Insulin-Fc fusion proteins and methods of use for treating cancer

Abstract

The present disclosure relates to fusion proteins, such as insulin-Fc fusion proteins, compositions thereof, and their use for treating cancer cells and cancer tumors in subjects in need thereof. After administration, the fusion proteins exhibit anti-tumor effects on the subject's cancer cells, such as downregulation of insulin receptor, downregulation of insulin-like growth factor 1 receptor (IGF1R), reduction of phosphorylated Akt, and combinations thereof. The present disclosure relates to methods for downregulating insulin-like growth factor 1 receptor (IGF1R) in subjects in need thereof, and methods for inhibiting the metabolism, growth, and / or proliferation of cancer cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 477,576, filed December 29, 2022, entitled INSULIN-Fc FUSION PROTEINS AND METHODS OF USE FOR TREATING CANCER, which is incorporated by reference in its entirety.

[0002] Sequence Listing The following application contains a sequence listing that was filed electronically as an XML file conforming to Standard ST.26 entitled "SequenceListing_ABC-048.xml," created on December 12, 2023, with a size of 60,412 bytes, the contents of which are incorporated herein by reference:

[0003] Technical Field The present disclosure relates to compositions of insulin-Fc fusion proteins and their use for treating cancer and cancer tumors. [Background technology]

[0004] background The following description of the background of the present technology is provided merely as an aid to understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] In 2020, 18 million cancer patients are predicted, resulting in annual costs exceeding $170 billion and approximately 600,000 deaths per year. While surgery remains the best available treatment, its coverage is often limited to the localized primary tumor and associated lymph nodes. Radiation therapy and chemotherapy, among other broad approaches, have long been used in combination with surgery to improve disease control or treat metastatic disease and are effective against many cancer types. However, both approaches are unable to distinguish between rapidly dividing normal and cancer cells, often resulting in side effects that often limit doses to below effective levels, and subsets of tumor cells are either intrinsically resistant or can acquire resistance to these treatments through various mechanisms. These limitations have prompted a shift in focus toward selectively targeting the genetic and biochemical drivers of disease. Summary of the Invention

[0006] Overview of this technology In one embodiment, the present technology provides a fusion protein comprising an insulin polypeptide and an Fc fragment having the following sequence: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 18) Disclosed is a fusion protein comprising:

[0007] In one embodiment, the present technology provides a fusion protein comprising an insulin polypeptide and an Fc fragment having the following sequence: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 20) Disclosed is a fusion protein comprising:

[0008] In embodiments, the fusion protein comprises an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide has the following sequence: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 6) wherein the fusion protein has a maximum insulin receptor binding IC50 ratio relative to recombinant human insulin (RHI) of 50 or less, 40 or less, 30 or less, or more preferably 20 or less.

[0009] In some embodiments, the Fc fragment of the fusion protein has the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32) The compound comprises:

[0010] In some embodiments, the Fc fragment of the fusion protein has the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 33) The compound comprises:

[0011] In some embodiments, the insulin polypeptide and the Fc fragment are linked by a linker, the linker having the following sequence: GGGGAGGGG (SEQ ID NO: 11). The compound comprises:

[0012] In some embodiments, the maximum insulin receptor binding IC of the fusion protein to recombinant human insulin (RHI) 50 The ratio is 50 or less, 40 or less, 30 or less, or more preferably 20 or less.

[0013] I C 50 The lower ratio embodiment is IC 50 Higher affinity for the insulin receptor than embodiments with higher ratios. Maximum insulin receptor binding IC for recombinant human insulin (RHI) 50Insulin-Fc fusion proteins that are dimeric with respect to the insulin polypeptide (e.g., two moles of insulin polypeptide chains per mole of insulin-Fc homodimer, e.g., two moles of insulin analog B chain and two moles of insulin analog A chain per mole of insulin-Fc homodimer) at a ratio of 50 or less, 40 or less, 30 or less, or more preferably 20 or less, exhibit greater insulin receptor downregulation (IR) than recombinant human insulin (RHI) (e.g., one mole of insulin polypeptide per mole of RHI, e.g., one mole of insulin B chain and one mole of insulin A chain per mole of RHI). Without being bound by any theory, it is believed that an insulin-Fc fusion protein that is (i) dimeric with respect to the insulin polypeptide (e.g., two insulin polypeptide chains per mole of insulin-Fc homodimer), and (ii) has the greatest insulin receptor binding IC for recombinant human insulin (RHI) is more likely to exhibit IR than an insulin-Fc fusion protein that is dimeric with respect to the insulin polypeptide (e.g., two insulin polypeptide chains per mole of insulin-Fc homodimer), and (ii) has the greatest insulin receptor binding IC for recombinant human insulin (RHI). 50 Insulin-Fc fusion proteins with a ratio of 50 or less, 40 or less, 30 or less, or more preferably 20 or less are hypothesized to exhibit the ability to down-regulate the insulin receptor (as shown in Figure 4). Down-regulation of the insulin receptor is a key aspect of the efficacy of preferred fusion proteins of the present technology. In embodiments, the insulin-Fc fusion proteins of the present technology exhibit reduced Fc(γ) receptor binding and reduced C1q binding, and do not generate anti-drug antibodies even with repeated use, making them a long-term treatment option for certain cancers. In particular, the insulin-Fc fusion proteins of the present technology are effective in reducing the size of tumors that overexpress the IGF1 receptor (IFG1R), a transmembrane receptor found on the cell surface. In embodiments, the insulin-Fc fusion proteins of the present technology can be used in combination with other anti-IGF1R therapies that may develop resistance through alternative signaling pathways.

[0014] In embodiments, the nucleic acid (cDNA) encoding the fusion protein of SEQ ID NO: 18 comprises the following nucleic acid sequence: ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCGACCTGGTGGAAGCTCTGGCTCTCGTGTGCGGCGAGCGGGGCTTCTTCTACACCGATCCCACTGGAGGCGGTCCACGCAGAGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCTGTACCAGCTGGAAAACTACTGCAATGGCGGAGGTGGTGCAGGAGGCGGTGGAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAGCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG(SEQ ID NO: 19)

[0015] In embodiments, the nucleic acid (cDNA) encoding the fusion protein of SEQ ID NO: 20 has the following nucleic acid sequence: ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCGACCTGGTGGAAGCTCTGGCTCTCGTGTGCGGCGAGCGGGGCTTCTTCTACACCGATCCCACTGGAGGCGGTCCACGCAGAGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCTGTACCAGCTGGAAAACTACTGCAATGGCGGAGGTGGTGCAGGAGGCGGTGGAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG(SEQ ID NO: 21) comprising

[0016] In some embodiments, cells can be engineered to express the fusion protein. The cells can be transfected with a nucleic acid encoding the fusion protein. In some examples, the cells are HEK293 cells or CHO cells.

[0017] In some embodiments, the fusion protein comprises a dimer of two identical monomers linked via a disulfide bond, eg, the fusion protein is a homodimer.

[0018] In some embodiments, the fusion protein comprises domains in the following orientation from N-terminus to C-terminus: (N-terminus)--insulin polypeptide--linker--Fc fragment--(C-terminus), and the insulin polypeptide comprises domains in the following orientation from N-terminus to C-terminus: (N-terminus)--B chain--C peptide--A chain-(C-terminus).

[0019] In one embodiment, the present technology discloses a pharmaceutical composition for inhibiting the metabolism, growth, and / or proliferation of cancer cells. In several embodiments, a pharmaceutical composition comprising an insulin-Fc fusion protein of the present technology exhibits reduced Fc(γ) receptor binding and reduced C1q binding, and does not generate anti-drug antibodies even with repeated use, making it a long-term treatment option for cancer tumors. In particular, a pharmaceutical composition comprising an insulin-Fc fusion protein of the present technology is effective in reducing the size of cancer tumors that overexpress IGF1R. In several embodiments, a pharmaceutical composition of an insulin-Fc fusion protein of the present technology can be used in combination with other anti-IGF1R therapies that may develop resistance through alternative signaling pathways.

[0020] In embodiments, the pharmaceutical composition comprises a fusion protein, e.g., an insulin-Fc fusion protein, dispersed in a pharmaceutically acceptable carrier (e.g., a buffer, e.g., a sodium phosphate buffer, e.g., a sodium phosphate and sodium chloride solution, e.g., a buffer solution optionally containing an additive, e.g., the additive is polysorbate-20 or polysorbate-80), the fusion protein comprising an insulin polypeptide and an Fc fragment linked by a linker, and having a maximum insulin receptor binding IC for recombinant human insulin (RHI). 50 The ratio is 50 or less, 40 or less, 30 or less, or more preferably 20 or less. In embodiments, the pharmaceutical composition comprises: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 6) The present invention relates to a fusion protein comprising an insulin polypeptide of the present invention.

[0021] In some embodiments, the pharmaceutical composition comprises a fusion protein, wherein the insulin polypeptide and the Fc fragment of the fusion protein are linked by a linker comprising the sequence GGGGAGGGG (SEQ ID NO: 11).

[0022] In embodiments, the pharmaceutical composition comprises a fusion protein dispersed in a pharmaceutically acceptable carrier, wherein the Fc fragment of the fusion protein has the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32) The compound comprises:

[0023] In embodiments, the pharmaceutical composition comprises a fusion protein dispersed in a pharmaceutically acceptable carrier, wherein the Fc fragment of the fusion protein has the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 33) The compound comprises:

[0024] In one embodiment, the present technology discloses a pharmaceutical composition for inhibiting the metabolism, growth, and / or proliferation of cancer cells, the pharmaceutical composition comprising a fusion protein dispersed in a pharmaceutically acceptable carrier, the fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are linked by a linker, and the fusion protein has the following sequence: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 18) The compound comprises:

[0025] In one embodiment, the present technology discloses a pharmaceutical composition for inhibiting the metabolism, growth, and / or proliferation of cancer cells, the pharmaceutical composition comprising a fusion protein dispersed in a pharmaceutically acceptable carrier, the fusion protein comprising an insulin polypeptide and an Fc fragment, wherein the insulin polypeptide and the Fc fragment are linked by a linker, and the fusion protein has the following sequence: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 20) The compound comprises:

[0026] In embodiments, the present technology discloses a pharmaceutical composition for use in treating cancer, the pharmaceutical composition comprising a fusion protein dispersed in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition exhibits an anti-tumor effect on cancer cells, the anti-tumor effect being selected from the group consisting of down-regulation of insulin receptor, down-regulation of insulin-like growth factor 1 receptor (IGF1R), reduction in phosphorylated Akt, or a combination thereof, compared to untreated control cancer cells.

[0027] In some embodiments, the present technology discloses a method for inhibiting the metabolism, growth, and / or proliferation of cancer cells or the growth of cancer tumors, comprising administering to a subject in need thereof an effective amount of a fusion protein or a pharmaceutical composition comprising an effective amount of the fusion protein dispersed in a pharmaceutically acceptable carrier. That is, the fusion protein inhibits the metabolism, growth, and / or proliferation of cancer cells or the growth of cancer tumors in the subject after administration of the fusion protein. In some embodiments, the fusion protein or a pharmaceutical composition comprising an effective amount of the fusion protein dispersed in a pharmaceutically acceptable carrier is administered to the subject under fasting conditions. In some embodiments, the subject has been diagnosed with a cancer selected from the group consisting of breast cancer, colorectal cancer, and melanoma.

[0028] In some instances, the fusion proteins of the present technology are effective in downregulating insulin-like growth factor 1 receptor (IGF1R) in a subject in need thereof, wherein IGF1R is downregulated in the subject following said administration, and the subject exhibits a reduction in tumor volume compared to an untreated control tumor.

[0029] In several instances, gene amplification of the insulin growth factor 1 receptor (IGF1R), which encodes the insulin-like growth factor 1 receptor, is a frequent phenomenon in many cancer types. The IGF axis, for which IGF1R is a surface cell receptor, is involved in cancer development, metastasis, and cancer treatment resistance. Among the molecules in the IGF axis, IGF1R is recognized as a promising target for cancer therapy due to its role as the primary receptor for insulin growth factor, as well as its frequent amplification and overexpression in various cancers and its established function in transmitting oncogenic signals.

[0030] Overexpression of IGF1R has been reported in several human cancer lines and is associated with poor prognosis in cancer patients. For example, in prostate cancer, IGF1R protein levels have been shown to be significantly higher in malignant epithelium than in benign prostate biopsies. In several cases of head and neck squamous cell carcinoma, patients with elevated IGF1R levels have been shown to have significantly lower overall and disease-specific survival.Multiple studies have demonstrated the expression of activated IGF1R in at least 50% of breast cancer cases (Yuan J, Yin Z, Tao K, Wang G, Gao J. Function of insulin-like growth factor 1 receptor in cancer resistance to chemotherapy. Oncol Lett. 2018 Jan;15(1):41-47. doi: 10.3892 / ol.2017.7276. Epub 2017 Oct 26. PMID: 29285186; PMCID: PMC5738696.; Alfaro-Arnedo, E., Lopez, IP, Pineiro-Hermida, S. et al. IGF1R acts as a cancer-promoting factor in the tumor microenvironment facilitating lung metastasis implantation and progression. Oncogene 41, 3625-3639 (2022).; Amutha P, Rajkumar T. Role of Insulin-like Growth Factor, Insulin-like Growth Factor Receptors, and Insulin-like Growth Factor-binding Proteins in Ovarian Cancer. Indian J Med Paediatr Oncol. 2017 Apr-Jun;38(2):198-206.; Sun Y, Sun X, Shen B. Molecular Imaging of IGF-1R in Cancer. Mol Imaging. 2017 Jan-Dec;16:1536012117736648. doi: 10.1177 / 1536012117736648. PMID: 29169312; PMCID: PMC5703088).

[0031] Examples of the most common cancers associated with IG1FR overexpression include uterine endometrial carcinoma, gastric adenocarcinoma, cutaneous melanoma, sarcoma, invasive breast cancer, ovarian serous cystadenocarcinoma, ovarian serous cystadenocarcinoma, adrenocortical carcinoma, esophageal adenocarcinoma, hepatocellular carcinoma, lung squamous cell carcinoma, bladder urothelial carcinoma, cervical squamous cell carcinoma, colorectal adenocarcinoma, lung adenocarcinoma, and pancreatic adenocarcinoma (Panpan Wang, Victor CY. Mak, Lydia WT. Cheung, Drugging IGF-1R in cancer: New insights and emerging opportunities, Genes & Diseases, 2022, ISSN 2352-3042). The insulin-Fc fusion protein of the present technology is expected to reduce tumor volume in cancer types that overexpress IG1FR.

[0032] In some embodiments, the subject may exhibit at least a 40% reduction in tumor volume after administration of the insulin-Fc fusion protein compared to fasted, untreated controls. In some embodiments, the subject may exhibit at least a 30% reduction in tumor volume after administration of the insulin-Fc fusion protein compared to fasted, untreated controls. In several embodiments, the first or second line cancer therapy is selected from the group consisting of a chemotherapeutic agent, a tamoxifen agonist, or an antibody against IGF1R.

[0033] In embodiments, the present technology discloses a method for inhibiting the metabolism, growth, and / or proliferation of cancer cells or cancer tumor growth, comprising administering an effective amount of a fusion protein or a pharmaceutical composition comprising the fusion protein dispersed in a pharmaceutically acceptable carrier, wherein the method exhibits an anti-tumor effect on the cancer cells, wherein the anti-tumor effect is selected from the group consisting of down-regulation of insulin receptor, down-regulation of insulin-like growth factor 1 receptor (IGF1R), reduction in phosphorylated Akt, or a combination thereof, compared to untreated control cancer cells.

[0034] In embodiments, the pharmaceutical composition comprising the fusion protein or the fusion protein dispersed in a pharmaceutically acceptable carrier is administered by intravenous injection, subcutaneous injection, or intratumoral injection. In embodiments, the pharmaceutical composition comprising the fusion protein or the fusion protein dispersed in a pharmaceutically acceptable carrier is administered as a bolus, infusion, or intravenous push. In embodiments, the pharmaceutical composition comprising the fusion protein or the fusion protein dispersed in a pharmaceutically acceptable carrier is administered via syringe or using a pump, pen, needle, or indwelling catheter.

[0035] The present invention is also directed to the use of the fusion protein of the present invention for the manufacture of a medicament for the treatment of cancer, preferably for the inhibition of cancer cell metabolism, growth, and / or proliferation.

[0036] Furthermore, the fusion protein can exhibit an anti-tumor effect on cancer cells in the subject after administration, wherein the anti-tumor effect is selected from the group consisting of down-regulation of insulin receptor, down-regulation of insulin-like growth factor 1 receptor (IGF1R), reduction in phosphorylated Akt, and combinations thereof, compared to untreated control cancer cells.

[0037] Ideally, the fusion protein is intended for use via intravenous injection, subcutaneous injection, or intratumoral injection, and / or can be administered as a bolus, drip, or intravenous push.Preferably, the fusion protein can be administered via a syringe, pump, pen, needle, or indwelling catheter.Furthermore, the fusion protein can be administered in combination with a first- or second-line cancer therapy selected from the group consisting of a chemotherapeutic agent, a tamoxifen agonist, or an antibody against IGF1R.

[0038] According to a preferred embodiment, the fusion protein can be administered to the subject at a dose of about 150 to about 1,500 micrograms per kilogram of body weight per day. Furthermore, the fusion protein can be administered to the subject under fasting or non-fasting conditions.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including the "Definitions," will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will become apparent from the following detailed description, examples, and claims. [Brief explanation of the drawings]

[0040] [Figure 1] FIG. 1 shows a schematic diagram of an example of an insulin-Fc fusion protein homodimer. [Figure 2] Figure 2 shows the "full amino acid sequence" of the fusion protein (SEQ ID NO: 18) containing the leader sequence of SEQ ID NO: 14, and the cDNA sequences of SEQ ID NO: 15 and SEQ ID NO: 19 correspond to the amino acid sequences of SEQ ID NO: 14 and SEQ ID NO: 18, respectively. [Figure 3] Figure 3 shows the "full amino acid sequence" of the fusion protein (SEQ ID NO: 20) containing the leader sequence of SEQ ID NO: 14, and the cDNA sequences of SEQ ID NO: 15 and SEQ ID NO: 21 correspond to the amino acid sequences of SEQ ID NO: 14 and SEQ ID NO: 20, respectively. [Figure 4] FIG. 4 shows Western blot images of HCT-116-bearing nude mice treated in vitro with medium alone, RHI, SEQ ID NO: 1, or SEQ ID NO: 47 at concentrations ranging from 0.05 to 500 nM. [Figure 5] FIG. 5 shows % fasting blood glucose for SEQ ID NO: 1 and porcine insulin NPH vehicle. [Figure 6] FIG. 6 shows the tumor volume ratio in HCT-116-bearing nude mice for SEQ ID NO:1. [Figure 7] FIG. 7 shows the tumor volume ratio in WM266.4-bearing nude mice for SEQ ID NO:1. [Figure 8] Figure 8 shows a side-by-side comparison of SEQ ID NO: 1 and SEQ ID NO: 47. A "*" represents a perfect homology in all sequences at a given sequence position, while a ":", "." or space refers to a conservative, moderate, or very different amino acid mutation in the sequence at a given sequence position, respectively. [Figure 9] Figure 9 shows a side-by-side alignment of SEQ ID NO: 1 and SEQ ID NO: 16. An "*" represents a perfect homology in all sequences at a given sequence position, while a ":", "." or space refers to a conservative, moderate, or very different amino acid mutation in the sequence at a given sequence position, respectively. [Figure 10] Figure 10 shows a side-by-side alignment of SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26. A "*" represents a perfect homology in all sequences at a given sequence position, while a ":", ".", or space refers to a conservative, moderate, or very different amino acid mutation in the sequence at a given sequence position, respectively. [Figure 11] Figure 11 shows a side-by-side comparison of SEQ ID NO: 1, SEQ ID NO: 28, and SEQ ID NO: 30. A "*" represents a perfect homology in all sequences at a given sequence position, while a ":", ".", or space refers to a conservative, moderate, or very different amino acid mutation in the sequence at a given sequence position, respectively. [Figure 12] Figure 12 shows a side-by-side alignment of SEQ ID NO: 1, SEQ ID NO: 18, and SEQ ID NO: 20. A "*" represents a perfect homology in all sequences at a given sequence position, while a ":", ".", or space refers to a conservative, moderate, or very different amino acid mutation in the sequence at a given sequence position, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description The present disclosure relates to compositions of fusion proteins, such as insulin-Fc fusion proteins, and their use to treat tumors.

[0042] definition As used herein, the articles "a" and "an" refer to one or more, e.g., at least one, of the grammatical object of the article. As used herein, the use of the words "a" or "an," when used with the term "comprising," can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0043] As used herein, "about" and "approximately" generally refer to an acceptable degree of error for the quantity measured given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value range.

[0044] As used herein, an amount of a molecule, compound, conjugate, or substance effective to treat a disorder (e.g., a disorder described herein), a "therapeutically effective amount," or an "effective amount" refers to the amount of a molecule, compound, conjugate, or substance that, upon single administration or multiple administrations to a subject, is effective to treat the subject or to cure, alleviate, relieve, or improve the condition of a subject having a disorder (e.g., a disorder described herein) beyond that expected in the absence of such treatment.

[0045] As used herein, the term "analog" refers to a compound or conjugate that has a similar chemical structure to another compound or conjugate, but differs in at least one aspect therefrom (e.g., a compound or conjugate described herein, e.g., insulin).

[0046] As used herein, the term "antibody" or "antibody molecule" refers to an immunoglobulin molecule (Ig), an immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule comprising an antigen-binding site that specifically binds, e.g., immunoreacts with, an antigen. As used herein, the term "antibody domain" refers to the variable or constant region of an immunoglobulin. As used herein, the term "antibody domain" refers to the variable or constant region of an immunoglobulin. Antibodies are described in the art as comprising several classes, e.g., IgA, IgM, or IgG in mammals (e.g., humans). Immunoglobulin classes can be further classified into different isotypes, such as IgGA, IgGB, IgGC, and IgGD in dogs and IgG1, IgG2, IgG3, and IgG4 in humans. Those skilled in the art will recognize that immunoglobulin isotypes of a given immunoglobulin class comprise different amino acid sequences, structures, and functional properties (e.g., different binding affinities for Fc(γ) receptors) from each other. By "specifically bind" or "immunoreact" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and has low affinity for, e.g., does not react with, other polypeptides.

[0047] As used herein, the term "insulin-Fc fusion protein" or "insulin-Fc protein" or "fusion protein" or "insulin-Fc fusion homodimer" refers to a protein comprising an insulin protein and an Fc fragment.

[0048] As used herein, the terms "biological activity," "activity," "biological activity," "potency," "biological activity efficacy," or "biological efficacy" refer to the extent to which an insulin-Fc fusion protein activates IR and / or results in a reduction in blood glucose levels in a target subject. As used herein, "in vitro activity" or "IR activity" refers to the affinity with which an insulin-Fc fusion protein binds to IR, generally the concentration at which the insulin-Fc fusion protein displaces half of an insulin reference standard from IR in a competitive binding assay (i.e., IC 50 As used herein, "in vivo activity" refers to the extent and duration of reduction in fasting blood glucose levels in a target subject following administration of an insulin-Fc fusion protein.

[0049] The fusion protein of this technology has an IC of over 1 50 It is noted that the binding to the insulin receptor is weaker than that to the RHI, as indicated by the ratio. The term "strong binding to the insulin receptor" refers to a relative comparison of the insulin receptor binding ratio to the RHI between different insulin-Fc fusion protein compounds. For example, IC 50 An insulin-Fc fusion protein with a ratio of 20 (referred to as "insulin-Fc fusion protein A") means that the insulin-Fc fusion protein binds to the insulin receptor 20 times less strongly than recombinant human insulin. However, the IC 50 Another insulin-Fc fusion protein (designated "insulin-Fc fusion protein A") with a ratio greater than 100 binds to the insulin receptor much weaker than the one designated "insulin-Fc fusion protein A." In this case, one can say "insulin-Fc fusion protein A exhibits strong binding to the insulin receptor," or "insulin-Fc fusion protein A exhibits stronger binding to the insulin receptor than a reference insulin-Fc fusion protein," where the reference insulin-Fc fusion protein has an IC 50 Alternatively, the ratio may be SEQ ID NO: 47 with a low IC of 142.50 This expression can also be used interchangeably to indicate that exemplary insulin-Fc fusion proteins of the present technology exhibit overall stronger binding to the insulin receptor than other insulin-Fc fusion proteins.

[0050] As used herein, the terms "biosynthesis," "recombinant synthesis," or "recombinantly produced" refer to the process of expressing an insulin-Fc fusion protein in a host cell by transfecting the cell with a nucleic acid molecule (e.g., a vector) encoding the insulin-Fc fusion protein (e.g., when the entire insulin-Fc fusion protein is encoded by a single nucleic acid molecule). Exemplary host cells include mammalian cells, such as HEK293 cells or CHO cells. The cells can be cultured using methods standard in the art, and the expressed insulin-Fc fusion protein can be harvested and purified from the cell culture using methods standard in the art.

[0051] As used herein, the term "cell surface receptor" refers to a molecule, such as a protein, that is generally found on the outer surface of the membrane of a cell and that interacts with a soluble molecule, e.g., a molecule circulating in the blood supply. In some embodiments, the cell surface receptor includes a hormone receptor (e.g., insulin hormone receptor or insulin receptor (IR)) or an Fc receptor that binds to the Fc fragment or Fc region of an antibody (e.g., an Fc(γ) receptor, e.g., Fc(γ)RI, or an Fc neonatal receptor, e.g., FcRn). As used herein, "in vitro activity" or "Fc(γ) receptor activity" or "Fc(γ) receptor binding" or "FcRn receptor activity" or "FcRn binding" refers to the binding affinity of an insulin-Fc fusion protein to an Fc receptor (e.g., Fc(γ) receptor or FcRn receptor), typically measured by the concentration of insulin-Fc fusion protein that causes the insulin-Fc fusion protein to reach half of its maximal binding (i.e., the EC50 value) as measured in an assay (e.g., an enzyme-linked immunosorbent assay (ELISA) assay) using the OD450nm value measured in a microplate reader. Alternatively, the binding affinity of an insulin-Fc fusion protein to an Fc receptor (e.g., Fc(γ) receptor or FcRn receptor) is measured in an enzyme-linked immunosorbent assay (ELISA) assay at a given concentration of insulin-Fc fusion protein by the OD450nm value obtained in a microplate reader.

[0052] As used herein, the term "C1q" or "complement component 1q" refers to a protein complex involved in the complement system, which is part of the innate immune system. C1q, together with C1r and C1s, forms the C1 complex. C1q plays a role in the presentation of specific antigens by dendritic cells to T cells and B cells.

[0053] As used herein, the term "fasting blood glucose level" or "FBGL" refers to the average blood glucose level in a target subject at the end of a food-deprived period and immediately prior to administration of an insulin-Fc fusion protein. As used herein, the term "percent fasting blood glucose level," "% fasting blood glucose level," or "%FBGL" refers to the ratio of a given blood glucose level to the fasting blood glucose level multiplied by 100.

[0054] As used herein, the terms "immunogenic" or "immunogenicity" refer to the ability of a given molecule (e.g., an insulin-Fc fusion protein of the present invention) to stimulate the immune system of a target subject, such that the target subject develops antibodies (i.e., anti-drug antibodies) that can specifically bind to the molecule after repeated administration of the molecule. As used herein, the terms "neutralizing," "neutralizing antibody," or "neutralizing anti-drug antibody" refer to the ability of an antibody to interfere with the biological activity of a compound in a target subject. As used herein, the terms "immunogenic epitope," "immunogenic hotspot," or "hotspot" refer to a mutation or epitope of a given molecule (e.g., an insulin-Fc fusion protein of the present invention) that is responsible for moderate or strong binding of anti-drug antibodies.

[0055] As used herein, the term "insulin reference standard" refers to either (i) native insulin from a mammal (e.g., human); (ii) an insulin polypeptide that does not contain an Fc fragment; or (iii) a standard therapeutic insulin (e.g., a commercially available insulin).

[0056] As used herein, the term "monomer" refers to a protein or fusion protein comprising a single polypeptide. In some embodiments, a "monomer" is a protein or fusion protein, e.g., a single polypeptide, comprising an insulin polypeptide and an Fc fragment polypeptide, where the insulin and Fc fragment polypeptides are linked by a peptide bond to form the single polypeptide. In some embodiments, the monomer is encoded by a single nucleic acid molecule.

[0057] As used herein, "N-terminus" refers to the beginning of a protein or polypeptide initiated by an amino acid containing a free amine group that is the α-amino group of the amino acid (e.g., a free amino that is covalently bonded to a carbon atom located adjacent to a second carbon atom, where the second carbon atom is part of the carbonyl group of the amino acid). As used herein, "C-terminus" refers to the end of a protein or polypeptide terminated by an amino acid containing a carboxylic acid group, where the carbon atom of the carboxylic acid group is located adjacent to the α-amino group of the amino acid.

[0058] As used herein, "pharmacodynamics" or "PD" generally refers to the biological effect of an insulin-Fc fusion protein in a subject. Specifically, as used herein, PD refers to the measure of the reduction in fasting blood glucose levels over time in a subject following administration of an insulin-Fc fusion protein.

[0059] As used herein, "pharmacokinetics" or "PK" generally refers to the characteristic interactions between an insulin-Fc fusion protein and a subject's body regarding absorption, distribution, metabolism, and excretion. Specifically, as used herein, PK refers to the concentration of an insulin-Fc fusion protein in a subject's blood or serum at a given time after administration of the insulin-Fc fusion protein. As used herein, "half-life" refers to the time required for the concentration of an insulin-Fc fusion protein in a subject's blood or serum to reach half of its original value, as calculated from a first-order exponential decay model of drug excretion. An insulin-Fc fusion protein with a larger "half-life" value exhibits a longer duration of action in a target subject.

[0060] As used herein, the terms "sequence identity," "sequence homology," "homology," or "identical" in the context of an amino acid or nucleotide sequence refer to the fact that when a specified contiguous segment of a variant's nucleotide or amino acid sequence is aligned and compared to the nucleotide or amino acid sequence of a reference sequence, the same nucleotides or amino acid residues are found in the variant and the reference sequence. Methods for sequence alignment and determining identity between sequences are known in the art and include the use of Clustal Omega, which orders, aligns, and compares sequences for similarity; this software highlights each sequence position, compares the entire sequence at that position, and assigns one of the following scores: *" (asterisk) indicates a sequence position with a single, perfectly conserved residue; ":" (colon) indicates conservation between groups of strongly similar features with a score greater than 0.5 in the Gonnet PAM 250 matrix; "." (period) indicates conservation between groups of weakly similar features with a score of 0.5 or less in the Gonnet PAM 250 matrix; "-" (dash) indicates a sequence gap, meaning that there is no local homology within a particular comparison set within a particular range of sequences; and an empty space " " indicates little or no sequence homology at that particular position across the compared sequences. See, e.g., Ausubel et al. (1995) Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978) in Atlas of Polypeptide Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC)). For optimal alignment of two nucleotide sequences, a contiguous segment of a variant nucleotide sequence may have nucleotide additions or deletions relative to the reference nucleotide sequence. Similarly, for optimal alignment of two amino acid sequences, a contiguous segment of a variant amino acid sequence may have amino acid residue additions or deletions relative to the reference amino acid sequence. In some embodiments, the contiguous segment used for comparison with a reference nucleotide sequence or reference amino acid sequence comprises 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. Correction for increased sequence identity due to the inclusion of gaps in the variant nucleotide or amino acid sequence can be performed by assigning gap penalties. Methods of sequence alignment are known in the art.

[0061] In some embodiments, the determination of percent identity or "homology" between two sequences is accomplished using a mathematical algorithm. For example, percent amino acid sequence identity is determined using the Smith-Waterman homology search algorithm, which uses an affine 6-gap search with a gap open penalty of 12 and a gap extension penalty of 2, and a BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is described in Smith and Waterman (1981) Adv. Appl. Math 2:482-489, which is incorporated herein by reference. In some embodiments, percent nucleotide sequence identity is determined using the Smith-Waterman homology search algorithm, which uses a gap open penalty of 25 and a gap extension penalty of 5. Such sequence identity determinations can be performed, for example, using TimeLogic's DeCypher Hardware Accelerator.

[0062] As used herein, the term "homology" is used to compare two or more proteins by, for example, determining common structural features and common spatial distribution of β-strands, helices, and folds. Homologous protein structures are therefore defined by spatial analysis. Measuring structural homology involves calculating spatial geometric-topological features. 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.

[0063] As used herein, the terms "subject" and "patient" are intended to include a human having a disease or disorder, e.g., a cancerous tumor, diabetes, or another disease or disorder described herein, or a normal subject.

[0064] As used herein, the term "titer" or "yield" refers to the amount of fusion protein product (e.g., an insulin-Fc fusion protein described herein) resulting from biosynthesis (e.g., in mammalian cells, e.g., HEK293 cells or CHO cells) 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 stated per volume of original cell culture. As used herein, the term "product yield" or "total protein yield" refers to the total amount of insulin-Fc fusion protein expressed by cells and purified via at least one affinity chromatography step (e.g., Protein A or Protein G), including insulin-Fc fusion protein monomers, insulin-Fc fusion protein homodimers, and higher-order molecular aggregates of insulin-Fc fusion protein homodimers. As used herein, the term "percent homodimer" or "% homodimer" refers to the fraction of a fusion protein product (e.g., an insulin-Fc fusion protein described herein) that is the desired homodimer. As used herein, the term "homodimer titer" refers to the product of the % homodimer and the total protein yield after the Protein A purification step, reported per volume of cell culture.

[0065] As used herein, the term "treating" a subject with a disease or disorder refers to administering to the subject a regimen, e.g., administration of a fusion protein, such as a fusion protein described herein, that cures, alleviates, relieves, relieves, alters, corrects, improves, or enhances at least one symptom of the disease or disorder. Treatment includes administering an amount effective to alleviate, alleviate, alter, improve, correct, alleviate, or affect the disease or disorder or a symptom of the disease or disorder. Treatment may inhibit the worsening or exacerbation of symptoms of the disease or disorder.

[0066] Fusion Protein Components and Structure The present disclosure relates to compositions of fusion proteins (i.e., insulin-Fc fusion proteins) comprising an insulin polypeptide linked directly or via a peptide linker to a species-specific Fc fragment, and their use for treating cancer in mammals. As used herein, the terms "fusion protein" and "insulin-Fc fusion protein" refer to proteins comprising two or more moieties (different proteins, polypeptides, cells, etc.) derived from different species covalently linked, for example, via a peptide bond. An insulin-Fc fusion protein can be covalently linked by (i) linking the genes encoding each moiety into a single nucleic acid molecule and (ii) expressing in a host cell (e.g., HEK or CHO) a protein encoded by the nucleic acid molecule as follows: (N-terminus)--insulin polypeptide--linker--Fc fragment--(C-terminus). A fully recombinant synthesis approach is preferred over separate synthesis of the insulin polypeptide and Fc fragment followed by chemical conjugation. The chemical conjugation step and subsequent purification step increase the complexity of production, reduce product yield, and increase costs.

[0067] As used herein, 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 a disulfide bond) to form a "homodimer" (as depicted in Figure 1). The disulfide bonds are depicted in Figure 1; the actual total number of disulfide bonds may be more or less than the number depicted in Figure 1. In embodiments, the homodimer is encoded by a single nucleic acid molecule and is produced recombinantly in a cell by first forming an insulin-Fc fusion protein monomer and then assembling two identical insulin-Fc fusion protein monomers into a homodimer upon further processing in the cell.

[0068] As used herein, the terms "multimer," "multimeric," or "multimeric state" refer to non-covalently associated forms of Fc fusion protein dimers that are in equilibrium with Fc fusion protein dimers or that may act as permanent aggregate versions of Fc fusion protein dimers (e.g., dimers of Fc fusion protein homodimers, trimers of Fc fusion protein homodimers, tetramers of Fc fusion protein homodimers, or higher order aggregates containing five or more Fc fusion protein homodimers). Multimeric forms of Fc fusion proteins are expected to have different physical activity, stability, or pharmacological activity than insulin-Fc fusion protein homodimers.

[0069] Insulin Polypeptide In some embodiments, the insulin-Fc fusion proteins described herein comprise an insulin polypeptide, e.g., insulin or an insulin analog. Insulin is a peptide hormone produced by the beta cells of the islets of Langerhans in the pancreas. Insulin acts by regulating glucose absorption from the blood. Upon stimulation, such as elevated protein and glucose levels, insulin is released from beta cells, binds to the insulin receptor, and initiates a signaling cascade that affects many aspects of mammalian metabolism. Disruption of this process is directly related to several diseases and conditions, particularly diabetes, insulinoma, insulin resistance, metabolic syndrome, and polycystic ovary syndrome.

[0070] The insulin analogs of the present disclosure may be related in structure to insulin but may 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 affect certain characteristics or properties of the insulin-Fc fusion protein construct. For example, the modifications or alterations described herein may affect the structure, stability, pH sensitivity, biological activity, or binding affinity to a cell surface receptor (e.g., insulin hormone receptor) of the insulin-Fc fusion protein construct compared to a reference standard.

[0071] The amino acid sequence of insulin is highly conserved throughout evolution, particularly among vertebrates. For example, naturally occurring dog and pig insulin differs from human insulin by only one amino acid, naturally occurring cormorant insulin differs from human insulin by only three amino acids, and naturally occurring feline insulin differs from human insulin by only four amino acids. As used herein, the terms "B chain or B chain analog," "C peptide" or "C chain," and "A chain or A chain analog" refer to peptide segments of the insulin polypeptide as shown in Figure 1. Insulin is a 51-amino acid hormone containing two peptide chains (i.e., the B chain and the A chain) linked via disulfide bonds (e.g., disulfide bonds formed by one or more B chain cysteine ​​side chain thiols and one or more A chain cysteine ​​side chain thiols). The A chain of insulin is 21 amino acids long, and the B chain of insulin is 30 amino acids long. In naturally occurring insulin, the A chain contains one intrachain disulfide bond formed by two A chain cysteine ​​side chain thiols.

[0072] As used herein, the term "insulin" or "insulin polypeptide" includes mature insulin, preproinsulin, proinsulin, and native insulin, or analogs thereof. In embodiments, an insulin polypeptide can be a full-length insulin polypeptide or a fragment thereof. In embodiments, an insulin polypeptide can comprise one or more fragments of mature insulin, preproinsulin, proinsulin, or native insulin.

[0073] Insulin is typically constructed as an N-terminus--B chain:C chain:A chain--C-terminus polypeptide, with the C chain being truncated to impart biological activity. For reference, the sequence of the entire human insulin molecule (i.e., human proinsulin), including the C chain, is shown below (the C chain is shown in bold): [ka]

[0074] Conversion of the single-chain insulin polypeptide to the biologically active two-chain polypeptide is normally accomplished within the beta cells of the islets of Langerhans prior to glucose-stimulated insulin secretion by two endoproteases: type I endoproteases PC1 and PC3, and PC2, which disrupt the C-peptide-B-chain bond, and type II endoprotease, which cleaves the C-peptide-A-chain bond at a precise site. However, cell lines used for the biosynthesis of therapeutic molecules such as insulin (e.g., bacterial, yeast, and mammalian (e.g., HEK and CHO) cell lines) lack this pathway, and therefore transformation must be performed after the single-chain polypeptide is expressed and harvested using chemical or enzymatic methods. A known technique for cleaving the C-chain after expression and harvest is to first modify the C-chain to terminate it at the lysine immediately preceding the N-terminus of the A-chain. The single-chain insulin polypeptide is then cleaved at the C-terminal lysine of the C chain and at the C-terminal lysine at position 29 from the N-terminus of the B chain using trypsin or an enzyme selected from the Lys-C family, which specifically cleaves peptide bonds at the C-terminus of lysine residues. In some cases, the resulting biologically active two-chain insulin is used without reattaching the truncated amino acid at position 30 from the N-terminus of the B chain; in other cases, the truncated amino acid at position 30 from the N-terminus of the B chain is added back to the molecule using additional enzymatic methods. This process works well with insulin because it contains only one lysine in its full-length two-chain polypeptide form.

[0075] Recombinant human insulin (referred to herein as "RHI") is a biologically active two-chain polypeptide comprising a B chain of SEQ ID NO:5:FVNQHLCGSHLVEALYLVCGERGFFYTPKT and an A chain of SEQ ID NO:9:GIVEQCCTSICSLYQLENYCN linked by two disulfide bonds derived from cysteine ​​residues (A7-B7 and A20-B19). The third disulfide is an intrachain disulfide bond derived from cysteine ​​residues (A6-A11) on the A chain. This structure of RHI is well known in the art (see, e.g., Brange, Jens, Gelanics of Insulin: The Physico-Chemical and Pharmaceutical Aspects of Insulin and Insulin Preparations (1987) Springer-Verlag Berlin Heidelberg, https: / / doi.org / 10.1007 / 978-3-662-02526-0).

[0076] However, this process cannot be used with the insulin-Fc fusion proteins included herein because all known Fc fragments contain multiple lysine residues. Therefore, enzymatic cleavage processes would digest the Fc fragment into non-functional portions, thereby eliminating the ability of the Fc fragment to prolong the in vivo action of the insulin polypeptide. Therefore, the insulin-Fc fusion proteins of the present invention must comprise an insulin polypeptide that does not require C-chain cleavage and therefore has biological activity in its single-chain form.

[0077] Many biologically active single-chain insulin polypeptides have been described in the art. In all cases, the single-chain insulin polypeptides contain a C chain of a specific length and composition, and A and B chains mutated at specific amino acid sites to achieve electrostatic balance, prevent aggregation, enhance IR binding and / or downstream signaling, and achieve a level of biological activity comparable to that of native two-chain insulin. Here, the position of the mutation on a peptide segment is designated using the name of the segment (e.g., B chain, C chain, A chain) and the number of amino acids counted from the N-terminus of the segment. For example, the designation "B10" refers to the 10th amino acid from the N-terminus of the amino acid sequence of the B chain. The designation "A8" refers to the 8th amino acid from the N-terminus of the A chain. Furthermore, if an amino acid at a particular position is mutated from its native form to a new amino acid, that position is designated with the single-letter amino acid code of the new amino acid. For example, B10D refers to an aspartic acid mutation at the 10th amino acid from the N-terminus of the amino acid sequence of the B chain, and A8H refers to a histidine mutation at the 8th amino acid from the N-terminus of the amino acid sequence of the A chain.

[0078] In some embodiments, the insulin polypeptides of the present disclosure comprise insulin analogs. Insulin analogs are closely related to the structure of insulin, but may contain modifications (e.g., structural modifications) to enhance certain functional aspects. In some embodiments, insulin analogs comprise variants or mutants of insulin. In some embodiments, insulin analogs comprise at least one amino acid substitution, deletion, or addition relative to insulin.

[0079] In some embodiments, modifications to the sequence or structure of insulin or an insulin analog (e.g., amino acid substitution, deletion, or addition, or chemical modification) can affect certain characteristics or properties of an insulin-Fc fusion protein (e.g., an insulin-Fc fusion protein described herein). For example, the modifications or alterations described herein can affect the structure, stability, pH sensitivity, biological activity, or binding affinity to a cell surface receptor (e.g., the insulin hormone receptor) of the insulin-Fc fusion protein. In some embodiments, amino acid substitution, addition, deletion, or chemical modification to insulin can affect the activity of an insulin analog relative to a reference standard.

[0080] In some embodiments, the insulin or insulin analog is a three-segment peptide comprising elements of a B chain, a C peptide, and an A chain. In other embodiments, the insulin-Fc fusion proteins described herein comprise an insulin polypeptide comprising a mutant insulin B chain, a C peptide, and / or an A chain.

[0081] In some embodiments, modifications to the insulin or insulin analog sequence (e.g., amino acid substitutions, deletions, or additions, or chemical modifications) may be to the insulin B chain, the insulin C peptide, the insulin A chain, or any combination thereof.

[0082] Fc fragment Insulin-Fc fusion proteins combine an insulin polypeptide with a human Fc region, as shown in Figure 1. These insulin-Fc fusion proteins are biologically produced in mammalian cells as a single chain, in which the insulin molecule is linked between the A and B chains by a short peptide sequence, and the use of the human Fc region serves to prolong its action in vivo.

[0083] The terms "Fc region," "Fc domain," "Fc polypeptide," or "Fc fragment" as used herein are used to define the C-terminal region of an immunoglobulin heavy chain. The Fc fragment, region, or domain may be a native-sequence Fc region or a variant / mutant Fc region. The boundaries of the Fc region of an immunoglobulin heavy chain can vary but generally comprise part or all of the heavy chain hinge region, the heavy chain CH2 region, and the heavy chain CH3 region. The hinge region of a human Fc fragment comprises an amino acid sequence linking the heavy chain CH1 domain and the heavy chain CH2 region, which contains one or more cysteines that form one or more inter-heavy chain disulfide bridges that form the Fc fusion protein homodimer from two identical but separate monomers of the Fc fusion protein. The hinge region may comprise all or part of a native or non-native amino acid sequence.

[0084] Fc receptor (FcR) refers to a receptor that binds to the Fc fragment or Fc region of an antibody. In some embodiments, the FcR is a native sequence human FcR. In some embodiments, the FcR binds to the Fc fragment or Fc region of an IgG antibody (a gamma receptor), including but not limited to receptors of the FcγRI, FcγRIIa, FcγRIIb, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., 1976 J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249) and is also responsible for extending the in vivo elimination half-life of antibodies and Fc fusion proteins. In embodiments, the Fc fragments described herein are capable of binding to a mammalian Fc(γ) receptor or Fc(Rn) receptor, for example, a human Fc(γ) receptor or a human Fc(Rn) receptor.

[0085] In some embodiments, the C-terminal lysine often found in native human IgG isotype Fc fragment amino acid sequences (i.e., the lysine corresponding to the last amino acid in the Fc fragment sequence) is deleted to prevent the inadvertent production of undesired amino acid sequence variants during manufacturing (e.g., mixing an Fc fragment containing a C-terminal lysine with an Fc fragment in which the C-terminal lysine has been deleted, which can occur during intracellular production of the desired protein) (Dick, LW., (2008) Biotechnol Bioeng. Aug 15;100(6) pp1132-43).

[0086] In some embodiments, the Fc fragment comprises the Fc region of a human immunoglobulin (e.g., IgG1), e.g., the hinge region, CH2 domain, and CH3 domain (or a fragment thereof). In some embodiments, the Fc fragment comprises the hinge region (or a fragment thereof) of human IgG1. In some embodiments, the Fc fragment comprises the Fc region of a human IgG1, e.g., the CH2 domain and CH3 domain (or a fragment thereof).

[0087] Linker In embodiments, the fusion proteins described herein comprise a linker, e.g., between one or more domains of the polypeptides, e.g., the fusion protein comprises a linker between the insulin polypeptide and the Fc fragment.

[0088] In some examples, the C-terminus of the insulin polypeptide is directly linked to the N-terminus of the Fc fragment (e.g., without a linker or no linker is present). In other examples, successful construction of a recombinantly produced insulin-Fc fusion protein requires a linker linking the insulin polypeptide and the Fc fragment. In embodiments, the linker is a peptide. In embodiments, the insulin-Fc fusion protein constructs described herein comprise a peptide linker comprising multiple amino acids (e.g., natural or unnatural amino acids) between the insulin polypeptide and the Fc fragment. In embodiments, the peptide linker can be encoded by a nucleic acid molecule, such that, for example, a single nucleic acid molecule can encode various peptides within the 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 affect the manufacturability of the insulin-Fc fusion protein construct (i.e., homodimer titer), chemical and enzymatic stability, biological activity, parameters that correlate with biological activity (i.e., EC50 value in the FcRn assay), and immunogenicity of the insulin-Fc fusion protein (Chen, X., Zaro, J., Shen, WC, Adv Drug Deliv Rev. 2013 October 15; 65(10): 1357-1369).

[0089] Fusion proteins Provided herein are fusion proteins, e.g., insulin-Fc fusion proteins. In some embodiments, the fusion protein comprises an insulin polypeptide, as described herein, e.g., in the "Insulin Polypeptides" section. In some embodiments, the fusion protein comprises an Fc fragment, as described herein, e.g., in the "Fc Fragment" section.

[0090] In some embodiments, the fusion protein comprises a linker, e.g., as described herein in the "Linker" section, between an insulin polypeptide, e.g., as described herein in the "Insulin Polypeptide" section, and an Fc fragment, e.g., as described herein in the "Fc Fragment" section.

[0091] In some embodiments, the insulin polypeptide comprises domains in the following orientation from N-terminus to C-terminus: (N-terminus)--B chain--C peptide--A chain--(C-terminus). The insulin polypeptide may be located N-terminal to the Fc domain.

[0092] In some embodiments, the fusion protein comprises domains in the following orientation, from N- to C-terminus: (N-terminus)--insulin polypeptide--linker--Fc fragment--(C-terminus) (e.g., (N-terminus)--B chain--C peptide--A chain--linker--Fc fragment--(C-terminus); or (N-terminus)--B chain--C peptide--A chain--linker--Fc fragment--(C-terminus)), as shown in Figure 1. In some embodiments, the fusion protein (also referred to as an insulin-Fc fusion protein) is composed of two identical insulin-Fc fusion proteins covalently linked via one or more disulfide bonds (shown as dotted lines in Figure 1; the actual total number of disulfide bonds may be more or less than the number shown in Figure 1). Each insulin-Fc fusion protein comprises a proinsulin-like insulin molecule containing an insulin B chain and an insulin A chain connected by a C chain between the B chain-C-terminal region and the A chain-NH2-terminal region (gray line in Figure 1), a linker between the A chain-C-terminal region and the Fc chain amino terminus, and the insulin-Fc fusion protein sequence terminating in the Fc-CH3 region-C-terminal region. Note that the B chain and A chain are also connected via two disulfide bonds (dotted lines in Figure 1). The A chain also contains an intramolecular disulfide bond (not shown in Figure 1).

[0093] Treatment of cancer tumors with long-term hypoglycemia Cancer cells consume more glucose than normal cells and metabolize glucose-derived pyruvate to lactate even in the presence of oxygen (the Warburg effect). Although aerobic glycolysis is less efficient (in terms of adenosine triphosphate production) than mitochondrial oxidative phosphorylation, which normal cells use to generate energy, it results in increased production of additional metabolites beneficial to proliferating cells, such as cancer cells. Because the Warburg effect is related to glucose uptake and utilization, it was hypothesized that ultra-long-acting basal insulin for the treatment of diabetes, which effectively lowers blood glucose levels over a prolonged period of time, would be useful in cancer treatment. Insulin analog mutants that can bind to and activate the insulin hormone receptor and utilize FcRn receptor recycling to extend their action, can achieve this goal by prolonging their interaction with insulin receptors present on the cell surface, helping to halt cancer cell growth and mitosis.

[0094] To achieve this goal, insulin-Fc fusion proteins containing mutations in the A and B chains and covalently linked to the IgG Fc region via a peptide linker were required. Variations of insulin-Fc fusion proteins were designed with the expectation that compounds with similar properties would be able to bind to and activate the insulin hormone receptor, do so with an acceptable low insulin receptor affinity EC50 ratio compared to the affinity of endogenous insulin, and take advantage of FcRn receptor recycling to substantially extend the compound's serum half-life. To achieve these results, the insulin-Fc fusion protein of SEQ ID NO: 1, for example, required the inclusion of a peptide sequence between the A and B chains, as well as specific mutations in the A and B chains themselves. In addition to exhibiting high affinity for the insulin receptor (as described in Example 6a), the insulin-Fc fusion protein of SEQ ID NO: 1 also demonstrated acceptable efficacy and sustained hypoglycemic activity in mice, as described in Example 8a and shown in Figure 5.

[0095] Unexpectedly, mice treated with the insulin-Fc fusion protein of SEQ ID NO: 1 showed no clinical signs of hypoglycemia, such as lethargy, loss of balance, convulsions, or loss of consciousness, even after prolonged fasting, despite having blood glucose (BG) levels significantly lower than normal (as shown in Figure 5 compared to the porcine insulin NPH vehicle). These unexpected results led to the hypothesis that the insulin-Fc fusion protein of SEQ ID NO: 1 may slow the growth of cancerous tumors by inducing "controlled hypoglycemia," thereby limiting the glucose available to cancer cells for aerobic glycolysis, while sparing other parts of the body. The insulin-Fc fusion protein of SEQ ID NO: 1 demonstrated the ability to downregulate insulin receptors in HCT-116 cells in vivo (as described in Example 7a and shown in Figure 4).

[0096] Studies in nude mice according to Example 9a, performed according to Example 10a using an in vivo model of a metastatic human melanoma cell line (WM266.4) in an HCT-116 xenograft model (HCT-116 cells are used in various biomedical studies, including colon cancer growth and corresponding inhibitors), demonstrated that the insulin-Fc fusion protein of SEQ ID NO: 1 was able to slow tumor growth compared to controls under fasting conditions. Animals treated with conventional NPH insulin showed no benefit from treatment under fasting conditions, despite similar fasting hypoglycemia levels after administration of both NPH and the insulin-Fc fusion protein of SEQ ID NO: 1, and higher doses of NPH were not feasible due to the frequent occurrence of life-threatening hypoglycemia in the mice. Unexpectedly, animals treated with the insulin-Fc fusion protein of SEQ ID NO: 1 without fasting still showed a significant reduction in tumor growth rate. This data indicated that prolonged hypoglycemia is not the mechanism by which the insulin-Fc fusion protein of SEQ ID NO: 1 inhibits cancer cell growth.

[0097] Treatment of cancer tumors by reducing insulin-like growth factor 1 receptor (IGF1R) The IGF1 receptor, or IGF1R, is a transmembrane receptor found on the cell surface. IGF1R is overexpressed in several tumor types, and as a result of its effects on tumor survival and growth in preclinical studies, several anti-IGF1R therapies are being developed for clinical trials. Although promising, the clinical success of these therapies has been limited, likely due to the development of resistance via alternative signaling pathways.

[0098] Furthermore, it is known that treating certain cancers with certain drugs (e.g., treating breast cancer with drugs such as tamoxifen) results in breast cancer cells with reduced or downregulated IFG1R. This downregulation of IFG1R ultimately allows cancer cells to become resistant to the drug through alternative signaling pathways (e.g., drug-induced resistance, or "tamoxifen-resistant" cancers or tumors), similar to what occurs with anti-IGF1R therapeutic approaches. This drug-induced resistance has been demonstrated in the laboratory using cancer cell lines (e.g., breast cancer cell line MCF-7 and tamoxifen-resistant breast cancer cell line MCF-7 (also known as MCF-7 TamR)).

[0099] IGF1R is activated by the hormone insulin-like growth factor 1 (IGF-1) and the related hormone insulin-like growth factor 2 (IGF-2). Binding of IGF-1 and IGF-2 to the cell surface IGF1R results in the autophosphorylation and activation of two distinct but overlapping pathways: PI3K-Ak and MAPK. The PI3K pathway is a cascade that leads to the phosphorylation and activation of the serine / threonine kinase Akt, which regulates cellular metabolism through the translocation of the glucose transporter GLUT4 to the cell surface. Fully activated Akt phosphorylates various intracellular proteins, mediating downstream responses including cell survival, growth, proliferation, cell migration, and angiogenesis. It also regulates cell survival through the inhibition of apoptosis, making it critical for tumor survival. Activation of the MAPK pathway leads to the activation of ERK1 / 2, resulting in increased cell proliferation, metastasis, and tumor growth.

[0100] Clinicians hope to treat certain cancers by reducing IGF1R levels in tumors, based on the hypothesis that IGF-1 and IGF-2 are less able to bind to IGF1R and induce downstream cell proliferation and tumor growth. Anti-IGF1R antibody therapy approaches have been attempted in clinical trials, many of which progressed to phase 3 clinical trials, but all programs were discontinued due to tumor resistance over time. Downregulation of IGF1R in response to binding of therapeutically administered anti-IGF1R antibodies and internalization of the receptor-antibody complex prevents rapid elimination of IGF-1 and IGF-2 via IGF1R, resulting in elevated serum concentrations of IGF-1 and IGF-2. Because IGF-1 and IGF-2 share structural similarity to insulin, high concentrations can activate the insulin receptor (IR), leading to IR-mediated binding and signaling. This undesired activation of IR / phospho-Akt signals tumors to continue growing. Thus, IR-mediated IGF-2 signaling is a potential mechanism of resistance to IFG1R treatment.

[0101] One way to disrupt IR-mediated IGF-2 signaling is to combine anti-IR antibodies with anti-IGF1R antibodies. However, because anti-IR antibodies downregulate IR, which leads to reduced insulin binding and undesirable hyperglycemia and insulin resistance, only a limited number of clinical candidates targeting IR have been developed.

[0102] Another approach focuses on short interfering RNA (siRNA) or RNA interference (RNAi). A promoter system can be used to deliver and express siRNA targeting IGF1R and reduce its expression in cells. This downregulation of IGF1R results in significant inhibition of cancer cell growth in vitro and in vivo in rodents. However, this approach can also lead to tumor growth and undesirable hyperglycemia due to increased IGF-1 and IGF-2 binding and IR activation. A further approach is to use small-molecule tyrosine kinase inhibitors (TKIs) that simultaneously target both the IGF1R and IR systems without blocking the receptors themselves or reducing their expression levels. However, because TKIs are small molecules, they lack specificity for IGF1R / IR and may disrupt other receptor systems, including receptors not involved in cancer cell metabolism, resulting in undesirable side effects and toxicity. Furthermore, TKIs also disrupt the IR pathway for glucose homeostasis, resulting in undesirable hyperglycemia. These various approaches support the assertion that formulations that downregulate both IGF1R and IR without the risk of unwanted side effects or hypo / hyperglycemia will significantly inhibit cancer cell growth and result in desirable antitumor efficacy.

[0103] Treatment of cancer tumors through down-regulation of insulin receptor (IR) To examine the relationship between in vitro IR binding and activation and tumor volume reduction in vivo, the IR activity of the insulin-Fc fusion protein of SEQ ID NO: 1 was tested in HCT-116 cells.

[0104] When tested according to the protocol in Example 7a, the insulin-Fc fusion protein of SEQ ID NO: 1 and RHI caused substantial downregulation of IR in HCT-116 cells. Unexpectedly, the insulin-Fc fusion protein of SEQ ID NO: 1 caused a more pronounced downregulation than RHI at all concentrations tested, suggesting that its bivalent homodimeric structure, unique insulin mutations, and / or Fc component differentiate its behavior at the receptor level. Furthermore, the insulin-Fc fusion protein of SEQ ID NO: 1 induced lower levels of Akt phosphorylation than RHI at all concentrations tested. Correlation of this data with in vitro IR binding suggests that strong IR binding (compared to the reference insulin-Fc fusion protein) is required for receptor downregulation. The effects of both the insulin-Fc fusion protein of SEQ ID NO: 1 and RHI on the MAPK pathway were much milder, with little change in phosphor-ERK1 / 2 expression after 72 hours of treatment.

[0105] Preliminary data indicate that, compared with insulin, the insulin-Fc fusion protein of SEQ ID NO: 1 slightly reduces activation of the PI3K pathway, which plays a key role in cancer cell metabolism and survival. However, unlike TKIs and anti-IR antibodies, the insulin-Fc fusion protein of SEQ ID NO: 1 is not an antagonist. It fully enables signaling through this pathway to regulate blood glucose levels, as shown in Figure 4 for Phospho S473 Akt, which demonstrates that insulin receptor pathway activation is maintained even when IR is downregulated. Thus, the insulin-Fc fusion protein of SEQ ID NO: 1 is unique among IR-targeting molecules in that it can inhibit tumor growth without causing hyperglycemia or insulin resistance. Therefore, the inventors hypothesized that the in vivo anti-tumor efficacy previously observed with the insulin-Fc fusion protein of sequence number 1 (and conversely, not observed with insulin NPH) may be related to one or more of the following effects on tumor cells: (i) downregulation of IR, (ii) downregulation of IGF1R, (iii) underactivation of the Akt pathway (e.g., reduced phosphorylated Akt in the presence of the insulin-Fc fusion protein of sequence number 1), (iv) downregulation of IR in combination with additional therapy that separately targets downregulation of IGF1R, or (v) downregulation of IR in tumors with low levels of IGF1R expression.

[0106] To determine whether specific mutations in the B and A chains of the insulin polypeptide contribute to the in vivo antitumor efficacy of the insulin-Fc fusion protein of SEQ ID NO: 1, an insulin-Fc fusion protein of SEQ ID NO: 47 was designed. As shown in Table A, the insulin-Fc fusion protein of SEQ ID NO: 47 comprises a B chain of SEQ ID NO: 49 (FVNQHLCGSHLVEALELVCGERGFHY) that retains the native histidine at B10 and an A chain of SEQ ID NO: 51 (GIVEQCCTSTCSLDQLENYC) that retains the native threonine at A8. The B and A chains are linked by a C peptide of SEQ ID NO: 50 (GGGGGGSGGGG). The complete insulin polypeptide of the fusion protein SEQ ID NO: 47 is shown below as SEQ ID NO: 53. FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYC (SEQ ID NO: 53)

[0107] The insulin polypeptide of SEQ ID NO: 53 was linked to the human IgG1 fragment of SEQ ID NO: 13 via the linker of SEQ ID NO: 52 (GGGGGGQGGGGQGGGGQGGGGG). DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 13)

[0108] The resulting insulin-Fc fusion protein of SEQ ID NO: 47 is shown below. FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 47)

[0109] FIG. 8 shows a side-by-side comparison of the insulin-Fc fusion protein of SEQ ID NO: 1 and the insulin-Fc fusion protein of SEQ ID NO: 47. * " represents a perfect homology in all sequences at a given sequence position, and ":", "." or a space refers to conservative, moderate, or very different amino acid mutations in the sequences at a given sequence position, respectively.

[0110] The insulin-Fc fusion protein of SEQ ID NO: 47 was produced according to Example 1a and yielded an acceptable protein titer of 196.4 mg / L. The insulin-Fc fusion protein of SEQ ID NO: 47 was purified according to Example 2a, and the structure of the insulin-Fc fusion protein of SEQ ID NO: 47 was confirmed according to Example 3a and characterized according to Example 4. The homodimer percentage was confirmed to be an acceptable 97.8% according to Example 5a.

[0111] In vitro IM-9 insulin receptor (IR) binding of the insulin-Fc fusion protein of SEQ ID NO: 47 was tested according to Example 6a. Unexpectedly, as described in Example 6a, the insulin-Fc fusion protein of SEQ ID NO: 47 exhibited very low affinity for the insulin receptor in vitro.

[0112] When tested according to the protocol in Example 7a, the insulin-Fc fusion protein of SEQ ID NO:47 unexpectedly did not induce greater downregulation of IR than RHI at any of the concentrations tested, as shown in Figure 4, even though the insulin-Fc fusion protein of SEQ ID NO:47 has the same bivalent homodimeric structure as the insulin-Fc fusion protein of SEQ ID NO:1. It was hypothesized that one or more of the unique insulin mutations B10D and A8H, present in the insulin-Fc fusion protein of SEQ ID NO:1 but absent in the insulin-Fc fusion protein of SEQ ID NO:47, are required to differentiate the behavior of the insulin-Fc fusion proteins at the insulin receptor level. Furthermore, the failure of the insulin-Fc fusion protein of SEQ ID NO:47 to induce lower levels of Akt phosphorylation than RHI at any of the concentrations tested (also shown in Figure 4), confirmed the correlation of this data with in vitro IR binding and confirmed that stronger IR binding (than the reference insulin-Fc fusion protein) is required for receptor downregulation.

[0113] To determine whether the specific mutations B10D on the B chain and A8H on the A chain of the insulin polypeptide contribute to the in vivo antitumor efficacy of the insulin-Fc fusion protein of SEQ ID NO: 1, an insulin-Fc fusion protein of the insulin-Fc fusion protein of SEQ ID NO: 16 was designed. As shown in Table A, the insulin Fc fusion protein of SEQ ID NO: 16 comprises a B chain of SEQ ID NO: 35 bearing the histidine to aspartic acid mutation (B10D) at B10 derived from the insulin-Fc fusion protein of SEQ ID NO: 1, a C chain peptide sequence of SEQ ID NO: 8, similar to the insulin-Fc fusion protein of SEQ ID NO: 1, and an A chain of SEQ ID NO: 10 bearing the threonine to histidine mutation (A8H) at A8 derived from the insulin-Fc fusion protein of SEQ ID NO: 1. FVNQHLCGSDLVEALALVCGEEGFFYTDPT (SEQ ID NO: 35) GGGPRR (SEQ ID NO: 8) GIVEQCCHSICSLYQLENYCN (SEQ ID NO: 10)

[0114] The resulting insulin polypeptide is shown in SEQ ID NO:41. FVNQHLCGSDLVEALALVCGEEGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 41)

[0115] The insulin polypeptide of SEQ ID NO: 41 is linked to the human IgG1 Fc fragment of SEQ ID NO: 13 via a peptide linker of SEQ ID NO: 12 (GGGGSGGGG). DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 13)

[0116] The resulting insulin-Fc fusion protein of SEQ ID NO: 16 is shown below. FVNQHLCGSDLVEALALVCGEEGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 16)

[0117] The insulin-Fc fusion protein of SEQ ID NO: 16 is produced according to Example 1b and purified according to Example 2b. The structure of the insulin-Fc fusion protein of SEQ ID NO: 16 is confirmed according to Example 3b and characterized according to Example 4. The homodimer percentage is determined according to Example 5b.

[0118] FIG. 9 shows a side-by-side comparison of the insulin-Fc fusion protein of SEQ ID NO: 1 and the insulin-Fc fusion protein of SEQ ID NO: 16. * " represents a perfect homology in all sequences at a given sequence position, and ":", "." or a space refers to conservative, moderate, or very different amino acid mutations in the sequences at a given sequence position, respectively.

[0119] Testing of the insulin-Fc fusion protein of SEQ ID NO: 16 according to Example 6 is expected to reveal that, similar to SEQ ID NO: 1, the insulin-Fc fusion protein of SEQ ID NO: 16 has strong affinity for the insulin receptor in vitro (where affinity or strength of binding to the insulin receptor is relative to a reference insulin-Fc fusion protein). When tested according to the protocol of Example 7b, the insulin-Fc fusion protein of SEQ ID NO: 16 is expected to cause substantial down-regulation of IR in HCT-116 cells at all concentrations tested and induce lower levels of Akt phosphorylation than RHI at all concentrations tested.

[0120] Studies in nude mice according to Example 9b carried out in the HCT-116 xenograft model and studies according to Example 10b using an in vivo model of a metastatic human melanoma cell line (WM266.4) are expected to demonstrate that the insulin-Fc fusion protein of SEQ ID NO: 16 can slow tumor growth compared to controls under fasting and non-fasting conditions.

[0121] In a further attempt to generate insulin-Fc fusion proteins with strong insulin receptor affinity (where affinity or strength of binding to the insulin receptor is relative to a reference insulin-Fc fusion protein) and the ability to down-regulate the insulin receptor in HCT-116 cells, two insulin-Fc fusion proteins were designed with the A-chain mutation A8H, in which the B-chain was linked directly to the A-chain without a C-chain peptide linker. The first of these insulin-Fc fusions was SEQ ID NO:28. As shown in Table A, the insulin Fc fusion protein of SEQ ID NO:28 comprises the B-chain of SEQ ID NO:39, which preserves the native histidine at B10, and the A-chain of SEQ ID NO:10, which has no C-chain peptide sequence and retains the threonine to histidine mutation (A8H) at A8 derived from SEQ ID NO:1. FVNQHLCGSHLVEALALVCGEEGFFYTDK (SEQ ID NO: 39) GIVEQCCHSICSLYQLENYCN (SEQ ID NO: 10)

[0122] The resulting insulin polypeptide is shown in SEQ ID NO:45. FVNQHLCGSHLVEALALVCGEEGFFYTDKGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 45)

[0123] The insulin polypeptide of SEQ ID NO: 45 is linked to the human IgG1 Fc fragment of SEQ ID NO: 34 via a peptide linker of SEQ ID NO: 11 (GGGGAGGGG). DQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 34)

[0124] The resulting insulin-Fc fusion protein of SEQ ID NO: 28 is shown below. FVNQHLCGSHLVEALALVCGEEGFFYTDKGIVEQCCHSICSLYQLENYCNGGGGAGGGGDQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 28)

[0125] A second insulin-Fc fusion in which the B chain was linked directly to the A chain without a C chain peptide linker was SEQ ID NO: 30. As shown in Table A, the insulin-Fc fusion protein of SEQ ID NO: 30 comprises a B chain of SEQ ID NO: 40 that preserves the native histidine at B10, no C chain peptide sequence, and an A chain of SEQ ID NO: 10 that retains the threonine to histidine mutation at A8 (A8H) from the insulin-Fc fusion protein of SEQ ID NO: 1. FVNQHLCGSHLVEALALVCGEEGFFYTDR (SEQ ID NO: 40) GIVEQCCHSICSLYQLENYCN (SEQ ID NO: 10)

[0126] The resulting insulin polypeptide is shown in SEQ ID NO:45. FVNQHLCGSHLVEALALVCGEEGFFYTDKGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 45)

[0127] The insulin polypeptide of SEQ ID NO: 45 is linked to the human IgG1 Fc fragment of SEQ ID NO: 34 via a peptide linker of SEQ ID NO: 11 (GGGGAGGGG). DQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 34)

[0128] The resulting insulin-Fc fusion protein of SEQ ID NO: 30 is shown below. FVNQHLCGSHLVEALALVCGEEGFFYTDRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 30)

[0129] The insulin-Fc fusion proteins of SEQ ID NO: 28 and SEQ ID NO: 30 were produced according to Example 1a. The yields obtained were acceptable and are shown in Table 1 below.

[0130] [Table 1]

[0131] The insulin-Fc fusion proteins of SEQ ID NO:28 and SEQ ID NO:30 are purified according to Example 2b, and the structures of the insulin-Fc fusion proteins of SEQ ID NO:28 and SEQ ID NO:30 are confirmed according to Example 3b and characterized according to Example 4. The homodimer percentage is determined according to Example 5b and is expected to be acceptable if it is greater than 80%.

[0132] FIG. 11 shows a side-by-side comparison of the insulin-Fc fusion protein of SEQ ID NO: 1, the insulin-Fc fusion protein of SEQ ID NO: 28, and the insulin-Fc fusion protein of SEQ ID NO: 30. * " represents a perfect homology in all sequences at a given sequence position, and ":", "." or a space refers to conservative, moderate, or very different amino acid mutations in the sequences at a given sequence position, respectively.

[0133] Testing of the insulin-Fc fusion proteins of SEQ ID NO:28 and SEQ ID NO:30 according to Example 6b is expected to reveal that, despite preserving the A8H mutation of the insulin-Fc fusion protein of SEQ ID NO:1, the insulin-Fc fusion proteins of SEQ ID NO:28 and SEQ ID NO:30 do not have strong affinity for the insulin receptor in vitro (where affinity or strength of binding to the insulin receptor is relative to the reference insulin-Fc fusion protein). It is hypothesized that either the B10D mutation or the presence of the C-chain peptide between the B and A chains is a necessary part of the insulin-Fc fusion protein structure. When tested according to the protocol of Example 7b, the insulin-Fc fusion proteins of SEQ ID NO:28 and SEQ ID NO:30 are not expected to cause substantial down-regulation of IR in HCT-116 cells at any of the concentrations tested, nor are they expected to induce levels of Akt phosphorylation lower than RHI at any of the concentrations tested.

[0134] Studies in nude mice according to Example 9b carried out in the HCT-116 xenograft model and studies according to Example 10b using an in vivo model of a metastatic human melanoma cell line (WM266.4) are expected to show that neither the insulin-Fc fusion protein of SEQ ID NO: 28 nor the insulin-Fc fusion protein of SEQ ID NO: 30 is able to slow tumor growth compared to controls under fasting or non-fasting conditions.

[0135] Given the predicted results of the insulin-Fc fusion proteins of SEQ ID NO:28 and SEQ ID NO:30, in a further attempt to generate an insulin-Fc fusion protein with strong insulin receptor affinity (where affinity or strength of binding to the insulin receptor is relative to a reference insulin-Fc fusion protein) and the ability to down-regulate the insulin receptor in HCT-116 cells, three insulin-Fc fusion proteins were designed in which the B-chain mutation B10D was restored in addition to the A-chain mutation A8H, and there was no C-chain peptide linker between the B-chain and A-chain, i.e., the B-chain was directly linked to the A-chain without a C-chain peptide linker. The first of these three insulin-Fc fusion proteins is SEQ ID NO:22. As shown in Table A, the insulin-Fc fusion protein of SEQ ID NO: 22 comprises a B chain of SEQ ID NO: 36 bearing a histidine to aspartic acid mutation at B10 (B10D) from the insulin-Fc fusion protein of SEQ ID NO: 1, no C chain peptide sequence, and an A chain of SEQ ID NO: 10 bearing a threonine to histidine mutation at A8 (A8H) from the insulin-Fc fusion protein of SEQ ID NO: 1. FVNQHLCGSDLVEALALVCGEEGFFYTDK (SEQ ID NO: 36) GIVEQCCHSICSLYQLENYCN (SEQ ID NO: 10)

[0136] The resulting insulin polypeptide is shown in SEQ ID NO:42. FVNQHLCGSDLVEALALVCGEEGFFYTDKGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 42)

[0137] The insulin polypeptide of SEQ ID NO: 42 is linked to the human IgG1 Fc fragment of SEQ ID NO: 34 via a peptide linker of SEQ ID NO: 11 (GGGGAGGGG). DQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 34)

[0138] The resulting insulin-Fc fusion protein of SEQ ID NO: 22 is shown below. FVNQHLCGSDLVEALALVCGEEGFFYTDKGIVEQCCHSICSLYQLENYCNGGGGAGGGGDQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 22)

[0139] The second of the three insulin-Fc fusion proteins that restores the B-chain mutation B10D in addition to the A-chain mutation A8H and lacks a C-chain peptide linker between the B and A chains is SEQ ID NO: 24. As shown in Table A, the insulin-Fc fusion protein of SEQ ID NO: 24 comprises a B-chain of SEQ ID NO: 37 bearing the histidine to aspartic acid mutation at B10 (B10D) from the insulin-Fc fusion protein of SEQ ID NO: 1, no C-chain peptide sequence, and an A-chain of SEQ ID NO: 10 bearing the threonine to histidine mutation at A8 (A8H) from the insulin-Fc fusion protein of SEQ ID NO: 1. FVNQHLCGSDLVEALALVCGEAGFFYTDK (SEQ ID NO: 37) GIVEQCCHSICSLYQLENYCN (SEQ ID NO: 10)

[0140] The resulting insulin polypeptide is shown in SEQ ID NO:43. FVNQHLCGSDLVEALALVCGEAGFFYTDKGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 43)

[0141] The insulin polypeptide of SEQ ID NO: 43 is linked to the human IgG1 Fc fragment of SEQ ID NO: 34 via a peptide linker of SEQ ID NO: 11 (GGGGAGGGG). DQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 34)

[0142] The resulting insulin-Fc fusion protein of SEQ ID NO: 24 is shown below. FVNQHLCGSDLVEALALVCGEAGFFYTDKGIVEQCCHSICSLYQLENYCNGGGGAGGGGDQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 24)

[0143] The third of the three insulin-Fc fusion proteins that restores the B-chain mutation B10D in addition to the A-chain mutation A8H and lacks a C-chain peptide linker between the B and A chains is SEQ ID NO: 26. As shown in Table A, the insulin-Fc fusion protein of SEQ ID NO: 26 comprises a B-chain of SEQ ID NO: 38 bearing a histidine to aspartic acid mutation at B10 (B10D) from SEQ ID NO: 1, no C-chain peptide sequence, and an A-chain of SEQ ID NO: 10 bearing a threonine to histidine mutation at A8 (A8H) from the insulin-Fc fusion protein of SEQ ID NO: 1. FVNQHLCGSDLVEALALVCGEEGFFYTDR (SEQ ID NO: 38) GIVEQCCHSICSLYQLENYCN (SEQ ID NO: 10)

[0144] The resulting insulin polypeptide is shown in SEQ ID NO:44. FVNQHLCGSDLVEALALVCGEEGFFYTDRGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 44)

[0145] The insulin polypeptide of SEQ ID NO: 44 is linked to the human IgG1 Fc fragment of SEQ ID NO: 34 via a peptide linker of SEQ ID NO: 11 (GGGGAGGGG). DQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 34)

[0146] The resulting insulin-Fc fusion protein of SEQ ID NO: 26 is shown below. FVNQHLCGSDLVEALALVCGEEGFFYTDRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 26)

[0147] Insulin-Fc fusion proteins of SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 26 were produced according to Example 1a. The yields obtained were acceptable and are shown in Table 2 below.

[0148] [Table 2]

[0149] The insulin-Fc fusion proteins of SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26 are purified according to Example 2b, and the structures of the insulin-Fc fusion proteins of SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26 are confirmed according to Example 3b and characterized according to Example 4. The homodimer percentage is determined according to Example 5b and is expected to be acceptable if it is greater than 80%.

[0150] FIG. 10 shows a side-by-side comparison of the insulin-Fc fusion proteins of SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26 with the insulin-Fc fusion protein of SEQ ID NO: 16. * " represents a perfect homology in all sequences at a given sequence position, and ":", "." or a space refers to conservative, moderate, or very different amino acid mutations in the sequences at a given sequence position, respectively.

[0151] Unexpectedly, testing of the insulin-Fc fusion proteins according to Example 6b with SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26 is expected to reveal that despite the conservation of both the B10D and A8H mutations of the insulin-Fc fusion protein of SEQ ID NO: 1, the insulin-Fc fusion proteins of SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26 do not have strong affinity for the insulin receptor in vitro (where the affinity or strength of binding to the insulin receptor is relative to the reference insulin-Fc fusion protein). The presence of the C chain peptide between the B and A chains appears to be a necessary part of the insulin-Fc fusion protein structure in order to obtain strong affinity for the insulin receptor (where the affinity or strength of binding to the insulin receptor is relative to the reference insulin-Fc fusion protein). When tested according to the protocol of Example 7b, the insulin-Fc fusion proteins of SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:26 are not expected to cause substantial downregulation of IR in HCT-116 cells at any of the concentrations tested, nor are they expected to induce lower levels of Akt phosphorylation than RHI at any of the concentrations tested.

[0152] Studies in nude mice according to Example 9b carried out in the HCT-116 xenograft model and studies according to Example 10b using an in vivo model of a metastatic human melanoma cell line (WM266.4) are expected to show that neither the insulin-Fc fusion protein of SEQ ID NO: 22, nor the insulin-Fc fusion protein of SEQ ID NO: 24, nor the insulin-Fc fusion protein of SEQ ID NO: 26 is able to slow tumor growth compared to controls under fasting or non-fasting conditions.

[0153] As previously shown, the insulin-Fc fusion protein of SEQ ID NO: 1 exhibits strong affinity for the insulin receptor (where the affinity or strength of binding to the insulin receptor is relative to a reference insulin-Fc fusion protein) and the ability to down-regulate the insulin receptor in HCT-116 cells, and the insulin-Fc fusion protein of SEQ ID NO: 16 is expected to achieve the same results. However, in previous studies of insulin-Fc fusion proteins in dogs and cats, it was unexpectedly found that the specific amino acid mutations B10D and A8H that conferred strong insulin receptor binding (where the affinity or strength of binding to the insulin receptor is relative to a reference insulin-Fc fusion protein) also resulted in the development of neutralizing anti-drug antibodies after repeated subcutaneous injections in target animals (e.g., dogs or cats). These anti-drug antibodies unacceptably reduced the ability of the insulin-Fc fusion protein to interact with the insulin receptor after multiple injections, rendering the relevant insulin-Fc fusion protein unusable for continuous therapy. Specifically, during the course of the present disclosure, it was discovered that the A8 to histidine and B10 to aspartic acid mutations account for the majority of anti-drug antibody specificity and therefore represent immunogenic "hot spots" (e.g., immunogenic epitopes) on the insulin polypeptide. The insulin-Fc fusion proteins of SEQ ID NO: 1 and SEQ ID NO: 16 are tested to assess their propensity to generate anti-drug antibodies according to Example 12. Significant inhibition of the anti-drug antibody signal is expected to be observed in drug-inhibited wells, indicating that the anti-drug antibodies are specific for the therapeutic compound.

[0154] The insulin-Fc fusion proteins of SEQ ID NO: 1 and SEQ ID NO: 16 are further analyzed for identification of immunogenic epitopes according to Example 13. By correlating the antibody concentrations obtained from the assay with the known amino acid composition of the coated insulin-Fc fusion protein library, it is determined that amino acid mutations of insulin at position 10 on the insulin B chain (B10D) and position 8 on the insulin A chain (A8H) account for some, most, or all of the total antibody signal in the assay and are expected to exhibit weak or strong binding to various insulin-Fc fusion protein homodimers. The amino acid mutations at position 10 on the insulin B chain (B10D) and position 8 on the insulin A chain (A8H) are expected to represent immunogenic "hot spots."

[0155] In a further attempt to preserve the efficacy of the insulin polypeptide of SEQ ID NO: 1, which has been shown to have strong affinity for the insulin receptor (where affinity or binding strength for the insulin receptor is relative to a reference insulin-Fc fusion protein), without the risk of anti-drug antibody generation, it is desirable to deglycosylate or prevent glycosylation of the Fc fragment during synthesis in host cells. Human IgG1 fragments contain a conserved asparagine (N)-glycosylation site in the CH2 domain of each heavy chain of the Fc region. Here, the notation used to refer to the conserved N-glycosylation site is "cNg." One way to remove attached carbohydrates from a synthesized insulin-Fc fusion protein is to mutate the cNg site to completely prevent carbohydrate attachment during production in host cells. Here, the notation used for the cNg mutation is cNg-(substituted amino acid). For example, if the asparagine at the cNg site is mutated to serine, this mutation is designated "cNg-S."

[0156] The insulin-Fc fusion protein of SEQ ID NO: 18 was designed by retaining the insulin polypeptide of the insulin-Fc fusion protein of SEQ ID NO: 1, which was shown to have strong affinity for the insulin receptor (in this case, affinity or strength of binding to the insulin receptor is relative to a reference insulin-Fc fusion protein). The insulin polypeptide of the insulin-Fc fusion protein of SEQ ID NO: 18 contains mutations in the B chain derived from native human RHI insulin (SEQ ID NO: 5). Specifically, B10 is mutated to aspartic acid (D), B16 is mutated to alanine (A), B28 is mutated to aspartic acid (D), and B29 is mutated to proline (P). The B chain of the insulin-Fc fusion protein of SEQ ID NO: 18 (SEQ ID NO: 7) is shown below. Additionally, there are mutations in the A chain of the insulin-Fc fusion protein of SEQ ID NO: 18 (specifically, A8 is mutated to histidine (H)), also shown below. FVNQHLCGSDLVEALALVCGERGFFYTDPT (SEQ ID NO: 7) GIVEQCCHSICSLYQLENYCN (SEQ ID NO: 10)

[0157] The B chain of the insulin-Fc fusion protein and the A chain of the insulin-Fc fusion protein of SEQ ID NO: 18 are linked via a C chain peptide comprising the amino acid sequence GGGPRR (SEQ ID NO: 8). The resulting insulin polypeptide is shown below. FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 6)

[0158] The Fc fragment of the insulin-Fc fusion protein of SEQ ID NO: 18 has an asparagine to serine mutation (cNg-S) at the conserved glycosylation site cNg. The Fc fragment of the insulin-Fc fusion protein of SEQ ID NO: 18 is SEQ ID NO: 32 and is shown below. DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32)

[0159] The Fc fragment (SEQ ID NO: 32) of the insulin-Fc fusion protein of SEQ ID NO: 18 is linked to the insulin polypeptide (SEQ ID NO: 6) of the insulin-Fc fusion protein of SEQ ID NO: 18 via the peptide linker GGGGAGGGG (SEQ ID NO: 11) to create the insulin-Fc fusion protein of SEQ ID NO: 18. The B10D and A8H mutations on the B and A chains, respectively, of the insulin polypeptide of the insulin-Fc fusion protein of SEQ ID NO: 18 have been demonstrated, through testing with other insulin-Fc fusion proteins, to be the necessary mutations for achieving insulin receptor binding affinity high enough to achieve downregulation of insulin receptors at the cell surface. Further in vivo studies are expected to demonstrate that the insulin-Fc fusion protein of SEQ ID NO: 18 achieves adequate glycemic control in vivo without causing hyperglycemia or hypoglycemia. In some examples, the required insulin receptor binding affinity is determined by measuring the IR binding IC of the fusion protein to the RHI, as described in detail in Example 6a. 50 This is achieved when the ratio is less than 20. The resulting insulin-Fc fusion protein of SEQ ID NO: 18 is shown below. FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 18)

[0160] In a further attempt to retain the efficacy of the insulin polypeptide of the insulin-Fc fusion protein of SEQ ID NO: 1, which was shown to have strong affinity for the insulin receptor (where affinity or strength of binding to the insulin receptor is relative to a reference insulin-Fc fusion protein), without the risk of anti-drug antibody generation, the insulin-Fc fusion protein of SEQ ID NO: 20 was designed. The insulin polypeptide of the insulin-Fc fusion protein of SEQ ID NO: 20 contains mutations in the B chain from native human RHI insulin (SEQ ID NO: 5). Specifically, B10 is mutated to aspartic acid (D), B16 is mutated to alanine (A), B28 is mutated to aspartic acid (D), and B29 is mutated to proline (P). The B chain of the insulin-Fc fusion protein of SEQ ID NO: 20 (SEQ ID NO: 7) is shown below. Additionally, there are mutations in the A chain of the insulin-Fc fusion protein of SEQ ID NO: 20 (specifically, A8 is mutated to histidine (H)), also shown below. FVNQHLCGSDLVEALALVCGERGFFYTDPT (SEQ ID NO: 7) GIVEQCCHSICSLYQLENYCN (SEQ ID NO: 10)

[0161] The B chain of the insulin-Fc fusion protein of SEQ ID NO: 20 and the A chain of the insulin-Fc fusion protein are linked via a C chain peptide comprising the amino acid sequence GGGPRR (SEQ ID NO: 8). The resulting insulin polypeptide is shown below. FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 6)

[0162] The Fc fragment of the insulin-Fc fusion protein of SEQ ID NO: 20 has an asparagine to glutamine mutation (cNg-Q) at the conserved glycosylation site cNg. The Fc fragment of the insulin-Fc fusion protein of SEQ ID NO: 20 is SEQ ID NO: 33 and is shown below. DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 33)

[0163] The Fc fragment of the insulin-Fc fusion protein of SEQ ID NO:20 (SEQ ID NO:33) is linked to the insulin polypeptide of the insulin-Fc fusion protein of SEQ ID NO:20 (SEQ ID NO:6) via the peptide linker GGGGAGGGG (SEQ ID NO:11) to create the insulin-Fc fusion protein of SEQ ID NO:20. The B10D and A8H mutations on the B and A chains, respectively, of the insulin polypeptide of the insulin-Fc fusion protein of SEQ ID NO:20 have been demonstrated, through testing with other insulin-Fc fusion proteins, to be the necessary mutations for achieving insulin receptor binding affinity high enough to achieve downregulation of insulin receptors at the cell surface. Further in vivo studies are expected to demonstrate that the insulin-Fc fusion protein of SEQ ID NO:20 achieves adequate glycemic control in vivo without causing hyperglycemia or hypoglycemia. In some examples, the required insulin receptor binding affinity is determined by measuring the IR binding IC of the fusion protein to the RHI, as described in detail in Example 6a. 50 This is achieved when the ratio is less than 20. The resulting insulin-Fc fusion protein of SEQ ID NO: 20 is shown below. FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 20)

[0164] The insulin-Fc fusion proteins of SEQ ID NO: 18 and SEQ ID NO: 20 were produced according to Example 1b and purified according to Example 2b. The structures of the insulin-Fc fusion proteins of SEQ ID NO: 18 and SEQ ID NO: 20 are confirmed according to Example 3b and characterized according to Example 4. The homodimer percentage of each insulin-Fc fusion protein is determined according to Example 5b. Testing of the insulin-Fc fusion proteins of SEQ ID NO: 18 and SEQ ID NO: 20 according to Example 6b is expected to reveal that, similar to the insulin-Fc fusion protein of SEQ ID NO: 1, the insulin-Fc fusion proteins of SEQ ID NO: 18 and SEQ ID NO: 20 have strong affinity for the insulin receptor in vitro (where the affinity or strength of binding to the insulin receptor is relative to the reference insulin-Fc fusion protein).

[0165] FIG. 12 shows a side-by-side comparison of the insulin-Fc fusion proteins of SEQ ID NO: 1, SEQ ID NO: 18, and SEQ ID NO: 20, the insulin-Fc fusion protein of SEQ ID NO: 28, and the insulin-Fc fusion protein of SEQ ID NO: 30. * " represents a perfect homology in all sequences at a given sequence position, and ":", "." or a space refers to conservative, moderate, or very different amino acid mutations in the sequences at a given sequence position, respectively.

[0166] Testing of the insulin-Fc fusion proteins of SEQ ID NO: 18 and SEQ ID NO: 20 according to Example 6b is expected to reveal that, similar to the insulin-Fc fusion protein of SEQ ID NO: 1, the insulin-Fc fusion proteins of SEQ ID NO: 18 and SEQ ID NO: 20 have strong affinity for the insulin receptor in vitro (where affinity or strength of binding to the insulin receptor is relative to a reference insulin-Fc fusion protein). When tested according to the protocol of Example 7b, the insulin-Fc fusion proteins of SEQ ID NO: 18 and SEQ ID NO: 20 are expected to cause substantial down-regulation of IR in HCT-116 cells at all concentrations tested and are expected to induce lower levels of Akt phosphorylation than RHI at all concentrations tested.

[0167] Studies in nude mice according to Example 9b, performed in the HCT-116 xenograft model, and studies according to Example 10b using an in vivo model of a metastatic human melanoma cell line (WM266.4), are expected to show that the insulin-Fc fusion proteins of SEQ ID NO: 18 and SEQ ID NO: 20 can slow tumor growth compared to controls under fasting or non-fasting conditions. Exemplary fusion proteins and their domains and sequences are shown in Table A.

[0168] The full-length sequences of the fusion proteins designed in this description and their corresponding cDNA sequences are shown below. SEQ ID NO:1: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:2 (cDNA sequence of SEQ ID NO:1): ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCGACCTGTGGAAGCTCTGGCTTCGTGTGCGGCGAGCGGGGCTTCTTCTACACCGATCCCACTGGAGGCGGTCCACGCAGAGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCTGTACCAGCTGGAAAACTACTGCAATGGCGGA GGTGGTGCAGGAGGCGGTGGAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTCTCCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCGTGATGCATGAGGCTCTGCACAACCACTACACGCCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG sequence number 16: FVNQHLCGSDLVEALALVCGEEGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 17 (cDNA sequence of SEQ ID NO: 16): ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCGACCTGTGGAAGCTCTGGCTCTCGTGTGCGGCGAGGGCTTCTTCTACACCGATCCCACTGGAGGCGGTCCACGCAGAGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCTGTACCAGCTGGAAAACTACTGCAATGGCGGA GGTGGTAGCGGAGGCGGTGGAGAAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTCTCCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCGTGATGCATGAGGCTCTGCACAACCACTACACGCCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG sequence number 18: FVNQHLCGSDLVEALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 19 (cDNA sequence of SEQ ID NO: 18): ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCGACCTGTGGAAGCTCTGGCTTCGTGTGCGGCGAGCGGGGCTTCTTCTACACCGATCCCACTGGAGGCGGTCCACGCAGAGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCTGTACCAGCTGGAAAACTACTGCAATGGCGGA GGTGGTGCAGGAGGCGGTGGAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTCTCCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAGCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCGTGATGCATGAGGCTCTGCACAACCACTACACGCCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG sequence number 20: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 21 (cDNA sequence of SEQ ID NO: 20): ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCGACCTGTGGAAGCTCTGGCTTCGTGTGCGGCGAGCGGGGCTTCTTCTACACCGATCCCACTGGAGGCGGTCCACGCAGAGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCTGTACCAGCTGGAAAACTACTGCAATGGCGGA GGTGGTGCAGGAGGCGGTGGAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTCTCCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATATGCCAAGACAAAGCCG CGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCGTGATGCATGAGGCTCTGCACAACCACTACACGCCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG sequence no. 22 FVNQHLCGSDLVEALALVCGEEGFFYTDKGIVEQCCHSICSLYQLENYCNGGGGAGGGGDQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 23 (cDNA sequence of SEQ ID NO: 22): ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCGACCTGTGGAAGCTCTGGCTCTCGTGTGCGGCGAGGGCTTCTTCTACACCGACAAGGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCTGTACCAGCTGGAAAACTACTGCAATGGCGGAGGTGGTGCAGGAGGC GGTGGAGACCAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGGAGC AGTACCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG sequence number 24: FVNQHLCGSDLVEALALVCGEAGFFYTDKGIVEQCCHSICSLYQLENYCNGGGGAGGGGDQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 25 (cDNA sequence of SEQ ID NO: 24): ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCGACCTGTGGAAGCTCTGGCTCTCGTGTGCGGCGAGGCAGGCTTCTTCACCGACAAGGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCTGTACCAGCTGGAAAACTACTGCAATGGCGGAGGTGGTGCAGGAGGC GGTGGAGACCAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGGAGC AGTACCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG sequence number 26: FVNQHLCGSDLVEALALVCGEEGFFYTDRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 27 (cDNA sequence of SEQ ID NO: 26): ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCGACCTGTGGAAGCTCTGGCTCTCGTGTGCGGCGAGGGCTTCTTCTACACCGACCGAGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCTGTACCAGCTGGAAAACTACTGCAATGGCGGAGGTGGTGCAGGAGGC GGTGGAGACCAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGGAGC AGTACCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG sequence number 28: FVNQHLCGSHLVEALALVCGEEGFFYTDKGIVEQCCHSICSLYQLENYCNGGGGAGGGGDQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 29 (cDNA sequence of SEQ ID NO: 28): ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCCACCTGTGGAAGCTCTGGCTCTCGTGTGCGGCGAGGGCTTCTTCTACACCGACAAGGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCTGTACCAGCTGGAAAACTACTGCAATGGCGGAGGTGGTGCAGGAGGC GGTGGAGACCAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGGAGC AGTACCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG sequence number 30: FVNQHLCGSHLVEALALVCGEEGFFYTDRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDQTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 31 (cDNA sequence of SEQ ID NO: 30): ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGCCCACCTGGTGGAAGCTCTGGCTCTCGTGTGCGGCGAGGGCTTCTTCTACACCGACAGGGGCATCGTGGAACAGTGCTGCCACTCCATCTGCTCCCGTACCAGCTGGAAAACTACTGCAATGGCGGAGGTGGTGCAGGAGGC GGTGGAGACCAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGGAGC AGTACCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG sequence number 47: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 48 (cDNA sequence of SEQ ID NO: 47): ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCTTCGTGAACCAGCACCTGTGCGGCTCCCACCTGGTGGAAGCTCTGGAACTCGTGTGCGGCGAGCGGGGCTTCCACTACGGGGGTGGCGGAGGAGGTTCTGGTGGCGGCGGAGGCATCGTGGAACAGTGCTGCACCTCCACCTGCTCCCTGGACCAGCTGGAAAACTACTGCGGTGGCGGAGGTGGTCAAGGAGGCGGTGGACAGGGTGGAGGTGGGCAGGGAGGAGGCGGGGGAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG

[0169] [Table 3-1] [Table 3-2] [Table 3-3]

[0170] The "full-length amino acid sequences" of the insulin-Fc fusion proteins of SEQ ID NO: 18 (Figure 2) and SEQ ID NO: 20 (Figure 3) include a leader sequence. In embodiments, the fusion proteins described herein do not include a leader sequence at the N-terminus. In embodiments, the fusion proteins described herein include a leader sequence, e.g., at the N-terminus. An exemplary leader sequence includes the amino acid sequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 14). In embodiments, the fusion proteins described herein are encoded by a nucleic acid molecule comprising a leader sequence, e.g., for expression (e.g., recombinant expression) in a cell (e.g., a eukaryotic cell, e.g., a mammalian cell). In embodiments, the leader sequence is part of the fusion protein within the cell, and then the leader sequence is cleaved via a process (e.g., an enzymatic process) during expression of the fusion protein into cell culture medium, e.g., in the cell or cell culture.

[0171] Exemplary nucleic acid sequences encoding leader sequences include the following nucleic acid sequences: ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCC (SEQ ID NO: 15)

[0172] In embodiments, the fusion proteins described herein are encoded by nucleic acid molecules that do not include a leader sequence.

[0173] In some embodiments, the fusion protein is in a preparation. In embodiments, the preparation has a percent dimer, e.g., homodimer, of the fusion protein of greater than about 50%, e.g., greater than about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, 95%, or about 100%. In embodiments, the percent dimer, e.g., homodimer, of the fusion protein preparation is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the percent homodimer is about 70% or greater (e.g., 80%, 85%, or 88% or greater), and can be increased to 90% or greater (e.g., 95%, 97%, 98%, 99%, or approaching 100%) using one or more processing steps (e.g., ion exchange chromatography, gel filtration, hydrophobic interaction chromatography, etc.). In some embodiments, the percent dimer, e.g., homodimer, in the preparation is determined by size exclusion chromatography (see Examples 5a and 5b), an analytical separation method that can distinguish between dimers, e.g., homodimers, and higher order non-covalent Fc-fusion protein aggregates (e.g., multimers). In some embodiments, the percent dimer, e.g., homodimer, is determined to be greater than 95%, e.g., as determined by size exclusion chromatography. In some embodiments, the percent dimer, e.g., homodimer, is determined to be greater than 99%, e.g., as determined by size exclusion chromatography. In some embodiments, insulin-Fc fusion proteins that have a substantially greater homodimer content than other insulin-Fc fusion proteins exhibit greater biological activity in subjects (eg, humans).

[0174] Fusion protein production In some embodiments, the fusion protein can be expressed by a vector such as those described in the Examples column.

[0175] Expression and purification In some embodiments, the fusion protein can be recombinantly expressed in, for example, eukaryotic cells, such as mammalian or non-mammalian cells. Exemplary mammalian cells used for expression include HEK cells, e.g., HEK293 cells, or CHO cells. In some embodiments, the cells are transfected with a nucleic acid molecule, e.g., a vector, encoding the fusion protein (e.g., the entire fusion protein is encoded by a single nucleic acid molecule). In other embodiments, the cells are transfected with multiple nucleic acid molecules, where each nucleic acid molecule encodes a different domain of the fusion protein. For example, one nucleic acid molecule can encode an insulin polypeptide and a different nucleic acid molecule can encode an Fc fragment. The cells can be cultured using standard methods in the art.

[0176] In some embodiments, the fusion protein is purified or isolated from the cells (e.g., by lysis of the cells). In other embodiments, the fusion protein is secreted by the cells, e.g., the fusion protein is purified or isolated from the cell culture medium in which the cells were grown. Purification of the fusion protein can involve the use of column chromatography, e.g., affinity chromatography, or separation methods involving size, charge, and / or affinity for a particular molecule. In several embodiments, purification of the fusion protein involves selecting for or enriching proteins having an Fc fragment, e.g., by using Protein A beads or a Protein A column, which allow proteins containing an Fc fragment to bind with high affinity to Protein A covalently bound to the Protein A beads at a neutral solution pH. The bound Fc-fusion protein is then eluted from the Protein A beads by changing a solution variable (e.g., decreasing the solution pH). Alternatively or additionally, other separation methods, such as ion exchange chromatography and / or gel filtration chromatography, can also be employed. In several embodiments, purification of the fusion protein further includes filtering or centrifuging the protein preparation. In embodiments, further purification of the fusion protein includes diafiltration, ultrafiltration, and filtration through porous membranes of various sizes, and final formulation with excipients.

[0177] The purified fusion protein can be characterized, for example, for purity, yield, structure, and / or activity, using various methods, such as absorbance at 280 nm (e.g., to determine yield), size exclusion or capillary electrophoresis (e.g., to determine molecular weight, percent aggregation, and / or purity), mass spectrometry (MS) and / or liquid chromatography (LC-MS) (e.g., to determine purity), and / or ELISA (e.g., to determine the extent of binding, e.g., affinity, to anti-insulin antibodies). Exemplary characterization methods are also described in the Examples section.

[0178] Functional characteristics of the fusion protein Described herein are methods for interacting with the human insulin receptor to reduce cancer tumor growth rates in mammals. These methods comprise administering a fusion protein (e.g., a fusion protein described herein) to a subject. In embodiments, the fusion proteins described herein can reduce glucose levels (e.g., blood glucose levels) after administration to a subject. In embodiments, the glucose-lowering activity of the fusion protein is greater than that of an insulin reference standard. In some embodiments, the duration of activity of the fusion protein can be measured by a reduction, e.g., a statistically significant reduction, in blood glucose relative to pre-administration levels.

[0179] In some embodiments, the duration of activity of the fusion protein (e.g., the time during which there is a statistically significant decrease in blood glucose levels in a subject relative to pre-administration levels) is greater than about 2 hours. In some embodiments, the duration of activity of the fusion protein (e.g., the time during which there is a statistically significant decrease in blood glucose levels in a subject relative to pre-administration levels) is greater than about 2 hours, 6 hours, 9 hours, 12 hours, 18 hours, 1 day, 1.5 days, 2 days, 2.2 days, 2.5 days, 3 days, 5 days, 7 days, 8 days, 9 days, 10 days, or more. In some embodiments, the duration of activity of the fusion protein (e.g., the time during which there is a statistically significant decrease in blood glucose levels in a subject relative to pre-administration levels) is greater than the duration of activity of an insulin reference standard or control formulation.

[0180] Pharmaceutical Compositions and Routes of Administration Provided herein is a pharmaceutical composition comprising the fusion protein described herein, which can be used to lower blood glucose in humans.The amount and concentration of the fusion protein in the pharmaceutical composition and the amount of the pharmaceutical composition administered to a subject can be selected based on clinically relevant factors, such as the medically relevant characteristics of the subject (for example, age, weight, sex, other medical conditions, etc.), the solubility of the compound in the pharmaceutical composition, the potency and activity of the compound, and the method of administration of the pharmaceutical composition.For further information on administration route and administration schedule, readers can refer to Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990, Volume 5, Chapter 25.3.

[0181] Formulations of the present disclosure include those suitable for parenteral administration. The phrases "parenteral administration" and "administered parenterally," as used herein, refer to modes of administration other than enteral and topical administration, usually by intravenous or subcutaneous injection.

[0182] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants, such as Tween-like surfactants. In some embodiments, the pharmaceutical compositions (e.g., as described herein) comprise a Tween-like surfactant, such as Tween-20 or Tween-80. In some embodiments, the pharmaceutical compositions (e.g., as described herein) comprise a Tween-like surfactant, such as Tween-80, at a concentration of about 0.001% to about 2%, or about 0.005% to about 0.1%, or about 0.01% to about 0.5%.

[0183] In some embodiments, the fusion protein is administered as a bolus, infusion, or intravenous push. In some embodiments, the fusion protein is administered via a syringe, pump, pen, needle, or indwelling catheter. Also, methods of delivery using rechargeable or biodegradable devices can be provided. For the controlled delivery of drugs, including proteinaceous biopharmaceuticals, various sustained-release polymeric devices have been developed and tested in vivo in recent years. Various biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants for sustained release of compounds at specific target sites.

[0184] dose The actual dosage level of the fusion protein can be varied to provide an amount of active ingredient effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend on a variety of factors, including the activity of the particular fusion protein, or ester, salt, or amide thereof, employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular fusion protein employed, the age, sex, weight, condition, health, and medical history of the person being treated, and factors well known in the medical field.

[0185] Generally, an appropriate dose of a fusion protein is the minimum amount of fusion protein effective to produce a therapeutic effect. Such an effective amount generally varies depending on the factors described above. In general, intravenous and subcutaneous doses of a fusion protein for a subject range from about 150 to about 1500 micrograms per kilogram of body weight per day.

[0186] The present disclosure contemplates formulating the fusion protein in any of the aforementioned pharmaceutical compositions and formulations. Furthermore, the present disclosure contemplates administration via any of the aforementioned administration routes. Those skilled in the art can select an appropriate formulation and administration route based on the condition being treated and the overall health, age, and size of the patient being treated. [Example]

[0187] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way.

[0188] Example 1a: Synthesis and production of insulin-Fc fusion proteins in HEK cells Insulin-Fc fusion proteins were synthesized as follows: The gene sequence of interest was constructed using proprietary software (LakePharma, Belmont, CA) and cloned into a high-expression mammalian vector. HEK293 cells were seeded into shake flasks 24 hours before transfection and grown in chemically defined, serum-free medium. DNA expression constructs encoding the insulin-Fc fusion proteins of interest were transiently transfected into 2 L of HEK293 cell suspension using Syd Labs' (Natick, MA) standard operating procedure for transient transfection. After 20 hours, cells were counted to determine viability and viable cell number, and titers were measured using a ForteBio® Octet® (Pall ForteBio LLC, Fremont, CA). Additional readings were taken during transient transfection production. Cultures were harvested after day 5.

[0189] Example 1b: Synthesis and production of insulin-Fc fusion proteins in HEK cells Insulin-Fc fusion proteins were synthesized as follows: The gene sequence of interest was constructed using proprietary software (LakePharma, Belmont, CA) and cloned into a high-expression mammalian vector. HEK293 cells were seeded into shake flasks 24 hours before transfection and grown in chemically defined, serum-free medium. DNA expression constructs encoding the insulin-Fc fusion proteins of interest were transiently transfected into 2 L of HEK293 cell suspension using Syd Labs' (Natick, MA) standard operating procedure for transient transfection. After 20 hours, cells were counted to determine viability and viable cell number, and titers were measured using a ForteBio® Octet® (Pall ForteBio LLC, Fremont, CA). Additional readings were taken during transient transfection production. Cultures were harvested after day 5.

[0190] Example 1c: Synthesis and production of insulin-Fc fusion protein in CHO cells The CHO cell line was originally derived from CHO-K1 (LakePharma, Belmont, CA), and the endogenous glutamine synthetase (GS) gene was knocked out by recombinant techniques using methods known in the art. Stable expression DNA vectors were designed, optimized for CHO expression and GS selection, and integrated into a high-expression mammalian vector (LakePharma, Belmont, CA). The sequence of each complete construct was verified before scale-up studies were initiated. Suspension-adapted CHO cells were cultured in a chemically defined medium (CD OptiCHO; Invitrogen, Carlsbad, CA) in a humidified 5% CO2 incubator at 37°C. Serum-free or other animal-derived products were used for CHO cell culture.

[0191] Approximately 80 million suspension-adapted CHO cells growing in logarithmic phase in CD OptiCHO medium are transfected by electroporation using the MaxCyte® STX® system (MaxCyte, Inc., Gaithersburg, MD) with 80 μg of DNA to generate stable CHO cell lines for each insulin-Fc fusion protein (the DNA construct contains the full-length sequence of the insulin-Fc fusion protein). After 24 hours, the transfected cells are counted and placed under selection for stable integration of the insulin-Fc fusion gene. The transfected cells are grown in shake flasks at 0.5 × 10 in CD OptiCHO selection medium containing 0-100 μM methionine sulfoximine (MSX). 6 Seed cells at a cell density of 1000 cells / mL and incubate at 37 °C, 5% CO. During the selection process, spin down the cells and resuspend them in fresh selection medium every 2-3 days until the CHO stable pool regains growth rate and viability. Monitor the cell culture for growth and titer.

[0192] 2.5 x 10 cells per mL 6 Cells were grown to a concentration of 15 x 10 per mL per vial. Viability remained above 95% when harvested for cell banking. Cells were then centrifuged and the cell pellet was resuspended in CD OptiCHO medium containing 7.5% dimethyl sulfoxide (DMSO). 6 The vials are frozen and stored in liquid nitrogen.

[0193] Small-scale production using CHO cells is performed as follows: Cells are scaled up for production at 37°C in CD OptiCHO growth medium containing 100 μM MSX, feeding every 2–4 days as needed, for approximately 14–21 days. CD OptiCHO growth medium is supplemented with glucose and additional amino acids as needed. Conditioned medium supernatant from a stable pool production run is clarified by centrifugation. The protein is applied to a Protein A (MabSelect, GE Healthcare, Little Chalfont, UK) column pre-equilibrated with binding buffer. Wash buffer is then passed through the column until the OD280 value (NanoDrop, Thermo Scientific) is at or near background levels. The insulin-Fc fusion protein is eluted with a low pH buffer, and the eluted fractions are collected and the OD280 value of each fraction is recorded. Fractions containing the desired insulin-Fc fusion protein are pooled and, optionally, further filtered using a 0.2 μM membrane filter.

[0194] This cell line is optionally further subcloned and monoclonalized, and optionally, high-titer insulin-Fc fusion protein-expressing clones are selected using limiting dilution, a method known to those skilled in the art. After obtaining a high-titer monoclonal insulin-Fc fusion protein-expressing cell line, production of the insulin-Fc fusion protein is achieved as described above in growth medium without MSX, or optionally in growth medium containing MSX, to obtain a cell culture supernatant containing CHO-produced recombinant insulin-Fc fusion protein. The MSX concentration is optionally increased over time to further select for clones capable of producing higher product titers.

[0195] Example 2a: Purification of insulin-Fc fusion protein Conditioned medium supernatant containing the secreted Fc fusion protein was collected from transiently transfected HEK production runs and clarified by centrifugation. The supernatant containing the insulin-Fc fusion protein of interest was applied to a Protein A column and washed with various wash buffers containing 0.15-0.50 M sodium chloride, followed by elution using a low pH solution. The eluted protein fractions were then pooled and buffer-exchanged into 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer. A final filtration step was performed using a 0.2 μm membrane filter. The final protein concentration was calculated from the solution optical density at 280 nm. Further purification by ion exchange chromatography (e.g., using anion or cation exchange beaded resins), gel filtration chromatography, or other methods was performed as needed. In some embodiments, insulin-Fc fusions were buffer exchanged via a Zeba gel filtration column (Thermo) into 50 mM sodium phosphate, pH 7.0 buffer, and purified via a Q-HP (Cytiva) ion exchange column operated in flow-through mode to remove molecular aggregates, host cell proteins, and host cell DNA. If necessary, the Q-HP step was followed by a buffer exchange via a Zeba gel filtration column (Thermo) into PBS buffer (25 mM sodium phosphate, 150 mM sodium chloride, pH 7.4).

[0196] As shown in Table 3, insulin-Fc fusions of the present technology synthesized in HEK293 cells demonstrated sufficient potency. It was determined that insulin-Fc fusion proteins of similar structure and composition to those of the present disclosure that exhibit Protein A purified titers of greater than 50 mg / L in transiently transfected HEK293 cells will exhibit higher, commercially viable CHO cell titers when the compounds are expressed using stably transfected CHO cells.

[0197] [Table 4]

[0198] Example 2b: Purification of insulin-Fc fusion protein Purification of insulin-Fc fusion proteins is performed as follows. Conditioned medium supernatant containing the secreted Fc fusion protein is collected from transiently transfected HEK, stably transfected HEK, or stably transfected CHO production runs and clarified by centrifugation. The supernatant containing the insulin-Fc fusion protein of interest is applied to a Protein A column, washed with various wash buffers containing 0.15–0.50 M sodium chloride, and then eluted using a low pH solution. The eluted protein fractions are then pooled and buffer-exchanged into 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer. A final filtration step is performed using a 0.2 μm membrane filter. The final protein concentration is calculated from the solution optical density at 280 nm. Optionally, further purification by ion exchange chromatography (e.g., using anion or cation exchange bead resins), gel filtration chromatography, or other methods is performed as needed. In some embodiments, insulin-Fc fusions are buffer exchanged via a Zeba gel filtration column (Thermo) into 50 mM sodium phosphate, pH 7.0 buffer, and purified via a Q-HP (Cytiva) ion exchange column operated in flow-through mode to remove molecular aggregates, host cell proteins, and host cell DNA. The Q-HP step is optionally followed by a buffer exchange via a Zeba gel filtration column (Thermo) into PBS buffer (25 mM sodium phosphate, 150 mM sodium chloride, pH 7.4).

[0199] Insulin-Fc fusion proteins of SEQ ID NO:18 and SEQ ID NO:20 synthesized in transiently transfected HEK293 cells exhibit Protein-A purified titers of greater than 50 mg / L. When stably transfected into CHO-K1 GSN cells (LakePharma, Belmont, CA), insulin-Fc fusion proteins of SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:47 are expected to exhibit stable pool titers of greater than 200 mg / mL and stable titers of greater than 500 mg / mL.

[0200] Example 3a: Structural confirmation by non-reduced and reduced CE-SDS A solution of purified insulin-Fc fusion protein dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer was subjected to capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) analysis on a LabChip® GXII (Perkin Elmer, Waltham, MA), and electropherograms were plotted. Samples were run against protein standards of known molecular weight (MW) under non-reducing conditions, and the elution peak represented the "apparent" MW of the insulin-Fc fusion protein homodimer.

[0201] Under reducing conditions (e.g., using β-mercaptoethanol to cleave the disulfide bonds of the fusion protein), the apparent MW of the resulting insulin-Fc fusion protein monomer is compared to half the molecular weight of the insulin-Fc fusion protein homodimer as a way to determine the likelihood of correct structural purity of the insulin-Fc fusion protein.

[0202] The major non-reducing and reducing peaks found by CE-SDS analysis of the insulin-Fc fusion proteins of SEQ ID NO: 1 and SEQ ID NO: 47 synthesized in HEK293 cells are shown in Table 4. The apparent MW of the resulting insulin-Fc fusion protein monomer was compared with that of the insulin-Fc fusion protein homodimer by twofold. The results in Table 4 indicate that the structural purity of the insulin-Fc fusion protein is likely to be correct.

[0203] [Table 5]

[0204] Example 3b: Structural confirmation by non-reduced and reduced CE-SDS A solution of purified insulin-Fc fusion protein dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer was subjected to capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) analysis on a LabChip® GXII (Perkin Elmer, Waltham, MA), and the electropherogram is plotted. Samples were run against protein standards of known molecular weight (MW) under non-reducing conditions, and the elution peak represented the "apparent" MW of the insulin-Fc fusion protein homodimer.

[0205] Under reducing conditions (e.g., using β-mercaptoethanol to cleave the disulfide bonds of the fusion protein), the apparent MW of the resulting insulin-Fc fusion protein monomer is compared to half the molecular weight of the insulin-Fc fusion protein homodimer as a way to determine the likelihood of correct structural purity of the insulin-Fc fusion protein.

[0206] For the insulin-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 30, the major non-reducing and reducing peaks found by CE-SDS analysis are expected to indicate a high likelihood of correct structural purity of the insulin-Fc fusion proteins.

[0207] Example 4: Sequence identification by LC-MS using deglycosylation To obtain an accurate estimate of insulin-Fc mass by mass spectrometry (MS), samples were first treated to remove native glycans that could interfere with MS analysis. 100 μL of 2.5 mg / mL insulin-Fc fusion protein dissolved in a buffer solution of 200 mM HEPES, 100 mM NaCl, and 50 mM NaOAc, pH 7.0, was first buffer-exchanged using a Zeba desalting column (ThermoFisher Scientific, Waltham, MA) into 0.1 M Tris, pH 8.0, containing 5 mM EDTA. 1.67 μL of PNGase F enzyme (Prozyme N-glycanase) was added to the solution to remove N-linked glycans present on the fusion protein, and the mixture was incubated overnight at 37°C in an incubator. The sample was then analyzed by LC-MS (Novatia, Newtown, PA), yielding the molecular weight of the desired homodimer without the glycans. This mass is then further corrected because the enzymatic treatment used to cleave the glycan from asparagine also deaminates the asparagine side chain to form aspartic acid, resulting in an overall increase of 2 Da in the enzymatically treated homodimer, corresponding to 1 Da of mass for each chain present in the homodimer. Therefore, the actual molecular weight is calculated by subtracting 2 Da from the measured mass to correct for enzymatic modification of the insulin-Fc fusion protein structure in the analyzed sample. The LC-MS predicted molecular weight data, predicted corrected mass data, and theoretical molecular weight (obtained using the Expasy MW / pI tool) for an exemplary insulin-Fc fusion protein are shown in Table 5.

[0208] [Table 6]

[0209] Example 5a: % Homodimer by Size Exclusion Chromatography Size-exclusion chromatography (SEC-HPLC) of insulin-Fc fusion proteins was performed at 280 nm using a Waters 2795HT HPLC (Waters Corporation, Milford, MA) coupled to a 2998 photodiode array. A sample of 100 μL or less containing the insulin-Fc fusion protein of interest was injected onto a MAbPac SEC-1, 5 μm, 4 × 300 mm column (ThermoFisher Scientific, Waltham, MA) operated 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, while unaggregated homodimers eluted at later retention times. The purity of the insulin-Fc fusion protein solution was confirmed in terms of the percentage of non-aggregated homodimers upon separation of the homodimer mixture from the aggregated multimeric homodimers via analytical SEC-HPLC. Table 6 shows the percentage of homodimers for insulin-Fc fusion proteins produced in HEK293 cells.

[0210] [Table 7]

[0211] Example 5b: % Homodimer by Size Exclusion Chromatography Size-exclusion chromatography (SEC-HPLC) of insulin-Fc fusion proteins was performed at 280 nm using a Waters 2795HT HPLC (Waters Corporation, Milford, MA) coupled to a 2998 photodiode array. A sample of 100 μL or less containing the insulin-Fc fusion protein of interest was injected onto a MAbPac SEC-1, 5 μm, 4 × 300 mm column (ThermoFisher Scientific, Waltham, MA) operated 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) elute at earlier retention times, while unaggregated homodimers elute at later retention times. The purity of the insulin-Fc fusion protein solution was confirmed in terms of the percentage of non-aggregated homodimers upon separation of the mixture of homodimers from aggregated multimeric homodimers via analytical SEC-HPLC.

[0212] The insulin-Fc fusion proteins of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30 are expected to exhibit a homodimer percentage of greater than 80% after the Protein A step of Example 2b. The insulin-Fc fusion proteins of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30 are expected to exhibit a homodimer percentage of greater than 90% after the Protein A step of Example 2b and an additional Q-HP ion exchange column step.

[0213] Example 6a: In vitro binding of insulin-Fc fusion proteins to the IM-9 insulin receptor (IR) at 4°C Human IM-9 cells (ATTC# CCL-159) expressing the human insulin receptor were cultured and maintained in complete RPMI 10% FBS medium to 70-80% confluence. IM-9 cell cultures were centrifuged at 250 × g (approximately 1000 rpm) for 10 minutes to pellet the cells. The cells were washed once with HBSS or PBS buffer and then diluted to 1 × 10 7 Cells were resuspended in cold FACS medium (HBSS / 2 mM EDTA / 0.1% sodium azide + 2% horse serum) to a concentration of 100 cells / mL and kept on ice (4°C) in FACS buffer for 20-30 minutes. Insulin-Fc protein, insulin, or insulin analogs (e.g., test compounds) were serially diluted 1:4 in FACS buffer (approximately 60 µL for each dilution) to 2x concentrations (800 nM, 400 nM, 100 nM, 25 nM, 6.25 nM, 1.57 nM, 0.39 nM) in 1.2 mL tubes and kept on ice at 4°C until ready for pipetting.

[0214] Biotinylated-RHI was diluted to 10 μg / mL in FACS staining medium at a 20x concentration (final concentration: 0.5 μg / mL). 50 μL of serially diluted test compound and 5 μL of 20x biotin-RHI were added to each well of a V-bottom microtiter plate, mixed, and placed on ice. Then, 45 μL of IM-9 cell suspension was added to each well using a multichannel pipette, mixed gently again, and incubated on ice for 30 minutes to competitively bind to the insulin receptor (IR) on the IM-9 cells. The cells were then washed twice with 250 μL of ice-cold FACS wash buffer (HBSS / 2 mM EDTA / 0.1% sodium azide + 0.5% horse serum) by centrifuging the V-bottom plate at 3000 rpm for 3 minutes and aspirating the supernatant. The cells were then resuspended in 50 μL of FACS medium containing 1:200 diluted streptavidin-PE (Life Technologies) and placed on ice for 20 min, washed once with 250 μL of ice-cold FACS buffer, and finally fixed with 4% paraformaldehyde for 10 min.

[0215] The cells were then transferred to FACS tubes and analyzed using a Guava 8-HT flow cytometer (Millipore). Binding of biotinylated-RHI to the insulin receptor was quantified by the median fluorescence intensity (MFI) of the cells on the FACS FL-2 channel and measured for each concentration of test compound. Control wells were labeled with biotinylated-RHI alone and used to calculate the percent inhibition of each test compound concentration. The percent inhibition of biotinylated-RHI binding on IM-9 cells by test compounds was plotted against the logarithmic concentration of each test compound, and the IC was calculated for each test compound using GraphPad Prism (GraphPad Software, La Jolla, CA). 50 The IC values ​​of the test compounds were calculated. 50 Lower values ​​reflected stronger binding to the insulin receptor (where affinity or binding strength to the insulin receptor is relative to a reference insulin-Fc fusion protein). A control compound, such as unlabeled recombinant human insulin (RHI), was also used as an internal standard to measure RHI IC. 50 The IC of the given compound is calculated. 50 can be compared (IC 50 (compound) / IC 50 (RHI)).IC 50 The lower the ratio, the higher the similarity of binding to RHI (stronger binding to insulin receptor), and the higher the IC 50 The higher the ratio, the weaker the binding to the insulin receptor than RHI. The inhibition of biotin-labeled insulin binding to the IM-9 insulin receptor by test compounds (IC 50 nM) and inhibition of biotin-labeled insulin binding to the IM-9 insulin receptor in RHI (IC 50 ; nM) was measured, and the IC 50 The ratio was calculated.

[0216] Current technology fusion proteins are IC 50Note that the binding to the insulin receptor is weaker than that to RHI, as indicated by a ratio greater than 1. The term "strong binding to insulin receptor" refers to a relative comparison of insulin receptor binding ratios to RHI between different insulin-Fc fusion protein compounds. For example, IC 50 An insulin-Fc fusion protein with a ratio of 20 (designated "insulin-Fc fusion protein A") means that the insulin-Fc fusion protein binds to the insulin receptor 20 times less strongly than recombinant human insulin. However, the IC 50 Another insulin-Fc fusion protein (designated "insulin-Fc fusion protein B") with a ratio greater than 100 binds much weaker to the insulin receptor than the one designated "insulin-Fc fusion protein A." In this case, one can say "insulin-Fc fusion protein A exhibits stronger binding to the insulin receptor," or "insulin-Fc fusion protein A exhibits stronger binding to the insulin receptor than a reference insulin-Fc fusion protein," where the reference insulin-Fc fusion protein exhibits stronger binding to the insulin receptor than an IC 50 The ratio may be SEQ ID NO: 47, where the ratio is 142. Alternatively, "Insulin-Fc fusion protein A has a low IC against RHI." 50 Such phrases can be used interchangeably to express that exemplary insulin-Fc fusion proteins of the present technology exhibit relatively strong binding to the insulin receptor compared to other insulin-Fc fusion proteins.

[0217] In vitro IM-9 insulin receptor binding of two batches of insulin-Fc fusion protein of SEQ ID NO: 1 and one batch of insulin-Fc fusion protein of SEQ ID NO: 47 is shown in Table 7. The results show that the insulin-Fc fusion protein of SEQ ID NO: 1 exhibits a higher affinity for the insulin receptor compared to RHI, and that the insulin-Fc fusion protein of SEQ ID NO: 47 has a much lower affinity for the insulin receptor.

[0218] [Table 8]

[0219] Example 6b: In vitro binding of insulin-Fc fusion proteins to the IM-9 insulin receptor (IR) at 4°C Human IM-9 cells (ATTC# CCL-159) expressing the human insulin receptor were cultured and maintained at 70-80% confluence in complete RPMI 10% FBS medium. The IM-9 cell culture was centrifuged at 250 × g (approximately 1000 rpm) for 10 minutes to pellet the cells. The cells were washed once with HBSS or PBS buffer and then collected at 1 × 10 7 Resuspend cells in cold FACS medium (HBSS / 2 mM EDTA / 0.1% sodium azide + 2% horse serum) to a concentration of 1000 cells / mL and keep on ice (4 °C) in FACS buffer for 20-30 min. Insulin-Fc protein, insulin, or insulin analogs (e.g., test compounds) are serially diluted 1:4 in FACS buffer (approximately 60 µL for each dilution) to 2x concentrations (800 nM, 400 nM, 100 nM, 25 nM, 6.25 nM, 1.57 nM, 0.39 nM) in 1.2 mL tubes. Keep tubes on ice at 4 °C until ready for pipetting.

[0220] Biotinylated-RHI was diluted to 10 μg / mL in FACS staining medium at a 20x concentration (final concentration: 0.5 μg / mL). 50 μL of each serially diluted test compound and 5 μL of 20x biotin-RHI were added to each well of a V-bottom microtiter plate, mixed, and placed on ice. 45 μL of IM-9 cell suspension was then added to each well with a multichannel pipette, mixed gently again, and incubated on ice for 30 minutes to allow competitive binding to the insulin receptor (IR) on the IM-9 cells. The cells were then washed twice with 250 μL of ice-cold FACS wash buffer (HBSS / 2 mM EDTA / 0.1% sodium azide + 0.5% horse serum) by centrifuging the V-bottom plate at 3000 rpm for 3 minutes and aspirating the supernatant. The cells were then resuspended in 50 μL of FACS medium containing 1:200 diluted streptavidin-PE (Life Technologies) and placed on ice for 20 min, washed once with 250 μL of ice-cold FACS buffer, and finally fixed with 4% paraformaldehyde for 10 min.

[0221] The cells were then transferred to FACS tubes and analyzed using a Guava 8-HT flow cytometer (Millipore). Binding of biotinylated-RHI to the insulin receptor was quantified by the median fluorescence intensity (MFI) of the cells on the FACS FL-2 channel, measured for each concentration of test compound. Control wells were labeled with biotinylated-RHI alone and used to calculate the percent inhibition of each test compound concentration. The percent inhibition of biotinylated-RHI binding on IM-9 cells by test compounds was plotted against the logarithmic concentration of each test compound, and the IC was calculated for each test compound using GraphPad Prism (GraphPad Software, La Jolla, CA). 50 Calculate the IC value of the test compound. 50 Lower values ​​reflect stronger binding to the insulin receptor. A control compound, such as unlabeled recombinant human insulin (RHI), was also used as an internal standard to measure the RHI IC. 50 The IC of the given compound is calculated. 50 can be compared with (IC 50 (compound) / IC 50(RHI)).IC 50 The lower the ratio, the higher the similarity of binding to RHI (stronger binding to insulin receptor), and the higher the IC 50 The higher the ratio, the weaker the binding to the insulin receptor than RHI. The inhibition of biotin-labeled insulin binding to the IM-9 insulin receptor by test compounds (IC 50 nM) and inhibition of biotin-labeled insulin binding to the IM-9 insulin receptor in RHI (IC 50 ; nM) was measured, and the IC 50 The meaning of the expression "binds more strongly to the insulin receptor" has been described and defined previously.

[0222] The insulin-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 were (IC 50 (compound) / IC 50 The insulin-Fc fusion proteins of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30 are expected to exhibit a ratio of (IC 1C 2C 3C 4C 5C 6C 7C 8C 9C 10C 11C 12C 13C 14C 15C 16C 17C 18C 19C 20C 21C 22D 23C 24D 25C 26D 28D 29C 30E 26D 28D 31C 29E 28C 32C 29E 29C 18C 20E 29C 20E 29C 33C 29E 29C 34C 29E 29C 29C 35C 29E 2 50 (compound) / IC 50 (RHI)) ratio was greater than 50, indicating weak binding to the insulin receptor.

[0223] Example 7a: Downregulation of IR in HCT-116 cells HCT-116 cells were treated in vitro with either RHI, insulin-Fc fusion protein of SEQ ID NO: 1, or insulin-Fc fusion protein of SEQ ID NO: 47 at multiple concentrations (0.05-500 nM). Expression levels of tumor IR, phospho-IR + phospho-IGF1R (e.g., "phospho-IR / IGF1R"), phospho-Akt (S473), pan Akt, and β-actin were measured by Western blot. Tumors were lysed in RIPA buffer, electrophoresed on SDS-PAGE gels, transferred to PVDF membranes using a dry blotting system, and probed for the aforementioned proteins using antibodies from Cell Signaling at 1:1000 and appropriate secondary antibodies known to those skilled in the art. Blots were imaged (cDigit blot scanner, Licor) and evaluated using Image Studio software (Licor).

[0224] Figure 4 shows a Western blot that demonstrates that after 72 hours of treatment, RHI caused observable downregulation of IR in HCT-116 cells. Unexpectedly, the insulin-Fc fusion protein of SEQ ID NO: 1 caused substantial downregulation of IR in HCT-116 cells at all concentrations tested. This is highlighted as 501 in Figure 4. Treatment with the insulin-Fc fusion protein of SEQ ID NO: 1 caused downregulation of IR even at very low concentrations. Furthermore, the insulin-Fc fusion protein of SEQ ID NO: 1 induced lower levels of Akt phosphorylation than RHI at all concentrations tested. This is an important finding, as Akt signaling is involved in cell proliferation and growth, and blockade of Akt signaling through the use of current cancer therapies has been found to correlate with slowed tumor growth. Unexpectedly, Figure 4 shows that the insulin-Fc fusion protein of SEQ ID NO: 47 did not cause downregulation of IR at any of the concentrations tested. This is highlighted as 503 in Figure 4. The insulin-Fc fusion protein of SEQ ID NO: 47 induces lower levels of Akt ph phosphorylation than RHI at all concentrations tested.

[0225] The Western blot in Figure 4 further shows that the insulin-Fc fusion protein of SEQ ID NO: 1, despite downregulating IR, exhibited measurable Phospho-IR / IGF1R. This is highlighted in Figure 4 as 502. This indicates that treatment with the insulin-Fc fusion protein of SEQ ID NO: 1 unexpectedly causes substantial downregulation of IR while simultaneously activating the signaling pathway to the insulin receptor (IR), allowing insulin uptake and reducing the risk of, or completely preventing, hyperglycemia associated with downregulation of IR in other therapeutic approaches (e.g., but not limited to, anti-IR antibodies). Figure 4 also shows that the insulin-Fc fusion protein of SEQ ID NO: 47 did not exhibit measurable Phospho-IR / IGF1R.

[0226] Example 7b: Downregulation of IR in HCT-116 cells HCT-116 cells were treated in vitro with either RHI, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30 insulin-Fc fusion proteins at multiple concentrations (0.05-500 nM). Expression levels of tumor IR, phospho-IR, phospho-IGF1R, phospho-Akt (S473), pan Akt, and β-actin were measured by Western blot. Tumors were lysed in RIPA buffer, electrophoresed on SDS-PAGE gels, transferred to PVDF membranes using a dry blotting system, and probed for the aforementioned proteins using antibodies from Cell Signaling at 1:1000 and appropriate secondary antibodies known to those skilled in the art. Blots were imaged (cDigit blot scanner, Licor) and evaluated using Image Studio software (Licor).

[0227] Western blots generated 72 hours after treatment are expected to show that insulin-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 cause substantial downregulation of IR in HCT-116 cells at all concentrations tested, even at very low concentrations. Furthermore, insulin-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 are expected to induce lower levels of Akt phosphorylation than RHI at all concentrations tested. Furthermore, insulin-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 are expected to exhibit measurable phospho-IR / IGF1R despite downregulating IR. Thus, treatment with insulin-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 is expected to unexpectedly cause substantial downregulation of IR while simultaneously activating the signaling pathway to the insulin receptor (IR) and allowing insulin uptake, reducing the risk of or completely preventing hyperglycemia associated with downregulation of IR in other therapeutic approaches (e.g., but not limited to, anti-IR antibodies).

[0228] Western blots generated after 72 hours of treatment with insulin-Fc fusion proteins of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30 are not expected to show measurable downregulation of insulin receptors, and under therapeutic conditions, the patient's own endogenous insulin would be expected to be able to bind to abundant insulin receptors (IR) and lead to tumor proliferation and growth, which is undesirable.

[0229] Example 8a: In vivo pharmacokinetics (PD) of fusion proteins in mice after a single dose A biologically active fusion protein construct of the insulin-Fc fusion protein of SEQ ID NO: 1 was synthesized according to Example 1a, and its effect on fasting blood glucose levels was evaluated as follows. Naive, non-fasting nude mice were used. On day 0, mice were injected with a single dose of a pharmaceutical composition containing a fusion protein homodimer of the insulin-Fc fusion protein of SEQ ID NO: 1 at a dose of 6 nmol / kg (equivalent to 0.39 mg Fc fusion protein / kg or 1.9 U / kg insulin on a molar basis) in a solution of 50 mM sodium dibasic phosphate, 150 mM sodium chloride, and 0.02% v / v Tween-80 pH 7.5. Blood samples were collected immediately before injection and at 15, 30, 45, 60, 120, 240, 360, 480 minutes and on days 1, 2, 3, 4, 5, 6, 7, and 8 after injection. Blood was drawn from the appropriate vein on day 0, immediately prior to injection, as well as at the remaining time points after treatment.

[0230] At each time point, a minimum of 0.1 mL of whole blood was drawn. Glucose level measurements were immediately determined using a glucose meter (ACCU-CHEK® Aviva Plus), which requires approximately one drop of blood. The mean % fasting blood glucose (%FBGL) from day 0 to day 8 is plotted in Figure 5, allowing the bioactivity of the fusion protein to be determined. Figure 5 shows that the insulin-Fc fusion protein of SEQ ID NO: 1 can lower blood glucose for a significant period of time with a single administration.

[0231] Example 8b: In vivo pharmacokinetics (PD) after a single dose of the fusion protein in mice The insulin-Fc fusion protein constructs of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:47 are synthesized according to Example 1b or Example 1c, and their effects on fasting blood glucose levels are evaluated as follows: Naive, non-fasting nude mice are used. On day 0, mice are injected with a single dose of a pharmaceutical composition containing the insulin-Fc fusion protein homodimer of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:47 in a solution of 50 mM sodium hydrogen phosphate, 150 mM sodium chloride, and 0.02% v / v Tween-80 pH 7.5 at a dose of 6 nmol / kg (equivalent to 0.39 mg Fc fusion protein / kg or 1.9 U / kg insulin on a molar basis). Blood is collected immediately prior to injection and at 15, 30, 45, 60, 120, 240, 360, 480 minutes after injection, and on days 1, 2, 3, 4, 5, 6, 7, and 8. Blood is collected from the appropriate vein on day 0, immediately prior to injection, and at the remaining time points after treatment.

[0232] At each time point, a minimum of 0.1 mL of whole blood is drawn. Glucose level measurements are immediately determined using a glucose meter (ACCU-CHEK® Aviva Plus), which requires approximately one drop of blood. The mean % fasting blood glucose (%FBGL) from day 0 to day 8 is plotted, allowing the bioactivity of the fusion protein to be determined.

[0233] Example 9a: Tumor volume in HCT-116 xenograft model in nude mice HCT-116 cells were cultured under sterile conditions at 37°C and 5% CO2 during logarithmic growth phase. On the day of inoculation, cells were harvested, washed with PBS, and resuspended at the appropriate concentration in a serum-free medium:Matrigel (1:1 vol:vol) mixture. Inoculations were performed on conscious, naive nude mice (n = 60 females) while they were manually restrained. 2 × 10 cells were inoculated into the dorsal right flank of the mice using a 28G needle. 6Cells were injected subcutaneously (SC) in a volume of 200 μL. The injection site was monitored until a tumor was visible / palpable. Once palpable, tumors were measured twice weekly using calipers. Tumor volume was determined using the maximum longitudinal diameter (length) and maximum transverse diameter (width) according to the following formula:

number

[0234] Tumors between 100 and 300 mm 3 When tumor volume reached 100 μg / kg, HCT-116 tumor-bearing mice were randomly assigned to four groups according to Table 8.

[0235] [Table 9]

[0236] Tumor dimensions were measured using calipers before the study, and tumor volume was estimated using the formula above. The tumor volume ratio (TVR) is defined as the ratio of the tumor volume on day X to the tumor volume on day 0. Mice were injected daily for six consecutive days with either vehicle, 150 μg / kg of SEQ ID NO: 1, or 2.5 U / kg of conventional NPH insulin, followed by one day without injections, and then fasted for 8–10 hours per day for a total of three weeks. As controls, parallel groups of treated and untreated mice were not fasted. Tumor volumes were measured before and after the study, and the measurements are shown in Table 9. The tumor volume ratio (TVR) is shown in Figure 6.

[0237] [Table 10]

[0238] Example 9b: Tumor volume in HCT-116 xenograft model in nude mice HCT-116 cells are cultured under sterile conditions at 37°C and 5% CO2 during logarithmic growth phase. On the day of inoculation, cells are harvested, washed with PBS, and resuspended at the appropriate concentration in a serum-free medium:Matrigel (1:1 vol:vol) mixture. Inoculations are performed in conscious, naive nude mice (n = 60 females) while manually restrained. 2 × 10 cells are inoculated into the dorsal right flank of the mice using a 28G needle. 6 Cells are injected subcutaneously (SC) in a volume of 200 μL. The injection site is monitored until a tumor is visible / palpable. Once palpable, the tumor is measured twice weekly using calipers. The maximum longitudinal diameter (length) and maximum transverse diameter (width) are used to determine the tumor volume according to the following formula:

number

[0239] Tumors between 100 and 300 mm 3 Once the tumor volume reaches 100 μg / kg, the HCT-116 tumor-bearing mice are randomly assigned to four groups according to Table 10.

[0240] [Table 11]

[0241] Tumor dimensions were measured using calipers before testing, and tumor volume was estimated using the formula above. The tumor volume ratio (TVR) was defined as the ratio of tumor volume on day X to tumor volume on day 0. Mice were injected daily for six consecutive days with either vehicle, 150 μg / kg of insulin-Fc fusion protein of SEQ ID NO: 47, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, or 2.5 U / kg of conventional NPH insulin, followed by one day without injections and fasting for 8-10 hours per day for a total of three weeks. As controls, parallel groups of treated and untreated mice were kept unfasted. Tumor volumes were measured before and after testing. The tumor volume ratio (TVR) of mice injected with insulin-Fc fusion protein of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 was expected to be at least 30% lower than that of the fasted control group. The tumor volume ratio (TVR) in mice injected with the insulin-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 is expected to be at least 40% lower than the non-fasted control group. The tumor volume ratio (TVR) in mice injected with the insulin-Fc fusion proteins of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 47 is expected to remain similar to the fasted and non-fasted control groups.

[0242] Example 10a: Tumor volume in a metastatic human melanoma cell line (WM266.4) WM266.4 cells were cultured under sterile conditions at 37°C and 5% CO2 during logarithmic growth phase. On the day of inoculation, cells were harvested, washed with PBS, and resuspended at the appropriate concentration in a serum-free medium:Matrigel (1:1 vol:vol) mixture. Inoculations were performed on conscious, naive nude mice (n = 60 females) while they were manually restrained. 2 × 10 cells were inoculated into the dorsal right flank of the mice using a 28G needle. 6 Cells were injected subcutaneously (SC) in a volume of 200 μL. The injection site was monitored until a tumor was visible / palpable. Once palpable, tumors were measured twice weekly using calipers. Tumor volume was determined using the maximum longitudinal diameter (length) and maximum transverse diameter (width) according to the following formula:

number

[0243] Tumors between 100 and 300 mm 3 Once tumor volume reached 1000 mg / kg, WM266.4 tumor-bearing mice were randomly assigned to four groups according to Table 11.

[0244] [Table 12]

[0245] Tumor dimensions were measured using calipers before the study, and tumor volume was estimated using the formula above. The tumor volume ratio (TVR) is defined as the ratio of the tumor volume on day X to the tumor volume on day 0. Mice were injected daily with either vehicle or 150 μg / kg of SEQ ID NO: 1 insulin-Fc fusion protein for 6 consecutive days, followed by one day without injections, and fasted for 8 to 10 hours per day for a total of 3 weeks. As controls, parallel groups of treated and untreated mice were not fasted. Tumor volumes were measured before and after the study, and the measurements are shown in Table 12. The tumor volume ratios (TVR) are shown in Figure 12.

[0246] [Table 13]

[0247] Example 10b: Tumor volume in a metastatic human melanoma cell line (WM266.4) WM266.4 cells are cultured under sterile conditions at 37°C and 5% CO2 during logarithmic growth phase. On the day of inoculation, cells are harvested, washed with PBS, and resuspended at the appropriate concentration in a serum-free medium:Matrigel (1:1 vol:vol) mixture. Inoculations are performed in conscious, naive nude mice (n = 60 females) while manually restrained. Mice are inoculated with 2 × 10 cells into the dorsal right flank using a 28G needle. 6Cells are injected subcutaneously (SC) in a volume of 200 μL. The injection site is monitored until a tumor is visible / palpable. Once palpable, tumors are measured twice weekly using calipers. The maximum longitudinal diameter (length) and maximum transverse diameter (width) are used to determine tumor volume according to the following formula:

number

[0248] Tumors between 100 and 300 mm 3 Once tumor volume reaches 1000 mg / kg, WM266.4 tumor-bearing mice are randomly assigned to four groups according to Table 13.

[0249] [Table 14]

[0250] Tumor dimensions were measured using calipers before testing, and tumor volume was estimated using the formula above. The tumor volume ratio (TVR) was defined as the ratio of tumor volume on day X to tumor volume on day 0. Mice were injected daily for six consecutive days with either vehicle or 150 μg / kg of insulin-Fc fusion protein of SEQ ID NO: 47, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, followed by one day without injections and fasting for 8-10 hours per day for a total of three weeks. As controls, parallel groups of treated and untreated mice were kept unfasted. Tumor volumes were measured before and after testing. The tumor volume ratio (TVR) of mice injected with insulin-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 was expected to be at least 50% lower than that of the fasted control group. The tumor volume ratio (TVR) of mice injected with insulin-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20 is expected to be at least 40% lower compared to non-fasted controls. Furthermore, SEQ ID NO: 18 and SEQ ID NO: 20 are expected to show greater efficacy in reducing tumor volume ratio compared to SEQ ID NO: 1 or SEQ ID NO: 16 due to their reduced immunogenicity due to reduced Fc(γ)R binding (measured as described in Example 14) and reduced C1q binding (measured as described in Example 15). The tumor volume ratio (TVR) of mice injected with insulin-Fc fusion proteins of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 47 is expected to remain comparable to fasted and non-fasted controls.

[0251] Example 11: Tumor volume of human breast cancer cell line (MCF-7L) and tamoxifen-resistant breast cancer cell line (MCF-7L TamR) in vivo without fasting MCF-7L or MCF-7L TamR (TamR = tamoxifen-resistant) cells differ from each other in that MCF-7L cells express significant levels of IR and IGF1R on their cell surface, as determined by Western blot analysis, whereas MCF-7L TamR cells express very low levels of IGF1R and significant levels of IR on their cell surface. These cells are cultured under sterile conditions at 37°C and 5% CO2 during logarithmic growth phase. On the day of inoculation, cells are harvested, washed with PBS, and resuspended at the appropriate concentration in serum-free medium. 1 x 10 cells per inoculation are injected into the second mammary fat pad of female nude mice. 6 Cells are used to implant mice bilaterally. Implants are placed on both the left and right sides of the mouse. The injection site is monitored until the tumor is visible / palpable. Once palpable, the tumor is measured twice weekly using calipers. The maximum longitudinal diameter (length) and maximum transverse diameter (width) are used to determine the tumor volume according to the following formula:

number

[0252] Tumors between 100 and 300 mm 3 When the tumor volume reaches 1000 mg / kg, the MCF-7L tumor-bearing mice are randomly assigned to groups according to Table 14.

[0253] [Table 15]

[0254] Tumor dimensions are measured using calipers before the study, and tumor volumes are estimated using the formula above. The tumor volume ratio (TVR) is defined as the ratio of the tumor volume at week X to the tumor volume at week 0. Mice are injected subcutaneously with vehicle or either vehicle or 100-200 μg / kg of insulin-Fc fusion protein of SEQ ID NO: 1, SEQ ID NO: 47, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, three times a week for up to 8 weeks. Tumor volumes for the vehicle and treatment groups are measured before and after the study, and expected measurements are shown in Table 15. Treatment with insulin-Fc fusion proteins of SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, both alone and in combination with tamoxifen, is expected to show significant efficacy. Furthermore, the insulin-Fc fusion proteins of SEQ ID NO: 18 and SEQ ID NO: 20 are expected to be more effective in reducing tumor volume ratio compared to the insulin-Fc fusion proteins of SEQ ID NO: 1 or SEQ ID NO: 16 due to their reduced immunogenicity due to reduced Fc(γ)R binding (measured as described in Example 14) and reduced C1q binding (measured as described in Example 15).

[0255] The insulin-Fc fusion proteins of SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20 are referred to as Group A in Table 15, and the insulin-Fc fusion proteins of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:47 are referred to as Group B in Table 15.

[0256] [Table 16]

[0257] MCF-7L TamR tumors are also studied in vivo by implanting MCF-7L TamR cells into female nude mice as described above. 3Once tumor volumes reach 100 μg / kg, MCF-7L TamR tumor-bearing mice are randomly assigned to groups according to Table 14 in a separate study. Tumor dimensions are measured using calipers before the study, and tumor volumes are estimated using the formula above. The tumor volume ratio (TVR) is defined as the ratio of the tumor volume at week X to the tumor volume at week 0. Mice are injected subcutaneously with vehicle or 100-200 μg / kg of either insulin-Fc fusion protein of SEQ ID NO: 1, SEQ ID NO: 47, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, three times a week for up to 8 weeks. Tumor volumes for the vehicle and treatment groups are measured before and after the study, and expected measurements are shown in Table 16. Treatment with insulin-Fc fusion proteins of SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, both alone and in combination with tamoxifen, is expected to show significant effects. Tumor volumes for vehicle and treatment groups were measured before and after the study, with expected measurements shown in Table 16, and treatment with insulin-Fc fusion proteins of SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 is expected to show significant efficacy compared to treatment with tamoxifen alone. Furthermore, insulin-Fc fusion proteins of SEQ ID NO: 18 and SEQ ID NO: 20 are expected to show greater efficacy in reducing tumor volume ratios compared to insulin-Fc fusion proteins of SEQ ID NO: 1 or SEQ ID NO: 16 due to their reduced immunogenicity due to decreased Fc(γ)R binding (measured as described in Example 14) and decreased C1q binding (measured as described in Example 15).

[0258] This is expected as MCF-7L TamR cells are tamoxifen resistant and therefore tamoxifen treatment alone should have little to no effect on tumor growth, whereas treatment with insulin-Fc fusion proteins of SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20, particularly at high doses, is expected to downregulate insulin receptors on MCF-7L TamR cells, resulting in slower tumor growth and reduced TVR compared to tamoxifen treatment alone.

[0259] [Table 17]

[0260] Example 12: Assay protocol for measuring anti-drug antibodies in human serum To measure ADA against test compounds, Maxisorp ELISA plates (Nunc) were coated overnight at 4°C with 10 μg / mL of the insulin-Fc fusion protein of interest diluted in coating buffer (carbonate-bicarbonate buffer, pH 9.6). To measure ADA against the insulin portion of insulin-Fc fusion proteins containing Fc fragments derived from human IgG, plates were coated with 30 μg / mL of purified insulin in coating buffer. Plates were then washed five times with PBST (PBS + 0.05% Tween 20) and blocked for at least 1 hour (or overnight) with SuperBlock blocking solution (ThermoFisher, Waltham, MA). To calculate ADA in human IgG units, strips were directly coated overnight at 4°C with 1:2 serial dilutions of human IgG (Jackson Immunoresearch Laboratories, West Grove, PA) at concentrations ranging from 300 to 4.69 ng / mL in carbonate-bicarbonate coating buffer, pH 9.6, and a seven-point pseudostandard curve was generated using this. Standard strip plates are also washed and blocked with SuperBlock blocking solution for at least 1 hour (or overnight).

[0261] Test serum samples are diluted 1:100 or greater (typically tested at 1:200) in PBST / SB / 20% HS sample dilution buffer (PBS + 0.1% Tween 20 + 10% SuperBlock + 20% horse serum) and added to insulin-Fc fusion protein-coated (or RHI-coated) strips at 100 μL / well in duplicate. Duplicate human IgG-coated standard strips are also added to each plate, filled with SuperBlock buffer. Plates are incubated at room temperature for 1 hour and then washed five times with PBST. For ADA detection, HRP-conjugated goat anti-cat IgG F(ab')2 (anti-cat IgG F(ab')2 reagent cross-reacts with human antibodies; Jackson Immunoresearch Laboratories, West Grove, PA) was diluted 1:10,000 in PBST / SB and added to both sample and standard wells at 100 μL / well five times. After washing once with deionized water, 100 μL / well of TMB substrate (Invitrogen, ThermoFisher Scientific, Waltham, MA) was added and incubated at room temperature in the dark for 15–20 minutes. The color development was then stopped by adding 100 μL / well of ELISA stop solution (Boston Bioproducts). The absorbance was read at 450 nm within 30 minutes using a SpattraMax plate reader. Anti-drug antibody concentrations were determined by interpolating the OD values ​​of the 4-PL pseudostandard curve using SoftMax Pro software (Molecular Devices, San Jose, CA).

[0262] To demonstrate the specificity of the detected ADA, an "inhibition" assay is performed. For the drug-inhibited ADA assay, serum samples are diluted 1:100 in PBST / SB / 20% HS buffer and mixed with an equal volume of 300 μg / mL of the relevant therapeutic compound (final sample dilution 1:200, final inhibitor compound 150 μg / mL). The mixture is incubated at room temperature for 30-40 minutes to allow binding of the anti-drug antibodies to the free inhibitor (i.e., therapeutic compound). After preincubation, 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 inhibitor compound are also tested on the sample plate, along with duplicate human IgG-coated standard strips. The remaining steps of the assay procedure are performed as described above. ADA measured in the drug-inhibited wells is compared to the non-inhibited ADA concentration to assess ADA specificity. If significant inhibition of the ADA signal is observed in the drug-inhibited wells, this indicates that the ADA is specific to the therapeutic compound.

[0263] Example 13: Assay Procedure for Identifying Immunogenic Epitopes Maxisorp ELISA microplates (Nunc) are coated with a library of insulin-Fc fusion protein homodimer compounds with known amino acid sequences, and these coated plates are blocked in a manner similar to that described for the anti-drug antibody ELISA assay in Example 12, except that each compound in the library is coated in a separate strip of ELISA microplate wells. The library compounds comprise a range of insulin-Fc fusion proteins with different insulin polypeptide amino acid compositions, including various B-, C-, and A-chain amino acid mutations, different linker compositions, and different Fc fragment compositions, including those of human origin. Separately, some plate strip wells are directly coated with 1:2 serial dilutions of human IgG (Jackson Immunoresearch Laboratories, West Grove, PA) to calculate anti-drug antibodies (ADAs) in human IgG units, respectively, as described in Example 12.

[0264] Sera from individual patients receiving repeated doses of insulin-Fc fusion proteins are first screened using an anti-drug antibody ELISA assay (Example 12). Serum samples that are moderately or highly positive in the assay described in Example 12 (e.g., moderate or high antibody titers) are serially diluted (1:200 to 1:8000) in PBST / SB / 20% HS sample dilution buffer (PBS + 0.1% Tween 20 + 10% SuperBlock + 20% horse serum) and added to plates coated with the insulin-Fc fusion protein compound library for 1 hour at room temperature. After incubation, the plates are washed five times with PBST. To detect human antibodies that may cross-react with the coated compound library, HRP-conjugated goat anti-dog IgG F(ab')2 (Jackson Immunoresearch Laboratories, West Grove, PA), which cross-reacts with dog IgG, was diluted 1:10,000 in PBST / SB and added to both sample and standard wells at 100 μL per well. The plate was washed five times with PBST and once with deionized water. The plate was then developed by adding 100 μL per well of TMB substrate (Invitrogen, ThermoFisher Scientific, Waltham, MA) and incubating for 15–20 minutes in the dark at room temperature. The color development was then stopped by adding 100 μL per well of ELISA stop solution (Boston Bioproducts, Ashland, MA). The absorbance was read at 450 nm within 30 minutes using a SpectraMax plate reader. The anti-compound cross-reactive antibody concentrations present in the serum samples are determined by interpolating the OD values ​​of the 4-PL pseudo-standard curve against the directly coated human IgG antibody control using SoftMax Pro software (Molecular Devices, San Jose CA).

[0265] By correlating the antibody concentrations obtained from the assay with the known amino acid composition of the coated insulin-Fc fusion protein library, it can be determined whether a particular amino acid mutation or epitope induces no antibody signal in the assay, or is responsible for inducing some, most, or all of the total antibody signal, indicating no, weak, or strong binding to various insulin-Fc fusion protein homodimers. Mutations or epitopes responsible for moderate or strong binding are referred to herein as immunogenic "hotspots."

[0266] Example 14: In vitro Fc(γ) receptor I binding affinity assay Binding of insulin-Fc fusion proteins to Fc(γ) receptor I at pH 7.4 was performed using an ELISA assay as follows. Because canine Fc(γ) receptor I is not commercially available, human Fc(γ) receptor I (i.e., rhFc(γ) receptor I) was used as a surrogate mammalian receptor. Insulin-Fc compounds were diluted to 10 μg / mL in sodium bicarbonate buffer, pH 9.6, and coated onto Maxisorp (Nunc) microtiter plates overnight at 4°C. The microplate strips were then washed five times with PBST (PBS / 0.05% Tween-20) buffer and blocked with Superblock blocking reagent (ThermoFisher). Serial dilutions of biotinylated rhFc(γ) receptor I (recombinant human Fc(γ)RI; R&D Systems) were prepared in PBST / 10% Superblock buffer from 6000 ng / mL to 8.2 ng / mL and loaded at 100 μL / well onto the insulin-Fc fusion protein-coated microplate strips. The microtiter plate was incubated at room temperature for 1 hour, after which the microplate strips were washed five times with PBST. Streptavidin-HRP diluted 1:10,000 in PBST / 10% Superblock buffer was then added at 100 μL / well. After a 45-minute incubation, the microplate strips were washed again five times with PBST. Bound Fc(γ) receptor I protein was revealed by adding TMB and stopped with ELISA stop reagent (Boston Bioproducts). Plates are read at 450 nm in an ELISA plate reader, and OD values ​​(proportional to the binding of rhFc(γ) receptor I to insulin-Fc protein) are plotted against the log concentration of rhFc(γ) receptor I added to each well, and binding curves are generated using GraphPad Prism software.

[0267] Example 15: In vitro C1q binding affinity assay Binding of insulin-Fc fusion proteins to complement component C1q at pH 7.4 was performed using an ELISA assay with human complement component C1q as follows: Insulin-Fc compounds were diluted to 10 μg / mL in sodium bicarbonate buffer, pH 9.6, and coated onto Maxisorp (Nunc) microtiter plates overnight at 4°C. The microplate strips were then washed five times with PBST (PBS / 0.05% Tween-20) buffer and blocked with Superblock blocking reagent (ThermoFisher). Serial dilutions of biotinylated complement component C1q (human complement component C1q; Sigma-Aldrich) were prepared in PBST / 10% Superblock buffer from 1000 ng / mL to 1.4 ng / mL and loaded at 100 μL / well onto the insulin-Fc fusion protein-coated microplate strips. The microtiter plate was incubated for 1 hour at room temperature, after which the microplate strips were washed five times with PBST, followed by the addition of 100 μL / well of streptavidin-HRP diluted 1:12,000 in PBST / 10% Superblock buffer. After a 45-minute incubation, the microplate strips were washed five times with PBST. Bound complement component C1q protein was revealed by the addition of TMB and stopped with ELISA stop reagent (Boston Bioproducts). The absorbance of the plate was read at 450 nm (OD450) using an ELISA plate reader, and the OD values ​​(proportional to the binding of complement component C1q to insulin-Fc protein) were plotted against the logarithmic concentration of complement component C1q added to each well, and a binding curve was generated using GraphPad Prism software. For groups of compounds with somewhat similar curves, the OD450 at one of the higher concentrations, e.g., at a complement component C1q concentration of 1000 ng / mL, can be used to identify differences between the coated insulin-Fc fusion protein compounds.

[0268] Example 16: Exemplary insulin-Fc fusion protein domains and sequences Exemplary insulin-Fc fusion protein domains and sequences used in the above examples are shown in Table A and in Figures 2 and 3.

[0269] equivalent In the claims, articles such as "a," "an," "the," etc. may mean one or more unless stated otherwise or clear from context. A claim or description including "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless stated otherwise or clear from context. The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which two or more, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0270] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, a claim that depends on another claim can be amended to include one or more limitations found in other claims that depend from the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of those elements is also disclosed, and any element can be deleted from the group. In general, when the present disclosure or aspects of the disclosure are referred to as comprising certain elements and / or features, it should be understood that certain embodiments of the present disclosure or aspects of the disclosure consist of or consist essentially of such elements and / or features. For the sake of brevity, those embodiments have not been specifically described in these terms herein. It should also be noted that the terms "comprise(s)," "comprising," "contain(s)," and "containing" are intended to be open, and their use permits the inclusion of additional elements or steps. When ranges are expressed, the endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume, in different embodiments of the present disclosure, any specific value or subrange within the stated range, to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

Claims

1. A fusion protein comprising an insulin polypeptide and an Fc fragment, The following sequence: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 18) A fusion protein comprising:

2. A fusion protein comprising an insulin polypeptide and an Fc fragment, The following sequence: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 20) A fusion protein comprising:

3. A fusion protein comprising an insulin polypeptide and an Fc fragment, The following sequence: FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 6) comprising A fusion protein having a maximum insulin receptor binding IC50 ratio relative to recombinant human insulin (RHI) of 50 or less, 40 or less, 30 or less, or more preferably 20 or less.

4. The Fc fragment of the fusion protein has the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32) The fusion protein of claim 3, comprising:

5. The Fc fragment of the fusion protein has the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 33) The fusion protein of claim 3, comprising:

6. the insulin polypeptide and the Fc fragment are linked by a linker; The linker has the sequence: GGGGAGGGG (SEQ ID NO: 11) 6. The fusion protein of claim 4 or 5, comprising:

7. A fusion protein according to any one of claims 1 to 6, which comprises a dimer of two identical monomers linked via a disulfide bond, e.g. is a homodimer.

8. The fusion protein has the following orientation from N-terminus to C-terminus: (N-terminus)--insulin polypeptide--linker--Fc fragment--(C-terminus) and a domain, The insulin polypeptide is oriented from N-terminus to C-terminus as follows: (N-terminus) --B chain --C peptide --A chain --(C-terminus) The fusion protein according to any one of claims 1 to 6, comprising a domain.

9. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 8 dispersed in a pharmaceutically acceptable carrier.

10. 10. A method for inhibiting the metabolism, growth, and / or proliferation of cancer cells, comprising administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1 to 8.

11. 10. A method of downregulating insulin-like growth factor 1 receptor (IGF1R) in a subject in need thereof, comprising administering an effective amount of the fusion protein of any one of claims 1 to 8, wherein following said administration IGF1R is downregulated in said subject and said subject exhibits a decrease in tumor volume compared to an untreated control tumor.

12. 12. The method of claim 10 or 11, wherein the fusion protein inhibits tumor growth in the subject after said administration.

13. 12. The method of claim 10 or 11, wherein the subject exhibits at least a 30% reduction in tumor volume after said administration compared to an untreated control tumor.

14. 12. The method of claim 10 or 11, wherein the fusion protein exhibits an anti-tumor effect on cancer cells in the subject after said administration, wherein said anti-tumor effect is selected from the group consisting of down-regulation of insulin receptor, reduction in phosphorylated Akt, and a combination thereof compared to untreated control cancer cells.

15. 12. The method of claim 10 or 11, wherein the fusion protein is administered via a route of administration selected from the group consisting of intravenous injection, subcutaneous injection, and intratumoral injection.

16. 12. The method of claim 10 or 11, wherein the fusion protein is administered as a bolus, infusion or intravenous push.

17. 12. The method of claim 10 or 11, wherein the fusion protein is administered via a syringe, pump, pen, needle, or indwelling catheter.

18. 12. The method of claim 10 or 11, wherein the fusion protein is administered to the subject at a dose of about 150 to about 1,500 micrograms per kilogram of body weight per day.

19. 12. The method of claim 10 or 11, wherein the fusion protein is administered in combination with a first or second line cancer therapy selected from the group consisting of a chemotherapeutic agent, a tamoxifen agonist, or an antibody against IGF1R.

20. 12. The method of claim 10 or 11, wherein the fusion protein is administered to the subject under fasting conditions.

21. 11. The method of claim 10, wherein the subject has been diagnosed with a cancer selected from the group consisting of breast cancer, colorectal cancer, and melanoma.

22. A fusion protein according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for use in a method for treating cancer in a subject, preferably inhibiting the metabolism, growth and / or proliferation of cancer cells.

23. 23. The fusion protein for use according to claim 22, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer and melanoma.

24. 24. The fusion protein for use according to claim 22 or 23, wherein the subject is a mammal.

25. 25. The fusion protein for use of claim 22, 23, or 24, wherein the fusion protein inhibits tumor growth in the subject after said administration.

26. 26. The fusion protein for use according to claim 22, 23, 24 or 25, wherein the subject exhibits at least a 30% reduction in tumor volume after said administration compared to an untreated control.

27. 27. The fusion protein for use according to any one of claims 22 to 26, wherein the fusion protein exhibits an anti-tumor effect on cancer cells in the subject after said administration, wherein the anti-tumor effect is selected from the group consisting of down-regulation of insulin receptor, down-regulation of insulin-like growth factor 1 receptor (IGF1R), reduction in phosphorylated Akt, and combinations thereof, compared to untreated control cancer cells.

28. The fusion protein for use according to any one of claims 22 to 27, wherein said fusion protein is administered by intravenous, subcutaneous or intratumoral injection.

29. The fusion protein for use according to any one of claims 22 to 28, wherein the fusion protein is administered as a bolus, infusion or intravenous push.

30. The fusion protein for use according to any one of claims 22 to 29, wherein the fusion protein is administered via a syringe, a pump, a pen, a needle or an indwelling catheter.

31. The fusion protein for use according to any one of claims 22 to 30, wherein the fusion protein is administered to the subject at a dose of about 150 to about 1,500 micrograms per kilogram of body weight per day.

32. 32. The fusion protein for use according to any one of claims 22 to 31, wherein the fusion protein is administered in combination with a first or second line cancer therapy selected from the group consisting of a chemotherapeutic agent, a tamoxifen agonist, or an antibody against IGF1R.

33. The fusion protein for use according to any one of claims 22 to 32, wherein the fusion protein is administered to said subject under fasting conditions.

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