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

Abstract

The present disclosure provides recombinantly produced ultra-long-acting insulin-Fc fusion proteins for use in the treatment of diabetes in dogs and cats. The insulin-Fc fusion proteins comprise an insulin polypeptide linked to an Fc fragment of canine or feline origin via a peptide linker. Based on the results obtained, developing a treatment that is low-cost to manufacture, exhibits sufficient in vivo bioactivity, exhibits extended long-term bioactivity, does not induce anti-drug antibodies, and substantially maintains efficacy over multiple administrations requires non-obvious combinations of insulin polypeptides, peptide linkers, and species-specific Fc fragments, as well as selective mutations in one or more of these components. Exemplary ultra-long-acting insulin-Fc fusion proteins, polynucleotides encoding these insulin-Fc fusion proteins, and pharmaceutical formulations of exemplary insulin-Fc fusion proteins are further provided along with methods of use and manufacture.

Description

Technology Field

[0001] Priority Claim and Related Applications

[0002] The present application is U.S. Provisional Application No. 62 / 837,188 filed on April 22, 2019; U.S. Provisional Application No. 62 / 827,809 filed on April 1, 2019; U.S. Provisional Application No. 62 / 824,176 filed on March 26, 2019; U.S. Provisional Application No. 62 / 781,378 filed on December 18, 2018; U.S. Provisional Application No. 62 / 781,368 filed on December 18, 2018; U.S. Provisional Application No. 62 / 774,682 filed on December 3, 2018; U.S. Provisional Application No. 62 / 743,358 filed on October 9, 2018; and October U.S. Provisional Application No. 62 / 740,735 filed on the 3rd, U.S. Provisional Application No. 62 / 719,347 filed on August 17, 2018, U.S. Provisional Application No. 62 / 702,167 filed on July 23, 2018, U.S. Provisional Application No. 62 / 698,648 filed on July 16, 2018, U.S. Provisional Application No. 62 / 696,645 filed on July 11, 2018, U.S. Provisional Application No. 62 / 693,814 filed on July 3, 2018, U.S. Provisional Application No. 62 / 692,507 filed on June 29, 2018, and U.S. Provisional Application filed on June 29, 2018 It relates to Patent No. 62 / 692,498 and claims the benefit of its priority. The contents of each of the aforementioned patent applications are incorporated herein by reference in their entirety.

[0003] The present technology relates to the composition of insulin-Fc fusion proteins for treating diabetes in companion animals, for example, dogs or cats, and the uses thereof. Background Technology

[0004] The following description of the background of the present technology is provided merely to aid in understanding the present technology and is not construed as describing or constituting prior art related to the present technology.

[0005] Diabetes is a chronic disease characterized by insulin deficiency and / or the ineffective use of insulin. Diabetic patients with absolute insulin deficiency are classified as having Type 1 or insulin-dependent diabetes mellitus (IDDM). Patients with Type 1 diabetes are thought to have a genetic predisposition combined with the immunological destruction of insulin-producing β-cells in the pancreas. In contrast, diabetic patients who can produce some insulin but have a relative deficiency due to insulin resistance or other dysfunction are classified as having Type 2 or non-insulin-dependent diabetes mellitus (NIDDM). Type 2 diabetes is associated with genetic predisposition, obesity, and certain medications.

[0006] When dogs or cats do not produce insulin or are unable to use it normally, blood glucose levels rise, leading to hyperglycemia. Dogs generally exhibit an atypical blood glucose phenotype very similar to human Type 1 diabetes. Dogs also sometimes display atypical blood glucose that shows strong similarities to human Type 2 diabetes. Female dogs may also exhibit transient insulin resistance during heat or pregnancy. In all cases, dogs are treated with chronic insulin injection therapy. Cats generally exhibit an atypical blood glucose phenotype that strongly resembles human Type 2 diabetes (i.e., insulin resistance); however, by the time a veterinarian diagnoses the disease, it progresses similarly to Type 1 diabetes (an inflammatory disease of the pancreas characterized by a significant loss of beta-cell mass), and cats become dependent on exogenous insulin. While some diabetic cats can be managed with dietary changes and oral medications, most diabetic cats receive chronic insulin injection therapy to maintain proper control. If left untreated, diabetes in dogs and cats can lead to weight loss, loss of appetite, vomiting, dehydration, motor dysfunction, coma, and even death.

[0007] In the United States, approximately 0.24% of dogs and 0.68% of cats suffer from diabetes. The current treatment for diabetes in dogs is Vetsulin for dogs. ® (Intervet Inc., dba MERCK Animal Health, Summit, NJ) and ProZinc for cats ®This includes the use of insulin, such as that prescribed by Boehringer Ingelheim Vetmedica (Duluth, Georgia), which is administered once or twice a day. The burden of frequent injections placed on owners often leads to poor adherence to treatment regimens and under-administration, worsening long-term health outcomes. In fact, the cost of insulin treatment and the practical reality of administering up to 14 injections per week to pets cause a significant proportion of owners to choose euthanasia for their pets as an alternative to intensive diabetes management. Therefore, there is a need for cost-effective and less burdensome treatment options for this disease. means of solving the problem

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

[0009] DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTIS KARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 16).

[0010] In some embodiments, the insulin polypeptide of the fusion protein comprises the following sequence:

[0011] FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3(sequence number: 6), where X1 is not D, X2 is not H, and X3 is absent or N.

[0012] In some embodiments, the insulin polypeptide of the fusion protein comprises the following sequence:

[0013] FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3(sequence number: 6), where X1 is H, X2 is T, and X3 is absent or N.

[0014] In some embodiments, the insulin polypeptide and Fc fragment of the fusion protein are connected by a linker such as a peptide linker comprising the following sequence:

[0015] GGGGGQGGGGQGGGGQGGGGG(Sequence No.: 14).

[0016] In the example, the fusion protein comprises the following sequence:

[0017] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNG TYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 32).

[0018] In the example, the fusion protein comprises the following sequence:

[0019] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNG TYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 34).

[0020] In one embodiment, 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, and wherein the Fc fragment comprises the following sequence:

[0021] DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTIS KARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 22).

[0022] In some embodiments, the insulin polypeptide of the fusion protein comprises the following sequence:

[0023] FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC(sequence number: 10), where X1 is not D and X2 is not H.

[0024] In some embodiments, the insulin polypeptide of the fusion protein comprises the following sequence:

[0025] FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC(sequence number: 10), where X1 is H and X2 is T.

[0026] In an example, the insulin polypeptide and the Fc fragment are connected by a linker such as a peptide linker comprising the following sequence:

[0027] GGGGGQGGGGQGGGGQGGGGG(Sequence No.: 14).

[0028] In the example, the fusion protein comprises the following sequence:

[0029] FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFS GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 36).

[0030] In one embodiment, the present disclosure provides a fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are linked by a linker such as a peptide linker, wherein the Fc fragment is of non-human animal origin and comprises the following sequence:

[0031] DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTIS KDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(sequence Number: 20).

[0032] In the example, the insulin polypeptide of the fusion protein comprises the following sequence:

[0033] FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3(sequence number: 6), where X1 is not D, X2 is not H, and X3 is not.

[0034] In the example, the insulin polypeptide of the fusion protein comprises the following sequence:

[0035] FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3(sequence number: 6), where X1 is H, X2 is T, and X3 is absent.

[0036] In an example, the insulin polypeptide and the Fc fragment are connected by a linker such as a peptide linker comprising the following sequence:

[0037] GGGGGQGGGGQGGGGQGGGGG(Sequence No.: 14).

[0038] In the example, the fusion protein comprises the following sequence:

[0039] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFN STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 38).

[0040] In one embodiment, 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, and wherein the Fc fragment comprises the following sequence:

[0041] DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTIS KDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(sequence Number: 23).

[0042] In the example, the insulin polypeptide of the fusion protein comprises the following sequence:

[0043] FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC(sequence number: 10), where X1 is not D and X2 is not H.

[0044] In the example, the insulin polypeptide of the fusion protein comprises the following sequence:

[0045] FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC(sequence number: 10), where X1 is H and X2 is T.

[0046] In an example, the insulin polypeptide and the Fc fragment are connected by a linker such as a peptide linker comprising the following sequence:

[0047] GGGGGQGGGGQGGGGQGGGGG(Sequence No.: 14).

[0048] In the example, the fusion protein comprises the following sequence:

[0049] FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSS TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(sequence Number: 40).

[0050] In some embodiments, the fusion protein described herein comprises a homodimer. In some embodiments, the percentage of homodimers in the fusion protein is 90% or more. In some embodiments, the fusion protein described herein is prepared using HEK293 cells, and the homodimer titer produced after purification using Protein A beads or a Protein A column is greater than 50 mg / L. In some embodiments, the insulin receptor IC50 for the fusion protein described herein is 5000 nM or less. In some embodiments, the serum half-life of the fusion protein described herein in the blood or serum of a target animal at administration is longer than about 3 days. In some embodiments, for the fusion protein described herein, the time at which there is a statistically significant decrease in blood glucose levels in the subject compared to pre-administration levels is 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, or 7 days or more.

[0051] In one embodiment, for the fusion protein described herein, the NAOC after the first subcutaneous injection in the target animal is greater than 150% FBGL·days·kg / mg. In another embodiment, for the fusion protein described herein, the ratio of the NAOC after the third week of subcutaneous injection of the fusion protein in the target animal to the NAOC after the first subcutaneous injection of the fusion protein in the target animal is greater than 0.50.

[0052] In an embodiment, the fusion protein described herein is formulated into a pharmaceutical composition. In an embodiment, the fusion protein is present in the pharmaceutical composition at a concentration of about 3 mg / mL or higher. In an embodiment, the composition is suitable for subcutaneous administration.

[0053] In one embodiment, a method for lowering the blood glucose level of a target animal is described, said method comprising the step of administering a physiologically effective amount of the fusion protein or a pharmaceutical composition thereof described herein to a patient. In an embodiment, the target animal is diagnosed with diabetes. In an embodiment, the target animal is a dog or a cat. In some embodiments, the fusion protein is administered subcutaneously. In some embodiments, the fusion protein is administered to the target animal daily, twice a week, or once a week. In an embodiment, the fusion protein is administered to the target animal once a week at a dosage between 0.025 and 0.5 mg / kg / week. In an embodiment, a cell engineered to express the fusion protein as described herein is described. In one example, the cell is transfected with a nucleic acid encoding the fusion protein. In one example, the cell is an HEK293 cell or a CHO cell.

[0054] In one embodiment, a cDNA encoding the fusion protein described herein is described. In an example, the cDNA comprises the following nucleic acid sequence:

[0055] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(서열 번호: 31).

[0056] In the example, the cDNA comprises the following nucleic acid sequence:

[0057] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcaacggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(서열 번호: 33).

[0058] In the example, the cDNA comprises the following nucleic acid sequence:

[0059] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(서열 번호: 35).

[0060] In the example, the cDNA comprises the following nucleic acid sequence:

[0061] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcaacagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag(서열 번호: 37).

[0062] In the example, the cDNA comprises the following nucleic acid sequence:

[0063] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcagcagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag(서열 번호: 39). 도면의 간단한 설명

[0064] Figure 1 illustrates a schematic representation of an exemplary insulin-Fc fusion protein homodimer. Figure 2 shows the average % fasting blood glucose levels from day 0 to day 3 for N = 3 dogs administered intravenously of the homodimer of sequence number: 42 at 0.2 mg / kg on day 0. Figure 3 illustrates a side-by-side sequence comparison of sequence numbers: 42, 44, 46, 48, and 50. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, moderate, or very different amino acid mutations across the sequence at a given sequence position, respectively. Figure 4 illustrates a side-by-side sequence comparison of sequence numbers: 42, 52, 54, and 56. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 5 shows the average % fasting blood glucose levels from day 0 to day 7 for N = 3 dogs intravenously administered 0.2 mg / kg of the homodimer of sequence number: 52 on day 0. Figure 6 shows the average % fasting blood glucose levels from day 0 to day 7 for N = 6 dogs administered subcutaneously of the homodimer of sequence number: 52 at 0.33 mg / kg on day 0. Figure 7 shows the mean anti-drug antibody titers (μg / mL) for N = 3 dogs administered subcutaneously with the homodimer of sequence number: 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). Figure 8 illustrates a side-by-side sequence comparison of sequence numbers: 58, 60, 62, and 64. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 9 shows the mean anti-drug antibody titer (μg / mL) for N = 1 dogs administered subcutaneously with the homodimer of sequence number: 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). Figure 10 shows the mean anti-drug antibody titer (μg / mL) for N = 1 dogs administered subcutaneously with the homodimer of sequence number: 66 at 0 days (0.33 mg / kg) and 14 days (0.16 mg / kg). Figure 11 shows the average % fasting blood glucose levels from day 0 to day 7 for N = 2 dogs administered 0.33 mg / kg of the homodimer of sequence number: 66 subcutaneously on day 0. Figure 12 illustrates a side-by-side sequence comparison of sequence numbers: 66, 68, 70, 72, 74, and 76. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 13 illustrates a side-by-side sequence comparison of sequence numbers: 66, 78, 80, 82, and 84. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 14 illustrates a side-by-side sequence comparison of sequence numbers: 66, 76, and 86. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 15 illustrates a side-by-side sequence comparison of sequence numbers: 66, 82, 84, and 88. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 16 illustrates a side-by-side sequence comparison of sequence numbers: 32, 34, 66, 90, 92, and 94. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 17 shows the average % fasting blood glucose levels from day 0 to day 7 for N = 1 dogs administered subcutaneously of the homodimer of sequence number: 34 at 0.16 mg / kg on day 0. Figure 18 shows the anti-drug antibody titers (μg / mL) for N = 1 dogs administered subcutaneously with the homodimer of sequence number: 34 at 0 (0.16 mg / kg), 14 (0.16 mg / kg), 28 (0.16 mg / kg), and 42 (0.16 mg / kg). Figure 19 shows the average % fasting blood glucose levels from day 0 to day 7 for N = 1 dogs administered subcutaneously of the homodimer of sequence number: 32 at 0.33 mg / kg on day 0. Figure 20 shows the mean % fasting blood glucose levels from day 0 to day 60 for N = 1 dogs administered subcutaneously with the homodimer of sequence number: 32 at 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). Figure 21 shows the anti-drug antibody titers (μg / mL) for N = 1 dogs administered subcutaneously with the homodimer of sequence number: 32 at 0 (0.33 mg / kg), 15 (0.16 mg / kg), 31 (0.16 mg / kg), and 45 (0.15 mg / kg). Figure 22 shows the average % fasting blood glucose levels from day 0 to day 7 for N = 1 dogs administered subcutaneously of 0.16 mg / kg of the homodimer of sequence number: 96 on day 0. Figure 23 shows the average % fasting blood glucose levels from day 0 to day 7 for N = 1 dogs administered subcutaneously of the homodimer of sequence number: 98 at 0.16 mg / kg on day 0. Figure 24 illustrates a side-by-side sequence comparison of sequence numbers 102 and 104. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 25 shows the % fasting blood glucose levels from day 0 to day 7 for N = 1 dogs administered subcutaneously with 0.16 mg / kg of the homodimer of sequence number: 102 on day 0, and the % fasting blood glucose levels from day 0 to day 7 for N = 1 dogs administered subcutaneously with 0.16 mg / kg of the homodimer of sequence number: 104 on day 0. Figure 26 shows the % fasting blood glucose levels for days 0 to 7 in N = 1 dogs subcutaneously administered the homodimer of sequence number: 36, in addition to the time at which food was provided to the dogs. Figure 27 shows the mean % fasting blood glucose levels from day 0 to day 7 for N = 3 cats administered subcutaneously of the homodimer of sequence number: 106 at 0.8 mg / kg on day 0. FIG. 28 illustrates a side-by-side sequence comparison of sequence numbers: 106, 108, 110, and 112. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 29 shows the mean anti-drug antibody titers (μg / mL) for N = 3 cats administered the homodimer of sequence number: 106 subcutaneously on day 0 (0.8 mg / kg), day 28 (0.6 mg / kg), day 35 (0.6 mg / kg), day 42 (0.6 mg / kg), and day 48 (0.8 mg / kg). FIG. 30 illustrates a side-by-side sequence comparison of sequence numbers: 108, 114, 116, and 118. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 31 illustrates a side-by-side sequence comparison of sequence numbers: 106, 112, and 122. "*" indicates complete homology across all sequences at a given sequence position, while ":", ".", or a space indicate conservative, intermediate, or very different amino acid mutations across sequences at a given sequence position, respectively. Figure 32 shows the % fasting blood glucose levels from day 0 to day 7 in N = 1 cats administered subcutaneously with the homodimer of sequence number: 122 at day 0 (0.16 mg / kg). Figure 33 shows the % fasting blood glucose levels for days 0 to 7 in N = 1 cat administered subcutaneously at day 0 (0.16 mg / kg) of the homodimer of sequence number: 38, in addition to the time of food delivery to the cat. Figure 34 shows the anti-drug antibody titers (μg / mL) for N = 1 cats administered subcutaneously with the homodimer of sequence number: 38 at 0 (0.16 mg / kg), 14 (0.16 mg / kg), 28 (0.11 mg / kg), and 42 (0.09 mg / kg). Figure 35 shows the % fasting blood glucose levels from day 0 to day 7 in N = 1 cats administered subcutaneously with the homodimer of sequence number: 124 at day 0 (0.16 mg / kg). Figure 36 shows the mean % fasting blood glucose levels from day 0 to day 7 for N = 3 cats administered subcutaneously with the homodimer of sequence number: 40 at day 0 (0.10 mg / kg). Figure 37 shows the mean % fasting blood glucose levels for days 7 to 14 in N = 3 cats administered subcutaneously with the homodimer of sequence number: 40 at 0.20 mg / kg for 7 days. FIG. 38 illustrates the "entire aa sequence" of a fusion protein (sequence number: 32) and its corresponding nucleic acid sequence (sequence number: 31). FIG. 39 illustrates the "entire aa sequence" of a fusion protein (sequence number: 34) and its corresponding nucleic acid sequence (sequence number: 33). FIG. 40 illustrates the "entire aa sequence" of a fusion protein (sequence number: 36) and its corresponding nucleic acid sequence (sequence number: 35). FIG. 41 illustrates the "entire aa sequence" of a fusion protein (sequence number: 38) and its corresponding nucleic acid sequence (sequence number: 37). FIG. 42 illustrates the "entire aa sequence" of a fusion protein (sequence number: 40) and its corresponding nucleic acid sequence (sequence number: 39). Specific details for implementing the invention

[0065] Insulin therapy requiring less frequent administration (e.g., once-weekly injection) is less burdensome to the owner, leading to better compliance, fewer cases of euthanasia, and better outcomes for the pet. For a given species (e.g., dog or cat), a molecule suitable for the very long-term treatment of diabetes must be able to be produced in mammalian cells, e.g., human embryonic kidney (HEK, e.g., HEK293) cells, while having an acceptable titer of the desired dimeric product (e.g., dimeric titer of 50 mg / L or more from transiently transfected HEK cells, 75 mg / L or more from transiently transfected HEK cells, 100 mg / L or more from transiently transfected HEK cells, etc.). Only candidates with a dimeric titer of 50 mg / L or more are considered useful in the present invention. This is because, according to experience, homodimeric titers below this level are unlikely to result in commercially produced homodimeric titers in Chinese hamster ovary (CHO) cells that meet the strictly low manufacturing cost requirements for veterinary products. Additionally, the molecule must bind to an insulin receptor with recognizable affinity (e.g., IC50 < 5000 nM, IC50 < 4000 nM, IC50 < 3000 nM, IC50 < 2500 nM, etc.) when measured in the 4°C IM-9 insulin receptor binding assay. Based on experience, only molecules exhibiting an insulin receptor activity IC50 value of less than 5000 nM are considered to possess the necessary bioactivity in the target species. The molecule must also demonstrate sustained bioactivity in vivo to justify less frequent administration (e.g., must demonstrate greater glucose-lowering activity for approximately 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).The molecule must also demonstrate an extended systemic residence time in target animals (e.g., the serum half-life must be at least 3 days). Bioactive efficacy and duration of bioactivity can be quantified by calculating the area under a % fasting blood glucose (% FBGL) curve normalized to % FBGL·days·kg / mg for a given dose of mg / kg (NAOC), as described in Example 11. NAOC increases with a greater decrease in % FBGL, which indicates increased bioactivity of the molecule, and takes longer for % FBGL to return to 100%, which indicates an increased duration of action of the insulin-Fc fusion protein. To be useful as described herein, the molecule must exhibit a sufficiently high NAOC value (e.g., preferably greater than 150% FBGL·days·kg / mg, more preferably greater than 200% FBGL·days·kg / mg, and even more preferably greater than 250% FBGL·days·kg / mg). Empirically, when the NAOC value is greater than 150% FBGL·days·kg / mg, the dosage requirement for the target species is sufficiently low to reach an acceptable treatment cost. Finally, to be useful for the treatment of chronic diseases such as diabetes, the molecule must not induce the production of anti-drug antibodies, particularly antibodies that neutralize the molecule's biological activity after repeated administration. Therefore, the molecule must demonstrate similar duration and bioactivity (i.e., NAOC) after multiple repeated administrations in target animals (e.g., the ratio of NAOC after the third week's subcutaneous injection to NAOC after the first week's subcutaneous injection of the molecule (i.e., NAOC ratio after the third administration (NAOCR))) in a preferred order of 0.50 or higher, 0.60 or higher, 0.70 or higher, 0.80 or higher, or 0.90 or higher.

[0066] Proposed ultra-long-acting insulin therapy for human clinical use involves insulin-Fc fusion proteins that use human Fc fragments to prolong their action in vivo. Since human Fc fragments are expected to be immunogenic and can induce the production of anti-drug antibodies in companion animals (e.g., dogs or cats), human Fc fragments must be replaced with species-specific (e.g., dogs or cats) Fc fragments. However, it was somewhat unexpectedly discovered that a simple exchange between human Fc fragments and species-specific (e.g., dogs or cats) Fc fragments does not produce products with acceptable homodimeric titers (e.g., homodimeric titers greater than 50 mg / L) or sufficiently high NAOC values ​​(e.g., NAOC greater than 150% FBGL·days·kg / mg). For example, in some cases, only specific isotypes of the Fc fragment (e.g., canine IgGB or feline IgG1b) produced insulin-Fc fusion proteins with sufficiently high homodimeric titers (e.g., homodimeric titers greater than 50 mg / L) and acceptable high NAOC values ​​(e.g., NAOC greater than 150% FBGL·days·kg / mg). In other cases, specific amino acids of the insulin polypeptide were found to be immunogenic in the target species, requiring site-directed mutations to find a relatively small number of examples that are both non-immunogenic and bioactive in the target species, while having acceptable high NAOC values ​​(e.g., NAOC values ​​greater than 150% FBGL·days·kg / mg) and NAOCR values ​​greater than 0.5 after third-week subcutaneous administration.In additional cases, when the Fc fragment was mutated to prevent glycosylation and further reduced 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 homodimeric titer (e.g., a homodimeric titer greater than 50 mg / L) and NAOC value (e.g., a NAOC value greater than 150% FBGL·days·kg / mg). Furthermore, it was found that additional mutations in the insulin component were required to produce these Fc-mutated, non-glycosylated insulin Fc-fusion proteins having the desired homodimeric titer (e.g., a homodimeric titer greater than 50 mg / L) and NAOC value (e.g., a NAOC value greater than 150% FBGL·days·kg / mg), while also achieving a NAOCR value greater than 0.5 after a third week of subcutaneous administration. Accordingly, the present invention provides a manufacturable high-purity, sustained-release, bioactive, non-immunogenic insulin-Fc fusion protein having an acceptable high homodimeric titer (e.g., a homodimeric titer greater than 50 mg / L), an NAOC value (e.g., an NAOC value greater than 150% FBGL·days·kg / mg), and an NAOCR value greater than 0.5 after third weekly subcutaneous administration, which is suitable for the treatment of diabetes in companion animals (e.g., dogs or cats), each comprising an insulin polypeptide, an Fc fragment, and a linker between the insulin polypeptide and the Fc fragment.

[0067] definition

[0068] As used herein, the articles "a" and "an" refer to one or more of the grammatical objects of the article, e.g., at least one. When used herein with the term "comprising," the use of the word "a" or "an" may mean "one," but may also be used to mean "one or more," "at least one," and "one or more."

[0069] As used herein, “approximately” and “roughly” generally refer to an acceptable degree of error for a measured quantity given the characteristics or precision of the measurement. An exemplary degree of error is within 20% of a given range of values, generally within 10%, and more generally within 5%.

[0070] As used herein, the amount of molecule, compound, conjugate, or substance effective for treating a disorder (e.g., a disorder described herein), the “therapeutic effective amount” or “effective amount” refers to the amount of molecule, compound, conjugate, or substance effective for treating a subject upon single or multiple administrations, or for healing, alleviating, remission, or improvement of a subject with a disorder (e.g., a disorder described herein) that would be expected without such treatment.

[0071] As used herein, the term "analogous" refers to a compound or conjugate that is similar to another compound or conjugate but has a different chemical structure in at least one aspect (e.g., a compound or conjugate as described herein, e.g., insulin).

[0072] As used herein, the terms “antibody” or “antibody molecule” mean an immunoglobulin molecule (Ig) or a molecule comprising an immunologically active portion of an immunoglobulin (Ig) molecule, namely, an antigen-binding site that specifically binds, for example, to an antigen and reacts immunely with it. As used herein, the term “antibody domain” means a variable or constant region of an immunoglobulin. As used herein, the term “antibody domain” means a variable or constant region of an immunoglobulin. It is noted in the art that antibodies include several classes, such as IgA, IgM, or IgG, for mammals (e.g., humans and cats). Classes of immunoglobulins may be further classified into different isotypes, such as IgGA, IgGB, IgGC, and IgGD for dogs or IgG1a, IgG1b, and IgG2 for cats. Those skilled in the art will recognize that immunoglobulin isotypes of a given class of immunoglobulins will have different amino acid sequences, structures, and functional characteristics (e.g., different binding affinities for Fc (gamma) receptors). “Specifically binds” or “immune responds” means that an antibody reacts with one or more antigen determinants of the desired antigen and has a low affinity for other polypeptides, for example, does not react with other polypeptides.

[0073] As used herein, the terms "area under the curve" or "AUC" refer to the integrated area under the % FBGL versus time curve for a subject after administration of a given dose of insulin-Fc fusion protein. As used herein, the terms "area under the curve" or "AOC" are used as a measure of the biological efficacy of insulin-Fc fusion protein such that AOC is equal to the difference between the total possible area under the % FBGL versus time curve and the AUC value. As used herein, "normalized area under the curve," "normalized AOC," or "NAOC" are the AOC values ​​divided by the actual dose of insulin-Fc fusion protein administered. As used herein, the terms "normalized AOC ratio" or "NAOCR" are the ratio of the NAOC resulting from a specific administration of insulin-Fc fusion protein to the NAOC resulting from the initial administration of insulin-Fc fusion protein in a series of administrations. Thus, NAOCR provides a measure of the change in the biological activity of insulin-Fc fusion protein after repeated administrations.

[0074] As used herein, the terms “bioactivity,” “activity,” “bioactivity,” “potency,” “bioactivity efficacy,” or “biological efficacy” refer to the extent to which an insulin-Fc fusion protein activates an insulin receptor and / or exerts a reduction in blood glucose levels in a target subject. As used herein, the terms “in vitro activity” or “insulin receptor activity” refer to the affinity of an insulin-Fc fusion protein to bind to an insulin receptor, which is generally measured by the concentration at which the insulin-Fc fusion protein replaces half of the insulin reference standard 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 a reduction in fasting blood glucose levels in a target subject following administration of an insulin-Fc fusion protein.

[0075] As used herein, the terms “biosynthesis,” “recombinant synthesis,” or “recombinant manufacturing” refer to the process by which an insulin-Fc fusion protein is expressed within a host cell by transfecting the cell with a nucleic acid molecule (e.g., a vector) that encodes the insulin-Fc fusion protein (e.g., the entire insulin-Fc fusion protein is encoded by a single nucleic acid molecule). Exemplary host cells include mammalian cells, e.g., HEK293 cells or CHO cells. Cells may be cultured using standard methods of the industry, and the expressed insulin-Fc fusion protein may be harvested and purified from the cell culture using standard methods of the industry.

[0076] As used herein, the term “cell surface receptor” generally refers to a molecule, such as a protein, found on the outer surface of a cell membrane and interacting with soluble molecules, for example, molecules circulating in the blood supply. In some embodiments, the cell surface receptor may include a hormone receptor (e.g., insulin hormone receptor or insulin receptor (IR)) or an Fc receptor that binds to an Fc fragment or the Fc region of an antibody (e.g., an Fc(gamma) receptor, e.g., Fc(gamma) receptor I, or an Fc neonatal receptor, e.g., FcRn). As used herein, the terms “in vitro activity” or “Fc(gamma) receptor activity” or “Fc(gamma) receptor binding” or “FcRn receptor activity” or “FcRn binding” refer to the affinity of an insulin-Fc fusion protein to bind to an Fc receptor (e.g., Fc(gamma) receptor or FcRn receptor), which is generally measured as the concentration of the insulin-Fc fusion protein that causes the insulin-Fc fusion protein to reach half of maximum binding (i.e., EC50 value) when measured in an analysis (e.g., enzyme-linked immunosorbent assay (ELISA) analysis) using an OD 450 nm value measured by a microplate reader.

[0077] As used herein, the terms "fasting blood glucose level" or "FBGL" refer to the average blood glucose level in the target subject immediately before the time when the insulin-Fc fusion protein is administered, after the period of no food administration has ended. As used herein, the terms "percent fasting blood glucose level," "% fasting blood glucose level," or "% FBGL" refer to the ratio of a given blood glucose level to the fasting blood glucose level multiplied by 100.

[0078] As used herein, the terms “immunogenic” or “immunogenic” refer to the ability of a given molecule (e.g., the insulin-Fc fusion protein of the present invention) to induce the immune system of a target subject to produce antibodies (i.e., anti-drug antibodies) that can specifically bind to the molecule after repeated administration of the molecule. As used herein, the terms “neutralization,” “neutralizing antibody,” or “neutralizing anti-drug antibody” refer to the ability of an antibody to interfere with the biological activity of a compound in a target subject. As used herein, the terms “immunogenic epitope,” “immunogenic hot spot,” or “hot spot” refer to a mutation or epitope of a given molecule (e.g., the insulin-Fc fusion protein of the present invention) responsible for intermediate or strong binding of anti-drug antibodies.

[0079] As used herein, the term “insulin reference standard” refers to any one of (i) naturally occurring insulin of mammals (e.g., humans, dogs, or cats); (ii) an insulin polypeptide that does not contain an Fc fragment; or (iii) a standard therapeutic insulin (e.g., commercially available insulin).

[0080] As used herein, the term "monomer" refers to a protein or fusion protein comprising a single polypeptide. In the embodiments, the "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 the Fc fragment polypeptide are joined by peptide bonds to form a single polypeptide. In the embodiments, the monomer is encoded by a single nucleic acid molecule.

[0081] As used herein, the term "N-terminus" refers to the beginning of a protein or polypeptide initiated by an amino acid containing a free amine group, which is the alpha-amino group of the amino acid (e.g., the free amino is covalently linked to a carbon atom located adjacent to the second carbon atom, where the second carbon atom is part of the carbonyl group of the amino acid).

[0082] The term "C-terminus" as used herein refers to the end of a protein or polypeptide terminated by an amino acid containing a carboxylic acid group, wherein the carbon atom of the carboxylic acid group is located adjacent to the alpha-amino group of the amino acid.

[0083] As used herein, the terms "pharmacokinetics" or "PD" generally refer to the biological effects of insulin-Fc fusion proteins in subjects. Specifically, herein, PD refers to the measurement of the decrease in fasting blood glucose levels over time in subjects following the administration of insulin-Fc fusion proteins.

[0084] As used herein, the terms "pharmacokinetics" or "PK" generally refer to the characteristic interactions between the insulin-Fc fusion protein and the subject's body in terms of absorption, distribution, metabolism, and excretion. Specifically, PK here refers to the concentration of the insulin-Fc fusion protein in the subject's blood or serum at a given time after administration of the insulin-Fc fusion protein. As used herein, the term "half-life" refers to the time required for the concentration of the insulin-Fc fusion protein in the 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 a larger "half-life" value exhibit a longer duration of action in the target subject.

[0085] The terms “sequence identity,” “sequence homology,” “homology,” or “identical” in amino acid or nucleotide sequences as used herein describe that when specific adjacent segments of a variant’s nucleotide sequence or amino acid sequence are aligned and compared with a reference sequence’s nucleotide sequence or amino acid sequence, identical nucleotide or amino acid residues are found within the variant and the reference sequence. Methods for aligning sequences and determining identity between sequences are known in the industry, including the use of Clustal Omega to organize, align, and compare sequences for similarity, wherein the software highlights each sequence position, compares all sequences at that position, and assigns one of the following scores: "*" (asterisk) indicates a sequence position with a single fully conserved residue; ":" (colon) indicates conservation between very similar characteristic groups with a score of 0.5 or higher in the Gonnet PAM 250 matrix; "."" (period) indicates conservation between weakly similar characteristic groups with a score of 0.5 or lower in the Gonnet PAM 250 matrix; "-" (dash) indicates a sequence gap, which means that local homology does not exist within a specific range of sequences or within a specific comparison set; and an empty space " " indicates little or no sequence homology for a specific position across the compared sequences. For example, see 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 alignment of two nucleotide sequences, adjacent segments of the variant nucleotide sequence may have additional nucleotides or deleted nucleotides relative to the reference nucleotide sequence. Similarly, for the optimal alignment of two amino acid sequences, adjacent segments of the variant amino acid sequence may have additional amino acid residues or deleted amino acid residues relative to the reference amino acid sequence. In some embodiments, adjacent segments used for comparison with the reference nucleotide sequence or reference amino acid sequence will contain at least 6, 10, 15, or 20 adjacent nucleotides or amino acid residues, and may contain 30, 40, 50, 100 or more nucleotides or amino acid residues. Correction for increased sequence identity related to the inclusion of gaps in the nucleotide sequence or amino acid sequence of the variant may be made by assigning a gap penalty. Sequence alignment methods are known in the art.

[0086] In the embodiment, the determination of the percentage of identity or "homology" between two sequences is achieved using a mathematical algorithm. For example, the percentage of identity of amino acid sequences is

[0087] The Smith-Waterman homology search algorithm is determined using an affine 6-gap search with a BLOSUM matrix 62 having a gap opening penalty of 12 and a gap extension penalty of 2. 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 example, the percent identity of the nucleotide sequence is determined using a Smith-Waterman homology search algorithm with a gap opening penalty of 25 and a gap extension penalty of 5. The determination of such sequence identity can be performed, for example, using TimeLogic's DeCypher hardware accelerator.

[0088] As used herein, the term "homology" is used to compare two or more proteins by identifying common structural features and common spatial distributions, for example, beta strands, helices, and folds. Therefore, homologous protein structures are defined by spatial analysis. To measure structural homology, geometric-topological features of space must be calculated. One approach used to generate and analyze three-dimensional (3D) protein structures is homology modeling (also known as comparative modeling or knowledge-based modeling), which operates by finding similar sequences based on the fact that 3D similarity reflects 2D similarity. Homologous structures do not imply sequence similarity as a prerequisite.

[0089] As used herein, the terms “subject” and “patient” are intended to include dogs and cats. Exemplary dog ​​and cat subjects include dogs and cats having a disease or disorder, e.g., diabetes or other diseases or disorders described herein, or normal subjects.

[0090] As used herein, the terms “potency” or “yield” refer to the amount of fusion protein product (e.g., the insulin-Fc fusion protein described herein) produced from biosynthesis (e.g., in mammalian cells, e.g., HEK293 cells or CHO cells) per volume of cell culture. The amount of product may be determined at any stage of the production process (e.g., before or after purification), but the yield or potency is always specified per volume of the original cell culture. As used herein, the terms “product yield” or “total protein yield” refer to the total amount of insulin-Fc fusion protein expressed by cells and purified through one or more affinity chromatography steps (e.g., protein A or protein G), which 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 terms “percentage of homodimers” or “% homodimers” refer to the proportion of the fusion protein product that is the desired homodimer (e.g., the insulin-Fc fusion protein described herein). As used herein, the term “homodimer titer” refers to the product of % homodimers and total protein yield reported after the protein A purification step per cell culture volume.

[0091] As used herein, the terms “treat” or “treating” refer to a therapy for treating a subject with a disease or disorder, wherein at least one symptom of the disease or disorder is treated, cured, alleviated, relieved, altered, rescued, improved, or enhanced by applying a therapy of administration of a fusion protein, such as the fusion protein described herein, to the subject. Therapeutics include administering an amount effective in alleviating, relieving, altering, rescuing, improving, enhancing, or affecting the disease or disorder or the symptoms of the disease or disorder. Therapeutics may prevent the exacerbation or worsening of the symptoms of the disease or disorder.

[0092] Insulin-Fc Fusion Protein Components and Structure

[0093] The present disclosure relates to a composition of a fusion protein (i.e., an insulin-Fc fusion protein) comprising an insulin polypeptide linked to a species-specific Fc fragment via a peptide linker, and its use for the treatment of 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 one or more parts from, for example, other sources (other proteins, polypeptides, cells, etc.) which are covalently linked via peptide bonds. The insulin-Fc fusion protein is covalently linked by (i) linking the genes encoding each part into a single nucleic acid molecule, and (ii) expressing the protein encoded by the nucleic acid molecule in a host cell (e.g., HEK or CHO) as follows [(N-terminus)--insulin polypeptide--linker--Fc fragment--(C-terminus)]. A fully recombinant synthetic approach is preferred over a method of synthesizing the insulin polypeptide and the Fc fragment separately and then chemically conjugating them. The chemical bonding step and subsequent purification process increase manufacturing complexity, reduce product yield, and increase costs.

[0094] As used herein, the term "dimer" refers to a protein or fusion protein comprising two covalently linked polypeptides. In the example, two identical polypeptides are covalently linked (e.g., via disulfide bonds) to form a "homodimer" (schematically shown in FIG. 1). Disulfide bonds are shown as dashed lines in FIG. 1; in practice, the total number of disulfide bonds may be greater or less than the number shown in FIG. 1. In the example, the homodimer is encoded by a single nucleic acid molecule, wherein the homodimer is recombinantly produced inside the cell by first forming an insulin-Fc fusion protein monomer and then assembling two identical insulin-Fc fusion protein monomers into a homodimer during further processing inside the cell.

[0095] As used herein, the terms “multimer,” “multimeric,” or “multimeric state” refer to a non-covalent form of an Fc fusion protein dimer that can be in equilibrium with the Fc fusion protein dimer or can function as a permanently aggregated version of the Fc fusion protein dimer (e.g., a dimer of the Fc fusion protein homodimer, a trimer of the Fc fusion protein homodimer, a tetramer of the Fc fusion protein homodimer, or a higher-order aggregate containing five or more Fc fusion protein homodimers). The Fc fusion protein in a multimeric form can be expected to have different physical, stable, or pharmacological activities than the insulin-Fc fusion protein homodimer.

[0096] Insulin polypeptide

[0097] Insulin polypeptides may be insulin or insulin analogs produced by β-cells in the islets of Langerhans within the pancreas, for example. Insulin functions by regulating the uptake of glucose from the blood. Upon specific stimuli, such as increased protein and glucose levels, insulin is released from β-cells and binds to insulin receptors (IR), initiating a signaling cascade that affects various aspects of mammalian (e.g., human, dog, or cat) metabolism. Disruption of this process is directly associated with several diseases, such as diabetes, insulinoma, insulin resistance, metabolic syndrome, and polycystic ovary syndrome. Insulin analogs of the present disclosure may be related to the structure of insulin but may include one or more modifications. In some embodiments, insulin analogs include at least one amino acid substitution, deletion, addition, or chemical modification to insulin that may affect specific functions or characteristics of the insulin-Fc fusion protein. For example, modifications or alterations described herein may affect the structure, stability, pH sensitivity, bioactivity, or binding affinity of the insulin-Fc fusion protein to a cell surface receptor (e.g., insulin hormone receptor) compared to the reference standard.

[0098] The amino acid sequence of insulin is strongly conserved throughout evolution, particularly in 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 peptide segments of the insulin polypeptide exemplified in FIG. 1. Insulin is a 51-amino acid hormone comprising two peptide chains (i.e., B-chain and A-chain) connected by disulfide bonds (e.g., disulfide bonds formed by one or more B-chain cysteine ​​side-chain thiols and one or more A-chain cysteine ​​side-chain thiols). The A-chain of insulin is 21 amino acids long, and the B-chain of insulin is 30 amino acids long. In the natural form of insulin, the A-chain contains a single intrachain disulfide bond formed by two A-chain cysteine ​​side-chain thiols. For reference, the sequences for the human insulin A-chain of Sequence No. 1 and the human insulin B-chain of Sequence No. 2 are as follows:

[0099] FVNQHLCGSHLVEALYLVCGERGFFYTPKT(Sequence No.: 1)

[0100] GIVEQCCTSICSLYQLENYCN(Sequence No.: 2)

[0101] As used herein, the terms “insulin” or “insulin polypeptide” include mature insulin, preproinsulin, proinsulin, and naturally occurring insulin or analogs thereof. In the embodiments, the insulin polypeptide may be a full-length insulin polypeptide or a fragment thereof. In the embodiments, the insulin polypeptide may comprise one or more fragments from mature insulin, preproinsulin, proinsulin, or naturally occurring insulin.

[0102] Insulin is generally composed of an N-terminal-B-chain:C-chain:A-chain-C-terminal polypeptide, where the C-chain is cleaved to make it bioactive. For reference, the sequence of the entire human insulin molecule containing the C-chain (i.e., human proinsulin) is shown below with the C-chain underlined.

[0103] FVNQHLCGSHLVEALYLVCGERGFFYTPKT RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR GIVEQCCTSICSLYQLENYCN(Sequence No.: 3)

[0104] The conversion of single-chain insulin polypeptides into bioactive 2-chain polypeptides typically occurs within β-cells of the islets of Langerhans prior to glucose-stimulated insulin secretion, mediated by two endoproteases—type I endoproteases PC1 and PC3—that disrupt the C-peptide-B chain linkage and PC2, and a type II endoprotease that cleaves the C-peptide-A chain linkage at the exact right site. However, cellular systems used for the biosynthesis of therapeutic molecules such as insulin (e.g., bacterial, yeast, and mammalian (e.g., HEK and CHO) cell systems) do not possess this pathway; therefore, transformation must be performed after the expression and harvesting of single-chain polypeptides using chemical or enzymatic methods. All known techniques for cleaving the C-chain after expression and harvest rely on first modifying the C-chain so that it terminates at lysine just before the N-terminus of the A-chain. Next, using an enzyme selected from the trypsin or Lys-C family that specifically clips peptide bonds at the C-terminus of lysine residues, the single-strand insulin polypeptide is cleaved at the C-terminal lysine of the C-chain and at the C-terminal lysine at the 29th position from the N-terminus of the B-chain. In some cases, the resulting bioactive 2-strand insulin is used without reattaching the amino acid clipped at the 30th position from the N-terminus of the B-chain, and in other cases, the clipped amino acid is added back to the molecule at the 30th position from the N-terminus of the B-chain using additional enzymatic methods. This process works well with insulin because the entire 2-strand polypeptide form contains only a single lysine. However, since all known Fc fragments contain multiple lysine residues, this process cannot be used for the insulin-Fc fusion protein contained herein. Therefore, the enzymatic cleavage process will decompose the Fc fragment into non-functional parts, thereby eliminating the ability of the raw Fc fragment to prolong the action of the insulin polypeptide in vivo.Therefore, the insulin-Fc fusion protein of the present invention must include an insulin polypeptide that does not require C-chain cleavage and thus has bioactivity in a single-chain form.

[0105] Numerous bioactive single-chain insulin polypeptides are described in the industry. In all cases, the single-chain insulin polypeptide comprises a C-chain of a specific length and composition, as well as A-chains and B-chains mutated at specific amino acid sites, to achieve electrostatic balance, prevent aggregation, and enhance insulin receptor (IR) binding and / or downstream signaling, so as to achieve bioactivity similar to that of natural 2-chain insulin. Here, the mutation location in the peptide segment is indicated using the name of the segment (e.g., B-chain, C-chain, A-chain) and the number of amino acids calculated from the N-terminus of the segment. For example, the notation "B16" refers to the 16th amino acid from the N-terminus of the B-chain amino acid sequence. The notation "A8" refers to the 8th amino acid from the N-terminus of the A-chain. Additionally, if an amino acid is mutated from its original form to a new amino acid at a specific location, a one-letter amino acid code for the new amino acid is added at that location. For example, B16A means an alanine mutation at the 16th amino acid from the N-terminus of the B-chain amino acid sequence, and A8H means a histidine mutation at the 8th amino acid from the N-terminus of the A-chain amino acid sequence.

[0106] In one example, a single-chain insulin analog having additional substitutions in the C-chain of the sequence GGGPRR, and in the A-chain and B-chain (Sequence No. 4), was developed by The Department of Biochemistry, Case Western Reserve University School of Medicine, University of Chicago (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 of the A-chain (i.e., A8), histidine is substituted with threonine; at position 10 of the B-chain (i.e., B10), aspartic acid is substituted with histidine; at position 28 of the B-chain (i.e., B28), aspartic acid is substituted with proline; At position 29 of the B-chain (i.e., B29), proline is substituted with lysine. Sequence number 4 is listed below along with each underlined non-natural amino acid:

[0107] FVNQHLCGS D LVEALYLVCGERGFFYT DP T GGGPRR GIVEQCC H SICSLYQLENYCN(SEQ number: 4)

[0108] In the example, alanine can be substituted with tyrosine at position 16 (i.e., B16) from the N-terminus of the B-chain of SEQ No. 4 to produce SEQ No. 5, as alanine substitution at this position is known to be less activating of 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 No. 5 is listed below along with each underlined non-natural amino acid:

[0109] FVNQHLCGS D LVEAL A LVCGERGFFYT DP T GGGPRR GIVEQCC H SICSLYQLENYCN(SEQ number: 5)

[0110] In some embodiments, it was unexpectedly discovered that specific amino acids of SEQ NO: 4 and SEQ NO: 5 induce the occurrence of a phenomenon that neutralizes anti-drug antibodies after repeated subcutaneous injections into target animals (e.g., dogs or cats). The anti-drug antibodies caused an unacceptable reduction in NAOC after multiple injections (e.g., an NAOCR value of less than 0.5 after the third injection) and rendered the associated insulin-Fc fusion protein viable. Specifically, it was discovered at the stage leading to the disclosure of the present invention that the A8 mutation for histidine and the B10 mutation for aspartic acid accounted for the majority of the anti-drug antibody specificity and thus exhibited immunogenic "hot spots" (e.g., immunogenic epitopes) on the insulin polypeptide. Accordingly, in a preferred embodiment, the insulin polypeptide does not contain histidine at position A8 of the insulin polypeptide or aspartic acid at position B10.

[0111] In one embodiment, simply retaining the A8 and B10 amino acids as natural threonine and histidine, respectively, eliminated the anti-drug antibody response, but the resulting insulin-Fc fusion protein was found to be inactive in the target species (e.g., NAOC less than 150% FBGL·days·kg / mg). Therefore, it was necessary to experiment with various A-chain, B-chain, and C-chain modifications to find a suitable solution. Most variants failed to achieve homodimeric titers of 50 mg / L or higher, and many variants that met this objective did not reach acceptable levels of bioactivity in the target species (e.g., acceptable NAOC values ​​of 150% FBGL·days·kg / mg). After screening over 120 variants, the following insulin polypeptide of sequence number: 6_NULL was considered suitable for achieving an allodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg / mg or greater in the target species, minimal immunogenicity, and a third-injection NAOCR value greater than 0.5 of the associated insulin-Fc fusion protein in the target species (non-natural amino acids are underlined, and natural amino acids marked with an underlined Z are deleted):

[0112] FVNQHLCGS X 1 LVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYC X 3 (Sequence number: 6_NULL)

[0113] Here, X1 is not D, X2 is not H, and X3 is absent or N.

[0114] In a specific embodiment, in SEQ ID NO: 6_NULL, X1 is H, X2 is T, and X3 is absent or N to produce the following SEQ ID NO: 7_NULL (non-natural amino acids are underlined, and natural amino acids indicated by an underlined Z are deleted):

[0115] FVNQHLCGSHLVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYC X 3 (Sequence number: 7_NULL)

[0116] Here, X3 is absent or N.

[0117] In a specific embodiment, in SEQ ID NO: 7_NULL, X3 is absent to produce the following SEQ ID NO: 8_NULL (non-natural amino acids are underlined, and natural amino acids indicated by an underlined Z are deleted):

[0118] FVNQHLCGSHLVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYC Z (Sequence number: 8_NULL)

[0119] In a specific embodiment, in sequence number: 7_NULL, X3 is N, and consequently, the following sequence number: 9_NULL (non-natural amino acids are underlined, and natural amino acids indicated by an underlined Z are deleted):

[0120] FVNQHLCGSHLVEAL E LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYCN(Sequence No.: 9_NULL)

[0121] In some embodiments, the Fc fragment was mutated to prevent glycosylation during synthesis and potentially reduce the immunogenicity of the insulin-Fc fusion protein produced in target animals (e.g., dogs or cats). Unexpectedly, it was discovered that in order to sufficiently produce an insulin-Fc fusion protein (e.g., a homodimeric titer of 50 mg / L or greater), and to achieve non-immunogenicity with an NAOC value of 150% FBGL·days·kg / mg or greater in the target species and an NAOCR value greater than 0.5 after the third injection in the target species, an interaction exists between the insulin polypeptide and the mutated Fc fragment, requiring another amino acid mutation in the insulin polypeptide. Specifically, it was discovered that mutating the B16 amino acid to alanine on the insulin polypeptide is required when it is linked to a specific, mutated, non-glycosylated Fc fragment, resulting in the following insulin polypeptide sequence number: 10_NULL (non-natural amino acids are underlined, and natural amino acids indicated by an underlined Z are deleted):

[0122] FVNQHLCGS X 1 LVEAL A LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYC Z (Sequence number: 10_NULL)

[0123] Here, X1 is not D, and X2 is not H.

[0124] In a specific embodiment, in SEQ ID NO: 10_NULL, X1 is H and X2 is T to produce the following SEQ ID NO: 11_NULL (non-natural amino acids are underlined, and natural amino acids indicated by an underlined Z are deleted):

[0125] FVNQHLCGSHLVEALA LVCGERGF H Y ZZZZGGGGGGSGGGG GIVEQCCTS T CSL D QLENYC Z (Sequence number: 11_NULL)

[0126] The following is a reconstruction of the presented sequence without the amino acid of the symbol Z removed from the insulin polypeptide sequence notation. Again, in all cases, non-natural amino acids are underlined. To avoid confusion, each original sequence containing the Z symbol is listed above the new sequence from which the Z symbol has been removed. Despite the two separate notations, the paired sequences represent exactly the same insulin polypeptide.

[0127] Sequence number: 6_NULL is re-displayed as follows:

[0128] FVNQHLCGS X 1 LVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYC X 3 (Sequence Number: 6)

[0129] Here, X1 is not D, X2 is not H, and X3 is absent or N.

[0130] Sequence number: 7_NULL is re-displayed as follows:

[0131] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYC X (Sequence Number: 7)

[0132] Here, X3 is absent or N.

[0133] Sequence number: 8_NULL is re-displayed as follows:

[0134] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYC (Sequence No.: 8)

[0135] Sequence number: 9_NULL is re-displayed as follows:

[0136] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYCN(Sequence No.: 9)

[0137] Sequence number: 10_NULL is re-displayed as follows:

[0138] FVNQHLCGS X 1 LVEAL A LVCGERGF H Y GGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYC (Sequence No.: 10)

[0139] Here, X1 is not D, and X2 is not H.

[0140] Sequence number: 11_NULL is re-displayed as follows:

[0141] FVNQHLCGSHLVEAL A LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYC (Sequence No.: 11)

[0142] Linker

[0143] The successful construction of a recombinant insulin-Fc fusion protein requires a linker that connects the insulin polypeptide to the Fc fragment. In the examples, the insulin-Fc fusion protein described herein comprises a peptide linker between the insulin polypeptide and an Fc fragment containing amino acids (e.g., natural or non-natural amino acids). In the examples, the peptide linker may be encoded by a nucleic acid molecule, for example, so that a single nucleic acid molecule can encode the peptide linker and the Fc fragment as well as various peptides within the insulin polypeptide. The selection of the peptide linker (e.g., length, composition, hydrophobicity, and secondary structure) may affect manufacturability (i.e., homodimeric titer), chemical and enzymatic stability, bioactivity (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 in the design of insulin-Fc fusion proteins for the purpose of improving homodimeric potency and bioactivity.

[0144]

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

[0146] GGGGAGGGG(Sequence No.: 12).

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

[0148] GGGGSGGGG(Sequence No.: 13).

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

[0150] GGGGGQGGGGQGGGGQGGGGG(Sequence No.: 14).

[0151] When constructing recombinant insulin-Fc fusion proteins using a peptide linker such as SEQ No. 14, attention must be paid to the potential for unwanted enzymatic cleavage between the C-terminus of the insulin A-chain and the N-terminus of the peptide linker. Cleavage of the linker and the Fc-fragment in the insulin polypeptide will prevent the insulin-Fc fusion protein from providing extended duration bioactivity. Known enzymatic cleavage sites exist between the asparagine-glycine bonds (Vlasak, J., Ionescu, R., (2011) MAbs Vol. 3, No. 3 pp 253-263). In many peptide linker embodiments containing the preferred peptide linker of SEQ No. 14, the N-terminal amino acid is glycine. Furthermore, the C-terminus of the insulin A-chain (i.e., the 21st amino acid from the N-terminus of the A-chain (i.e., A21)) is asparagine. Therefore, to eliminate the potentially enzymatically cleavable asparagine-glycine bond that would form between the A-chain and the peptide linker, A21 asparagine is omitted from the insulin polypeptides of SEQ No. 8, SEQ No. 10, and SEQ No. 11. Unexpectedly, an insulin-Fc fusion protein constructed from the insulin polypeptide of SEQ No. 9, which retains asparagine at the C-terminus of the A-chain, demonstrates feasibility in mammalian cells with an acceptable homodimeric titer (i.e., homodimeric titer greater than 50 mg / L), acceptable bioactivity in vivo (i.e., NAOC greater than 150% FBGL·days·kg / mg in target animals), and sustained bioactivity levels after multiple administrations (i.e., NAOCR value greater than 0.5 after a 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 the insulin-Fc fusion protein containing an asparagine-glycine link between the insulin polypeptide and the peptide linker, at least for the insulin-Fc fusion protein containing the Fc fragment sequence disclosed herein.

[0152] Fc short story

[0153] The terms “Fc fragment,” “Fc region,” “Fc domain,” or “Fc polypeptide” are used herein to define the C-terminal region of an immunoglobulin heavy chain. The Fc fragment, region, domain, or polypeptide may be a natural sequence Fc region or a variant / mutant Fc region. The boundaries of the Fc region of the immunoglobulin heavy chain may vary, but generally include part 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 the dog or cat Fc fragment connects the CH1 domain of the heavy chain to the CH2 region of the heavy chain and includes an amino acid sequence containing one or more cysteines that forms one or more inter-heavy chain disulfide bridges, thereby forming a homodimer of the Fc fusion protein from two identical but distinct monomers of the Fc fusion protein. The hinge region may include all or part of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence.

[0154] Fc receptors (FcRs) refer to receptors that bind to the Fc region of an Fc fragment or antibody. In the examples, FcRs are natural sequences of dog or cat FcRs. In the examples, FcRs bind to the Fc region (gamma receptor) of an Fc fragment or IgG antibody and include, without limitation, Fc(gamma) receptor I, Fc(gamma) receptor IIa, Fc(gamma) receptor IIb, and Fc(gamma) receptor III subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). "FcRs" also include neonatal receptor FcRn, which is responsible for the transfer of maternal IgG molecules to the fetus (Guyer et al., 1976 J. Immunol., 117:587; and Kim et al., 1994 J. Immunol., 24:249) and also for the extended in vivo clearance half-life of antibodies and Fc-fusion proteins. In the example, FcRs of human origin are used in vitro (e.g., in an analysis) to measure the binding of an insulin-Fc fusion protein containing an Fc fragment of canine or cat origin to evaluate their FcR binding characteristics. Those skilled in the art will understand that mammalian FcRs from one species (e.g., FcRs of human origin) may sometimes bind to an Fc fragment of a second species (e.g., FcRs of canine or cat origin) in vitro. In the example, FcRs of canine origin are used in vitro (e.g., in an analysis) to measure the binding of an insulin-Fc fusion protein containing an Fc fragment of canine or cat origin to evaluate their FcR binding characteristics. Those skilled in the art will understand that a mammalian FcR from one species (e.g., an FcR of canine origin) can in vitro bind an insulin-Fc fusion protein containing an Fc fragment of the same species (e.g., canine origin) and, from time to time, an insulin-Fc fusion protein containing an Fc fragment of another mammalian species (e.g., cat origin).

[0155] In the example, the Fc fragment comprises the Fc region of mammalian IgG (e.g., hinge region, CH2 domain and CH3 domain), e.g., canine IgGA Fc fragment (SEQ No.: 15), canine IgGB Fc fragment (SEQ No.: 16), canine IgGC Fc fragment (SEQ No.: 17), or canine IgGD Fc fragment (SEQ No.: 18) or feline IgG1a fragment (SEQ No.: 19), feline IgG1b Fc fragment (SEQ No.: 20), or feline IgG2 Fc fragment (SEQ No.: 21). In the example, the C-terminal lysine, which is often found in the natural dog or cat IgG isotype Fc fragment amino acid sequence (i.e., lysine representing the last amino acid of the Fc fragment sequence), is omitted to prevent the accidental production of unwanted amino acid sequence variants during manufacturing (e.g., when the C-terminal lysine is omitted, the Fc fragment containing the C-terminal lysine becomes mixed with the Fc fragment, which can occur during the production of the desired protein within the cell) (Dick, LW., (2008) Biotechnol Bioeng. Aug 15; 100(6) pp1132-43). Accordingly, in the example, the dog and cat Fc fragment sequences lacking C-terminal lysine are as follows:

[0156] RCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTI SKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG(sequence Number: 15)

[0157] DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 16)

[0158] CNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG(서열 번호: 17)

[0159] CISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG(서열 번호: 18)

[0160] DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(서열 번호: 19)

[0161] DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(서열 번호: 20)

[0162] GEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(서열 번호: 21)

[0163] Replacing human Fc with canine IgGA is desirable for minimizing any unwanted immunogenicity in dogs because the Fc (gamma) effector function of the IgGA isotype in dogs is lacking (it is very similar to the human IgG2 isotype in humans). However, in an example comprising the insulin polypeptide of SEQ No. 5 and the peptide linker of SEQ No. 12, it was unexpectedly discovered that the insulin-Fc fusion protein containing the canine IgGA fragment (SEQ No. 15) was highly aggregated to a low titer of the desired homodimer (i.e., a homodimer titer of less than 50 mg / L). Furthermore, this compound was non-biologically active in dogs due to high aggregation levels (e.g., low % homodimer) (i.e., the NAOC value was less than 150% FBGL·days·kg / mg). Despite mutating the insulin polypeptide of SEQ No. 5, the canine IgGA Fc fragment (SEQ No. 15), and / or the linker, there were no examples based on the canine IgGA Fc fragment that possessed sufficiently low levels of aggregation and sufficiently high titers of the desired homodimer. Meanwhile, replacing the canine IgGA Fc fragment (SEQ No. 15) with the canine IgGB Fc fragment (SEQ No. 16) produced a much less aggregated compound with relatively high titers of the desired homodimer. Furthermore, the compound containing the insulin polypeptide of SEQ No. 5 and the canine IgGB Fc fragment (SEQ No. 16) was bioactive in dogs and exhibited glucose-lowering bioactivity over several days (i.e., the NAOC value was greater than 150% FBGL·days·kg / mg).

[0164] A preference for the canine IgGB Fc fragment over the canine IgGA Fc fragment was confirmed in an example comprising the insulin polypeptide of SEQ No. 8 and the peptide linker of SEQ No. 14, both of which differ significantly from the insulin polypeptide of SEQ No. 5 and the peptide linker of SEQ No. 12. Insulin-Fc fusion proteins comprising the insulin polypeptide of SEQ No. 8 and the peptide linker of SEQ No. 14 were synthesized using Fc fragments from canine IgGA (SEQ No. 15), canine IgGB (SEQ No. 16), canine IgGC (SEQ No. 17), or canine IgGD (SEQ No. 18) immunoglobulins. Using conventional purification methods, only compounds containing canine IgGA and canine IgGB exhibited distinct protein yields. However, as before, the canine IgGA version of the compound exhibited high aggregation with low levels of bioactivity, whereas the canine IgGB version of the compound showed low aggregation (i.e., high % homodimer), high titers of the desired homodimer (i.e., homodimer titers greater than 50 mg / L), and significant levels of long-term glucose-reducing bioactivity in dogs (i.e., NAOC values ​​greater than 150% FBGL·days·kg / mg). Using an alternative purification method, the canine IgGC version of the compound was recovered with low levels of aggregation, but due to low affinity for the FcRn receptor, it exhibited only minimal bioactivity in dogs (i.e., NAOC values ​​less than 150% FBGL·days·kg / mg). Therefore, regarding the canine-specific product, canine IgGB (Sequence No. 16) is the preferred Fc fragment for all insulin-Fc fusion proteins used in dogs, regardless of the choice of insulin polypeptide.

[0165] Replacing human Fc with feline IgG2 is desirable for minimizing any unwanted immunogenicity in cats because the Fc (gamma) effector function of the IgG2 isotype in cats is lacking (which is very similar to the human IgG2 isotype in humans). Unlike in the case of dogs, in the example containing the insulin polypeptide of SEQ No. 4, it was found that the insulin-Fc fusion protein containing the feline IgG2 fragment (SEQ No. 21) and the feline IgG1b fragment (SEQ No. 21) exhibited a similarly high yield while having low aggregation (i.e., a homodimeric titer of 50 mg / L or higher) and significant insulin receptor affinity (i.e., an insulin receptor IC50 value of less than 5000 nM). However, unexpectedly, when the insulin polypeptide was changed to SEQ NO: 7, the insulin-Fc fusion protein containing the feline IgG2 fragment (SEQ NO: 21) was inactive in cats (i.e., the NAOC was less than 150% FBGL·days·kg / mg), whereas the insulin-Fc fusion protein containing the feline IgG1b fragment (SEQ NO: 20) exhibited low aggregation (i.e., high % homodimer), high titers of the desired homodimer (i.e., homodimer titers greater than 50 mg / L), and significant levels of long-term glucose-lowering bioactivity (i.e., NAOC values ​​greater than 150% FBGL·days·kg / mg) in cats. Therefore, with respect to the cat-specific product, when the insulin polypeptide sequence contains SEQ NO: 7, the feline IgG1b fragment (SEQ NO: 20) is the preferred Fc fragment.

[0166] Given that canine IgGB and feline IgG1b isotypes interact with their respective species-specific Fc(gamma) receptors with higher affinity than their canine IgGA and feline IgG2 isotype counterparts, there may or may not be a risk of unwanted immunogenicity after repeated injections. One method to reduce Fc(gamma) interactions involves deglycosylation of the Fc fragment during synthesis in host cells or preventing glycosylation. Each IgG fragment contains a conserved asparagine (N)-glycosylation site in the CH2 domain of each heavy chain of the Fc region. Here, the notation used to refer to the conserved N-glycosylation site is "cNg". One method to remove attached glycans from the synthesized insulin-Fc fusion protein is to mutate the cNg site during production in host cells to completely prevent glycan attachment. Here, the notation used to describe the cNg mutation is cNg-(substituted amino acid). For example, if the asparagine in the cNg site is mutated into serine, this mutation is denoted as "cNg-S".

[0167] The absolute position of the cNg site from the N-terminus of the B-chain of the insulin-Fc fusion protein depends on the length of the insulin polypeptide, the length of the linker, and any omitted amino acids in the Fc fragment preceding the cNg site. Here, the notation used to designate the absolute position of the cNg site in a given insulin-Fc fusion protein sequence (as calculated from the N-terminus of the B-chain of the insulin-Fc fusion protein) is “NB(number)”. For example, if the cNg site is found at the 151st amino acid position calculated from the N-terminus of the B-chain, the absolute position of this site is designated as cNg-NB151. As an additional example, if the cNg site is found at the 151st amino acid position calculated from the N-terminus of the B-chain and the asparagine at this site is mutated to serine, this mutation is designated as “cNg-NB151-S”.

[0168] In an example comprising the insulin polypeptide of SEQ No. 5 and canine IgGB Fc fragments having cNg-Q, cNg-S, cNg-D, and cNg-K mutations, it was unexpectedly found that only compounds containing cNg-K and cNg-S mutations exhibited essential homodimeric titers of 50 mg / L or higher and the lowest Fc(gamma)RI binding affinity. Meanwhile, in an example comprising the insulin polypeptide of SEQ No. 8 and canine IgGB Fc fragments having cNg-S mutations, it was unexpectedly found that the resulting compounds were significantly less bioactive in dogs compared to their natural canine IgGB Fc-containing counterparts (i.e., NAOC values ​​were significantly lower for counterparts containing natural glycosylation site amino acids, e.g., cNg-N). When the B16 amino acid was mutated to alanine as described above for insulin polypeptide sequence number: 11, bioactivity was unexpectedly restored in the cNg-S mutant (i.e., the NAOC value increased significantly). Taken together, there was an unexpected and significant interaction between the selection of the cNg mutation and the insulin polypeptide composition, and consequently, experiments were required to confirm a preferred embodiment. In a specific embodiment, a canine IgGB Fc mutant containing the cNg-S mutation is preferred, and the sequence having the underlined cNg-S is indicated as follows:

[0169] DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 22)

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

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

[0172] Insulin-Fc fusion protein

[0173] An insulin-Fc fusion protein comprising an insulin polypeptide, an Fc fragment, and a linker between the insulin polypeptide and the Fc fragment is provided herein. In an example, the insulin polypeptide comprises a domain extending from the N-terminus to the C-terminus: (N-terminus)--B-chain--C-chain--A-chain--(C-terminus). In an example, the insulin polypeptide is located on the N-terminal side of the Fc fragment. In an example, the fusion protein comprises a domain extending from the N-terminus C to the 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)), as illustrated in FIG. 1:

[0174] In a preferred embodiment, the preferred non-immunogenic, bioactive insulin polypeptide of SEQ NO: 6 is combined with the preferred canine IgGB Fc fragment of SEQ NO: 16 using the preferred linker of SEQ NO: 14 to produce a family of highly homodimeric titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion proteins of SEQ NO: 24, which exhibits a homodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg or greater in dogs, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ NO: 24 with non-natural amino acids underlined:

[0175] FVNQHLCGS X 1 LVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYC X 3 GGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKA LPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 24)

[0176] Here, X1 is not D, X2 is not H, and X3 is absent or N.

[0177] In a preferred embodiment comprising SEQ NO: 24, X1 is H, X2 is T, and X3 is absent or N. The choice produces a highly homodimeric titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ NO: 25, which exhibits a homodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg or greater in dogs, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ NO: 25 with non-natural amino acids underlined:

[0178] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYC X 3 GGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKA LPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 25)

[0179] Here, X3 is absent or N.

[0180] In a preferred embodiment, X3 is absent at SEQ NO: 25 to produce a highly homodimeric titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ NO: 32, which exhibits a homodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg or greater in dogs, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ NO: 32 with non-natural amino acids underlined:

[0181] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVN NKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 32)

[0182] In a preferred embodiment, X3 is N in SEQ NO: 25 to produce a highly homodimeric titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ NO: 34, which exhibits a homodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg or greater in dogs, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ NO: 34 with non-natural amino acids underlined:

[0183] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKV NNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 34)

[0184] In a preferred embodiment, the preferred non-glycosylated, cNg-S mutant canine IgGB Fc fragment of SEQ No. 22 is combined with the preferred B16A mutant insulin polypeptide sequence of SEQ No. 10 using the preferred linker of SEQ No. 10 to produce the highly homodimeric titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein family of SEQ No. 26, which exhibits a homodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg or greater in dogs, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ No. 25 with non-natural amino acids underlined:

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

[0186] Here, X1 is not D, and X2 is not H.

[0187] In a preferred embodiment, X1 in SEQ NO: 26 is H and X2 is T, thereby producing a highly homodimeric titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ NO: 36, which exhibits a homodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg or greater in dogs, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ NO: 36 with non-natural amino acids underlined:

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

[0189] In a preferred embodiment, the preferred non-immunogenic, bioactive insulin polypeptide of SEQ No.: 6, which is absent from X3, is combined with the preferred feline IgG1b Fc fragment of SEQ No.: 20 using the preferred linker of SEQ No.: 14 to produce the highly homodimeric titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein family of SEQ No.: 27, which exhibits a homodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg or greater in cats, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in cats. The following shows SEQ No.: 27 with non-natural amino acids underlined:

[0190] FVNQHLCGS X 1 LVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCC X 2 S T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVN SKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG Number: 27)

[0191] Here, X1 is not D, and X2 is not H.

[0192] In a preferred embodiment, in SEQ NO: 27, X1 is H and X2 is T, thereby producing a highly homodimeric titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ NO: 38, which exhibits a homodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg or greater in cats, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in cats. The following shows SEQ NO: 38 with non-natural amino acids underlined:

[0193] FVNQHLCGSHLVEAL E LVCGERGF H Y GGGGGGSGGGG GIVEQCCTS T CSL D QLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVN SKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG Number: 38)

[0194] In a preferred embodiment, the preferred non-glycosylated, cNg-S mutant feline IgG1b Fc fragment of SEQ No. 23 is combined with the preferred B16A mutant insulin polypeptide sequence of SEQ No. 10 using the preferred linker of SEQ No. 10 to produce the highly homodimeric titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein family of SEQ No. 28, which exhibits a homodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg or greater in cats, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in cats. The following shows SEQ No. 28 with non-natural amino acids underlined:

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

[0196] Here, X1 is not D, and X2 is not H.

[0197] In a preferred embodiment, in SEQ NO: 28, X1 is H and X2 is T, thereby producing a highly homodimeric titer-producing, non-aggregating, bioactive, non-immunogenic insulin-Fc fusion protein of SEQ NO: 40, which exhibits a homodimeric titer of 50 mg / L or greater, an NAOC value of 150% FBGL·days·kg or greater in cats, and an NAOCR value greater than 0.5 after the third injection in a series of repeated injections in cats. The following shows SEQ NO: 40 with non-natural amino acids underlined:

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

[0199] In some embodiments, the insulin-Fc fusion 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, for example, a leader sequence at the N-terminus. An exemplary leader sequence includes the amino acid sequence MEWSWVFLFFLSVTTGVHS (Sequence No.: 30). In some embodiments, the insulin-Fc fusion protein described herein is encoded by a nucleic acid molecule containing the leader sequence for expression (e.g., recombinant expression) in a cell (e.g., eukaryotic, e.g., mammalian cell). In certain embodiments, the leader sequence is cleaved during expression, for example, in a cell culture. An exemplary nucleic acid sequence encoding the leader sequence includes the following nucleic acid sequence:

[0200] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcc (SEQ ID NO: 29).

[0201] Additionally, nucleic acid sequences (e.g., cDNA) encoding insulin-Fc fusion proteins of sequence numbers: 032, 034, 036, 038 and 040 are disclosed herein.

[0202] In an example containing the insulin-Fc fusion protein of SEQ ID NO: 32, the nucleic acid sequence (leader sequence is underlined) is as follows:

[0203] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(서열 번호: 31)

[0204] In an example containing the insulin-Fc fusion protein of SEQ ID NO: 34, the nucleic acid sequence (leader sequence is underlined) is as follows:

[0205] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcaacggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(서열 번호: 33)

[0206] In an example containing the insulin-Fc fusion protein of SEQ ID NO: 36, the nucleic acid sequence (leader sequence is underlined) is as follows:

[0207] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(서열 번호: 35)

[0208] In an example containing the insulin-Fc fusion protein of SEQ ID NO: 38, the nucleic acid sequence (leader sequence is underlined) is as follows:

[0209] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcaacagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag(서열 번호: 37)

[0210] In an example containing the insulin-Fc fusion protein of SEQ ID No. 40, the nucleic acid sequence (leader sequence is underlined) is as follows:

[0211] atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcagcagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgacccagagcccgggatag(서열 번호: 39)

[0212] Insulin-Fc fusion protein production

[0213] In the examples, the fusion protein can be expressed by the cell as described in more detail in the example section.

[0214] Expression and Purification

[0215] In the examples, the insulin-Fc fusion protein may be recombinantly expressed in, for example, eukaryotic cells, for example, mammalian cells or non-mammalian cells. Exemplary mammalian cells used for expression include HEK cells (e.g., HEK293 cells) or CHO cells. CHO cells may be subdivided into various strains or subclasses (e.g., CHO DG44, CHO-M, and CHO-K1), and some of these cell strains may be genetically engineered for optimal use with specific types of nucleic acid molecules (e.g., vectors containing DNA) or specific cell growth medium compositions as described in the examples section. In the examples, cells are transfected with a nucleic acid molecule (e.g., a vector) encoding the insulin-Fc fusion protein (e.g., the entire insulin-Fc fusion protein is encoded by a single nucleic acid molecule). In the example, HEK293 cells are transfected with a vector encoding an insulin-Fc fusion protein, but only result in transient expression of the insulin-Fc fusion protein for a certain period (e.g., 3, 4, 5, 7, 10, 12, 14 days or more) before the host cells cease expressing a significant level of the insulin-Fc fusion protein (i.e., transient transfection). HEK293 cells transiently transfected with a nucleic acid sequence encoding an insulin-Fc fusion protein often enable faster production of the recombinant protein, which facilitates the creation and screening of multiple insulin-Fc fusion protein candidates. In the example, CHO cells are transfected with a vector that permanently integrates into the host cell DNA and induces consistent and permanent expression of the insulin-Fc fusion protein (i.e., stable transfection) as long as the cells are properly cultured.CHO cells and CHO cell lines stably transfected with nucleic acids encoding an insulin-Fc fusion protein often take longer to develop but often produce higher protein yields and enable easier manufacturing of low-cost products (e.g., products for use in the veterinary pharmaceutical market). Cells and cell lines can be cultured using standard methods of the industry. In a preferred embodiment, HEK cells containing any one of the cDNA sequences having SEQ 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 Nos. 31, 33, 35, 37, and 39 are used to express the insulin-Fc fusion protein.

[0216] In some embodiments, the insulin-Fc fusion protein is purified or separated from the cell (e.g., by lysis of the cell). In other embodiments, the insulin-Fc fusion protein is purified or separated from the cell culture medium in which the cell is secreted by the cell and in which the cell grows. Purification of the insulin-Fc fusion protein may involve using column chromatography (e.g., affinity chromatography) or other separation methods based on differences in size, charge, and / or affinity for a specific molecule. In an embodiment, purification of the insulin-Fc fusion protein involves selecting or concentrating the protein containing the Fc fragment by using, for example, protein A beads or a protein A column, which causes the protein containing the Fc fragment to bind to protein A covalently bound to the protein A beads with high affinity at a neutral solution pH. The bound insulin-Fc fusion protein may be eluted from the protein A beads by a change in solution variables (e.g., a decrease in solution pH). Other separation methods, such as ion exchange chromatography and / or gel filtration chromatography, may also be used alternatively or additionally. In the examples, the purification of the insulin-Fc fusion protein further comprises filtration or centrifugation of the protein preparation. In the examples, further purification of the insulin-Fc fusion protein includes dialysis filtration through porous membranes of various sizes, ultrafiltration and filtration, as well as final formulation with excipients.

[0217] Purified insulin-Fc fusion proteins can be characterized for, for example, purity, protein yield, structure, and / or activity using various methods, e.g., absorbance at 280 nm (e.g., to determine protein yield), size-exclusion or capillary electrophoresis (e.g., to determine molecular weight, aggregation percentage, and / or purity), mass spectrometry (MS) and / or liquid chromatography (LC-MS) (e.g., to determine purity and / or glycosylation), and / or ELISA (e.g., to determine the degree of binding to an anti-insulin antibody, e.g., affinity). Exemplary characterization methods are also described in an example section.

[0218] In the example, the protein yield of the insulin-Fc fusion protein produced from transiently transfected HEK cells and Protein A purification is greater than 5 mg / L, 10 mg / L, or 20 mg / L. In the preferred example, the protein yield of the insulin-Fc fusion protein produced from transiently transfected HEK cells and Protein A purification is 50 mg / L or higher (e.g., 60 mg / L or higher, 70 mg / L or higher, 80 mg / L or higher, 90 mg / L or higher, 100 mg / L or higher). In the example, the % isodimer of the insulin-Fc fusion protein produced from transiently transfected HEK cells and Protein A purification is 70% or higher (e.g., 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher). In the example, the isomer titer of the insulin-Fc fusion protein produced after transiently transfected HEK cells and protein A purification, calculated as the product between the insulin-Fc fusion protein yield and % isomer, is 50 mg / L or higher (e.g., 60 mg / L or higher, 70 mg / L or higher, 80 mg / L or higher, 90 mg / L or higher, 100 mg / L or higher). Only candidates having an isomer titer of 50 mg / L or higher were considered useful in the present invention, because experience shows that isomer titers below this level make it impossible to obtain commercially viable titers in CHO cells that meet the strictly low manufacturing cost requirements for veterinary products.

[0219] In an example, the protein yield of the insulin-Fc fusion protein produced from stably transfected CHO cells (e.g., CHO cell line or CHO cell clone) and Protein A purification is 100 mg or more of insulin-Fc fusion protein per L (e.g., mg / L of culture medium). In a preferred example, the protein yield of the insulin-Fc fusion protein produced from stably transfected CHO cells (e.g., CHO cell line or CHO cell clone) and Protein A purification is 150 mg or more of insulin-Fc fusion protein / L of culture medium (e.g., 200 mg / L or more, 300 mg / L or more, 400 mg / L or more, 500 mg / L or more, 600 mg / L or more). In the example, the % homodimer of the insulin-Fc fusion protein after production from stably transfected CHO cells (e.g., CHO cell line or CHO cell clone) and Protein A purification is 70% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more). In the example, the homodimer titer of the insulin-Fc fusion protein after production from stably transfected CHO cells (e.g., CHO cell line or CHO cell clone) and Protein A purification, calculated as the product between the insulin-Fc fusion protein yield and the % homodimer, is 150 mg / L or more (e.g., 200 mg / L or more, 300 mg / L or more, 400 mg / L or more, 500 mg / L or more, 600 mg / L or more).

[0220] Functional characteristics of insulin-Fc fusion protein

[0221] A method for interacting with an insulin receptor to lower blood glucose in companion animals (e.g., dogs or cats) is described herein, wherein the method comprises administering an insulin-Fc fusion protein, e.g., the fusion protein described herein, to a subject. In some embodiments, the subject is diagnosed with diabetes mellitus (e.g., canine diabetes or feline diabetes).

[0222] In the examples, the insulin-Fc fusion protein described herein binds to an insulin receptor with significant affinity as measured by IC50 (e.g., IC50 of less than 5000 nM, IC50 of less than 4000 nM, IC50 of less than 3000 nM, IC50 of less than 2500 nM) in the 4°C IM-9 insulin receptor binding assay described in Example 7. Based on experience, only compounds exhibiting an insulin receptor activity IC50 value of less than 5000 nM were considered to exhibit bioactivity in the target species. Generally, higher affinity insulin receptor binding (i.e., lower IC50 value) is desirable. However, it is widely known that the clearance of insulin and insulin analogs (e.g., the insulin polypeptide described herein) is regulated primarily through insulin receptor internalization and intracellular degradation following binding to the insulin receptor. Therefore, insulin-Fc fusion proteins with too high an insulin receptor binding affinity (i.e., too low an IC50) are removed from circulation too quickly, which can result in a shorter duration of the desired period of bioactivity for lowering glucose in target animals.

[0223] In the embodiments, the insulin-Fc fusion protein described herein can lower glucose levels (e.g., blood glucose levels) in a subject after administration. In the embodiments, the glucose-lowering activity of the insulin-Fc fusion protein is greater than that of the insulin reference standard. In some embodiments, the duration of activity of the insulin-Fc fusion protein may be measured by a decrease in fasting blood glucose levels compared to the fasting blood glucose level before administration, e.g., a statistically significant decrease. In the embodiments, the duration of activity of the insulin-Fc fusion protein (e.g., the time during which the fasting blood glucose level in the subject decreases statistically significantly compared to the level before administration) is longer than about 2 hours. In the embodiments, the duration of activity of the insulin-Fc fusion protein (e.g., the time during which the blood glucose level in the subject decreases statistically significantly compared to the level before administration) is 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 more, or longer. In the example, the insulin-Fc fusion protein is long-lasting (e.g., has a long half-life in serum).

[0224] In an example, the serum half-life of the insulin-Fc fusion protein in a target animal (e.g., a dog or a cat) is longer than that of the insulin reference standard or control formulation. In an example, the serum half-life of the insulin-Fc fusion protein (e.g., in the subject's blood at the time of administration) in a target animal (e.g., a dog or a cat) is longer than about 2 hours. In an example, the serum half-life of the insulin-Fc fusion protein in a target animal (e.g., a dog or a cat) is about 0.5 days, 1 day, 2 days, or 2.5 days. In a preferred example, the serum half-life of the insulin-Fc fusion protein in a target animal (e.g., a dog or a cat) is about 3 days or more.

[0225] In the example, the combination of efficacy and duration of bioactivity can be quantified by calculating the area under the % fasting blood glucose (% FBGL) curve normalized to a given dose of mg / kg (NAOC) in units of % FBGL·days·kg / mg. In the example, the NAOC of the insulin-Fc fusion protein is 150% FBGL·days·kg / mg or greater (e.g., 200% FBGL·days·kg / mg or greater, 250% FBGL·days·kg / mg or greater, or greater). Once again based on experience, at NAOC values ​​greater than 150% FBGL·days·kg / mg, the dose requirement for the target species will be sufficiently low to achieve an acceptable treatment cost. In the example, the NAOC of the insulin-Fc fusion protein must be maintained after repeated administration in the target species (i.e., the ratio of the NAOC after the third administration to the NAOC after the first administration of the insulin-Fc fusion protein is 0.50 or higher (e.g., 0.60 or higher, 0.70 or higher, 0.80 or higher, 0.90 or higher, or higher)).

[0226] In some embodiments, the insulin-Fc fusion protein described herein binds to an Fc(gamma) receptor having a lower affinity than the insulin-Fc fusion protein reference standard measured according to Example 8. In some embodiments, the ratio of the affinity of the insulin-Fc fusion protein to the Fc(gamma) receptor of the insulin-Fc fusion protein to the insulin-Fc fusion protein reference standard is less than 0.50 (e.g., less than 0.40, less than 0.30, less than 0.20).

[0227] Characteristics of treatment methods and subject selection

[0228] A method for treating diabetes (e.g., canine diabetes or feline diabetes) is described herein, and said method comprises administering an insulin-Fc fusion protein (e.g., the insulin-Fc fusion protein described herein) to a subject.

[0229] In the embodiments, the reference standard used in any method described herein comprises a reference treatment or reference therapy. In some embodiments, the reference comprises a standard care formulation for the treatment of diabetes (e.g., a standard care formulation for canine diabetes or a standard care formulation for feline diabetes). In some embodiments, the reference standard is a commercially available insulin or insulin analog. In some embodiments, the reference standard comprises long-acting insulin, medium-acting insulin, short-acting insulin, rapid-acting insulin, short-acting insulin, medium-acting insulin, and long-acting insulin. In some embodiments, the reference standard is Vetsulin ® , Prozinc ® , Insulin NPH, Insulin Glargine (Lantus ® It includes ) or recombinant human insulin.

[0230] In the embodiments, the reference standard used in any method described herein includes the results of a diabetes treatment regimen (e.g., a canine diabetes treatment regimen or a feline diabetes treatment regimen), e.g., the results described herein.

[0231] In the example, the reference standard is the level of a marker (e.g., blood glucose or fructosamine) in the subject prior to the initiation of treatment, e.g., the insulin-Fc fusion protein therapy described herein, if the subject has diabetes. In the example, the blood glucose level of the pet (e.g., dog or cat) is 200 mg / dL or higher (e.g., 250 mg / dL, 300 mg / dL, 350 mg / dL, 400 mg / dL or higher) prior to the start of treatment. In the example, the fructosamine levels of the companion animal (e.g., dog or cat) are 250 micromolar / L, 350 micromolar / L or higher (e.g., 400 micromolar / L, 450 micromolar / L, 500 micromolar / L, 550 micromolar / L, 600 micromolar / L, 650 micromolar / L, 700 micromolar / L, 750 micromolar / L or higher) prior to the start of treatment. In the example, the reference standard is a measure of the presence, progression, or severity of the disease. In the example, if the subject has diabetes, the reference standard is a measure of the presence or severity of disease symptoms prior to the start of treatment, e.g., the insulin-Fc fusion protein treatment described herein.

[0232] Pharmaceutical composition and route of administration

[0233] Pharmaceutical compositions containing the insulin-Fc fusion protein described herein, which may be used to lower blood glucose in companion animals (e.g., dogs or cats), are provided herein. The amount and concentration of the insulin-Fc fusion protein in the pharmaceutical composition, as well as the amount of the pharmaceutical composition administered to the subject, may be selected based on the subject's medically relevant characteristics (e.g., age, weight, sex, other medical conditions, etc.), the solubility of the compound in the pharmaceutical composition, the efficacy and activity of the compound, and the mode of administration of the pharmaceutical composition. For details regarding the route and mode of administration, refer to Chapter 25.3, Vol. 5, Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press, 1990.

[0234] The formulations of the present disclosure include formulations suitable for parenteral administration. As used herein, the terms “parenteral administration” and “parenteral administration” generally refer to a mode of administration other than intestinal and local administration, by intravenous or subcutaneous injection.

[0235] Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity may be maintained, for example, by the use of a coating material such as lecithin, the maintenance of the required particle size in the case of a dispersion, and the use of a surfactant, for example, a Tween-like surfactant. In some embodiments, the pharmaceutical composition (e.g., as described herein) comprises a Tween-like surfactant, for example, polysorbate-20, Tween-20, or Tween-80. In some embodiments, the pharmaceutical composition (e.g., as described herein) comprises a Tween-like surfactant, e.g., Tween-80, at a concentration of about 0.001% to about 2%, or about 0.005% to about 0.1%, or about 0.01% to about 0.5%.

[0236] In some embodiments, the concentration of insulin-Fc fusion protein in the aqueous carrier is about 3 mg / mL. In some embodiments, the concentration of insulin-Fc fusion protein in the aqueous carrier is about 6 mg / mL. In some embodiments, the concentration of insulin-Fc fusion protein in the aqueous carrier is about 8 mg / mL, 9 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, or higher.

[0237] In some embodiments, the insulin-Fc fusion protein is administered by a bolus, injection, or intravenous push. In some embodiments, the fusion protein is administered via a syringe injection, pump, pen, needle, or internal catheter. In some embodiments, the insulin-Fc fusion protein is administered by a subcutaneous bolus injection. The delivery method may also be provided by a rechargeable or biodegradable device. In recent years, various sustained-release polymer devices have been developed and tested in vivo for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. By using various biocompatible polymers (including hydrogels) including biodegradable and non-degradable polymers, implants can be formed for the sustained release of compounds at specific target sites.

[0238] Dosage

[0239] The actual dosage level of the insulin-Fc fusion protein may vary to obtain an amount of the active ingredient effective in achieving a desired therapeutic response for a specific subject (e.g., a dog or a cat). The selected dosage level will depend on various factors, including the activity of the specific fusion protein used, or its esters, salts, or amides; the route of administration; the timing of administration; the excretion rate of the specific compound used; the duration of treatment; other drugs, compounds, and / or substances used in conjunction with the specific fusion protein; and the age, sex, weight, condition, general health status, and medical history of the subject, as well as similar factors known to the medical community.

[0240] Generally, the appropriate dosage of the insulin-Fc fusion protein will be the amount that is the lowest effective dosage to produce a therapeutic effect. This effective dosage generally depends on the factors described above. Generally, the intravenous and subcutaneous dosage of the insulin-Fc fusion protein for dogs or cats will be in the range of about 0.001 to about 1 mg per kg of body weight per day (e.g., mg / kg), e.g., about 0.001 to 1 mg / kg / day, about 0.01 to 0.1 mg / kg / day, about 0.1 to 1 mg / kg / day, or about 0.01 to 1 mg / kg / day. In another embodiment, the fusion protein is administered at a dosage of 0.025 to 4 mg per kg of body weight per week, e.g., 0.025 to 0.5 mg / kg / week.

[0241] The present disclosure considers formulations of insulin-Fc fusion proteins in any of the aforementioned pharmaceutical compositions and formulations. Additionally, the present disclosure considers administration via any of the aforementioned routes of administration. Those skilled in the art may select appropriate formulations and routes of administration based on the condition to be treated and the overall health, age, and size of the patient to be treated.

[0242] Example of implementation

[0243] The technology is further explained by the following embodiments, which should not be construed as limiting in any way.

[0244] General Methods, Analysis and Materials

[0245] Example 1: Synthesis and preparation method of insulin-Fc fusion protein in HEK293 cells

[0246] The insulin-Fc fusion protein was synthesized as follows. The gene sequence of interest was constructed using proprietary software (LakePharma, Belmont, CA) and cloned into a high-expression mammalian vector. HEK293 cells were seeded into shake flasks 24 hours prior to transfection and grown using serum-free, chemically defined medium. The DNA expression construct encoding the insulin-Fc fusion protein of interest was transiently transfected into the HEK293 cell suspension using standard operating procedures for transient transfection (LakePharma, Belmont, CA). After 20 hours, cells were counted to determine viability and the number of surviving cells, and titers were calculated using ForteBio ® Octet ® Measured by Pall ForteBio LLC, Fremont, CA. Additional readings were performed through transient transfection production runs. Cultures were harvested on day 5 or later.

[0247] Example 2: Synthesis and preparation method of insulin-Fc fusion protein in CHO cells

[0248] The CHO cell line was originally derived from CHO-K1 (LakePharma, Belmont, CA), and the endogenous glutamine synthase (GS) gene was knocked out by recombinant technology using methods known to the industry. Stable expression DNA vectors were designed and optimized for CHO expression and GS selection, and incorporated into high-expression mammalian vectors (LakePharma, Belmont, CA). The sequences of each completed construct were verified prior to starting scale-up experiments. Suspension-adapted CHO cells were cultured in chemically defined medium (CD OptiCHO; Invitrogen, Carlsbad, CA) in humidified 5% CO2 incubators at 37°C. No serum or other animal-derived products were used for CHO cell culture.

[0249] Approximately 80 million suspension-adapted CHO cells grown in CD OptiCHO medium during the exponential growth phase, MaxCyte with 80 μg DNA ® STX ® Stable CHO cell lines for each insulin-Fc fusion protein were generated by transfection via electroporation using a system (MaxCyte, Inc., Gaithersburg, MD) (DNA constructs contained the full-length sequence of the insulin-Fc fusion protein). After 24 hours, transfected cells were counted and subjected to screening for stable integration of the insulin-Fc fusion gene. Transfected cells were seeded in CD OptiCHO selection medium containing 0–100 μM methionine sulfoxymine (MSX) at a cell density of 0.5 x 10⁶ cells / mL in shaker flasks and cultured at 37°C with 5% CO₂. During the screening process, cells were rotated and resuspended in fresh screening medium every 2–3 days until the CHO stable pool recovered its growth rate and viability. Cell cultures were monitored for growth and titer.

[0250] Cells were grown to 2.5 x 10⁶ cells per mL. At the time of harvest for cell banking, the viability was over 95%. Subsequently, the cells were centrifuged, and the cell pellet was resuspended in CD OptiCHO medium containing 7.5% dimethyl sulfoxide (DMSO) to achieve a cell count of 15 x 10⁶ cells per mL in each vial. The vials were cryopreserved for storage in liquid nitrogen.

[0251] Small-scale production was performed using CHO cells as follows. Cells were expanded for production in CD OptiCHO growth medium containing 100 μM MSX at 37°C and fed every 2–4 days as needed with CD OptiCHO growth medium supplemented with the necessary glucose and additional amino acids for approximately 14–21 days. The regulated medium supernatant harvested from stable pool production runs was clarified by centrifugation. Proteins were run on a Protein A (MabSelect, GE Healthcare, Little Chalfont, United Kingdom) column pre-equilibrated with binding buffer. Then, wash buffer was passed through the column until the OD280 value (NanoDrop, Thermo Scientific) was measured at or near background levels. Insulin-Fc fusion proteins were eluted using low pH buffer, the eluted fractions were collected, and the OD280 value of each fraction was recorded. The fraction containing the target insulin-Fc fusion protein was collected and optionally further filtered using a 0.2 μM membrane filter.

[0252] Cell lines were selectively subcloned for monoclonal expression and selectively further selected for high-tit insulin-Fc-fusion protein-expressing clones using a dilution restriction method known to those skilled in the art. After obtaining high-tit monoclonal insulin-Fc fusion protein expressing cell lines, production of insulin-Fc fusion protein was achieved as described above in growth medium without MSX or, optionally, in growth medium with MSX, to obtain cell culture supernatants containing recombinant, CHO-produced insulin-Fc fusion protein. The MSX concentration was selectively increased over time to exert additional selectivity for clones capable of yielding higher product titers.

[0253] Example 3: Purification of Insulin-Fc Fusion Protein

[0254] Purification of the insulin-Fc fusion protein was performed as follows. The supernatant of the conditioned medium containing the secreted insulin-Fc fusion protein was harvested from transient or stable transfected HEK production runs and purified by centrifugation. The supernatant containing the desired insulin-Fc fusion protein was run through a Protein A or Protein G column and eluted using a low pH gradient. Optionally, the recovery of the insulin-Fc fusion protein could be enhanced by reloading the initial Protein A or Protein G column eluent into a second Protein A or Protein G column. Subsequently, the eluted fraction containing the desired protein was collected, and the buffer was exchanged with 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer. A final filtration step was performed using a 0.2 µm membrane filter. The final protein concentration was calculated from the solution optical density at 280 nm. Further selective purification by ion exchange chromatography (e.g., using anion exchange bead resin or cation exchange bead resin), gel filtration chromatography, or other methods was performed as needed.

[0255] Example 4: Structural confirmation by non-reducing and reduced CE-SDS

[0256] Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) analysis was performed on a LabChip in a solution of purified insulin-Fc fusion protein dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer. ® The procedure was performed on GXII (Perkin Elmer, Waltham, MA) and the electrophoresis was plotted. Under non-reducing conditions, the samples were run against known molecular weight (MW) protein standards, and the elution peak indicates the 'explicit' MW of the insulin-Fc fusion protein homodimer.

[0257] Under reducing conditions (e.g., breaking the disulfide bonds of the insulin-Fc fusion homodimer using beta-mercaptoethanol), 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 as a method to determine the structural purity of the insulin-Fc fusion protein.

[0258] Example 5: Sequence identification by LC-MS through glycan removal

[0259] To obtain an accurate estimate of insulin-Fc mass via mass spectrometry (MS), the sample is first processed to remove naturally occurring glycans that may interfere with MS analysis. 100 μL of 2.5 mg / mL insulin-Fc fusion protein dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer is first buffer-exchanged with 0.1 M Tris, pH 8.0 buffer containing 5 mM EDTA using a Zeba desalting column (Pierce, ThermoFisher Scientific, Waltham, MA). To remove N-linked glycans present in the fusion protein (e.g., glycans bound to the side chain of asparagine located at the cNg-N site), 1.67 μL of PNGase F enzyme (Prozyme N-glycanase) is added to this solution, and the mixture is incubated overnight at 37°C in an incubator. Next, the sample is analyzed via LC-MS (NovaBioassays, Woburn, MA) to obtain the molecular weight of the molecule corresponding to the desired homodimer without glycan. This mass is then further modified because the enzymatic process used to cleave the glycan from cNg-asparagine deaminates the asparagine side chain to form aspartic acid, thereby yielding an enzymatically treated homodimer of 2 Da in total, which corresponds to a mass of 1 Da for each chain present in the homodimer. Therefore, the actual molecular weight is the value obtained by subtracting 2 Da from the measured mass to correct for the enzymatic modification of the insulin-Fc fusion protein structure in the analysis sample.

[0260] Example 6: % homodimers by size-exclusion chromatography

[0261] Size-exclusion chromatography (SEC-HPLC) of insulin-Fc fusion proteins was performed using a Waters 2795HT HPLC (Waters Corporation, Milford, MA) connected to a 2998 photodiode array at a wavelength of 280 nm. Less than 100 μL of a sample containing the insulin-Fc fusion protein of interest was injected into a MAbPac SEC-1, 5 μm, 4×300 mm column (ThermoFisher Scientific, MA, Waltham) having a mobile phase containing 0 mM sodium phosphate, 300 mM NaCl, and 0.05% w / v sodium azide (pH 6.2) and operating at a flow rate of 0.2 mL / min. The MAbPac SEC-1 column operates according to the molecular size separation principle. Therefore, larger soluble insulin-Fc aggregates (e.g., polymers of insulin-Fc fusion protein homodimers) eluted at earlier retention times, while non-aggregated homodimers eluted at later retention times. The purity of the insulin-Fc fusion protein solution was confirmed with respect to the percentage of non-aggregated homodimers when separating the mixture of homodimers from the aggregated polydimers via analytical SEC-HPLC.

[0262] Example 7: In vitro binding of exemplary insulin-Fc fusion protein to IM-9 insulin receptor at 4°C

[0263] Human IM-9 cells (ATTC# CCL-159) expressing the human insulin receptor were cultured and maintained at 70–80% confluency in full RPMI 5% FBS medium. The IM-9 cell cultures were centrifuged at 250×g (~1000 rpm) for 10 minutes to pellet the cells. The cells were washed once with HBSS or PBS buffer and stained 8×10⁶ cells in cold FACS staining medium (HBSS / 2 mM EDTA / 0.1% Na-azide + 4% horse serum). 6Resuspended at a cell / mL concentration and stored on ice or at 4°C until the test solution was prepared. Insulin-Fc protein was diluted in FACS buffer at a 2-fold concentration in a 1:3 serial dilution in 1.2 mL tubes (approximately 60 μL volume for each dilution) and kept on ice until the solution was ready for pipetting.

[0264] Biotinylated-RHI was diluted to a concentration of 1.25 μg / mL in FACS staining medium. 40 μL of the 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 slow vortexing, and placed on ice. 40 μL of IM-9 cell suspension (8×10⁶ 6 Cells / mL) were added to each well using a multi-channel pipette, gently mixed, and incubated on ice for 30 minutes to enable competitive binding to insulin receptors in IM-9 cells. Subsequently, the cells were washed twice with 275 μL of ice-cold FACS wash buffer (HBSS / 2 mM EDTA / 0.1% Na-azide + 0.5% horse serum) by centrifuging the V-bottom plate at 3000 rpm for 3 minutes and aspirating the supernatant. Then, the cells were resuspended on ice for 20 minutes in 40 μL of FACS staining medium containing 1:100 diluted streptavidin-PE (Life Technologies). Finally, the cells were washed once with 275 μL of ice-cold FACS buffer and fixed with 3% paraformaldehyde for 10 minutes at room temperature. Next, the cells were washed once with 275 μL of ice-cold FACS buffer and resuspended in 250 μL of FACS buffer for analysis.

[0265] V-bottom plates containing cells were analyzed on a Guava 8-HT flow cytometer (Millipore). Biotinylated-RHI binding to the insulin receptor was quantified by the central fluorescence intensity (MFI) of the cells on the FACS FL-2 channel for each concentration of the test compound. Control wells were labeled only with biotinylated-RHI and were used to calculate the inhibition rate (%) occurring at each test compound concentration. The inhibition rate (%) of biotinylated-RHI binding by the test compound in IM-9 cells was plotted against the log concentration of the test compound, and the resulting IC50 values ​​were calculated for the test compound using GraphPad Prism (GraphPad Software, La Jolla, CA). Thus, lower IC50 values ​​of the test compound indicate higher levels of biotinylated-RHI inhibition at lower concentrations, which represents stronger binding of the insulin-Fc fusion protein to the insulin receptor. A control compound, such as unlabeled recombinant human insulin (RHI), was also used as an internal standard to generate the RHI IC50, to which the IC50 of a given compound could be ratioed (IC50(compound) / IC50(RHI)). A lower IC50 ratio indicates more similar binding to RHI (stronger binding to the insulin receptor), whereas a higher IC50 ratio indicates weaker binding to the insulin receptor compared to RHI.

[0266] Example 8: In vitro analysis of Fc(gamma) receptor I binding affinity

[0267] Binding of the insulin-Fc fusion protein to Fc(gamma) receptor I at pH 7.4 was performed using an ELISA assay as follows. Since neither canine nor feline Fc(gamma) receptor I is commercially available, human Fc(gamma) receptor I (i.e., rhFc(gamma) receptor I) was used as a surrogate mammalian receptor. The insulin-Fc compound was diluted to 10 μg / mL in sodium bicarbonate buffer at pH 9.6 and coated onto Maxisorp (Nunc) microtiter plates overnight at 4°C. Afterward, the microplate strips were washed five times with PBST (PBS / 0.05% Tween-20) buffer and blocked with a Superblock blocking agent (ThermoFisher). Serial dilutions of biotinylated rhFc(gamma) receptor I (recombinant human Fc(gamma)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 microplate strips coated with insulin-Fc fusion protein. After incubating the microplates at room temperature for 1 hour, the microplate strips were washed 5 times with PBST, and then 100 μL / well of streptavidin-HRP diluted 1:10000 in PBST / 10% Superblock buffer was loaded. After incubating for 45 minutes, the microplate strips were washed again 5 times with PBST. TMB was added to express the bound Fc(gamma) receptor I protein and stopped with ELISA stop reagent (Boston Bioproducts). The plate was read at 450 nm using an ELISA plate reader, and the OD values ​​(proportional to the binding of rhFc(gamma) receptor I to insulin-Fc protein) were plotted against the log concentration of rhFc(gamma) receptor I added to each well to generate binding curves using GraphPad Prism software.

[0268] Example 9: In vitro measurement of insulin-Fc fusion protein affinity for canine FcRn receptors

[0269] The in vitro binding affinity of insulin-Fc fusion proteins containing Fc fragments of canine or feline IgG origin for canine FcRn receptors was measured using ELISA technology performed at a solution pH of 5.5. A slightly acidic pH is a favorable binding environment for Fc fragment-containing molecules to bind to the FcRn receptor. In vivo, cells express FcRn on their surfaces and inside endosomes. As molecules containing Fc fragments enter the cell via natural processes (e.g., pinocytosis or endocytosis), the pH changes to a lower pH in the endosome, where the FcRn receptor binds to Fc fragment-containing molecules degraded in the endosome-lysosome compartment, thereby allowing these molecules to be returned to the cell surface where the pH is near neutral (e.g., pH 7.0–7.4). A neutral pH does not favor binding to the FcRn receptor, allowing the Fc-fragments containing the molecules to be released back into circulation. This is the primary mechanism by which Fc fragment-containing molecules exhibit a long-term circulating pharmacokinetic half-life in vivo.

[0270] Insulin-Fc fusion proteins containing Fc fragments of canine or feline origin were diluted to 10 µg / ml in sodium bicarbonate pH 9.6 buffer and double-coated onto Maxisorb ELISA plate strips for 1-2 hours at room temperature. Then, the strips were washed four times with PBST (PBS / 0.1% Tween-20) buffer and blocked with a superblock blocking reagent (ThermoFisher). The strips for FcRn binding were washed twice again with pH 5.5 MES / NaCl / Tween (50 mM MES / 150 mM NaCl / 0.1% Tween-20) buffer before adding the FcRn reagent (biotinylated canine FcRn; Immunitrack). Since feline FcRn reagents were not found to be commercially available, insulin-Fc fusion proteins containing canine Fc fragments or feline Fc fragments were analyzed for binding to canine FcRn. Serial dilutions (1:3X dilution) of biotinylated FcRn reagents were prepared at concentrations ranging from 1000 ng / ml to 0.45 ng / ml in pH 5.5 MES / NaCl / Tween / 10% Superblock buffer and loaded onto strips coated with insulin-Fc fusion protein compounds at a rate of 100 μL / well using a multichannel pipette. Subsequently, the assay plates were incubated at room temperature for 1 hour. The FcRn binding strips were washed four times with pH 5.5 MES / NaCl / Tween buffer and then loaded with 100 μL / well streptavidin-HRP diluted 1:10000 in pH 5.5 MES / NaCl / 10% Superblock buffer. After incubating for 45 minutes, the strip was washed four times again with pH 5.5 MES / NaCl / Tween buffer. Finally, TMB was added to confirm the conjugated biotinylated canine FcRn reagent, and the color development was stopped with an ELISA stop reagent. The plate was read on an ELISA plate reader at a wavelength of 450 nm.OD values ​​(proportional to the binding of dog-FcRn to the insulin-Fc fusion protein test compound) were plotted against the log concentration of FcRn added to each well to generate binding curves using GraphPad Prism software. EC50 values ​​were calculated for each binding curve to compare different compounds.

[0271] Example 10: Generalized procedure for determining in vivo pharmacokinetics (PD) after a single administration of insulin Fc-fusion protein in dogs or cats.

[0272] The effects of insulin-Fc fusion proteins on fasting blood glucose levels were evaluated as follows. N = 1, 2, or 3 healthy, antibody-free dogs weighing approximately 10–15 kg or cats weighing approximately 5 kg were administered one by one for each insulin-Fc fusion protein. Animals were also observed twice daily for signs of anaphylaxis, lethargy, distress, and pain, and optionally, for some compounds, subcutaneous injection treatment was continued for an additional 3 weeks or more to observe whether the glucose-lowering ability of the compound decreased over time. This is a key signal for potential induction of neutralization of anti-drug antibodies. On day 0, the animal received a single injection via intravenous or subcutaneous administration of a pharmaceutical composition containing 1 to 10 mg / mL of insulin Fc-fusion protein homodimer 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 0.02-1.00 mg / mL of a bacterial inhibitor (e.g., phenol, m-cresol, or methylparaben) at a solution pH between 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 on a molar basis). Blood was collected from a suitable vein on day 0, immediately before and after injection at 15, 30, 45, 60, 120, 240, 360, and 480 minutes, and on days 1, 2, 3, 4, 5, 6, and 7.

[0273] At each time point, at least 1 mL of whole blood was collected. Blood glucose levels were measured using a blood glucose meter (ACCU-CHEK ® It was measured immediately using Aviva Plus, and about one drop of blood was required. To evaluate the bioactivity of the given insulin-Fc fusion protein, the average % fasting blood glucose level (% FBGL) from day 0 to day 7 was plotted.

[0274] Example 11: Generalized procedure for measuring in vivo pharmacokinetics (PD) after repeated administration of insulin-Fc fusion protein in dogs or cats.

[0275] The effect of insulin-Fc fusion proteins on blood glucose levels upon repeated injections was evaluated as follows. Healthy, antibody-free dogs or cats weighing approximately 5 to 20 kg were used, and insulin-Fc fusion proteins were administered to each animal. Animals were observed twice daily for signs of anaphylaxis, lethargy, distress, pain, and other adverse side effects, and optionally, for some compounds, an additional 2 to 5 subcutaneous injection treatments were continued to observe whether the glucose-lowering ability of the compound decreased over time, indicating the potential presence of in vivo neutralizing anti-drug antibodies. On day 0, animals received a single subcutaneous injection of a pharmaceutical composition containing 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 0.02-1.00 mg / mL of a bacterial inhibitor (e.g., phenol, m-cresol, or methylparaben) at a solution pH between 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 on a molar basis). Blood was collected from a suitable vein on day 0, immediately before and after injection at 15, 30, 45, 60, 120, 240, 360, and 480 minutes, and on days 1, 2, 3, 4, 5, 6, and 7.

[0276] Subsequent subcutaneous injections were not administered more frequently than once a week, and in some cases, injections were given at different intervals based on the pharmacokinetics of the given insulin-Fc fusion protein preparation. Subsequent injections for each insulin-Fc fusion protein were adjusted to higher or lower doses according to the proven pharmacokinetics of the insulin-Fc fusion protein. For example, if the dose of the first injection on day 0 was found to be ineffective in lowering blood glucose, the level of the subsequent dose of the injected insulin-Fc fusion protein was increased. Similarly, if the dose of the first injection on day 0 was found to lower blood glucose in an excessively strong manner, the level of the subsequent dose of the injected insulin-Fc fusion protein was decreased. Additionally, it was found that intermediate or final doses could be adjusted in a similar manner as needed. For each dose, blood was collected from a suitable vein immediately before injection and at 15, 30, 45, 60, 120, 240, 360, and 480 minutes after injection, and on days 1, 2, 3, 4, 5, 6, and 7 (and optionally day 14). At least 1 mL of whole blood was collected at each time point. Blood glucose levels were measured using a blood glucose meter (ACCU-CHEK ® It was measured immediately using Aviva Plus, and about one drop of blood was required. The average % fasting blood glucose level (% FBGL) obtained from the entire study was plotted against time to determine the bioactivity of the fusion protein.

[0277] To determine the bioactivity of each dose, an area-over-the-curve (AOC) analysis was performed as follows. After constructing the % FBGL versus time data, the data was entered into data analysis software (GraphPad Prism, GraphPad Software, San Diego, CA). The software first performed an area-under-the-curve (AUC) analysis, which was then used to integrate the area under the % FBGL versus time curve for each dose. To convert the AUC data into the desired AOC data, the following equation was used: AOC = TPA - AUC; where TPA is the total possible area obtained by multiplying each dose duration (e.g., 7 days, 14 days, etc.) by 100% (where 100% represents y = 100% of the % FBGL versus time curve). For example, if the dose life is 7 days and the calculated AUC is 500% FBGL·day, the following is provided for the AOC: AOC = (100% FBGL x 7 days) - (500% FBGL·day) = 200% FBGL·day. To obtain the AOC value for Injection 1, Injection 2, Injection 3, etc., an analysis can be performed on each dose injected as a series of injection doses.

[0278] As previously discussed, since the dosage of insulin-Fc fusion proteins can vary, it is often more convenient to normalize all AOC values ​​calculated for a given insulin-Fc fusion protein to the specific dosage of that insulin-Fc fusion protein. This allows for a convenient comparison of the glucose-lowering efficacy of insulin-Fc fusion proteins across multiple injections, even if dosage levels change depending on the injections in a given study. The normalized AOC (NAOC) for a given dosage is calculated as follows: NAOC = AOC / D, which has units of % FBGL·days·kg / mg; where D is the actual dosage administered to the animal in mg / kg. NAOC values ​​can be calculated for each injection in a series of injections for a given animal and averaged across groups of animals administered the same insulin-Fc fusion protein formulation.

[0279] The NAOC ratio (NAOCR) can also be calculated for each injection in a series of injections for a given animal by taking the NAOC value for each injection (e.g., injection 1, 2, 3,… N) and dividing each NAOC for a given injection by the NAOC at 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 in vivo activity over a series of N doses (e.g., NAOCR for the Nth dose greater than 0.5), or whether the in vivo glucose-lowering activity of the given insulin-Fc fusion protein has lost a significant portion of its efficacy over the course of N doses, indicating the potential formation of neutralizing anti-drug antibodies in vivo (e.g., NAOCR for the Nth dose less than 0.5 over the course of N doses). In a preferred embodiment, the ratio of NAOC after the third subcutaneous injection to NAOC after the first subcutaneous injection is greater than 0.5 (i.e., the NAOCR of the third subcutaneous injection is greater than 0.5).

[0280] Example 12: Generalized procedure for in vivo pharmacokinetic (PK) measurement in dog and cat serum

[0281] An assay was constructed as follows to measure the concentration of insulin-Fc fusion proteins containing canine isotype Fc fragments in canine serum. The assay involves 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, then detected by an HRP-conjugated anti-canine IgG Fc-specific antibody, followed by the use of a TMB substrate system for color development. Maxisorp ELISA plates (Nunc) are coated with 5 μg / ml of the anti-insulin mAb clone D6C4 (Biorad) in coating buffer (pH = 9.6 sodium carbonate-biocarbonate buffer) overnight at 4°C. The plates are then washed five times with PBST (PBS + 0.05% Tween 20) and blocked with a superblock blocking solution (ThermoFisher) at room temperature for at least one hour (or overnight at 4°C). Test serum samples are diluted 1:20 in PBST / SB / 20% HS sample dilution buffer (PBS + 0.1% Tween 20 + 10% Superblock + 20% horse serum). To create standard curves, the insulin-Fc fusion protein of interest is diluted in sample dilution buffer (PBST / SB / 20% HS) + 5% pooled Beagle serum (BioIVT) at a 1:2.5 serial dilution to concentrations ranging from 200 ng / ml to 0.82 ng / ml. Standard and diluted serum samples are double-layered into blocked plates at 100 μl / well and incubated at room temperature for 1 hour. After incubation, samples and standards are washed 5 times with PBST. Dilute the HRP-conjugated goat anti-dog IgG Fc(Sigma) detection antibody to about 1:15,000 in PBST / SB / 20% HS buffer, add 100 μl to each well, and incubate in the dark at room temperature for 45 minutes.The plate is washed 5 times with PBST and 1 time with deionized water, and developed by adding 100 μl / well TMB (Invitrogen) at room temperature for 8-10 minutes. Then, 100 μl / well ELISA stop solution (Boston Bioproducts) is added to stop color development, and the absorbance is 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 is calculated by interpolating from the 4-PL curve using SoftMaxPro software.

[0282] Similarly, an assay was constructed to measure the concentration of an insulin-Fc fusion protein containing a feline isotype Fc fragment in feline serum as follows. This assay includes a sandwich ELISA format in which the therapeutic compound in the serum sample is captured by an anti-insulin / proinsulin mAb coated on an ELISA plate, then detected by an HRP-conjugated goat anti-feline IgG Fc-specific antibody, followed by the use of a TMB substrate system for color development.

[0283] Maxisorp ELISA plates (Nunc) are coated with 5 μg / ml of the anti-insulin mAb clone D6C4 (Biorad) in coating buffer (pH = 9.6 sodium carbonate-biocarbonate buffer) overnight at 4°C. Then, the plates are washed 5 times with PBST (PBS + 0.05% Tween 20) and blocked with Superblock blocking solution (ThermoFisher) at room temperature for at least 1 hour (or overnight at 4°C). Test serum samples are diluted 1:20 in PBST / SB / 20% HS sample dilution buffer (PBS + 0.1% Tween 20 + 10% Superblock + 20% horse serum). To construct a standard curve, the insulin-Fc fusion protein of interest is diluted in sample dilution buffer (PBST / SB / 20% HS) + normal cat serum (Jackson Immunoresearch) at a 1:2.5 serial dilution to a concentration range of 200 ng / ml to 0.82 ng / ml. Standard and diluted serum samples are double-layered at 100 μl / well into blocked plates and incubated at room temperature for 1 hour. After incubation, samples and standards are washed 5 times with PBST. The HRP-conjugated goat anti-feline IgG Fc (Bethyl Lab) detection antibody is diluted approximately 1:20,000 in PBST / SB / 20% HS buffer, 100 μl is added to each well, and incubated in the dark at room temperature for 45 minutes. The plate is washed five times with PBST and once with deionized water, and developed by adding 100 μl / well TMB (Invitrogen) at room temperature for 8-10 minutes. Then, 100 μl / well ELISA stop solution (Boston Bioproducts) is added to stop color development, and the absorbance is 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 is calculated by interpolating from the 4-PL curve using SoftMaxPro software.

[0284] Example 13: Analysis protocol for measuring anti-drug antibodies in dog serum

[0285] Maxisorp ELISA plates (Nunc) are coated with 10 μg / mL of the insulin-Fc fusion protein of interest diluted in coating buffer (pH = 9.6 carbonate-biocarbonate buffer) at 4°C overnight to measure ADA for the test compound. To measure ADA for the insulin portion of the insulin-Fc fusion protein containing the canine IgG-origin Fc fragment, the plates are coated with 30 μg / mL of purified insulin in coating buffer. Then, the plates are washed 5 times with PBST (PBS + 0.05% Tween 20) and blocked with Superblock blocking solution (ThermoFisher, Waltham MA) for at least 1 hour (or overnight). To calculate the ADA of canine IgG units, strips are directly coated with a 1:2 serial dilution of canine IgG (Jackson Immunoresearch Laboratories, West Grove, PA) in a pH = 9.6 Carb-Biocarb coating buffer at a concentration of 300–4.69 ng / ml overnight at 4°C, which is used to create a 7-point pseudo-standard curve. Standard strip plates are also washed and blocked with a superblock blocking solution for at least 1 hour (or overnight).

[0286] Test serum samples are diluted to at least 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 in duplicate at 100 μL / well to insulin-Fc fusion protein-coated (or RHI-coated) strips. Duplicate strips of canine IgG-coated standard strips are also added to each plate and filled with PBST / SB (PBS + 0.1% Tween 20 + 10% Superblock) buffer at 100 μL / well. Plates are incubated at room temperature for 1 hour, then washed 5 times with PBST. For the detection of ADA, HRP-conjugated goat anti-cat IgG F(ab')2 (anti-cat IgG F(ab')2 reagent cross-reacts with canine antibodies; Jackson Immunoresearch Laboratories, West Grove, PA) was diluted 1:10000 in PBST / SB, added at 100 μL / well to both sample and standard wells, and incubated at RT in the dark for 45 minutes. The plates were washed 5 times with PBST, then washed once with deionized water, followed by the addition of 100 μL / well TMB substrate (Invitrogen, ThermoFisher Scientific, Waltham MA) and development at room temperature in the dark for 15-20 minutes. Then, 100 μL / well of ELISA stop solution (Boston Bioproducts) was added to stop color development, and the absorbance was read at 450 nm using a SpectraMax plate reader within 30 minutes. The concentration of anti-drug antibodies is determined by interpolating the OD values ​​of a 4-PL pseudo-standard curve using SoftMax Pro Software (Molecular Devices, San Jose CA).

[0287] To verify the specificity of the detected ADA, an "inhibition" assay is performed. In the drug inhibition ADA assay, a serum sample is diluted 1:100 in PBST / SB / 20% HS buffer, mixed with an equal volume of 300 μg / mL of the relevant therapeutic compound (a final sample dilution of 1:200 and 150 μg / mL of the final inhibitor compound), and incubated at room temperature for 30–40 minutes to allow the anti-drug antibody to bind to the free inhibitor (i.e., the therapeutic compound). After pre-incubation, the sample is added in duplicate at 100 μL / well to insulin-Fc fusion protein-coated (or RHI-coated) strips. The sample diluted 1:200 in PBST / SB / 20% HS buffer without the inhibitor compound is tested on a sample plate along with duplicate strips of dog IgG-coated standards. The remaining steps of the assay procedure are performed as described above. ADA measured in the drug-inhibition well is matched with non-inhibition ADA concentration to evaluate the specificity of ADA. If significant inhibition of the ADA signal is observed in the drug-inhibition well, it means that ADA is specific to the therapeutic compound.

[0288] Example 14: Analysis protocol for measuring anti-drug antibodies in cat serum

[0289] Maxisorp ELISA plates (Nunc) are coated with 10 μg / mL of the insulin-Fc fusion protein of interest diluted in coating buffer (pH = 9.6 carbonate-biocarbonate buffer) overnight at 4°C to measure ADA for the Fc fusion protein containing the Fc fragment of feline IgG origin. To measure ADA for the insulin portion of the insulin-Fc fusion protein, the plates are coated with 30 μg / mL of purified insulin in coating buffer. Then, the plates are washed 5 times with PBST (PBS + 0.05% Tween 20) and blocked with Superblock blocking solution (ThermoFisher, Waltham MA) for at least 1 hour (or overnight). To calculate the ADA of feline IgG units, strips are directly coated with a 1:2 serial dilution of feline IgG (Jackson Immunoresearch Laboratories, West Grove, PA) in a sodium carbonate-sodium bicarbonate coating buffer at a concentration of 300–4.69 ng / ml at 4°C overnight, which is used to create a 7-point pseudo-standard curve. Standard strip plates are also washed and blocked with a superblock blocking solution for at least 1 hour (or overnight).

[0290] Test serum samples are diluted to at least 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 in duplicate at 100 μL / well to insulin-Fc fusion protein-coated (or RHI-coated) strips. Duplicate strips of feline IgG-coated standard strips are added to each plate and filled with PBST / SB (PBS + 0.1% Tween 20 + 10% Superblock) buffer at 100 μL / well. Plates are incubated at room temperature for 1 hour, then washed 5 times with PBST. For the detection of ADA, HRP-conjugated goat anti-feline IgG F(ab') 2 (Jackson Immunoresearch Laboratories, West Grove, PA) was diluted in PBST / SB at a ratio of 1:10000, added to both sample and standard wells at a rate of 100 μL / well, and incubated in the dark at room temperature for 45 minutes. The plates were washed 5 times with PBST and 1 time with deionized water, and developed by adding 100 μL / well of TMB substrate (Invitrogen) in the dark at room temperature for 15-20 minutes. Then, 100 μL / well of ELISA stop solution (Boston Bioproducts, Ashland, MA) was added to stop color development, and the absorbance was read at 450 nm using a SpectraMax plate reader within 30 minutes. The anti-drug antibody concentration is determined by interpolating the OD values ​​of a 4-PL pseudo-standard curve using SoftMax Pro Software (Molecular Devices, San Jose CA).

[0291] Example 15: Analysis procedure for identifying immunogenic epitopes

[0292] Maxisorp ELISA microplates (Nunc) are coated with a library of insulin-Fc fusion protein homodimeric compounds having known amino acid sequences, and the coated plates are blocked in a manner similar to that described in anti-drug antibody ELISA assay Examples 13 and 14, except that each compound in the library is coated on a separate individual strip of the ELISA microplate well. The compounds in the library include various insulin-Fc fusion proteins having different insulin polypeptide amino acid compositions, including various B-chain, C-chain, and A-chain amino acid mutations, including some of human origin, different linker compositions, and different Fc fragment compositions. Separately, some plate strip wells are directly coated with a 1:2 serial dilution of canine or feline IgG (Jackson Immunoresearch Laboratories, West Grove, PA) to count anti-drug antibodies (ADA) in canine or feline IgG units, as described in Examples 13 and 14, respectively.

[0293] Serum obtained from individual dogs or cats administered repeated doses of insulin-Fc fusion protein is first screened using an anti-drug antibody ELISA assay (Example 13 for dogs and Example 14 for cats). Serum samples showing intermediate or high positivity (e.g., intermediate or high antibody titers) in the assay of Example 13 or Example 14 are 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 an insulin-Fc fusion protein compound library at room temperature for 1 hour. After incubation, the plates are washed 5 times with PBST. For the detection of canine or feline antibodies capable of cross-reacting to a 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, is diluted to :10000 in PBST / SB, added at 100 μL / well to both sample and standard wells, and incubated at RT in the dark for 45 minutes. The plates are washed 5 times with PBST and 1 time with deionized water, and developed by adding 100 μL / well TMB substrate (Invitrogen, ThermoFisher Scientific, Waltham MA) for 15-20 minutes at room temperature in the dark. Then, 100 μL / well of ELISA stop solution (Boston Bioproducts, Ashland MA) is added to stop color development, and absorbance is read at 450 nm using a SpectraMax plate reader within 30 minutes.The concentration of anti-compound cross-reactive antibodies present in serum samples is determined by interpolating the OD values ​​of a 4-PL pseudo-standard curve for direct-coated dog or cat IgG antibody controls using SoftMax Pro Software (Molecular Devices, San Jose CA).

[0294] By correlating the antibody concentration obtained from the analysis with the known amino acid composition of the coated insulin-Fc fusion protein library, it is possible to determine whether a specific amino acid mutation or epitope causes no, some, most, or all of the total antibody signal in the analysis [which indicates no, weak, or strong binding to various insulin-Fc fusion protein homodimers]. Mutations or epitopes causing intermediate or strong binding are referred herein to as immunogenic "hot spots".

[0295] Example 16: Design process for obtaining an insulin-Fc fusion protein having high homodimeric titers and acceptable levels of acute and repeated-dose bioactivity in target species

[0296] The process for achieving the design objectives described in the detailed description of the present invention consists of the following steps. First, the insulin polypeptide of SEQ ID NO: 4 or SEQ ID NO: 5 is combined with a species-specific Fc fragment and linker of a specific IgG isotype, so that the resulting insulin-Fc fusion protein is most likely to yield a long-acting bioactive product with minimal immunogenicity (e.g., a species-specific IgG isotype having minimal Fc(gamma) receptor I binding is selected). A DNA sequence encoding the desired fusion protein is prepared and cloned into a vector (LakePharma, San Carlos, CA), and then HEK cells are transiently transfected using the vector according to the procedure described in Example 1. Next, the insulin-Fc fusion protein is purified according to Example 3, and the total protein yield and % homodimer are measured according to Example 6. Only candidates with a homodimeric titer of 50 mg / L or higher were considered acceptable, as titers below this level could not yield commercially viable potencies that meet the very low manufacturing cost requirements for veterinary products. Subsequently, the selected insulin-Fc fusion proteins were screened for indicators of bioactivity through in vitro insulin receptor binding studies, as described in Example 7. Based on experience, only compounds exhibiting an IR activity IC50 value of less than 5000 nM were considered to possess bioactivity in the target species. While in vitro IR IC50 values ​​are a useful qualitative screening tool, the use of human IM-9 cells expressing human insulin receptors may fail to capture some small differences in affinity between canine or feline IRs and human IRs. Furthermore, factors other than insulin receptor binding may influence the bioactivity of the compounds in vivo (e.g., affinity for canine or feline FcRn to enable an extended pharmacokinetic elimination half-life in vivo).Therefore, selected insulin-Fc fusion proteins acceptable in terms of manufacturing and IR activity IC50 values ​​were further screened for bioactivity in animals of interest (e.g., dogs or cats) to filter out any materials with less than the desired efficacy and / or duration of bioactivity (e.g., NAOC less than 150% FBGL·days·kg / mg). Once again, experience suggests that at NAOC values ​​greater than 150% FBGL·days·kg / mg, the dosage requirements in the target species would be sufficiently low to reach an acceptable therapeutic cost. Finally, an additional evaluation criterion, rarely mentioned in the industry, was added. As discussed in more detail in the examples below, many insulin-Fc fusion protein examples exhibiting acceptable NAOC levels in the target species after the first administration unexpectedly fail to maintain those levels of bioactivity after repeated administration. Furthermore, in most cases, the decrease in bioactivity upon repeated administration in the target species is associated with the development of neutralizing anti-drug antibodies. The inability to maintain this propensity and activity to generate anti-drug antibodies makes these insulin-Fc fusion proteins impractical for use in treating chronic diseases such as canine diabetes or feline diabetes. Therefore, only insulin-Fc fusion proteins that possess a minimal level of anti-drug antibodies and exhibit an acceptable level of repeated-dose bioactivity (e.g., a NAOCR value greater than 0.50 for the third dose compared to the first dose) were considered permissible for use in the present invention.

[0297] Result—Insulin-FC fusion protein containing the Fc fragment

[0298] Example 17: Canine insulin-Fc fusion protein containing canine Fc IgGA isotype

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

[0300] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVV SVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG (sequence Number: 42)

[0301] The insulin-Fc fusion protein of SEQ 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 reduced CE-SDS according to Example 4, and its sequence was further identified by glycan-removed LC-MS according to Example 5. The % homodimer content was measured by size-exclusion chromatography according to Example 6 and was determined to be 24%, indicating a high level of homodimer aggregates. Consequently, the resulting homodimer titer was only 5 mg / L. In summary, the production of the insulin-Fc fusion protein of SEQ No. 42 in HEK cells resulted in high levels of aggregates and a low homodimer titer (5 mg / L), which did not meet the design objective of a homodimer titer of 50 mg / L or higher.

[0302] Nevertheless, the insulin-Fc fusion protein of SEQ No. 42 was evaluated for bioactivity. First, the insulin receptor binding of the insulin-Fc fusion protein of SEQ No. 42 was measured according to Example 7, yielding an IC50 value of 2,733 nM, which indicates that the compound is likely bioactive in vivo (i.e., an IC50 of less than 5,000 nM).

[0303] Next, the in vivo pharmacokinetics (PD) of the insulin-Fc fusion protein of SEQ No. 42 were measured after administering the compound once intravenously to N = 3 dogs according to Example 10. Figure 2 shows the % fasting blood glucose levels of SEQ No. 42 as a function of time. The NAOC for SEQ No. 42 was calculated as 105% FBGL·days·kg / mg according to the procedure of Example 11. The in vivo half-life of SEQ No. 42 was calculated to be less than 1 day using the method of Example 12. The relatively low NAOC is likely the result of a large amount of aggregates (i.e., low % homodimers) in the sample, but the soluble homodimers remaining in circulation have a pharmacokinetic elimination half-life of less than 1 day, which is considered insufficient to support once-weekly administration.

[0304] Example 18: Mutation of the Fc fragment region of an insulin-Fc fusion protein containing dog IgGA isotypes

[0305] In an attempt to increase the % homodimer content, improve bioactivity, and increase the half-life of the insulin-Fc fusion protein of SEQ No. 42, a mutation was inserted into the CH3 region of the Fc fragment to prevent intermolecular binding (e.g., intermolecular Fc fragment-Fc fragment interactions) and to promote stronger binding to the FcRn receptor (e.g., higher affinity for FcRn) to increase recirculation and systemic circulation times. The insulin-Fc fusion protein below was synthesized in HEK cells according to Example 1, purified according to Example 3, and tested according to Examples 4-7 shown below. The sequence alignment and amino acid sequence differences of SEQ Nos. 44, 46, 48, and 50 for SEQ No. 42 are shown in FIG. 3.

[0306] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVV SVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHEALHSHYTQKSLSLSPG(sequence Number: 44)

[0307] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHETLQSHYTDLSLSHSPG(서열 번호: 46)

[0308] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQSHYTDLSLSHSPG(서열 번호: 48)

[0309] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHETLQNHYTDLSLSHSPG(서열 번호: 50)

[0310] Insulin-Fc fusion proteins based on canine IgGA variants are listed in Table 2, along with corresponding protein yields, % dimers, and dimer titers. The results show that various mutations in the IgGA Fc fragment resulted in highly aggregated proteins with very low dimer titers of less than 5 mg / L, instead of improving % dimers and dimer titers. Therefore, the in vivo bioactivity and pharmacokinetics of the compounds could not be evaluated.

[0311]

[0312] Example 19: Canine insulin-Fc fusion protein using a different canine Fc fragment isotype

[0313] As previously mentioned, canine IgGA is considered a preferred isotype for the Fc fragment to produce non-immunogenic insulin-Fc fusion proteins for dogs because it lacks Fc(gamma) I effector function in dogs (much like the human IgG2 isotype in humans). However, insulin-Fc fusion proteins prepared from the canine IgGA Fc fragment exhibited high aggregation and had unacceptably low homodimeric titers and unacceptably low levels of bioactivity and duration of action. Therefore, Fc fragments of other canine IgG isotypes (canine IgGB of SEQ No. 16, canine IgGC of SEQ No. 17, and canine IgGD of SEQ No. 18) were evaluated as substitutes for the canine IgGA Fc fragment of the insulin-Fc fusion of SEQ No. 42. Three insulin-Fc fusion proteins containing Fc fragments based on canine IgGB, IgGC, and IgGD isotypes were synthesized using the same insulin polypeptide of SEQ No. 5 and the peptide linker of SEQ No. 12, as used to prepare the insulin-Fc fusion protein of SEQ No. 42. The proteins were prepared in HEK293 cells according to Example 1. The insulin-Fc fusion proteins were purified using a Protein A column according to Example 3. The structures of the insulin-Fc fusion proteins were confirmed by non-reducing and reduced CE-SDS according to Example 4, and the sequences were further identified by glycan-removed LC-MS according to Example 5. % homodimers were measured by size-exclusion chromatography according to Example 6. These sequences are shown below, and a sequence alignment comparison for SEQ No. 42 is shown in Fig. 4.

[0314] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 52)

[0315] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG(서열 번호: 54)

[0316] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG(서열 번호: 56)

[0317] The generated protein yields, % homodimers, and homodimer titers are provided in Table 3. Unexpectedly, only the insulin-Fc fusion protein of Sequence No. 52, containing an Fc fragment based on a canine IgGB isotype, demonstrated a homodimer titer meeting the design criteria of 50 mg / L or higher. The insulin-Fc fusion protein of Sequence No. 54, containing an Fc fragment based on a canine IgGC isotype, produced no compounds, and the insulin-Fc fusion protein of Sequence No. 56, containing an Fc fragment based on a canine IgGD isotype, showed a significant protein yield but exhibited a high level of aggregation, resulting in an unacceptably low homodimer titer.

[0318] In vitro insulin receptor binding to the insulin-Fc fusion proteins of SEQ No. 52 and SEQ No. 56 was tested according to the procedure of Example 7. The insulin-Fc fusion protein of SEQ No. 56 showed an IC50 of 5000 nM or higher, which indicates that the compound is highly unlikely to exhibit bioactivity in vivo. However, the insulin-Fc fusion protein of SEQ No. 52 showed an IC50 of 28 nM, which indicates that the sequence may exhibit bioactivity in vivo.

[0319]

[0320] Example 20: In vivo efficacy of an insulin-Fc fusion protein comprising the insulin polypeptide of SEQ No. 5 having an IgGB isotype Fc fragment

[0321] Given the promising homodimeric titer and insulin receptor activity results in Example 19, the insulin-Fc fusion protein of SEQ No. 52 was intravenously injected into each of N = 3 healthy, antibody-free Beagle dogs weighing approximately 10 kg, and then tested for in vivo bioactivity according to Example 10. In a separate experiment, the compound was administered subcutaneously to N = 3 naive Beagle dogs. Figure 5 shows % FBGL versus time for a single intravenous administration of the insulin-Fc fusion protein of SEQ No. 52, and Figure 6 shows % FBGL versus time for a single subcutaneous administration of the insulin-Fc fusion protein of SEQ No. 52, both of which demonstrate that the insulin-Fc fusion protein of SEQ No. 52 is significantly bioactive in dogs.

[0322] 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 intravenously injected insulin-Fc fusion protein SEQ No. 52 was 399% FBGL·days·kg / mg, which was 3.8 times the NAOC of the intravenously injected insulin-Fc fusion protein SEQ No. 42. This indicates significantly increased bioactivity compared to the insulin-Fc fusion protein containing the canine IgGB Fc fragment versus the insulin-Fc fusion protein containing the canine IgGA Fc fragment. The NAOC of the subcutaneously injected insulin-Fc fusion protein SEQ No. 52 was 366% FBGL·days·kg / mg, which shows a level of bioactivity through subcutaneous administration that is similar to that obtained through intravenous administration.

[0323] Example 21: In vivo immunogenicity screening of an insulin-Fc fusion protein comprising the insulin polypeptide of SEQ No. 5 having an IgGB isotype Fc fragment

[0324] Next, the bioactivity of the insulin-Fc fusion protein of sequence number: 52 upon repeated subcutaneous administration was tested in dogs according to the method described in Example 11. N = 3 animals were administered subcutaneously on days 0, 35, and 42, and % FBGL was measured for a 7-day window after each administration according to Example 11. NAOC and NAOCR were calculated for each repeated subcutaneous injection according to the procedure of Example 11. As illustrated in Table 4, repeated subcutaneous administration in dogs unexpectedly showed a significant decrease in bioactivity upon the third administration, as measured by a significant decrease in NAOCR (i.e., the NAOC for the third injection was only 0.40 or 40% of the NAOC for the first injection).

[0325]

[0326] Without being bound by specific descriptions, the cause of the significant decrease in the bioactivity of the insulin-Fc fusion protein of SEQ No. 52 after a third repeated subcutaneous administration in dogs was presumed to be due to the development of anti-drug antibodies that neutralize bioactivity. Anti-drug antibodies may be directed against the insulin polypeptide, linker, or Fc-fragment portion of the insulin-Fc fusion protein. Immunogenic responses manifest as interactions between antigen-presenting cells, T-helper cells, B-cells, and related cytokines, which can generate endogenous antibodies against the drug (e.g., anti-drug antibodies). Binding antibodies are all isotypes capable of binding to the insulin-Fc fusion protein and can be detected in immunoassays as described in Example 13. Neutralizing antibodies that inhibit the functional activity of the insulin-Fc fusion protein are generally directed against the epitope required for bioactivity. To evaluate whether this was true, serum collected before the administration of each dose and at the end of the experiment as described in Examples 11 and 12 was tested to quantify the level of anti-drug antibodies according to Example 13. As shown in Figure 7, the level of anti-drug antibodies actually increased with multiple subcutaneous administrations of the compound, which indicates that the production of neutralizing anti-drug antibodies may be the cause of the decrease in NAOCR after the third injection of the insulin Fc-fusion protein of SEQ No. 52.

[0327] Example 22: A non-glycosylated insulin-Fc fusion protein comprising the insulin polypeptide of SEQ No. 5 having a canine IgGB isotype Fc fragment to reduce the potential risk of immunogenicity

[0328] As shown in Examples 19 and 20, the insulin-Fc fusion protein of SEQ No. 52 exhibited acceptable % homodimeric content, homodimeric titer, and bioactivity in dogs; however, its use for chronic diseases such as diabetes is hindered by a decrease in bioactivity due to repeated subcutaneous administration (Example 21) and the generation of anti-drug antibodies (Example 21). Without being bound by a specific theory, one possible cause of the generation of anti-drug antibodies and the decrease in bioactivity is the increased interaction between the canine IgGB Fc fragment and various receptors of the canine immune system (e.g., Fc(gamma) receptors, e.g., Fc(gamma) RI). Nevertheless, the canine IgGB isotype was the only one of four canine IgG isotypes that produced an insulin-Fc fusion protein that met the design goals of manufacturability and single-dose bioactivity when used with the Fc fragment (Example 16). As described in the detailed description of the present invention, one method for reducing Fc(gamma) interactions involves mutating the cNg site of the Fc fragment to prevent glycosylation during synthesis in host cells. Accordingly, to reduce the binding affinity of the Fc fragment to the in vivo Fc(gamma) receptor, as measured by binding in the in vitro human Fc(gamma) RI assay described in Example 8, a cNg site mutation was made in the Fc fragment region of SEQ No. 52. Verification of glycan deficiency was performed using the LC-MS method of Example 5, but with the PNGase F treatment step omitted. The position of the cNg site in the insulin-Fc fusion protein of SEQ No. 52 is cNg-NB139.Mutations for SEQ No. 52 include SEQ No. 58, which contains a mutation in cNg-NB139-Q; SEQ No. 60, which contains a mutation in cNg-NB139-S; SEQ No. 62, which contains a mutation in cNg-NB139-D; and SEQ No. 64, which contains a mutation in cNg-NB139-K. The full amino acid sequences of the cNg-mutated insulin-Fc fusion proteins are listed below (NB139 positions are underlined), and the resulting sequence alignment is shown in Fig. 8 (cluster omega).

[0329] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF Q GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 58)

[0330] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 60)

[0331] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF D GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 62)

[0332] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF K GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 64)

[0333] 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 proteins was confirmed by non-reducing and reduced CE-SDS according to Example 4, and their sequences were further identified by LC-MS with glycan removal according to Example 5. % dimers were measured by size-exclusion chromatography according to Example 6. As shown in Table 5, the dimer titers of the insulin-Fc fusion proteins of sequence number: 60, sequence number: 62, and sequence number: 64 met the design objectives, but the insulin-Fc fusion protein of sequence number: 58, which unexpectedly contains the cNg-NB139-Q mutation, did not meet the design objectives for dimer titers.

[0334]

[0335] To determine which of the remaining three compounds was most likely to exhibit reduced immunogenicity, Fc(gamma) receptor binding was measured according to the procedure of Example 8. Low Fc(gamma) receptor binding is most likely to correlate with minimal immunogenicity. Table 6 compares the Fc(gamma) receptor binding of these insulin-Fc fusion proteins with the Fc(gamma) receptor binding of the insulin-Fc fusion protein of SEQ No. 52; unexpectedly, the insulin-Fc fusion protein of SEQ No. 62, containing the cNg-D mutation, exhibits Fc(gamma) receptor binding activity approximately twice that of the insulin-Fc fusion proteins of SEQ No. 60, containing the cNg-S mutation, and SEQ No. 64, containing the cNg-K mutation. Therefore, only the insulin-Fc fusion proteins containing the latter two compounds, containing the cNg-S and cNg-K mutations, were considered suitable for bioactivity testing upon repeated administration in dogs.

[0336]

[0337] Example 23: Evaluation of in vivo bioactivity and immunogenicity of the insulin polypeptide of SEQ No. 5 having non-glycosylated cNg-K and cNg-S IgGB isotype Fc fragments

[0338] To determine whether the insulin-Fc fusion protein of SEQ No. 60 containing the cNg-S mutation improved bioactivity upon repeated administration in dogs, the compound was administered subcutaneously to N = 1 dog 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 the blood glucose to a safe level. The NAOC for the first injection was 191% FBGL·days·kg / mg, which indicates that the insulin-Fc fusion protein of SEQ No. 60 is satisfactorily bioactive in vivo. NAOC and NAOCR were also measured for each subsequent dose according to the general procedure of Example 11, calculated from the time the dose was administered until immediately before the next dose was administered. The NAOC and NAOCR shown in Table 7 illustrate that the insulin-Fc fusion protein of sequence number: 60 exhibited significantly reduced NAOCR at doses 3 and 4 of the 4-dose regimen. Therefore, although the insulin-Fc fusion protein of sequence number: 60 containing the cNg-S mutation had a 4-fold lower Fc(gamma)RI binding than the insulin-Fc fusion protein of sequence number: 52, it could not demonstrate bioactivity at repeated doses in dogs.

[0339]

[0340] To determine whether the insulin-Fc fusion protein of SEQ No. 64 containing the cNg-K mutation improves the bioactivity of repeated doses in dogs, the compound was administered subcutaneously to N = 1 dog 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 the blood glucose to a safe level. The NAOC for the first injection was 449% FBGL·days·kg / mg, indicating that the insulin-Fc fusion protein of SEQ No. 64 is satisfactorily bioactive in vivo. The pharmacokinetic profile of the compound was also measured using ELISA according to the method of Example 12, and a 2-compartment model was fitted to the data to determine its elimination half-life, which was approximately 0.9 days. NAOC and NAOCR were also measured for each subsequent dose according to the general procedure of Example 11, calculated from the time the dose was administered until immediately before the next dose was administered. The NAOC and NAOCR shown in Table 8 illustrate that the insulin-Fc fusion protein of sequence number: 64 maintained an NAOCR greater than 0.6 over four doses. Thus, the insulin-Fc fusion protein of sequence number: 64, which unexpectedly contained a cNg-K mutation, was the only non-glycosylated mutant of the insulin-Fc fusion protein of sequence number: 52, and consequently, the bioactivity of repeated doses in dogs was significantly improved.

[0341]

[0342] Levels of anti-drug and anti-insulin antibodies were also measured during the treatment course (28 days) and for an additional 2 weeks according to Example 13. Figure 9 shows that although the insulin-Fc fusion protein of sequence number: 64 still produced anti-drug antibodies upon repeated subcutaneous administration in dogs, the anti-drug antibody titers were much lower than those produced by the insulin-Fc fusion protein of sequence number: 52 (Example 19).

[0343] Example 24: Screening of dog serum containing anti-drug antibodies and identification of potential immunogenic epitopes at the B10D and A8H positions of insulin polypeptide

[0344] Mutating the cNg site of the canine IgGB Fc fragment to Lys (i.e., cNg-K) improved the bioactivity of repeated doses of an insulin-fusion protein containing the insulin polypeptide of SEQ No. 5 and the peptide linker of SEQ No. 12 (Example 23). However, the resulting insulin-Fc fusion protein of SEQ No. 64 still elicited anti-drug antibodies (Example 23). Therefore, it was hypothesized that the insulin polypeptide of SEQ No. 5 might unexpectedly contain a specific epitope (i.e., an immunogenic "hot spot") to which the canine immune system is directed. Accordingly, the binding specificity of antibodies present in the serum sample described in Example 13 was evaluated according to the general procedure of Example 15. Analysis of antibody-containing serum samples from repeated administration of the insulin-Fc fusion protein of sequence number: 52 to a coated insulin-Fc fusion protein library (Example 19) revealed the presence of two unexpected primary "hot spots" within the insulin polypeptide sequence of sequence number: 5: an aspartic acid mutation at the 10th position from the N-terminus of the B-chain (i.e., B10), and, separately, a histidine mutation at the 8th position from the N-terminus of the A-chain (i.e., A8). The results suggest that insulin-Fc fusion proteins containing an insulin polypeptide amino acid composition containing these two specific amino acid mutations are likely to be immunogenic in dogs, and thus may generate anti-drug antibodies that neutralize bioactivity after repeated injections. Therefore, it was determined that an insulin polypeptide not containing B10 aspartic acid and A8 histidine is preferred for insulin-Fc fusion proteins that need to be administered repeatedly to dogs over a long period (e.g., treatment of canine diabetes).

[0345] Example 25: An insulin-Fc fusion protein comprising the insulin polypeptide of SEQ No. 5, and a non-glycosylated canine IgGB isotype Fc fragment in which the B10D and A8H mutations of the insulin polypeptide are restored to their natural composition to reduce the potential risk of immunogenicity.

[0346] To evaluate whether replacing the "hot spot" mutation would improve the immunogenicity and repeated-dose bioactivity of an insulin-Fc fusion protein comprising the insulin polypeptide of SEQ No. 5 and a canine IgGB isotype fragment, an exemplary insulin-Fc fusion protein (SEQ No. 66) was synthesized in which the B10 and A8 amino acids of the insulin polypeptide were restored to the natural histidine and threonine compositions (SEQ No. 125), respectively. The non-natural amino acids are listed below underlined.

[0347] FVNQHLCGSHLVEAL A LVCGERGFFYT DP T GGGPRR GIVEQCCTSICSLYQLENYCN(Sequence No.: 125)

[0348] Furthermore, considering the additional potential benefits of non-glycosylated cNg mutants, the insulin-Fc fusion protein of Sequence No. 66 contains a cNg-Q mutant. The full amino acid sequence of the insulin-Fc fusion protein of Sequence No. 66 is as follows:

[0349] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFQGTYRVVS VLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 66)

[0350] Insulin-Fc fusion protein of sequence number: 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 reduced CE-SDS according to Example 4, and the sequence was further identified by LC-MS after removing glycans according to Example 5. The % isomer measured by size-exclusion chromatography according to Example 6 was 98.0%, which indicates that there are relatively no aggregates in the protein.

[0351] Despite a relatively low homodimeric titer of 21 mg / L, the insulin-Fc fusion protein of sequence number: 66 was evaluated in vivo for bioactivity and immunogenicity in dogs according to the procedures of Examples 11-13, respectively. Figure 10 shows that the restoration of the B10D and A8H mutations in the insulin-Fc fusion protein of sequence number: 66 to their natural amino acids (i.e., B10H and A8T) significantly reduced the immunogenicity of the parent compound (sequence number: 52).

[0352] However, as shown in Fig. 11, the insulin-Fc fusion protein of sequence number: 66 containing natural B10 and A8 amino acids was not bioactive (i.e., NAOC was essentially 0).

[0353] Example 26: Attempt to incorporate additional B-chain and A-chain mutations into the insulin polypeptide of SEQ No. 125 to improve the bioactivity of an associated insulin-Fc fusion protein containing a dog IgGB Fc fragment

[0354] The fact that the insulin-Fc fusion protein of SEQ No. 66 did not produce anti-drug antibodies (Example 25) compared to the insulin-Fc fusion protein of SEQ No. 52 (Example 20) provides strong evidence for the theory that the B10D and A8H mutations in the insulin polypeptide of SEQ No. 5 are likely immunogenic epitopes responsible for the production of anti-drug antibodies. However, the lack of in vivo efficacy of the insulin-Fc fusion protein of SEQ No. 66 compared to that of SEQ No. 52 also indicates that these two amino acid mutations are responsible for achieving an acceptable level of bioactivity. The lack of in vivo efficacy for the insulin-Fc fusion protein of SEQ No. 66 is associated with a high IC50 (shown in Table 9 below) measured by insulin receptor binding analysis according to the method of Example 7. Therefore, additional efforts were required to increase the bioactivity of the insulin-Fc fusion protein (i.e., to reduce the insulin receptor binding assay IC50 value to less than 5000 nM, more preferably less than 4000 nM, or even more preferably less than 3000 nM) while maintaining a low level of immunogenicity by retaining natural B10 and A8 amino acids in the insulin polypeptide.

[0355] It is known that various parts of the insulin B-chain and A-chain are required for strong binding to IR (Hubbard SR, "Structural biology: Insulin meets its receptor", Nature. 2013; 493(7431):171-172). Therefore, parts of the B-chain or A-chain were modified while maintaining B10 and A8 identical to those in natural insulin and keeping the C-chain and peptide linker constant. Some of these 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 reduced CE-SDS according to Example 4, and their sequences were further identified by glycan-removed LC-MS according to Example 5. Their % homodimer content was measured by size-exclusion chromatography according to Example 6, and insulin receptor binding affinity was measured according to Example 7. Their sequences are as follows. The resulting sequence alignment for sequence number: 66 is shown in FIG. 12 (cluster omega).

[0356] FVNQHLCGSHLVQALYLVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSV LPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 68)

[0357] FVNQHLCGSELVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 70)

[0358] FVNQHLCGSHLVEALALVCGEAGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 72)

[0359] FVNQHLCGSHLVEALALVCGERGFYYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 74)

[0360] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSV LPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 76)

[0361]

[0362] In only three cases (Sequence Nos. 68, 70, and 74), the proposed mutation improved IR binding (i.e., lowered the IC50 value) compared to Sequence No. 66. However, no mutation occurred in compounds meeting the manufacturing design objectives of homodimeric titers of 50 mg / L or higher, and in some cases, the mutation significantly reduced manufacturing feasibility (e.g., homodimeric titers of less than 20 mg / L).

[0363] Example 27: Attempt to incorporate a C-chain mutation into the insulin polypeptide of SEQ No. 125 to improve the bioactivity of an associated insulin-Fc fusion protein containing a dog IgGB Fc fragment

[0364] The results obtained in Example 26 show that all attempts to mutate the A-chain and B-chain of the insulin polypeptide of SEQ No. 125 resulted in unacceptable low HEK homodimer titers of the associated insulin-Fc fusion protein (i.e., homodimer titers of 25 mg / L or less). Therefore, further experiments were required. In this example, the C-chain composition of the insulin polypeptide of SEQ No. 125 was mutated by making it longer or increasing its flexibility. Natural insulin (e.g., human insulin) has been shown to undergo significant conformational changes, including movements of B-chain and A-chain folding, when binding to insulin receptors (e.g., Menting, et al., Nature, 2013; 493(7431):pp241-245). Unlike the insulin polypeptide of the present invention, natural insulin can freely undergo such conformational changes at the insulin receptor, because it is a 2-chain polypeptide in its natural form and is connected only by two disulfide bonds without a C-chain that restricts the mobility of the A-chain and B-chain. Without being bound by any specific theory, the C-chain contained within the insulin polypeptide of SEQ No. 125 is not flexible enough (e.g., amino acid composition and sequence that does not allow easy movement between the B-chain and the A-chain) and / or is too short (e.g., insufficient amino acids between the C-terminus of the B-chain and the N-terminus of the A-chain), which prevents the insulin polypeptide from undergoing the necessary molecular conformational changes required for strong binding to the insulin receptor. Accordingly, several insulin-Fc fusion proteins were synthesized based on the insulin-Fc fusion protein of sequence number: 66 having a variation in the insulin polypeptide C-chain as listed below, and consequently, the sequence alignment for sequence number: 66 is shown in FIG. 13 (cluster omega).

[0365] FVNQHLCGSHLVQALYLVCGERGFFYTDPTQRGGGGGQRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 78)

[0366] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGGGGSGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 80)

[0367] FVNQHLCGSHLVEALALVCGERGFFYTDPGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 82)

[0368] FVNQHLCGSHLVEALALVCGERGFFYTPGGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSV LPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 84)

[0369]

[0370] 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 reduced CE-SDS according to Example 4, and their sequences were further identified by glycan-removed LC-MS according to Example 5. Their % isomer content was measured by size-exclusion chromatography according to Example 6, and insulin receptor binding affinity was measured according to Example 7. In only one case containing the longest C-chain (GGGGGGSGGGG) (Sequence No. 80), the C-chain mutation significantly improved insulin receptor binding affinity (IC50 < 3000 nM) compared to the insulin-Fc fusion protein of Sequence No. 66. However, none of these C-chain mutated insulin-Fc fusion proteins exhibited an isomer titer greater than the preparation design target of 50 mg / L. In fact, in one case (sequence number: 78), the C-chain mutation resulted in an unexpectedly low homodimeric titer.

[0371] Example 28: An attempt to improve bioactivity by incorporating a peptide linker mutation into an insulin-Fc fusion protein containing the insulin polypeptide of SEQ No. 125 and a canine IgGB Fc fragment.

[0372] Without being bound by a specific theory, another possible reason for the poor insulin receptor binding of the insulin-Fc fusion protein of sequence number 66 was thought to be the steric disturbance between the insulin polypeptide and the insulin receptor caused by the proximity of the much larger Fc fragment molecule attached to the insulin polypeptide via a peptide linker. Shorter peptide linkers or tighter-folded peptide linkers were thought to potentially exacerbate this problem, while longer peptide linkers or peptide linkers that resist folding themselves (e.g., linkers with greater molecular stiffness) could mitigate this problem by creating more space between the insulin polypeptide and the Fc fragment. Increased space between the insulin polypeptide and the Fc fragment would also increase the distance between the insulin receptor and the Fc fragment, thereby reducing interference during insulin receptor binding. The peptide linker of sequence number 12 (i.e., GGGGAGGGG) used to construct the insulin-Fc fusion protein of sequence number 66 was hypothesized to be potentially too short or / or too flexible, as the amino acids containing the linker do not contain any side chains (i.e., contain only glycine and alanine amino acids). Therefore, to test this hypothesis, two different insulin-Fc fusion protein variants of the insulin-Fc fusion protein of sequence number 66 were synthesized. The insulin-Fc fusion protein of sequence number 76 contains the same peptide linker used to construct the insulin-Fc fusion protein of sequence number 66, but the insulin polypeptide lacked asparagine at the 21st position from the N-terminus of the A chain (i.e., A21) (i.e., des-A21). This specific mutation was incorporated to determine whether the splicing between the A chain and the peptide linker affects the protein yield and / or bioactivity of the molecule.Another insulin-Fc fusion protein of SEQ No. 86 contains a peptide linker that is more than twice the length used to construct this des-A21N A-chain mutation and the insulin-Fc fusion protein of SEQ No. 66. In this long peptide linker, alanine is disfavored and is instead replaced by glutamine containing a polar amide side chain. The glutamine substitution was expected to increase the hydrophilicity of the peptide linker and potentially prevent the linker from refolding against itself. The sequence is presented below, along with the resulting sequence alignment for SEQ No. 66 (cluster omega) shown in Fig. 14.

[0373] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 86)

[0374] FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSV LPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 76)

[0375]

[0376] Two 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 reduced CE-SDS according to Example 4, and their sequences were further identified by glycan-removed LC-MS according to Example 5. Their % homodimer content was measured by size-exclusion chromatography according to Example 6, and insulin receptor binding affinity was measured according to Example 7. The incorporation of longer peptide linkers of different compositions (GGGGGGQGGGGQGGGGQGGGGG for SEQ No. 86 versus GGGGAGGGG for SEQ No. 66) enhanced insulin receptor binding, as measured by a significant decrease in IC50 values, suggesting that longer linkers may be a strategy to increase insulin receptor binding for different insulin-Fc fusion proteins. However, the incorporation of longer linkers still failed to improve the homodimer potency beyond the manufacturing design target of 50 mg / L or higher.

[0377] Example 29: Attempt to delete the B-chain portion of the insulin polypeptide of SEQ No. 125 to improve the homodimeric titer of an associated insulin-Fc fusion protein containing a dog IgGB Fc fragment

[0378] The results of Example 28 demonstrate that the peptide linker can be modified to increase the insulin receptor binding affinity of the insulin-Fc fusion protein of SEQ No. 66, which contains natural B10 and A8 amino acids. However, the peptide linker mutation did not increase the homodimeric titer sufficiently to meet the manufacturing design objectives. Since homodimeric titer is a function of several properties, including intracellular synthesis and intracellular processing, it was assumed that the insulin-Fc molecules likely self-coupled (i.e., aggregated) during and after synthesis, either within the molecule between the two monomers of the homodimeric or between molecules of two or more separated homodimerics. This aggregation would result in unacceptably low homodimeric titers obtained from the cell culture supernatant during the production processes described in Examples 1, 3, and 6. These potential interactions between insulin-Fc fusion protein molecules are partly due to the known tendency of insulin to self-couple and form aggregates. One method known in the industry to reduce the tendency of insulin to self-bind involves mutating amino acids near the C-terminus of the B-chain. For example, insulin lispro (B28K; B29P mutation) and insulin aspart (B28D mutation) are well-known commercial 2-chain insulins containing non-natural B-chain mutations that prevent binding and aggregation, producing insulin in a primarily monomeric form in solution. Another approach to preventing aggregation involves the deletion of amino acid structures. For example, despentapeptide insulin (DPPI; Brange J., Dodson GG, Edwards J., Holden PH, Whittingham JL 1997b. The 2-chain insulin known as the “insulin fibril model (despentapeptide insulin) derived from the X-ray crystal structure of monomeric insulin” (see Protein 27 507-516) is identical to natural 2-chain human insulin except that five C-terminal amino acids (YTPKT) of the B-chain are removed. Although DPPI has a lower binding affinity for insulin receptors compared to natural 2-chain human insulin, it is completely monomeric in solution, which means there is no significant binding or aggregation between DPPI molecules. Therefore, to reduce the possibility of intramolecular and intermolecular self-binding and to improve the homodimeric titers of the insulin-Fc fusion protein, several variants of the insulin-Fc fusion protein of SEQ No. 66 were constructed using partial B-chain amino acid cleavages and B-chain amino acid mutations, as described above for DPPI, insulin lispro, and insulin aspart. The sequence is presented below, along with the resulting sequence alignment for sequence number: 66 (cluster omega) shown in Fig. 15.

[0379] FVNQHLCGSHLVEALALVCGERGFFYTDPGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVS VLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 82)

[0380] FVNQHLCGSHLVEALALVCGERGFFYTPGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 84)

[0381] FVNQHLCGSHLVEALALVCGERGFFYTQGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 88)

[0382]

[0383] 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 reduced CE-SDS according to Example 4, and their sequences were further identified by glycan-removed LC-MS according to Example 5. Their % dimer content was measured by size-exclusion chromatography according to Example 6, and their insulin receptor binding affinity was measured according to Example 7. The dimer titers of the resulting compounds were uniquely and significantly increased in one case (Sequence No. 82), but unexpectedly, insulin receptor affinity was enhanced for all mutated compounds (Sequence Nos. 82, 88, and 84).

[0384] Example 30: An attempt to further improve homodimeric titer and bioactivity by binding B-chain, C-chain, and A-chain mutations, B-chain cleavage, and linker mutations to the insulin-Fc fusion protein of SEQ No. 66.

[0385] As shown in Examples 26, 27, 28, and 29, there is no single strategy for successfully incorporating an insulin polypeptide containing non-immunogenic natural B10 and A8 amino acids into a dog IgGB Fc fragment to form an insulin-Fc fusion protein having acceptable insulin receptor activity and homodimeric titer. Therefore, the concepts of a longer C-chain, a longer peptide linker, and C-terminal amino acid cleavage of the B-chain were combined. Additionally, to potentially further reduce self-binding and aggregation tendencies, additional point mutations were introduced into the natural insulin hydrophobic amino acid residue sites using less hydrophobic amino acids, including those having negatively or positively charged side groups at physiological pH. Exemplary mutations include variations from tyrosine to alanine, tyrosine to glutamic acid, isoleucine to threonine, and phenylalanine to histidine. Additionally, to simplify the analysis, in all cases, the cNg site of the canine IgGB Fc fragment was restored to natural asparagine. The sequences for these insulin-Fc fusion protein variants are presented below, along with the resulting sequence alignment for sequence number: 66 (cluster omega) shown in Fig. 16.

[0386] FVNQHLCGSHLVEALELVCGERGFFYTPKTGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNG TYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 90)

[0387] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNHGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 92)

[0388] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 34)

[0389] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 32)

[0390] FVNQHLCGSHLVEALELVCGERGFFYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 94)

[0391]

[0392] 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 reduced CE-SDS according to Example 4, and their sequences were further identified by glycan-removed LC-MS according to Example 5. Their % homodimer content was measured by size-exclusion chromatography according to Example 6, and insulin receptor binding affinity was measured according to Example 7. The results show that a combination of reducing the hydrophobicity of specific B-chain and A-chain amino acids, using longer and more flexible C-peptide sequences, cleaving several C-terminal B-chain amino acids, and using longer peptide linkers can yield several useful insulin-Fc fusion proteins that meet the design criteria for minimum homodimer titer and insulin receptor binding activity. Sequence numbers 92, 34, 32 and 94 (368d), (366d), (218d), and (375d) show more desirable insulin receptor IC50 values ​​(less than 3000 nM) and more desirable HEK homodimer titer values ​​(greater than 100 mg / L) than Sequence number 66 or Sequence number 90. Surprisingly, changing only a few amino acids improves insulin receptor affinity several-fold, and in the case of the insulin-Fc fusion protein of Sequence number 32, a dramatic increase in homodimer titer was observed compared to the original insulin-Fc fusion protein of Sequence number 66.

[0393] Example 31: In vivo bioactivity, bioactivity at repeated doses, and immunogenicity of an insulin-Fc fusion protein constructed from the insulin polypeptide of SEQ No. 7, the peptide linker of SEQ No. 14, and the canine IgGB Fc fragment of SEQ No. 16

[0394] Given the positive homodimeric titers and insulin receptor binding activity results from Example 30, the two most promising insulin-Fc fusion proteins (SEQ Nos. 32 and 34) were tested in dogs to evaluate their bioactivity and immunogenicity at repeated doses. Each compound contains the longer, more hydrophilic peptide linker of SEQ No. 14 and the more manufacturable and less aggregating canine IgGB Fc fragment of SEQ No. 16. Most importantly, both insulin-Fc fusion proteins contain an insulin polypeptide having putatively less immunogenic natural B10 and A8 amino acids (i.e., generic SEQ No. 7). In the case of the insulin-Fc fusion protein of SEQ No. 34, asparagine is present at position A21 (i.e., the insulin polypeptide contains SEQ No. 9). In the case of the insulin-Fc fusion protein of sequence number 32, asparagine is absent at position A21 (i.e., the insulin polypeptide contains sequence number 8).

[0395] The in vivo bioactivity of the insulin-Fc fusion protein of SEQ No. 34 was tested in N = 1 dog according to the procedure of Example 10. The results shown in Fig. 17 for a single subcutaneous administration demonstrate that the insulin-Fc fusion protein of SEQ No. 34 is indeed bioactive in vivo with an NAOC of 1076% FBGL·days·kg / mg calculated according to the procedure of Example 11. The pharmacokinetic profile of the insulin-Fc fusion protein of SEQ No. 34 was measured by the method of Example 12 using ELISA, and a 2-compartment model was fitted to the data to determine its elimination half-life of 3.5 days.

[0396] Next, the bioactivity of repeated doses was evaluated by continuously administering the insulin-Fc fusion protein of SEQ No. 34 to N = 1 dogs on days 14, 28, and 42 after the initial injection, following the procedure of Example 8. When the dogs' % FBGL dropped too low, food was provided to raise blood glucose levels to a safe level. NAOC and NAOCR were measured for each subsequent dose according to the general procedure of Example 11, calculated from the time of dose administration until immediately before the next dose was administered. The NAOC and NAOCR shown in Table 14 demonstrate that the insulin-Fc fusion protein of SEQ No. 34 maintained an NAOCR greater than 0.8 over four doses, satisfying the design objective for the bioactivity of repeated doses.

[0397]

[0398] The immunogenicity of the insulin-Fc fusion protein of sequence number: 34 was tested according to the procedure of Example 13. Figure 18 shows that the insulin-Fc fusion protein of sequence number: 34 does not exhibit apparent immunogenicity in vivo, along with the maintenance of in vivo bioactivity through repeated dose experiments.

[0399] The insulin-Fc fusion protein of SEQ No. 32, which lacks asparagine at A21 of the insulin polypeptide chain, was evaluated for repeated dose bioactivity in dogs. The compound was administered subcutaneously to N = 1 dog on days 0, 14, 28, and 42 according to the procedure of Example 11. When the dogs' % FBGL dropped too low, food was provided to raise blood glucose to a safe level. The NAOC for the first injection was an impressive 2278% FBGL·days·kg / mg, demonstrating that the insulin-Fc fusion protein of SEQ No. 32 is satisfactorily bioactive in vivo with nearly twice the efficacy of the insulin-Fc fusion protein of SEQ No. 34. The pharmacokinetic profile of the insulin-Fc fusion protein was measured using ELISA according to the method of Example 12, and a 2-compartment model was fitted to the data to determine an elimination half-life of 4.1 ± 0.7 days. Figures 19 and 20 show a single-dose blood glucose control and a multiple-dose, multi-dose blood glucose control for animals that received the homodimer of SEQ No. 32. AOC and NAOCR were measured for each subsequent dose according to the general procedure of Example 11, calculated from the time the dose was administered until immediately before the next dose was administered. The NAOC and NAOCR shown in Table 15 demonstrate that the insulin-Fc fusion protein of SEQ No. 32 maintained an NAOCR of 1.0 or higher over four doses, satisfying the bioactive design objectives for repeated doses described in Example 16.

[0400] The immunogenicity of the insulin-Fc fusion protein of sequence number: 32 was tested according to the procedure of Example 13. Figure 21 shows that the insulin-Fc fusion protein of sequence number: 32 maintained bioactivity in vivo through repeated dose experiments and did not exhibit apparent immunogenicity in vivo.

[0401]

[0402] As discussed in the detailed description 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). In the insulin polypeptide of SEQ No. 8 included in the insulin-Fc fusion protein of SEQ No. 32 together with the peptide linker of SEQ No. 14, omitting asparagine at the 21st amino acid of the A chain (i.e., A21) 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 No. 34 includes the peptide linker of SEQ No. 14 and the insulin polypeptide of SEQ No. 8 retaining asparagine at A21. Therefore, the insulin-Fc fusion protein of SEQ No. 34 would have been expected to be enzymatically digested in vivo after subcutaneous administration or during synthesis. However, rather unexpectedly, the insulin-Fc fusion protein of SEQ No. 34 could be produced in HEK cells with acceptable homodimeric titers and showed acceptable bioactivity in vivo without signs of enzymatic digestion that impairs bioactivity.

[0403] Example 32: Identification of a canine IgGB isotype Fc fragment for optimal manufacturability and in vivo efficacy of an insulin-Fc fusion protein comprising the preferred insulin polypeptide of SEQ No. 8 and the preferred peptide linker of SEQ No. 14

[0404] Even after discovering a new insulin polypeptide and peptide linker combination that produces a non-immunogenic, high-yield, high-purity, and highly bioactive insulin-Fc fusion protein as described in Examples 30 and 31, the question remained whether the canine IgGB Fc fragment was still the preferred isotype in terms of homodimeric potency and bioactivity, as in the case of the insulin-Fc fusion protein in Examples 19 and 20. Therefore, additional insulin-Fc fusion proteins were designed, in which the insulin polypeptide (SEQ No. 8) and peptide linker (SEQ No. 14) of the insulin-Fc fusion protein of SEQ No. 32 were maintained, and the canine IgGB Fc fragment of SEQ No. 16 was replaced with the canine IgGA Fc fragment of SEQ No. 15, the canine IgGC Fc fragment of SEQ No. 17, or the canine IgGD Fc fragment of SEQ No. 18. The sequences for these generated insulin-Fc fusion protein variants are as follows:

[0405] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNG TYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(sequence Number: 32)

[0406] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG(서열 번호: 96)

[0407] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG(서열 번호: 98)

[0408] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG(서열 번호: 100)

[0409] Insulin-Fc fusion proteins were prepared in HEK293 cells according to Example 1 and purified using a Protein A or Protein G column according to Example 3. Their structures were confirmed by non-reducing and reduced CE-SDS according to Example 4. Sequences were further identified by glycan-removed LC-MS according to Example 5. Their % homodimer content was measured by size-exclusion chromatography according to Example 6, and insulin receptor binding affinity was measured according to Example 7. Additionally, 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 Sequence No. 32, containing the canine IgGB Fc fragment, showed the highest homodimer titer among these sequences. The insulin-Fc fusion protein of SEQ No. 96, containing a canine IgGA Fc fragment, exhibited poor homodimeric titers when purified using a Protein A column; however, when purified using a Protein G column, the homodimeric titers were significantly improved, exceeding the design target of 50 mg / L or higher. The same was true for the insulin-Fc fusion protein of SEQ No. 98, containing a canine IgGC Fc fragment. The insulin-Fc fusion protein of SEQ No. 100, containing a canine IgGD Fc fragment, produced no compounds when purified with a Protein A or Protein G column. Thus, as demonstrated in the insulin-Fc fusion protein of SEQ No. 52, containing a different insulin polypeptide (SEQ No. 5) and peptide linker (SEQ No. 12), canine IgGB was a desirable Fc fragment for homodimeric titers (see Example 19).

[0410]

[0411] The in vivo bioactivity of the insulin-Fc fusion protein of SEQ No. 96, containing a canine IgGA Fc fragment purified via protein G, was tested according to the procedure of Example 10. The results shown in Figure 22 indicate that the insulin-Fc fusion protein of SEQ No. 96 is only slightly bioactive in vivo, as the NAOC calculated according to Example 11 is only 174% FBGL·days·kg / mg.

[0412] The in vivo bioactivity of the insulin-Fc fusion protein of SEQ No. 98, containing a canine IgGC Fc fragment purified via protein G, was tested according to the procedure of Example 10. The results shown in Figure 23 indicate that the insulin-Fc fusion protein of SEQ No. 98 is only slightly bioactive in vivo, as the NAOC calculated according to Example 11 is only 39% FBGL·days·kg / mg.

[0413] Accordingly, as demonstrated in the insulin-Fc fusion protein of SEQ ID NO: 52 containing different insulin polypeptides (SEQ ID NO: 5) and peptide linkers (SEQ ID NO: 12), canine IgGB is a preferred Fc fragment with respect to bioactivity (see Examples 19 and 20 and Table 16 above).

[0414] Example 33: A non-glycosylated insulin-Fc fusion protein comprising the insulin polypeptide of SEQ No. 8, the peptide linker of SEQ No. 14, and a canine IgGB Fc fragment to reduce the potential risk of immunogenicity

[0415] While the insulin-Fc fusion protein of SEQ No. 32 meets all design objectives (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 more), and if this occurs, the use of the insulin-Fc fusion protein for the treatment of diabetes may be difficult. As described in the detailed description of the invention and in Examples 21 and 22, one possible cause of reduced bioactivity after repeated administration is the generation of neutralizing anti-drug antibodies due to unwanted interactions between the canine IgGB Fc fragment and the canine immune system. However, the results shown in Example 32 unexpectedly demonstrate that the canine IgGB isotype was the only choice among the four canine IgG isotypes that yielded the desired manufacturability and bioactivity. Therefore, additional Fc mutations were sought to achieve a non-glycosylated insulin-Fc fusion protein with low Fc(gamma)RI receptor binding, which should reduce the risk of long-term chronic immunogenicity.

[0416] As described in the detailed description of the present invention, one method for reducing Fc(gamma)RI interaction involves mutating the cNg site of the Fc fragment to prevent glycosylation during synthesis in host cells. Accordingly, a cNg site mutation was made in the Fc fragment region of SEQ ID NO: 32 to reduce the binding affinity of the Fc fragment to the in vivo Fc(gamma) receptor, as measured by binding in the in vitro human Fc(gamma)RI 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 for SEQ ID NO: 32 include SEQ ID NO: 104, which contains the cNg-NB151-S mutation, and SEQ ID NO: 102, which contains the same cNg-NB151-S mutation and the NB119-A mutation. NB119-A was incorporated in an additional attempt to reduce interaction with Fc(gamma)RI, as described by Lo, M. et al. in "Effector attenuation substitution to maintain antibody stability and reduce toxicity in live strain," J. Biol. Chem. (2017), pp. 1-20, specifically for use in mouse antibodies. The full amino acid sequence of the resulting insulin-Fc fusion protein is listed below, along with the (cluster omega) sequence alignment shown in Fig. 24 (NB119 and NB151 sites are underlined for clarity).

[0417] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVV A LDPEDPEVQISWFVDGKQMQTAKTQPREEQF SGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 102)

[0418] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 104)

[0419] 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 reduced CE-SDS according to Example 4, and their sequences were further identified by glycan-removed LC-MS according to Example 5. Their % homodimer content was measured by size-exclusion chromatography according to Example 6, and insulin receptor binding affinity was measured according to Example 7. As shown in Table 17, incorporation of the cNg-NB151-S mutant into the Fc fragment reduced the % homodimer content, indicating an unacceptably high level of aggregation (i.e., the % homodimer content dropped just below 70%).

[0420]

[0421] The in vivo bioactivity of the insulin-Fc fusion proteins of sequence number: 102 and sequence number: 104 was tested in N = 1 dog, each according to the procedure of Example 10. The results shown in Fig. 25 for a single subcutaneous dose show that both compounds are significantly less bioactive in vivo than the insulin-Fc fusion protein of sequence number: 32 (NAOC for sequence number: 104 = 574% FBGL·days·kg / mg; NAOC for sequence number: 102 = 921% FBGL·days·kg / mg). The results indicate that incorporating the cNg-NB151-S mutation into the Fc fragment to produce a non-glycosylated version of the insulin-Fc fusion protein of sequence number: 32 unexpectedly reduced the in vivo bioactivity of the resulting compounds.

[0422] In an attempt to reduce the aggregation and improve the bioactivity of the insulin-Fc fusion protein of sequence number 104 containing a cNg-NB151-S site mutation, various insulin-polypeptide B-chain variants were investigated along with mutations in the region thought to be responsible for aggregation. 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 according to Example 4 by CE-SDS non-reduction and reduction, and their sequences were further identified by LC-MS with glycan removal according to Example 5. Their % isomer content was measured by size-exclusion chromatography according to Example 6. Among the tested B-chain variants, one insulin Fc-fusion protein (Sequence No. 36) containing a substitution from tyrosine to alanine at the 16th amino acid from the N-terminus of the B-chain (i.e., B16) was found to have a high homodimeric titer (105 mg / L) with unexpectedly low aggregation (99% homodimeric), and consequently had a homodimeric titer of 104 mg / L. 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. The pharmacokinetic profile of the insulin-Fc fusion protein of Sequence No. 36 was measured using ELISA in the manner of Example 12, and a 2-compartment model was fitted to the data to determine its elimination half-life of 4.1 ± 0.7 days. The sequence of sequence number 36 is shown below (B16A and cNg-NB151-S mutations are underlined for clarity).

[0423] FVNQHLCGSHLVEAL ALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF S GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(서열 번호: 36)

[0424] Next, the insulin-Fc fusion protein of SEQ No. 36 was evaluated for repeated dose bioactivity in dogs. The compound was administered subcutaneously to N = 1 dog on days 0, 7, 14, and 28 according to the procedure of Example 11. When the dogs' % FBGL dropped too low, food was provided to raise the blood glucose to a safe level. Unexpectedly, compared to the insulin-Fc fusion protein of SEQ No. 104, the NAOC for the first injection of the insulin-Fc fusion protein of SEQ No. 36 containing the B16A mutation was significantly higher (1185% FBGL·days·kg / mg). The in vivo bioactivity plot for the first dose is shown in Fig. 26. The pharmacokinetic profile of the compound was also measured using ELISA in the manner of Example 12, and a 2-compartment model was fitted to the data to determine its elimination half-life of 3.5 days. NAOC and NAOCR were also measured for each subsequent dose according to the general procedure of Example 11, calculated from the time the dose was administered until immediately before the next dose was administered. The NAOC and NAOCR shown in Table 18 demonstrate that the insulin-Fc fusion protein of SEQ No. 36 maintained an NAOCR of 0.6 or higher over four administrations, satisfying the bioactive design objectives for repeated doses. Taken together, the results indicate that it was necessary to mutate the insulin B-chain sequence to obtain a suitable non-glycosylated cNg-S variant of SEQ No. 32. Therefore, the insulin polypeptide of SEQ No. 11 was preferred for the non-glycosylated insulin-Fc fusion protein containing a cNg-mutated canine IgGB Fc fragment.

[0425]

[0426] Finally, the selected compounds were tested for their potential to interact with the immune system by measuring Fc(gamma) receptor binding activity according to the procedure of Example 8. Table 19 compares the Fc(gamma) receptor I binding of these insulin-Fc fusion proteins with the Fc(gamma) receptor binding of the insulin-Fc fusion protein of SEQ ID NO: 52. It can be seen that the non-glycosylated insulin-Fc fusion protein (achieved via the cNg-S mutation) exhibited the lowest Fc(gamma) receptor binding ratio relative to SEQ ID NO: 52.

[0427]

[0428] Example 34: Exemplary CHO-based production execution using a preferred insulin-Fc fusion protein comprising an Fc fragment of canine IgGB origin prepared via a stably transfected CHO cell line

[0429] Isolated CHO cell lines stably transfected with a vector encoding SEQ ID NO: 32 or SEQ ID NO: 36 were constructed as described in Example 2. A 14-day production run (0.5-2.0 L medium scale) was seeded at 0.5 million cells / mL in an incubator-shaker set to 37°C and 5% carbon dioxide, and the run was performed as described in Example 2 above, except that CD OptiCHO replaced Dynamis as the growth medium (ThermoFisher) and Efficient Feed C (ThermoFisher) was used as the feed. Starting on the 3rd day of the production run, the feed was added at 3% v / v, and on the 4th day, the shake flask temperature was adjusted to 32°C and the incubator-shaker carbon dioxide concentration was lowered from 5% to 2%. During the run, the cells increased to 8-14 million cells / mL, and on day 14, the production run was harvested, the cells were removed, and the culture supernatant was purified and tested to obtain insulin-Fc fusion proteins as described in Examples 3, 4, 5, and 6. Table 20 lists the production data obtained from the production run using stably transfected CHO cell lines.

[0430]

[0431] Example 35: Exemplary CHO-based production execution using a preferred insulin-Fc fusion protein containing an Fc fragment of canine IgGB origin via a stably transfected CHO cell line

[0432] A CHO cell line stably transfected with a vector encoding sequence number 34 was constructed as described in Example 2. A 14-day production run (0.5-2.0 L medium scale) was seeded at 0.5 million cells / mL in an incubator shaker set to 37°C and 5% carbon dioxide, and the run was performed as described in Example 2 above, except that CD OptiCHO replaced Dynamis as the growth medium (ThermoFisher) and Efficient Feed C (ThermoFisher) was used as the feed. Starting on day 3 of the production run, the feed was added at 3% v / v, and on day 4, the shaker flask temperature was adjusted to 32°C and the incubator shaker carbon dioxide concentration was lowered from 5% to 2%. On the 14th, the production run was harvested, the cells were removed, and the culture supernatant was purified and tested to obtain insulin-Fc fusion proteins as described in Examples 3, 4, 5, and 6. The produced production run provides a protein yield of 200 mg / L or more, 95% or more of the homodimer, and a homodimer titer of 190 mg / L or more of SEQ No.: 34.

[0433] Result—Insulin-FC fusion protein containing the feline Fc fragment

[0434] Example 36: Insulin-Fc fusion protein containing a feline IgG2 isotype Fc fragment

[0435] To develop a product suitable for use in cats, an attempt was made to produce an insulin-Fc fusion protein containing the insulin polypeptide sequence of SEQ No. 4 and the feline IgG2 isotype Fc fragment (SEQ No. 4) using the peptide linker of SEQ No. 13 with the following amino acid sequence:

[0436] FVNQHLCGSDLVEALYLVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVS VLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(sequence Number: 106)

[0437] The insulin-Fc fusion protein of sequence number 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 reduced CE-SD according to Example 4, and the sequence was further identified by glycan-removed LC-MS according to Example 5. The % homodimer of the resulting compound, measured by size-exclusion chromatography according to Example 6, was 88%. The homodimer titer of the resulting compound was only 20 mg / L, which is because HEK cells were unable to produce the product in high yield (i.e., the protein yield after protein purification was only 23 mg / L). In summary, the production of the insulin-Fc fusion protein of sequence number 106 in HEK cells resulted in moderate levels of aggregation and a low homodimer titer of 20 mg / L, which did not meet the design objective of a homodimer titer of 50 mg / L or higher.

[0438] Nevertheless, the insulin-Fc fusion protein of SEQ No. 106 was evaluated for bioactivity. First, the insulin receptor binding of the insulin-Fc fusion protein of SEQ No. 106 was measured according to Example 7, yielding an IC50 value of 22 nM, which indicates that the compound is likely bioactive in vivo (i.e., an IC50 of less than 5000 nM).

[0439] Next, the in vivo pharmacokinetics (PD) of the insulin-Fc fusion protein of SEQ No. 106 were measured after a single subcutaneous administration of the compound at a dose of 0.8 mg / kg to N = 3 cats according to Example 10. Figure 27 shows the % fasting blood glucose level for the insulin-Fc fusion protein of SEQ No. 106 (161c) as a function of time. The NAOC for the insulin-Fc fusion protein was calculated as 215% FBGL·days·kg / mg according to the procedure of Example 11. Surprisingly, unlike the insulin-Fc fusion protein for dogs of SEQ No. 42, which contains the insulin polypeptide of SEQ No. 5 and the peptide linker of SEQ No. 12, the insulin-Fc fusion protein for cats of SEQ No. 106 was found to be much less aggregative and much more bioactive in the target animals.

[0440] Since the NAOC was acceptable and pharmacokinetic data supported once-weekly administration, additional subcutaneous doses were administered to cats on days 28, 35, 42, and 49, and % FBGL was measured for a 7-day window after each administration according to Example 11. NAOC and NAOCR were calculated for each repeated subcutaneous injection according to the procedure of Example 11. As illustrated in Table 21, repeated subcutaneous administration to cats resulted in a significant decrease in bioactivity by the third dose, as measured by a significant decrease in NAOCR (i.e., the NAOC for the third injection was only 0.40 or 40% of the NAOC for the first injection, and the NAOC for the fourth injection was only 0.10 or 10% of the NAOC for the first injection). After repeated administration in cats, the significant decrease in the bioactivity of the insulin-Fc fusion protein of sequence number: 106 was similar to that observed for the insulin-Fc fusion protein of sequence number: 52 in dogs as shown in Example 20.

[0441]

[0442] Example 37: Evaluation of Insulin Polypeptide Mutations for Protein Yield, Purity, and Insulin Receptor Activity and Selection of Feline IgG1b or IgG2 Fc Fragments

[0443] In an attempt to increase the % homodimer content and protein yield of the insulin-Fc fusion protein of SEQ No. 106, a mutation was inserted into the sequence of the insulin polypeptide B-chain (e.g., B16A mutation) and the peptide linker. Additionally, the feline IgG1b Fc fragment (SEQ No. 20) was evaluated in addition to the feline IgG2 Fc fragment (SEQ No. 21) used to construct the insulin-Fc fusion protein of SEQ No. 106. The resulting insulin-Fc fusion protein sequences are presented below, along with the sequence alignment generated for SEQ No. 106 (cluster omega) shown in Fig. 28.

[0444] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(서열 번호: 108)

[0445] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(서열 번호: 110)

[0446] FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(서열 번호: 112)

[0447] 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 reduced CE-SDS according to Example 4, and their sequences were further identified by glycan-removed LC-MS according to Example 5. Their % dimer content was measured by size-exclusion chromatography according to Example 6, and insulin receptor binding affinity was measured according to Example 7. Insulin-Fc fusion protein variants are listed in Table 22 along with their corresponding protein yields, % dimers, and dimer titers. The results show that when various mutations combined with the feline IgG1b isotype Fc fragment to produce the insulin-Fc fusion protein of SEQ No. 108, they produced much higher protein yields, but the resulting proteins were more aggregated (e.g., lower % dimers than SEQ No. 106). This was surprising because feline IgG1b is functionally more similar to the canine IgGB Fc fragment isotype, which is a highly desirable Fc isotype for the production of canine insulin-Fc fusion protein (Example 32). Among the mutant feline compositions containing feline IgG2 isotypes, the inclusion of the B16A mutation of the insulin polypeptide B-chain (i.e., SEQ No. 110 and SEQ No. 112) improved protein yield and homodimeric titer. However, the mutant linker present in SEQ No. 110 (i.e., GGGGAGGGG) appears to provide an additional doubling in protein yield and homodimeric titer compared to SEQ No. 112.

[0448]

[0449] Example 38: In vivo immunogenicity screening after repeated subcutaneous administration of an insulin-Fc fusion protein comprising the insulin polypeptide of SEQ No. 4 having a feline IgG2 isotype Fc fragment

[0450] Without being bound by specific descriptions, the cause of the significant decrease in the bioactivity of the insulin-Fc fusion protein of SEQ No. 106 after four repeated subcutaneous administrations in cats was presumed to be due to the development of anti-drug antibodies that neutralize its bioactivity (Example 36). Anti-drug antibodies may be directed against the insulin polypeptide, linker, or Fc-fragment portion of the insulin-Fc fusion protein. Immunogenic responses manifest as interactions between antigen-presenting cells, T-helper cells, B-cells, and related cytokines, which can generate endogenous antibodies against the drug (e.g., anti-drug antibodies). Binding antibodies are any isotype capable of binding to the insulin-Fc fusion protein and can be detected in immunoassays as described in Example 14. Neutralizing antibodies that inhibit the functional activity of the insulin-Fc fusion protein are generally directed against the bioactive site. To evaluate whether this was true, serum collected before the administration of each dose and at the end of the experiment described in Example 11 was tested to quantify the level of anti-drug antibodies according to Example 14. As shown in Fig. 29, the level of anti-drug antibodies actually increased with multiple subcutaneous administrations of the compound, which indicates that the production of neutralizing anti-drug antibodies may be the cause of the decrease in NAOCR after the fourth injection of the insulin Fc-fusion protein of SEQ No. 106.

[0451] Example 39: Screening of cat serum containing anti-drug antibodies and identification of potential immunogenic epitopes at the B10D and A8H positions of insulin polypeptide

[0452] As observed for sequence number 52 in dogs (Example 20), the bioactivity of repeated doses of the insulin-fusion protein of sequence number 106, containing the insulin polypeptide of sequence number 4 and the peptide linker of sequence number 13, still produced anti-drug antibodies (Example 38). Therefore, it was hypothesized that the insulin polypeptide of sequence number 4 may contain a specific epitope (i.e., an immunogenic "hot spot") to which the cat's immune system unexpectedly targets. Accordingly, the binding specificity of the antibodies present in the serum sample described in Example 38 was evaluated according to the general procedure of Example 15. Analysis of antibody-containing cat serum samples from repeated administration of the insulin-Fc fusion protein of sequence number: 106 (Example 38) to a coated insulin-Fc fusion protein library revealed the unexpected presence of two primary "hot spots" within the insulin polypeptide sequence of sequence number: 4: a B10D site mutation (i.e., an aspartic acid mutation at the 10th position from the N-terminus of the B-chain (i.e., B10)) and, separately, a A8H site mutation (i.e., a histidine mutation at the 8th position from the N-terminus of the A-chain (i.e., A8)). The results suggest that the insulin-Fc fusion protein containing an insulin polypeptide amino acid composition containing these two specific amino acid mutations is likely immunogenic in cats, and therefore may generate anti-drug antibodies that neutralize bioactivity after repeated injections. Therefore, insulin polypeptides not containing B10D and A8H were determined to be preferred for insulin-Fc fusion proteins that need to be administered repeatedly to cats over a long period (e.g., to treat feline diabetes).

[0453] Example 40: Insulin-Fc fusion protein comprising the insulin polypeptide of SEQ No. 4, and glucosylated and non-glucosylated feline IgG1b and IgG2 isotype Fc fragments in which the B10, A8, and other sites of the insulin polypeptide are further mutated to reduce the potential risk of immunogenicity.

[0454] To evaluate whether replacing the "hot spot" mutation would improve the immunogenicity and repeated-dose bioactivity of an insulin-Fc fusion protein containing the insulin polypeptide of SEQ No. 4 and a feline IgG2 isotype fragment, exemplary insulin-Fc fusion proteins of SEQ No. 114, 116, and 118 were synthesized in which 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 with alanine (i.e., B16A), as in the case of the insulin polypeptide of SEQ No. 5 used in many canine insulin-Fc fusion proteins. The A21N site of natural insulin was also deleted. In this example, other insulin polypeptide amino acids were mutated to make the structure more similar to natural feline insulin (e.g., B30A, A8A, A10V, and A18H). The sequence of the generated insulin polypeptide (sequence number: 120) is listed below with the non-natural amino acids for feline insulin underlined.

[0455] FVNQHLCGSHLVEAL A LVCGERGFFYT DP A GGGPRR GIVEQCCASVCSLYQLEHYC(Sequence No.: 120)

[0456] In addition, considering the additional potential benefits of the non-glycosylated cNg mutants discussed in Examples 22 and 33, two of the evaluated insulin-Fc fusion proteins (Sequence Nos: 116 and 118) contain cNg-S mutants. The full amino acid sequences of the insulin-Fc fusion proteins are presented below, along with the resulting sequence alignment for Sequence No. 108 (cluster omega) shown in Fig. 30.

[0457] FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSP(서열 번호: 114)

[0458] FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(서열 번호: 116)

[0459] FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVALGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(서열 번호: 118)

[0460] 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 reduced CE-SDS according to Example 4, and their sequences were further identified by glycan-removed LC-MS according to Example 5. Their % homodimer content was measured by size-exclusion chromatography according to Example 6, and insulin receptor binding affinity was measured according to Example 7. Table 23 below shows the compatibilities and in vitro IR binding parameters for the generated compounds.

[0461]

[0462] Unexpectedly, all three insulin-Fc fusion proteins exhibited significantly lower protein yields compared to the insulin-Fc fusion protein of sequence number: 108. In fact, although the insulin receptor binding affinity (IC50) was sufficiently high (IC50 of 707 nM), the insulin-Fc fusion protein of sequence number: 116 provided almost no protein yield. The insulin-Fc fusion protein of sequence number: 118 provided unacceptably low protein yields and homodimeric titers, and was considered non-biogenic in vivo due to high IR binding IC50 values ​​greater than 5000 nM. The protein of sequence number: 114 also provided unacceptably low protein yields and much lower insulin receptor binding affinity (higher IR IC50 values) compared to the insulin-Fc fusion protein of sequence number: 108.

[0463] Example 41: Insulin-Fc fusion protein comprising the insulin polypeptide of SEQ ID No. 8, the linker of SEQ ID No. 14, and a feline IgG2 isotype Fc fragment

[0464] In an attempt to obtain an acceptable protein yield of an insulin-Fc fusion protein containing an insulin polypeptide sequence free of immunogenic "hotspot" mutations (i.e., B10D and A8H), lessons were learned from the concurrent and parallel development of canine insulin-Fc fusion proteins, which demonstrated that using the insulin polypeptide of SEQ No. 8 and the peptide linker of SEQ No. 14 on a canine IgGB isotype Fc fragment resulted in high protein yield, homodimeric titers, and acceptable IR binding affinities. Accordingly, a feline insulin-Fc fusion protein was constructed using the insulin polypeptide of SEQ No. 8 and the peptide linker of SEQ No. 14 on the feline IgG2 Fc fragment of SEQ No. 21, generating the following sequence:

[0465] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNS TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(sequence Number: 122)

[0466] The sequence alignment of sequence number 122 with sequence number 106 and 112, which are the sequences of Example 37, is shown in FIG. 31 (cluster omega).

[0467]

[0468] The insulin-Fc fusion protein of sequence number 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 reduced CE-SDS according to Example 4. The sequence was further identified by glycan-removed LC-MS according to Example 5. Its % isomer content was measured by size-exclusion chromatography according to Example 6, and insulin receptor binding affinity was measured according to Example 7. FcRn receptor binding affinity was measured according to Example 9. The protein yield was 146 mg / L, and the % isomer content was measured as 99%, yielding an isomer titer of 145 mg / L that meets the design objectives. The IR binding affinity IC50 value was 2,536 nM, indicating that the compound is likely bioactive in vivo. The EC50 value of the FcRn receptor binding affinity was 3114 ng / mL. Therefore, the insulin-Fc fusion protein of sequence number: 122 was a potential candidate for further in vivo testing.

[0469] Example 42: In vivo bioactivity of an insulin-Fc fusion protein constructed from the insulin polypeptide of SEQ ID NO: 8, the peptide linker of SEQ ID NO: 14, and the feline IgG2 Fc fragment of SEQ ID NO: 21

[0470] The insulin-Fc fusion protein of SEQ No. 122 was tested for in vivo bioactivity according to Example 10. A healthy, antibody-free cat weighing approximately 5 kg was used. On Day 0, the cat received a single injection of a pharmaceutical composition containing the insulin Fc-fusion protein of SEQ No. 122. Blood was collected from a suitable vein on Day 0, immediately before the injection, and at 15, 30, 45, 60, 120, 240, 360, and 480 minutes after the injection, and on Days 1, 2, 3, 4, 5, 6, and 7. If the subject's blood glucose level dropped to a dangerous level, food and / or glucose injections were provided to prevent symptomatic hypoglycemia.

[0471] Figure 32 shows the % FBGL for a single dose, which unexpectedly indicates that the insulin-Fc fusion protein of sequence number: 122 was only slightly bioactive in vivo (essentially 0% FBGL·days·kg / mg NAOC). This result was surprising, particularly because the insulin-Fc fusion protein did not aggregate (i.e., had a high % homodimer content) and the molecule exhibited IR affinity in a range similar to that of the canine insulin-Fc fusion protein that showed significant bioactivity in dogs (Example 31). Since there was insufficient bioactivity at the first dose, repeated administration was not performed.

[0472] Example 43: Evaluation of the substitution of feline IgG1b for the feline IgG2 Fc fragment regarding the yield, purity, bioactivity, and immunogenicity of an insulin-Fc fusion protein comprising the insulin polypeptide of SEQ No. 8 and the peptide linker of SEQ No. 14

[0473] As the long-acting insulin research programs for dogs and cats were conducted in parallel, some of the lessons learned from the canine insulin-Fc fusion protein research program were applied to the feline insulin-Fc protein research program. One key takeaway from the canine insulin-Fc program was how the selection of different IgG isotype Fc fragments (e.g., canine IgGA, canine IgGB, canine IgGC, and canine IgGD isotypes) dramatically alters manufacturing and in vivo efficacy performance. Therefore, the feline IgG2 Fc fragment (SEQ No. 122) was replaced with the feline IgG1b Fc fragment (SEQ No. 20) while retaining the insulin polypeptide (SEQ No. 8) and the peptide linker (SEQ No. 14), allowing for the following amino acid sequence:

[0474] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNS TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG(sequence Number: 38)

[0475] The insulin-Fc fusion protein of sequence number 38 was synthesized in HEK293 cells according to the procedure of Example 1 and purified using a Protein A column according to Example 3. Its 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. At this stage, the protein yield was 158 mg / L. % homodimer for the sequence was measured by size-exclusion chromatography according to Example 6 and determined to be 99.5%, resulting in a homodimer titer of 157 mg / L that met the manufacturing design objectives. The in vitro IM-9 insulin receptor binding IC50 value, measured according to Example 7, was 2398 nM, which also met the design objectives. The FcRn receptor binding affinity EC50 value was measured according to Example 9 and was found to be 1552 ng / mL.

[0476] Subsequently, the insulin-Fc fusion protein of SEQ No. 38 was tested for in vivo bioactivity according to Example 10. A healthy, antibody-free cat weighing approximately 5 kg received a single subcutaneous injection of a pharmaceutical composition containing the insulin-Fc-fusion protein of SEQ No. 38 at a dose of 0.16 mg insulin-Fc fusion protein / kg. Blood was collected from a suitable vein on Day 0, immediately before the injection, and at 15, 30, 45, 60, 120, 240, 360, and 480 minutes after the injection, and on Days 1, 2, 3, 4, 5, 6, and 7. If the subject's blood glucose dropped to a dangerous level, food and / or glucose injections were provided to prevent symptomatic hypoglycemia.

[0477] Figure 33 shows % FBGL after the first administration. Animals were fed regularly to prevent symptomatic hypoglycemia, which indicated that the insulin-Fc fusion protein of SEQ No. 38 was significantly bioactive in vivo, with an NAOC of 1838% FBGL·days·kg / mg. The pharmacokinetic profile of the compound was also measured using ELISA in the manner of Example 12, and a 2-compartment model was fitted to the data to determine its elimination half-life of 6.3 ± 0.5 days. The difference in bioactivity (in vitro and in vivo) between the insulin-Fc fusion protein of SEQ No. 38 and the insulin-Fc fusion protein of SEQ No. 122 unexpectedly demonstrates that when the insulin polypeptide sequence is modified as in SEQ No. 8, the feline IgG1b isotype is preferred over the feline IgG2 isotype for the Fc fragment.

[0478] Since NAOC was acceptable and pharmacokinetic data supported once-weekly administration, additional subcutaneous doses were administered to cats on days 14, 28, and 42, and % FBGL was measured for a 7-day window following each administration according to Example 11. NAOC and NAOCR were calculated for each repeated subcutaneous injection according to the procedure of Example 11. As illustrated in Table 25, the insulin-Fc fusion protein of Sequence No. 38 exhibited acceptable bioactivity in vivo after multiple administrations.

[0479]

[0480] In addition, to test for the presence of any anti-drug antibody and to quantify its levels according to Example 14, serum was collected once a week before administration of each dose and for 2 weeks after the end of the experiment. As shown in Fig. 34, there was no measurable increase in anti-drug antibodies above baseline after multiple administrations of the compound. Therefore, in order to obtain a feline insulin-Fc fusion protein candidate (e.g., SEQ No. 38) that meets the design criteria of acceptable homodimeric titer, in vivo bioactivity, and sustained bioactivity after repeated weekly injections to cats, it was necessary to replace the insulin polypeptide of SEQ No. 4 with the insulin polypeptide of SEQ No. 8 and use the feline IgG1b Fc fragment of SEQ No. 20 instead of the feline IgG2 Fc fragment of SEQ No. 21.

[0481] Example 44: A non-glycosylated insulin-Fc fusion protein comprising the insulin polypeptide of SEQ No. 8, the peptide linker of SEQ No. 14, and the feline IgG1b Fc fragment to reduce the potential risk of immunogenicity

[0482] While the insulin-Fc fusion protein of SEQ No. 38 meets all design objectives (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 more), and if this occurs, it may make the use of this insulin-Fc fusion protein for the treatment of diabetes difficult. As described in the detailed description of the invention, one possible cause of reduced bioactivity after repeated administration is the generation of neutralizing anti-drug antibodies due to unwanted interactions between the feline IgG1b Fc fragment and the feline immune system. However, the results shown in Example 43 unexpectedly demonstrate that the feline IgG1b isotype is preferable to the feline IgG2 isotype, which is less immunogenic in relation to in vivo bioactivity. Therefore, additional Fc mutations were sought to achieve a non-glycosylated insulin-Fc fusion protein with low Fc(gamma)RI receptor binding that should reduce the risk of long-term chronic immunogenicity.

[0483] As described in the detailed description of the present invention, one method for reducing Fc(gamma)RI interaction involves mutating the cNg site of the Fc fragment to prevent glucosylation during synthesis in host cells. Accordingly, to reduce the binding affinity of the Fc fragment to the in vivo Fc(gamma) receptor, as measured by binding in the in vitro human Fc(gamma)RI assay described in Example 8, a cNg site mutation was created in the Fc fragment region of SEQ ID NO: 38. The location of the cNg site in the insulin-Fc fusion protein of SEQ ID NO: 38 is cNg-NB151. Again, using learning 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 full amino acid sequence of the resulting insulin-Fc fusion protein is listed below (cNg-NB151-S is underlined for clarity):

[0484] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 124)

[0485] The insulin-Fc fusion protein of sequence number 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. At this stage, the protein yield was 202 mg / L. The % homodimer for the sequence was measured by size-exclusion chromatography according to Example 6 and was determined to be 99%, yielding a homodimer titer of 200 mg / L that meets the manufacturing design target. However, the in vitro IM-9 insulin receptor binding IC50 value measured according to Example 7 was greater than 5000 nM, which is outside the design target for in vitro bioactivity. The FcRn receptor binding affinity EC50 value was measured according to Example 9 and was 6922 ng / mL.

[0486] Although the insulin-Fc fusion protein of SEQ No. 124 did not meet the design objectives for insulin receptor binding, its in vivo bioactivity was tested according to Example 10. A healthy, antibody-free cat weighing approximately 5 kg was used. On Day 0, the cat received a single injection of a pharmaceutical composition containing the insulin-Fc-fusion protein of SEQ No. 124 at a dose of 0.16 mg / kg. On Day 0, blood was collected from a suitable vein immediately before and after the injection at 15, 30, 45, 60, 120, 240, 360, and 480 minutes, and on Days 1, 2, 3, 4, 5, 6, and 7. If the subject's blood glucose dropped to a dangerous level, food and / or glucose injections were provided to prevent symptomatic hypoglycemia.

[0487] Figure 35 shows the % FBGL for a single administration, which indicates that the insulin-Fc fusion protein of sequence number: 124 has a NAOC of 65% FBGL·days·kg / mg, indicating that it is only slightly bioactive in vivo. Since there was insufficient bioactivity at the first administration, repeated administration was not performed.

[0488] Unexpectedly, it was found that reversing the insulin polypeptide sequence of SEQ No. 124 so that the 16th amino acid from the N-terminus of the B-chain (i.e., B16) is mutated from tyrosine to alanine (i.e., B16A), as in the case of Example 33 for the insulin Fc-fusion protein of SEQ No. 36, resulted in the resulting insulin-Fc fusion protein of SEQ No. 40 becoming bioactive. The amino acid sequence of the resulting insulin-Fc fusion protein is as follows (B16A and cNg-NB151-S mutations are underlined for clarity):

[0489] FVNQHLCGSHLVEAL A LVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQF S STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 40)

[0490] The insulin-Fc fusion protein of sequence number 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 reduced CE-SDS according to Example 4. The sequence was further identified by glycan-removed LC-MS according to Example 5. The protein yield at this stage was 174 mg / L. The % homodimer for the sequence was measured by size-exclusion chromatography according to Example 6 and was determined to be 98.9%, yielding a homodimer titer of 172 mg / L that meets the manufacturing design criteria. The in vitro IM-9 insulin receptor binding IC50 value of 4635 nM, measured according to Example 7, also meets the design objectives. Fc(gamma) receptor activity was measured according to Example 8 and was found to be about 4 times less than that obtained for the insulin-Fc fusion protein of Sequence No. 38 using the same procedure, indicating that the insulin-Fc fusion protein interacts 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.

[0491] Next, the insulin-Fc fusion protein of SEQ No. 40 was tested for in vivo bioactivity according to Example 11. A healthy, antibody-free cat weighing approximately 5 kg was used. On days 0, 7, and 21, the cat received a single subcutaneous injection of a pharmaceutical composition containing the insulin-Fc-fusion protein of SEQ No. 40 at a dose of 0.1 mg insulin-Fc fusion protein / kg. On day 0, blood was collected from a suitable vein immediately before and 15, 30, 45, 60, 120, 240, 360, and 480 minutes after the injection, and on days 1, 2, 3, 4, 5, 6, and 7. If the subject's blood glucose dropped to a dangerous level, food and / or glucose injections were provided to prevent symptomatic hypoglycemia.

[0492] Figure 36 shows the % FBGL after the first administration, indicating that the insulin-Fc fusion protein of SEQ No. 40 is bioactive in vivo, yielding a NAOC of 159% FBGL·days·kg / mg for a subcutaneous dose of 0.1 mg insulin-Fc fusion protein / kg. A second, higher subcutaneous dose of 0.2 mg insulin-Fc fusion protein / kg provided a much higher NAOC of 702% FBGL·days·kg / mg, as shown in Figure 37. Pharmacokinetic profiles were measured using ELISA in the manner of Example 12, and a 2-compartment model was fitted to the data to determine a elimination half-life longer than 3 days. These results demonstrate that, in contrast to the results obtained with the insulin-Fc fusion protein of SEQ No. 124, the same compound containing tyrosine in B16 instead of alanine was only very weakly bioactive at nearly the same dose (0.16 mg insulin-Fc fusion protein / kg). Therefore, for a non-glycosylated insulin-Fc fusion protein containing a cNg-mutated feline IgG1b Fc fragment, the insulin polypeptide of sequence number: 11 was preferred.

[0493] To analyze repeatable in vivo activity after multiple administrations, the insulin-Fc fusion protein of SEQ No. 40 was additionally administered to cats on days 7, 21, and 35. When the % FBGL of the cats dropped too low, food was provided to the cats 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, calculated from the time the dose was administered until immediately before the next dose was administered. The NAOC and NAOCR presented in Table 26 exemplify the in vivo in vivo activity of the insulin-Fc fusion protein of SEQ No. 40 after multiple administrations.

[0494]

[0495] In addition, serum was collected before administration of each dose and at the end of the experiment to test for the presence of any anti-drug antibody according to Example 14 and to quantify its level. 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 manufacturing and bioactive design criteria with significantly reduced Fc(gamma) receptor activity, it was necessary not only to mutate cNg to serine but also to mutate the insulin polypeptide B16 amino acid to alanine.

[0496] Example 45: Exemplary CHO-based production execution using a preferred insulin-Fc fusion protein comprising an Fc fragment of feline IgG1b origin prepared via a stably transfected CHO cell line

[0497] A CHO cell line stably transfected with a vector encoding sequence number 38 was constructed as described in Example 2 above. A 14-day production run (0.5-2.0 L medium scale) in a feed-batch shake flask was seeded at 0.5 million cells / mL in an incubator-shaker set to 37°C and 5% carbon dioxide, and the run was performed as described in Example 2 above, except that CD OptiCHO replaced Dynamis as the growth medium (ThermoFisher) and Efficient Feed C (ThermoFisher) was used as the feed. Starting from day 3 of the production run, the feed was added at 3% v / v, and on day 4, the shake flask temperature was adjusted to 32°C and the incubator-shaker carbon dioxide concentration was lowered from 5% to 2%. During the run, cell density was increased to 8-14 million cells / mL, and on day 14, the production run was harvested, cells were removed, the culture supernatant was purified, and characterized to obtain insulin-Fc fusion proteins as described in Examples 3, 4, 5, and 6. Table 27 lists the production data for the insulin-Fc fusion proteins obtained through these stably transfected CHO cell line production runs.

[0498]

[0499] Example 46: Exemplary CHO-based production execution using a preferred insulin-Fc fusion protein of feline IgG1b origin prepared via a stably transfected CHO cell line

[0500] A CHO cell line stably transfected with a vector encoding sequence number 40 was constructed as described in Example 2 above. A 14-day production run (0.5-2.0 L medium scale) was seeded at 0.5 million cells / mL in an incubator shaker set to 37°C and 5% carbon dioxide, and the run was performed as described in Example 2 above, except that CD OptiCHO replaced Dynamis as the growth medium (ThermoFisher) and Efficient Feed C (ThermoFisher) was used as the feed. Starting on day 3 of the production run, the feed was added at 3% v / v, and on day 4, the shake flask temperature was adjusted to 32°C and the incubator shaker carbon dioxide concentration was lowered from 5% to 2%. On the 14th, the production run was harvested, the cells were removed, the culture supernatant was purified, and the insulin-Fc fusion protein was characterized to be obtained as described in Examples 3, 4, 5, and 6. The resulting production run provides a protein yield of 200 mg / L or more, 95% or more of the dimer, and a dimer titer of 190 mg / L or more of SEQ No.: 40.

[0501] Example 47: Exemplary insulin-Fc fusion protein domain and sequence

[0502] The exemplary insulin-Fc fusion protein amino acid sequence and corresponding DNA sequence used in the above example are shown in FIGS. 38, 39, 40, 41 and 42.

[0503] Equivalents

[0504] In the claims, articles such as "a," "an," and "the" may mean one or more unless otherwise indicated or otherwise evident from the context. Claims or descriptions containing "or" between one or more group members are deemed to have one, one or more, or all of the group members present or adopted in connection with a given product or process, unless otherwise indicated or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present, used, or otherwise related to a given product or process. The present disclosure includes embodiments in which one or more or all of the group members are present, used, or otherwise related to a given product or process.

[0505] Additionally, the present disclosure includes all variations, combinations, and permutations in which one or more limitations, elements, phrases, and descriptive terms from one or more of the listed claims are introduced into other claims. For example, a claim dependent on another claim may be modified to include one or more limitations found in other claims dependent on the same base claim. For example, in a Markush group format, where elements are listed, each subgroup of elements is also disclosed, and all elements may be removed from the group. Generally, where the disclosure or an aspect of the disclosure is referred to as including specific elements and / or features, it should be understood that a specific embodiment of the disclosure or an aspect of the disclosure is composed of or essentially composed of such elements and / or features. For the sake of simplification, such embodiments are not described in detail in this specification. Also, it should be noted that the terms “include,” “comprising,” “containing,” and “containing” are intended to be open, and their use allows for the inclusion of additional elements or steps. Where a scope is provided, an endpoint is included. Additionally, unless otherwise indicated or evident from the context and the understanding of those skilled in the art, values ​​expressed as a range may be assumed to be any specific value or sub-range within the range mentioned in other embodiments of the present disclosure up to one-tenth of the lower limit unit of the range, unless otherwise specified in the context.

Claims

Claim 1 A fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are linked by a linker such as a peptide linker, and wherein the Fc fragment comprises the following sequence: DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (Sequence No.: 22). Claim 2 In claim 1, the insulin polypeptide comprises a fusion protein having the following sequence: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (sequence number: 10), wherein X1 is not D and X2 is not H. Claim 3 In claim 1, the insulin polypeptide comprises a fusion protein having the following sequence: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (Sequence No. 10), where X1 is H and X2 is T. Claim 4 In claim 1, the insulin polypeptide and the Fc fragment are linked by a peptide linker comprising the following sequence: GGGGGQGGGGQGGGGQGGGGG (Sequence No. 14). Claim 5 In claim 1, the fusion protein comprises the following sequence: FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (Sequence No.: 36). Claim 6 In any one of claims 1 to 5, the fusion protein is a homodimeric fusion protein. Claim 7 delete Claim 8 In any one of claims 1 to 5, the fusion protein is prepared using HEK293 cells, and the homodimeric titer produced after purification using protein A beads or protein A columns is greater than 50 mg / L. Claim 9 In any one of claims 1 to 5, the insulin receptor IC for the fusion protein 50 A fusion protein with a g of 5000 nM or less. Claim 10 A fusion protein according to any one of claims 1 to 5, wherein the serum half-life of the fusion protein in the blood or serum of a target animal at the time of administration is longer than 3 days (+ / -5%). Claim 11 A fusion protein according to any one of claims 1 to 5, wherein the time of a statistically significant decrease in blood glucose levels in the subject compared to the level before administration is longer than 2 hours, 6 hours, 9 hours, 12 hours, 18 hours, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. Claim 12 A fusion protein according to any one of claims 1 to 5, wherein the NAOC is greater than 150% FBGL·days·kg / mg after the first subcutaneous injection into the target animal. Claim 13 In Clause 12, the ratio of NAOC after the third weekly subcutaneous injection of the fusion protein to the NAOC after the first subcutaneous injection of the fusion protein in the target animal is greater than 0.50 for the fusion protein. Claim 14 A composition for treating diabetes, comprising a fusion protein of any one of claims 1 to 5. Claim 15 In claim 14, the fusion protein is present in the composition at a concentration of 3 mg / mL (+ / -5%) or higher. Claim 16 In claim 15, the composition is a composition suitable for subcutaneous administration. Claim 17 A method for lowering the blood glucose level of a target dog, comprising the step of administering a physiologically effective amount of a fusion protein of any one of claims 1 to 5 or a pharmaceutical composition thereof to a dog. Claim 18 In claim 17, the method by which the target dog is diagnosed with diabetes. Claim 19 In claim 17, a method of administering the fusion protein subcutaneously. Claim 20 In claim 19, a method in which the fusion protein is administered to the target dog daily, twice a week, or once a week. Claim 21 A method according to claim 20, wherein the fusion protein is administered to a target dog once a week at a dosage of 0.025 to 0.5 mg / kg / week. Claim 22 An isolated host cell engineered to express a fusion protein of any one of claims 1 to 5. Claim 23 In claim 22, the host cell is an isolated host cell transfected with a nucleic acid encoding the fusion protein. Claim 24 In claim 23, the host cell is an isolated host cell that is a HEK293 cell or a CHO cell. Claim 25 cDNA encoding a fusion protein of any one of claims 1 to 5. Claim 26 In claim 25, the cDNA comprises the following nucleic acid sequencecDNA:atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag(서열 번호: 35) Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete

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