Fusion protein, its use, pharmaceutical composition, cell, cDNA, recombinant cell, and its method of preparation.

Description

Fusion protein, its use, pharmaceutical composition, cell, cDNA, recombinant cell, and its method of preparation. Separated from BR112020026777-5, filed on June 28, 2019. Priority of Related Orders.

[001] This application claims the benefit of priority and relates to U.S. Patent Provisional Application No. 62 / 837,188, filed April 22, 2019, U.S. Patent Provisional Application No. 62 / 827,809, filed April 1, 2019, U.S. Patent Provisional Application No. 62 / 824,176, filed March 26, 2019, U.S. Patent Provisional Application No. 62 / 781,378, filed December 18, 2018, U.S. Patent Provisional Application No. 62 / 781,368, filed December 18, 2018, U.S. Patent Provisional Application No. 62 / 774,682, filed December 3, 2019. December 2018, to U.S. Provisional Patent Application No. 62 / 743,358, filed October 9, 2018, to U.S. Provisional Patent Application No. 62 / 740,735, filed October 3, 2018, to U.S. Provisional Patent Application No. 62 / 719,347, filed August 17, 2018, to U.S. Provisional Patent Application No. 62 / 702.This application incorporates the content of U.S. Patent Application No. 167, filed July 23, 2018, U.S. Provisional Application No. 62 / 698,648, filed July 16, 2018, U.S. Provisional Application No. 62 / 696,645, filed July 11, 2018, U.S. Provisional Application No. 62 / 693,814, filed July 3, 2018, U.S. Provisional Application No. 62 / 692,507, filed June 29, 2018, and U.S. Provisional Application No. 62 / 692,498, filed June 29, 2018. The content of each of the aforementioned patent applications is incorporated herein by reference. Petition 870260049851, dated 05 / 25 / 2026, page 11 / 693 2 / 180 Field of Invention

[002] The present technology relates to insulin-Fc fusion protein compositions and their use in treating diabetes in companion animals, for example, dogs or cats. Background of the Invention

[003] The following description of the background of the present technology is provided merely to assist in understanding the present technology and is not admitted to describe or establish the prior art of the present technology.

[004] Diabetes is a chronic condition characterized by insulin deficiency and / or ineffective insulin utilization. Diabetics with absolute insulin deficiency are classified as having type 1 or insulin-dependent diabetes mellitus (IDDM). Type 1 diabetics are believed to have a genetic predisposition combined with immune destruction of the insulin-producing cells (3) of the pancreas. In comparison, diabetics who can still 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 related to genetic predisposition, obesity, and certain medications.

[005] When a dog or cat does not produce insulin or cannot use it normally, blood sugar levels rise, resulting in hyperglycemia. Dogs generally exhibit an atypical glycemia phenotype with strong similarities to human type 1 diabetes. Dogs also occasionally exhibit atypical glycemia with strong similarities to type 2 diabetes in humans. Female dogs may also develop temporary insulin resistance during estrus or pregnancy. In all cases, dogs are treated with chronic insulin injection therapy. Cats generally exhibit a phenotype of Petition 870260049851, dated 05 / 25 / 2026, page 12 / 693 3 / 180 atypical blood glucose levels with strong similarities to human type 2 diabetes (i.e., insulin resistance), but by the time the disease is diagnosed by a veterinarian, it progresses to resemble a type 1 diabetes condition (inflammatory disease of the pancreas with significant loss of beta cell mass), and the cat becomes dependent on exogenous insulin. Some diabetic cats can be treated with dietary changes and oral medication, but most diabetic cats receive chronic insulin injection therapy to maintain proper regulation. If left untreated, diabetes in dogs and cats can lead to weight loss, loss of appetite, vomiting, dehydration, problems with motor function, coma, and even death.

[006] Approximately 0.24% of dogs and about 0.68% of cats in the United States are affected by diabetes. Current therapies for diabetes in dogs and cats include the use of insulin, such as Vetsulin® for dogs (Intervet Inc., dba MERCK Animal Health, Summit, NJ) and ProZinc® for cats (Boehringer Ingelheim Vetmedica, Duluth, Georgia), which are administered once or twice daily. The burden of frequent injections on owners often results in non-compliance with the treatment regimen and underdosing, leading to poor long-term health outcomes. In fact, the cost of insulin therapy and the practicality of administering doses to their pets up to 14 times a week lead a significant percentage of owners to choose euthanasia for their pets as an alternative to intensive diabetes treatment. Therefore, there is a need for economical and less costly treatment options for this disease. Brief Description of the Present Technology

[007] In one aspect, the present disclosure provides a fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and Fc fragment are linked by a Petition 870260049851, dated 05 / 25 / 2026, page 13 / 693 4 / 180 ligand, such as a peptide ligand, and wherein the Fc fragment is of non-human animal origin and comprises the following sequence: DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPE VQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGK QFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSL TCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKL SVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 16). In some embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCSLDQLENYCX3 (SEQ ID NO: 6), wherein X1 is not D, X2 is not H, and X3 is absent or is N. In some embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCSLDQLENYCX3 (SEQ ID NO: 6), wherein X1 is H, X2 is T, and X3 is absent or is N. In embodiments, the insulin polypeptide and the Fc-binding fragment are connected by an insulin ligand, such as a peptide ligand, comprising the sequence GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

[008] In embodiment examples, fusion protein comprises the sequence FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGK QMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKAL PSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDI DVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRG DTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 32). In embodiment examples, the fusion protein comprises the sequence Petition 870260049851, dated 05 / 25 / 2026, page 14 / 693 5 / 180 FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGDCPKCPAPE MLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDG KQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNK ALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPP DIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 34).

[009] In one aspect, the present disclosure provides a fusion protein comprising an insulin polypeptide and an Fc fragment, characterized in that the insulin polypeptide and the Fc fragment are linked by a linker, such as a peptide linker, wherein the Fc fragment comprises the sequence: DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPE TCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKL SVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 22). In some embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is not D and X2 is not H. In some embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCS LDQLENYC (SEQ ID NO: 10), wherein X1 is H and X2 is T. In embodiments, the insulin polypeptide and the Fc fragment are connected by a linker, such as a peptide linker, comprising the sequence GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14). Petition 870260049851, dated 05 / 25 / 2026, page 15 / 693 6 / 180

[010] In embodiment examples, the fusion protein comprises the sequence FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCWVDLDPEVQISWFVDGKQ MQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALP SPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDID VEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGD TFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 36).

[011] In one aspect, the present disclosure provides a fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are linked by a linker, such as a peptide linker, and wherein the Fc fragment is of non-human animal origin and comprises the sequence DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSD VQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEF KCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVT CLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSV DRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 20). In embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCSLDQLENYCX3 (SEQ ID NO: 6), wherein X1 is not D, X2 is not H, and X3 is absent. In embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCSLDQLENYCX3 (SEQ ID NO: 6), wherein X1 is H, X2 is T, and X3 is absent. In embodiments, the insulin polypeptide and the Fc fragment are connected by a linker, such as a peptide linker, comprising the following sequence Petition 870260049851, dated 05 / 25 / 2026, page 16 / 693 7 / 180 GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

[012] In embodiment examples, the fusion protein comprises the sequence FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPPPEM LGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQ VYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPS PIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAV EWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTY TCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 38).

[013] In one aspect, the present disclosure provides a fusion protein comprising an insulin polypeptide and an Fc fragment, characterized in that the insulin polypeptide and the Fc fragment are linked by a linker, such as a peptide linker, wherein the Fc fragment comprises the sequence DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSD VQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEF KCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVT CLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSV DRSRWQRGNTYTCSVSHEALHS HHTQKSLTQSPG (SEQ ID NO: 23). In embodiments, the insulin polypeptide of the fusion protein comprises the sequence FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCS LDQLENYC (SEQ ID NO: 10), wherein X1 is not D and X2 is not H. In some embodiments, the insulin polypeptide comprises the following sequence FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCS LDQLENYC (SEQ ID NO: 10), where X1 is H and X2 is T. In embodiments, the insulin polypeptide and the Fc fragment are connected by a linker, such as a peptide linker, understanding Petition 870260049851, dated 05 / 25 / 2026, page 17 / 693 8 / 180 dendo a sequência GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

[014] In embodiment examples, the fusion protein comprises the sequence FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPPPEM LGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQ VYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPS PIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAV EWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTY TCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 40).

[015] In some aspects, the fusion proteins described in the present invention comprise a homodimer. In the embodiments, the homodimer percentage of the fusion protein is greater than 90%. In the embodiments, the fusion proteins described in the present invention are made using HEK293 cells and the resulting homodimer titer after purification using Protein A beads or a Protein A column is greater than 50 mg / L. In the embodiments, the insulin receptor IC50 for the fusion proteins described in the present invention is less than or equal to 5000 nM. In the embodiments, the serum half-life of the fusion proteins described in the present invention in the blood or serum of a target animal after administration is greater than about 3 days.In embodiments, for the fusion proteins described in the present invention, the time during which there is a statistically significant decrease in a subject's blood glucose level relative to a pre-dose level is greater 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.

[016] In some respects, for the fusion proteins described in the present invention, NAOC after the first subcutaneous injection in a Petition 870260049851, dated 05 / 25 / 2026, page 18 / 693 9 / 180 target animal is greater than 150%FBGL days kg / mg. In exemplary embodiments, for the fusion proteins described in the present invention, the ratio between the NAOC after the third weekly subcutaneous injection of the fusion proteins into the target animal and the NAOC after the first subcutaneous injection of the fusion protein into the target animal is greater than 0.50.

[017] In some respects, the fusion proteins described in the present invention are formulated as a pharmaceutical composition. In embodiments, in the pharmaceutical composition, the fusion protein is present at a concentration of about 3 mg / mL or greater. In embodiments, the composition is suitable for subcutaneous administration.

[018] In some aspects, a method is described for lowering the blood glucose level of a target animal, which method comprises administering a physiologically effective amount of a fusion protein as described in the present invention or a pharmaceutical composition of said fusion protein to the patient. In embodiments, the target animal is diagnosed with diabetes. In embodiments, the target animal is a dog or cat. In some embodiments, the fusion protein is administered subcutaneously. In some embodiments, the fusion protein is administered daily, twice weekly, or once weekly to the target animal. In some examples, the fusion protein is administered once weekly to the target animal at a dose between 0.025 and 0.5 mg / kg / week. In some aspects, a cell manipulated to express a fusion protein as described in the present invention is described.In some examples, the cell is transfected with a nucleic acid that codes for the fusion protein. In others, the cell is either a HEK293 cell or a CHO cell.

[019] In one aspect, a cDNA encoding a fusion protein is described, as described in this document. In Petition 870260049851, dated 05 / 25 / 2026, page 19 / 693 10 / 180 exemplos de realização, o cDNA compreende a sequência de ácido nucleico atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccag cacctgtg cggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggt ggcggag gaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggacca gctggaaa actactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggagg aggcggg ggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcc caagccc aaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatccc gaagacc ccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccc cgggaag agcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaa ggggaag caattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaag gccaggg gccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaaca cagtcagc cttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggcca gcaggag cccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctac agcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggc tttgcataa ccactacacccaggagagcctgtcccacagcccggatag (SEQ ID NO: 31). Petition 870260049851, of 25 / 05 / 2026, p. 20 / 693 11 / 180

[020] Em exemplos de realização, o cDNA compreende a sequênciade ácido nucleico atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccag cacctgtg cggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggt ggcggag gaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggacca gctggaaa actactgcaacggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcaggg aggaggc gggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttcc ctcccaag cccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggat cccgaag accccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaa ccccggg aagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggct gaagggg aagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagc aaggcca ggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaaga acacagtc agccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacgg ccagcag gagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcct ctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacg aggctttgc ataaccactacacccaggagagcctgtcccacagcccggatag (SEQ ID NO: 33). Petition 870260049851, of 25 / 05 / 2026, p. 21 / 693 12 / 180

[021] Em exemplos de realização, o cDNA compreende a sequênciade ácido nucleico atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccag cacctgtg cggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggt ggcggag gaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggacca gctggaaa actactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggagg aggcggg ggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcc caagccc aaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatccc gaagacc ccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccc cgggaag agcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaag gggaagc aattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaagg ccagggg ccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacac agtcagcc ttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccag caggagc ccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctac agcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggc tttgcataa ccactacacccaggagagcctgtcccacagcccggatag (SEQ ID NO: 35), Petition 870260049851, of 25 / 05 / 2026, p. 22 / 693 13 / 180

[022] Em exemplos de realização, o cDNA compreende a sequênciade ácido nucleico atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccag cacctgtg cggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggt ggcggag gaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggacca gctggaaa actactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggagg aggcggg ggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgccc aagcccaa ggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagac gactctga cgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggag gagcagtt caacagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaa agagttca agtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaa gggccagc cgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggtt agcgtga catgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacc cgagccc gagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagc aggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttg cacag ccaccacactcagaagagtctgacccagagcccgggatag (SEQ ID NO: 37).

[023] In embodiment examples, cDNA comprises the nucleic acid sequence Petition 870260049851, dated 05 / 25 / 2026, p. 23 / 693 14 / 180 atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttctggaaccag cacctgtg cggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggt ggcggag gaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggacca gctgggaaa actactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggagg aggcggg ggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttccccgcc aagcccaa ggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagac gactctga cgtgcagatcacctggttcgtagacaacccaggtttacactgccaagaccagtcccagggag gagcagtt cagcagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaa agagttca agtgtaaggtgaacagcaagagcctgcccagccccattgaaaggacatcagcaaggacaa gggccagc cgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcagagaacaaggtt agcgtga catgcctgatcgagggtttctaccccagcacatcgccgtggagtgggaaatcaccggccaacc cgagccc gagaacaactacaggaccactccgcaactggacagcgacgggacctacttctgtatagatc aggctgag cgtggaccggagcaggcaggggcaacacctacactttcagcgtgagccacgaggccttg cacag ccaccacactcagaagagtctgacccagagcccgggatag(SEQ ID NO: 39). Brief Description of the Figures

[024] FIG. 1A shows a schematic representation of an exemplary homodimeric insulin-Fc fusion protein. Petition 870260049851, dated 05 / 25 / 2026, p. 24 / 693 15 / 180

[025] FIG. 2 shows the mean % of fasting blood glucose levels from Day 0 to Day 3 for N = 3 dogs that received intravenously on Day 0 0.2 mg / kg of the homodimer of SEQ ID NO: 42.

[026] FIG. 3 illustrates a side-by-side sequence comparison of SEQ ID NOs: 42, 44, 46, 48 and 50. * represents complete homology in all sequences at a given sequence position, while . or spaces refer to conservative, moderate or very different amino acid mutations in the sequences at a given sequence position, respectively.

[027] FIG. 4 illustrates a side-by-side sequence comparison of SEQ ID NOs: 42, 52, 54 and 56. * represents complete homology in all sequences at a given sequence position, while :, . or spaces refer to conservative, moderate or very different amino acid mutations in the sequences at a given sequence position, respectively.

[028] FIG. 5 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for N = 3 dogs that received intravenously on Day 0 0.2 mg / kg of the SEQ ID NO: 52 homodimer.

[029] FIG. 6 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for N = 6 dogs that received subcutaneous injection on Day 0 at 0.33 mg / kg of the SEQ ID NO: 52 homodimer.

[030] FIG. 7 shows the mean antidrug antibody titers (ug / mL) for N = 3 dogs that received subcutaneously 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) the homodimer of SEQ ID NO: 52.

[031] FIG. 8 illustrates a side-by-side sequence comparison of SEQ IDs 58, 60, 62, and 64. * represents complete homology across all sequences at a given sequence position, while :, ., or spaces refer to amino acid mutations. Petition 870260049851, dated 05 / 25 / 2026, p. 25 / 693 16 / 180 conservative, moderate, or very different in sequences at a given sequence position, respectively.

[032] FIG. 9 shows the mean antidrug antibody titers (ug / mL) for N = 1 dog that received subcutaneously on day 0 (0.33 mg / kg), day 7 (0.50 mg / kg), day 14 (0.50 mg / kg), day 21 (0.50 mg / kg), the homodimer of SEQ ID NO: 64.

[033] FIG. 10 shows the mean antidrug antibody titers (ug / mL) for a dog (N = 1) that received subcutaneously on day 0 (0.33 mg / kg), and day 14 (0.16 mg / kg) the homodimer of SEQ ID NO: 66.

[034] FIG. 11 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for N = 2 dogs that received subcutaneously on Day 0 0.33 mg / kg of the SEQ ID NO: 66 homodimer.

[035] FIG. 12 illustrates a side-by-side sequence comparison of SEQ ID NOs: 66, 68, 70, 72, 74 and 76. * represents complete homology in all sequences at a given sequence position, while :, . or spaces refer to conservative, moderate or very different amino acid mutations in the sequences at a given sequence position, respectively.

[036] FIG. 13 illustrates a side-by-side sequence comparison of SEQ ID NOs: 66, 78, 80, 82 and 84. * represents complete homology in all sequences at a given sequence position, while :, . or spaces refer to conservative, moderate or very different amino acid mutations in the sequences at a given sequence position, respectively.

[037] FIG. 14 illustrates a side-by-side sequence comparison of SEQ ID NOs: 66, 76, and 86. * represents complete homology in all sequences at a given sequence position, while :, . or spaces refer to conservative, moderate, or very different amino acid mutations in the sequences at a given sequence position, respectively. Petition 870260049851, dated 05 / 25 / 2026, p. 26 / 693 17 / 180

[038] FIG. 15 illustrates a side-by-side sequence comparison of SEQ ID NOs: 66, 82, 84, and 88. * represents complete homology in all sequences at a given sequence position, while . or spaces refer to conservative, moderate, or very different amino acid mutations in the sequences at a given sequence position, respectively.

[039] FIG. 16 illustrates a side-by-side sequence comparison of SEQ ID NOs: 32, 34, 66, 90, 92 and 94. * represents complete homology in all sequences at a given sequence position, while :, . or spaces refer to conservative, moderate or very different amino acid mutations in the sequences at a given sequence position, respectively.

[040] FIG. 17 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for a dog (N = 1) treated subcutaneously on Day 0 at 0.16 mg / kg with the SEQ ID NO: 34 homodimer.

[041] FIG. 18 shows the mean antidrug antibody titers (ug / mL) for a dog (N = 1) that received subcutaneously on day 0 (0.16 mg / kg), day 14 (0.16 mg / kg), day 28 (0.16 mg / kg) and day 42 (0.16 mg / kg) the homodimer of SEQ ID NO: 34.

[042] FIG. 19 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for a dog (N = 1) treated subcutaneously on Day 0 at 0.33 mg / kg with the SEQ ID NO: 32 homodimer.

[043] FIG. 20 shows the mean % of fasting blood glucose levels from Day 0 to Day 60 for a dog (N = 1) treated subcutaneously on Day 0 (0.33 mg / kg), Day 15 (0.16 mg / kg), Day 31 (0.16 mg / kg) and Day 45 (0.15 mg / kg) with the homodimer of SEQ ID NO: 32.

[044] FIG. 21 shows the mean antidrug antibody titers (ug / mL) for a dog (N = 1) that received subcutaneous administration on day 0 (0.33 mg / kg), day 15 (0.16 mg / kg), day 31 (0.16 mg / kg) and day 45 (0.15 mg / kg) of the homodimer of SEQ ID NO: 32. Petition 870260049851, dated 05 / 25 / 2026, p. 27 / 693 18 / 180

[045] FIG. 22 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for a dog (N = 1) treated subcutaneously on Day 0 at 0.16 mg / kg with the SEQ ID NO: 96 homodimer.

[046] FIG. 23 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for a dog (N = 1) treated subcutaneously on Day 0 at 0.16 mg / kg with the SEQ ID NO: 98 homodimer.

[047] FIG. 24 illustrates a side-by-side sequence comparison of SEQ IDs NOS: 102 and 104. * represents complete homology in all sequences at a given sequence position, while :,. or spaces refer to conservative, moderate, or very different amino acid mutations in the sequences at a given sequence position, respectively.

[048] FIG. 25 shows the % of fasting blood glucose levels from day 0 to day 7 for N = 1 dog treated subcutaneously on day 0 with 0.16 mg / kg with the SEQ ID NO: 102 homodimer, and % of fasting blood glucose levels from day 0 to day 7 for N = 1 dog treated subcutaneously on day 0 with 0.16 mg / kg with the SEQ ID NO: 104 homodimer.

[049] FIG. 26 shows the % of fasting blood glucose levels from Day 0 to Day 7 for N = 1 dog treated subcutaneously with the SEQ ID NO: 36 homodimer, in addition to the times the dog was fed.

[050] FIG. 27 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for N = 3 cats that received subcutaneous injection on Day 0 at 0.8 mg / kg of the homodimer with SEQ ID NO: 106.

[051] FIG. 28 illustrates a side-by-side sequence comparison of SEQ IDs NOS: 106, 108, 110, and 112. * represents complete homology across all sequences at a given sequence position, while :, ., or spaces refer to amino acid mutations. Petition 870260049851, dated 05 / 25 / 2026, p. 28 / 693 19 / 180 conservative, moderate, or very different in sequences at a given sequence position, respectively.

[052] FIG. 29 shows the mean antidrug antibody titers (g / mL) for N = 3 cats that received subcutaneous administration 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) of the homodimer with SEQ ID NO: 106.

[053] FIG. 30 illustrates a side-by-side sequence comparison of SEQ ID NOs: 108, 114, 116 and 118. * represents complete homology in all sequences at a given sequence position, while :, . or spaces refer to conservative, moderate or very different amino acid mutations in the sequences at a given sequence position, respectively.

[054] FIG. 31 illustrates a side-by-side sequence comparison of SEQ ID NOs: 106, 112, and 122. * represents complete homology in all sequences at a given sequence position, while :, ., or spaces refer to conservative, moderate, or very different amino acid mutations in the sequences at a given sequence position, respectively.

[055] FIG. 32 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for a hato (N = 1) who received subcutaneously on Day 0, 0.16 mg / kg of the homodimer of SEQ ID NO: 122.

[056] FIG. 33 shows the % of fasting blood glucose levels from Day 0 to Day 7 for a cat (N = 1) treated subcutaneously on day 0 (0.16 mg / kg) with the SEQ ID NO: 38 homodimer, in addition to the times the cat was fed.

[057] FIG. 34 shows the mean antidrug antibody titers (ug / mL) for a cat (N = 1) that received subcutaneously on day 0 (0.16 mg / kg), day 14 (0.16 mg / kg), day 28 (0.11 mg / kg) and day 42 (0.09 mg / kg) the homodimer of SEQ ID NO: 38. Petition 870260049851, dated 05 / 25 / 2026, p. 29 / 693 20 / 180

[058] FIG. 35 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for a cat (N = 1) that received subcutaneously on Day 0, 0.16 mg / kg of the homodimer of SEQ ID NO: 124.

[059] FIG. 36 shows the mean % of fasting blood glucose levels from Day 0 to Day 7 for N = 3 cats that received subcutaneous administration on Day 0 (0.10 mg / kg) with the SEQ ID NO: 40 homodimer.

[060] FIG. 37 shows the mean % of fasting blood glucose levels from Day 7 to Day 14 for N = 3 cats that received subcutaneous administration on Day 7 (0.20 mg / kg) with the SEQ ID NO: 40 homodimer.

[061] FIG. 38 illustrates the complete amino acid sequence of a fusion protein (SEQ ID NO: 32) and its corresponding nucleic acid sequence (SEQ ID NO: 31).

[062] FIG. 39 illustrates the complete amino acid sequence of a fusion protein (SEQ ID NO: 34) and its corresponding nucleic acid sequence (SEQ ID NO: 33).

[063] FIG. 40 illustrates the complete amino acid sequence of a fusion protein (SEQ ID NO: 36) and its corresponding nucleic acid sequence (SEQ ID NO: 35).

[064] FIG. 41 illustrates the complete amino acid sequence of a fusion protein (SEQ ID NO: 38) and its corresponding nucleic acid sequence (SEQ ID NO: 37).

[065] FIG. 42 illustrates the complete amino acid sequence of a fusion protein (SEQ ID NO: 40) and its corresponding nucleic acid sequence (SEQ ID NO: 39). DETAILED DESCRIPTION OF THE INVENTION

[066] An insulin treatment that requires less frequent dosing (e.g., injections once a week) would be less costly for owners, leading to better adherence, Petition 870260049851, dated 05 / 25 / 2026, p. 30 / 693 21 / 180 fewer cases of euthanasia, and better outcomes for pets. For a given species (e.g., dog or cat), a suitable molecule for an ultralong-acting diabetes treatment must be manufactured in mammalian cells, e.g., human embryonic kidney cells (HEK, e.g., HEK293), with an acceptable titer of the desired homodimer product (e.g., homodimer titer greater than 50 mg / L of transiently transfected HEK cells, greater than 75 mg / L of transiently transfected HEK cells, or greater than 100 mg / L of transiently transfected HEK cells, etc.).Only candidates with a homodimer titer greater than 50 mg / L are considered useful in the present invention, as experience has shown that homodimer titers lower than this level are unlikely to result in the commercial production of homodimer in Chinese hamster ovary (CHO) cells that meet the low-cost manufacturing requirements for veterinary products. Furthermore, the molecule must bind to the insulin receptor with a notable affinity (e.g., IC50 less than 5000 nM, IC50 less than 4000 nM, IC50 less than 3000 nM, IC50 less than 2500 nM, etc.) as measured in the IM-9 insulin receptor binding assay at 4°C. Based on experimentation, only molecules exhibiting insulin receptor activity with IC50 values ​​below 5000 nM are considered likely to meet the bioactivity requirement in the species in question.The molecule must also demonstrate sustained bioactivity in vivo (e.g., demonstrate glucose-lowering activity exceeding 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 more) to justify less frequent dosing. The molecule must also demonstrate prolonged residence time in the target animal system (e.g., Petition 870260049851, dated 05 / 25 / 2026, page 31 / 693 22 / 180 example, the serum half-life must be greater than 3 days or more). The bioactive potency and duration of bioactivity can be quantitatively represented by calculating the area under the curve of the percentage of fasting glucose (%FBGL) normalized to a given dose in mg / kg (NAOC) with units of %FBGL days kg / mg as described in Example 11. NAOC increases with a greater drop in %FBGL, which is the case where the molecule demonstrates increased bioactivity, and when %FBGL takes longer to return to 100%, which is the case where the insulin-Fc fusion protein demonstrates increased duration of action. To be useful, as described in this document, a molecule must demonstrate a sufficiently high NAOC value (e.g., preferably an NAOC greater than 150 %FBGL days kg / mg, more preferably an NAOC greater than 200 %FBGL days kg / mg, and even more preferably an NAOC greater than 250 %FBGL days kg / mg).Based on experiments, at NAOC values ​​greater than 150%FBGL days kg / mg, the dose requirements in the target species will be sufficiently low to achieve an acceptable treatment cost. Finally, to be useful for the treatment of a chronic disease, such as diabetes, the molecule should not induce the production of anti-drug antibodies, especially antibodies that neutralize the bioactivity of the molecule, after repeated administration. Therefore, the molecule should demonstrate similar duration and extent of bioactivity (i.e., NAOC) after multiple repeated doses in the target animal (e.g., the ratio of NAOC after the third weekly subcutaneous injection to the NAOC after the first weekly subcutaneous injection of the molecule (i.e., the NAOC ratio (NAOCR) after the third dose) is, in order of preference, greater than 0.50, greater than 0.60, greater than 0.70, greater than 0.80, or greater than 0.90 or more). Petition 870260049851, dated 05 / 25 / 2026, page 32 / 693 23 / 180

[067] The proposed treatments with ultralong-acting insulin for human clinical use comprise an insulinFc fusion protein that makes use of a human Fc fragment to prolong its action in vivo. As a human Fc fragment is expected to be immunogenic and consequently capable of inducing the production of anti-drug antibodies in companion animals (e.g., dogs or cats), the human Fc fragment must be replaced by a species-specific Fc fragment (e.g., canine or feline). However, it has been found, quite unexpectedly, that a simple exchange between the human Fc fragment and the species-specific Fc fragment (e.g., canine or feline) does not yield a product with an acceptable homodimer titer (e.g., a homodimer titer greater than 50 mg / L) or a sufficiently high NAOC value (e.g., an NAOC greater than 150 %FBGL days kg / mg).For example, in some cases, only one specific isotype (e.g., canine IgGB or feline IgG1b) for the Fc fragment resulted in an insulin-Fc fusion protein with a sufficiently high homodimer titer (e.g., a homodimer titer greater than 50 mg / L) and an acceptably high NAOC value (e.g., an 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, thus requiring site-directed mutations to find the relatively small number of realizations that were non-immunogenic and bioactive in the target species with acceptably high NAOC (e.g., NAOC values ​​greater than 150 %FBGL.days.kg / mg) and NAOCR values ​​after the third week of subcutaneous dosing that were greater than 0.5.In other cases, when the Fc fragments were mutated to prevent glycosylation and thus further reduce the immunogenicity of the insulin-Fc fusion protein, it was unexpectedly found that only... Petition 870260049851, dated 05 / 25 / 2026, page 33 / 693 24 / 180 specific amino acid mutations in the Fc fragment lead to the desired homodimer titers (e.g., homodimer titers greater than 50 mg / L) and NAOC values ​​(e.g., NAOC values ​​greater than 150 %FBGL days kg / mg). Furthermore, it was found that an additional mutation in the insulin component was necessary to produce these mutated and non-glycosylated insulin-Fc fusion proteins with the desired homodimer titers (e.g., homodimer titers greater than 50 mg / L) and NAOC values ​​(e.g., NAOC values ​​greater than 150 %FBGL days kg / mg), while simultaneously achieving NAOCR values ​​greater than 0.5 after the third weekly subcutaneous dose.Therefore, the invention provides high-purity, long-acting, bioactive, and non-immunogenic insulin-Fc fusion proteins that can be manufactured with acceptably high homodimer titers (e.g., homodimer titers greater than 50 mg / L), NAOC values ​​(e.g., NAOC values ​​greater than 150 %FBGL days kg / mg), and NAOCR values ​​after the third weekly subcutaneous dose greater than 0.5, 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. DEFINITIONS

[068] As used in this invention, the articles "a" and "an" refer to one or more of the grammatical object of the article. The use of the words "a" or "an" when used in conjunction with the term comprising in this document may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more of one."

[069] As used in the present invention, about and approximately generally mean an acceptable degree of error for the Petition 870260049851, dated 05 / 25 / 2026, p. 34 / 693 25 / 180 measured quantity, given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), normally within 10%, and most typically within 5% of a given range of values.

[070] As used in the present invention, an amount of a molecule, compound, conjugate or substance effective for treating a disorder (for example, a disorder described in the present invention), therapeutically effective amount or effective amount refers to an amount of the molecule, compound, conjugate or substance that is effective, through single or multiple dose(s) administration to a subject, in treating a subject, or in curing, relieving, attenuating or improving a subject with a disorder (for example, a disorder described in the present invention) beyond what would be expected in the absence of such treatment.

[071] As used in the present invention, the term analogue refers to a compound or conjugate (for example, a compound or conjugate as described in the present invention, for example, insulin) having a chemical structure similar to that of another compound or conjugate, but differing from it in at least one respect.

[072] As used in this application, the term antibody or antibody molecule refers to an immunoglobulin (Ig) molecule, immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule containing an antigen-binding site that specifically binds to, and for example, immunoreacts with, an antigen. As used in this document, the term antibody domain refers to a variable or constant region of an immunoglobulin. As used in this document, the term antibody domain refers to a variable or constant region of an immunoglobulin. It is documented in the art that antibodies comprise Petition 870260049851, dated 05 / 25 / 2026, page 35 / 693 26 / 180 various classes, for example, IgA, IgM, or IgG in the case of mammals (e.g., humans and felines). Immunoglobulin classes can be classified into different isotypes, such as IgGa, IgGb, IgGc, and IgGd for canines, or IgG1a, IgG1b, and IgG2 for felines. Those skilled in the art will recognize that immunoglobulin isotypes of a given immunoglobulin class will comprise different amino acid sequences, structures, and functional properties (e.g., different binding affinities for Fc(gamma) receptors). "Specifically binds" or "immunoreacts with" means that the antibody reacts with one or more antigenic determinants of the desired antigen and has a lower affinity for other polypeptides, e.g., does not react with other polypeptides.

[073] As used in the present invention, the term area-under-curve or AUC refers to the integrated area under the curve of %FBGL versus time curve for a subject after a given dose of insulin-Fc fusion protein is administered. As used in this document, the term area under the curve or AOC is used as a measure of the biological potency of an insulin-Fc fusion protein such that the AOC is equal to the difference between the total possible area under the curve of %FBGL vs. time curve and the AUC value. As used in this document, the normalized area under the curve, normalized AOC or NAOC is the AOC value divided by the actual dose of insulin-Fc fusion protein administered. As used in this document, the term normalized AOC ratio or NAOCR is the ratio of the NAOC resulting from a particular administration of an insulin-Fc fusion protein to the NAOC resulting from the first administration of an insulin-Fc fusion protein in a series of administrations.NAOCR thus provides a measure of the change in the biological activity of an insulin-Fc fusion protein after repeated administrations. Petition 870260049851, dated 05 / 25 / 2026, page 36 / 693 27 / 180

[074] As used in the present invention, the term bioactivity, activity, biological activity, potency, bioactive potency, or biological potency refers to the extent to which an insulin-Fc fusion protein activates the insulin receptor and / or exerts a reduction in blood glucose levels in a target subject. As used in the present invention, in vitro activity or activity on the insulin receptor refers to the affinity with which an insulin-Fc fusion protein binds to the insulin receptor and is typically measured by the concentration at which an insulin-Fc fusion protein displaces half of an insulin reference standard from the insulin receptor in a competitive binding assay (i.e., IC50). As used in the present invention, in vivo activity refers to the extent and duration of the reduction in the fasting blood glucose level of a target subject after administration of an insulin-Fc fusion protein.

[075] As used in the present invention, the term biosynthesis, recombinant synthesis or recombinantly made refers 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., vector) encoding the insulin-Fc fusion protein (e.g., where 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. The cells can be cultured using standard methods in the art and the expressed insulin-Fc fusion protein can be collected and purified from the cell culture using standard methods in the art.

[076] As used in the present invention, the term cell surface receptor refers to a molecule such as a protein, generally found on the outer surface of the cell membrane and which interacts with soluble molecules, for example, molecules that Petition 870260049851, dated 05 / 25 / 2026, page 37 / 693 28 / 180 circulate in the blood supply. In some embodiments, a cell surface receptor may include a hormone receptor (e.g., an insulin hormone receptor or insulin receptor (IR)) or an Fc receptor that binds to an Fc fragment or Fc region of an antibody (e.g., Fc(gamma) receptor, e.g., Fc(gamma)I receptor, or a neonatal Fc receptor, e.g., FcRn).As used in the present invention, in vitro activity or activity on the Fc(gamma) receptor or binding to the Fc(gamma) receptor or activity on the FcRn receptor or binding to the FcRn refers to the affinity with which an insulin-Fc fusion protein binds to the Fc receptor (e.g., Fc(gamma) receptor or FcRn receptor) and is typically measured by the concentration of an insulin-Fc fusion protein that causes the insulin-Fc fusion protein to reach half of its maximum binding (i.e., EC50 value) as measured in an assay (e.g., an enzyme-linked immunosorbent assay (ELISA)) using OD450 nm values ​​measured on a microplate reader.

[077] As used in the present invention, the term fasting blood glucose level or FBGL refers to the average blood glucose level in a target subject at the end of a period during which no food is administered and immediately before the time when an insulin-Fc fusion protein is administered. As used in this document, the term percentage fasting blood glucose level, % fasting blood glucose level or %FBGL refers to the ratio of a given blood glucose level to the fasting blood glucose level multiplied by 100.

[078] As used in the present invention, the term immunogenic or immunogenicity refers to the ability of a particular molecule (for example, an insulin-Fc fusion protein of the present invention) to elicit the immune system of a target subject in such a way Petition 870260049851, dated 05 / 25 / 2026, page 38 / 693 29 / 180 that after repeated administrations of the molecule, the subject develops antibodies capable of specifically binding to the molecule (i.e., anti-drug antibodies). As used in the present invention, the term neutralizing, neutralizing antibodies, or anti-drug neutralizing antibodies refers to the ability of antibodies to interfere with the biologically active compound in the target object. As used in the present invention, the terms immunogenic epitopes, immunogenic hotspots, or simply hot spots, refer to mutations or epitopes of a given molecule (for example, an insulin-Fc fusion protein of the present invention) that are responsible for moderate or strong binding of anti-drug antibodies.

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

[080] As used in the present invention, the term monomer refers to a protein or a fusion protein comprising a single polypeptide. In embodiments, the monomer is a protein or a fusion protein, for example, a single polypeptide comprising an insulin polypeptide and an Fc fragment polypeptide, wherein the insulin and Fc fragment polypeptides are joined by peptide bonds to form the single polypeptide. In embodiments, the monomer is encoded by a single nucleic acid molecule.

[081] As used in the present invention, N-terminal refers to the beginning of a protein or polypeptide that is initiated by an amino acid containing a free amino group which is the alpha-amino group of Petition 870260049851, dated 05 / 25 / 2026, p. 39 / 693 30 / 180 amino acid (for example, the free amino that is covalently linked to a carbon atom that is located adjacent to a second carbon atom, wherein the second carbon atom is part of the carbonyl group of the amino acid). As used in the present invention, C-terminal refers to the end of a protein or polypeptide that is terminated by an amino acid containing a carboxylic acid group, wherein the carbon atom of the carboxylic acid group is located adjacent to the alpha-amino group of the amino acid.

[082] As used in the present invention, pharmacodynamics or PD generally refers to the biological effects of an insulin-Fc fusion protein in a subject. Specifically, here PD refers to the measurement of the reduction in fasting blood glucose levels over time in a subject following administration of an insulin-Fc fusion protein.

[083] As used in the present invention, pharmacokinetics or PK generally refers to the characteristic interactions of an insulin-Fc fusion protein and the subject's body in terms of absorption, distribution, metabolism, and excretion. Specifically, here PK refers to the concentration of an insulin-Fc fusion protein in the blood or serum of a subject at a given time after administration of the insulin-Fc fusion protein. As used in the present invention, half-life refers to the time elapsed for the concentration of the insulin-Fc fusion protein in the blood or serum of a subject to reach half of its original value calculated from a first-order exponential decay model for drug elimination. Insulin-Fc fusion proteins with higher half-life values ​​demonstrate a longer duration of action in the target subject.

[084] The terms sequence identity, sequence homology, homology or identical in amino acid or nucleotide sequences, used in the present invention, describe that the Petition 870260049851, dated 05 / 25 / 2026, p. 40 / 693 31 / 180 The same nucleotides or amino acid residues are found within variant and reference sequences when a specified contiguous segment of the variant nucleotide sequence or amino acid sequence is aligned and compared with the reference sequence nucleotide sequence or amino acid sequence. Methods for sequence alignment and for determining identity between sequences are known in the state of the art, including the use of Clustal Omega, which organizes, aligns, and compares sequences by similarity, where the software highlights each sequence position and compares it across all sequences at that position and assigns one of the following scores: an * (asterisk) for sequence positions that have a single fully conserved residue; : (colon) indicates conservation between groups of strongly similar properties with a score greater than 0.5 in the Gonnet PAM 250 matrix; and a .(Dot) indicates conservation between groups of weakly similar properties with a score less than or equal to 0.5 in the Gonnet PAM 250 matrix; a - (dash) indicates a sequence gap, meaning that there is no local homology within a given set of comparisons within a certain sequence range, and an empty space indicates little or no sequence homology for that particular position among the compared sequences. See, for example, Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing & 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 regard to the optimal alignment of two nucleotide sequences, the contiguous segment of the variant nucleotide sequence may have additional nucleotides or nucleotides deleted relative to the reference nucleotide sequence.Similarly, for the purpose of optimal alignment of two amino acid sequences, the... Petition 870260049851, dated 05 / 25 / 2026, p. 41 / 693 The 32 / 180 contiguous segment 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, the contiguous segment used for comparison with the reference nucleotide sequence or reference amino acid sequence will comprise at least 6, 10, 15, or 20 contiguous nucleotides or amino acid residues and may be 30, 40, 50, 100, or more nucleotides or amino acid residues. Corrections may be made for greater sequence identity associated with the inclusion of gaps in the variant nucleotide sequence or amino acid sequence by assigning gap penalties. Sequence alignment methods are known in the art.

[085] In embodiments, the determination of the percentage of identity or homology between two sequences is performed using a mathematical algorithm. For example, the percentage of identity of an amino acid sequence is determined using the Smith-Waterman homology search algorithm using an affine 6 gap search with a gap open penalty of 12 and a gap extension penalty of 2, and using the BLOSUM 62 matrix. The Smith-Waterman homology search algorithm is described in Smith and Waterman (1981) Adv. Appl. Math 2:482-489, incorporated herein by reference. In embodiments, the percentage of identity of a nucleotide sequence is determined using the Smith-Waterman homology search algorithm, using a gap open penalty of 25 and a gap extension penalty of 5.Such sequence identity determination can be performed using, for example, TimeLogic's DeCypher Hardware Accelerator. Petition 870260049851, dated 05 / 25 / 2026, page 42 / 693 33 / 180

[086] As used in the present invention, the term homology is used to compare two or more proteins by locating common structural features and common spatial distribution of, for example, beta strands, helices, and folds (folds). Consequently, the structures of homologous proteins are defined by spatial analyses. Measuring structural homology involves computing the geometric-topological features of a space. One approach used to generate and analyze three-dimensional (3D) protein structures is homology modeling (also called comparative modeling or knowledge-based modeling), which works by finding similar sequences based on the fact that 3D similarity reflects 2D similarity. Homologous structures do not imply sequence similarity as a necessary condition.

[087] As used in the present invention, the terms subject and patient are intended to include canine and feline animals. Exemplary canine and feline subjects include dogs and cats with a disease or disorder, for example, diabetes or another disease or disorder described in the present invention, or normal subjects.

[088] As used in the present invention, the term titer or yield refers to the amount of a fusion protein product (e.g., an insulin-Fc fusion protein described in the present invention) resulting from biosynthesis (e.g., in a mammalian cell, e.g., in a HEK293 cell or CHO cell) per volume of cell culture. The amount of product can be determined at any stage of the production process (e.g., before or after purification), but the yield or titer is always determined per volume of the original cell culture. As used in the present invention, the term product yield or total protein yield refers to the total amount of insulin-Fc fusion protein expressed per cell and purified by at least one Petition 870260049851, dated 05 / 25 / 2026, page 43 / 693 34 / 180 affinity chromatography step (e.g., protein A or protein G) and includes insulin-Fc fusion protein monomers, insulin-Fc fusion protein homodimers, and higher-order molecular aggregates of insulin-Fc fusion protein homodimers. As used in the present invention, the term homodimer percent or % homodimer refers to the proportion of a fusion protein product (e.g., an insulin-Fc fusion protein described in the present invention) that is the desired homodimer. As used in the present invention, the term homodimer titer refers to the product of the % homodimer and the total protein yield after the purification step in Protein A disclosed per cell culture volume.

[089] As used in the present invention, the terms treat or treatment of a patient suffering from a disease or disorder refer to subjecting the individual to a treatment regimen, for example, the administration of a fusion protein such as a fusion protein described in the present invention, such that at least one symptom of the disease or disorder is cured, healed, relieved, attenuated, altered, remedied, or improved. Treatment includes administering an amount effective to relieve, alleviate, alter, remedy, improve, or affect the disease or disorder, or the symptoms of the disease or disorder. Treatment may inhibit the deterioration or worsening of a symptom of a disease or disorder. Components and Structure of the Insulin-Fc Fusion Protein

[090] The present disclosure relates to a fusion protein composition (i.e., an insulin-Fc fusion protein) comprising an insulin polypeptide linked via a peptide linker to a species-specific Fc fragment, and to its use for treating diabetes in companion animals (e.g., dogs or cats). As used in the present invention, the terms fusion protein and insulin-Fc fusion protein refer to a protein Petition 870260049851, dated 05 / 25 / 2026, page 44 / 693 35 / 180 comprising more than one part, for example, from different sources (e.g., different proteins, polypeptides, cells, etc.), covalently linked by peptide bonds. Insulin-Fc fusion proteins are covalently linked by (i) linking the genes encoding each part into a single nucleic acid molecule and (ii) expression in a host cell (e.g., HEK or CHO) of the protein for which the nucleic acid molecule encodes as follows: (N-terminal)-insulin polypeptide - linker - Fc fragment - (C-terminal). The fully recombinant synthesis approach is preferred over methods where the insulin polypeptide and Fc fragments are synthesized separately and then chemically conjugated. The chemical conjugation step and subsequent purification process increase manufacturing complexity, reduce product yield, and increase costs.

[091] As used in the present invention, the term dimer refers to a protein or fusion protein comprising two covalently linked polypeptides. In embodiments, two identical polypeptides are covalently linked (e.g., via disulfide bonds) forming a homodimer (represented in a diagram in FIG. 1). The disulfide bonds are shown as dashed lines in FIG. 1; the total number of disulfide bonds in reality may be greater or less than the number shown in FIG. 1. In embodiments, the homodimer is encoded by a single nucleic acid molecule, wherein the homodimer is recombinantly made within a cell by first forming insulin-Fc fusion protein monomers and then assembling two identical insulin-Fc fusion protein monomers into the homodimer after further processing within the cell.

[092] As used in the present invention, the terms multimeric, multimeric state or multimeric state refer to non-multimeric forms Petition 870260049851, dated 05 / 25 / 2026, page 45 / 693 36 / 180 covalently associated Fc fusion protein dimers that may be in equilibrium with Fc fusion protein dimers or may act as permanently aggregated versions of Fc fusion protein dimers (e.g., Fc fusion protein homodimer dimers, Fc fusion protein homodimer trimers, Fc fusion protein homodimer tetramers, or higher-order aggregates containing five or more Fc fusion protein homodimers). Multimeric forms of Fc fusion proteins may be expected to have different physical, stability, or pharmacological activities than insulin-Fc fusion protein homodimers. Polypeptide Insulin

[093] An insulin polypeptide may be, for example, an insulin analogue or insulin produced by cells (3 in the islets of Langerhans in the pancreas. Insulin functions by regulating the uptake of glucose from the blood. Upon a stimulus, such as increased protein and glucose levels, insulin is released from the cells ( and binds to the insulin receptor (IR), initiating a signaling cascade that affects many aspects of mammalian metabolism (e.g., human, canine, or feline). Disruption of this process is directly related to several diseases, notably diabetes, insulinoma, insulin resistance, metabolic syndromes, and polycystic ovary syndrome. The insulin analogues of this disclosure may be related to the structure of insulin and may also contain one or more modifications.In some embodiments, the insulin analog comprises at least one amino acid substitution, deletion, addition, or chemical modification relative to insulin, which may impact a particular feature or characteristic of the insulin-Fc fusion protein. For example, the modifications or alterations described in the present invention may impact the structure, stability, pH sensitivity, bioactivity, or binding affinity of the insulin-Fc fusion protein. Petition 870260049851, dated 05 / 25 / 2026, page 46 / 693 37 / 180 a cell surface receptor (e.g., an insulin hormone receptor) relative to a reference standard.

[094] The amino acid sequence of insulin is strongly conserved throughout evolution, particularly in vertebrates. For example, native canine insulin differs by only one amino acid from human insulin, and native feline insulin differs by only four amino acids from human insulin. As used in this document, the terms B chain, C-peptide, or C-chain and A-chain refer to the peptide segments of an insulin polypeptide as illustrated in FIG. 1. Insulin is a 51-amino acid hormone containing two peptide chains (i.e., a B chain and an A chain) connected by disulfide bonds (e.g., disulfide bonds formed by one or more cysteine ​​side-chain thiols of the B chain and one or more cysteine ​​side-chain thiols of the A chain). The A chain of insulin is 21 amino acids long, and the B chain of insulin is 30 amino acids long.In its native form, insulin, chain A, contains an intrachain disulfide bond formed by two cysteine ​​side-chain thiols of chain A. For reference, the sequences for human insulin chain A with SEQ ID NO: 1 and human insulin chain B with SEQ ID NO: 2 are shown below: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID. NO: 1) GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 2)

[095] As used in the present invention, the term insulin or insulin polypeptide encompasses mature insulin, preproinsulin, proinsulin, and naturally occurring insulin or analogues thereof. In embodiments, an insulin polypeptide may be a full-length insulin polypeptide or a fragment thereof. In embodiments, an insulin polypeptide may comprise one or more fragments of mature insulin, preproinsulin, proinsulin, or naturally occurring insulin. Petition 870260049851, dated 05 / 25 / 2026, page 47 / 693 38 / 180

[096] Insulin is normally constructed as an N-terminal B-chain:C-chain:A-chain -C-terminal polypeptide, wherein the C-chain is cleaved in order to make it bioactive. For reference purposes, the sequence of the entire human insulin molecule including the C-chain (i.e., human proinsulin) is shown below with the C-chain underlined: FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAG SLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 3)

[097] The transformation of a single-chain insulin polypeptide into a bioactive two-chain polypeptide is normally carried out within the beta cells of the islets of Langerhans, prior to glucose-stimulated insulin secretion, by two endoproteases: Type I endoproteases, PC1 and PC3, which disrupt the binding of the C-peptide B chain and PC2, and a Type II endoprotease, which cleaves the binding of the C-peptide A chain at the precise sites. However, cellular systems used for the biosynthesis of therapeutic molecules such as insulin (e.g., bacteria, yeast, and mammalian cellular systems (e.g., HEK and CHO)) do not possess this pathway, and therefore, the transformation must occur after the expression and collection of the single-chain polypeptide using chemical or enzymatic methods.Well-known techniques for cleaving the C chain after expression and collection are based on first modifying the C chain so that it terminates in a lysine, just before the N-terminus of the A chain. Then, using an enzyme selected from the trypsin or Lys-C families, which specifically cleaves peptide bonds at the C-terminus of lysine residues, the single-chain insulin polypeptide is cleaved at the C-terminus lysine of the C chain and at the lysine at the 29th position from the C-terminus end of the B chain. In some cases, the two resulting bioactive insulin chains are used without replacing the amino acid cut at the 30th position from. Petition 870260049851, dated 05 / 25 / 2026, page 48 / 693 39 / 180 of the N-terminal of the B chain, and in some cases the amino acid cut at the 30th position of the N-terminal of the B chain is added back to the molecule using an additional enzymatic method. This process works well with insulin because it contains only one lysine in its entire two-chain polypeptide form. However, this process cannot be used on the insulin-Fc fusion proteins contained herein, as all known Fc fragments contain multiple lysine residues. The enzymatic cleavage process would therefore digest the Fc fragment into non-functional parts, thus eliminating the ability of the Fc fragment to prolong the action of the insulin polypeptide in vivo. Therefore, an insulin-Fc fusion protein of the present invention must comprise an insulin polypeptide that does not require cleavage of the C chain and is therefore bioactive in its single-chain form.

[098] Several bioactive single-chain insulin polypeptides have been described in the art. In all cases, the single-chain insulin polypeptides contain the C chain of specific length and composition, as well as the A and B chains mutated at specific amino acid sites in order to achieve electrostatic balance, avoid aggregation, and increase binding to the insulin receptor (IR) and / or downstream signaling to achieve bioactivity at levels comparable to those of native two-chain insulin. Here, the locations (sites) of mutations in peptide segments are annotated using the segment name (e.g., B chain, C chain, A chain) and the number of amino acids counting from the N-terminus of the segment. For example, the annotation B16 refers to the 16th amino acid from the N-terminus of the amino acid sequence of the B chain. The annotation A8 refers to the 8th amino acid from the N-terminus of the A chain.Furthermore, if an amino acid mutates from its native form to a new amino acid at a specific location, the location is appended with the one-letter amino acid code for the new amino acid. For example, B16A. Petition 870260049851, dated 05 / 25 / 2026, p. 49 / 693 40 / 180 refers to an alanine mutation at the 16th amino acid position from the N-terminus of the B amino acid sequence, and A8H refers to a histidine mutation at the 8th amino acid position from the N-terminus of the A amino acid sequence.

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

[0100] In embodiment examples, alanine can be replaced by tyrosine at position 16 from the N-terminal of the B chain (i.e., B16) in SEQ ID NO: 4 to produce SEQ ID NO: 5, as an alanine substitution at this position is known to be less able to activate insulin-specific T cells (Alleva, DG, Gaur, A., Jin, L., Wegmann, D., Gottlieb, PA, Pahuja, A., Johnson, EB, Motheral, T., Putnam, A., Crowe, PD, Ling, N., Boehme, SA, Conlon, PJ, (2002) Diabetes Vol. SEQ ID NO: 5 is listed below with each of the non-native amino acids underlined: Petition 870260049851, dated 05 / 25 / 2026, p. 50 / 693 41 / 180 FVNQHLCGSDLVEALALVCGERGFFYTDPTGGPRRGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 5)

[0101] In some embodiments, it was unexpectedly found that specific amino acids in SEQ ID NO: 4 and SEQ ID NO: 5 led to the development of neutralizing antidrug antibodies after repeated subcutaneous injections in the target animal (e.g., dog or cat). The antidrug antibodies led to an unacceptable reduction in NAOC after multiple injections (e.g., a NAOCR value after the third injection of less than 0.5), rendering the associated insulin-Fc fusion proteins non-viable. Specifically, it was found in the steps leading to the invention of the present disclosure that the A8 mutation for histidine and the B10 mutation for aspartic acid accounted for the vast majority of the antidrug antibody specificity and therefore represented immunogenic hot spots (e.g., immunogenic epitopes) in the insulin polypeptide.Therefore, in preferred embodiments, the insulin polypeptide does not contain histidine at position A8 or aspartic acid at position B10 of the insulin polypeptide.

[0102] In one embodiment, it was confirmed that simply maintaining amino acids A8 and B10 as in the native forms of threonine and histidine, respectively, eliminates the anti-drug antibody response, but the resulting insulin-Fc fusion protein is not bioactive in the target species (e.g., NAOC less than 150 %FBGL days kg / mg). Therefore, it was necessary to experiment with several variations of chain A, chain B, and chain C to find a suitable solution. Most variants failed to achieve homodimer titers greater than 50 mg / L, and many of those that met these objectives did not achieve acceptable levels of bioactivity in the target species (e.g., acceptable NAOC values ​​of more than 150 %FBGL days kg / mg). Having screened more than 120 variants, the following Petition 870260049851, dated 05 / 25 / 2026, p. 51 / 693 42 / 180 insulin polypeptide with SEQ ID NO: 6_NULL was considered suitable in relation to obtaining homodimer titers greater than 50 mg / L, NAOC values ​​in target species greater than 150 %FBGL days kg / mg, minimum immunogenicity, and NAOCR values ​​after the third injection in target species greater than 0.5 of the associated insulin-Fc fusion proteins (non-native amino acids underlined and excluded native amino acids represented with an underlined Z): FVNQHLCGSX1LVEALELVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3 (SEQ ID NO: 6_NULL) where X1 is not D, X2 is not H, and X3 is absent or is N.

[0103] In specific embodiment examples, in SEQ ID NO: 6_NULL, X1 represents H, X2 is T, and X3 is absent or is N, resulting in the following SEQ ID NO: 7_NULL (with non-native amino acids underlined and excluded native amino acids represented with an underlined Z): FVNQHLCGSHLVEALELVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCTST CSLDQLENYCX3 (SEQ ID NO: 7_NULL) where X3 is absent or is N.

[0104] In a specific embodiment, in SEQ ID NO: 7_NULL, X3 is missing resulting in the following SEQ ID NO: 8_NULL (with non-native amino acids underlined and deleted native amino acids represented with an underlined Z): FVNQHLCGSHLVEALELVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCTST CSLDQLENYCZ (SEQ ID NO: 8_NULL)

[0105] In a specific embodiment, in SEQ ID NO: 7_NULL, X3 is N resulting in the following SEQ ID NO: 9_NULL (with non-native amino acids underlined and deleted native amino acids represented with an underlined Z): FVNQHLCGSHLVEALELVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCTST CSLDQLENYCN (SEQ ID NO: 9_NULL) Petition 870260049851, dated 05 / 25 / 2026, p. 52 / 693 43 / 180

[0106] In some embodiments, the Fc fragment was mutated to prevent glycosylation during synthesis and potentially reduce the immunogenicity of the resulting insulin-Fc fusion protein in the target animal (e.g., dog or cat). Unexpectedly, it was found that there was an interaction between the insulin polypeptide and the mutated Fc fragment, such that another amino acid mutation was required in the insulin polypeptide to make the insulin-Fc fusion protein sufficiently manufacturable (e.g., with a homodimer titer greater than 50 mg / L) and non-immunogenic with a NAOC value in the target species greater than 150 %FBGL days kg / mg and a NAOCR value after the third injection in the target species greater than 0.5.Specifically, it was found that the mutation of amino acid B16 to an alanine in the insulin polypeptide was necessary when it was linked to specific, mutated, and non-glycosylated Fc fragments, resulting in the following insulin polypeptide SEQ ID NO: 10_NULL (with non-native amino acids underlined and deleted native amino acids represented with an underlined Z): FVNQHLCGSX1LVEALALVCGERGFHYZZZZGGGGGSGGGG GIVEQCCX2S TCSLDQLENYCZ (SEQ ID NO: 10_NULL) where X1 is not D and X2 is not H.

[0107] In a specific embodiment, in SEQ ID NO: 10_NULL, X1 is H and X2 is T resulting in the following SEQ ID NO: 11_NULL (with non-native amino acids underlined and deleted native amino acids represented with an underlined Z): FVNQHLCGSHLVEALALVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCTST CSLDQLENYCZ - SEQ ID NO: 11_NULL

[0108] The following are re-presentations of the sequences shown above, but with the amino acids missing from the Z symbol removed from the insulin polypeptide sequence notation. Again, in all cases the non-native amino acids are underlined. For Petition 870260049851, dated 05 / 25 / 2026, p. 53 / 693 44 / 180 To avoid confusion, each original sequence containing Z symbols is listed above the new sequence with the Z symbols removed. Despite the two separate notations, the paired sequences refer to exactly the same insulin polypeptide. SEQ ID NO: 6_NULL reformulated as: FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCSLDQLENYCXs (SEQ ID NO: 6) where X1 is not D, X2 is not H, and X3 is missing or is N. SEQ ID NO: 7_NULL reformulated as: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLD QLENYCX3 (SEQ ID NO: 7) where X3 is missing or is N. SEQ ID NO: 8_NULL reformulated as: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYC (SEQ ID NO: 8) SEQ ID NO: 9_NULL reformulated as: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLD QLENYCN (SEQ ID NO: 9) SEQ ID NO: 10_NULL reformulated as: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCSL DQLENYC (SEQ ID NO: 10) where X1 is not D and X2 is not H. SEQ ID NO: 11_NULL reformulated as: FVNQHLCGSH LVEALALVCG AND RG FHYGGGGGSGGGG GIVEQCCT STCSLDQLENYC (SEQ ID NO: 11) Binder

[0109] The successful construction of a recombinant insulin-Fc fusion protein requires a linker that connects the insulin polypeptide to the Fc fragment. In embodiments, an insulin-Fc fusion protein described in this invention comprises a peptide linker between the insulin polypeptide and the Fc fragment comprising amino acids (e.g., natural amino acids or Petition 870260049851, dated 05 / 25 / 2026, page 54 / 693 45 / 180 non-natural). In embodiments, the peptide linker can be encoded by a nucleic acid molecule, for example, so that a single nucleic acid molecule can encode the various peptides within an insulin polypeptide, as well as the peptide linker and the Fc fragment. The choice of peptide linker (e.g., length, composition, hydrophobicity, and secondary structure) can impact the manufacturability (i.e., homodimer 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. October 15, 2013; 65(10): 1357-1369). Table 1 lists several linkers used in the design of an insulin-Fc fusion protein with the aim of improving homodimer titer and bioactivity. Table 1 Peptide linking the AE chain to the Fc fragment of a protein. Insulin-Fc Fusion GGGGAGGGG GGGGSGGGG GGGGGAGGGG GGGGSGGGGSGGGGSGGGG GGGGKGGGGKGGGGKGGGG GGGGGQGGGGQGGGGQGGGGG GGGGGAGGGGAGGGGAGGGGG SGGGGQGGGGQGGGQGGGGG HGGGGQGGGGQGGGGQGGGGG PGGGGGQGGGGQGGGGQGGGGG In examples of implementation, the peptide linker comprises the sequence: GGGGAGGGG (SEQ ID NO: 12). Petition 870260049851, dated 05 / 25 / 2026, page 55 / 693 46 / 180 In another example of embodiment, the peptide linker comprises the sequence: GGGGSGGGG (SEQ ID NO: 13). In preferred embodiments, the peptide linker comprises the sequence: GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

[0110] When constructing a recombinant insulin-Fc fusion protein made with a peptide linker such as that of SEQ ID NO: 14, attention must be paid to the possibility of unwanted enzymatic cleavage between the C-terminal of the insulin A chain and the N-terminal of the peptide linker. Cleavage of the linker and the Fc fragment of the insulin polypeptide would render the insulin-Fc fusion protein incapable of providing a prolonged duration of bioactivity. There is a known enzymatic cleavage site between asparagine-glycine linkages (Vlasak, J., Ionescu, R., (2011) MAbs Vol. 3, No. 3 pp. 253-263). In many examples of peptide linkage implementation, including the preferred peptide linker of SEQ ID NO: 14, the N-terminal amino acid is glycine. Furthermore, the C-terminal of the A chain of insulin, that is (the 21st amino acid of the N-terminal of the A chain (i.e., A21)) is asparagine.Therefore, asparagine A21 is omitted in the insulin polypeptides of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 11 to eliminate the potentially enzymatically cleavable asparagine-glycine linkage that would form between the A chain and the peptide linker. Unexpectedly, an insulin-Fc fusion protein constructed from the insulin polypeptide of SEQ ID NO: 9, which retains asparagine at the C-terminal of the A chain, demonstrates fabrication capability in mammalian cells with an acceptable homodimer titer (i.e., a homodimer titer greater than 50 mg / L), acceptable in vivo bioactivity (i.e., NAOC greater than 150 %FBGL days kg / mg in the target animal), and sustained levels of bioactivity after multiple doses (i.e., NAOCR values ​​after the third injection in the target animal above 0.5). The results indicate that... Petition 870260049851, dated 05 / 25 / 2026, page 56 / 693 47 / 180 Contrary to expectations based on previous teachings, there is no risk of enzymatic cleavage or deactivation of insulin-Fc fusion proteins containing the asparagine-glycine linkage between the insulin polypeptide and the peptide linker, at least for insulin-Fc fusion proteins comprising the Fc fragment sequences disclosed herein. Fragment Fc

[0111] The terms Fc fragment, Fc region, Fc domain, or Fc polypeptide are used herein to define a C-terminal region of an immunoglobulin heavy chain. The Fc fragment, region, domain, or polypeptide may be a native sequence Fc region or a variant / mutant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, they generally comprise part or all of the heavy chain hinge region, the CH2 region of the heavy chain, and the CH3 region of the heavy chain. The hinge region of a dog or cat Fc fragment comprises amino acid sequences that connect the CH1 domain of the heavy chain to the CH2 region of the heavy chain and contains one or more cysteines that form one or more inter-heavy chain disulfide bridges to form a homodimer of an Fc fusion protein from two identical but separate monomers of the Fc fusion protein.The hinge region may comprise all or part of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence.

[0112] An Fc receptor (FcR) refers to a receptor that binds to an Fc fragment or Fc region of an antibody. In embodiments, FcR is a native canine or feline FcR sequence. In embodiments, FcR is one that binds to an Fc fragment or Fc region of an IgG antibody (a gamma receptor) and includes, without Petition 870260049851, dated 05 / 25 / 2026, p. 57 / 693 48 / 180 limitation, Fc(gamma) receptor I, Fc(gamma) receptor IIa, Fc(gamma) receptor IIb, and Fc(gamma) receptor III subclasses, including allelic variants and alternative splicing forms of these receptors. FcR also includes the 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 is also responsible for the prolonged in vivo elimination half-life of antibodies and Fc fusion proteins in vivo. In embodiments, human-derived FcRs are used in vitro (e.g., in an assay) to measure the binding of insulin-Fc fusion proteins comprising canine- or feline-derived Fc fragments, in order to assess their FcR binding properties.Those skilled in the art will understand that mammalian FcRs of one species (e.g., FcRs of human origin) are sometimes capable of in vitro binding to Fc fragments of a second species (e.g., FcRs of canine or feline origin). In embodiments, canine-derived FcRs are used in vitro (e.g., in an assay) to measure the binding of insulin-Fc fusion proteins comprising canine or feline-derived Fc fragments, in order to assess their FcR binding properties. Those skilled in the art will understand that mammalian FcRs of one species (e.g., FcRs of canine origin) are capable of in vitro binding to insulin-Fc fusion proteins comprising Fc fragments of the same species (e.g., of canine origin) and sometimes also to insulin-Fc fusion proteins comprising Fc fragments originating from another mammalian species (e.g., of feline origin).

[0113] In embodiment examples, the Fc fragment comprises the Fc region (e.g., hinge region, CH2 domain, and CH3 domain) of a mammalian IgG, for example, an Fc fragment of canine IgGA (SEQ ID NO: 15), an Fc fragment of canine IgGB (SEQ ID NO: Petition 870260049851, dated 05 / 25 / 2026, page 58 / 693 49 / 180 16), an Fc fragment of canine IgGC (SEQ ID NO: 17), or an Fc fragment of canine IgGD (SEQ ID NO: 18) or a fragment of feline IgG1a (SEQ ID NO: 19), an Fc fragment of feline IgG1b (SEQ ID NO: 20), or an Fc fragment of feline IgG2 (SEQ ID NO: 21). In embodiments, the C-terminal lysine that is frequently found in native canine or feline IgG isotype Fc amino acid sequence fragments (i.e., the lysine that represents the last amino acid of the Fc fragment sequence) is omitted to avoid the accidental production of unwanted amino acid sequence variants during manufacturing (e.g., Fc fragments containing the C-terminal lysine become mixed with Fc fragments in which the C-terminal lysine is omitted, which can occur during the production of the desired protein within cells (Dick, LW, (2008) Biotechnol Bioeng. August 15; 100 (6) pp. 1132-43).Therefore, in the realization examples, the sequences of the canine and feline Fc fragment without a C-terminal lysine are: RCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGRED PEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTG KEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTV SITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYS KLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG (SEQ ID NO: 15) DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPE TCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKL SVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 16) CNNCPPCCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENP EVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGK QFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTL Petition 870260049851, dated 05 / 25 / 2026, page 59 / 693 50 / 180 TCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSK LSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG (SEQ ID NO: 17) CISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEV QISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEF KCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLT CLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLS VDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG (SEQ ID NO: 18) DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSD VQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEF KCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVT CLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSV DRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 19) DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSD VQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEF KCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVT CLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSV DRSRWQRGNTYTCSVSHEALHS HHTQKSLTQSPG (SEQ ID NO: 20) GEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDS NVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKE FKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSV TCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLS VDRSHWQRGNTYTCSVSHEAL HSHHTQKSLTQSPG (SEQ ID NO: 21)

[0114] Replacing human Fc with canine IgGa is preferable to minimize any unwanted immunogenicity in dogs due to the absence of the Fc(gamma) effector function of the IgGa isotype in dogs (very similar to the human IgG2 isotype in humans). However, in an example embodiment containing the insulin polypeptide of SEQ ID NO: Petition 870260049851, dated 05 / 25 / 2026, page 60 / 693 51 / 180 and the peptide linker of SEQ ID NO: 12, it was unexpectedly found that the insulin-Fc fusion protein comprising the canine IgGa fragment (SEQ ID NO: 15) was highly aggregated with low titers of the desired homodimer (i.e., homodimer titers less than 50 mg / L). Furthermore, the compound was not bioactive in dogs (i.e., the NAOC value was less than 150 %FBGL days kg / mg), presumably due to its high level of aggregation (i.e., low % homodimer). Despite the mutation of the insulin polypeptide of SEQ ID NO: 5, the canine IgGA Fc fragment (SEQ ID NO: 15) and / or the linker, there was no example of a realization based on the canine IgGA Fc fragment with a sufficiently low degree of aggregation and a sufficiently high titer of the desired homodimer.On the other hand, replacing the canine IgGA Fc fragment (SEQ ID NO: 15) with the canine IgGB Fc fragment (SEQ ID NO: 16) yielded a compound with significantly less aggregate and a comparatively high titer of the desired homodimer. Furthermore, the compound containing the insulin polypeptide from SEQ ID NO: 5 and the canine IgGB Fc fragment (SEQ ID NO: 16) was bioactive in dogs, exhibiting glucose-lowering bioactivity over several days (i.e., the NAOC value was greater than 150%FBGL days kg / mg).

[0115] The preference for the canine IgGB Fc fragment over the canine IgGA Fc fragment was confirmed in embodiments containing the insulin polypeptide of SEQ ID NO: 8 and the peptide linker of SEQ ID NO: 14, both of which differ considerably from the insulin polypeptide of SEQ ID NO: 5 and the peptide linker of SEQ ID NO: 12. Insulin-Fc fusion proteins containing the insulin polypeptide of SEQ ID NO: 8 and the peptide linker of SEQ ID NO: 14 were synthesized using canine IgGA (SEQ ID NO: 15), canine IgGB (SEQ ID NO: 16), canine IgGC (SEQ ID NO: 17), or canine IgGD (SEQ ID NO: 18) Fc fragments. Using the method of Petition 870260049851, dated 05 / 25 / 2026, page 61 / 693 52 / 180 conventional purification, only the compounds comprising canine IgGA and canine IgGB showed any appreciable protein yield. However, as before, the canine IgGA version of the compound was highly aggregated with low levels of bioactivity, while the canine IgGB version of the compound exhibited a low degree of aggregation (i.e., high % homodimer), a high titer of the desired homodimer (i.e., a homodimer titer greater than 50 mg / L), and appreciable levels of long-lasting glucose-lowering bioactivity in dogs (i.e., the NAOC value was greater than 150 %FBGL days kg / mg). Using an alternative purification method, the canine IgGC version of the compound was recovered with low degrees of aggregation but was minimally bioactive in dogs (i.e., the NAOC value was less than 150 %FBGL days kg / mg), presumably due to its low affinity for the FcRn receptor.Therefore, with respect to a specific product for dogs, canine IgGB (SEQ ID NO: 16) is the preferred Fc fragment for all insulin-Fc fusion proteins used in dogs, regardless of the choice of insulin polypeptide.

[0116] Replacing the human Fc fragment with that of feline IgG2 is preferable to minimize any undesirable immunogenicity in cats due to the absence of the Fc(gamma) effector function of the IgG2 isotype in cats (very similar to the human IgG2 isotype in humans). Unlike the case with dogs, in embodiments containing the insulin polypeptide of SEQ ID NO: 4, the insulin-Fc fusion protein comprising a feline IgG2 fragment (SEQ ID NO: 21) and a feline IgG1b fragment (SEQ ID NO: 20) showed similarly high yield with low degrees of aggregation (i.e., homodimer titers greater than 50 mg / L) and appreciable affinity for the insulin receptor (i.e., insulin receptor IC50 values ​​less than 5000 nM). However, unexpectedly, when the insulin polypeptide was changed to SEQ ID NO: 7, the insulin-Fc fusion protein Petition 870260049851, dated 05 / 25 / 2026, page 62 / 693 The 53 / 180 fragment comprising the feline IgG2 fragment (SEQ ID NO: 21) was not bioactive in cats (i.e., NAOC was less than 150 %FBGL days kg / mg), while the insulin-Fc fusion protein comprising the feline IgG1b fragment (SEQ ID NO: 20) exhibited a low degree of aggregation (i.e., high % homodimer), a high titer of the desired homodimer (i.e., a homodimer titer greater than 50 mg / L), and appreciable levels of long-term glucose-lowering bioactivity in cats (i.e., the NAOC value was greater than 150 %FBGL days kg / mg). Therefore, with respect to a cat-specific product, the feline IgG1b fragment (SEQ ID NO: 20) is the preferred Fc fragment when the insulin polypeptide sequence comprises SEQ ID NO: 7.

[0117] Considering that canine IgGB and feline IgG1b isotypes interact with their respective species-specific Fc(gamma) receptors with higher affinities than their canine IgGA and feline IgG2 isotype counterparts, this may or may not pose a risk of undesirable immunogenicity after repeated injections. One method to reduce the Fc(gamma) interaction involves deglycosylation or prevention of glycosylation of the Fc fragment during synthesis in the host cell. Each IgG fragment contains a conserved (N)-glycosylation site of asparagine in the CH2 domain of each heavy chain of the Fc region. Here, the annotation used to refer to the conserved N-glycosylation site is cNg. One way to remove the bound glycans from a synthesized insulin-Fc fusion protein is by mutating the cNg site to completely prevent glycan binding during production in the host cell. Here, the annotation used to describe a cNg mutation is cNg-(substituted amino acid).For example, if asparagine at the cNg site is mutated to serine, this mutation is denoted as cNg-S. Petition 870260049851, dated 05 / 25 / 2026, p. 63 / 693 54 / 180

[0118] The absolute position of the cNg site at the N-terminal end of the B chain of the insulin-Fc fusion protein varies depending on the length of the insulin polypeptide, the length of the ligand, and any amino acids omitted in the Fc fragment before the cNg site. Here, the notation used to refer to the absolute position of the cNg site in a given insulin-Fc fusion protein sequence (measured counting from the N-terminal end 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 counted from the N-terminal end of the B chain, the absolute position of this site is referred to as GNC-NB151. As another example, if the cNg site is found at the 151st amino acid position counted from the N-terminal end of the B chain, and the asparagine at this site is mutated to serine, this mutation is known as cNgNB151-S.

[0119] In embodiments containing the insulin polypeptide from SEQ ID NO: 5 and the canine IgGB Fc fragment with the cNg-Q, cNg-S, cNg-D, and cNg-K mutations, it was unexpectedly found that only the compound containing the CNG-K and CNG-S mutations exhibited the higher homodimer titer requirement of 50 mg / L and lower Fc(gamma)RI binding affinities. On the other hand, in an embodiment containing the insulin polypeptide from SEQ ID NO: 8 and the canine IgGB Fc fragment with the cNg-S mutation, it was unexpectedly found that the resulting compound was significantly less bioactive in dogs compared to the native counterpart containing canine IgGB Fc (i.e., the NAOC value was significantly lower for the counterpart containing the native glycosylation site amino acid, e.g., cNg-N).Bioactivity was unexpectedly restored in the cNg-S mutant form (i.e., the NAOC value increased significantly) when amino acid B16 was mutated to alanine, as described above for the insulin polypeptide SEQ ID NO: 11. Taken. Petition 870260049851, dated 05 / 25 / 2026, pp. 64 / 693 55 / 180 taken together, there is an unexpected and significant interaction between the choice of gNc mutation and the insulin polypeptide composition, so experimentation is needed to identify preferred embodiments. In specific embodiments, the Fc of canine IgGB mutant containing the cNg-S mutation is preferred and the sequence with cNg-S underlined is shown as: DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPE VQISWFVDGKQMQTAKTQPREEQFSGTYRWSVLPIGHQDWLKGKQ FTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLT CLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLS VDKSRWQRGDTFICAVMHEALH NHYTQESLSHSPG (SEQ ID NO: 22) In specific embodiments, the Fc of feline IgGlb mutant containing the cNg-S mutation is preferred: DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSD VQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEF KCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVT CLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSV DRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 23) Insulin-Fc Fusion Proteins

[0120] The present invention provides insulin-Fc fusion proteins comprising an insulin polypeptide, an Fc fragment, and a linking agent between the insulin polypeptide and the Fc fragment. In embodiments, the insulin polypeptide comprises domains in the following N-terminal to C-terminal orientation: (N-terminal) - B chain - C chain - A chain - (C-terminal). In embodiments, the insulin polypeptide is located on the N-terminal side of the Fc fragment. In embodiments, the fusion protein comprises domains in the following N- to C-terminal orientation: (N-terminal) - insulin polypeptide - linker - Fc fragment - (C-terminal) (e.g., Petition 870260049851, dated 05 / 25 / 2026, page 65 / 693 56 / 180 (N-terminal)) - chain B - chain C - chain A - ligand - Fc fragment (C-terminal)) as illustrated in FIG. 1

[0121] In preferred embodiments, the preferably non-immunogenic insulin-active polypeptide of SEQ ID NO: 6 is combined with the canine IgGB Fc fragment of SEQ ID NO: 16, using the preferred ligand of SEQ ID NO: 14 to produce a family of non-aggregated, bioactive, non-immunogenic, high-yielding insulin-Fc fusion proteins of SEQ ID NO: 24 that exhibit homodimer titers greater than 50 mg / L, NAOC values ​​greater than 150 %FBGL days kg / mg in dogs, and NAOCR values ​​greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ ID NO: 24 with non-native amino acids underlined: FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCC (SEQ ID NO: 24) where X1 is not D, X2 is not H, and X3 is either absent or N.

[0122] In preferred embodiments comprising SEQ ID NO: 24, X1 is H, X2 is T, and X3 is absent or is N. The selections yield non-aggregated, bioactive, non-immunogenic, high-yielding Fc-insulin fusion proteins of SEQ ID NO: 25 exhibiting homodimer titers greater than 50 mg / L, NAOC values ​​greater than 150 %FBGL days kg / mg in dogs, and NAOCR values ​​greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ ID NO: 25 with non-native amino acids underlined: Petition 870260049851, dated 05 / 25 / 2026, pp. 66 / 693 57 / 180 FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCX3GGGGGQGGGGQGGGGQGGGGGDCPKCPAP EMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVD GKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN KALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFP PDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 25) where X3 is absent or is N.

[0123] In preferred embodiments, X3 is absent in SEQ ID NO: 25 to produce non-aggregated, bioactive, non-immunogenic insulin-Fc fusion proteins with a high homodimer yield of SEQ ID NO: 32 that exhibits a homodimer titer greater than 50 mg / L, NAOC value greater than 150 %FBGL days kg / mg in dogs, and NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ ID NO: 32 with non-native amino acids underlined: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEVQISWFVDGK QMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKAL PSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDI DVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRG DTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 32)

[0124] In preferred embodiments, X3 is N in SEQ ID NO: 25 to produce non-aggregated, bioactive, non-immunogenic insulin-Fc fusion proteins with a high homodimer yield of SEQ ID NO: 34 that exhibits a homodimer titer greater than 50 mg / L, a NAOC value greater than 150 %FBGL days kg / mg in dogs, and a NAOCR value greater than 0.5 after the third injection in a series of injections. Petition 870260049851, dated 05 / 25 / 2026, page 67 / 693 58 / 180 repeated in dogs. The following shows the SEQ ID NO: 34 with non-native amino acids underlined: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPE MLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDG KQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNK ALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPP DIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 34)

[0125] In preferred embodiments, the preferred non-glycosylated canine IgGB Fc fragment with the cNg-S mutation from SEQ ID NO: 22 is combined with the preferred B16A mutated insulin polypeptide sequence from SEQ ID NO: 10, using the preferred ligand from SEQ ID NO: 14 to produce a family of non-aggregated, bioactive, non-immunogenic, high-yielding insulin-Fc fusion proteins from SEQ ID NO: 26 that exhibits homodimer titers greater than 50 mg / L, NAOC values ​​greater than 150 %FBGL days kg / mg in dogs, and NAOCR values ​​greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ ID NO: 26 with non-native amino acids underlined: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCC KALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFP PDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 26) where X1 is not D and X2 is not H. Petition 870260049851, dated 05 / 25 / 2026, pp. 68 / 693 59 / 180

[0126] In a preferred embodiment, X1 is H and X2 is T in SEQ ID NO: 26 to produce non-aggregated, bioactive, non-immunogenic insulin-Fc fusion proteins with a high homodimer yield of SEQ ID NO: 36 exhibiting a homodimer titer greater than 50 mg / L, NAOC value greater than 150 %FBGL days kg / mg in dogs, and NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ ID NO: 36 with non-native amino acids underlined: FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGK QMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKAL PSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDI DVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRG DTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 36)

[0127] In preferred embodiments, the bioactive insulin polypeptide, preferably non-immunogenic, of SEQ ID NO: 6, in which X3 is absent, is combined with the feline IgG1b Fc fragment of SEQ ID NO: 20, using the preferred ligand of SEQ ID NO: 14 to produce a family of non-aggregated, bioactive, non-immunogenic, high-yielding insulin-Fc fusion proteins of SEQ ID NO: 27 that exhibit homodimer titers greater than 50 mg / L, NAOC values ​​greater than 150 %FBGL days kg / mg in felines, and NAOCR values ​​greater than 0.5 after the third injection in a series of repeated injections in felines. The following shows SEQ ID NO: 27 with non-native amino acids underlined: FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCC Petition 870260049851, dated 05 / 25 / 2026, pp. 69 / 693 60 / 180 LPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPS DIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQR GNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 27) where X1 is not D and X2 is not H.

[0128] In a preferred embodiment, X1 is H and X2 is T in SEQ ID NO: 27 to produce non-aggregated, bioactive, non-immunogenic insulin-Fc fusion proteins with a high homodimer yield of SEQ ID NO: 38 exhibiting a homodimer titer greater than 50 mg / L, NAOC value greater than 150 %FBGL days kg / mg in felines, and NAOCR value greater than 0.5 after the third injection in a series of repeated injections in felines. The following shows SEQ ID NO: 38 with non-native amino acids underlined: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPPPEM LGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQ VYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPS PIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAV EWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTY TCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 38)

[0129] In preferred embodiments, the preferred non-glycosylated, non-aggregated feline IgG1b Fc fragment with the cNg-S mutation, from SEQ ID NO: 23, is combined with the preferred B16A mutated insulin polypeptide sequence from SEQ ID NO: 10, using the preferred ligand from SEQ ID NO: 14, to produce a family of non-aggregated, bioactive, non-immunogenic, high-yielding Fc-insulin fusion proteins from SEQ ID NO: 28 that exhibit homodimer titers greater than 50 mg / L, NAOC values ​​greater than 150 %FBGL days kg / mg in cats, and NAOCR values ​​greater than 0.5 after the third injection in a series of repeated injections in cats. Petition 870260049851, dated 05 / 25 / 2026, page 70 / 693 61 / 180 The following shows SEQ ID NO: 28 with non-native amino acids underlined: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCC PSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIA VEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTY TCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 28) where X1 is not D and X2 is not H.

[0130] In a preferred embodiment, X1 is H and X2 is T in SEQ ID NO: 28 to produce non-aggregated, bioactive, non-immunogenic insulin-Fc fusion proteins with a high homodimer yield of SEQ ID NO: 40 exhibiting a homodimer titer greater than 50 mg / L, NAOC value greater than 150 %FBGL days kg / mg in felines, and NAOCR value greater than 0.5 after the third injection in a series of repeated injections in dogs. The following shows SEQ ID NO: 40 with non-native amino acids underlined: FVNQHLCGSH LVEALALVCG AND RG FHYGGGGGSGGGG GIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEM LGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQ (SEQ ID NO: 40)

[0131] In some embodiments, an insulin-Fc fusion protein described in the present invention does not include a leader amino acid sequence in the N-terminal portion. In other embodiments, an insulin-Fc fusion protein described in the present invention includes a leader sequence, for example, in the N-terminal portion. Petition 870260049851, dated 05 / 25 / 2026, p. 71 / 693 The exemplary 62 / 180 leader sequence includes the amino acid sequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 30). In some embodiments, an insulin-Fc fusion protein described in the present invention is encoded by a nucleic acid molecule comprising a leader sequence, for example, for expression (e.g., recombinant expression) in cells (e.g., eukaryotic cells, e.g., mammalian cells). In certain embodiments, the leader sequence is cleaved, for example, in cell culture, during expression. An exemplary nucleic acid sequence encoding a leader sequence includes the nucleic acid sequence: atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcc (SEQ ID NO: 29).

[0132] Nucleic acid sequences (e.g., cDNA) encoding insulin-Fc fusion proteins are also disclosed with SEQ ID numbers: 032, 034, 036, 038, and 040.

[0133] In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO: 32, the nucleic acid sequence (underlined leader sequence) is: atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccag cacctgtg cggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggt ggcggag gaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggacca gctggaaa actactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggagg aggcggg ggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcc caagccc aaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatccc gaagacc Petition 870260049851, dated 05 / 25 / 2026, p. 72 / 693 63 / 180 ccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccc cgggaag agcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaa ggggaag caattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaag gccaggg gccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaaca cagtcagc cttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggcca gcaggag cccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctac agcaaatt gagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggc tttgcataa ccactacacccaggagagcctgtcccacagccccggatag (SEQ ID NO: 31)

[0134] In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO: 34, the nucleic acid sequence (underlined leader sequence) is: atggaatggagctgggtctctctcttctctgtcgtaacgactggtgtccactccttcgtcgtcg cacctgtg cggctcccacctggtggaagctctggaactcgtgcggcgagcggggcttccactacgggggt ggcggag gaggttctggtggcggccggcggcgcccctcctcccgcgcg gctggaaa actactgcaacggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcaggg aggaggc ggggagactgccccaagtgcccgctccgagatgctgggcggacccagcgtgttcatcttc ctcccaag Petition 870260049851, of 25 / 05 / 2026, p. 73 / 693 64 / 180 cccaaggacacactgctgatcgccaggaccccggaggtgcgtggtggtggacctggat cccgaag acccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaa cccggg aagagcagttcaacggcac gaagggg aagcaattcacatgcaaggttaataaaggccctgcccagccccatcgagaggaccatcagc aaggcca ggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaactgagcaaga acacagtc agccttacttgcctgatcaaggacttctc gagcccgagagcaagtataggaccacaccgcccaactggacgaggacggaagcttacttcct ctacagca aattgagcgttgacaaaagcaggtggcgcgaggcgacaccttcatctgcgccgtgatgcacg aggctttgc ataaccactacacccaggagagcctgtcccagcagc (SEQ13:353 NO) In the exemplary embodiment comprising the insulin-Fc fusion protein of SEQ ID NO: 36,the nucleic acid sequence (underlined leader sequence) is: gaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggacca gctggaaa actactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggagg aggcggg ggagactgccccaagtgccccgcccc paperccc, Petition 870260049851, of 25 / 05 / 2026, p. 74 / 693 65 / 180 aaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatccc gaagacc ccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccc cgggaag agcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaag gggaagc aattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaagg ccagggg ccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacac agtcagcc ttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccag caggagc ccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctac agcaaatt gagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggc tttgcataa ccactacacccaggagagcctgtcccacagccccggatag (SEQ ID NO: 35)

[0136] In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO: 38, the nucleic acid sequence (underlined leader sequence) is: atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccag cacctgtg cggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggt ggcggag gaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggacca gctggaaa actactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggagg aggcggg ggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgccc aagcccaa Petição 870260049851, de 25 / 05 / 2026, pág. 75 / 693 66 / 180 ggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagac gactctga cgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggag gagcagtt caacagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaa agagttca agtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaa gggccagc cgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggtt agcgtga catgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacc cgagccc gagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagc aggctgag cgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggcct tgcacag ccaccacactcagaagagtctgacccagagcccgggatag (SEQ ID NO: 37)

[0137] In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO: 40, the nucleic acid sequence (underlined leader sequence) is: atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccag cacctgtg cggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggt ggcggag gaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggacca gctggaaa actactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggagg aggcggg ggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgccc aagcccaa Petição 870260049851, de 25 / 05 / 2026, pág. 76 / 693 67 / 180 ggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagac gactctga cgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggag gagcagtt cagcagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaa agagttca agtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaa gggccagc cgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggtt agcgtga catgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacc cgagccc gagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagc aggctgag cgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggcct tgcacag ccaccacactcagaagagtctgacccagagcccgggatag (SEQ ID NO: 39). Production of Insulin-Fc Fusion Protein

[0138] In embodiment examples, a fusion protein can be expressed by a cell, as described in more detail in the Examples section. Expression and purification

[0139] In some embodiments, an insulin-Fc fusion protein can be recombinantly expressed, for example, in a eukaryotic cell, e.g., mammalian cell or non-mammalian cell. Exemplary mammalian cells used for expression include HEK cells (e.g., HEK293 cells) or CHO cells. CHO cells can be subdivided into several strains or subclasses (e.g., CHO DG44, CHO-H, and CHO-K1 cells), and some of these strains can be genetically engineered for use. Petition 870260049851, dated 05 / 25 / 2026, page 77 / 693 68 / 180 optimum with a particular type of nucleic acid molecule (e.g., a vector comprising DNA) or a particular cell growth medium composition, as described in the Examples section. In embodiments, cells are transfected with a nucleic acid molecule, e.g., vector, encoding the insulin-Fc fusion protein (e.g., where the entire insulin-Fc fusion protein is encoded by a single nucleic acid molecule). In embodiments, HEK293 cells are transfected with a vector encoding the insulin-Fc fusion protein, but only results in temporary expression of the insulin-Fc fusion protein for a period of time (e.g., 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days, or more) before the host cell stops expressing appreciable levels of the insulin-Fc fusion protein (i.e., transient transfection).HEK293 cells that are transiently transfected with nucleic acid sequences encoding insulin-Fc fusion proteins often allow for faster production of recombinant proteins, facilitating the production and screening of various insulin-Fc fusion protein candidates. In embodiments, CHO cells are transfected with a vector that is permanently incorporated into the host cell's DNA and leads to consistent and permanent expression (i.e., stable transfection) of the insulin-Fc fusion protein, provided the cells are cultured appropriately.CHO cells and CHO cell lines that are stably transfected with nucleic acids encoding insulin-Fc fusion proteins often take longer to grow, but frequently produce higher protein yields and are more suitable for manufacturing low-cost products (e.g., products for use in the veterinary pharmaceutical market). The cells and cell lines can be cultured using standard state-of-the-art methods. Examples of... Petition 870260049851, dated 05 / 25 / 2026, page 78 / 693 In preferred embodiments 69 / 180, HEK cells comprising any of the cDNA sequences of SEQ ID NOs: 31, 33, 35, 37, and 39 are used to express insulin-Fc fusion proteins. In preferred embodiments, CHO cells comprising any of the cDNA sequences of SEQ ID NOs: 31, 33, 35, 37, and 39 are used to express insulin-Fc fusion proteins.

[0140] In some embodiments, the insulin-Fc fusion protein is purified or isolated from cells (e.g., by cell lysis). In other embodiments, the insulin-Fc fusion protein is secreted by cells and purified or isolated from the cell culture medium in which the cells were grown. Purification of the insulin-Fc fusion protein may involve the use of column chromatography (e.g., affinity chromatography) or the use of other separation methods based on differences in size, charge, and / or affinity for certain molecules.In exemplary embodiments, the purification of insulin-Fc fusion protein involves the selection or enrichment of proteins containing an Fc fragment, for example, using Protein A beads or a Protein A column that causes proteins containing an Fc fragment to become highly affine bound in neutral pH solution to Protein A covalently conjugated to the Protein A beads. The bound insulin-Fc fusion protein can then be eluted from the Protein A beads by a change in a solution variable (e.g., a decrease in the pH of the solution). Other separation methods, such as ion-exchange chromatography and / or gel filtration chromatography, may also be employed alternatively or additionally. In exemplary embodiments, the purification of insulin-Fc fusion protein further comprises filtration or centrifugation of the protein preparation. In exemplary embodiments, further purification of insulin-Fc fusion protein... Petition 870260049851, dated 05 / 25 / 2026, page 79 / 693 70 / 180 comprises diafiltration, ultrafiltration and filtration through porous membranes of various sizes, as well as final formulation with excipients.

[0141] Purified insulin-Fc fusion protein can be characterized, for example, as to purity, protein yield, structure and / or activity, using a variety of methods, for example, absorbance at 280 nm (e.g., to determine protein yield), capillary or size exclusion electrophoresis (e.g., to determine molecular weight, percentage of aggregation and / or purity), mass spectrometry (MS) and / or liquid chromatography (LCMS) (e.g., to determine purity and / or glycosylation), and / or ELISA (e.g., to determine the extent of binding, e.g., affinity, to an anti-insulin antibody). Exemplary characterization methods are also described in the Examples section.

[0142] In embodiments, the protein yield of an insulin-Fc fusion protein after production in transiently transfected HEK cells and purification to protein A is greater than 5 mg / L, 10 mg / L, or 20 mg / L. In preferred embodiments, the protein yield of an insulin-Fc fusion protein after production in transiently transfected HEK cells and purification to protein A is greater than 50 mg / L (e.g., greater than 60 mg / L, greater than 70 mg / L, greater than 80 mg / L, greater than 90 mg / L, greater than 100 mg / L). In embodiment examples, the % homodimer of an insulin-Fc fusion protein after production in transiently transfected HEK cells and purification into protein A is greater than 70% (e.g., greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%).In embodiment examples, the homodimer titer of an insulin-Fc fusion protein after production in transiently transfected HEK cells and purified into protein A, calculated as the... Petition 870260049851, dated 05 / 25 / 2026, page 80 / 693 71 / 180 The product between the insulin-Fc fusion protein yield and the % homodimer is greater than 50 mg / L (e.g., greater than 60 mg / L, greater than 70 mg / L, greater than 80 mg / L, greater than 90 mg / L, greater than 100 mg / L). Only candidates with a homodimer titer greater than 50 mg / L were considered useful in the present invention, as experience has shown that homodimer titers lower than this level are unlikely to result in commercially productive titers in CHO cells that meet the low-cost manufacturing requirements for veterinary products.

[0143] In embodiments, the protein yield of an insulin-Fc fusion protein after production in stably transfected CHO cells (e.g., CHO cell lines or CHO cell clones) and purification of protein A is greater than 100 mg of insulin-Fc fusion protein per L (e.g., mg / L of culture medium). In preferred embodiments, the protein yield of an insulin-Fc fusion protein after production in stably transfected CHO cells (e.g., CHO cell lines or CHO cell clones) and purification of protein A is greater than 150 mg of insulin-Fc fusion protein / L of culture medium (e.g., greater than 200 mg / L, greater than 300 mg / L, greater than 400 mg / L, greater than 500 mg / L, greater than 600 mg / L or more).In the embodiments, the % homodimer of an insulin-Fc fusion protein after production in stably transfected CHO cells (e.g., CHO cell lines or CHO cell clones) and purification of protein A is greater than 70% (e.g., greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%). In embodiments, the homodimer titer of an insulin-Fc fusion protein after production in stably transfected CHO cells (e.g., CHO cell lines or CHO cell clones) and... Petition 870260049851, dated 05 / 25 / 2026, page 81 / 693 72 / 180 purification in protein A, calculated as the product between the insulin-Fc fusion protein yield and the % homodimer is greater than 150 mg / L (e.g., greater than 200 mg / L, greater than 300 mg / L, greater than 400 mg / L, greater than 500 mg / L, greater than 600 mg / L or more). Functional Characteristics of Insulin-Fc Fusion Proteins

[0144] Methods are described for interacting with insulin receptors to lower blood glucose in companion animals (e.g., dogs or cats), wherein the method comprises administering to a subject an insulin-Fc fusion protein, for example, a fusion protein described in the present invention. In some embodiments, the subject has been diagnosed with diabetes (e.g., canine diabetes or feline diabetes).

[0145] In some embodiments, an insulin-Fc fusion protein described in the present invention binds to the insulin receptor with appreciable affinity, as measured by the IC50 in the IM-9 4°C insulin receptor binding assay described in Example 7 (e.g., IC50 less than 5000 nM, IC50 less than 4000 nM, IC50 less than 3000 nM, IC50 less than 2500 nM). Based on the experiment, only compounds exhibiting insulin receptor activity with IC50 values ​​less than 5000 nM are considered likely to exhibit bioactivity in the species in question. In general, higher affinity insulin receptor binding (i.e., with lower IC50 values) is preferred.However, it is well known that the clearance of insulin and its analogues (e.g., insulin polypeptides described in the present invention) is governed primarily by binding to the insulin receptor followed by internalization and degradation of the insulin receptor in the cell. Therefore, insulin-Fc fusion proteins with very high insulin receptor binding affinity (i.e., very low IC50) can be cleared very rapidly from the body. Petition 870260049851, dated 05 / 25 / 2026, page 82 / 693 73 / 180 circulation, resulting in a shorter than desired duration of glucose-lowering bioactivity in the target animal.

[0146] In embodiments, an insulin-Fc fusion protein described in the present invention is capable of lowering glucose levels (e.g., blood glucose levels) after administration to a subject. In embodiments, the glucose-lowering activity of the insulin-Fc fusion protein is greater than that of an insulin reference standard. In some embodiments, the duration of activity of the insulin-Fc fusion protein can be measured by a decrease, for example, a statistically significant decrease, in fasting blood glucose relative to a pre-dose fasting blood glucose level. In embodiments, the duration of activity of the insulin-Fc fusion protein (e.g., the time during which there is a statistically significant decrease in the fasting blood glucose level in a subject relative to a pre-dose level) is greater than about 2 hours.In embodiments, the duration of activity of the insulin-Fc fusion protein (e.g., the time during which there is a statistically significant decrease in blood glucose level in a subject relative to a pre-dose level) is greater than approximately 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. In embodiments, the insulin-Fc fusion protein is long-acting (e.g., has a long half-life, e.g., in serum).

[0147] In embodiments, the serum half-life of insulin-Fc fusion protein in the target animal (e.g., dog or cat) is greater than that of a reference insulin standard or control formulation. In embodiments, the serum half-life of insulin-Fc fusion protein (e.g., in a subject's blood after administration) in the target animal (e.g., dog or cat) is greater than approximately Petition 870260049851, dated 05 / 25 / 2026, p. 83 / 693 74 / 180 of 2 hours. In embodiments, the serum half-life of insulin-Fc fusion protein in the target animal (e.g., dog or cat) is approximately 0.5 days, 1 day, 2 days, or 2.5 days. In preferred embodiments, the serum half-life of insulin-Fc fusion protein in the target animal (e.g., dog or cat) is approximately 3 days or more.

[0148] In embodiments, the combination of potency and duration of bioactivity can be quantified by calculating the area under the curve of fasting blood glucose percentage (%FBGL) normalized to a given dose in mg / kg (NAOC) with units of %FBGL days kg / mg. In embodiments, the NAOC of insulin-Fc fusion protein is greater than 150 %FBGL days kg / mg (e.g., greater than 200 %FBGL days kg / mg, greater than 250 %FBGL days kg / mg or more). Again, based on experience, at NAOC values ​​greater than 150 %FBGL days kg / mg, the dose requirements in the target species will be sufficiently low to achieve an acceptable treatment cost. In embodiments, the NAOC of insulin-Fc fusion protein should be maintained after repeated dosing in the target species (i.e., the ratio of NAOC after the third dose to NAOC after the first dose of insulin-Fc fusion protein is greater than 0.50, for example, greater than 0.60, greater than 0.70).greater than 0.80, greater than 0.90 or more).

[0149] In some embodiments, the insulin-Fc fusion protein described in the present invention binds to the Fc(gamma) receptor with an affinity that is less than that of a reference standard insulin-Fc fusion protein as measured according to Example 8. In some embodiments, the ratio of the Fc(gamma) receptor affinity of the insulin-Fc fusion protein to that of a reference standard insulin-Fc fusion protein is less than 0.50 (e.g., less than 0.40, less than 0.30, less than 0.20). Treatment Methods and Subject Selection Characteristics Petition 870260049851, dated 05 / 25 / 2026, p. 84 / 693 75 / 180

[0150] The present invention describes methods for treating diabetes (for example, canine diabetes or feline diabetes), which methods comprise administering an insulin-Fc fusion protein (for example, an insulin-Fc fusion protein described in the present invention) to a subject.

[0151] In embodiments, a reference standard used in any method described herein comprises a reference treatment or reference therapy. In some embodiments, the reference comprises a standard care agent for the treatment of diabetes (e.g., a standard care agent for canine diabetes or a standard care agent for feline diabetes). In some embodiments, the reference standard is a commercially available insulin or insulin analogue. In some embodiments, the reference standard comprises a long-acting insulin, intermediate-acting insulin, short-acting insulin, rapid-acting insulin, short-acting insulin, intermediate-acting insulin, and long-acting insulin. In some embodiments, the reference standard comprises Vetsulin®, Prozinc®, NPH insulin, insulin glargine (Lantus®), or recombinant human insulin.

[0152] In embodiments, a reference standard used in any described method includes an outcome, for example, the outcome described herein, of a diabetes therapy (for example, a canine diabetes therapy or a feline diabetes therapy).

[0153] In embodiments, a reference standard is a level of a marker (e.g., blood glucose or fructosamine) in the subject before the start of a therapy, for example, an insulin-Fc fusion protein therapy described in the present invention; where the subject has diabetes. In embodiments, the blood glucose level in a companion animal (e.g., dog or cat) is greater than 200 mg / dL (e.g., greater than 250 mg / dL, 300 mg / dL, 350 mg / dL). Petition 870260049851, dated 05 / 25 / 2026, p. 85 / 693 76 / 180 mg / dL, 400 mg / dL or more) before the start of therapy. In embodiments, the fructosamine level in a companion animal (e.g., dog or cat) is greater than 250 micromoles / L, 350 micromoles / L (e.g., greater than 400 micromoles / L, 450 micromoles / L, 500 micromoles / L, 550 micromoles / L, 600 micromoles / L, 650 micromoles / L, 700 micromoles / L, 750 micromoles / L or more) before the start of therapy. In embodiments, a reference standard is a measure of the presence, progression, or severity of the disease. In embodiments, a baseline is a measure of the presence or severity of disease symptoms prior to the initiation of therapy, for example, therapy with the insulin-Fc fusion protein described in the present invention, for example, when the subject has diabetes. Pharmaceutical Compositions and Routes of Administration

[0154] Pharmaceutical compositions containing an insulin-Fc fusion protein described in the present invention are provided that can be used to lower blood glucose in companion animals (e.g., dogs or cats). The amount and concentration of the insulin-Fc fusion protein in the pharmaceutical compositions, as well as the amount of the pharmaceutical composition administered to a subject, can be selected based on clinically relevant factors, such as medically relevant characteristics of the subject (e.g., age, weight, gender, other medical conditions and the like), the solubility of the compounds in the pharmaceutical compositions, the potency and activity of the compounds, and the mode of administration of the pharmaceutical compositions. For more information on Routes of Administration and Dosage, the reader should consult Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of the Editorial Board), Pergamon Press 1990.

[0155] The formulations in this disclosure include those suitable for parenteral administration. The administration phrases Petition 870260049851, dated 05 / 25 / 2026, page 86 / 693 77 / 180 parenteral and administered parenterally, as used in the present invention, mean modes of administration other than enteral and topical administration, generally by intravenous or subcutaneous injection.

[0156] Examples of suitable aqueous and non-aqueous vehicles that can be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the necessary particle size in the case of dispersion, and by the use of surfactants, for example, surfactants similar to Tween. In some embodiments, the pharmaceutical composition (for example, as described in the present invention) comprises a Tween-type surfactant, for example, polysorbate 20, Tween-20, or Tween-80.In some embodiments, the pharmaceutical composition (for example, as described in the present invention) comprises a Tween-type surfactant, for example, Tween-80, at a concentration between about 0.001% and about 2%, or between about 0.005% and about 0.1%, or between about 0.01% and about 0.5%.

[0157] In some embodiments, the concentration of insulin-Fc fusion protein in the aqueous transporter (vehicle) is about 3 mg / mL. In some embodiments, the concentration of insulin-Fc fusion protein in the aqueous transporter is about 6 mg / mL. In some embodiments, the concentration of insulin-Fc fusion protein in the aqueous transporter is about 8 mg / mL, 9 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL or more.

[0158] In some embodiments, insulin-Fc fusion protein is administered as a bolus, infusion, or boost. Petition 870260049851, dated 05 / 25 / 2026, page 87 / 693 78 / 180 intravenous. In some embodiments, the fusion protein is administered by injection using a syringe, pump, pen, needle, or indwelling catheter. In some embodiments, the insulin-Fc fusion protein is administered as a bolus, infusion, or intravenous injection. Delivery methods may also be provided by rechargeable or biodegradable devices. Several slow-release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including protein biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the prolonged release of a compound at a specific target site. Dosages

[0159] Actual dosage levels of insulin-Fc fusion protein may be varied in order to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular subject (e.g., dog or cat). The dosage level selected will depend on a variety of factors, including the activity of the particular fusion protein employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the excretion rate of the particular compound to be employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the fusion protein employed, the age, sex, weight, condition, general health and previous medical history of the subject to be treated, and similar factors also known in the medical arts.

[0160] In general, an adequate dose of an insulin-Fc fusion protein will be the amount that is the lowest effective dose to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Generally, intravenous and subcutaneous doses of insulin-Fc fusion protein for a dog or cat will vary. Petition 870260049851, dated 05 / 25 / 2026, p. 88 / 693 79 / 180 from about 0.001 to about 1 mg per kilogram (e.g., mg / kg) of body weight per day, for example, about 0.001 to 1 mg / kg / day, about 0.01 to 0.1 mg / kg / day, about 0.1 to 1 mg / kg / day, or about 0.01 to 1 mg / kg / day. In other embodiments, the fusion protein is administered at a dose between 0.025 and 4 mg per kilogram of body weight per week, for example, between 0.025 and 0.5 mg / kg / week.

[0161] This disclosure covers the formulation of insulin-Fc fusion protein in any of the pharmaceutical compositions and preparations mentioned above. Furthermore, this disclosure covers administration via any of the aforementioned routes of administration. A person skilled in the art may select the appropriate formulation and route of administration based on the condition being treated and the general health, age, and size of the patient being treated. Examples

[0162] The present technology is further illustrated by the following Examples, which should not be interpreted as limiting the invention. General Methods, Tests and Materials EXAMPLE 1 Synthesis and production methods of an insulin-FC fusion protein in HEK293 cells.

[0163] Insulin-Fc fusion proteins were synthesized as follows. A gene sequence of interest was constructed using proprietary software (LakePharma, Belmont, CA) and cloned into a high-expression mammalian vector. HEK293 cells were seeded in a shake flask for 24 hours before transfection and grown using chemically defined serum-free medium. A DNA expression construct encoding the fusion protein was created. Petition 870260049851, dated 05 / 25 / 2026, page 89 / 693 80 / 180 insulin-Fc was transiently transfected into a 2 L suspension of HEK293 cells using the standard operating procedure (LakePharma, Belmont, CA) for transient transfection. After 20 hours, cells were counted to determine viability and viable cell count, and titer was measured with the FortéBio® Octeto® kit (Pall FortéBio LLC, Fremont, CA). Additional readings were taken throughout the transient transfection production cycle. The culture was collected on or after day 5. EXAMPLE 2 Synthesis and production methods of an insulin-FC fusion protein in CHO cells.

[0164] A CHO cell line was originally derived from CHO-K1 (LakePharma, Belmont, CA), and the endogenous glutamine synthetase (GS) genes were deleted by recombinant technology using methods known in the art. Stable expression DNA vectors were designed and optimized for CHO expression and GS selection and incorporated into a vector for high-expression mammalian cells (LakePharma, Belmont, CA). The sequence of each complete construct was confirmed before the start of expansion experiments. CHO cells adapted for suspension culture were grown in a humidified atmosphere with 5% CO2 at 37°C in a chemically defined medium (CD OptiCHO™, Invitrogen, Carlsbad, CA). No serum or other animal-derived products were used in the CHO cell culture.

[0165] Approximately 80 million CHO cells adapted for suspension culture, growing in CD OptiCHO™ medium during the exponential growth phase, were transfected by electroporation using the MaxCyte® STX® system (MaxCyte, Inc., Gaithersburg, MD) with 80 Mg of DNA to create a stable CHO cell line for each insulin-Fc fusion protein (DNA construct). Petition 870260049851, dated 05 / 25 / 2026, page 90 / 693 81 / 180 contains the complete sequence of the insulin-Fc fusion protein). After twenty-four hours, the transfected cells were counted and placed under selection for stable integration of the insulin-Fc fusion genes. The transfected cells were seeded in CD OptiCHO selection medium containing 0-100 µM methionine sulfoximine (MSX) at a cell density of 0.5 x 10⁶ cells / mL in a shaker flask and incubated at 37°C with 5% CO₂. During a selection process, the cells were centrifuged and resuspended in fresh selection media every 2-3 days until the pool of stable CHO cells recovered its growth rate and viability. The cell culture was monitored for growth and titer.

[0166] The cells were cultured to a density of 2.5 * 106 cells per mL. At the time of collection for the cell bank, viability was above 95%. The cells were then centrifuged and the cell pellet was resuspended in CD OptiCHO medium with 7.5% dimethyl sulfoxide (DMSO) to a cell count of 15 * 106 cells per mL per vial. The vials were cryopreserved by storage in liquid nitrogen.

[0167] A small-scale production was carried out using CHO cells as follows. Cells were scaled up 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 glucose and additional amino acids as needed for approximately 14-21 days. The supernatant of the conditioned media collected from the stable production pool was clarified by centrifugation. The protein was passed through a Protein A column (MabSelect, GE Healthcare, Little Chalfont, UK) pre-equilibrated with binding buffer. Wash buffer was then passed through the column until the OD280 value (NanoDrop, Thermo Scientific) was measured to be at the level or Petition 870260049851, dated 05 / 25 / 2026, page 91 / 693 82 / 180 close to background levels. The insulin-Fc fusion protein was eluted using a low pH elution buffer, and fractions were collected, and the OD280 value of each fraction was recorded. Fractions containing the target insulin-Fc fusion protein were pooled and optionally further filtered using a 0.2 µM membrane filter.

[0168] The cell line was optionally subcloned for monoclonality and optionally further selected for clones with high expression of insulin-Fc fusion protein using the limiting dilution method, a method known to those skilled in the art. After obtaining a monoclonal cell line expressing insulin-Fc fusion protein at high titers, insulin-Fc fusion protein production was performed as described above in growth medium without MSX, or optionally in growth medium containing MSX, to obtain a cell culture supernatant containing recombinant insulin-Fc fusion protein produced with CHO. The MSX concentration was optionally increased over time to exert additional selectivity for clones capable of yielding higher product titers. Example 3 Purification of an Insulin-Fc Fusion Protein

[0169] The purification of an insulin-Fc fusion protein was performed as follows. Supernatants of conditioned media containing the secreted insulin-Fc fusion protein were collected from stably or transiently transfected HEK cell production cultures and clarified by centrifugation. The supernatant containing the desired insulin-Fc fusion protein was passed through a Protein A or Protein G column and eluted using a low pH gradient. Optionally, the recovery of insulin-Fc fusion proteins could be enhanced by reloading the initial Protein A or Protein G eluent back onto a second column. Petition 870260049851, dated 05 / 25 / 2026, page 92 / 693 83 / 180 Protein A or Protein G. Subsequently, the eluted fractions were pooled and the buffer was changed to 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0. A final filtration step was performed using a 0.2 µm membrane filter. The final protein concentration was calculated from the optical density of the solution at 280 nm. Optional further purification by ion-exchange chromatography (e.g., using an anion-exchange or cation-exchange resin bead), gel filtration chromatography, or other methods was performed as needed. Example 4 CE-SDS Structural Confirmation Under Reducing and Non-Reducing Conditions

[0170] Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) analysis was performed on a LabChip® GXII (Perkin Elmer, Waltham, MA) in a solution of a purified insulin-Fc fusion protein dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0, and the electropherogram was plotted. Under non-reducing conditions, the sample was run against known molecular weight (MW) protein standards, and the elution peak represented the 'apparent' MW of the insulin-Fc fusion protein homodimer.

[0171] Under reducing conditions (e.g., using beta-mercaptoethanol to break disulfide bonds of the insulin-Fc fusion homodimer), the apparent molecular weight of the resulting insulin-Fc fusion protein monomer was compared with half the molecular weight of the insulin-Fc fusion protein homodimer as a way to determine that the structural purity of the insulin-Fc fusion protein is likely correct. Example 5 Sequence Identification by LC-MS with Glycan Removal Petition 870260049851, dated 05 / 25 / 2026, p. 93 / 693 84 / 180

[0172] To obtain an accurate estimate of insulin-Fc mass via mass spectrometry (MS), the sample is first treated to remove naturally occurring glycan that could interfere with MS analysis. 100 µL of an insulin-Fc fusion protein at 2.5 mg / mL in 200 mM HEPES buffer, 100 mM NaCl, 50 mM NaOAc, pH 7.0, are first buffered with 0.1 M Tris, pH 8.0 containing 5 mM EDTA using a Zeba desalting column (Pierce, ThermoFisher Scientific, Waltham, MA). 1.67 µL of PNGase F (Prozyme N-glucanase) enzyme is added to this solution in order to remove the N-linked glycan present in the fusion protein (e.g., glycan linked to the asparagine side chain located at the cNg-N site), and the mixture is incubated at 37°C overnight in an incubator. The sample is then analyzed by LCMS (NovaBioassays, Woburn, MA) resulting in a molecular mass of the molecule that corresponds to the desired homodimer without the glycan.This mass is then further corrected, since the enzymatic process used to cleave the glycan from asparagine cNg also deaminates the asparagine side chain to form an aspartic acid, and in doing so the enzymatically treated homodimer gains a total of 2 Da, corresponding to a mass of 1 Da for each chain present in the homodimer. Therefore, the actual molecular mass is the measured mass minus 2 Da to correct for the enzymatic modification of the insulin-Fc fusion protein structure in the analytical sample. Example: 6% Homodimer by Size Exclusion Chromatography

[0173] Size exclusion chromatography (SEC-HPLC) of insulin-Fc fusion proteins was performed using a Waters 2795HT HPLC system (Waters Corporation, Milford, MA) with a 2998 Photodiode array at a wavelength of 280 nm. 100 µL or less of a sample containing an insulin-Fc fusion protein of interest was injected onto a MAbPac SEC-1, 5 µm, 4 x 300 column. Petition 870260049851, dated 05 / 25 / 2026, page 94 / 693 An 85 / 180 mm (ThermoFisher Scientific, Waltham, MA) column was operating at a flow rate of 0.2 mL / min with a mobile phase comprising 50 mM sodium phosphate, 300 mM NaCl, and 0.05% w / v sodium azide, pH 6.2. The MAbPac SEC-1 column operates on the principle of molecular size separation. Thus, larger soluble insulin-Fc aggregates (e.g., multimers of insulin-Fc fusion protein homodimers) eluted at earlier retention times, and non-aggregated homodimers eluted at later retention times. By separating the homodimer mixture from aggregated multimeric homodimers using analytical SEC-HPLC, the purity of the insulin-Fc fusion protein solution was determined in terms of the percentage of non-aggregated homodimer. Example 7 In Vitro Binding of an Insulin-Fc Fusion Protein to the IM-9 Insulin Receptor at 4°C

[0174] Human IM-9 cells (ATTC# CCL-159) expressing the human insulin receptor were cultured and maintained in RPMI medium with 5% complete SBF at 70-80% confluence. IM-9 cell cultures were centrifuged at 250*g (~1000 rpm) for 10 min to pellet the cells. The cells were washed once with HBSS or PBS buffer, resuspended in cold FACS staining medium (HBSS / 2mM EDTA / 0.1% Na-azide + 4% horse serum) at a concentration of 8 * 106 cells / mL and kept on ice or at 4°C until test solutions were made. The insulin-Fc protein was diluted in FACS buffer in serial 1:3 dilutions as 2 concentrations in 1.2 mL tubes (approx. 60 µL volume of each dilution), and the solutions were kept cold on ice until ready for pipetting.

[0175] Biotinylated RHI was diluted in FACS staining medium to a concentration of 1.25 µg / mL. 40 µL of the serially diluted test compound and 8 µL of Biotin-RHI at 1.25 µg / mL were added to each well. Petition 870260049851, dated 05 / 25 / 2026, p. 95 / 693 86 / 180 of a V-bottom microtiter plate, mixed in a slow vortex mixer, and placed on ice. 40 µL of an IM-9 cell suspension (8 * 10⁶ cells / mL) were then added to each well by multichannel pipette, mixed again gently, and incubated on ice for 30 min to allow competitive binding to the insulin receptor on IM9 cells. The cells were then washed twice with 275 µL of ice-cold FACS washing buffer (HBSS / EDTA 2 mM / Na-azide 0.1% + horse serum 0.5%) by centrifuging the V-bottom well plates at 3000 rpm for 3 min and aspirating the supernatant. The cells were then resuspended in 40 µL of FACS staining medium containing streptavidin-PE diluted 1:100 (Life Technologies) for 20 min on ice. The cells were then washed once with 275 µL of ice-cold FACS buffer and finally fixed with 3% paraformaldehyde for 10 min at room temperature.The cells were then washed once with 275 µL of ice-cold FACS buffer and resuspended in 250 µL of FACS buffer for analysis.

[0176] V-bottom plates containing cells were then analyzed on a Guava 8-HT flow cytometer (Millipore). Biotinylated RHI binding to the insulin receptor was quantified by the mean fluorescence intensity (MFI) of the cells in the FACS channel FL-2 for each concentration of the test compound. Control wells were labeled with biotinylated RHI only and were used to calculate the percentage (%) of inhibition resulting from each concentration of the test compound. The % inhibition by test compounds of biotinylated RHI binding in IM-9 cells was plotted against the concentrations (in log) of the test compound, and the resulting IC50 values ​​were calculated using GraphPad Prism software (GraphPad Software, La Jolla, CA) for the test compounds. Thus, lower IC50 values ​​for the test compound indicate higher levels of biotinylated RHI inhibition at lower concentrations, indicating strong binding. Petition 870260049851, dated 05 / 25 / 2026, page 96 / 693 87 / 180 insulin-Fc fusion protein for the insulin receptor. A control compound, such as unlabeled recombinant human insulin (RHI), was also used as an internal standard to generate an IC50 of RHI against which an IC50 of a given compound could be compared (IC50 (compound) / IC50 (RHI)). Lower IC50 ratios have more similar binding to RHI (stronger binding to the insulin receptor), while higher IC50 ratios have weaker binding to the insulin receptor relative to RHI. Example 8 In Vitro Fc(gamma)I Receptor Binding Affinity Assay

[0177] The binding of insulin-Fc fusion protein to the Fc(gamma)I receptor at pH 7.4 was conducted using an ELISA assay as follows. Since neither the canine Fc(gamma)I receptor nor the feline Fc(gamma)I receptor is commercially available, the human Fc(gamma)I receptor (i.e., rhFc(gamma)I receptor) was used as a surrogate mammalian receptor. The insulin-Fc compounds were diluted to 10 µg / mL in sodium bicarbonate buffer at pH 9.6 and coated onto Maxisorp microtiter plates (Nunc) overnight at 4°C, then the microplate strips were washed 5 times with PBST (PBS / Tween-20 at 0.05%) buffer and blocked with Superblock blocking reagent (ThermoFisher). Serial dilutions of biotinylated rhFc(gamma)I receptor (recombinant human Fc(gamma)RI; R&D Systems) were prepared in PBST / 10% Superblock buffer from 6,000 ng / mL to 8.2 ng / mL and loaded at 100 µL / well onto insulin-Fc fusion protein-coated microplate strips.The microtiter plate was incubated for 1 hour at room temperature, and then the microplate strips were washed 5 times with PBST and loaded with 100 µL / well of streptavidin-HRP diluted 1:10,000 in PBST / 10% Superblock buffer. After incubation for 45 min, the microplate strips were again washed 5 times with PBST. Petition 870260049851, dated 05 / 25 / 2026, page 97 / 693 88 / 180 TMB was added to reveal the bound Fc(gamma)I receptor proteins, and the reaction was stopped with ELISA stop reagent (Boston Bioproducts). Plate reading was performed on an ELISA plate reader at 450 nm, and OD values ​​(proportional to rhFc(gamma)I receptor binding to insulin-Fc protein) were plotted against the log concentrations of rhFc(gamma)I receptor added to each well to generate binding curves using GraphPad Prism software. Example 9 In Vitro Measurement of the Affinity of Insulin-Fc Fusion Protein for the Canine Fcrn Receptor

[0178] The in vitro binding affinity of insulinFc fusion proteins containing Fc fragments of IgG of canine or feline origin to the canine FcRn receptor was measured using an ELISA technique conducted at a pH of 5.5. The slightly acidic pH is the preferred binding environment for molecules containing Fc fragments to bind to the FcRn receptor. In vivo, cells express FcRn on their surfaces and internally in endosomes. As molecules containing Fc fragments are brought into the cell via natural processes (e.g., pinocytosis or endocytosis), the pH changes to a lower pH in the endosomes, where the FcRn receptor binds to molecules containing Fc fragments that would otherwise be degraded in endosome-lysosomal compartments, thus allowing these molecules to be recycled back to the cell surface where the pH is closer to neutral (e.g., pH 7.0–7.4).Neutral pH discourages binding to the FcRn receptor and allows the release of molecules containing the Fc fragment back into the circulation. This is a primary mechanism by which molecules containing Fc fragments exhibit prolonged circulating pharmacokinetic half-lives in vivo. Petition 870260049851, dated 05 / 25 / 2026, page 98 / 693 89 / 180

[0179] Insulin-Fc fusion proteins comprising canine or feline Fc fragments were diluted to 10 µg / mL in sodium bicarbonate buffer pH 9.6 and coated in duplicate onto Maxisorb ELISA strips for 1–2 hours at room temperature. The strips were then washed 4 times with PBST buffer (PBS / 0.1% Tween-20) and blocked with Superblock blocking reagent (ThermoFisher). The strips for FcRn binding were then washed again twice with MES / NaCl / Tween-20 buffer, pH 5.5 (MES 50 mM / NaCl 150 mM / Tween-20 0.1%) before the addition of the FcRn reagent (biotinylated canine FcRn; Immunitrack). Since no commercially available feline FcRn reagent was found, insulin-Fc fusion proteins containing either a canine Fc fragment or a feline Fc fragment were used in the assay for binding to canine FcRn.Serial dilutions (1:3X dilutions) of biotinylated FcRn reagent were prepared in MES / NaCl / Tween / 10% Superblock buffer pH 5.5 at concentrations from 1000 ng / mL to 0.45 ng / mL and loaded at 100 µL / well using a multichannel pipette onto strips coated with insulin-Fc fusion protein compounds. The final assay was then incubated for 1 hour at room temperature. The FcRn-binding strips were washed 4 times with MES / NaCl / Tween buffer pH 5.5 and then loaded with 100 µL / well of streptavidin-HRP diluted 1:10000 in MES / NaCl / Superblock buffer 10%, pH 5.5. After incubation for 45 minutes, the strips were washed again 4 times with MES / NaCl / Tween buffer pH 5.5. TMB was finally added to reveal the bound biotinylated canine FcRn reagent, and color development was stopped with the ELISA stop reagent. The plate was read on an ELISA plate reader at a wavelength of 450 nm.Optical density (OD) values ​​(proportional to the binding of canine FcRn to insulin-Fc fusion protein test compounds) were plotted against log concentrations of FcRn. Petition 870260049851, dated 05 / 25 / 2026, page 99 / 693 90 / 180 values ​​were added to each well to generate binding curves using GraphPad Prism software. EC50 values ​​for each binding curve were calculated for comparison between the different compounds. Example 10 Generalized Procedure for Determining In Vivo Pharmacodynamics (PD) Following a Single Administration of Insulin-Fc Fusion Proteins

[0180] Insulin-Fc fusion proteins were evaluated for their effects on fasting blood glucose levels as follows. N = 1, 2, 3 or more healthy, antibody-naive dogs weighing approximately 10-15 kg or cats weighing approximately 5 kg were used for each insulin-Fc fusion protein. The animals were also observed twice daily for signs of anaphylaxis, lethargy, distress, pain, etc., and, optionally for some compounds, treatment was continued with three or more additional weekly subcutaneous injections to observe whether the glucose-lowering capacity of the compounds decreased over time, a key sign of the potential induction of neutralizing anti-drug antibodies.On day 0, animals received a single intravenous or subcutaneous injection of a pharmaceutical composition containing an insulin-Fc fusion protein homodimer at a concentration between 1 and 10 mg / mL in a sodium hydrogen phosphate solution between 10-50 mM, 50-150 mM sodium chloride, 0.005-0.05% v / v Tween-80, and optionally a bacteriostat (e.g., phenol, m-cresol, or methylparaben) at a concentration between 0.02-1.00 mg / mL, at a solution pH between 7.0-8.0, at a dose of 0.08-0.80 mg of 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 of insulin equivalent on a molar basis). On day 0, blood was collected from a vein. Petition 870260049851, dated 05 / 25 / 2026, page 100 / 693 91 / 180 adequate immediately before injection and at 15, 30, 45, 60, 120, 240, 360 and 480 min and at 1, 2, 3, 4, 5, 6, and 7 days after injection.

[0181] At least 1 mL of whole blood was collected at each time point. The glucose level reading was taken immediately using a glucometer (ACCU-CHEK® Aviva Plus), which required approximately one drop of blood. The mean % of fasting blood glucose levels (%FBGL) from day 0 to day 7 were plotted graphically to assess the bioactivity of a specific insulin-Fc fusion protein. Example 11 Generalized Procedure for Determining In Vivo Pharmacodynamics (PD) Following Repeated Administrations of Insulin-Fc Fusion Proteins in Dogs or Cats.

[0182] Insulin-Fc fusion proteins were evaluated for their effects on fasting blood glucose levels after repeated injections as follows. Healthy, antibody-naive dogs or cats weighing approximately 5 to 20 kg were used, and each animal received doses of an insulin-Fc fusion protein. The animals were observed twice daily for signs of anaphylaxis, lethargy, distress, pain, and other negative side effects, and optionally, for some compounds, treatment was continued for up to two to five additional subcutaneous injections to observe whether the glucose-lowering capacity of the compounds decreased over time, indicating a possible presence of neutralizing antidrug antibodies in vivo.On day 0, the animals received a single subcutaneous injection of a pharmaceutical composition containing an insulin-Fc fusion protein homodimer in a solution of 10-50 mM sodium hydrogen phosphate, 50-150 mM sodium chloride, 0.0050.05% v / v Tween-80, and optionally a bacteriostat (e.g., phenol, m-cresol, or methylparaben) at a concentration between 0.02. Petition 870260049851, dated 05 / 25 / 2026, page 101 / 693 92 / 180 1.00 mg / mL, in a solution pH between 7.0-8.0, at a dose of 0.080-0.80 mg of insulin-Fc fusion protein / kg (or approximately equivalent to 1.2-12.3 nmol / kg or approximately equivalent to 0.44 U / kg of insulin equivalent on a molar basis). On day 0, blood was collected from a suitable vein immediately before injection and at 15, 30, 45, 60, 120, 240, 360, and 480 min and at 1, 2, 3, 4, 5, 6, and 7 days after injection.

[0183] Subsequent subcutaneous injections were given no more frequently than once a week, and in some cases, injections were given at different intervals based on the pharmacodynamics of a given insulin-Fc fusion protein formulation. Subsequent injections for each insulin-Fc fusion protein were adjusted to higher or lower doses depending on the demonstrated pharmacodynamics of the insulin-Fc fusion protein. For example, if the dose of a first injection on day 0 was found to be ineffective in reducing blood glucose, subsequent dose levels of the injected insulin-Fc fusion protein were adjusted upwards. Similarly, if the dose of a first injection on day 0 was found to lower glucose too strongly, then the subsequent dose of the injected insulin-Fc fusion protein was adjusted downwards.It was also found that intermediate or final doses could be adjusted similarly 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 min and at 1, 2, 3, 4, 5, 6, and 7 days (and optionally 14 days) after injection. At least 1 mL of whole blood was collected at each time point. Glucose level readings were taken immediately using a glucometer (ACCUCHEK® Aviva Plus), requiring approximately one drop of blood. The mean percentage of fasting blood glucose (%FBGL) throughout the study was plotted against the... Petition 870260049851, dated 05 / 25 / 2026, page 102 / 693 93 / 180 time, which allows the bioactivity of a fusion protein to be determined.

[0184] To determine the bioactivity of each dose, an area under the curve (AOC) analysis was conducted as follows. After constructing the %FBGL versus time data, the data were then entered into data analysis software (GraphPad Prism, GraphPad Software, San Diego, CA). The software was first used to conduct an area under the curve (AUC) analysis to integrate the area under the %FBGL vs. 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 lifetime dose (e.g., 7 days, 14 days, etc.) by 100% (where 100% represents y = 100% of the %FBGL vs. time curve). For example, given a dose half-life of 7 days and a calculated AUC of 500%FBGL / day, the following results for AOC: AOC = (100%FBGL x 7 days) - (500%FBGL / day) = 200%FBGL / day.The analysis can be performed for each injected dose in a series of injected doses to obtain the AOC values ​​for injection 1, injection 2, injection 3, etc.

[0185] Because insulin-Fc fusion protein doses can vary as discussed earlier, it is often more convenient to normalize all calculated AOC values ​​for a given insulin-Fc fusion protein to a particular dose of that insulin-Fc fusion protein. This allows for convenient comparison of the glucose-lowering potency of an insulin-Fc fusion protein across multiple injections, even if dose levels change across injections in a given study. The normalized AOC (NAOC) for a given dose is calculated as follows: NAOC = AOC / D with units of %FBGL days kg / mg; where D is the actual dose injected into the animal in mg / kg. NAOC values ​​can be calculated for each Petition 870260049851, dated 05 / 25 / 2026, page 103 / 693 94 / 180 injection is a series of injections for a given animal and may be an average among a group of animals that received the same insulin-Fc fusion protein formulation.

[0186] The NAOC ratio (NAOCR) can also be calculated for each injection in a series of injections for a given animal, taking the NAOC values ​​for each injection (e.g., injections 1, 2, 3,... N) and dividing each NAOC for a given injection by the NAOC from injection 1 as follows: NAOCR = (NAOC (Nés / ma injection) / NAOC (injection 1)).When evaluating the NAOCR of a given insulin-Fc fusion protein homodimer formulation for the nth injection in a series of injections, it is possible to determine whether the / nv / vo glucose-lowering activity of a given insulin-Fc fusion protein has substantially retained its bioactivity over a series of N doses (e.g., NAOCR for the nth dose greater than 0.5) or whether the / n vivo glucose-lowering activity of a given insulin-Fc fusion protein has lost a substantial portion of its potency (e.g., NAOCR of the nth dose is less than 0.5) over a course of N doses, indicating the potential formation of neutralizing / nv / vo antidrug antibodies. In preferred embodiments, the ratio of NAOC after the third subcutaneous injection to the 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). Example 12 Generalized Procedure for the In Vivo Determination of Pharmacokinetics (PK) in Canine and Feline Serum

[0187] An assay was constructed to measure the concentrations of insulin-Fc fusion proteins comprising Fc fragments of a canine isotype in canine serum as follows. The assay comprises a sandwich ELISA format in which the therapeutic compounds Petition 870260049851, dated 05 / 25 / 2026, page 104 / 693 95 / 180 serum samples were captured by an anti-insulin / proinsulin monoclonal antibody (mAb) coated onto ELISA plates and then detected by an antibody specific for the Fc portion of canine IgG conjugated with HRP followed by the use of a TMB substrate system for color development. Maxisorp ELISA plates (Nunc) are coated with the anti-insulin mAb clone D6C4 (Biorad) in coating buffer (pH = 9.6 sodium carbonate-sodium bicarbonate buffer) at 5 µg / mL overnight at 4°C. The plates are then washed 5x with PBST (PBS + Tween 20 at 0.05%) and blocked for a minimum of one hour at room temperature (or overnight at 4°C) with Superblock blocking solution (ThermoFisher). As amostras de soro teste são diluídas a 1:20 em PBST / SB / tampão de diluição de amostra HS 20% (PBS + Tween 20 0,1% + Superblock 10% + soro de cavalo 20%).To create a standard curve, the insulin-Fc fusion protein of interest is diluted in sample dilution buffer (PBST / SB / 20% HS) + 5% pooled beagle serum (BiolVT) from a concentration range of 200 ng / mL down to 0.82 ng / mL in serial 1:2.5 dilutions. Standards and diluted serum samples are added to blocked plates at 100 µL / well in duplicate and incubated for 1 hour at room temperature. After incubation, samples and standards are washed 5x with PBST. HRP-conjugated goat anti-Fc detection antibody (Sigma) diluted approximately 1:15,000 in PBST / SB / 20% HS buffer and 100 µL is added to all wells and incubated for 45 minutes at room temperature in the dark. The plates are washed 5 times with PBST and once with deionized water and developed by adding 100 uL / well of TMB (Invitrogen) for 8-10 minutes at room temperature.The color development is then stopped by the addition of 100 µL / well of ELISA Stop Solution (Boston Bioproducts) and the absorbance is read at 450 nm using a SpectraMax plate reader (Molecular Devices) in 30 minutes. Petition 870260049851, dated 05 / 25 / 2026, page 105 / 693 96 / 180 concentrations of insulin-Fc fusion protein compounds in the samples are calculated by interpolation onto a 4-PL curve using SoftMaxPro software.

[0188] Similarly, an assay was constructed to measure the concentrations of insulin-Fc fusion proteins comprising Fc fragments of a feline isotype in cat serum as follows. The assay comprises a sandwich ELISA format in which the therapeutic compounds in serum samples were captured by an anti-insulin / proinsulin mAb coated onto ELISA plates and then detected by a goat antibody specific for the Fc portion of feline IgG conjugated with HRP followed by the use of a TMB substrate system for color development. Maxisorp ELISA plates (Nunc) are coated with the anti-insulin mAb clone D6C4 (Biorad) in coating buffer (pH = 9.6 sodium carbonate-sodium bicarbonate buffer) at 5 ug / mL overnight at 4°C. The plates are then washed 5 times with PBST (PBS + Tween 20 at 0.05%) and blocked for a minimum of one hour at room temperature (or overnight at 4°C) with Superblock blocking solution (ThermoFisher).Test serum samples are diluted 1:20 in PBST / SB / HS 20% sample dilution buffer (PBS + Tween 20 0.1% + Superblock 10% + horse serum 20%). To make a standard curve, the insulinFc fusion protein compound of interest is diluted in sample dilution buffer (PBST / SB / 20% HS) + 5% cat serum (Jackson Immunoresearch) from a concentration range of 200 ng / mL to 0.82 ng / mL in serial 1:2.5 dilutions. Standards and diluted serum samples are added to blocked plates at 100 µL / well in duplicate and incubated for 1 hour at room temperature. After incubation, samples and standards are washed 5x with PBST. The goat antibody anti-Fc feline IgG conjugated with HRP (Bethyl Lab) diluted approximately 1:20:00,000 in PBST / SB / 20% SH buffer. Petition 870260049851, dated 05 / 25 / 2026, page 106 / 693 97 / 180 and 100 µL is added to all wells and incubated for 45 minutes at room temperature in the dark. The plates are washed 5x with PBST and once with deionized water and revealed by adding 100 µL / well of TMB (Invitrogen) for 8-10 minutes at room temperature. Color development is then stopped by adding 100 µL / well of ELISA Stop Solution (Boston Bioproducts) and the absorbance is read at 450 nm using a SpectraMax plate reader (Molecular Devices) in 30 minutes. The concentrations of insulin-Fc fusion protein compounds in the samples are calculated by interpolation on a 4-PL curve using SoftMaxPro software. Example 13 Test Protocol for Measuring Antidrug Antibodies in Canine Serum

[0189] Maxisorp ELISA plates (Nunc) are coated with the insulin-Fc fusion protein of interest diluted in coating buffer (pH = 9.6 carbonate-biocarbonate buffer) at 10 µg / mL overnight at 4°C for measurement of anti-drug antibodies (ADAs) against the test compound. To measure ADAs against the insulin portion of the insulin-Fc fusion protein containing an Fc fragment of canine IgG origin, the plates are coated with purified insulin at 30 µg / mL in coating buffer. The plates are then washed 5x with PBST (PBS + Tween 20 at 0.05%) and blocked for at least 1 hour (or overnight) with Superblock blocking solution (ThermoFisher, Waltham MA).To calculate ADAs in canine IgG units, strips are directly coated with serial 1:2 dilutions of canine IgG (Jackson Immunoresearch Laboratories, West Grove, PA) in Carb-Biocarb coating buffer pH = 9.6 at concentrations between 300-4.69 ng / mL overnight at 4°C and used to create a 7-point pseudo-standard curve. Standard plate strips are also used. Petition 870260049851, dated 05 / 25 / 2026, page 107 / 693 98 / 180 washed and blocked with Superblock blocking solution for at least 1 hour (or overnight).

[0190] Test serum samples are diluted to a concentration greater than or equal to 1:100 (normally tested as 1:200) in 20% PBST / SB / HS sample dilution buffer (PBS + 0.1% Tween 20 + 10% Superblock + 20% horse serum), and then samples are added to coated Fc-insulin fusion protein (or coated RHI) at 100 µL / well in duplicate. Standard canine IgG strips in duplicate are also added to each plate and filled with PBST / SB buffer (PBS + 0.1% Tween 20 + 10% Superblock) at 100 µL / well. Plates are incubated for 1 hour at room temperature and then washed 5x with PBST. For the detection of ADAs, goat anti-F(ab')2 feline IgG antibody (anti-F(ab')2 feline IgG reagent cross-reacts with canine antibodies; Jackson Immunoresearch Laboratories, West Grove, PA), which is diluted 1:10000 in PBST / SB and added at 100 µL / well to the sample and standard wells, was incubated for 45 minutes at room temperature in the dark.The plates are washed 5 times with PBST and then once with deionized water, and then the reaction is developed by adding 100 µL / well of TMB substrate (Invitrogen, ThermoFisher Scientific, Waltham, MA) for 15-20 minutes at room temperature in the dark. Color development is then stopped by adding 100 µL / well of ELISA Stop Solution (Boston Bioproducts), and the absorbance is read at 450 nm using a SpectraMax plate reader in 30 minutes. The antidrug antibody concentration is determined by interpolating the OD values ​​on the 4-PL pseudo-standard curve using the SoftMax Pro Software program (Molecular Devices, San Jose, CA).

[0191] To demonstrate the specificity of the detected ADAs, an inhibition assay is performed. In the ADA inhibition assay, serum samples are diluted 1:100 in PBST / SB / 20%HS buffer and Petition 870260049851, dated 05 / 25 / 2026, page 108 / 693 99 / 180 samples are mixed with an equal volume of 300 µg / mL of the relevant therapeutic compound (final sample dilution of 1:200 and final inhibitor compound at 150 µg / mL) and incubated for 30–40 minutes at room temperature to allow antidrug antibodies to bind to the free inhibitor (i.e., the therapeutic compound). After pre-incubation, the samples are added to insulin-Fc fusion protein-coated (or RHI-coated) strips at 100 µL / well in duplicate. Samples diluted 1:200 in PBST / SB / 20%HS buffer without the inhibitor compound are also tested on sample plates along with canine IgG-coated standard strips in duplicate. The remaining steps of the assay procedure are performed as described above. ADAs measured in drug-inhibited wells are combined with uninhibited ADA concentrations to assess ADA specificity.If significant inhibition of ADA signals is observed in the drug-inhibited wells, this means that the ADAS are specific to the therapeutic compound. Example 14 Test Protocol for Measuring Antidrug Antibodies in Feline Serum

[0192] Maxisorp ELISA plates (Nunc) are coated with insulin-Fc fusion protein of interest diluted in coating buffer (pH = 9.6 carbonate-biocarbonate buffer) at 10 µg / mL overnight at 4°C for measurement of ADAs against insulin-Fc fusion protein containing an Fc fragment of feline IgG origin. To measure ADAs against the insulin portion of insulin-Fc fusion protein, plates are coated with purified insulin at 30 µg / mL in coating buffer. Plates are then washed 5x with PBST (PBS + Tween 20 at 0.05%) and blocked for at least 1 hour (or overnight) with Superblock blocking solution (ThermoFisher, Waltham MA). To calculate ADAs in feline IgG units, the strips are Petition 870260049851, dated 05 / 25 / 2026, page 109 / 693 100 / 180 strips were directly coated with serial 1:2 dilutions of feline IgG (Jackson Immunoresearch Laboratories, West Grove, PA) in sodium carbonate-sodium bicarbonate coating buffer pH = 9.6 at concentrations between 300-4.69 ng / mL overnight at 4°C and used to create a 7-point pseudo-standard curve. The standard plate strips were also washed and blocked with Superblock blocking solution for at least 1 hour (or overnight).

[0193] Test serum samples are diluted to a concentration greater than or equal to 1:100 (normally tested as 1:200) in 20% PBST / SB / HS sample dilution buffer (PBS + 0.1% Tween 20 + 10% Superblock + 20% horse serum), and then samples are added to coated Fc-insulin fusion protein (or coated RHI) at 100 µL / well in duplicate. Duplicate strips of standard feline IgG are also added to each plate and filled with PBST / SB buffer (PBS + 0.1% Tween 20 + 10% Superblock) at 100 µL / well. Plates are incubated for 1 hour at room temperature and then washed 5x with PBST. For the detection of ADAs, goat anti-F(ab')2 feline IgG antibodies conjugated with HRP (Jackson Immunoresearch Laboratories, West Grove PA) are diluted in PBST / SB by a dilution factor of 1:10000 and added at 100 µL / well to standard samples and wells and incubated for 45 minutes at room temperature in the dark.The plates are washed 5 times with PBST and then once with deionized water, and then the reaction is developed by adding 100 µL / well of TMB substrate (Invitrogen) for 15–20 minutes at room temperature in the dark. Color development is then stopped by adding 100 µL / well of ELISA Stop Solution (Boston Bioproducts, Ashland MA) and the absorbance is read at 450 nm using a SpectraMax plate reader in 30 minutes. The antidrug antibody (ADA) concentration is determined by interpolating the OD values ​​on the 4-PL pseudo-standard curve. Petition 870260049851, dated 05 / 25 / 2026, page 110 / 693 101 / 180 using the SoftMax Pro Software program (Molecular Devices, San Jose CA). Example 15 Test Procedure for Identification of Immunogenic Epitope

[0194] Maxisorp ELISA microplates (Nunc) were coated with a library of homodimeric insulin-Fc fusion protein compounds with known amino acid sequences, and the coated plates are blocked in a manner similar to the procedure described in the antidrug antibody ELISA assay of Examples 13 and 14, except that each compound in the library is coated onto a separate individual well strip of the ELISA microplate. The compounds in the library comprise a range of insulin-Fc fusion proteins with different insulin polypeptide amino acid compositions, including various B-chain, C-chain, and A-chain amino acid mutations, different ligand compositions, and different Fc fragment compositions, including some of human origin.Separately, some wells of the plate strip are directly coated with 1:2 serial dilutions of canine or feline IgG (Jackson Immunoresearch Laboratories, West Grove, PA) to calculate anti-drug antibodies (ADA) in canine or feline IgG units, respectively, as described in Examples 13 and 14.

[0195] Serum obtained from individual dogs or cats receiving repeated doses of an insulin-Fc fusion protein is first screened in an ELISA assay for antidrug antibodies (Example 13 for dogs and Example 14 for cats). Serum samples demonstrating moderate or high positivity (e.g., moderate or high antibody titers) in the assays 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% Petition 870260049851, dated 05 / 25 / 2026, page 111 / 693 102 / 180 horse serum) and added to plates coated with the insulin-Fc fusion protein compound library for 1 hour at room temperature. After incubation, the plates are washed 5 times with PBST. For the detection of canine or feline antibodies capable of cross-reacting with the coated compound library, goat anti-F(ab')2 feline IgG antibodies conjugated with HRP (Jackson Immunoresearch Laboratories, West Grove PA), which cross-reacts to both canine and feline IgGs, are diluted in PBST / SB to 1:10,000 and added at 100 µL / well to the sample and standard wells and incubated for 45 min at room temperature in the dark. The plates were washed 5 times with PBST and once with deionized water, and the color reaction was developed by adding 100 µL / well of TMB substrate (Invitrogen, ThermoFisher Scientific, Waltham MA) for 15-20 min at room temperature in the dark.The color development is then stopped by the addition of 100 µL / well of ELISA Stop Solution reagent (Boston Bioproducts, Ashland, MA) and the absorbance is read at 450 nm using a SpectraMax plate reader in 30 min. The concentrations of cross-reacting anti-compound antibodies present in the serum samples are determined by interpolating the OD values ​​on the 4-PL pseudo-standard curve against directly coated canine or feline IgG antibody controls using SoftMax Pro software (Molecular Devices, San Jose, CA).

[0196] By correlating the antibody concentrations resulting from the assay with the known amino acid compositions of the insulin-Fc coated fusion protein library, it can be determined whether certain amino acid mutations or epitopes are responsible for causing no, some, most, or all total antibody signals in the assay, indicating no binding, weak binding, or strong binding to various insulin-Fc fusion protein homodimers. The mutations or epitopes responsible for moderate or Petition 870260049851, dated 05 / 25 / 2026, p. 112 / 693 103 / 180 strong are referred to here as immunogenic hot spots. Example 16 Design Process for Obtaining Insulin-Fc Fusion Proteins with High Homodimer Titers and Acceptable Levels of Bioactivity in Acute and Repeated Doses in Target Species

[0197] The process for achieving the project objectives described in the Detailed Description of the Invention comprises the following steps. First, the insulin polypeptide of SEQ ID NO: 4 or SEQ ID NO: 5 was combined with a species-specific Fc fragment of a particular IgG isotype and a ligand so that the resulting insulin-Fc fusion protein would likely produce a long-acting bioactive product with minimal immunogenicity (e.g., a species-specific IgG isotype was chosen with minimal binding to the Fc(gamma)I receptor). The DNA sequence encoding the desired fusion protein was prepared, cloned into a vector (LakePharma, San Carlos, CA), and the vector was then used to transfect HEK cells according to the procedure described in Example 1. The insulin-Fc fusion protein was then purified according to Example 3, and the overall protein yield and % homodimer were measured according to Example 6.Only candidates with a homodimer titer greater than 50 mg / L were considered acceptable, as titers below this level are unlikely to result in commercially viable production titers that meet the strictly low-cost manufacturing requirements for veterinary products. Subsequently, the selected insulin-Fc fusion proteins were screened for bioactivity indicators through in vitro insulin receptor binding studies, as described in Example 7. Based on the experiment, only compounds exhibiting insulin receptor (IR) activity with IC50 values ​​below 5000 nM are considered acceptable. Petition 870260049851, dated 05 / 25 / 2026, page 113 / 693 104 / 180 probability of exhibiting bioactivity in the species in question. Although the in vitro IC50 value of IR is a useful qualitative screening tool, this approach uses human IM-9 cells that express the human insulin receptor and, consequently, cannot capture some of the small differences in affinity between canine or feline IR and human IR. Furthermore, factors other than insulin receptor binding can influence the bioactivity of a compound in vivo (e.g., affinity for canine or feline FcRn to allow for prolonged pharmacokinetic elimination half-life in vivo).Therefore, the selected insulin-Fc fusion proteins that were acceptable from an IC50 value standpoint for IR activity and manufacturing were further screened for bioactivity in the animal of interest (e.g., dog or cat) to screen for any materials with less than the desired potency and / or duration of bioactivity (e.g., NAOC less than 150 %FBGL days kg / mg). Again, based on experience, at NAOC values ​​greater than 150 %FBGL days kg / mg, the dose requirements in the target species will be sufficiently low to achieve an acceptable treatment cost. Lastly, an additional evaluation criterion was added that is rarely or never mentioned in the state of the art.As discussed in more detail in the examples below, many realizations of insulin-Fc fusion protein that exhibit acceptable NAOC levels in target species after the first dose unexpectedly fail to maintain this level of bioactivity after repeated doses. Furthermore, in most cases, the reduction in bioactivity from repeated doses in target species is correlated with the development of neutralizing antidrug antibodies. This propensity to generate antidrug antibodies and the failure to maintain activity make these insulin-Fc fusion proteins impractical for use in the treatment of chronic diseases such as canine diabetes or feline diabetes. For this reason, Petition 870260049851, dated 05 / 25 / 2026, page 114 / 693 105 / 180 only insulin-Fc fusion proteins that exhibit acceptable levels of bioactivity in repeated doses (e.g., NAOCR values ​​greater than 0.50 for the third dose relative to the first dose) with minimal levels of antidrug antibodies were considered acceptable for use in the present invention. Results - Insulin-Fc Fusion Proteins Comprising a Canine Fc Fragment Example 17 Canine Insulin-Fc Fusion Protein Comprising Canine IgGA Isotype Fc

[0198] An attempt was made to produce an insulin-Fc fusion protein comprising the insulin polypeptide sequence of SEQ ID NO: 5 and the Fc fragment of the canine IgGA isotype (SEQ ID NO: 15) using the peptide linker of SEQ ID NO: 12. The complete amino acid sequence (full length) for the resulting insulinFc fusion protein is as follows: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSI CSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKP KDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQ QFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPPIERTISKARGR AHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEP ERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETL QNHYTDLSLSHSPG (SEQ ID NO: 42)

[0199] The insulin-Fc fusion protein of SEQ ID NO: 42 was synthesized in HEK cells according to Example 1 and purified according to Example 3. The protein yield was 22 mg / L after the purification step in protein A. The structure of the insulin-Fc fusion protein was confirmed according to Example 4 by CE-SDS under reducing and non-reducing conditions, and the sequence was further identified by LC-MS with glycan removal according to Example 5. The % homodimer was measured by exclusion chromatography. Petition 870260049851, dated 05 / 25 / 2026, page 115 / 693 106 / 180 by size according to Example 6 and determined to be 24%, indicating a high degree of homodimer aggregation. The resulting homodimer titer was therefore only 5 mg / L. In summary, the fabrication of the insulin-Fc fusion protein from SEQ ID NO: 42 in HEK cells resulted in a high level of aggregation and a low homodimer titer (5 mg / L), which did not meet the design objective of a homodimer titer greater than 50 mg / L.

[0200] However, the insulin-Fc fusion protein of SEQ ID NO: 42 was evaluated for bioactivity. Firstly, the receptor binding of the insulin-Fc fusion protein of SEQ ID NO: 42 measured according to Example 7, which results in an IC50 value of 2733 nM, indicates that the compound is likely to be bioactive in vivo (i.e., an IC50 less than 5,000 nM).

[0201] Next, the in vivo pharmacodynamics (PD) of the insulin-Fc fusion protein of SEQ ID NO: 42 was measured after a single intravenous administration of the compound in N = 3 dogs, according to Example 10. FIG. 2 shows the percentage of fasting blood glucose level of SEQ NO: 42 as a function of time. The NAOC for SEQ ID NO: 42 was calculated to be 105%FBGL days kg / mg according to the procedure of Example 11. The in vivo half-life of SEQ ID NO: 42 was calculated to be less than one day using the method of Example 12. The relatively low NAOC was probably the result of the large amount of aggregates in the sample (i.e., low % homodimer), but the soluble homodimer that still remained in circulation had only a pharmacokinetic elimination half-life of less than one day, which was considered unlikely to support a single once-weekly administration. Example 18 Mutations in the Fc Fragment Region of Insulin-Fc Fusion Proteins Comprising the Canine IgGA Isotype Petition 870260049851, dated 05 / 25 / 2026, page 116 / 693 107 / 180

[0202] In an attempt to increase the % homodimer content, improve bioactivity, and increase the half-life of the insulin-Fc fusion protein of SEQ ID NO: 42, mutations were inserted into the Fc fragment of the CH3 region to try to prevent intermolecular association (e.g., Fc fragment-Fc fragment interactions between molecules) and encourage stronger binding to the FcRn receptor (e.g., higher affinity for FcRn) to increase recycling and systemic circulation time. The following insulin-Fc fusion proteins that were synthesized in HEK cells according to Example 1, purified according to Example 3, and tested according to Examples 4-7, are shown below. The sequence alignment of SEQ ID NOs: 44, 46, 48, and 50 against SEQ ID NO: 42 and the differences in amino acid sequences are shown in Fig. 3. FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPK DILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQ FNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRA HKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPE RKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHEALHS HYTQKSLSLSPG (SEQ ID NO: 44) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPK DILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQ FNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRA HKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPE RKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHETLQS HYTDLSLSHSPG (SEQ ID NO: 46) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPK DILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQ Petição 870260049851, de 25 / 05 / 2026, pág. 117 / 693 108 / 180 FNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRA HKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPE RKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQ SHYTDLSLSHSPG (SEQ ID NO: 48) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPK DILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQ FNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRA HKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPE RKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHETLQN HYTDLSLSHSPG (SEQ ID NO: 50)

[0203] Canine IgGA variant-based insulin-Fc fusion proteins are listed in Table 2, along with the corresponding protein yields, % homodimers, and homodimer titers. The results show that several mutations in the IgGA Fc fragment, instead of improving the % homodimers and homodimer titers, gave rise to highly aggregated proteins with extremely low homodimer titers less than 5 mg / L. Thus, the in vivo bioactivity and pharmacokinetics of the compounds could not be evaluated. Table 2 HOMODIMER TITLES FOR SEQUENCES USING A CH3 REGION OF FC FRAGMENT OF IGGA CANINA MUTATED SEQ ID NO: Protein yield (mg / L) % homodimer Homodimer titer (mg / L) SEQ ID NO: 42 22 24% 5 SEQ ID NO: 44 33 0% 0 SEQ ID NO: 46 57 0% 0 SEQ ID NO: 48 67 0% 0 Petition 870260049851, dated 05 / 25 / 2026, p. 118 / 693 109 / 180 SEQ ID NO: 50 80 0% 0 EXAMPLE 19 Canine insulin-FC fusion protein using FC fragment from other canine isotypes.

[0204] As described above, canine IgGA is believed to be the preferred isotype for the Fc fragment to produce non-immunogenic insulin-Fc fusion protein for dogs due to its lack of effector function of canine Fc(gamma)I (very similar to the IgG2 isotype in humans). However, insulin-Fc fusion proteins manufactured with a canine IgGa Fc fragment were highly aggregated with an unacceptably low homodimer titer and acceptably low levels of bioactivity and duration of action. Therefore, Fc fragments of other canine IgG isotypes (canine IgGB of SEQ ID NO: 16), canine IgGC of SEQ ID NO: 17 and canine IgGD of SEQ ID NO: 18) were evaluated as replacements for the canine IgGA Fc fragment of the insulin-Fc fusion protein of SEQ ID NO: 42.The three insulin-Fc fusion proteins containing Fc fragments based on canine IgGB, IgGC, and IgGD isotypes were synthesized using the same insulin polypeptide from SEQ ID NO: 5 and the peptide linker from SEQ ID NO: 12 as were used to make the insulin-Fc fusion protein from SEQ ID NO: 42. The proteins were fabricated in HEK293 cells according to Example 1. The insulin-Fc fusion proteins were then purified using a Protein A column according to Example 3. The structure of the insulin-Fc fusion protein was confirmed according to Example 4 by CE-SDS under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The % homodimer was measured by size exclusion chromatography according to Example 6. Their sequences are shown below and the sequence alignment comparison against SEQ ID NO: 42 is shown in Fig. 4:. Petition 870260049851, of 25 / 05 / 2026, p. 119 / 693 110 / 180 FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTL LIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTACTQPREEQFN GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKKARGQAH QPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPES KYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQGDTFICAVMHEALHNH YTQSPG: ID52: NO FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGCNNCPCPGCGLLGGPSVFIFPPKPKDI LVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQS NGTYRVVVVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAH QPNVYVLPPSRDEMSKNTVTLTCVKDFFPPEIDVEWQSNGQQEPE SKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHSHN HYPSQIQ: IDQ 54) NO FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGCISPCPVPESLGGPSVFIFPPKPKDILRI TRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTY RVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSV YVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHT TAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDL SLSHSPG (SEQ ID NO: 56)

[0205] The resulting protein yield, % homodimer, and homodimer titers are provided in Table 3. Unexpectedly, only the insulin-Fc fusion protein from SEQ ID NO: 52 comprising an Fc fragment based on the canine IgGB isotype demonstrated a homodimer titer that met the design criteria, i.e., above 50 mg / L. The insulin-Fc fusion protein from SEQ ID NO: 54 comprising an Fc fragment based on the canine IgGC isotype did not yield any compounds, and the insulin-Fc fusion protein from SEQ ID NO: 56 comprising an Fc fragment based on the IgGD isotype Petition 870260049851, dated 05 / 25 / 2026, page 120 / 693 The 111 / 180 canine sample demonstrated an appreciable protein yield, but with a high degree of aggregation and therefore an unacceptably low homodimer titer.

[0206] In vitro insulin receptor binding to insulin-Fc fusion proteins from SEQ ID NO: 52 and SEQ ID NO: 56 was tested according to the procedure in Example 7. The insulin-Fc fusion protein from SEQ ID NO: 56 demonstrated an IC50 greater than 5,000 nM, indicating that the compound was highly unlikely to show bioactivity in vivo. However, the insulin-Fc fusion protein from SEQ ID NO: 52 demonstrated an IC50 of 28 nM, indicating that this sequence was likely bioactive in vivo. Table 3 Homodimer Titers for Sequences Using Fc Fragments of Native Canine IgGB, IgGC, and IgGD SEQ ID NO: IgG Fragment Protein Yield (mg / L) % Homodimer Homodimer Titer (mg / L) IC50 of IR Binding (nM) SEQ ID NO: 42 (Example 17) IgGA 21 24% 5 2,733 SEQ ID NO: 52 IgGB 80 93% 74 28 SEQ ID NO: 54 IgGC 0 0% 0 DNM* SEQ ID NO: 56 IgGD 134 12% 16 >5000 *DNM = not measured EXAMPLE 20 In Vivo Efficacy of an Insulin-Fc Fusion Protein Comprising the Insulin Polypeptide of SEQ ID NO: 5 with an Fc Isotype Fragment IgGB Canine

[0207] Due to the promising results of homodimer titer and insulin receptor activity obtained in Example 19, Petition 870260049851, dated 05 / 25 / 2026, page 121 / 693 The insulin-Fc fusion protein from SEQ ID NO: 52 was tested for in vivo bioactivity according to Example 10 following intravenous injection into each of N = 3 healthy, antibody-naive Beagle dogs weighing approximately 10 kg. In a separate experiment, the compound was administered subcutaneously to N = 3 naive Beagle dogs. FIG. 5 shows the %FBGL versus time for a single intravenous administration of the insulin-Fc fusion protein from SEQ ID NO: 52, and FIG. 6 shows the %FBGL vs. time for a single subcutaneous administration of the insulin-Fc fusion protein from SEQ ID NO: 52, both demonstrating that the insulin-Fc fusion protein from SEQ ID NO: 52 is significantly bioactive in dogs.

[0208] The NAOC was calculated according to the procedure of Example 11 to determine the relative bioactivity and duration of action of the insulin-Fc fusion protein. The NAOC of the intravenously injected insulin-Fc fusion protein from SEQ ID NO: 52 was 399 %FBGL days kg / mg, a result that was 3.8 times that of the NAOC of the intravenously injected insulin-Fc fusion protein from SEQ ID NO: 42, illustrating a significantly increased bioactivity of the insulin-Fc fusion protein comprising the canine IgGB Fc fragment compared to the insulin-Fc fusion protein comprising the canine IgGA Fc fragment. The NAOC of the subcutaneously administered insulin-Fc fusion protein from SEQ ID NO: 52 was 366 %FBGL days kg / mg, demonstrating a level of bioactivity by subcutaneous administration similar to the level obtained by intravenous administration. Example 21 In Vivo Immunogenicity Screening After Repeated Subcutaneous Dosing of Insulin-Fc Fusion Protein Comprising Insulin Polypeptide of SEQ ID NO: 5 with an Fc Isotype Fragment IgGB Canine Petition 870260049851, dated 05 / 25 / 2026, page 122 / 693 113 / 180

[0209] Next, repeated-dose subcutaneous bioactivity of the insulin-Fc fusion protein from SEQ ID NO: 52 was tested in dogs according to the method described in Example 11. N = 3 animals were administered subcutaneously on day 0, day 35, and day 42, and %FBGL was measured for the 7-day window after each dose according to Example 11. NAOC and NAOCR were calculated according to the procedure in Example 11 for each repeated subcutaneous injection. As illustrated in Table 4, repeated subcutaneous dosing in dogs unexpectedly revealed a significant drop in bioactivity by the third dose, 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). TABLE 4 NAOC per dose and NAOCR for repeated doses of Seq ID NO: 52 Number of Injections of SEQ ID NO: 52 NAOC (%FBGL^days*g / mg) NAOCR (proportional to week 1) 1 330 1.0 2 339 1.1 3 115 0.4

[0210] Without being linked to any particular explanation, it has been postulated that the cause of the significant reduction in the bioactivity of the insulin-Fc fusion protein of SEQ ID NO: 52, after the third repeated subcutaneous dose in dogs, was due to the development of antidrug antibodies that neutralize the biological activity of fusion proteins. Antidrug antibodies can be directed against the insulin polypeptide, ligand, or Fc fragments of an insulin-Fc fusion protein. The immunogenic response manifests as interactions between antigen-presenting cells, helper T cells, and B cells. Petition 870260049851, dated 05 / 25 / 2026, page 123 / 693 114 / 180 and its associated cytokines, which can lead to the production of endogenous antibodies against the drug (e.g., antidrug antibodies). Binding antibodies are all isotypes capable of binding to the insulin-Fc fusion protein and these can be detected in an immunoassay, as described in Example 13. Neutralizing antibodies that inhibit the functional activity of the insulin-Fc fusion protein are generally directed against an epitope that is necessary for bioactivity. To assess whether this was the case, sera collected before the administration of each dose, and at the end of the experiment described in Examples 11 and 12, were tested to quantify the levels of antidrug antibodies according to Example 13. As shown in FIG.7, antidrug antibody levels did in fact increase with multiple subcutaneous administrations of the compound, indicating that the generation of neutralizing antidrug antibodies was the likely cause for the reduction in NAOCR after the third injection of insulin-Fc fusion protein from SEQ ID NO: 52. Example 22 Non-Glycosylated Insulin-Fc Fusion Protein Comprising Insulin Polypeptide of SEQ ID NO: 5 with Canine IgGB Isotype Fc Fragments to Reduce Potential Immunogenicity Risk

[0211] As shown in Examples 19 and 20, the insulin-Fc fusion protein of SEQ ID NO: 52 showed acceptable % homodimer content, homodimer titer, and bioactivity in dogs; however, its use for a chronic disease, such as diabetes, is compromised by the reduction in bioactivity (Example 21) and generation of antidrug antibodies (Example 21) caused by repeated subcutaneous administration. Without being linked to any specific theory, the possible cause of the generation of antidrug antibodies and the reduction in bioactivity is the increased interaction of the Fc fragment of canine IgGB. Petition 870260049851, dated 05 / 25 / 2026, page 124 / 693 115 / 180 with numerous receptors of the canine immune system (e.g., Fc(gamma) receptors, e.g., FcyRI). However, the canine IgGB isotype was the only isotype among the four canine IgG isotypes that, when used for the Fc fragment, resulted in an insulin-Fc fusion protein that met the objectives of the single-dose bioactivity and productivity study design (Example 16). As described in the Detailed Description of the Invention, a method for reducing interaction with Fc(gamma)RI involves mutating the cNg site of the Fc fragment to prevent glycosylation during synthesis in the host cell. Therefore, mutations were made to the cNg site in the Fc fragment region of SEQ ID NO: 52 to reduce the binding affinity of the Fc fragment to Fc(gamma) receptors in vivo, as measured by an in vivo human Fc(gamma)RI binding assay described in Example 8.The verification of glycan deficiency was performed using the LC-MS method of Example 5, but omitting the treatment step. PNGase F. The position of the cNg site on the insulin-Fc fusion protein at SEQ ID NO: 52 is cNg-NB139. Mutations for SEQ ID NO: 52 included SEQ ID NO: 58 comprising a cNg-NB139-Q mutation, SEQ ID NO: 60 comprising a cNg-NB139-S mutation, SEQ ID NO: 62 comprising a cNg-NB139-D mutation, and SEQ ID NO: 64 comprising a cNg-NB139-K mutation. The complete amino acid sequences of the insulin-Fc fusion proteins mutated at the cNg sites are listed below (with the NB139 position underlined), and the resulting sequence alignments are shown in Fig. 8 (Omega Cluster): FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGGPSVFIFPPKPKDTL LIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFQ GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAH QPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPES Petição 870260049851, de 25 / 05 / 2026, pág. 125 / 693 116 / 180 KYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPG (SEQ ID NO: 58) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTL LIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFS GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAH QPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPES KYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPG (SEQ ID NO: 60) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTL LIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFD GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAH QPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPES KYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPG (SEQ ID NO: 62) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGGPSVFIFPPKPKDTL LIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFK GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAH QPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPES KYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPG (SEQ ID NO: 64)

[0212] Insulin-Fc fusion proteins were fabricated in HEK293 cells according to Example 1 and purified using a Protein A column according to Example 3. The structure of the insulin-Fc fusion protein was confirmed according to Example 4 by CESDS under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The % homodimer was measured by Petition 870260049851, dated 05 / 25 / 2026, page 126 / 693 117 / 180 size exclusion chromatography according to Example 6. As shown in Table 5, the homodimer titers of the insulin-Fc fusion proteins from SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64 met the objective of the study design, while unexpectedly the insulin-Fc fusion protein from SEQ ID NO: 58 containing the cNg-NB139-Q mutation did not meet the objective for homodimer titer. TABLE 5 HOMODIMER TITLES FOR CNG VARIATIONS OF SEQ ID NO: 52 SEQ ID NO: cNg Mutation Protein Yield (mg / L) % Homodimer Homodimer Titer (mg / L) SEQ ID NO: 58 cNg-Q 37 98% 36 SEQ ID NO: 60 cNg-S 77 98% 75 SEQ ID NO: 62 cNg-D 88 98% 86 SEQ ID NO: 64 cNg-K 68 98% 67

[0213] To determine which of the three remaining compounds was most likely to exhibit reduced immunogenicity, receptor binding Fc(gamma) was measured according to the procedure in Example 8. Low Fc(gamma) receptor binding is more 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 from SEQ ID NO: 52, unexpectedly demonstrating that the insulin-Fc fusion protein from SEQ ID NO: 62, containing the cNg-D mutation, exhibits Fc(gamma) receptor binding activity that is approximately twice that of the insulin-Fc fusion proteins from SEQ ID NO: 60, containing the cNg-S mutation, and SEQ ID NO: 64, containing the cNg-K mutation. Therefore, only the insulin-Fc fusion proteins comprising the latter two compounds containing the cNg-S and cNg-K mutations are considered. Petition 870260049851, dated 05 / 25 / 2026, page 127 / 693 118 / 180 were considered adequate for repeated-dose bioactivity testing in dogs. TABLE 6 FC(GAMMA) RECEIVER CONNECTION FOR CNG VARIATIONS OF SEQ ID NO: 52 SEQ ID NO: Mutation cNg OD450nm Log[Fc(gamma) RI (ng / mL) OD450nm Minus Assay Background Value Ratio for SEQ ID NO: 52 SEQ ID NO: 52 Native cNg 0.386 0.323 1.00 SEQ ID NO: 60 cNg-S 0.140 0.077 0.24 SEQ ID NO: 62 cNg-D 0.204 0.141 0.44 SEQ ID NO: 64 cNg-K 0.126 0.063 0.20 Assay background value (without compound) N / A 0.063 0.000 N / A EXAMPLE 23 Evaluation of the bioactivity and immunogenicity in vivo of a polypeptide. INSULIN OF SEQ ID NO: 5 WITH FC FRAGMENTS OF CANINE IGB ISOTYPE CNG-K AND CNG-S NOT GLYCOSYLATED

[0214] To determine whether the insulin-Fc fusion protein from SEQ ID NO: 60, containing the cNg-S mutation, improved repeated-dose bioactivity performance in dogs, the compound was administered subcutaneously to N = 1 dog on day 0, day 7, day 14, and day 28 according to the procedure of Example 11. When the dog's %FBGL dropped too low, the dog was given food to raise blood glucose to a safe level. The NAOC for the first injection was 191 %FBGL kg / mg, showing that the insulin-Fc fusion protein from SEQ ID NO: 60 was satisfactorily bioactive in vivo. The 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 given. Petition 870260049851, dated 05 / 25 / 2026, page 128 / 693 119 / 180 administered. The NAOC and NAOCR shown in Table 7 illustrate that the insulin-Fc fusion protein from SEQ ID NO: 60 exhibited a NAOCR that decreased significantly at doses 3 and 4 of a four-dose regimen. Therefore, the insulin-Fc fusion protein from SEQ ID NO: 60, containing the cNg-S mutation, was unable to demonstrate repeated-dose bioactivity in dogs, despite having low Fc(gamma)RI binding, i.e., four times lower than that of the insulin-Fc fusion protein from SEQ ID NO: 52. TABLE 7 NAOC PER DOSE FOR REPEATED DOSES OF SEQ ID NO: 60 Number of Injections of SEQ ID NO: 60 NAOC (%FBGL^days*g / mg) NAOCR 1 191 1.0 2 240 1.3 3 0 0.0 4 39 0.2

[0215] To determine whether the insulin-Fc fusion protein from SEQ ID NO: 64, containing the cNg-K mutation, improved repeated-dose bioactivity performance in dogs, the compound was administered subcutaneously to N = 1 dog on day 0, day 7, day 14, and day 28 according to the procedure of Example 11. When the dog's %FBGL dropped too low, the dog was given food to raise blood glucose to a safe level. The NAOC for the first injection was 449 %FBGL kg / mg days, showing that the insulin-Fc fusion protein from SEQ ID NO: 64 was satisfactorily bioactive in vivo. The pharmacokinetic profile of the compound was also measured by the method of Example 12 using ELISA, and a two-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. Petition 870260049851, dated 05 / 25 / 2026, page 129 / 693 120 / 180 administered until immediately before the next dose is administered. The NAOC and NAOCR shown in Table 8 illustrate that the insulin-Fc fusion protein of SEQ ID NO: 64 maintains a NAOCR greater than 0.6 across the four doses. Therefore, unexpectedly, the insulin-Fc fusion protein of SEQ ID NO: 64, containing the cNg-K mutation, was the only non-glycosylated mutant insulin-Fc fusion protein of SEQ ID NO: 52 that resulted in significantly improved repeated-dose bioactivity in dogs. TABLE 8 NAOC PER DOSE FOR REPEATED DOSES OF SEQ ID NO: 64 Number of Injections of SEQ ID NO: 64 NAOC (%FBGL-days-kg / mg) NAOCR 1 449 1.0 2 361 0.8 3 259 0.6 4 638 1.4

[0216] Antidrug and anti-insulin antibody levels were also measured throughout the course of treatment (28 days) and for two more weeks according to Example 13. FIG. 9 illustrates that the insulin-Fc fusion protein of SEQ ID NO: 64 still generated antidrug antibodies with repeated subcutaneous administration in dogs, but the antidrug antibody titers were much lower than those generated by the insulin-Fc fusion protein of SEQ ID NO: 52 (Example 19). Example 24 Screening of Canine Serum Containing Antidrug Antibodies and Identification of Potential Immunogenic Epitopes at Positions B10D and A8H Polypeptide Insulin

[0217] Mutation of the cNg site of the canine IgGB Fc fragment to a Lys (i.e., cNg-K) improved bioactivity at repeated doses. Petition 870260049851, dated 05 / 25 / 2026, page 130 / 693 121 / 180 of the insulin-Fc fusion protein comprising the insulin polypeptide of SEQ ID NO: 5 and the peptide linker of SEQ ID NO: 12 (Example 23), but the insulin-Fc fusion protein resulting from SEQ ID NO: 64 still gave rise to antidrug antibodies (Example 23). It was therefore hypothesized that the insulin polypeptide of SEQ ID NO: 5 may unexpectedly contain specific epitopes (i.e., immunogenic hot spots) against which the dog's immune system is directed. Therefore, the binding specificity of the antibodies present in the serum samples described in Example 13 was evaluated according to the general procedure of Example 15.Analysis of serum samples containing antibodies from repeated dosing of insulin-Fc fusion protein from SEQ ID NO: 52 (Example 19) against the coated insulin-Fc fusion protein library demonstrated that there were unexpectedly two primary hot spots present in the insulin polypeptide sequence from SEQ ID NO: 5: the aspartic acid mutation at the 10th N-terminal position of the B chain (i.e., B10), and, separately, the histidine mutation at the 8th N-terminal position of the A chain (i.e., A8). The results suggest that insulin-Fc fusion proteins comprising insulin polypeptide amino acid compositions containing these two particular amino acid mutations are likely immunogenic in dogs and therefore will likely give rise to anti-drug antibodies that neutralize bioactivity after repeated injections.Therefore, it was determined that insulin polypeptides that do not contain aspartic acid B10 and histidine A8 are preferred for insulin-Fc fusion proteins that need to be administered repeatedly in dogs for long periods in the long term (e.g., to treat canine diabetes). Example 25 Insulin-Fc Fusion Protein comprising Insulin Polypeptide of SEQ ID NO: 5 and a Canine IgGB Fragment Petition 870260049851, dated 05 / 25 / 2026, page 131 / 693 122 / 180 Glycosylated form in which the B10D and A8H mutations of the insulin polypeptide are restored to their native compositions to reduce the potential risk of immunogenicity.

[0218] To evaluate whether replacing hot spot mutations would improve the immunogenicity and bioactivity in repeated doses of insulin-Fc fusion proteins comprising the insulin polypeptide of SEQ ID NO: 5 and the Fc fragment of the canine IgGB isotype, an exemplary insulin-Fc fusion protein (SEQ ID NO: 66) was synthesized in which amino acids B10 and A8 of the insulin polypeptide were restored to their native histidine and threonine compositions, respectively (SEQ ID NO: 125) listed below with the non-native amino acids underlined). FVNQHLCGSHLVEALALVCGERGFFYTDPTGGPRRGIVEQCCTSIC SLYQLE NYCN (SEQ ID NO: 125)

[0219] Additionally, due to the potential additional benefits of the non-glycosylated cNg mutant forms, the insulin-Fc fusion protein of SEQ ID NO: 66 contained the cNg-Q mutation. The complete amino acid sequence of the insulin-Fc fusion protein of SEQ ID NO: 66 is provided below: FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSIC SLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGGPSVFIFPPKPKDTL LIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFQ GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAH QPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPES KYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPG (SEQ ID NO: 66)

[0220] The insulin-Fc fusion protein of SEQ ID NO: 66 was fabricated 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 Petition 870260049851, dated 05 / 25 / 2026, page 132 / 693 The 123 / 180 sequence was confirmed according to Example 4 by CE-SDS under reducing and non-reducing conditions, and the sequence was further identified by LC-MS with glycan removal according to Example 5. The % of homodimer measured by size exclusion chromatography according to Example 6 was 98.0%, indicating that the protein was relatively free of aggregates.

[0221] Despite the relatively low homodimer titer of 21 mg / L, the insulin-Fc fusion protein of SEQ ID NO: 66 was evaluated in dogs for bioactivity and immunogenicity in vivo according to the procedures of Examples 11-13, respectively. FIG. 10 demonstrates that restoring the B10D and A8H mutations to their native amino acids (i.e., B10H and A8T) in the insulin-Fc fusion protein of SEQ ID NO: 66 significantly reduced the immunogenicity of the original compound (SEQ ID NO: 52).

[0222] However, as shown in Fig. 11, the insulin-Fc fusion protein of SEQ ID NO: 66 containing the native amino acids B10 and A8 was not bioactive (i.e., the NAOC was essentially zero). Example 26 Attempts to Incorporate Additional B-Chain and α-Chain Mutations into the Insulin Polypeptide of SEQ ID NO: 125 to Improve the Bioactivity of Insulin-Fc Associated Fusion Proteins Containing the Canine IgGB Fc Fragment

[0223] The fact that the insulin-Fc fusion protein of SEQ ID NO: 66 does not generate antidrug antibodies (Example 25) compared to the insulin-Fc fusion protein of SEQ ID NO: 52 (Example 20) provides strong evidence for the theory that the B10D and A8H mutations in the insulin polypeptide of SEQ ID NO: 5 are likely the immunogenic epitopes responsible for the production of antidrug antibodies. Petition 870260049851, dated 05 / 25 / 2026, page 133 / 693 124 / 180 However, the lack of in vivo potency of the insulin-Fc fusion protein from SEQ ID NO: 66 compared to that of SEQ ID NO: 52 indicates that these two amino acid mutations are also responsible for achieving acceptable levels of bioactivity. The lack of in vivo potency for the insulin-Fc fusion protein from SEQ ID NO: 66 correlates with its high IC50 (shown in Table 9 below) as measured by the insulin receptor binding assay according to the method of Example 7. Therefore, additional efforts were needed to increase the bioactivity of the insulin-Fc fusion protein (i.e., decrease the IC50 value in the insulin receptor binding assay to less than 5000 nM, or more preferably less than 4000 nM, or even more preferably less than 3000 nM) while maintaining a low degree of immunogenicity, retaining the native B10 and A8 amino acids in the insulin polypeptide.

[0224] It is well known that several portions 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, portions of the B chain or the A chain were modified while B10 and A8 were kept the same as those of native insulin, and the C chain and peptide linker were kept constant. Many of these insulin-Fc fusion proteins were fabricated 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 under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The percentages (%) of homodimer content were measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinities were measured according to Example 7. Petition 870260049851, dated 05 / 25 / 2026, page 134 / 693 125 / 180 Their sequences are shown below, and the resulting sequence alignments against SEQ ID NO: 66 are shown in Fig. 12 (Clustal Omega). FVNQHLCGSHLVQALYLVCGERGFFYTDPTGGGPRRGIVEQCCTSIC SLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLI ARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQP SVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKY RTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QESLSHSPG (SEQ ID NO: 68) FVNQHLCGSELVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSIC SLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLI ARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQP SVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKY RTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QESLSHSPG (SEQ ID NO: 70) FVNQHLCGSHLVEALALVCGEAGFFYTDPTGGGPRRGIVEQCCTSIC SLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLI ARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQP SVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKY RTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QESLSHSPG (SEQ ID NO: 72) FVNQHLCGSHLVEALVCGERGFYYTDPTGGGPRRGIVEQCCTSIC SLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLI ARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTACTQPREEQFSGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQP SVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKY Petition 870260049851, of 25 / 05 / 2026, p. 135 / 693 126 / 180 RTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QESLSHSPG (SEQ ID NO: 74) FVNQHLCGSHLVEALVCGERGFFYTDPTGGGPRRGIVEQCCTSIC SLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLI ARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTACTQPREEQFSGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQP SVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKY RTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHYTSE QNHYTSE (IDHSPGQ 76) IDHSPGQ: Table 9 % OF HOMODIMER, HOMODIMER TITERS, AND IC50 IR VALUES FOR VARIOUS SEQ ID NOS. SEQ ID NO: % homodimer HEK homodimer titer (mg / L) IC50 IR (nM) SEQ ID NO: 66 98.0% 21 >5000 SEQ ID NO: 68 97.6% 9 2624 SEQ ID NO: 70 81.4% 17 633 SEQ ID NO: 72 99.1% 22 >5000 SEQ ID NO: 74 96.6% 25 2402 SEQ ID NO: 76 98.0% 6 >5000

[0225] In only three cases (SEQ ID NOs: 68, 70, and 74) did the proposed mutations improve IR binding (i.e., there was a decrease in the IC50 value), compared with SEQ ID NO: 66. However, none of the mutations resulted in compounds that satisfied the study design objective of producing a higher homodimer titer, above 50 mg / L, and, in some cases, the mutations led to significantly reduced production (e.g., homodimer titer less than 20 mg / L). Example 27 Petition 870260049851, dated 05 / 25 / 2026, p. 136 / 693 127 / 180 Attempts to Incorporate C-Chain Mutations into the Insulin Polypeptide of SEQ ID NO: 125 to Improve the Bioactivity of Insulin-Fc Associated Fusion Proteins Containing the Canine IgGB Fc Fragment

[0226] The results obtained in Example 26 show that all attempts to mutate the A and B chains of the insulin polypeptide from SEQ ID NO: 125 resulted in unacceptably low homodimer titers in HEK cells of the associated insulin-Fc fusion (i.e., homodimer titers less than or equal to 25 mg / L). Therefore, further experimentation was needed. In the present example, the C chain composition of an insulin polypeptide from SEQ ID NO: 125 was mutated, making it larger or increasing its flexibility. Native insulin (e.g., human insulin) has been shown to undergo a significant conformational change involving movement of the B chain and folding of the A chain as it binds to the insulin receptor (e.g., as described by Menting, et al., Nature, 2013; 493 (7431): pp. 241-245).Native insulin, unlike the insulin polypeptides of the present invention, is freely able to undergo this conformational change at the insulin receptor, since it is a double-chain polypeptide in its native form, linked only by two disulfide bonds without the C chain restricting the mobility of the A and B chains. Without being limited by any particular theory, it was hypothesized that the C chain contained in the insulin polypeptide of SEQ ID NO: 125 was too inflexible (e.g., an amino acid composition and sequence that does not allow easy movement between the B chain and the A chain) and / or too short (e.g., insufficient amino acids between the C-terminal of the B chain and the N-terminal of the A chain), thus preventing the insulin polypeptide from undergoing the necessary change in molecular form required for strong binding to the insulin receptor.Therefore, several insulin-Fc fusion proteins were synthesized based on the insulin-Fc fusion protein from SEQ ID NO: 66 with variations in the C chain of the insulin polypeptide. Petition 870260049851, dated 05 / 25 / 2026, page 137 / 693 128 / 180 as shown below with the resulting sequence alignments against SEQ ID NO: 66 shown in Fig. 13 (Clustal Omega). FVNQHLCGSHLVQALYLVCGERGFFYTDPTQRGGGGGQRGIVEQC CTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKP KDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREE QFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARG QAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQE PESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEAL HNHYTQESLSHSPG (SEQ ID NO: 78) FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGGGGSGGGGGIVE QCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPP KPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPR EEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKA RGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQ QEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHE ALHNHYTQESLSHSPG (SEQ ID NO: 80) FVNQHLCGSHLVEALALVCGERGFFYTDPGGGGGGGGGIVEQCCT SICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKD TLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQF SGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPE SKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPG (SEQ ID NO: 82) FVNQHLCGSHLVEALALVCGERGFFYTPGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGPSVFIFPPKPKDTL LIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFS GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAH QPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPES KYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPG (SEQ ID NO: 84) Petition 870260049851, dated 05 / 25 / 2026, page 138 / 693 129 / 180 Table 10 shows the percentage of homodimers, homodimer titers, and IC50 IR values ​​for various sequence ID numbers. SEQ ID NO: % homodimer Homodimer titer in HEK (mg / L) IC50 IR (nM) SEQ ID NO: 66 98.0% 21 >5000 SEQ ID NO: 78 94.0% 8 4176 SEQ ID NO: 80 99.6% 37 1609 SEQ ID NO: 82 98.3% 42 >5000 SEQ ID NO: 84 98.6% 33 4720

[0227] Insulin-Fc fusion proteins were fabricated 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 under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The % homodimer content was measured by size exclusion chromatography according to Example 6, and insulin receptor binding affinities were measured according to Example 7. In only one case, (SEQ ID NO: 80) comprising the longer C chain (GGGGGGGSGGGG), the C chain mutation significantly improved the insulin receptor binding affinity (IC50 less than 3000 nM) compared to the insulin receptor binding affinity of the Fc fusion protein from SEQ ID NO: 66.However, none of the C-chain mutated insulin-Fc fusion proteins exhibited a homodimer titer greater than the study's production target of 50 mg / L. In fact, in one case (SEQ ID NO: 78) the C-chain mutation unexpectedly led to significantly lower homodimer titers. Petition 870260049851, dated 05 / 25 / 2026, page 139 / 693 130 / 180 Example 28 Attempts to Incorporate Mutations in the Peptide Ligand in Insulin-Fc Fusion Proteins Containing the Insulin Polypeptide of SEQ ID NO: 125 and the Canine IgGB Fc Fragment to Improve Bioactivity

[0228] Without being tied to any specific theory, it has been suggested that another possible reason for the weak insulin receptor binding of the insulin-Fc fusion protein of SEQ ID NO: 66 involved steric hindrance between the insulin polypeptide and the insulin receptor, resulting from the proximity of the much larger Fc fragment molecule bound to the insulin polypeptide by the peptide linker. It was believed that shorter peptide linkers or more tightly folded peptide linkers potentially exacerbated this problem, while longer peptide linkers or peptide linkers that are resistant to folding (coiling) upon themselves (e.g., linkers with more molecular rigidity) could alleviate this problem by creating more space between the insulin polypeptide and the Fc fragment.The increased space between the insulin polypeptide and the Fc fragment would also increase the distance between the insulin receptor and the Fc fragment, leading to less interference during insulin receptor binding. It was hypothesized that the peptide linker of SEQ ID NO: 12 (i.e., GGGGAGGGG) used to construct the insulin-Fc fusion protein of SEQ ID NO: 66 is potentially too short and / or too flexible, as the amino acids comprising the linker do not contain side chains (i.e., they contain only the amino acids glycine and alanine). Therefore, to test this hypothesis, two other variants of the insulin-Fc fusion protein were synthesized from the insulin-Fc fusion protein of SEQ ID NO: 66. The insulin-Fc fusion protein of SEQ ID NO: 76 contained the same peptide linker that was used to construct the insulin-Fc fusion protein of SEQ ID NO: 66, but with... Petition 870260049851, dated 05 / 25 / 2026, page 140 / 693 131 / 180 is an insulin polypeptide in which asparagine at the N-terminal position of the A chain (i.e., A21) is absent (i.e., des-A21). This particular mutation was incorporated to see if the junction between the A chain and the peptide linker affects the protein yield and / or bioactivity of the molecule. The other insulin-Fc fusion protein from SEQ ID NO: 86 contains this mutation in the A chain (des-A21N) and a peptide linker that is twice as long as the peptide linker used to construct the insulin-Fc fusion protein from ID SEQ NO: 66. In this longer peptide linker, alanine is defavored and instead replaced by glutamine, which contains a polar amide side chain. The glutamine substitutions were expected to increase the hydrophilic nature of the peptide linker and potentially prevent the linker from folding in on itself.The sequences are shown below with the resulting sequence alignments against SEQ ID NO: 66 shown in Fig. 14 (Clustal Omega). FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSIC SLYQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLG PSVFIFPPKPKDTLLIARTPEVTCVVVDDLDPEDPEVQISWFVDGKQMQ TAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPI ERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVE WQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFI CAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 86) FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSIC SLYQLENYCGGGGAGGGGDCPKCPAPEMLGGGPSVFIFPPKPKDTLLI ARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQP SVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKY RTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QESLSHSPG (SEQ ID NO: 76) Petition 870260049851, dated 05 / 25 / 2026, page 141 / 693 132 / 180 Table 11 % OF HOMODIMER, HOMODIMER TITLES, AND IC50 IR VALUES FOR VARIOUS SEQ ID NOS. SEQ ID NO: % of homodimer Homodimer titer em HEK (mg / L) IC50 IR (nM) SEQ ID NO: 66 98.0% 21 >5000 SEQ ID NO: 66 98.0% 6 >5000 SEQ ID NO: 66 99.6% 11 1281

[0229] The two insulin-Fc fusion proteins were fabricated 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 under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. Their % homodimer content was measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinities were measured according to Example 7. The incorporation of a long peptide linker of different composition (GGGGGQGGGGQGGGGQGGGGG for SEQ ID NO: 86 vs.GGGGAGGGG for SEQ ID NO: 66) improved binding, as measured by the significant reduction in IC50 values ​​for the insulin receptor, indicating that longer ligands may be a strategy to increase insulin receptor binding in other insulin-Fc fusion proteins. However, incorporation of a longer ligand did not yet improve homodimer titers above the manufacturing study target of 50 mg / L. Petition 870260049851, dated 05 / 25 / 2026, page 142 / 693 133 / 180 Example 29 Attempts to Delete Portions of the B Chain of the Insulin Polypeptide of SEQ ID NO: 125 to Improve the Homodimer Titer of Insulin-Fc Associated Fusion Proteins Containing the Fc Fragment of Canine IgGB

[0230] 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 ID NO: 66, which contains the native amino acids B10 and A8. However, the mutation of the peptide linker failed to increase the homodimer titer sufficiently to meet the manufacturing design objective. Since the homodimer titer is a function of several properties, including intracellular synthesis and processing within cells, it was hypothesized that perhaps the insulin-Fc molecule would self-associate (i.e., aggregate) intramolecularly during and after synthesis between the two homodimer monomers or intermolecularly between two or more separate homodimers. This aggregation would lead to unacceptably low homodimer titers obtained from cell culture supernatants during the production process described in Examples 1, 3, and 6.This potential interaction between insulin-Fc fusion protein molecules may be due, in part, to insulin's known propensity to self-associate and form aggregates. A known method in the art to reduce insulin's propensity to self-associate involves mutating amino acids near the C-terminal end of the B chain. For example, insulin lispro (B28K; B29P mutations) and insulin aspart (B28D mutation) are well-known commercial two-chain insulins with mutations in the non-native B chain that prevent association and aggregation, thus resulting in a predominantly monomeric form of insulin in solution. Another approach to prevent this... Petition 870260049851, dated 05 / 25 / 2026, p. 143 / 693 134 / 180 Aggregation involves structural deletions of amino acids. For example, a two-chain insulin known as depentapeptide insulin (DPPI; see Brange J., Dodson GG, Edwards J., Holden PH, Whittingham JL 1997b. A model of insulin fibrils derived from the xray crystal structure of a monomeric protein (depentapeptide insulin) '27 507-516), is identical to native two-chain human insulin except that the five C-terminal amino acids of the B chain (YTPKT) are removed. DPPI has lower binding affinity for the insulin receptor compared to native two-chain human insulin, but is completely monomeric in solution, meaning there is no significant association or aggregation between DPPI molecules.Therefore, in an attempt to decrease the potential for intramolecular and intermolecular self-association and improve the homodimer titer of the insulin-Fc fusion protein, several variants of the insulin-Fc fusion protein of SEQ ID NO: 66 were constructed using partial B-chain amino acid truncations and B-chain amino acid mutations as described above for DPPI, insulin lispro, and insulin aspart. The sequences are shown below with the resulting sequence alignments against SEQ ID NO: 66 shown in Fig. 15 (Clustal Omega). FVNQHLGCGSHLVEALVCGERGFFYTDPGGGGGGGGGIVEQCCT SICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKD TLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTACTQPREEQF SGTYRVVVVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPE SKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYHSEQ: IDQ 82: NO FVNQHLCGSHLVEALVCGERGFFYTPGGGGGGGGGIVEQCCCTSI CSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTL LIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTACTQPREEQFS Petition 870260049851, of 25 / 05 / 2026, p. 144 / 693 135 / 180 GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKKARGQAH QPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPES KYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTSQHESQ: ID84: NO FVNQHLCGSHLVEALALVCGERGFFYTQGGGGGGGGIVEQCCTSI CSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTL LIARTPEVTCVVVDLDPEVQISWFVDGKQMQTAKTQPREEQFS GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAH QPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPES KYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPG (SEQ ID NO: 88) Table 12 % of Homodimer, Homodimer Titers, and IC50 IR Values ​​for Various Seq ID Nos. SEQ ID NO: % homodimer Homodimer titer in HEK (mg / L) IC50 IR (nM) SEQ ID NO: 66 98.0% 21 >5000 SEQ ID NO: 82 98.3% 42 1915 SEQ ID NO: 88 99.4% 22 2195 SEQ ID NO: 84 98.6% 33 1930

[0231] Insulin-Fc fusion proteins were fabricated 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 under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The percentages (%) of homodimer content were measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinities were measured. Petition 870260049851, dated 05 / 25 / 2026, page 145 / 693 136 / 180 according to Example 7. The homodimer titer of the resulting compounds was only significantly increased in one case (SEQ ID NO: 82), but unexpectedly, insulin receptor affinity was improved for all mutated compounds (SEQ ID NOs: 82, 88 and 84). Example 30 Attempts to combine mutations in the B chain, C chain, and α chain, truncation of the B chain, and Fc-Insulin Fusion Protein-binding mutations from SEQ ID NO: 66 to further improve homodimer titration and bioactivity.

[0232] As shown in Examples 26, 27, 28, and 29, no single strategy successfully incorporated an insulin polypeptide comprising the non-immunogenic native amino acids B10 and A8 with the Fc fragment of canine IgGB to form an insulin-Fc fusion protein with activity on the insulin receptor and acceptable homodimer titers. Therefore, the concepts of a longer C chain, a longer peptide linker, and truncation of the C-terminal amino acids of the B chain were combined. In addition, to potentially further decrease the tendency for self-association and aggregation, additional point mutations were introduced to the hydrophobic amino acid residue sites of native insulin using less hydrophobic amino acids, including those with side groups that are negatively or positively charged at physiological pH.Examples of such mutations include alterations of tyrosine to alanine, tyrosine to glutamic acid, isoleucine to threonine, and phenylalanine to histidine. Furthermore, to simplify the analysis, in all cases the cNg site of the canine IgGB Fc fragment was restored to its native asparagine. The sequences for these variant insulin-Fc fusion proteins are shown below with sequence alignments. Petition 870260049851, dated 05 / 25 / 2026, page 146 / 693 137 / 180 resulting against SEQ ID NO: 66 shown in Fig. 16 (Clustal Omega). FVNQHLCGSHLVEALELVCGERGFFYTPKTGGSGGGGIVEQCCTS TCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEML GGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQ MQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALP SPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDID VEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGD TFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 90) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCNHGGGGQGGGGQGGGGQGGGGGDCPKCPAPE MLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDG KQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNK ALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPP DIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 92) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPE MLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDG KQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNK ALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPP DIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 34) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGK QMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKAL PSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDI DVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRG DTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 32) Petition 870260049851, dated 05 / 25 / 2026, page 147 / 693 138 / 180 FVNQHLCGSHLVEALELVCGERGFFYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEVQISWFVDGK QMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKAL PSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDI DVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRG DTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 94) Table 13 % OF HOMODIMER, HOMODIMER TITLES, AND IC50 IR VALUES FOR VARIOUS SEQ ID NOS. SEQ ID NO: % of homodimer Homodimer titer in HEK (mg / L) IC50 of IR binding (nM) SEQ ID NO: 66 98.0% 21 >5000 SEQ ID NO: 90 97.9% 69 3869 SEQ ID NO: 92 99.5% 101 554 SEQ ID NO: 34 99.7% 107 1247 SEQ ID NO: 94 99.7% 128 2043 SEQ ID NO: 32 99.4% 187 2339

[0233] Insulin-Fc fusion proteins were fabricated 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 under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The percentages (%) of homodimer content were measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinities were measured according to Example 7. The results show that a combination of decreasing the hydrophobicity of certain amino acids of the B chain and A chain, using larger and more peptide C sequences Petition 870260049851, dated 05 / 25 / 2026, page 148 / 693 Flexible 139 / 180 amino acid truncations of the B-chain, and the use of a larger peptide linker resulted in several useful insulin-Fc fusion proteins that meet the design criteria regarding minimum homodimer titer and insulin receptor binding activity. SEQ ID NOs: 92, 34, 32 and 94 (368d), (366d), (218d) and (375d) showed more preferable IC50 values ​​for insulin receptor (less than 3,000 nM) and more preferable homodimer titer values ​​in HEK cells (titers greater than 100 mg / L) than SEQ ID NO: 66 or SEQ ID NO: 90. Surprisingly, the change of just a few amino acids leads to a multifaceted improvement in insulin receptor affinity and, in the case of the insulin-Fc fusion protein of SEQ ID NO: 32, a dramatic increase in homodimer titer compared to the original insulin-Fc fusion protein of SEQ ID NO: 66. Example 31 In Vivo Bioactivity, Repeated-Dose Bioactivity, and Immunogenicity of Insulin-Fc Fusion Proteins Constructed from the Insulin Polypeptide of SEQ ID NO: 7, the Peptide Linker of SEQ ID NO: 14, and the Canine IgGB Fc Fragment of SEQ ID NO: 16

[0234] Due to the positive homodimer titer and insulin receptor binding activity results of Example 30, two of the most promising insulin-Fc fusion proteins (SEQ ID NOs: 32 and 34) were tested in dogs to evaluate bioactivity and immunogenicity at repeated doses. Each compound comprises the longer, hydrophilic peptide linker of SEQ ID NO: 14 and the more fabricable and less aggregated canine IgGB Fc fragment of SEQ ID NO: 16. Most importantly, both insulin-Fc fusion proteins comprise insulin polypeptides with the putatively less immunogenic native amino acids B10 and A8 (i.e., generally from SEQ ID NO: 7). In the case of the insulin-Fc fusion protein of SEQ ID NO: 34, the Petition 870260049851, dated 05 / 25 / 2026, page 149 / 693 140 / 180 asparagine at position A21 is present (i.e., the insulin polypeptide comprises SEQ ID NO: 9). In the case of the insulinFc fusion protein of SEQ ID NO: 3 2, asparagine at position A21 is absent (i.e., the insulin polypeptide comprises SEQ ID NO: 8).

[0235] The in vivo bioactivity of the insulin-Fc fusion protein from SEQ ID 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 dose demonstrate that the insulin-Fc fusion protein from SEQ ID NO: 34 is indeed bioactive in vivo, with a NAOC of 1.076 %FBGL days kg / mg calculated according to the procedure of Example 11. The pharmacokinetic profile of the insulin-Fc fusion protein from SEQ ID NO: 34 was measured by the method of Example 12 using ELISA, and a two-compartment model was fitted to the data to determine its elimination half-life, which was approximately 3.5 days.

[0236] Repeated-dose bioactivity was then assessed by continuing to administer subcutaneous insulin-Fc fusion protein from SEQ ID NO: 34 to N = 1 dog on day 14, day 28, and day 42 after the initial injection according to the procedure in Example 8. When the dog's %FBGL dropped too low, the dog was given food to raise blood glucose to a safe level. NAOC and NAOCR were measured for each subsequent dose according to the general procedure in 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 14 illustrate that insulin-Fc fusion protein from SEQ ID NO: 34 maintains a NAOCR greater than 0.8 across the four doses, thus satisfying the study design objective of exhibiting repeated-dose bioactivity. Petition 870260049851, dated 05 / 25 / 2026, page 150 / 693 141 / 180 Table 14 NAOC per dose for repeated doses of SEQ ID NO: 34 Injection No. Day NAOC (%FBGL days kg / mg) NAOCR 1 0 1076 1.0 2 14 1005 0.9 3 28 900 0.8 4 42 838 0.8

[0237] The immunogenicity of the insulin-Fc fusion protein from SEQ ID NO: 34 was tested according to the procedure of Example 13. FIG. 18 demonstrates that the insulin-Fc fusion protein from SEQ ID NO: 34 does not exhibit apparent in vivo immunogenicity according to the maintenance of in vivo bioactivity throughout the repeated-dose experiment.

[0238] The insulin-Fc fusion protein of SEQ ID NO: 32, with asparagine in A21 of the insulin polypeptide chain absent, was also evaluated for repeated-dose bioactivity performance in dogs. The compound was administered subcutaneously to N = 1 dog on day 0, day 14, day 28, and day 42 according to the procedure of Example 11. When the dog's %FBGL dropped too low, the dog was given food to raise blood glucose to a safe level. The NAOC for the first injection was an impressive value of 2278 %FBGL days kg / mg, which shows that the insulin-Fc fusion protein from SEQ ID NO: 32 was satisfactorily bioactive in vivo, at almost twice the potency of the insulin-Fc fusion protein from SEQ ID NO: 34. The pharmacokinetic profile of the insulin-Fc fusion protein was measured by the method of Example 12 using ELISA, and a two-compartment model was fitted to the data to determine its elimination half-life, which was 4.1 ± 0.7 days. Figs.Figures 19 and 20 show single-dose blood glucose control and multi-week multi-dose blood glucose control for animals that received the SEQ ID homodimer. Petition 870260049851, dated 05 / 25 / 2026, page 151 / 693 142 / 180 NO: 32. 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 15 illustrate that the insulin-Fc fusion protein of SEQ ID NO: 32 maintains a NAOCR greater than or equal to 1.0 across the four doses, thus satisfying the study design objective of exhibiting repeated-dose bioactivity described in Example 16.

[0239] The immunogenicity of the insulin-Fc fusion protein from SEQ ID NO: 32 was tested according to the procedure of Example 13. FIG. 21 demonstrates that the insulin-Fc fusion protein from SEQ ID NO: 32 does not exhibit apparent in vivo immunogenicity according to the maintenance of in vivo bioactivity throughout the repeated-dose experiment. TABLE 15 NAOC PER DOSE FOR REPEATED DOSES OF SEQ ID NO: 32 Injection No. Day NAOC (%FBGL^days^g / mg) NAOCR 1 0 2278 1.0 2 14 4029 1.8 3 28 3450 1.5 4 42 3257 1.4

[0240] As discussed in the Detailed Description of the invention, there is a known enzymatic cleavage site between asparagine-glycine linkages (Vlasak, J., Ionescu, R., (2011) MAbs Vol. 3, No. 3 pp. 253-263). The omission of asparagine at the 21st amino acid in the A chain (i.e., A21) in the insulin polypeptide of SEQ ID NO: 8 contained in the insulin-Fc fusion protein of SEQ ID NO: 32 with the peptide linker of SEQ ID NO: 14, eliminates the possibility of enzymatic cleavage of the linkage. Petition 870260049851, dated 05 / 25 / 2026, p. 152 / 693 143 / 180 asparagine-glycine between the C-terminal of the A chain and the N-terminal of the peptide linker. However, the insulin-Fc fusion protein of SEQ ID NO: 3 4 comprises the peptide linker of SEQ ID NO: 14 and the insulin polypeptide of SEQ ID NO: 8, which holds the asparagine in A21. Therefore, it would be expected that the insulin-Fc fusion protein of SEQ ID NO: 34 would be enzymatically digested during synthesis or in vivo after subcutaneous administration. However, instead, the insulin-Fc fusion protein of SEQ ID NO: 34 was unexpectedly produced in HEK cells with an acceptable homodimer titer and demonstrated acceptable in vivo bioactivity without signs of enzymatic digestion that could compromise its bioactivity. Example 32 Confirmation of the Canine IgGB Isotype Fc Fragment for Optimal In Vivo Manufacturing Ability and In Vivo Efficacy of the Insulin-Fc Fusion Protein Comprising the Preferred Insulin Polypeptide of SEQ ID NO: 8 and the Preferred Peptide Linker of SEQ ID NO: 14

[0241] Having observed a novel combination of insulin polypeptides and peptide linkers resulting in non-immunogenic, high-yield, high-purity, and highly bioactive insulin-Fc fusion proteins, as described in Examples 30 and 31, it remained to be seen whether the canine IgGB Fc fragment was still the preferred isotype with respect to homodimer titer and bioactivity, as was the case with the insulin-Fc fusion proteins in Examples 19 and 20. Therefore, additional insulin-Fc fusion proteins were designed in which the insulin polypeptide (SEQ ID NO: 8) and the peptide linker (SEQ ID NO: 14) of the insulin-Fc fusion protein of SEQ ID NO: 32 were kept constant, and the canine IgGB Fc fragment of SEQ ID NO: 16 was replaced by the canine IgGA Fc fragment of SEQ ID NO: 15, the canine IgGC Fc fragment of SEQ ID NO: 17, or Fc fragment of canine IgGD from SEQ ID Petition 870260049851, dated 05 / 25 / 2026, page 153 / 693 144 / 180 NO: 18. The sequences for these variants resulting from the insulin-Fc fusion protein are shown below: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEVQISWFVDGK QMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKAL PSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDI DVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRG DTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 32) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGRCTDTPPCPV PEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVD GKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHI DLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYP PDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRW QQGDPFTCAVMHETLQNHYTDLSLSHSPG (SEQID NO: 96) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGGGGGGGGGCNNCPCPGC GLLGGPSVFIFPPKPKDILVTARTPTVVVDLDPENPEVQISWFVD SKQVQTANTQPREEQSNGTYRVVVLPIGHQDWLSGKQFKCKVNN KALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCVKDFF PPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSR WQRSGALSEQHSEQHTFIFH ID NO: 98) FVNQHLGCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGQGGGGGGGGCISPCPVPESL GGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEV HTACTQPREQQFNSTYRVVVVLPIEHQDWLTGKEFKCRVNHIGLPSP IERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDV EWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDLTSQLDSQLDSYFLYSKLSVDKSRWQQGDLTSQLDSQLDSYFLYSKLSVDKSRWQQGDLTSQLDSQLDSYFLYSKLSVDKSRWQQGDLTSQLDSQLD: 100) Petition 870260049851, of 25 / 05 / 2026, p. 154 / 693 145 / 180

[0242] Insulin-Fc fusion proteins were fabricated in HEK293 cells according to Example 1 and purified using Protein A or Protein G columns according to Example 3. Their structures were confirmed according to Example 4 by CE-SDS under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The percentages (%) of homodimer content were measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinities were measured according to Example 7. In addition, the affinities of the insulin-Fc fusion protein for the canine FcRn receptor were measured according to Example 8. As shown in Table 16, the insulin-Fc fusion protein from SEQ ID NO: 32 comprising the canine IgGB Fc fragment demonstrated the highest homodimer titer of these sequences.The insulin-Fc fusion protein from SEQ ID NO: 96 comprising the Fc fragment of canine IgGA exhibited a poor homodimer titer when purified using a Protein A column; however, when purified using a Protein G column, the homodimer titer was significantly improved, exceeding the study target of titers greater than 50 mg / L. The same was true for the insulin-Fc fusion protein from SEQ ID NO: 98 comprising the Fc fragment of canine IgGC. The insulin-Fc fusion protein of SEQ ID NO: 100 comprising the canine IgGD Fc fragment did not yield any compound when purified with a Protein A or Protein G column. Therefore, as demonstrated with the insulin-Fc fusion protein of SEQ ID NO: 52 containing a different insulin polypeptide (SEQ ID NO: 5) and peptide linker (SEQ ID NO: 12), the canine IgGB Fc fragment was preferred with respect to homodimer titer (see Example 19). Petition 870260049851, dated 05 / 25 / 2026, p. 155 / 693 146 / 180 Table 16 Homodimer Titers, IR Binding, and FcRN Binding for Sequences Using Fc Fragments of Native Canine IgGA, IgGB, IgGC, and IgGD SEQ ID NO: Fc Fragment of IgG Isotype Protein Yield - Protein A / (Protein G) (mg / L) % of homodimer Protein A / (Protein G) Homodimer Titer (mg / L) IC50 of Binding to IR (nM) EC50 of Binding to FcRn (ng / mL) NAOC at first dose (%FBGL days / kg / mg) 32 IgGB 187 / (DNM) 99% / (DNM) 185 2339 599 2278 96 IgGA 10 / (69) 45% / (91%) 62* 2586# 1610 174 98 IgGC 0 / (86) 0% / (94%) 81* 2084* >200000 39 100 IgGD 0 / (0) (DNM) / (DNM) 0 DNM DNM DNM DNM = not measured; # = purified by protein A; * = purified by protein G.

[0243] The in vivo bioactivity of the insulin-Fc fusion protein from SEQ ID NO: 96 comprising the canine IgGA Fc fragment that was purified via Protein G was tested according to the procedure of Example 10. The results illustrated in FIG. 22 show that the insulin-Fc fusion protein from SEQ ID NO: 96 is only slightly bioactive in vivo with a NAOC of only 174 %FBGL days kg / mg calculated according to Example 11.

[0244] The in vivo bioactivity of the insulin-Fc fusion protein from SEQ ID NO: 98 comprising the Fc fragment of canine IgGC was purified via G protein and tested according to the procedure of Example 10. The results illustrated in FIG. 23 show that the insulin-Fc fusion protein from SEQ ID NO: 98 is only slightly bioactive. Petition 870260049851, dated 05 / 25 / 2026, page 156 / 693 147 / 180 in vivo with a NAOC of only 39%FBGL days kg / mg calculated according to Example 11.

[0245] Therefore, as demonstrated with the insulin-Fc fusion protein of SEQ ID NO: 52 containing a different insulin polypeptide (SEQ ID NO: 5) and peptide linker (SEQ ID NO: 12), the canine IgGB Fc fragment was the preferred Fc fragment with respect to bioactivity (see Examples 19 and 20 and Table 6 above). Example 33 Non-glycosylated Insulin-Fc Fusion Proteins Comprising the Insulin Polypeptide of SEQ ID NO: 8, the Peptide Linker of SEQ ID NO: 14, and the Canine IgGB Fc Fragment to Reduce the Potential Risk of Immunogenicity

[0246] Although the insulin-Fc fusion protein of SEQ ID NO: 32 satisfies all the objectives of the study design (Example 16), there may or may not be a risk of immunogenicity over prolonged treatment periods (e.g., 6 months, 1 year, 2 years or more), which could compromise the use of this insulin-Fc fusion protein for the treatment of diabetes should this occur. As described in the Detailed Description of the Invention and in Examples 21 and 22, a possible cause of reduced bioactivity after repeated doses is the undesirable interaction of the canine IgGB Fc fragment with the dog's immune system resulting in the production of neutralizing anti-drug antibodies. However, the results shown in Example 32 demonstrate that, unexpectedly, the canine IgGB isotype was the only option of the four canine IgG isotypes that produced the desired manufactureability and bioactivity.Therefore, other Fc mutations have been explored to achieve non-glycosylated insulin-Fc fusion proteins with low binding to the Fc(gamma)RI receptor, which should reduce the risk of long-term chronic immunogenicity. Petition 870260049851, dated 05 / 25 / 2026, page 157 / 693 148 / 180

[0247] As described in the Detailed Description of the Invention, a method for reducing Fc(gamma)RI interaction involves mutating the cNg site of the Fc fragment to prevent glycosylation during synthesis in the host cell. Therefore, mutations were made to the cNg site in the Fc fragment region of SEQ ID NO: 32 to reduce the binding affinity of the Fc fragment to Fc(gamma) receptors in vivo, as measured by an in vivo human Fc(gamma)RI binding assay described in Example 8. The position of the cNg site on the insulin-like fusion protein Fc of SEQ ID NO: 32 is cNg-NB151. Mutations in SEQ ID NO: 32 included SEQ ID NO: 104 comprising a cNg-NB151S mutation and SEQ ID NO: 102 comprising the same cNgNB151-S mutation, as well as an NB119-A mutation. NB119-A was incorporated in a further attempt to reduce the interaction with Fc(gamma)RI, as previously described for use in mouse antibodies by Lo, M. et al.Effector attenuating substitutions that maintain antibody stability and reduce toxicity in mice, J. Biol. Chem. (2017), pp. 1-20. The complete amino acid sequences of the resulting insulin-Fc fusion proteins are listed below (NB119 and NB151 sites underlined for clarity), along with their sequence alignments (Clustal Omega) which are shown in Fig. 24. FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGK QMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKAL PSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDI DVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRG DTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 102) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDDLDPEDPEVQISWFVDGK Petition 870260049851, dated 05 / 25 / 2026, page 158 / 693 149 / 180 QMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKAL PSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDI DVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRG DTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 104)

[0248] Insulin-Fc fusion proteins were fabricated 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 under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The percentages (%) of homodimer content were measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinities were measured according to Example 7. As shown in Table 17, the incorporation of cNg-NB151-S mutations into the Fc fragment decreased the % homodimer, indicating an unacceptably high level of aggregation (i.e., the % homodimer dropped to just above 70%). TABLE 17 HOMODIMER TITRATES FOR NON-GLYCOSYLATED INSULIN-FC FUSION PROTEINS OF SEQ ID NO: 102 AND 104 SEQ ID NO: IgG Fragment Relevant Mutations Protein Yield (mg / L) % Homodimer Homodimer Titer (mg / L) IC50 of Binding to IR (nM) 32 IgGB cNg-NB151-N 187 99% 185 2339 102 IgGB cNg-NB151-S, NB119-A 78 73% 57 3093 104 IgGB cNgNB151-S 130 71% 93 2302

[0249] The in vivo bioactivity of the insulin-Fc fusion proteins of SEQ ID NO: 102 and SEQ ID NO: 104 was tested in N = 1 dog according to Petition 870260049851, dated 05 / 25 / 2026, page 159 / 693 150 / 180 with the procedure of Example 10. The results shown in Fig. 25 for a single subcutaneous dose demonstrate that both compounds were significantly less bioactive in vivo than the insulin-Fc fusion protein of SEQ ID NO: 32 (NAOC for SEQ ID NO: 104 = 574 %FBGL days kg / mg; NAOC for SEQ ID NO: 102 = 921 %FBGL days kg / mg). The results indicate that the incorporation of cNg-NB151-S mutations into the Fc fragment to produce non-glycosylated versions of the insulin-Fc fusion protein of SEQ ID NO: 32 unexpectedly decreased the in vivo bioactivity of the resulting compounds.

[0250] In an attempt to decrease the degree of aggregation and improve the bioactivity of the insulin-Fc fusion protein of SEQ ID NO: 104, containing the cNg-NB151-S mutation site, several B-chain variants of the insulin polypeptide were investigated with mutations in the region considered responsible for aggregation. Insulin-Fc fusion proteins were fabricated 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 under reducing and non-reducing conditions, and the sequences were additionally identified by LC-MS with glycan removal according to Example 5. Their % homodimer content was measured by size exclusion chromatography according to Example 6.Among the B-chain variants tested, it was unexpectedly found that the insulin-Fc fusion protein (SEQ ID NO: 36) containing a tyrosine to alanine substitution at the 16th amino acid from the N-terminus of the B-chain (i.e., B16) has high homodimer titers (105 mg / L) with low aggregation (99% homodimer), resulting in a homodimer titer of 104 mg / L. Insulin receptor binding measured according to Example 7 was acceptable with an IC50 value of 2,040 nM. The EC50 value of FcRn receptor binding affinity measured according to... Petition 870260049851, dated 05 / 25 / 2026, p. 160 / 693 151 / 180 Example 9 was 1194 ng / mL. The pharmacokinetic profile of the insulin-Fc fusion protein of SEQ ID NO: 36 was measured by the method of Example 12 using ELISA, and a two-compartment model was fitted to the data to determine its elimination half-life, which was 4.1 ± 0.7 days. The sequence of SEQ ID NO: 36 is shown below (B16A and cNg-NB151-S mutations underlined for clarity). FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPAPEM LGGPSVFIFPPKPKDTLUARTPEVTCVVVDLDPEVQISWFVDGK QMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKAL PSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDI DVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRG DTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 36)

[0251] The insulin-Fc fusion protein from SEQ ID NO: 36 was then evaluated for repeated-dose bioactivity performance in dogs. The compound was administered subcutaneously to N = 1 dog on day 0, day 7, day 14, and day 28 according to the procedure in Example 11. When the dog's %FBGL dropped too low, the dog was given food to raise blood glucose to a safe level. Unexpectedly, compared with the insulin-Fc fusion protein from SEQ ID NO: 104, the NAOC for the first injection of the insulin-Fc fusion protein from SEQ ID NO: 36 containing the B16A mutation was significantly higher (1185 %FBGL days / kg / mg). The bioactivity graph of the first dose in vivo is shown in Fig. 26. The pharmacokinetic profile of the compound was also measured by the method of Example 12 using ELISA, and a two-compartment model was fitted to the data to determine its elimination half-life, which was 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. Petition 870260049851, dated 05 / 25 / 2026, page 161 / 693 152 / 180 to be administered. The NAOC and NAOCR shown in Table 18 illustrate that the insulin-Fc fusion protein of SEQ ID NO: 36 maintains a NAOCR greater than or equal to 0.6 across the four doses, thus satisfying the objective of the study design to exhibit repeated-dose bioactivity. 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 ID NO: 32. Therefore, the insulin polypeptide of SEQ ID NO: 11 was preferred over non-glycosylated insulin-Fc fusion proteins comprising canine IgGB Fc fragments with a mutated cNg site. TABLE 18 NAOC PER DOSE FOR REPEATED DOSES OF SEQ ID NO: 36 Injection No. Day NAOC (%FBGL^days^g / mg) NAOCR 1 0 1185 1.0 2 7 954 0.8 3 14 764 0.6 4 28 991 0.8

[0252] Finally, the selected compounds were tested for the likelihood of interacting with the immune system by measuring their Fc(gamma) receptor binding activities according to the procedure of Example 8. Table 19 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 ID NO: 52. It can be observed that the non-glycosylated insulin-Fc fusion proteins (achieved through a mutation in the cNg-S site) exhibited a lower Fc(gamma) receptor binding ratio for SEQ ID NO: 52. Petition 870260049851, dated 05 / 25 / 2026, p. 162 / 693 153 / 180 Table 19 Connection to the Fc(gamma) Receiver for cNg Variations of SEQ ID NO: 52 SEQ ID NO: Species / Fc Isotype Glycosylation mutation OD450nm at an Fc(gamma) concentration RI of 3000 (ng / mL) OD450nm Minus Assay Background Value Ratio to SEQ ID NO: 52 SEQ ID NO: 52 Canine / native IgGB cNg 0.428 0.371 1.00 SEQ ID NO: 32 Canine / native IgGB cNg 0.368 0.311 0.84 SEQ ID NO: 96 Canine / native IgGA cNg 0.253 0.196 0.53 SEQ ID NO: 104 Canine / IgGB cNg-S 0.175 0.118 0.32 SEQ ID NO: 102 Canine / IgGB cNg-S and NB-119-A 0.166 0.109 0.29 SEQ ID NO: 36 Canine / IgGB cNg-S and B16A 0.177 0.120 0.32 EXAMPLE 34 EXAMPLES OF CHO CELL-BASED PRODUCTION EXECUTIONS USING PREFERRED INSULIN-FC FUSION PROTEINS COMPRISING CANINE IgG FC FRAGMENTS PRODUCED IN STABLY TRANSFECTED CHO CELL LINES

[0253] Stably transfected CHO cell lines with vectors encoding SEQ ID NO: 32, or SEQ ID NO: 36 were constructed as described in Example 2. 14-day production cultures in fed-batch flasks (medium scale 0.5-2.0 L) were seeded at 0.5 million cells / mL in a shaker incubator set at 37°C and Petition 870260049851, dated 05 / 25 / 2026, p. 163 / 693 154 / 180 5% carbon dioxide, and cultures were conducted as described in Example 2, except that the CD OptiCHO medium was replaced with Dynamis as the growth medium (ThermoFisher) and Efficient Feed C (ThermoFisher) was used as a supplement. Supplementation was added at 3% v / v starting on day 3 of production, and on day 4, the temperature of the shaker flask was set to 32°C and the carbon dioxide concentration of the shaker-incubator was reduced from 5% to 2%. During the run, cells increased to between 8-14 million cells / mL, and on Day 14 the production was harvested to remove cells, and the culture supernatant was purified and tested for insulin-Fc fusion protein as described in Examples 3, 4, 5, and 6. / Table 20 describes the production data obtained from runs with the stably transfected CHO cell line. TABLE 20 HOMODIMER TITRATES FOR INSULIN-FC FUSION PROTEINS GLYCOSYLATED WITH SEQ ID NO: 32 AND SEQ ID NO: 36 SEQ ID NO: Protein yield (mg / L) % homodimer Homodimer titer (mg / L) SEQ ID NO: 32 485 99.3% 482 SEQ ID NO: 36 260 99.0% 257 EXAMPLE 35 EXAMPLES OF CHO CELL-BASED PRODUCTION EXECUTIONS USING PREFERRED INSULIN-FC FUSION PROTEINS COMPRISING CANINE IgG FC FRAGMENTS PRODUCED IN STABLY TRANSFECTED CHO CELL LINES

[0254] A stably transfected CHO cell line with vectors encoding SEQ ID NO: 34 is constructed as described in Example 2. 14-day production cultures in flasks in Petition 870260049851, dated 05 / 25 / 2026, page 164 / 693 155 / 180 fed-batch systems (medium scale 0.5-2.0 L) are seeded at 0.5 million cells / mL in a shaker incubator set at 37°C and 5% carbon dioxide, and cultures are conducted as described in Example 2, except that CD OptiCHO medium is replaced with Dynamis as the growth medium (ThermoFisher) and Efficient Feed C (ThermoFisher) is used as a supplement. The supplement is added at 3% v / v starting on day 3 of production, and on day 4, the temperature of the shaker flask is set to 32°C and the carbon dioxide concentration of the shaker incubator is reduced from 5% to 2%. On Day 14, the production run is collected to remove the cells, and the culture supernatant is purified and tested for the insulin-Fc fusion protein as described in Examples 3, 4, 5, and 6.The resulting production of SEQ ID NO: 34 gives a protein yield above 200 mg / L, homodimer content above 95% and homodimer titer above 190 mg / L. Results - Insulin-Fc Fusion Proteins Comprising a Feline Fc Fragment Example 36 Insulin-Fc Fusion Protein Comprising an Fc Fragment of Feline IgG2 Isotype

[0255] To develop a product suitable for use in cats, an attempt was made to produce an insulin-Fc fusion protein comprising the insulin polypeptide sequence of SEQ ID NO: 4 and the Fc fragment of the cat IgG2 isotype (SEQ ID NO: 21) with the following amino acid sequence: FVNQHLCGSDLVEALYLVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDT LSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFN STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPH EPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENN Petition 870260049851, dated 05 / 25 / 2026, page 165 / 693 156 / 180 YQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHH TQKSLTQSPG (SEQ ID NO: 106)

[0256] The insulin-Fc fusion protein of SEQ ID NO: 106 was synthesized in HEK cells according to Example 1 and purified according to Example 3. The structure of the insulin-Fc fusion protein was confirmed according to Example 4 by CE-SDS under reducing and non-reducing conditions, and the sequence was further identified by LC-MS with glycan removal according to Example 5. The % homodimer of the resulting compound, measured by size exclusion chromatography according to Example 6, was 88%. The resulting homodimer titer was only 20 mg / L, resulting from the inability of HEK cells to produce the product in high yield (the protein yield after protein purification was only 23 mg / L).In summary, the fabrication of the insulin-Fc fusion protein from SEQ ID NO: 106 in HEK cells resulted in a moderate level of aggregates and a low homodimer titer of 20 mg / L, which did not meet the design objective of a homodimer titer greater than 50 mg / L.

[0257] However, the insulin-Fc fusion protein of SEQ ID NO: 106 was evaluated for bioactivity. Firstly, the receptor binding of the insulin-Fc fusion protein of SEQ ID NO: 106 measured according to Example 7, which results in an IC50 value of 22 nM, indicates that the compound is likely to be bioactive in vivo (i.e., an IC50 less than 5,000 nM).

[0258] Next, the in vivo pharmacodynamics (PD) of the insulin-Fc fusion protein of SEQ ID NO: 106 was measured after a single subcutaneous administration of the compound in N = 3 cats at a dose of 0.8 mg / Kg, according to Example 10. FIG. 27 shows the percentage of fasting blood glucose level for the insulin-Fc fusion protein of SEQ NO: 106 (106c) as a function of time. The NAOC for the insulin-Fc fusion protein was calculated to be 215%FBGL days Petition 870260049851, dated 05 / 25 / 2026, page 166 / 693 157 / 180 kg / mg according to the procedure of Example 11. Surprisingly, unlike the insulin-Fc fusion protein analogue for dogs of SEQ ID NO: 42 comprising the insulin polypeptide of SEQ ID NO: 5 and the peptide linker of SEQ ID NO: 12, the insulin-Fc fusion protein for cats of SEQ NO: 106 was found to be much less aggregated and significantly more bioactive in the target animal.

[0259] Since the NAOC was acceptable and the pharmacokinetic data were favorable to weekly administration, cats received additional subcutaneous doses on day 28, day 35, day 42, and day 49, and %FBGL was measured for the 7-day window after each dose according to Example 11. NAOC and NAOCR were calculated according to the procedure of Example 11 for each repeated subcutaneous injection. As illustrated in Table 21, repeated subcutaneous dosing in cats unexpectedly revealed a significant drop in bioactivity by the third dose, 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).The significant decrease in bioactivity for the insulin-Fc fusion protein of SEQ ID NO: 106 after repeated dosing in cats was similar to that observed for the insulin-Fc fusion protein of SEQ ID NO: 52 in dogs shown in Example 20. TABLE 21 NAOC PER DOSE FOR REPEATED DOSES OF SEQ ID NO: 106 Injection No. Day NAOC (%FBGL^days^g / mg) NAOCR 1 0 215 1.0 2 28 161 0.7 3 35 120 0.6 4 42 80 0.4 5 49 21 0.1 Petition 870260049851, dated 05 / 25 / 2026, page 167 / 693 158 / 180 EXAMPLE 37 Evaluation of mutations in insulin polypeptides and the choice of FC fragments of IgG1B or IgG2 from cats in relation to protein yield, purity, and activity on the insulin receptor.

[0260] In an attempt to increase the % homodimer content and protein yield of the insulin-Fc fusion protein of SEQ ID NO: 106, mutations were inserted into the B-chain sequences of the insulin polypeptide (e.g., the B16A mutation) and peptide linker. In addition, the Fc fragment of cat IgG1b (SEQ ID NO: 20) was evaluated in addition to the Fc fragment of cat IgG2 (SEQ ID NO: 21) which was used to construct the insulin-Fc fusion protein of SEQ ID NO: 106. The resulting insulin-Fc fusion protein sequences are shown below with the resulting sequence alignments against SEQ ID NO: 106 shown in Fig. 28 (Clustal Omega). FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGSGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTL SISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNS TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEP QVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYR TTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQ KSLTQSPG (SEQ ID NO: 108) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDT LSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFN STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPH EPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENN YQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHH TQKSLTQSPG (SEQ ID NO: 110) Petição 870260049851, de 25 / 05 / 2026, pág. 168 / 693 159 / 180 FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSIC SLYQLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDT LSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFN STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPH EPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENN YQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHH TQKSLTQSPG (SEQ ID NO: 112)

[0261] Insulin-Fc fusion proteins were fabricated 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 under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. Homodimer content percentages (%) were measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinities were measured according to Example 7. The variant insulin-Fc fusion proteins are listed in Table 22, along with the protein yields, % homodimers, and corresponding homodimer titers.The results show that the various mutations, when combined with the Fc fragment of the feline IgG1b isotype to produce the insulin-Fc fusion protein of SEQ ID NO: 108, gave rise to a much higher protein yield, but the resulting protein was more aggregated (i.e., lower % of homodimer than that of SEQ ID NO: 106). This was surprising, as feline IgG1b is more similar in function to the Fc fragment of the canine IgGB isotype, which was the highly preferred Fc isotype for the production of canine insulin-Fc fusion proteins (Example 32). Of the mutated compositions for felines containing the feline IgG2 isotype, those comprising a B16 mutation in the B chain of the insulin polypeptide (i.e., SEQ ID NO: 110 and SEQ ID NO: 106) showed the highest percentage of mutations. Petition 870260049851, dated 05 / 25 / 2026, pp. 169 / 693 160 / 180 112) led to improved protein yield and homodimer titers. However, the mutated ligand present in SEQ ID NO: 110 (i.e., GGGGAGGGG) appears to have provided an additional doubling in protein yield and homodimer titer compared to SEQ ID NO: 112. Table 22 Production and Binding to IR for Insulin-Fc Fusion Proteins Using Fc Fragments of Feline IgG1b and IgG2 SEQ ID NO: IgG Fragment Protein Yield (mg / L) % Homodimer Homodimer Titer (mg / L) IC50 of Binding to IR (nM) 106 IgG2 23 88.0% 20 22 108 IgG1b 127 49.0% 62 62 110 IgG2 122 89.7% 109 41 112 IgG2 64 80.4% 51 53 EXAMPLE 38 In vivo immunogenicity screening after repeated subcutaneous doses of insulin fusion protein FC comprising insulin polypeptide of sequence ID no. 4 with an FC fragment of feline IgG2 isotype.

[0262] Without being linked to any particular explanation, it has been postulated that the cause of the significant reduction in the bioactivity of the insulin-Fc fusion protein of SEQ ID NO: 106, after the fourth repeated subcutaneous dose in cats (Example 36), was due to the development of antidrug antibodies that neutralize the biological activity of the fusion proteins. Antidrug antibodies can be directed against the insulin polypeptide, ligand, or Fc fragments of an insulin-Fc fusion protein. The immunogenic response manifests as interactions between antigen-presenting cells, helper T cells, B cells, and their associated cytokines, which can lead to the production of endogenous antibodies against the drug (e.g., antidrug antibodies). Petition 870260049851, dated 05 / 25 / 2026, page 170 / 693 161 / 180 macos). Binding antibodies are all isotypes capable of binding to the insulin-Fc fusion protein and these can be detected in an immunoassay, as described in Example 14. Neutralizing antibodies that inhibit the functional activity of the insulin-Fc fusion protein are generally directed against a biologically active site. To assess whether this was the case, serum collected before administration of each dose, and at the end of the experiment described in Example 11, was tested to quantify antidrug antibody levels according to Example 14. As shown in FIG. 29, antidrug antibody levels did indeed increase with multiple subcutaneous administrations of the compound, indicating that the generation of neutralizing antidrug antibodies was the likely cause for the reduction in NAOCR after the fourth injection of the insulin-Fc fusion protein from SEQ ID NO: 106. EXAMPLE 39 Screening of feline serum containing antidrug antibodies and identification of potential immunogenic epitopes at positions B10D and A8H of the insulin polypeptide.

[0263] As observed for SEQ ID NO: 52 in dogs (Example 20), the bioactivity of repeated doses of the fusion protein of SEQ ID NO: 106 comprising the insulin polypeptide of SEQ ID NO: 4 and the peptide linker of SEQ ID NO: 13 gave rise to anti-drug antibodies (Example 38). It was therefore hypothesized that the insulin polypeptide of SEQ ID NO: 4 may unexpectedly contain specific epitopes (i.e., immunogenic hot spots) against which the cat's immune system is directed. Therefore, the binding specificity of the antibodies present in the serum samples described in Example 38 was evaluated according to the general procedure of Example 15. Analysis of cat serum samples containing antibodies from the dosage Petition 870260049851, dated 05 / 25 / 2026, page 171 / 693 Repeated 162 / 180 assays of the insulin-Fc fusion protein from SEQ ID NO: 106 (Example 38) against the coated insulin-Fc fusion protein library demonstrated that there were unexpectedly two primary hot spots present in the insulin polypeptide sequence from SEQ ID NO: 4: the B10D site mutation (i.e., the aspartic acid mutation at the 10th position of the N-terminal B chain (i.e., B10)), and, separately, the A8H site mutation (i.e., the histidine mutation at the 8th position of the N-terminal A chain (i.e., A8)). The results suggest that insulin-Fc fusion proteins comprising insulin polypeptide amino acid compositions containing these two particular amino acid mutations are likely immunogenic in cats and therefore will likely give rise to anti-drug antibodies that neutralize bioactivity after repeated injections.Therefore, it was determined that insulin polypeptides that do not contain B10D and A8H are preferred for insulin-Fc fusion proteins that need to be administered repeatedly in cats for long periods in the long term (e.g., to treat diabetes). Example 40 Fc-Insulin Fusion Proteins comprising the Insulin Polypeptide of SEQ ID NO: 4 and Fc Fragments of Glycosylated and Non-Glycosylated Feline IgGIb and IgG2 Isotypes in which B10, A8 and Other Sites of the Insulin Polypeptide Are Mutated to Reduce the Risk of Potential Immunogenicity

[0264] To evaluate whether replacing hot spot mutations would improve the immunogenicity and bioactivity of insulin-Fc fusion proteins comprising the insulin polypeptide from SEQ ID NO: 4 and the Fc fragment of the feline IgG2 isotype in repeated doses, exemplary insulin-Fc fusion proteins from SEQ ID NOs: 114, 116, and 118 were synthesized in which amino acids B10 and A8 of the insulin polypeptide were restored to their native histidine and alanine compositions, Petition 870260049851, dated 05 / 25 / 2026, page 172 / 693 163 / 180 respectively, and the histidine in B16 was replaced by alanine (i.e., B16A) as was the case with the insulin polypeptide of SEQ ID NO: 5 used for many of the canine insulin-Fc fusion proteins. The A21N site of native insulin was also deleted. For this example, other amino acids of the insulin polypeptide were mutated to make the structure more similar to native feline insulin (e.g., B30A, A8A, A10V, and A18H). The resulting insulin polypeptide sequence (SEQ ID NO: 120) is listed below with the amino acids not active for feline insulin underlined. FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQL EHYC (SEQ ID NO: 120)

[0265] Furthermore, due to the potential additional benefits of the non-glycosylated cNg mutants discussed in Examples 22 and 33, two of the insulin-Fc fusion proteins evaluated (SEQ ID NOs: 116 and 118) contain the cNg-S mutation. The complete amino acid sequences of the insulin-Fc fusion proteins are shown below with the resulting sequence alignments against SEQ ID NO: 108 shown in Fig. 30 (Clustal Omega). FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASV CSLYQLEHYCGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDT LSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFN STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPH EPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENN YQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHH TQKSLTQSP (SEQ ID NO: 114) FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASV CSLYQLEHYCGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDT LSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFS STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPH EPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENN Petição 870260049851, de 25 / 05 / 2026, pág. 173 / 693 164 / 180 YQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHH TQKSLTQSPG (SEQ ID NO: 116) FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASV CSLYQLEHYCGGGGAGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTL SISRTPEVTCLVVALGPDDSDVQITWFVDNTQVYTAKTSPREEQFSS TYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEP QVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYR TTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQ KSLTQSPG (SEQ ID NO: 118)

[0266] Insulin-Fc fusion proteins were fabricated 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 under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The percentages (%) of homodimer content were measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinities were measured according to Example 7. Table 23 below illustrates the fabrication capability and in vitro IR binding parameters for the resulting compounds. Table 23 Production and binding to IR for insulin-FC fusion proteins using FC FRAGMENTS OF FELINE IGG1B AND IGG2 SEQ ID NO: IgG Fragment Protein Yield (mg / L) % Homodimer Homodimer Titer (mg / L) IC50 of Binding to IR (nM) 108 IgG1b 127 48.6% 62 62 118 IgG1b 18 97.5% 18 >5000 114 IgG2 25 90.5% 23 3,480 116 IgG2 1 73.0% 1 707 Petition 870260049851, dated 05 / 25 / 2026, page 174 / 693 165 / 180

[0267] Unexpectedly, all three insulin-Fc fusion proteins gave much lower protein yields compared to the insulin-Fc fusion protein yield from SEQ ID NO: 108. In fact, although it had a sufficiently high insulin receptor binding affinity (IC50 of 707 nM), the insulin-Fc fusion protein from SEQ ID NO: 116 provided almost no protein production. The insulin-Fc fusion protein from SEQ ID NO: 118 achieved an unacceptably low protein yield and homodimer titer and was considered unlikely to be bioactive in vivo due to the high IC50 value of IR binding greater than 5000 nM. The protein from SEQ ID NO: 114 also achieved an unacceptably low protein yield and an even lower insulin receptor binding affinity (higher IC50 value) compared to the insulin-Fc fusion protein from SEQ ID NO: 108. Example 41 Insulin-Fc Fusion Protein Comprising Insulin Polypeptide of SEQ ID NO: 8, Ligand of SEQ ID NO: 14 and an Fc Fragment of Feline IgG2 Isotype

[0268] In an attempt to obtain an acceptable protein yield from an insulin-Fc fusion protein comprising an insulin polypeptide sequence without the immunogenic 'hot spot' mutations (i.e., B10D and A8H), learnings were obtained from the simultaneous and parallel development of canine insulin-Fc fusion proteins that showed that the use of an insulin polypeptide from SEQ ID NO: 8 and a peptide linker from SEQ ID NO: 14 in a canine IgGB isotype Fc fragment resulted in high protein titers and homodimers and acceptable IR binding affinity. Therefore, a feline insulin-Fc fusion protein was constructed using the insulin polypeptide from SEQ ID NO: 8 and the peptide linker from SEQ ID NO: 14 in Petition 870260049851, dated 05 / 25 / 2026, page 175 / 693 166 / 180 a fragment of cat IgG2 Fc from SEQ ID NO: 21 to produce the following sequence: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGGEGPKCPVPE IPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNT EMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLP SAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDI AVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGN TYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 122)

[0269] The alignments between the sequence with SEQ ID NO: 122 against the sequences with SEQ ID NOs: 106 and 112 from Example 37 are shown in Fig. 31 (Clustal Omega). Table 24 Production and binding to IR for insulin-FC fusion proteins using FC FRAGMENTS OF FELINE IGG1B AND IGG2 SEQ ID NO: IgG Fragment Protein Yield (mg / L) % Homodimer Homodimer Titer (mg / L) IC50 of Binding to IR (nM) 106 IgG2 23 88.0% 20 22 112 IgG2 64 80.4% 51 53 122 IgG2 146 99.0% 145 2,536

[0270] The insulin-Fc fusion proteins of SEQ ID NO: 122 were fabricated 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 under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The percentages (%) of homodimer content were measured by size exclusion chromatography according to Example 6, and their insulin receptor binding affinities were determined. Petition 870260049851, dated 05 / 25 / 2026, page 176 / 693 167 / 180 were measured according to Example 7. The FcRn receptor binding affinity was measured according to Example 9. The protein yield was 146 mg / L, and the % homodimer was determined to be 99%, resulting in a homodimer titer of 145 mg / L, which meets the manufacturing design objective. The IC50 value of the IR binding affinity was 2,536 nM, indicating that the compound is likely bioactive in vivo. The EC50 value of the FcRn receptor binding affinity was 3,114 ng / mL. Therefore, the insulin-Fc fusion protein of SEQ ID NO: 122 was a potential candidate for further in vivo testing. Example 42: In vivo bioactivity of an insulin-fc fusion protein constructed from insulin polypeptide (seq id no: 8), peptide ligand (seq id no: 14), and cat IgG2 fc fragment (seq id no: 21).

[0271] The insulin-Fc fusion protein of SEQ ID NO: 122 was tested for bioactivity in vivo according to Example 10. A healthy, antibody-naive 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 ID NO: 122. On day 0, blood was collected from a suitable vein immediately before injection and at 15, 30, 45, 60, 120, 240, 360, and 480 min and at 1, 2, 3, 4, 5, 6, and 7 days after injection. If the subject's blood glucose fell to levels considered dangerous, dextrose injections were administered and / or food was provided to prevent symptomatic hypoglycemia.

[0272] Fig. 32 shows the %FBGL for a single administration, showing that, unexpectedly, the insulin-Fc fusion protein of SEQ ID NO: 122 was only marginally bioactive in vivo (NAOC of essentially 0 %FBGL days / kg / mg). This result was surprising. Petition 870260049851, dated 05 / 25 / 2026, page 177 / 693 168 / 180 tooth, especially because the insulin-Fc fusion protein did not aggregate (i.e., it had a high % homodimer content), and the molecule exhibited an affinity for IR in a range similar to that of canine insulin-Fc fusion proteins that have been found to exhibit significant bioactivity in dogs (Example 31). Due to the lack of bioactivity on the first administration, repeated administrations were not performed. Example 43: Evaluation of the substitution of the feline IgG1 fc fragment by feline IgG2 on the yield, purity, bioactivity, and immunogenicity of an Insulin-Fc Fusion Protein comprising the Insulin polypeptide of SEQ ID NO: 8 and the Peptide Linker of SEQ ID NO: 14

[0273] As the long-acting insulin research programs in dogs and cats were conducted in parallel, some of the learnings from the canine insulin-Fc fusion protein research program were applied to the feline insulin-Fc fusion protein research program. A key learning from the canine insulin-Fc research program was how selecting Fc fragments from different IgG isotypes (e.g., canine IgGA, canine IgGB, canine IgGC, and canine IgGD isotypes) led to dramatically different manufacturing performance and in vivo efficacy. Therefore, the feline IgG2 Fc fragment from SEQ ID NO: 122 was replaced by the feline IgG1b Fc fragment from SEQ ID NO: 20, retaining the insulin polypeptide from SEQ ID NO: 8 and the peptide linker from SEQ ID NO: 14, resulting in the following amino acid sequences: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPPPEM LGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQ VYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPS PIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAV Petition 870260049851, dated 05 / 25 / 2026, page 178 / 693 169 / 180 EWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTY TCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 38)

[0274] The insulin-Fc fusion protein of SEQ ID NO: 38 was synthesized in HEK293 cells according to the procedure of Example 1 and purified using a Protein A column according to Example 3. The structure was confirmed according to Example 4 by LC-MS under reducing and non-reducing conditions, and the sequences were further identified by LC-MS with glycan removal according to Example 5. The protein yield was 158 mg / L at this stage. The % homodimer for the sequence was measured by size exclusion chromatography according to Example 6 and was determined to be 99.5%, resulting in a homodimer titer of 157 mg / L, which meets the project objective. The IC50 value of binding to the IM-9 insulin receptor in vitro, measured according to Example 7, was 2398 nM, which also meets the project objective. The EC50 value of the binding affinity to the FcRn receptor was measured according to Example 9, and was 1552 ng / mL.

[0275] The insulin-Fc fusion protein of SEQ ID NO: 38 was then tested for bioactivity in vivo according to Example 10. A healthy, antibody-naive cat weighing approximately 5 kg received a single subcutaneous injection of a pharmaceutical composition containing the insulin-Fc fusion protein of SEQ ID NO: 38 at a dose of 0.16 mg of insulin-Fc fusion protein / kg. On day 0, blood was collected from a suitable vein immediately before injection and at 15, 30, 45, 60, 120, 240, 360, and 480 min and at 1, 2, 3, 4, 5, 6, and 7 days after injection. If the subject's blood glucose fell to levels considered dangerous, dextrose injections were administered and / or food was provided to prevent symptomatic hypoglycemia.

[0276] Figure 33 shows the %FBGL after the first administration. The food was given to the animal regularly to avoid hypoglycemia. Petition 870260049851, dated 05 / 25 / 2026, page 179 / 693 170 / 180 symptomatic, illustrating that the insulin-Fc fusion protein from SEQ ID NO: 38 was significantly bioactive in vivo with a NAOC of 1.838 %FBGL days kg / mg. The pharmacokinetic profile of the compound was also measured by the method of Example 12 using ELISA, and a two-compartment model was fitted to the data to determine its elimination half-life, which was 6.3 ± 0.5 days. The difference in biological activity (in vitro and in vivo) between the insulin-Fc fusion protein from SEQ ID NO: 38 and that from SEQ ID NO: 122 demonstrates that, unexpectedly, the feline IgG1b isotype is preferred over the feline IgG2 isotype for the Fc fragment when the insulin polypeptide sequence is modified as in SEQ ID NO: 8.

[0277] Since NAOC was acceptable and pharmacokinetic data supported once-weekly administration, the cat received additional subcutaneous doses on day 14, day 28, and day 42, and %FBGL was measured for the 7-day window after each dose according to Example 11. NAOC and NAOCR were calculated according to the procedure in Example 11 for each repeated subcutaneous injection. As illustrated in Table 25, the insulin-Fc fusion protein of SEQ ID NO: 3 8 demonstrates acceptable bioactivity in vivo after multiple dose administration. TABLE 25 NAOC PER DOSE, FOR REPEATED DOSES OF SEQ ID NO: 38 Injection No. Day NAOC %FBGL^days^g / mg) NAOCR 1 0 1838 1.0 2 14 1431 0.8 3 28 1900 1.0 4 42 2400 1.3

[0278] In addition, serum was collected before administration of each dose and once a week for two weeks after the end of the experiment in order to test for the presence and quantify the levels of Petition 870260049851, dated 05 / 25 / 2026, page 180 / 693 171 / 180 any antidrug antibodies according to Example 14. As shown in Fig. 34, there was no measurable increase in antidrug antibodies above baseline values ​​after multiple administrations of the compound. Therefore, to obtain a candidate feline insulin-Fc fusion protein (e.g., from SEQ ID NO: 38) that met acceptable homodimer titer, in vivo bioactivity, and sustained bioactivity criteria after repeated weekly injections in cats, it was necessary to replace the insulin polypeptide from SEQ ID NO: 4 with the insulin polypeptide from SEQ ID NO: 8 and use the feline IgG1b Fc fragment from SEQ ID NO: 20 instead of the feline IgG2 Fc fragment from SEQ ID NO: 21. Example 44 Non-glycosylated Insulin-Fc Fusion Proteins Comprising the Insulin Polypeptide of SEQ ID NO: 8, the Peptide Linker of SEQ ID NO: 14, and the Feline IgGIb Fc Fragment to Reduce the Potential Risk of Immunogenicity

[0279] Although the insulin-Fc fusion protein of SEQ ID NO: 38 satisfies all the objectives of the study design (Example 43), there may or may not be a risk of immunogenicity over prolonged treatment periods (e.g., 6 months, 1 year, 2 years or more), which could compromise the use of this insulin-Fc fusion protein for the treatment of diabetes, should this occur. As described in the Detailed Description of the Invention, a possible cause of reduced bioactivity after repeated doses is the undesirable interaction of the feline IgG1b Fc fragment with the cat's immune system, resulting in the production of neutralizing antidrug antibodies. However, the results shown in Example 43 unexpectedly demonstrate that the feline IgG1b isotype was preferable to the less immunogenic feline IgG2 isotype with respect to in vivo bioactivity. Therefore, other Fc mutations were explored to obtain insulin-Fc fusion proteins. Petition 870260049851, dated 05 / 25 / 2026, page 181 / 693 172 / 180 non-glycosylated proteins with low binding to the Fc(gamma)RI receptor, which should reduce the risk of long-term chronic immunogenicity.

[0280] As described in the Detailed Description of the Invention, a method for reducing interaction with Fc(gamma)RI involves mutating the cNg site of the Fc fragment to prevent glycosylation during synthesis in the host cell. Therefore, mutations were made to the cNg site in the Fc fragment region of SEQ ID NO: 38 to reduce the binding affinity of the Fc fragment to Fc(gamma) receptors in vivo, as measured by an in vivo human Fc(gamma)RI binding assay described in Example 8. The position of the cNg site on the insulin fusion protein Fc of SEQ ID NO: 38 is cNg-NB151. Again, capitalizing on the learnings from the canine insulin-Fc fusion proteins described in Example 33, a cNg-NB151-S mutation was introduced into the Fc fragment of SEQ ID NO: 38. The complete amino acid sequence of the resulting insulin-Fc fusion protein is listed below (cNg-NB151-S underlined for clarity): FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGDCPKCPPPEM LGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQ VYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPS PIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAV EWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTY TCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 124)

[0281] The insulin-Fc fusion protein of SEQ ID NO: 124 was synthesized in HEK293 cells according to the procedure of Example 1 and purified using a Protein A column according to Example 3. The structure of the insulin-Fc fusion protein was confirmed according to Example 4 by LC-MS under reducing and non-reducing conditions, and the sequence was further identified by LC-MS with glycan removal according to Example 5. The yield of Petition 870260049851, dated 05 / 25 / 2026, page 182 / 693 The 173 / 180 protein concentration was 202 mg / L at this stage. The percentage of homodimer for the sequence was measured by size exclusion chromatography according to Example 6 and was determined to be 99%, resulting in a homodimer titer of 200 mg / L, which meets the project objective. However, the IC50 value of in vitro binding to the IM-9 insulin receptor, measured according to Example 7, was greater than 5000 nM, which is outside the study objective for in vitro bioactivity. The EC50 value of binding affinity to the FcRn receptor was measured according to Example 9 and was 6,922 ng / mL.

[0282] Although the insulin-Fc fusion protein from SEQ ID NO: 124 did not meet the objectives of binding to the insulin receptor, it was tested for bioactivity in vivo according to Example 10. A healthy, antibody-naive 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 from SEQ ID NO: 124 at a dose of 0.16 mg of insulin-Fc fusion protein / kg. On day 0, blood was collected from a suitable vein immediately before injection and at 15, 30, 45, 60, 120, 240, 360, and 480 min and at 1, 2, 3, 4, 5, 6, and 7 days after injection. If the subject's blood glucose fell to levels considered dangerous, dextrose injections were administered and / or food was provided to prevent symptomatic hypoglycemia.

[0283] Fig. 35 shows the %FBGL for a single administration, illustrating that the insulin-Fc fusion protein of SEQ ID NO: 124 was poorly bioactive in vivo, with a NAOC of 65 %FBGL days kg / mg. Due to the lack of bioactivity in the first administration, repeated administrations were not performed.

[0284] Unexpectedly, as was the case in Example 33 for the canine insulin Fc fusion protein of SEQ ID NO: 36, a mutation was found in the insulin polypeptide sequence of SEQ ID NO: 124 such that the 16th N-terminal amino acid of the B chain (B16) was mutated from Petition 870260049851, dated 05 / 25 / 2026, p. 183 / 693 The 174 / 180 tyrosine to alanine mutation (i.e., B16A) rendered the resulting insulin-Fc fusion protein bioactive, with SEQ ID NO: 40. The amino acid sequence of the resulting insulin-Fc fusion protein is shown below (B16A and cNg-NB151-S mutations underlined for clarity): FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEM LGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQ VYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPS PIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAV EWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTY TCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 40)

[0285] The insulin-Fc fusion protein of SEQ ID NO: 40 was synthesized in HEK293 cells according to the procedure of Example 1 and purified using a Protein A column according to Example 3. The structure of the insulin-Fc fusion protein was confirmed according to Example 4 by CE-SDS under reducing and non-reducing conditions, and the sequence was further identified by LC-MS with glycan removal according to Example 5. The protein yield was 174 mg / L at this stage. The % homodimer for the sequence was measured by size exclusion chromatography according to Example 6 and was determined to be 98.9%, resulting in a homodimer titer of 172 mg / L which meets the design criterion. The IC50 value of binding to the IM-9 insulin receptor in vitro, measured according to Example 7, also meets the study objective.The activity on the Fc(gamma) receptor was measured according to Example 8 and found to be approximately four times lower than the activity obtained for the insulin-Fc fusion protein of SEQ ID NO: 38 using the same procedure, thus indicating that the insulin-Fc fusion protein is less likely to interact adversely with the cat's immune system. The EC50 value of affini. Petition 870260049851, dated 05 / 25 / 2026, page 184 / 693 175 / 180 The FcRn receptor binding capacity was measured according to Example 9 and was 8.157 ng / mL.

[0286] The insulin-Fc fusion protein of SEQ ID NO: 40 was tested for bioactivity in vivo according to Example 11. A healthy, antibody-naive 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 ID 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 injection and at 15, 30, 45, 60, 120, 240, 360, and 480 min and at 1, 2, 3, 4, 5, 6, and 7 days after injection. If the subject's blood glucose fell to levels considered dangerous, dextrose injections were administered and / or food was provided to prevent symptomatic hypoglycemia.

[0287] Fig. 36 shows the %FBGL after the first administration, illustrating that the insulin-Fc fusion protein of SEQ ID NO: 40 is bioactive in vivo with a NAOC of 159 %FBGL days kg / mg for a subcutaneous dose of 0.1 mg of insulin-Fc fusion protein / kg. A second, higher subcutaneous dose of 0.2 mg of insulin-Fc fusion protein / kg gave a much higher NAOC of 702 %FBGL days kg / mg, shown in Fig. 37. The pharmacokinetic profile is measured by the method of Example 12 using ELISA, and a two-compartment model is fitted to the data to determine its elimination half-life, which is greater than 3 days. These results contrast with those obtained with the insulin-Fc fusion protein from SEQ ID NO: 124, which showed that the same compound comprising a tyrosine at B16 instead of an alanine was only weakly bioactive at approximately the same dose (0.16 mg of insulin-Fc fusion protein / kg). Therefore, the insulin polypeptide from SEQ ID NO: 11 was preferred for Petition 870260049851, dated 05 / 25 / 2026, page 185 / 693 176 / 180 non-glycosylated insulin-Fc fusion proteins comprising Fc fragments of feline IgG1b with a mutated cNg site.

[0288] To analyze reproducible bioactivity after multiple dose administration, the cat received an additional dose of the insulin-Fc fusion protein from SEQ ID NO: 40 on day 7, day 21, and day 35. When the cat's %FBGL dropped too low, the cat was fed food 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 illustrate that the insulin-Fc fusion protein from SEQ ID NO: 40 is bioactive in vivo after multiple dose administration. TABLE 26 NAOC PER DOSE FOR REPEATED DOSES OF SEQ ID NO: 40 Injection No. Day NAOC (%FBGL^days^g / mg) NAOCR 1 0 159 1.0 2 7 702 4.4 3 21 462 2.9 4 35 670 4.2

[0289] In addition, serum was collected before administration of each dose and at the end of the experiment in order to test for the presence and quantify the levels of any antidrug antibodies according to Example 14. There is no measurable increase in antidrug antibodies above baseline values ​​after multiple administrations of the compound. Therefore, in order to obtain a feline insulin-Fc fusion protein that met the manufacturing design criteria and bioactivity with significantly reduced activity on the Fc(gamma) receptor, it was necessary not only to mutate cNg to serine, but also to mutate amino acid B16 to alanine in the insulin polypeptide. Petition 870260049851, dated 05 / 25 / 2026, page 186 / 693 177 / 180 Example 45 Examples of CHO Cell-Based Production Runs Using Preferred Insulin-Fc Fusion Proteins Comprising Feline IgG1b Fc Fragments Produced in Stably Transfected CHO Cell Lines

[0290] A stably transfected CHO cell line with vectors encoding SEQ ID NO: 38 was constructed as described in Example 2 above. 14-day production cultures in fed-batch flasks (medium scale 0.5-2.0 L) were seeded at 0.5 million cells / mL in a shaker incubator set at 37°C and 5% carbon dioxide, and the cultures were conducted as described in Example 2, except that CD OptiCHO medium was replaced with Dynamis as growth medium (ThermoFisher) and Efficient Feed C (ThermoFisher) was used as a supplement. Supplementation was added at 3% v / v starting on day 3 of production, and on day 4, the temperature of the shaker flask was adjusted to 32°C and the carbon dioxide concentration of the shaker incubator was reduced from 5% to 2%.During the run, the cell density increased to between 8-14 million cells / mL, and on Day 14 the production was harvested to remove the cells, and the culture supernatant was purified and characterized to obtain the insulin-Fc fusion protein as described in Examples 3, 4, 5, and 6. Table 27 describes the manufacturing data for the insulin-Fc fusion protein obtained through these production cultures with stably transfected CHO cell lines. Table 27 HOMODIMER TITRATES FOR NON-GLYCOSYLATED INSULIN-FC FUSION PROTEINS SEQ ID NO: 38 SEQ ID NO: Protein yield (mg / L) % homodimer Homodimer titer (mg / L) SEQ ID NO: 38 633 96.3% 610 Petition 870260049851, dated 05 / 25 / 2026, page 187 / 693 178 / 180 EXAMPLE 46 EXAMPLES OF CHO CELL-BASED PRODUCTION EXECUTIONS USING FELINE IGGIB-PREFERRED INSULIN-FC FUSION PROTEINS PRODUCED IN STABLY TRANSFECTED CHO CELL LINES

[0291] A stably transfected CHO cell line with vectors encoding SEQ ID NO: 40 is constructed as described in Example 2 above. 14-day production cultures in fed-batch flasks (medium scale 0.5-2.0 L) are seeded at 0.5 million cells / mL in a shaker incubator set at 37°C and 5% carbon dioxide, and the cultures are conducted as described in Example 2 above, except that CD OptiCHO medium is replaced with Dynamis as growth medium (ThermoFisher) and Efficient Feed C (ThermoFisher) is used as a supplement. Supplementation is added at 3% v / v starting on day 3 of production, and on day 4, the temperature of the shaker flask is adjusted to 32°C and the carbon dioxide concentration of the shaker incubator is reduced from 5% to 2%.On Day 14, the production run is collected to remove the cells, and the culture supernatant is purified and characterized to obtain the insulin-Fc fusion protein as described in Examples 3, 4, 5, and 6. The resulting production of SEQ ID NO: 40 give...

Claims

CLAIMS 1. Fusion protein, characterized in that it comprises an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are linked by a ligand, such as a peptide ligand, wherein the Fc fragment comprises the following sequence: DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSD VQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEF KCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVT CLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSV DRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 23); and wherein the insulin peptide comprises a B chain and an A chain, wherein said insulin polypeptide comprises an alanine substitution at B16.

2. Fusion protein, according to claim 1, characterized in that chain B and chain A are connected to a chain C, comprising the following sequence: GGGGGGSGGGG (SEQ ID NO: 133).

3. Fusion protein, according to claim 1 or 2, characterized in that the insulin polypeptide comprises the following sequence: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX 2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is not D and X2 is not H.

4. Fusion protein, according to claim 1 or 2, characterized in that the insulin polypeptide comprises the following sequence: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is H, and X2 is T.

5. Fusion protein, according to any one of claims 1 to 4, characterized in that the insulin polypeptide and the Fc fragment are linked by a linker, such as a peptide linker, comprising the following sequence: GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

6. Fusion protein, according to claim 1, characterized in that the insulin polypeptide comprises the following sequence: FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEM LGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQ VYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPS PIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAV EWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTY TCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 40).

7. Fusion protein, according to any one of claims 1 to 6, characterized in that it is a homodimer.

8. Fusion protein, according to claim 7, characterized in that the homodimer percentage of the fusion protein is greater than 90%.

9. Fusion protein, according to any one of claims 1 to 6, characterized in that the fusion protein is made using HEK293 cells, and the resulting homodimer titer after purification using protein A beads or a protein A column is greater than 50 mg / L.

10. Pharmaceutical composition, characterized in that it comprises the fusion protein, as defined in any one of claims 1 to 9, dispersed in a vehicle.

11. Pharmaceutical composition, according to claim 10, characterized in that the fusion protein is present in the pharmaceutical composition at a concentration of approximately 3 mg / mL or higher of the fusion protein in said vehicle. Petition 870260049851, dated 05 / 25 / 2026, p. 192 / 693 3 / 6 12. Use of the fusion protein, as defined in any one of claims 1 to 9, or of a pharmaceutical composition thereof, characterized in that it is for the preparation of a medicament to reduce the blood glucose level of a target animal, wherein the target animal is a cat.

13. Use according to claim 12, characterized in that the target animal is diagnosed with diabetes.

14. Use according to claim 12 or 13, characterized in that the fusion protein is administered subcutaneously.

15. Use according to claim 14, characterized in that the fusion protein is administered daily, twice weekly, or once weekly to the target animal.

16. Use, according to claim 15, characterized in that the fusion protein is administered once a week to the target animal, at a dose between 0.025 and 0.5 mg / kg / week.

17. A cell, characterized in that it is manipulated to express a fusion protein, as defined in any one of claims 1 to 9.

18. A cell according to claim 17, characterized in that it is transfected with a nucleic acid encoding the fusion protein.

19. Cell according to claim 18, characterized in that it is either a HEK293 cell or a CHO cell.

20. cDNA, characterized in that it encodes a fusion protein, as defined in any one of claims 1 to 9.

21. cDNA, according to claim 20, characterized in that it comprises the following nucleic acid sequence: atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccag cacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttcccta Petition 870260049851, dated 05 / 25 / 2026, p.193 / 693 4 / 6 cgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccac ctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggaca gggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgc tgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggaccc ccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttc gtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcagcagcac atacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaag tgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagg gccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaa caaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaa atcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcg acgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaaca cctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgaccc agagcccgggatag (SEQ ID NO: 39).

22. Fusion protein, according to any one of claims 1 to 9, characterized in that the fusion protein has domains in the N- to C-terminal orientation: (N-terminal)—insulin polypeptide—ligand—Fc fragment—(C-terminal).

23. Recombinant cell, characterized in that it comprises a nucleic acid encoding a fusion protein, wherein said fusion protein comprises 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 comprises the following sequence: DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSD VQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEF KCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVT CLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSV DRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 23); Petition 870260049851, dated 05 / 25 / 2026, page 194 / 693 5 / 6 in which the insulin peptide comprises a B chain and an A chain, and in which said insulin polypeptide comprises an alanine substitution at B16.

24. Recombinant cell, according to claim 23, characterized in that the insulin polypeptide comprises the following sequence: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is not D, and X2 is not H.

25. Recombinant cell, according to claim 23, characterized in that the insulin polypeptide comprises the following sequence: FVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCC X2STCSLDQLENYC (SEQ ID NO: 10), wherein X1 is H, and X2 is T.

26. Recombinant cell, according to claim 23, characterized in that the nucleic acid comprises a cDNA encoding said fusion protein, wherein said fusion protein comprises the following sequence: FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGGDCPKCPPPEM LGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQ VYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPS PIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAV EWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTY TCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 40).

27. Recombinant cell, according to claim 23, characterized in that the cDNA comprises the following nucleic acid sequence: atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccag cacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttcccca Petition 870260049851, dated 05 / 25 / 2026, p.195 / 693 6 / 6 cgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccac ctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggaca gggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgc tgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggaccc ccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttc gtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcagcagcac atacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaag tgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagg gccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaa caaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaa atcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcg acgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaaca cctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgaccc agagcccgggatag (SEQ ID NO: 39).

28. Recombinant cell, according to any one of claims 23 to 27, characterized in that the cell can be either HEK293 cells or CHO cells.

29. Recombinant cell, according to any one of claims 23 to 27, characterized in that it is a stably transfected recombinant cell.

30. A method for preparing a recombinant cell, as defined in any one of claims 23 to 29, characterized in that it comprises: transfecting a host cell with a nucleic acid encoding said fusion protein, wherein said fusion protein is expressed in said recombinant cell after said transfection step.

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