SEQ ID NO:16) were bioactive in dogs, demonstrating glucose-lowering bioactivity over multiple days (i.e., NAOC values ​​greater than 150% FBGL·day·kg / mg).

[0174] 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), the superiority of the canine IgGB Fc fragment over the canine IgGA Fc fragment was demonstrated. Insulin-Fc fusion proteins containing the insulin polypeptide of SEQ ID NO: 8 and the peptide linker of SEQ ID NO: 14 were synthesized using Fc fragments from 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) immunoglobulins. Using conventional purification methods, only 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, the 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, for dog-specific products, canine IgGB (SEQ ID NO: 16) is the preferred Fc fragment for all insulin-Fc fusion proteins used in dogs, regardless of the insulin polypeptide selected.

[0175] Since the IgG2 isotype lacks Fc(γ) effector function in cats (very similar to the IgG2 isotype in humans), it is preferred to replace the 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, had low aggregation (i.e., homodimer titers greater than 50 mg / L) and appreciable insulin receptor affinity (i.e., insulin receptor IC50 values ​​less than 5000 nM). However, unexpectedly, when the insulin polypeptide was altered to SEQ ID NO: 7, the insulin-Fc fusion protein comprising a feline IgG2 fragment (SEQ ID NO: 21) had no biological activity in cats (i.e., NAOC less than 150% FBGL·day·kg / mg), whereas the insulin-Fc fusion protein comprising a feline IgG1b fragment (SEQ ID NO: 20) exhibited low aggregation (i.e., high homodimer %), high titers of the desired homodimers (i.e., homodimer titers greater than 50 mg / L), and appreciable levels of long-term glucose-lowering biological activity in cats (i.e., NAOC values ​​greater than 150% FBGL·day·kg / mg). Therefore, for cat-specific products, the feline IgG1b fragment (SEQ ID NO: 20) is the preferred Fc fragment when the insulin polypeptide sequence comprises SEQ ID NO: 7.

[0176] Given 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. One method for reducing Fc (γ) interactions involves deglycosylation of the Fc fragment during synthesis in the host cell or preventing its 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". One method for removing attached glycans from a synthetic insulin-Fc fusion protein is to mutate the cNg site to prevent glycans from attaching together during production in the host cell. In this article, the symbol used to describe the cNg mutation is cNg-(substituted amino acid). For example, if the asparagine at the cNg site is mutated to serine, the mutation is named "cNg-S".

[0177] The absolute position of the cNg site from the N-terminus of the B chain of the insulin-Fc fusion protein varies depending on the length of the insulin polypeptide, the length of the linker, and any omitted amino acids in the Fc fragment before the cNg site. Herein, 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 a cNg site is found at the 151st amino acid position, as counted 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 a cNg site is found at the 151st amino acid position, as counted 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".

[0178] In the embodiment comprising an insulin polypeptide of SEQ ID NO: 5 and a canine IgGB Fc fragment having cNg-Q, cNg-S, cNg-D, and cNg-K mutations, it was unexpectedly found that compounds containing only the cNg-K and cNg-S mutations exhibited the requisite homodimer titers greater than 50 mg / L and the lowest Fc(γ)RI binding affinity. In another aspect, in the embodiment comprising an insulin polypeptide of SEQ ID NO: 8 and a canine IgGB Fc fragment having a cNg-S mutation, it was unexpectedly found that the resulting compounds had significantly lower bioactivity in dogs compared to their counterparts containing native canine IgGB Fc (i.e., significantly lower NAOC values ​​for counterparts containing native glycosylation site amino acids (e.g., cNg-N)). When the B16 amino acid was mutated to alanine, 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, necessitating experimentation to identify preferred embodiments. In a specific embodiment, a canine IgGB Fc mutant containing the cNg-S mutation is preferred, and the sequence of cNg-S is underlined as follows:

[0179] DCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF SGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:22)

[0180] In a specific embodiment, a feline IgG1b Fc mutant containing the cNg-S mutation is preferred: DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ IDNO:23)

[0181] Insulin-Fc fusion protein

[0182] Provided herein are insulin-Fc fusion proteins 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 domains in the following orientation from N-terminus to C-terminus: (N-terminus)--B chain--C chain--A chain--(C-terminus). In an embodiment, the insulin polypeptide is located on the N-terminal side of the Fc fragment. In an embodiment, as ​ 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)).

[0183] 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:

[0184] FVNQHLCGS X1 LVEAL E LVCGERGF H Y ​ GIVEQCC X2 S T CSL D QLENYC X3 GGGGGQGGGGQGGGGQGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKA LPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:24)

[0185] Where X1 is not D, X2 is not H, and X3 does not exist or is N.

[0186] In a preferred embodiment comprising SEQ ID NO:24, X1 is H, X2 is T, and X3 is absent or N. Such selection results in 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, an 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, with the unnatural amino acids underlined:

[0187] FVNQHLCGSHLVEAL E LVCGERGF H Y ​ GIVEQCCTS T CSL D QLENYC X3 GGGGGQGGGGQGGGGQGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKA LPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:25)

[0188] X3 does not exist or is N.

[0189] In a preferred embodiment, X3 is absent from SEQ ID NO: 25 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 greater than 50 mg / L, an NAOC value greater than 150% FBGL·day·kg / mg in dogs, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. SEQ ID NO: 32 is shown below, with the unnatural amino acids underlined:

[0190] FVNQHLCGSHLVEAL E LVCGERGF H Y ​ GIVEQCCTS T CSL DQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVN NKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:32)

[0191] In a preferred embodiment, X3 in SEQ ID NO: 25 is N, to produce a high homodimer titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ ID NO: 34, which exhibits a homodimer titer greater than 50 mg / L, an NAOC value greater than 150% FBGL·day·kg / mg in dogs, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. SEQ ID NO: 34 is shown below, with the unnatural amino acids underlined:

[0192] FVNQHLCGSHLVEAL E LVCGERGF H Y ​ GIVEQCCTS T CSL D QLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVN NKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:34)

[0193] 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 in a series of repeated injections in dogs. SEQ ID NO: 26 is shown below, with the unnatural amino acids underlined:

[0194] FVNQHLCGS X1 LVEAL A LVCGERGF H Y ​ GIVEQCC X2 S T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:26)

[0195] Where X1 is not D, and X2 is not H.

[0196] In a preferred embodiment, in SEQ ID NO: 26, X1 is H and X2 is T, to produce a high homodimer titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ ID NO: 36, which exhibits a homodimer titer greater than 50 mg / L, an NAOC value greater than 150% FBGL·day·kg / mg in dogs, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. SEQ ID NO: 36 is shown below, with the unnatural amino acids underlined:

[0197] FVNQHLCGSHLVEAL A LVCGERGF H Y ​ GIVEQCCTS T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:36)

[0198] In a preferred embodiment, the preferred non-immunogenic, biologically active insulin polypeptide of SEQ ID NO: 6 (where X3 is absent) is combined with the preferred feline IgG1b Fc fragment of SEQ ID NO: 20 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: 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, with the unnatural amino acids underlined:

[0199] FVNQHLCGS X1 LVEAL E LVCGERGF H Y ​ GIVEQCC X2 S T CSL DQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVN SKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:27)

[0200] Where X1 is not D, and X2 is not H.

[0201] In a preferred embodiment, in SEQ ID NO: 27, X1 is H and X2 is T to produce a high homodimer titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ ID NO: 38, which exhibits a homodimer titer greater than 50 mg / L, an 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, with the unnatural amino acids underlined:

[0202] FVNQHLCGSHLVEAL E LVCGERGF H Y ​ GIVEQCCTS T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVN SKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:38)

[0203] 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, with the unnatural amino acids underlined:

[0204] FVNQHLCGS X1 LVEAL A LVCGERGF H Y ​ GIVEQCC X2 S T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:28)

[0205] Where X1 is not D, and X2 is not H.

[0206] In a preferred embodiment, in SEQ ID NO: 28, X1 is H and X2 is T, to produce a high homodimer titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ ID NO: 40, which exhibits a homodimer titer greater than 50 mg / L, an NAOC value greater than 150% FBGL·day·kg / mg in cats, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in cats. SEQ ID NO: 40 is shown below, with the unnatural amino acids underlined:

[0207] FVNQHLCGSHLVEAL A LVCGERGF H Y ​ GIVEQCCTS T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S S

[0208] TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYP

[0209] SDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:40)

[0210] In some embodiments, the insulin-Fc fusion protein described herein does not include a leader amino acid sequence at the N-terminus. 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 leader sequences include the following nucleic acid sequences:

[0211] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcc (SEQ ID NO: 29).

[0212] 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.

[0213] In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO: 32, the nucleic acid sequence (leader sequence is underlined) is:

[0214] ​ ttcgtgaaccagcacctgt

[0215] gcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggagg

[0216] ttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgc

[0217] ggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagt

[0218] gccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgc

[0219] caggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtg

[0220] gatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtg

[0221] tgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccag

[0222] ccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagg

[0223] gaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagt

[0224] ggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagcta

[0225] cttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:31).

[0226] In the example of the insulin-Fc fusion protein containing SEQ ID NO:34, the nucleic acid sequence (the leader sequence is underlined) is:

[0227] ​ ttcgtgaaccagcacctgt

[0228] gcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggagg

[0229] ttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgc

[0230] aacggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgcccca

[0231] agtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgat

[0232] cgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttc

[0233] gtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtga

[0234] gtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcc

[0235] cagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatct

[0236] agggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtgg

[0237] agtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaag

[0238] ctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:33).

[0239] In the example of the insulin-Fc fusion protein containing SEQ ID NO:36, the nucleic acid sequence (the leading sequence is underlined) is:

[0240] ​ ttcgtgaaccagcacctgt

[0241] gcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggagg

[0242] ttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgc

[0243] ggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagt

[0244] gccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgc

[0245] caggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtg

[0246] gatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttctcaggcacctacagggtggtgagtg

[0247] tgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccag

[0248] ccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagg

[0249] gaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagt

[0250] ggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagcta

[0251] cttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(SEQ ID NO:35)。

[0252] In the example of the insulin-Fc fusion protein containing SEQ ID NO:38, the nucleic acid sequence (the leader sequence is underlined) is:

[0253] ​ ttcgtgaaccagcacctgt

[0254] gcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggagg

[0255] ttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgc

[0256] ggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaat

[0257] gtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattag

[0258] caggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgta

[0259] gacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcaacagcacatacagggtggtgagcg

[0260] ttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccag

[0261] ccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacag

[0262] gaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagt

[0263] gggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggaccta

[0264] cttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag(SEQ ID NO:37)。

[0265] In the example of the insulin-Fc fusion protein containing SEQ ID NO:40, the nucleic acid sequence (the leader sequence is underlined) is:

[0266] ​ ttcgtgaaccagcacctgt

[0267] gcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggagg

[0268] ttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgc<原子

[0269] ggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaat

[0270] gtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattag

[0271] caggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgta It should be noted that there may be some inaccuracies in the translation of the specific biological sequence content, and it is recommended to verify and correct it in combination with relevant biological knowledge. And the "原子0000763" in the translation of ID=20 seems to be an incorrect expression, which may need to be adjusted according to the correct content.

[0272] gacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcagcagcacatacagggtggtgagcg

[0273] ttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccag

[0274] ccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacag

[0275] gaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagt

[0276] gggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggaccta

[0277] cttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag (SEQ ID NO: 39).

[0278] Production of Insulin-Fc Fusion Protein

[0279] In embodiments, the fusion protein can be expressed by a cell, as described in more detail in the Examples section.

[0280] Expression and purification

[0281] In an embodiment, insulin-Fc fusion protein can be for example recombinantly expressed in eukaryotic cells (for example mammalian cells or non-mammalian cells). Exemplary mammalian cells for expression include HEK cells (for example, HEK293 cells) or Chinese hamster ovary celI. Chinese hamster ovary celI can be subdivided into various strains or subclasses (for example 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 (for example, carriers comprising DNA) or specific cell growth medium compositions, as described in the examples section. In an embodiment, cells are transfected with nucleic acid molecules (for example, carriers) encoding insulin-Fc fusion protein (for example, wherein whole 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 temporary expression of insulin-Fc fusion protein in a period of time (for example, 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days or longer) before the host cell stops expressing the insulin-Fc fusion protein (i.e. transient transfection) of considerable level. HEK293 cells transiently transfected with the nucleic acid sequence encoding insulin-Fc fusion protein generally allow for more rapid production of recombinant protein, which helps to prepare and screen multiple insulin-Fc fusion protein candidates. In an embodiment, Chinese hamster ovary celIs (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 cell is suitably cultured. Chinese hamster ovary celIs and Chinese hamster ovary celIs stably transfected with the nucleic acid 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 (for example, products for the veterinary drug market). The standard method culture cells and cell lines in this area can be used. In a preferred embodiment, HEK cells containing any one of the cDNA sequences having SEQ ID NOs: 31, 33, 35, 37, and 39 are used to express the insulin-Fc fusion protein. In a preferred embodiment, CHO cells containing any one of the cDNA sequences having SEQ ID NOs: 31, 33, 35, 37, and 39 are used to express the insulin-Fc fusion protein.

[0282] 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 in size, charge and / or affinity to certain molecules. In an embodiment, the purification of insulin-Fc fusion protein relates to selection or enrichment of proteins containing Fc fragments, for example, by using protein A beads or protein A columns, which make the protein containing Fc fragments become bound to the protein A covalently conjugated to protein A beads with high affinity at neutral solution pH. Then, the combined insulin-Fc fusion protein can be eluted from 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 filtration or centrifugal protein preparations. In the Examples, further purification of the insulin-Fc fusion protein comprises diafiltration, ultrafiltration, and filtration through porous membranes of various sizes, and final formulation with excipients.

[0283] Purified insulin-Fc fusion protein can be characterized using a variety of methods, such as purity, protein yield, structure and / or activity, such as absorbance at 280 nm (e.g., to determine protein yield), size exclusion or capillary electrophoresis (e.g., to determine molecular weight, percent aggregation 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.

[0284] In embodiments, the protein yield of the insulin-Fc fusion protein after production in transiently transfected HEK cells and purification by Protein A is greater than 5 mg / L, 10 mg / L, or 20 mg / L. In preferred embodiments, the protein yield of the insulin-Fc fusion protein after production in transiently transfected HEK cells and purification by Protein A 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 embodiments, the homodimer % of the insulin-Fc fusion protein after production in transiently transfected HEK cells and purification by Protein A 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 the insulin-Fc fusion protein after production in transiently transfected HEK cells and protein A purification (calculated as the product of the insulin-Fc fusion protein yield 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 a homodimer titer greater than 50 mg / L are considered useful in the present invention, as experience has shown that homodimer titers below this level are unlikely to produce commercial production titers in CHO cells that meet the stringent low manufacturing cost requirements for veterinary products.

[0285] In an embodiment, the protein yield of the insulin-Fc fusion protein after production in stably transfected CHO cells (e.g., a CHO cell line or a CHO cell clone) and purification by Protein A is greater than 100 mg of insulin-Fc fusion protein / L (e.g., mg / L culture medium). In a preferred embodiment, the protein yield of the insulin-Fc fusion protein after production in stably transfected CHO cells (e.g., a CHO cell line or a CHO cell clone) and purification by Protein A is greater than 150 mg of 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 embodiments, the insulin-Fc fusion protein is produced in stably transfected CHO cells (e.g., CHO cell lines or CHO cell clones) and has a homodimer % 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%) after protein A purification. In embodiments, the insulin-Fc fusion protein is produced in stably transfected CHO cells (e.g., CHO cell lines or CHO cell clones) and has a homodimer titer (calculated as the product of the insulin-Fc fusion protein yield and the homodimer %) 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).

[0286] Functional characteristics of insulin-Fc fusion protein

[0287] Described herein are methods for interacting with the insulin receptor to lower blood glucose in a companion animal (e.g., a dog or cat), wherein the methods comprise 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).

[0288] In the embodiments, the insulin-Fc fusion proteins described herein bind 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 less than 5000nM, IC50 less than 4000nM, IC50 less than 3000nM, IC50 less than 2500nM). As a rule of thumb, compounds that only exhibit an insulin receptor activity IC50 value of less than 5000nM are considered 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 primarily controlled by binding to the insulin receptor, followed by insulin receptor internalization and degradation within the cell. Therefore, insulin-Fc fusion proteins with too high an insulin receptor binding affinity (i.e., too low an IC50) may be cleared from the circulation too quickly, resulting in a duration less than the desired duration of glucose-lowering biological activity in the target animal.

[0289] In an embodiment, insulin-Fc fusion protein as herein described 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 time 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 time 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).

[0290] In an embodiment, the serum half-life of the insulin-Fc fusion protein in the target animal (e.g., dog or cat) is longer than the serum half-life of an insulin reference standard or control formulation. In an embodiment, the serum half-life of the insulin-Fc fusion protein (e.g., in the subject's blood upon administration) in the target animal (e.g., dog or cat) is longer than about 2 hours. In an embodiment, the serum half-life of the 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 the insulin-Fc fusion protein in the target animal (e.g., dog or cat) is about 3 days or longer.

[0291] In embodiments, the combination of potency and duration of biological activity can be quantified by calculating the area under the curve (NAOC) normalized to the percentage of fasting glucose (%FBGL) for a given dose, expressed in mg / kg, where the units are %FBGL·day·kg / mg. In embodiments, 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, as a rule of thumb, at NAOC values ​​greater than 150%FBGL·day·kg / mg, the dosage requirements in the target species will be sufficiently low to achieve an acceptable cost of treatment. In embodiments, the NAOC of the insulin-Fc fusion protein must be maintained after repeated dosing 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)).

[0292] In some embodiments, the insulin-Fc fusion proteins described herein bind to the Fc(γ) receptor with a lower affinity than a reference standard insulin-Fc fusion protein, 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 insulin-Fc fusion protein is less than 0.50 (e.g., less than 0.40, less than 0.30, less than 0.20).

[0293] Treatment methods and characteristics of subject selection

[0294] 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).

[0295] 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 an insulin analog. 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.

[0296] In embodiments, the 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.

[0297] In an embodiment, a reference standard is the level of a marker (e.g., blood glucose or fructosamine) in a subject before starting therapy (e.g., an 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., a 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., a 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 greater). In an embodiment, a 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).

[0298] Pharmaceutical compositions and routes of administration

[0299] Provided herein is a pharmaceutical composition comprising insulin-Fc fusion protein as herein described, which can be used to reduce the blood sugar in a companion animal (e.g., dog or cat). The amount and concentration of insulin-Fc fusion protein in the pharmaceutical composition and the amount of the pharmaceutical composition used to the subject can be selected based on clinically relevant factors, such as the medically relevant characteristics (e.g., age, body 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 administration mode of the pharmaceutical composition. For further information on route of administration and dosage regimen, the reader can refer to Chapter 25.3 (Corwin Hansch; Chairman of Editorial Board) in Volume 5 of Comprehensive Medicinal Chemistry, Pergamon Press 1990.

[0300] The 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.

[0301] The example of suitable aqueous carrier and non-aqueous carrier that can be used for pharmaceutical composition of the present disclosure includes water, ethanol, polyol (such as glycerol, propylene glycol, polyethylene glycol etc.) and suitable mixture thereof, vegetable oil (such as olive oil) and injectable organic ester (such as ethyl oleate).For example, by using coating material such as lecithin, by maintaining required particle size in the case of dispersion, and by using surfactant such as tween-like surfactant, suitable fluidity can be maintained.In certain embodiments, pharmaceutical composition (for example, as described herein) comprises tween-like surfactant, for example polysorbate-20, tween-20 or tween-80.In certain embodiments, pharmaceutical composition (for example, as described herein) comprises tween-like surfactant, for example tween-80, its concentration is 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%.

[0302] In some embodiments, the concentration of the insulin-Fc fusion protein in the aqueous carrier is about 3 mg / mL. In some embodiments, the concentration of the insulin-Fc fusion protein in the aqueous carrier is about 6 mg / mL. In some embodiments, the concentration of the 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.

[0303] In certain embodiments, insulin-Fc fusion protein is administered in the form of a pill, infusion or intravenous push. In certain embodiments, the 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 a rechargeable or biodegradable device. In recent years, a variety of slow-release polymer devices have been developed and tested in vivo for the 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.

[0304] dose

[0305] The actual dosage level of the insulin-Fc fusion protein can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject (e.g., a 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.

[0306] In some embodiments, the insulin-Fc fusion protein is administered in an amount of 0.025 to 400 mg / day, or in an amount of 0.05 mg / day to 0.1 mg / day. In some embodiments, the insulin-Fc fusion protein is administered in an amount of 0.025 to 400 mg / day, or in an amount of 0.05 mg / day to 0.1 mg / day. In some embodiments, the insulin-Fc fusion protein is administered in an amount of 0.025 to 400 mg / day, or in an amount of 0.05 mg / day to 0.1 mg / day. In some embodiments, the insulin-Fc fusion protein is administered in an amount of 0.025 to 400 mg / day, or in an amount of 0.05 mg / day to 0.1 mg / day.

[0307] The present disclosure contemplates the formulation of insulin-Fc fusion proteins in any of the aforementioned pharmaceutical compositions and formulations. In addition, the present disclosure contemplates administration via any of the aforementioned routes of administration. Those skilled in the art can select appropriate formulations and routes of administration based on the overall health, age, and size of the patient being treated and the disorder being treated.

[0308] Examples

[0309] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way.

[0310] General methods, assays, and materials

[0311] ​

[0312] 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. DNA expression constructs encoding the target insulin-Fc fusion protein were transiently transfected into a suspension of HEK293 cells using the standard operating procedure for transient transfection (LakePharma, Belmont, CA). After 20 hours, cells were counted to determine viability and viable cell count, and the expression of the constructs was determined by Titers were measured using a PCR amplification kit (Pall Forte Bio LLC, Fremont, CA). Additional readings were taken throughout the transient transfection production run. Cultures were harvested on or after day 5.

[0313] ​

[0314] 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 techniques 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. Suspension-adapted CHO cells were cultured in a chemically defined medium (CDOptiCHO; Invitrogen, Carlsbad, CA) at 37°C in a humidified 5% CO2 incubator. No serum or other animal-derived products were used in the culture of CHO cells.

[0315] Approximately 80 million suspension-adapted CHO cells grown in CDOptiCHO medium in exponential growth phase were electroporated using Stable CHO cell lines were established by transfection with 80 μg of DNA for each insulin-Fc fusion protein using the MaxCyte system (MaxCyte, Inc., Gaithersburg, MD). The DNA construct contained the full-length sequence of the insulin-Fc fusion protein. After 24 hours, the transfected cells were counted and placed under screening conditions for stable integration of the insulin-Fc fusion gene. The transfected cells were cultured in shake flasks at 0.5 × 10 6The cells were seeded at a cell density of 10 cells / mL into CDOptiCHO selection medium containing 0-100 μM methionine sulfoxide (MSX) and incubated at 37°C in the presence of 5% CO2. During the selection process, the cells were spun down and resuspended in fresh selection medium every 2-3 days until the CHO stable pool recovered its growth rate and viability. The cell culture was monitored for growth and titer.

[0316] Cells were grown to 2.5 × 10 6 cells / mL. At the time of cell bank harvest, the viability was greater than 95%. The cells were then centrifuged and the cell pellet was resuspended in CDOptiCHO medium containing 7.5% dimethyl sulfoxide (DMSO) to a cell count of 15 × 10 6 Vials were cryopreserved at 4 °C for storage in liquid nitrogen.

[0317] CHO cells are used to amplify production on a small scale as follows. In the CD OptiCHO growth medium comprising 100 μM MSX, cells are amplified in proportion at 37 ° C for production, and fed once every 2-4 days as needed, wherein the CD OptiCHO growth medium is supplemented with glucose and other amino acids for approximately 14-21 days as needed. The conditioned medium supernatant harvested from the stable pool production run 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. Wash buffer is then passed through the chromatographic column until OD280 value (NanoDrop, ThermoScientific) is measured and is equal to or close to background level. Low pH buffer is used to elute the insulin-Fc fusion protein, elution fractions are collected, and the OD280 value of each fraction is recorded. The fractions containing target insulin-Fc fusion protein are merged, and optionally further filtered using a 0.2 μM filter membrane.

[0318] The cell line is optionally further subcloned into single clones using limiting dilution (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, insulin-Fc fusion protein production is completed as described above in growth medium without MSX or, optionally, in growth medium containing MSX to obtain a cell culture supernatant containing recombinant CHO-derived insulin-Fc fusion protein. Over time, the MSX concentration is optionally increased to provide additional selection for clones capable of producing higher product titers.

[0319] Example 3: Purification of Insulin-Fc Fusion Protein

[0320] The purification of insulin-Fc fusion protein is carried out as follows. From the HEK of transient or stable transfection, produce and run the conditioned medium supernatant that contains secretory insulin-Fc fusion protein in the crops, and clarify by centrifugation.The supernatant containing required insulin-Fc fusion protein is run on protein A or protein G column, and uses low pH gradient elution.Optionally, the rate of recovery of insulin-Fc fusion protein can be improved by initial protein A or protein G column eluent being reloaded onto second protein A or protein G column again.Then, the fraction of the wash-out containing required protein is merged, and 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.Calculate final protein concentration according to the solution optical density at 280nm place.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.

[0321] Example 4: Structural confirmation by non-reducing and reducing CE-SDS.

[0322] exist Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) analysis of a solution of purified insulin-Fc fusion protein dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer was performed in a GXII (PerkinElmer, Waltham, MA), and electropherograms were drawn. Under non-reducing conditions, 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.

[0323] As a possible means of determining 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).

[0324] Example 5: Sequence Identification by Glycan-Removed LC-MS

[0325] In order to obtain an accurate estimate of insulin-Fc mass by mass spectrometry (MS), first process sample to remove the naturally occurring polysaccharides that may interfere with MS analysis.First, use Zeba desalting columns (Pierce, ThermoFisherScientific, Waltham, MA) by 100 μ L of 2.5 mg / mL insulin-Fc fusion protein to be dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer, buffer is exchanged into 0.1 M Tris, pH 8.0 buffer comprising 5 mM EDTA. 1.67 μ L of PNGaseF enzyme (Prozyme N-glycanase) is added to this solution to remove the polysaccharides (for example, polysaccharides connected to the side chains of the asparagine positioned at cNg-N sites) present in the fusion protein, and the mixture is incubated overnight at 37 DEG C in an incubator. The sample was then analyzed by LC-MS (Nova Bioassays, Woburn, MA), yielding a molecular mass corresponding to the desired homodimer 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. Therefore, the actual molecular mass is the measured mass minus 2 Da to correct for enzymatic modifications to the insulin-Fc fusion protein structure in the analyzed sample.

[0326] Example 6: Homodimer % Determination by Size Exclusion Chromatography

[0327] 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 MAb Pac SEC-1, 5 μm, 4 × 300 mm column (Thermo Fisher Scientific, Waltham, MA) operated at a flow rate of 0.2 mL / min, and wherein the mobile phase comprised 50 mM sodium phosphate, 300 mM NaCl, and 0.05% w / v sodium azide (pH 6.2). The MAb Pac SEC-1 column works based 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 mixture of homodimers was separated from the aggregated multimeric homodimers by analytical SEC-HPLC.

[0328] Example 7: In vitro IM-9 insulin receptor binding of exemplary insulin-Fc fusion proteins at 4°C

[0329] Human IM-9 cells (ATTC#CCL-159) expressing the human insulin receptor were cultured and maintained at 70-80% confluence in complete RPMI 5% FBS medium. IM-9 cell cultures were centrifuged at 250 × g (approximately 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 / 2 mM EDTA / 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 preparation of the test solution. Insulin-Fc protein was diluted in FACS buffer at a 2× concentration in a 1:3 serial dilution in 1.2 mL tubes (approximately 60 μL volume per dilution) and the solution was kept on ice until ready to be pipetted.

[0330] Biotinylated RHI was diluted to a concentration of 1.25 μg / mL in FACS staining medium. 40 μL of serially diluted test compound 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 cells) was then added to each well using a multichannel pipette. 6Cells / mL) were added, gently mixed again, and incubated on ice for 30 min to allow competitive binding to the insulin receptor on IM-9 cells. The V-shaped bottom plate was then centrifuged at 3000 rpm for 3 min, and the supernatant was aspirated and the cells were washed twice with 275 μL of ice-cold FACS wash buffer (EDTA / 0.1% sodium azide+0.5% horse serum in HBSS / 2mM). The cells were then resuspended in 40 μL of FACS staining medium containing 1:100 diluted streptavidin-PE (Life Technologies) and placed on ice for 20 min. The cells were then washed once with 275 μL of ice-cold FACS buffer and finally fixed at room temperature for 10 min with 3% paraformaldehyde. The cells were then washed once with 275 μL of ice-cold FACS buffer and resuspended in 250 μL of FACS buffer for analysis.

[0331] The V-shaped bottom plate containing cell is then analyzed on Guava8-HT flow cytometer (Millipore).For the test compound of every kind of concentration, by the median fluorescence intensity (MFI) of cell on FACSFL-2 channel, the biotinylated RHI of insulin receptor is combined and quantified.Control wells are only labeled with biotinylated RHI, and are used to calculate the inhibition percentage (%) produced by every kind of test compound concentration.The inhibition % that test compound is combined with the biotinylated RHI on IM-9 cell has been drawn for the logarithmic concentration of test compound, and GraphPadPrism (GraphPad Software, LaJolla, CA) has been used to calculate the IC50 value of the gained of test compound.Therefore, the lower IC50 value of test compound shows that biotinylated RHI inhibition level is higher under lower concentrations, and this shows that insulin-Fc fusion protein is more strongly combined with insulin receptor.Control compound (such as unlabeled recombinant human insulin (RHI)) is also used as internal standard, to generate RHIIC50, can infer the IC50 (IC50 (compound) / IC50 (RHI)) of given compound accordingly. 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.

[0332] Example 8: In vitro Fc(γ) receptor I binding affinity assay

[0333] Binding of insulin-Fc fusion protein to Fc(γ) receptor I was determined using an ELISA assay at pH 7.4 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. The microtiter plates were then washed five times with PBST (PBS / 0.05% Tween-20) buffer and blocked with Superblock blocking reagent (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 plate strips coated with insulin-Fc fusion protein. The microtiter plate was incubated at room temperature for 1 hour, after which the microtiter plate strips were washed 5 times with PBST and then loaded with 100 μL / well of a 1:10,000 dilution of streptavidin-HRP in PBST / 10% Superblock buffer. After incubation for 45 min, the microtiter plate strips were washed again 5 times with PBST. TMB was added to reveal bound Fc(γ) receptor I protein and terminated with ELISA stop reagent (Boston Bioproducts). The plate was read at 450 nm in an ELISA plate reader, 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.

[0334] Example 9: In vitro measurement of insulin-Fc fusion protein affinity for canine FcRn receptor

[0335] The in vitro binding affinity of insulin-Fc fusion proteins containing Fc fragments derived from canine or feline IgG to the canine FcRn receptor was measured using an ELISA technique performed at a solution pH of 5.5. A slightly acidic pH is a preferred binding environment for Fc fragment-containing molecules to bind to the FcRn receptor. In vivo, cells express FcRn on their surface and within endosomes. When Fc fragment-containing molecules enter cells through natural processes (e.g., pinocytosis or endocytosis), the pH shifts to a lower pH within the endosomes, where the FcRn receptor binds to Fc fragment-containing molecules that would otherwise be degraded in the endosomal-lysosomal compartment, allowing these molecules to recirculate back to the cell surface, where the pH is closer to neutral (e.g., pH 7.0-7.4). Neutral pH is unfavorable for binding to the FcRn receptor and allows the Fc fragment-containing molecules to be released back into the circulation. This is the primary mechanism by which Fc fragment-containing molecules exhibit prolonged circulating pharmacokinetic half-lives in vivo.

[0336] Insulin-Fc fusion proteins containing canine or feline Fc fragments were diluted to 10 μg / ml in sodium bicarbonate buffer at pH 9.6 and coated in duplicate on Maxisorb ELISA plate strips for 1-2 hours at room temperature. The strips were then washed four 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 (50 mM MES / 150 mM NaCl / 0.1% Tween-20) before adding the FcRn reagent (biotinylated canine FcRn; Immunitrack). Since no commercially available feline FcRn reagents were found, the binding of insulin-Fc fusion proteins containing canine or feline Fc fragments to canine FcRn was determined. Serial dilutions of the biotinylated FcRn reagent were prepared in MES / NaCl / Tween / 10% Superblock buffer, pH 5.5, at concentrations ranging from 1000 ng / ml to 0.45 ng / ml (1:3X dilutions) and loaded onto the strips coated with the insulin-Fc fusion protein compound at 100 μL / well using a multichannel pipette. The assay plate was then incubated at room temperature for 1 hour. The FcRn-binding strips were washed four times with MES / NaCl / Tween buffer, pH 5.5, and then loaded with 100 μL / well of streptavidin-HRP diluted 1:10,000 in MES / NaCl / 10% Superblock buffer, pH 5.5. After incubation for 45 minutes, the strips were washed again four times with MES / NaCl / Tween buffer, pH 5.5. TMB was added to reveal the bound biotinylated canine FcRn reagent, and color development was stopped with ELISA stop reagent. Plates were read in an ELISA plate reader at a wavelength of 450 nm. GraphPad Prism software was used to plot OD values ​​(proportional to the binding of canine-FcRn to insulin-Fc fusion protein test compounds) relative to the logarithmic concentration of FcRn added to each well to generate binding curves. EC50 values ​​were calculated for each binding curve to compare the differences between different compounds.

[0337] Example 10: Determination of in vivo pharmacodynamics following a single administration of insulin Fc-fusion protein in dogs or cats (PD) general procedures.

[0338] The effects of insulin-Fc fusion proteins on fasting blood glucose levels were evaluated as follows. N = 1, 2, 3, or more healthy, antibody-naive dogs weighing approximately 10-15 kg or cats weighing approximately 5 kg were used, one for each insulin-Fc fusion protein. Animals were also observed twice daily for signs of allergic reactions, lethargy, distress, pain, and other symptoms. Treatment was optionally continued with three additional subcutaneous injections per week or more for some compounds to observe whether the compound's glucose-lowering ability diminished over time, 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 insulin Fc-fusion protein homodimers 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 methylparaben) at a concentration of 0.02-1.00 mg / mL, administered intravenously or subcutaneously 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 equivalents 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, as well as on days 1, 2, 3, 4, 5, 6, and 7 after injection.

[0339] For each time point, at least 1 mL of whole blood was collected. Immediately use a blood glucose meter ( 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.

[0340] Example 11: Repeated administration of insulin-Fc fusion protein to determine in vivo General Procedures for Pharmacodynamics (PD) .

[0341] 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. Optionally, for some compounds, treatment was continued for up to 2 to 5 additional subcutaneous injections to observe whether the compound's glucose-lowering ability decreased over time, indicating the possible presence of neutralizing anti-drug antibodies. 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 1.2-12.3 nmol / kg or approximately 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.

[0342] Subsequent subcutaneous injection frequency is no more than once a week, and in some cases, according to the pharmacodynamics of given insulin-Fc fusion protein preparation, injection is given at different intervals.According to the pharmacodynamics of proven insulin-Fc fusion protein, 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 invalid in reducing blood sugar, the dosage level of the insulin-Fc fusion protein injected subsequently is adjusted upwards.In a similar manner, if it is found that the dosage of the first injection on the 0th day is too strong to reduce glucose, the dosage level of the insulin-Fc fusion protein injected subsequently is adjusted downwards.It has also been found that interim dose or final dose can be adjusted in a similar manner as needed.For each dosage, immediately before injection and after injection 15,30,45,60,120,240,360 and 480 minutes and 1,2,3,4,5,6,7 days (and optionally 14 days) collect blood from a suitable vein.For each time point, at least 1mL of whole blood was collected.Use blood glucose meter ( Glucose level readings were measured using an Aviva Plus glucometer, which requires approximately one drop of blood. The mean fasting blood glucose level (%FBGL) over the entire study period was plotted against time, which allowed the biological activity of the fusion protein to be determined.

[0343] To determine the bioactivity of each dose, an area under the curve (AOC) analysis was performed as follows. After constructing the % FBGL versus time data, the data was then entered into 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 period (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 period and a calculated AUC of 500% FBGL·day, the following result for AOC is 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.

[0344] Since the dose of insulin-Fc fusion protein may vary as described above, it is often more convenient to normalize all calculated AOC values ​​for a given insulin-Fc fusion protein to a specific dose of that 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 varies between injections in a given study. The normalized AOC (NAOC) for a given dose is calculated as follows: NAOC = AOC / D, where the units are % 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 can be averaged for a group of animals receiving the same insulin-Fc fusion protein formulation.

[0345] 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 follows: NAOCR = (NAOC (Nth injection) / NAOC (injection 1)). By evaluating the NAOCR of a given insulin-Fc homodimer fusion protein formulation for the Nth injection in a series of injections, it can be determined whether the in vivo glucose-lowering activity of the given insulin-Fc fusion protein has substantially maintained 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 the given insulin-Fc fusion protein has lost 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 preferred embodiments, 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).

[0346] Example 12: General Procedure for Determining In Vivo Pharmacokinetics (PK) in Canine and Feline Serum

[0347] An assay was constructed as follows to measure the concentration of an insulin-Fc fusion protein comprising an Fc fragment of a canine isotype in canine serum. The assay comprises a sandwich ELISA format in which the therapeutic compound in the serum sample is captured by an anti-insulin / proinsulin monoclonal antibody (mAb) coated on an ELISA plate and then detected by an HRP-conjugated anti-canine IgG Fc-specific antibody, followed by development using a TMB substrate system. Maxisorp ELISA plates (Nunc) were coated with anti-insulin mAb clone D6C4 (Biorad) in 5 μg / ml coating buffer (pH = 9.6 sodium carbonate-biological sodium carbonate buffer) at 4°C overnight. The plates were then washed five 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 generate a standard curve, the target insulin-Fc fusion protein was diluted in sample dilution buffer (PBST / SB / 20% HS) + 5% pooled beagle serum (BioIVT) at concentrations ranging from 200 ng / ml to 0.82 ng / ml in 1:2.5 serial dilutions. Standards and diluted serum samples were added to the blocked plate in duplicate at 100 μl / well and incubated at room temperature for 1 hour. 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 100 μl 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 1 time 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 450 nm using a SpectraMax plate reader (Molecular Devices) within 30 minutes. The concentration of the insulin-Fc fusion protein compound in the sample was calculated using SoftMax Pro software by interpolation on the 4-PL curve.

[0348] Similarly, a determination was constructed as follows for measuring the concentration of insulin-Fc fusion proteins comprising 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 HRP-conjugated goat anti-feline IgG Fc-specific antibody, followed by development using a TMB substrate system. Maxisorp ELISA plates (Nunc) were coated with anti-insulin mAb clone D6C4 (Biorad) in 5 μg / ml coating buffer (pH = 9.6 sodium carbonate-biological sodium carbonate buffer) at 4°C overnight. 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 prepare a standard curve, the target insulin-Fc fusion protein compound was diluted in a 1:2.5 serial dilution in sample dilution buffer (PBST / SB / 20% HS) + 5% normal cat serum (Jackson Immunoresearch) with a concentration range of 200ng / ml to 0.82ng / ml. The 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 IgGFc (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 1 time 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 450 nm using a SpectraMax plate reader (Molecular Devices) within 30 minutes. The concentration of the insulin-Fc fusion protein compound in the sample was calculated using SoftMax Pro software by interpolation on the 4-PL curve.

[0349] Example 13: Assay Protocol for Measuring Anti-Drug Antibodies in Canine Serum

[0350] 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) and overnight at 4°C for measuring ADA against test compounds. In order 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). In order to calculate ADA expressed in canine IgG units, the strips were directly coated overnight at 4°C with a 1:2 serial dilution of canine IgG in a Carb-Biocarb coating buffer 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).

[0351] The test serum samples were diluted to greater than or equal to 1:100 (typically 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 insulin-Fc fusion protein coated (or RHI coated) strips in duplicate at 100 μL / well. Repeat strips of canine IgG coated standard strips 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 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 then washed once with deionized water and then developed for 15-20 minutes in the dark at room temperature by adding 100 μL / well of TMB substrate (Invitrogen, Thermo Fisher Scientific, Waltham MA). 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 a 4-PL pseudo-standard curve using SoftMax Pro software (Molecular Devices, San Jose CA).

[0352] 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 were performed as described above. The ADA measured in the drug-inhibited wells was matched with non-inhibited ADA concentrations to assess 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.

[0353] Example 14: Assay Protocol for Measuring Anti-Drug Antibodies in Feline Serum

[0354] Maxisorp ELISA plates (Nunc) were coated with the target insulin-Fc fusion protein diluted at 10 μg / mL in coating buffer (pH = 9.6 carbonate-biocarbonate buffer) and overnight at 4°C for measuring ADA against insulin-Fc fusion proteins containing Fc fragments derived from feline IgG. 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 five 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 as feline IgG units, strips were directly coated overnight at 4°C with a 1:2 serial dilution of canine IgG (Jackson Immunoresearch Laboratories, West Grove PA) at concentrations ranging from 300 to 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).

[0355] The test serum samples were diluted to greater than or equal to 1:100 (typically 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 coated (or RHI coated) strips of insulin-Fc fusion protein 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 plates were 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 with a factor of 1: 10000 and added to sample wells and standard wells with 100 μL / wells and incubated in the dark for 45 minutes at room temperature. The plate was washed 5 times with PBST and washed once with deionized water, and developed 15-20 minutes in the dark by adding 100 μL / wells of TMB substrate (Invitrogen). The color development was then stopped by adding 100 μL / wells of ELISA stop solution (Boston Bioproducts, Ashland MA), and absorbance was read at 450 nm using a SpectraMax plate reader within 30 minutes. Anti-drug antibody concentration was determined by interpolating the OD values ​​in the 4-PL pseudo standard curve using SoftMax Pro software (Molecular Devices, San Jose CA).

[0356] Example 15: Assay Procedure for Immunogenic Epitope Identification

[0357] 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 anti-drug antibody ELISA assays, except that each compound in the library was coated on a separate strip of ELISA microplate wells. The compounds in the library comprised a series 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 human-derived compositions. Additionally, as described in Examples 13 and 14, some 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.

[0358] Serum obtained from individual dogs or cats that received repeated doses 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 were moderately or highly positive in the assays 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 plates coated with the library of insulin-Fc fusion protein compounds and left at room temperature for 1 hour. After incubation, the plates were washed five times with PBST. To detect canine or feline antibodies that cross-react with the coated compound library, HRP-conjugated goat anti-feline IgG F(ab')2 (Jackson Immunoresearch Laboratories, West Grove PA), which cross-reacts with 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 in the dark at room temperature for 15-20 min by adding 100 μL / well of TMB substrate (Invitrogen, ThermoFisherScientific, Waltham MA). 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).

[0359] 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 various insulin-Fc fusion protein homodimers. Mutations or epitopes responsible for moderate or strong binding are referred to herein as immunogenic "hotspots."

[0360] Example 16: Acute and repeated testing for obtaining high homodimer titers and acceptable levels in target species Design process of insulin-Fc fusion protein with bioactive drug delivery

[0361] 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 insulin-Fc fusion protein obtained is most likely to produce a long-acting biologically active product with minimum immunogenicity (for example, selecting the species-specific IgG isotype with minimum Fc (γ) receptor I binding). The DNA sequence encoding the required fusion protein is prepared, cloned into a vector (LakePharma, SanCarlos, CA), and then the vector is used for transient transfection of HEK cells according to the procedure described in Example 1. The insulin-Fc fusion protein is then purified according to Example 3, and total protein yield and homodimer % are measured according to Example 6. Only candidates with homodimer titers greater than 50 mg / L are considered to be acceptable because the titer below this level is unlikely to produce a commercial production titer that meets the strict low production cost requirements of veterinary products. The bioactivity index of the selected insulin-Fc fusion protein is then screened by in vitro insulin receptor binding studies as described in Example 7. As a rule of thumb, only compounds exhibiting IR activity IC50 values ​​less than 5000 nM are considered likely to exhibit bioactivity in the target species. While 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 minor differences in affinity between canine or feline IR and human IR. Furthermore, factors other than insulin receptor binding may influence a compound's in vivo bioactivity (e.g., affinity for canine or feline FcRn to allow for a prolonged in vivo pharmacokinetic elimination half-life). Therefore, selected insulin-Fc fusion proteins that are acceptable from the perspective of production and IR activity IC50 values ​​are further screened for bioactivity in the target species (e.g., dog or cat) to identify any materials with bioactivity below the desired potency and / or duration (e.g., NAOC below 150% FBGL·day·kg / mg). Similarly, as a rule of thumb, at NAOC values ​​greater than 150% FBGL·day·kg / mg, the dose requirement in the target species will be sufficiently low to achieve an acceptable therapeutic cost. Finally, an additional evaluation criterion was added, which is rarely mentioned in the art. As discussed in more detail in the examples below, multiple examples of insulin-Fc fusion proteins that exhibited acceptable NAOC levels in target species after a first dose unexpectedly failed to maintain this level of bioactivity after repeated doses. Furthermore, 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 these insulin-Fc fusion proteins unsuitable for treating chronic diseases, such as canine or feline diabetes.Therefore, only insulin-Fc fusion proteins that exhibit acceptable levels of repeat-dose 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.

[0362] Results - Insulin-Fc fusion protein containing a canine Fc fragment

[0363] Example 17: Canine Insulin-Fc Fusion Protein Comprising a Canine FcIgGA Isotype

[0364] 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:

[0365] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPV

[0366] PEPLGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLP

[0367] IEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQ

[0368] SNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG (SEQ ID NO: 42).

[0369] 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, homodimer % was measured by size exclusion chromatography and was determined to be 24%, indicating a high degree of homodimer aggregates. Therefore, the resulting homodimer titer was only 5 mg / L. In short, the insulin-Fc fusion protein of SEQ ID NO:42 produced in HEK cells produced a high level of aggregates and a low level of homodimer titer (5 mg / L), which does not meet the design goal of a homodimer titer greater than 50 mg / L.

[0370] However, the biological activity of the insulin-Fc fusion protein of SEQ ID NO: 42 was evaluated. First, 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 less than 5000 nM).

[0371] 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 was calculated to be 105% FBGL·day·kg / mg. The in vivo half-life of SEQ ID NO: 42 was 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 amount 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.

[0372] Example 18: Mutation of the Fc fragment region of an insulin-Fc fusion protein comprising a canine IgGA isotype

[0373] In order to increase the homodimer content % of the insulin-Fc fusion protein of SEQ ID NO: 42, improve its biological activity and prolong its half-life, mutations were inserted into the CH3 region of the Fc fragment to try 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.

[0374] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPV

[0375] PEPLGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLP

[0376] IEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHEALHSHYTQKSLSLSPG(SEQ ID NO:44)

[0377] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPV

[0378] PEPLGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLP

[0379] IEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHETLQSHYTDLSLSHSPG(SEQ ID NO:46)

[0380] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPV

[0381] PEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLP

[0382] IEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQSHYTDLSLSHSPG(SEQ ID NO:48)

[0383] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPV

[0384] PEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLP

[0385] IEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHETLQNHYTDLSLSHSPG(SEQ ID NO:50)

[0386] Table 2 lists insulin-Fc fusion proteins based on canine IgGA variants, along with the corresponding protein yield, homodimer % and homodimer titer. The results indicate that various mutations in the IgGAFc fragment did not improve homodimer % and homodimer titer, but instead produced highly aggregated proteins with very low homodimer titers below 5 mg / L. Therefore, the in vivo bioactivity and pharmacokinetics of these compounds could not be assessed.

[0387]

[0388] Example 19: Canine Insulin-Fc Fusion Proteins Using Other Canine Fc Fragment Isotypes

[0389] As described above, canine IgGA is considered to be the preferred isotype for the production of non-immunogenic insulin-Fc fusion proteins for dogs using Fc fragments because it lacks Fc (γ) I effector function in canines (very similar to the human IgG2 isotype in humans). However, insulin-Fc fusion proteins prepared with canine IgGAFc 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 with the insulin-Fc fusion protein for preparing SEQ ID NO:42 and the peptide linker of SEQ ID NO:12, three kinds of insulin-Fc fusion proteins comprising Fc fragments based on canine IgGB, IgGC and IgGD isotypes were synthesized. According to Example 1, protein was prepared in HEK293 cells. Then, according to Example 3, protein A column was used to purify the insulin-Fc fusion protein. According to Example 4, the structure of the insulin-Fc fusion protein was confirmed by non-reducing and reducing CE-SDS, 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 they are compared relative to the sequence alignment of SEQ ID NO:42. Figure 4 It shows:

[0390] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEM

[0391] LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGH

[0392] QDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:52)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTY RVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG(SEQ FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG (SEQ ID NO: 56) The resulting protein yield, homodimer % and homodimer titer 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 homodimer titers meeting 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 considerable protein production, but had a high degree of aggregation and, therefore, an unacceptably low homodimer titer.

[0393] 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 exhibited an IC50 greater than 5000 nM, indicating that this compound is highly unlikely to exhibit in vivo biological activity. However, the insulin-Fc fusion protein of SEQ ID NO: 52 exhibited an IC50 of 28 nM, indicating that this sequence may have in vivo biological activity.

[0394]

[0395] *DNM = Not Measured

[0396] Example 20: Contains SEQ ID Insulin peptide of NO:5 and insulin of canine IgGB isotype Fc fragment In vivo efficacy of Fc fusion proteins

[0397] 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 following 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 the % 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 demonstrating that the insulin-Fc fusion protein of SEQ ID NO: 52 has significant biological activity in dogs.

[0398] The NAOC was calculated according to the procedure of Example 11 to determine the relative bioactivity 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 is 3.8 times the NAOC of the insulin-Fc fusion protein of SEQ ID NO:42 injected intravenously, indicating that the bioactivity of the insulin-Fc fusion protein containing the canine IgGBFc fragment is significantly increased compared to the insulin-Fc fusion protein containing the canine IgGAFc 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 bioactivity achieved by subcutaneous administration is similar to that achieved by intravenous administration.

[0399] Example 21: Repeated subcutaneous administration of a drug containing SEQ ID NO:5 insulin polypeptide and canine IgGB isotype In vivo immunogenicity screening of insulin-Fc fusion proteins after Fc fragment

[0400] 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 was measured over a 7-day window after each administration 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).

[0401]

[0402] Without being bound by any particular explanation, it is hypothesized 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 neutralized 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 that are 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, serum collected before administration of each dose and at the end of the experiments described in Examples 11 and 12 was 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 was the possible reason for the decrease in NAOCR after the third injection of the insulin Fc-fusion protein of SEQ ID NO: 52.

[0403] Example 22: Contains SEQ ID No. 5 insulin peptide and canine IgGB isotype Fc fragment non-glycosylated Insulin-Fc fusion protein to reduce the potential risk of immunogenicity

[0404] As shown in Examples 19 and 20, the insulin-Fc fusion protein of SEQ ID NO: 52 showed acceptable homodimer content %, homodimer titer, and bioactivity in dogs; however, its use for chronic diseases such as diabetes was compromised by reduced bioactivity (Example 21) and the production of anti-drug antibodies (Example 21) with repeated subcutaneous administration. Without being bound by any particular theory, one possible reason for the production of anti-drug antibodies and reduced bioactivity 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 bioactivity (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 omitting the PNGaseF treatment step. 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 FIG. Figure 8 The resulting sequence alignment (ClustalOmega) is shown in:

[0405] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF QGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:58)

[0406] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:60)

[0407] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF D GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:62)

[0408] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF K GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:64)

[0409] 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 glycan-removed LC-MS 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.

[0410]

[0411] To determine which of the remaining three compounds was 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. Unexpectedly, it was demonstrated that the insulin-Fc fusion protein of SEQ ID NO: 62 containing the cNg-D mutation exhibited approximately twice the Fc (γ) receptor binding activity 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 was considered suitable for repeated administration bioactivity testing in dogs.

[0412]

[0413] Example 23: SEQ ID NO: 10 with non-glycosylated cNg-K and cNg-S canine IgGB isotype Fc fragments ID NO: Evaluation of the in vivo bioactivity and immunogenicity of 5 insulin peptides

[0414] To determine whether the insulin-Fc fusion protein of SEQ ID NO:60 containing the cNg-S mutation improves repeat-dose bioactivity performance in dogs, the compound was administered subcutaneously to N=1 dogs on days 0, 7, 14, and 28 according to the procedure of Example 11. When the dog's %FBGL dropped too low, food was provided 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 dose were also measured according to the general procedure of Example 11, starting from the time of dose administration until just before the next dose was administered. The NAOC and NAOCR shown in Table 7 indicate that the insulin-Fc fusion protein of SEQ ID NO:60 exhibited 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 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 dosing biological activity in dogs.

[0415]

[0416] To determine whether the insulin-Fc fusion protein of SEQ ID NO: 64 containing the cNg-K mutation improves the repeat dosing 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 dog's %FBGL dropped too low, food was provided to the dog to raise blood glucose 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 (which was approximately 0.9 days). The NAOC and NAOCR of each subsequent dose were also measured according to the general procedure of Example 11, starting from the time of dose administration until just before the next dose was administered. The NAOC and NAOCR values ​​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 that significantly improved repeat-dose bioactivity in dogs.

[0417]

[0418] According to Example 13, anti-drug and anti-insulin antibody levels were also measured throughout the treatment course (28 days) and for an additional two weeks. Figure 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).

[0419] 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

[0420] Mutating the cNg position of the canine IgGB Fc fragment to Lys (i.e., cNg-K) did improve the repeated-dose bioactivity of an insulin-fusion protein comprising an insulin polypeptide of SEQ ID NO: 5 and a 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 was hypothesized that the insulin polypeptide of SEQ ID NO: 5 might unexpectedly contain a specific epitope (i.e., an immunogenic "hotspot") that is targeted by the canine 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 the insulin-Fc fusion protein of SEQ ID NO: 52 (Example 19) to a coated insulin-Fc fusion protein library revealed the unexpected presence of two major "hotspots" within 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). These results suggest that insulin-Fc fusion proteins containing an insulin polypeptide amino acid composition containing these two specific amino acid mutations may be immunogenic in dogs and, therefore, may generate anti-drug antibodies that neutralize the biological activity after repeated injections. Therefore, it was determined that insulin polypeptides that do not contain the B10 aspartic acid and the A8 histidine are preferred for use in insulin-Fc fusion proteins that require long-term repeated administration in dogs (e.g., for the treatment of canine diabetes).

[0421] Example 25: Contains SEQ ID NO:5 insulin peptide and non-glycosylated canine IgGB isotype Fc fragment The insulin-Fc fusion protein of the segmented insulin polypeptide is restored to its natural composition by the B10D and A8H mutations to reduce the immune Potential risks of originality

[0422] To evaluate whether replacing the "hotspot" mutations would improve the immunogenicity and repeat-dosing bioactivity of an insulin-Fc fusion protein comprising the 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).

[0423] FVNQHLCGSHLVEAL A LVCGERGFFYT DP T GGGPRR GIVEQCCTSICSLYQLENYCN(SEQ ID NO:125)

[0424] In addition, considering the additional potential benefits of the non-glycosylated cNg mutant, the insulin-Fc fusion protein of SEQ ID NO: 66 contains the cNg-Q mutation. The complete amino acid sequence of the insulin-Fc fusion protein of SEQ ID NO: 66 is given below:

[0425] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCNGGGGAGGGGDCPKCPAPEM

[0426] LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFQGTYRVVSVLPIGH

[0427] QDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:66)

[0428] 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 glycan-removed LC-MS according to Example 5. The homodimer percentage measured by size exclusion chromatography according to Example 6 was 98.0%, indicating that the protein was relatively free of aggregates.

[0429] 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. Figure 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 indeed significantly reduced the immunogenicity of the parent compound (SEQ ID NO: 52).

[0430] However, if Figure 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).

[0431] Example 26: Attempts to incorporate additional B-chain and A-chain mutations into SEQ ID In the insulin peptide of NO:125, Improve the biological activity of related insulin-Fc fusion proteins containing canine IgGBFc fragments

[0432] 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 the 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.

[0433] As is well known, each part of insulin B chain and A chain is necessary for strong binding to IR (Hubbard SR, " Structural biology: Insul in meets its receptor ", Nature.2013; 493 (7431): 171-172). Therefore, the part of B chain or A chain is modified while keeping B10 and A8 identical to native insulin and C chain and peptide linker constant. According to Example 1, several of these insulin-Fc fusion proteins were prepared in HEK293 cells, and according to Example 3, protein A column purification was used. According to Example 4, their structure was confirmed by non-reducing and reducing CE-SDS, and according to Example 5, the sequence was further identified by LC-MS removing polysaccharides. According to Example 6, their homodimer content % was measured by size exclusion chromatography, and their insulin receptor binding affinity was measured according to Example 7. Their sequences are shown below, and the resulting sequences are aligned for SEQ ID NO:66. Figure 12 Shown in (ClustalOmega).

[0434] FVNQHLCGSHLVQALYLVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEML

[0435] GGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQ

[0436] DWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:68)FVNQHLCGSELVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:68) IDNO:70)FVNQHLCGSHLVEAALALVCGEAGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQIDNO:72)FVNQHLCGSHLVEALALVCGERGFYYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYR VVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:74)FVNQHLCGSHHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYR VVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:76)

[0437]

[0438]

[0439] In only three cases (SEQ ID NOs: 68, 70, and 74) did the proposed mutations improve IR binding (i.e., reduce IC50 values) compared to SEQ ID NO: 66. However, none of these mutations resulted in compounds that met the production design target of a homodimer titer 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).

[0440] Example 27: Attempt to incorporate C-chain mutations into SEQ ID NO:125 insulin peptide to improve the canine Biological activity of insulin-Fc fusion proteins related to animal IgGBFc fragments

[0441] The results obtained in Example 26 show that the attempts of the insulin polypeptide A chain and B chain of all mutations SEQ ID NO:125 result in unacceptable low HEK homodimer titers (that is, homodimer titers are less than or equal to 25 mg / L) of related insulin-Fc fusions. Therefore, it is necessary to perform 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 native insulin (such as human insulin) undergoes significant conformational changes when combined with the insulin receptor, including movement of the B chain and A chain folding (for example, as described in Menting et al., Nature, 2013; 493 (7431): pp241–245). Different from the insulin polypeptide of the present invention, native insulin can freely undergo this conformational change at the insulin receptor because it is a double-chain polypeptide of its native form, connected only by two disulfide bonds, without the C chain to limit the mobility of the 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 do 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 C chain of the insulin polypeptide are shown below, wherein the resulting sequences are aligned with SEQ ID NO: 66 in Figure 13 Shown in (ClustalOmega).

[0442] FVNQHLCGSHLVQALYLVCGERGFFYTDPTQRGGGGGQRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAP

[0443] EMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPI

[0444] GHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQID NO:78)

[0445] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGGGGSGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCP

[0446] APEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVL

[0447] PIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:80)

[0448] FVNQHLCGSHLVEALALVCGERGFFYTDPGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEM

[0449] LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGH

[0450] QDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:82)FVNQHLCGSHHLVEALALVCGERGFFYTPGGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYR VVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:84)

[0451]

[0452] 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 through 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.

[0453] Example 28: Attempt to incorporate peptide linker mutations into a peptide containing SEQ ID NO:125 insulin polypeptide and canines IgGBFc fragments were incorporated into insulin-Fc fusion proteins to enhance biological activity

[0454] 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 via a peptide linker. Shorter peptide linkers or more tightly folded peptide linkers are thought to exacerbate this problem, while longer peptide linkers or peptide linkers that are self-resistant to folding (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 of SEQ ID NO:12 (i.e., GGGGAGGGG) used to construct the insulin-Fc fusion protein of SEQ ID NO:66 is hypothesized to be possibly 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 that used to construct the insulin-Fc fusion protein of SEQ ID NO:66, but with an insulin polypeptide in which the asparagine at position 21 from the N-terminus of the A chain (i.e., A21) is absent (i.e., des-A21). This specific mutation was incorporated to see if the connection between the A chain and the peptide linker affects protein production and / or the biological activity of the molecule. The other insulin-Fc fusion protein of SEQ ID NO:86 contains this des-A21 N 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 was deprecated and replaced with 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 Figure 14 Shown in (ClustalOmega).

[0455] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGGQGGGGQGGGGQGGGG

[0456] GDCKPKPAPEMLGGPSVFIFPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGT

[0457] YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPP

[0458] DIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:86)

[0459] FVNQHLCGSHLVVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEML

[0460] GGPSVFIFPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQ

[0461] DWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:76)

[0462]

[0463]

[0464] According to example 1, two insulin-Fc fusion proteins were prepared in HEK293 cells and purified using 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. The longer peptide linkers (GGGGGQGGGGQGGGGGGGG of SEQ ID NO:86 relative to GGGGAGGGG of SEQ ID NO:66) incorporated with different compositions did improve insulin receptor binding, as measured by the significant reduction in IC50 values, which suggests that longer linkers may be strategies for increasing the insulin receptor binding of other insulin-Fc fusion proteins. However, incorporating longer linkers still did not improve homodimer titers to a production design target greater than 50 mg / L.

[0465] Example 29: Attempting to delete SEQ ID NO:125 insulin polypeptide B chain portion to increase the content of canine Homodimer titer of insulin-Fc fusion protein related to IgGBFc fragment

[0466] 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 improve the homodimer titer to a level sufficient to meet the production design target. Since homodimer titer is a function of several properties (including intracellular synthesis and intracellular processing), it is assumed that the insulin-Fc molecule may undergo intramolecular self-association (i.e., aggregation) between the two monomers of the homodimer during and after synthesis, or perform intermolecular self-association between two or more independent homodimers. This aggregation will result in obtaining unacceptable low homodimer titers 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 of reducing the self-association tendency of insulin known in the art includes mutating the amino acids near the C-terminal end of the B chain. For example, insulin lispro (B28K; B29P mutation) and insulin aspart (B28D mutation) are well-known commercial double-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 for preventing aggregation relates to amino acid structure deletion. For example, the double-chain insulin (DPPI) referred to as depentapeptide insulin; See Brange J., Dodson G.G., Edwards J., Holden P.H., Whittingham J.L. 1997b. " A model of insulin fibrils derived from the x-ray crystal structure of a monomeric insulin (despentapeptide insulin) " Proteins 27507–516) is identical to natural double-chain human insulin, except that the five C-terminal amino acids of the B chain (YTPKT) are removed. Compared with native 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 improve 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, insulin lispro and insulin aspart. The sequences are shown below, wherein the sequence alignment relative to the resulting sequence of SEQ ID NO:66 is shown in FIG. Figure 15 Shown in (Clustal Omega).

[0467] FVNQHLCGSHLVVEALALVCGERGFFYTDPGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEM

[0468] LGGPSVFIFPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGH

[0469] QDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:82)FVNQHLCGSHLVEAALALVCGERGFFYTPGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQ IDNO:84)FVNQHLCGSHLVEAALALVCGERGFFYTQGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ IDNO:88)

[0470]

[0471] 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 their sequences were further identified by LC-MS after 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, the insulin receptor affinity was improved for all mutant compounds (SEQ ID NOs: 82, 88, and 84).

[0472] Example 30: Attempt to combine B chain, C chain and A chain mutations, B chain truncation and linker mutations with SEQ ID No. 66 pancreatic islets Combined with Fc fusion protein to further improve homodimer titer and biological activity

[0473] As shown in Examples 26, 27, 28 and 29, no single strategy has successfully incorporated an insulin polypeptide comprising non-immunogenic natural B10 and A8 amino acids with a canine IgGBFc fragment to form an insulin-Fc fusion protein with acceptable insulin receptor activity and homodimer titer. Therefore, the concepts of longer C chains, longer peptide linkers and truncation of the C-terminal amino acids of the B chain were combined. In addition, in order to potentially further reduce the tendency of self-association and aggregation, additional point mutations were introduced into the natural insulin hydrophobic amino acid residue sites using lower hydrophobic amino acids (including amino acids with negatively charged or positively charged side groups at physiological pH). Exemplary mutations include tyrosine to alanine, tyrosine to glutamic acid, isoleucine to threonine and phenylalanine to histidine. In addition, to simplify the analysis, in all cases, the cNg site of the canine IgGBFc fragment was restored to its natural asparagine. The sequences of these insulin-Fc fusion protein variants are shown below, wherein the resulting sequence alignment relative to SEQ ID NO:66 is shown in FIG. Figure 16 Shown in (ClustalOmega).

[0474] FVNQHLCGSHLVEALELVCGERGFFYTPKTGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:90)

[0475] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNHGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:92)

[0476] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:34)

[0477] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:32)

[0478] FVNQHLCGSHLVEALELVCGERGFFYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:94)

[0479]

[0480] 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. The results showed 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 resulted in several useful insulin-Fc fusion proteins that met the design criteria for minimum homodimer titer and insulin receptor binding activity. SEQ ID NOs: 92, 34, 32, and 94 (368d), (366d), (218d), and (375d) showed 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.

[0481] Example 31: From SEQ ID NO:7 insulin polypeptide, SEQ ID Peptide linker and SEQ ID NO:14 ID No. 16 In vivo bioactivity, repeated administration bioactivity and immunohistochemistry of insulin-Fc fusion protein constructed from canine IgGBFc fragment Epidemiogenicity

[0482] 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 comprises 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 comprise insulin polypeptides with natural B10 and A8 amino acids that are assumed to be less immunogenic (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 comprises 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 comprises SEQ ID NO: 8).

[0483] 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. Figure 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.

[0484] Repeated dose bioactivity was then assessed by subcutaneously administering the insulin-Fc fusion protein of SEQ ID NO: 34 to N = 1 dog on days 14, 28, and 42 after the initial injection, according to the procedure of Example 8. When the dog's % FBGL dropped too low, food was provided to raise blood glucose to a safe level. NAOC and NAOCR were measured for each subsequent dose according to the general procedure of Example 11, starting from the time of the dose administration until just before the next dose was administered. The NAOC and NAOCR shown in Table 14 indicate that the insulin-Fc fusion protein of SEQ ID NO: 34 maintained a NAOCR greater than 0.8 throughout the four doses, thus meeting the design target for repeated dose bioactivity.

[0485]

[0486] The immunogenicity of the insulin-Fc fusion protein of SEQ ID NO: 34 was tested according to the procedure of Example 13. Figure 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 the maintenance of in vivo biological activity throughout the repeated administration experiment.

[0487] The insulin-Fc fusion protein of SEQ ID NO: 32 (wherein the asparagine at A21 of the insulin polypeptide chain is deleted) was also evaluated for repeated dosing bioactivity in dogs. According to the procedure of Example 11, the compound was administered subcutaneously to N=1 dog on days 0, 14, 28 and 42. When the dog's %FBGL dropped too low, food was provided to the dog to raise blood glucose 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 bioactivity, which was almost twice the potency of the insulin-Fc fusion protein of SEQ ID NO: 34. The pharmacokinetic profile of the insulin-Fc fusion protein was 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 4.1±0.7 days). Figure 19 and 20 Single-dose glycemic control and multiple-dose, multi-week glycemic control for animals receiving the homodimer of SEQ ID NO: 32 are shown. NAOC and NAOCR were also measured for each subsequent dose according to the general procedure of Example 11, starting from the time of dose administration and continuing until just before the next dose was administered. The NAOC and NAOCR shown in Table 15 indicate that the insulin-Fc fusion protein of SEQ ID NO: 32 maintained a NAOCR greater than or equal to 1.0 over the four doses, thus meeting the repeat-dose bioactivity design goal described in Example 16.

[0488] The immunogenicity of the insulin-Fc fusion protein of SEQ ID NO: 32 was tested according to the procedure of Example 13. Figure 21 This indicates that the insulin-Fc fusion protein of SEQ ID NO: 32 exhibits no significant immunogenicity in vivo, which is consistent with the maintenance of in vivo biological activity throughout the repeated administration experiment.

[0489]

[0490] As discussed in the specific embodiments of the present invention, known enzymatic cleavage sites exist between asparagine-glycine bonds (Vlasak, J., Ionescu, R., (2011) MAbs Vol. 3, No. 3 pp 253-263). Omitting the asparagine at the 21st amino acid (i.e., A21) of the A chain in the insulin polypeptide of SEQ ID NO: 8 and the peptide linker of SEQ ID NO: 14 contained in the insulin-Fc fusion protein of SEQ ID NO: 32 eliminates the possibility of enzymatic 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, which contains the peptide linker of SEQ ID NO: 14 and the insulin polypeptide of SEQ ID NO: 8, retains the 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. Unexpectedly, however, the insulin-Fc fusion protein of SEQ ID NO: 34 can be produced in HEK cells with acceptable homodimer titers and exhibits acceptable bioactivity in vivo, with no evidence of impairment of its bioactivity by enzymatic digestion.

[0491] Example 32: For a ID NO:8 preferred insulin polypeptide and SEQ ID Preferred peptide of NO:14 Optimal Manufacturability and In Vivo Efficacy of Canine IgGB Isotype Fc Fragments with Linkers for Insulin-Fc Fusion Proteins recognize

[0492] As described in Examples 30 and 31, after discovering that novel insulin polypeptide and peptide linker combinations produced non-immunogenic, high-yield, high-purity, and highly bioactive insulin-Fc fusion proteins, a question remained as to whether the canine IgGB Fc fragment was still the preferred isotype in terms of homodimer titer and bioactivity, as was the case with 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 remained constant, and the canine IgGB Fc fragment of SEQ ID NO: 16 was replaced with 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:

[0493] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGG

[0494] GGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNG

[0495] TYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFP

[0496] PDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:32)

[0497] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGG

[0498] GGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQ

[0499] FNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIK

[0500] DFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG(SEQ ID NO:96)

[0501] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGG

[0502] GGCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSN

[0503] GTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEI ISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFF

[0504] PPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG(SEQ ID NO:98)

[0505] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGG

[0506] GGCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNS

[0507] TYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFF

[0508] PPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG(SEQ ID NO:100)

[0509] Insulin-Fc fusion proteins were prepared in HEK293 cells according to Example 1 and purified using either Protein A or Protein G columns according to Example 3. Their structures were confirmed by non-reducing and reducing CE-SDS according to Example 4, and their sequences were further identified by glycan-removing LC-MS according to Example 5. Their homodimer content was determined by size exclusion chromatography according to Example 6, and their insulin receptor binding affinities were measured according to Example 7. Furthermore, the affinity of the insulin-Fc fusion proteins for the canine FcRn receptor was measured according to Example 8. As shown in Table 16, the insulin-Fc fusion protein of SEQ ID NO: 32, comprising the 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, comprising the canine IgGAFc fragment, exhibited poor homodimer titers; however, when purified using a Protein G column, the homodimer titer was significantly improved, exceeding the design target of greater than 50 mg / L. The same was true for the insulin-Fc fusion protein of SEQ ID NO: 98, which contains the Fc fragment of canine IgG. The insulin-Fc fusion protein of SEQ ID NO: 100, which contains the Fc fragment of canine IgGD, did not yield any compound when purified using either Protein A or Protein G columns. 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).

[0510]

[0511] DNM = not measured; # = purified by protein A; = Purified by Protein G.

[0512] The in vivo biological activity of the insulin-Fc fusion protein of SEQ ID NO: 96 comprising a canine IgGAFc fragment purified by Protein G was tested according to the procedure of Example 10. Figure 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.

[0513] The in vivo bioactivity of the insulin-Fc fusion protein of SEQ ID NO: 98 comprising a canine IgGC Fc fragment was tested by protein G purification according to the procedure of Example 10. Figure 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.

[0514] Thus, canine IgGB is the preferred Fc fragment with respect to 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).

[0515] Example 33: Contains SEQ ID NO:8 insulin polypeptide, SEQ ID NO:14 peptide linker and canine IgGB Non-glycosylated Fc fragment of insulin-Fc fusion protein to reduce the potential risk of immunogenicity

[0516] While 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 extended treatment periods (e.g., 6 months, 1 year, 2 years, or longer), which could compromise the use of this insulin-Fc fusion protein for the treatment of diabetes (if such a scenario occurs). As described in the specific embodiments of the present invention and in Examples 21 and 22, one possible reason for the reduced bioactivity after repeated dosing is the undesirable interaction of the canine IgGB Fc fragment with the dog's immune system, leading to 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.

[0517] 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 was 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, only used 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 below. Figure 24 As shown:

[0518] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVV A LDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:102)

[0519] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGG

[0520] GGDCPKCPAPEMLGGPSVFIFPPPKPKDTTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S G

[0521] TYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFP

[0522] PDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:104)

[0523] 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 their 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. 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%).

[0524]

[0525] According to the procedure of Example 10, the in vivo biological activities of the insulin-Fc fusion proteins of SEQ ID NO: 102 and SEQ ID NO: 104 were tested in N=1 dogs, respectively. Figure 25The results of a single subcutaneous administration shown in demonstrate that both compounds exhibited significantly lower in vivo bioactivity than 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 incorporating the cNg-NB151-S mutation into the Fc fragment to generate a non-glycosylated version of the insulin-Fc fusion protein of SEQ ID NO: 32 unexpectedly reduced the in vivo bioactivity of the resulting compound.

[0526] 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, which had mutations in the region believed to be responsible for aggregation. The 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. In the B chain variants tested, it was unexpectedly found that a kind of insulin Fc- fusion protein (SEQ ID NO: 36) was found to contain tyrosine to alanine substitution at the 16th amino acid place (i.e. B16) of the N-terminal of the B chain. This insulin Fc- fusion protein has 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 (for clarity, the B16A and cNg-NB151-S mutations are underlined).

[0527] FVNQHLCGSHLVEAL A LVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGG

[0528] GGDCPKCPAPEMLGGPSVFIFPPPKPKDTTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S G

[0529] TYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFP

[0530] PDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:36)

[0531] 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 administered subcutaneously to N=1 dog on days 0, 7, 14 and 28. When the dog's %FBGL dropped too low, food was provided to the dog to raise blood glucose 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 first administration in vivo bioactivity diagram is shown in FIG. Figure 26 14. The pharmacokinetic profile of the compound was 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 3.5 days). The NAOC and NAOCR of each subsequent administration were also measured according to the general procedure of Example 11, and the time from the administration of the dose was calculated until just before the next dose was administered. 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 repeated administration bioactivity design target. 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 IgGBFc fragment comprising cNg mutation, the insulin polypeptide of SEQ ID NO:11 is preferred.

[0532]

[0533] Finally, the selected compounds were tested for their potential to interact with the immune system 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. As can be seen, the non-glycosylated insulin-Fc fusion protein (achieved by the cNg-S mutation) exhibited the lowest Fc(γ) receptor binding ratio with SEQ ID NO: 52.

[0534]

[0535] 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

[0536] As described in Example 2, construct the isolated CHO cell line of the vector stably transfected with encoding SEQ ID NO:32 or SEQ ID NO:36. The fed-batch shake flask of 14-day production run (0.5-2.0L culture medium scale) was inoculated with 500,000 cells / mL in an incubator-shaker set at 37°C and 5% carbon dioxide, and operated as described in above-mentioned Example 2, except that Dynamis was replaced by CDOptiCHO as growth medium (ThermoFisher) and high-efficiency 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-shaker carbon dioxide concentration was reduced to 2% from 5%. During operation, cells increased to 8,000,000-14,000,000 cells / mL, and production run was harvested on the 14th day to remove cells, and purification and test culture supernatant were used to obtain the insulin-Fc fusion protein as described in Examples 3, 4, 5 and 6. Table 20 describes production data obtained from production runs using stably transfected CHO cell lines.

[0537]

[0538] Example 35: Using a stably transfected CHO cell line containing a canine IgGB-derived Fc fragment Exemplary CHO-based production runs of preferred insulin-Fc fusion proteins

[0539] As described in Example 2, the Chinese hamster ovary celI (CHO) cell line of the carrier stably transfected with coding SEQ ID NO:34 is constructed.The fed batch shake flask of 14 days production operation (0.5-2.0L culture medium scale) is inoculated in the incubator-shake flask being set at 37 ℃ and 5% carbon dioxide with 500,000 cells / mL, and is run as described in Example 2, difference is to replace Dynamis as growth medium (ThermoFisher) and use efficient feed C (ThermoFisher) as feed with CDOptiCHO.From production operation the 3rd day, add feed with 3%v / v, and at the 4th day, shake flask temperature is adjusted to 32 ℃, and incubator-shake flask carbon dioxide concentration is reduced to 2% from 5%.At the 14th day, harvest and produce operation to remove cell, and purification and test culture supernatant are obtained as insulin-Fc fusion rotein as described in Example 3,4,5 and 6. The resulting production runs gave protein yields greater than 200 mg / L, greater than 95% homodimer, and homodimer titers of SEQ ID NO: 34 greater than 190 mg / L.

[0540] Results - Insulin-FC fusion protein containing feline FC fragment

[0541] Example 36: Insulin-Fc fusion protein comprising an Fc fragment of the feline IgG2 isotype

[0542] To develop a product suitable for cats, attempts were 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:

[0543] FVNQHLCGSDLVEALYLVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGGEGPKCPVPE

[0544] IPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPIL

[0545] HQDWLKGKEFKCKVNSSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQID NO:106)

[0546] 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 resulting compound was measured by size exclusion chromatography according to Example 6 to be 88%. The homodimer titer of the resulting compound 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 moderate level of aggregates and a low homodimer titer of 20 mg / L, which did not meet the design goal of a homodimer titer greater than 50 mg / L.

[0547] Nevertheless, the biological activity of the insulin-Fc fusion protein of SEQ ID NO: 106 was evaluated. First, 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 less than 5000 nM).

[0548] 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. Figure 27 The percentage of fasting blood glucose levels for the insulin-Fc fusion protein of SEQ ID 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, which comprises the insulin polypeptide of SEQ ID NO: 5 and the peptide linker of SEQ ID NO: 12, the insulin-Fc fusion protein of SEQ ID NO: 106 for cats was found to have much less aggregation and significantly higher biological activity in the target animals.

[0549] Because NAOC is acceptable and pharmacokinetic data support weekly administration once, therefore according to example 11, at the 28th day, the 35th day, the 42nd day and the 49th day, give cat extra subcutaneous dose, and measure %FBGL 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 cat shows the significant attenuation of biological activity during the third administration, as measured by the significant reduction of NAOCR (i.e., the NAOC of the third injection is only 0.40 or 40% of the NAOC of the first injection, and the NAOC of the fourth injection is only 0.10 or 10% of the NAOC of the first injection).After repeated administration in cat, the significant decline of the biological activity of the insulin-Fc fusion protein of SEQ ID NO:106 and the biological activity decline of the insulin-Fc fusion protein of SEQ ID NO:52 shown in example 20 observed in dog are similar.

[0550]

[0551] 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

[0552] 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 shown in FIG. Figure 28 Shown in (Clustal Omega).

[0553] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGDCPKCPPPEM

[0554] LGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILH

[0555] QDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ IDNO:108)FVNQHLCGSDLVEAALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ IDNO:110)

[0556] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGEGGPKCPVPE

[0557] IPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPIL

[0558] HQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQID NO:112)

[0559] Insulin-Fc fusion protein was prepared in HEK293 cells according to Example 1 and purified using Protein A column according to Example 3. According to Example 4, their structure was confirmed by non-reducing and reducing CE-SDS, and according to Example 5, the sequence was 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 yield, homodimer % and homodimer titer 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., lower homodimer % than SEQ ID NO:106). This is surprising because feline IgG1b is more similar in function to the canine IgGB Fc fragment isotype, which is a highly preferred Fc isotype for producing canine insulin-Fc fusion proteins (Example 32). Among feline compositions containing mutations of the feline IgG2 isotype, 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 fold increase in protein yield and homodimer titer compared to SEQ ID NO: 112.

[0560]

[0561] Example 38: Repeated subcutaneous administration of a drug containing SEQ ID NO:4 insulin peptide and feline IgG2 isotype In vivo immunogenicity screening of insulin-Fc fusion proteins after Fc fragment

[0562] 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: 106 is 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 manifests 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 that can bind to insulin-Fc fusion proteins, and these antibodies can be detected in immunoassays as described in Example 14. Neutralizing antibodies that inhibit the functional activity of insulin-Fc fusion proteins are generally directed against biologically active sites. To assess whether this is the case, according to Example 14, serum collected before administering each dose and at the end of the experiment described in Example 11 was tested to quantify the level of anti-drug antibodies. As Figure 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 the possible reason for the decrease in NAOCR.

[0563] Example 39: Screening of feline sera for anti-drug antibodies and identification of sites B10D and A8H of the insulin polypeptide Potentially immunogenic epitopes

[0564] 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 "hotspot") that the feline immune system targets. 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 administration of the insulin-Fc fusion protein of SEQ ID NO: 106 (Example 38) against a coated insulin-Fc fusion protein library revealed the unexpected presence of two major "hotspots" within the insulin polypeptide sequence of SEQ ID NO: 4: a mutation at the B10D site (i.e., an aspartic acid mutation at position 10 from the N-terminus of the B chain (i.e., B10)) and, respectively, a mutation at the A8H site (i.e., a histidine mutation at position 8 from the N-terminus of the B chain (i.e., A8)). These results suggest that insulin-Fc fusion proteins containing insulin polypeptide amino acid compositions containing these two specific amino acid mutations may be immunogenic in cats and, therefore, may generate anti-drug antibodies that neutralize biological activity after repeated injections. Therefore, it was determined that insulin polypeptides that do not contain B10D and A8H are preferred for use in insulin-Fc fusion proteins that require long-term, repeated administration in cats (e.g., for the treatment of feline diabetes).

[0565] Example 40: Contains SEQ ID NO:4 insulin peptide 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

[0566] To evaluate whether replacing "hotspot" mutations would improve the immunogenicity and repeat-dose bioactivity of an insulin-Fc fusion protein comprising an insulin polypeptide of SEQ ID NO: 4 and a feline IgG2 isotype fragment, 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 compositions, respectively, and the histidine at B16 was replaced by alanine (i.e., B16A), as was the case with the insulin polypeptide of SEQ ID NO: 5 for various canine insulin-Fc fusion proteins. The A21N site of native insulin was also deleted. For this example, other insulin polypeptide amino acids were mutated to make their structure more similar to native feline insulin (e.g., B30A, A8A, A10V, and A18H). The sequence of the resulting insulin polypeptide (SEQ ID NO: 120) is listed below, with the non-natural amino acids of feline insulin underlined.

[0567] FVNQHLCGSHLVEAL A LVCGERGFFYT DP A GGGPRR GIVEQCCASVCSLYQLEHYC (SEQ ID NO: 120) 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 sequence of the insulin-Fc fusion protein is shown below, with the resulting sequence alignment relative to SEQ ID NO: 108 being at Figure 30 Shown in (ClustalOmega).

[0568] FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVS VLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSP(SEQ IDNO:114)

[0569] FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGGEGPPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFSSTYRVVSV LPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ IDNO:116)

[0570] FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVALGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSV LPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ IDNO:118)

[0571] 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 their sequences were further identified by LC-MS after 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. Table 23 below shows the manufacturability and in vitro IR binding parameters of the resulting compounds.

[0572]

[0573] Insulin-Fc fusion protein of SEQ ID NO:108 is compared, and all three kinds of insulin-Fc fusion protein provide much lower protein yield.In fact, although it has enough high insulin receptor binding affinity (IC50 is 707nM), the insulin-Fc fusion protein of SEQ ID NO:116 almost does not produce protein yield.The insulin-Fc fusion protein of SEQ ID NO:118 provides unacceptable low protein yield and homodimer titer, and is considered to be unlikely to have biological activity in vivo because its high IR in conjunction with IC50 value higher than 5000nM.Compared with the insulin-Fc fusion protein of SEQ ID NO:108, the albumen of SEQ ID NO:114 has also produced unacceptable low protein yield and much lower insulin receptor binding affinity (higher IRIC50 value).

[0574] Example 41: Contains SEQ ID NO:8 insulin polypeptide, SEQ ID The linker of NO:14 is the same as that of feline IgG2 Insulin-Fc fusion protein

[0575] In an attempt to obtain acceptable protein yields for an insulin-Fc fusion protein comprising an insulin polypeptide sequence devoid of 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. Therefore, 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 yield the following sequence:

[0576] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNS TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:122)

[0577] exist Figure 31 A sequence alignment of SEQ ID NO: 122 relative to Example 37 sequences SEQ ID NO: 106 and 112 is shown in (ClustalOmega).

[0578]

[0579] 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. Its structure was confirmed by non-reducing and reducing CE-SDS according to Example 4, and its sequence was further identified by glycan-removed LC-MS according to Example 5. Its homodimer content was measured by size exclusion chromatography according to Example 6, and its insulin receptor binding affinity was measured according to Example 7. Its FcRn receptor binding affinity was measured according to Example 9. The protein yield was 146 mg / L, and the homodimer content was determined to be 99%, resulting in a homodimer titer of 145 mg / L, which met the production design target. The IR binding affinity IC50 value was 2,536 nM, indicating that this 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.

[0580] Example 42: By SEQ ID NO:8 insulin polypeptide, SEQ ID Peptide linker and SEQ ID NO:14 ID No. 21 In vivo biological activity of insulin-Fc fusion protein constructed from feline IgG2Fc fragment

[0581] 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 approximately 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, as well as on days 1, 2, 3, 4, 5, 6, and 7 after injection. If the subject's blood sugar dropped to a dangerous level, food and / or glucose injections were given to prevent symptomatic hypoglycemia.

[0582] Figure 32 The % FBGL for a single administration is shown, demonstrating that, unexpectedly, the insulin-Fc fusion protein of SEQ ID NO: 122 has only weak bioactivity in vivo (NAOC is essentially 0% FBGL·day·kg / mg). This result is surprising, particularly since 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 bioactivity in dogs (Example 31). Due to the lack of bioactivity upon the first administration, no repeat administration was performed.

[0583] Example 43: Evaluation of feline IgG1b substituted for feline IgG2 Fc fragments comprising SEQ ID NO:8 pancreatic islets peptide and SEQ ID Yield, purity, biological activity and immunogenicity of insulin-Fc fusion protein with peptide linker NO:14 Impact

[0584] Since the long-acting insulin research projects for dogs and cats were conducted in parallel, some of the knowledge gained from the canine insulin-Fc fusion protein research project was applied to the feline insulin-Fc protein research project. An important learning gained from the canine insulin-Fc research project was 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:

[0585] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGG

[0586] GGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNS

[0587] TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYP

[0588] SDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:38)

[0589] 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. The structure was confirmed by non-reducing and reducing LC-MS according to Example 4, and the sequence was further identified by glycan-removed LC-MS according to Example 5. The protein yield at this stage was 158 mg / L. The homodimer % of the sequence was measured by size exclusion chromatography according to Example 6 and was determined to be 99.5%, resulting in a homodimer titer of 157 mg / L, which 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.

[0590] The in vivo biological activity of the insulin-Fc fusion protein of SEQ ID NO:38 was then tested according to Example 10. Healthy, antibody-naive cats weighing approximately 5 kg received a single subcutaneous injection of a pharmaceutical composition containing the insulin Fc-fusion protein of SEQ ID NO:38 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 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. If the experimenter's blood sugar dropped to a dangerous level, food and / or glucose injection was given to prevent symptomatic hypoglycemia.

[0591] Figure 33Shown is the %FBGL after first administration. Animal food is given regularly to prevent symptomatic hypoglycemia, which illustrates that the insulin-Fc fusion protein of SEQ ID NO:38 has significant biological activity in vivo, wherein NAOC is 1838%FBGL·day·kg / mg. Also by the method of Example 12, ELISA is used to measure the pharmacokinetic profile of this compound, and a two-compartment model is fitted to the data to determine its elimination half-life (which is 6.3 ± 0.5). The biological activity difference (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 shows that when the insulin polypeptide sequence is modified as described in SEQ ID NO:8, for Fc fragment, feline IgG1 b isotype is more preferred than feline IgG2 isotype.

[0592] Since NAOC is acceptable and pharmacokinetic data support weekly administration, additional subcutaneous administration was given to cats according to Example 11 on days 14, 28, and 42, and % FBGL was measured over a 7-day window 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 exhibited acceptable biological activity in vivo.

[0593]

[0594] 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. Figure 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 met the design criteria of acceptable homodimer titer, in vivo bioactivity, and sustained bioactivity after repeated weekly injections in cats, it was 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.

[0595] Example 44: Contains SEQ ID NO:8 insulin polypeptide, SEQ ID NO:14 peptide linker and feline Non-glycosylated insulin-Fc fusion protein of IgG1bFc fragment to reduce the potential risk of immunogenicity

[0596] While 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 extended treatment periods (e.g., 6 months, 1 year, 2 years, or longer), which could compromise the use of the insulin-Fc fusion protein for treating diabetes (if such a scenario occurs). As described in the specific embodiments of the invention, one possible reason for the reduction in bioactivity after repeated dosing is the undesirable interaction of the feline IgG1b Fc fragment with the feline 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 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.

[0597] As described in a specific embodiment of the present invention, a method for reducing Fc(γ)RI interactions comprises 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: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 was 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):

[0598] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGG

[0599] GGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S S

[0600] TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYP

[0601] SDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:124)

[0602] 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. The structure of the insulin-Fc fusion protein was confirmed by non-reducing and reducing LC-MS according to Example 4, and the sequence was further identified by glycan-removed LC-MS according to Example 5. 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 for in vitro biological activity. The FcRn receptor binding affinity EC50 value was measured according to Example 9 and was 6922 ng / mL.

[0603] Although the insulin-Fc fusion protein of SEQ ID NO: 124 did not meet the insulin receptor binding design goals, its in vivo biological activity was tested according to Example 10. Healthy, antibody-naive cats weighing approximately 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: 124 at a dose of 0.16 mg insulin-Fc fusion protein / kg. On day 0, blood was collected from an appropriate vein before injection and at 15, 30, 45, 60, 120, 240, 360, and 480 minutes, as well as on days 1, 2, 3, 4, 5, 6, and 7 after injection. If the subject's blood glucose dropped to a dangerous level, food and / or glucose injections were given to prevent symptomatic hypoglycemia.

[0604] Figure 35 The % FBGL of a single administration is shown, indicating that the insulin-Fc fusion protein of SEQ ID NO: 124 has only limited bioactivity in vivo, with a NAOC of 65% FBGL·day·kg / mg. Due to the lack of bioactivity after the first administration, repeated administration was not performed.

[0605] Unexpectedly, as with the canine insulin Fc-fusion protein of SEQ ID NO:36 in Example 33, it was discovered that mutating the insulin polypeptide sequence of SEQ ID NO:124 such that the 16th amino acid from the N-terminus of the B chain (B16) was mutated from tyrosine to alanine (i.e., B16A) resulted in the resulting insulin Fc-fusion protein of SEQ ID NO:40 being 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):

[0606] FVNQHLCGSHLVEAL A LVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGG

[0607] GGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S S

[0608] TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYP

[0609] SDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:40)

[0610] 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 with glycan removal 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, also meeting the design goals. Fc(γ) receptor activity was measured according to Example 8 and was found to be approximately 4-fold lower than the activity obtained for the insulin-Fc fusion protein of SEQ ID NO:38 using the same procedure, suggesting that the insulin-Fc fusion protein may interact less adversely with the feline immune system. The FcRn receptor binding affinity EC50 value was measured according to Example 9 and was 8157 ng / mL.

[0611] The in vivo biological activity of the insulin-Fc fusion protein of SEQ ID NO:40 was then tested according to Example 11. Healthy, antibody-naive cats weighing approximately 5 kg were used. On Day 0, Day 7, and Day 21, cats were given a single subcutaneous injection of a pharmaceutical composition containing the insulin-Fc fusion protein of SEQ ID NO:40 at a dosage of 0.1 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 after injection, as well as on Days 1, 2, 3, 4, 5, 6, and 7. If the subject's blood sugar dropped to a dangerous level, food and / or glucose injection was given to prevent symptomatic hypoglycemia.

[0612] Figure 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 Figure 3716mg insulin-Fc fusion protein / kg) . The pharmacokinetic profiles were 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 is greater than 3 days). These results were contrasted with the results obtained using the insulin-Fc fusion protein of SEQ ID NO: 124, which showed that the same compound comprising the tyrosine at B16 rather than alanine had only very weak biological activity at approximately the same dosage (0.16mg insulin-Fc fusion protein / kg). Therefore, for the non-glycosylated insulin-Fc fusion protein of the feline IgG1 b Fc fragment comprising the cNg mutation, the insulin polypeptide of SEQ ID NO: 11 is preferred.

[0613] 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 other dosage was given. When the %FBGL of cat declined too low, food was provided to cat to raise blood glucose 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 from the administration of the dosage was calculated until just before the next dosage was used. 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.

[0614]

[0615] In addition, according to Example 14, serum was collected before each dose and at the end of the experiment to test for the presence and quantify the levels of any anti-drug antibodies. 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 design standards for production and biological activity 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.

[0616] Example 45: Use of Fc fragments containing feline IgG1b derived proteins produced by stably transfected CHO cell lines Exemplary CHO-based production runs of preferred insulin-Fc fusion proteins

[0617] As described in above-mentioned example 2, construct the CHO cell line of the carrier stably transfected with encoding SEQ ID NO:38.The fed batch shake flask of 14 days production operation (0.5-2.0L culture medium scale) is inoculated in the incubator-shake flask that is set as 37 DEG C and 5% carbon dioxide with 500,000 cells / mL, and is run as described in above-mentioned example 2, except that, replace Dynamis as growth medium (ThermoFisher) with CDOptiCHO and use efficient feed C (ThermoFisher) as feed.From the 3rd day of production operation, add feed with 3%v / v, and at the 4th day, shake flask temperature is adjusted to 32 DEG C, and incubator-shake flask carbon dioxide concentration is reduced to 2% from 5%.During operation, cell density increases to 8,000,000-14,000,000 cells / mL, and at the 14th day, harvest production operation to remove cell, and purify culture supernatant and characterize to obtain insulin-Fc fusion protein as described in example 3,4,5 and 6. Table 27 describes the production data of insulin-Fc fusion protein obtained by these stably transfected CHO cell line production runs.

[0618]

[0619] Example 46: Preferred pancreatic islets derived from feline IgG1b produced using a stably transfected CHO cell line Exemplary CHO-based production run of adenovirus-Fc fusion protein

[0620] As described in above-mentioned example 2, construct the Chinese hamster ovary celI cell line with the carrier stably transfected of coding SEQ ID NO:40.The fed batch shake flask of 14 days production operation (0.5-2.0L culture medium scale) is inoculated in the incubator-shake flask being set at 37 DEG C and 5% carbon dioxide with 500,000 cells / mL, and run as described in above-mentioned example 2, difference is, replace Dynamis as growth medium (ThermoFisher) and use efficient feed C (ThermoFisher) as feed with CDOptiCHO.From production operation the 3rd day, add feed with 3%v / v, and at the 4th day, shake flask temperature is adjusted to 32 DEG C, and incubator-shake flask carbon dioxide concentration is reduced to 2% from 5%.At the 14th day, harvest production operation to remove cell, and purified culture supernatant and characterize to obtain 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 SEQ ID NO:40 greater than 190 mg / L.

[0621] Example 47: Exemplary Insulin-Fc Fusion Protein Domains and Sequences

[0622] The exemplary insulin-Fc fusion protein amino acid sequences and corresponding DNA sequences used in the above examples are Figure 38 、 39 , 40, 41 and 42 are shown.

[0623] equivalent

[0624] 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 a 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.

[0625] In addition, the present disclosure includes all variations, combinations and permutations in which 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. Where elements are present in the form of a list (e.g., in the form of Markush groups), each subgroup of elements is also disclosed, and any element can be removed from the group. It should be understood that, in general, where the present disclosure or aspects of the present disclosure are referred to as comprising specific elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure are comprised of or substantially comprised of these elements and / or features. For 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. Where a range is given, the endpoint is included. Furthermore, unless otherwise indicated or apparent from the context and understanding of one of ordinary skill in the art, in various embodiments of the present disclosure, values ​​expressed as ranges may take on any specific value or sub-range within the range, up to the tenth of the unit on the lower limit of the range, unless the context clearly indicates otherwise.

Claims

1. A fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are connected by a linker such as a peptide linker, wherein the Fc fragment comprises the following sequence: DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISK DKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:23).

2. The fusion protein according to claim 1, wherein the insulin polypeptide comprises the following sequence: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is not D, and X2 is not H.

3. The fusion protein according to claim 1, wherein the insulin polypeptide comprises the following sequence: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is H, and X2 is T.

4. The fusion protein according to claims 1 to 3, wherein the insulin polypeptide and the Fc fragment are connected by a linker, such as a peptide linker, comprising the following sequence: GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

5. The fusion protein according to claim 1, wherein the fusion protein comprises the following sequence: FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSST YRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(SEQ ID NO:40). The fusion protein according to claim 1 , wherein the fusion protein is a homodimer. The fusion protein according to claim 6 , wherein the homodimer percentage of the fusion protein is greater than 90%.

8. The fusion protein according to any one of claims 1 to 5, wherein the fusion protein 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.

9. The fusion protein according to any one of claims 1 to 5, wherein the insulin receptor IC50 of the fusion protein is less than or equal to 5000 nM.

10. The fusion protein of any one of claims 1 to 5, wherein upon administration, the fusion protein has a serum half-life in the blood or serum of a target animal that is longer than about 3 days.

11. according to the fusion protein described in any one of claims 1 to 5, wherein blood glucose level has a statistically significant reduction time relative to pre-dose level in the subject longer 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 longer time.

12. The fusion protein according to any one of claims 1 to 5, wherein the NAOC after the first subcutaneous injection in the target animal is greater than 150% FBGL·day·kg / mg.

13. The fusion protein of claim 12, 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.

14. The fusion protein according to any one of claims 1 to 5, wherein the fusion protein is formulated into a pharmaceutical composition.

15. The pharmaceutical composition of claim 14, wherein the fusion protein is present in the pharmaceutical composition at a concentration of about 3 mg / mL or higher.

16. The pharmaceutical composition according to claim 15, wherein the composition is suitable for subcutaneous administration. 17 . A method for lowering the blood glucose level of a target cat, comprising administering to the target cat a physiologically effective amount of the fusion protein according to claim 1 or a pharmaceutical composition thereof.

18. The method of claim 17, wherein the target cat is diagnosed with diabetes.

19. The method of claim 17 or 18, wherein the fusion protein is administered subcutaneously.

20. The method of claim 19, wherein the fusion protein is administered to the target cat daily, twice a week, or once a week.

21. The method of claim 20, wherein the fusion protein is administered to the target cat once a week at a dose of 0.025 to 0.5 mg / kg / week.

22. A cell engineered to express the fusion protein of any one of claims 1 to 5.

23. The cell of claim 22, wherein the cell is transfected with a nucleic acid encoding the fusion protein.

24. The cell according to claim 23, wherein the cell is a HEK293 cell or a CHO cell.

25. A cDNA encoding the fusion protein of any one of claims 1 to 5.

26. The cDNA according to claim 25, wherein the cDNA comprises the following nucleic acid sequence: atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcagcagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag(SEQ ID NO:39)。 27. Use of the fusion protein or pharmaceutical composition thereof according to any one of claims 1 to 5 in the preparation of a medicament for lowering the blood glucose level of a target cat.

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