Insulin-FC fusion protein

Abstract

This invention relates to a fusion protein comprising an insulin receptor agonist fused to the human IgG2 Fc region, and to the use of such a fusion protein in the treatment of diabetes. The fusion protein of this invention has a long-acting profile and is useful for providing long-term basal glucose control.

Description

[Technical Field]

[0001] This invention relates to a fusion protein for use in the treatment of diabetes. More specifically, this invention relates to a fusion protein comprising an insulin receptor agonist fused to the human IgG2 Fc region, and to the use of such a protein in the treatment of diabetes. The fusion protein of this invention has a long-acting profile and is useful in providing sustained basal glucose control and suppression of glucose production in the liver. [Background technology]

[0002] Diabetes mellitus is a chronic disorder characterized by hyperglycemia resulting from a deficiency in insulin secretion, insulin action, or both. Type 1 diabetes mellitus is characterized by little or no insulin secretion, and patients with type 1 diabetes mellitus require insulin for survival. Insulin therapy for type 1 diabetes generally involves the use of two types of exogenously administered insulin: rapid-acting mealtime insulin delivered by bolus injection and long-acting basal insulin administered once or twice daily to control blood glucose levels between meals. Type 2 diabetes mellitus is characterized by elevated blood glucose levels resulting from insulin deficiency, insulin resistance, excessive glucose production in the liver, and / or all of the above contributions. Treatment for patients with type 2 diabetes typically begins with prescribed weight loss, exercise, and a diabetic diet. However, if these measures fail to control elevated blood glucose levels, oral medications and incretin therapy may be necessary, such as glucagon-like peptide-1 (GLP-1) receptor agonists and / or dipeptidyl peptidase 4 (DPP-4) inhibitors that increase incretin levels. If these medications remain insufficient, insulin therapy is considered. Patients with type 2 diabetes whose disease has progressed to the point of requiring insulin therapy generally begin with once-daily injections of long-acting basal insulin, although in some cases, rapid-acting insulin injections with meals may be included as appropriate.

[0003] Several types of basal insulin suitable for once-daily administration are currently available, including insulin glargine (marketed under the trademark LANTUS®), insulin detemir (marketed under the trademark LEVEMIR®), and insulin degludec (marketed under the trademark TRESIBA®). All of these insulins are suitable for once-daily administration.

[0004] Many diabetic patients are unwilling or unable to follow treatment regimens involving daily injections. To improve acceptance and compliance, research is being conducted to identify insulin products that have a longer duration of action and therefore require fewer injections than currently available insulin products.

[0005] International Publication No. 2016 / 178905 describes a fusion protein containing an insulin receptor agonist fused to a human IgG Fc region that has a long-acting profile for providing sustained basal glucose regulation.

[0006] Nevertheless, there is still a need for additional options for basal insulin therapy, including therapies with improved chemical stability suitable for long-term storage at room temperature. [Overview of the project] [Means for solving the problem]

[0007] This disclosure provides a fusion protein comprising an insulin receptor agonist fused to a human IgG2 Fc region, the human IgG2 Fc region comprising modification of the N residue at position 389 to an amino acid selected from the group consisting of S, Q, E, D, and A.

[0008] Homodimers of the two fusion proteins of this disclosure are also provided.

[0009] A method for treating patients with diabetes mellitus is also provided, which includes administering a therapeutically effective amount of the fusion protein of this disclosure to a patient in need of treatment.

[0010] Fusion proteins of this disclosure for use in therapeutic purposes are also provided.

[0011] Furthermore, the use of the fusion protein of this disclosure in the manufacture of pharmaceuticals is also provided.

[0012] Polynucleotides encoding the fusion proteins of this disclosure are also provided.

[0013] A process for producing the fusion protein of this disclosure is also provided, and this process is The method includes the steps of culturing mammalian host cells containing a polynucleotide encoding the fusion protein of the present disclosure under conditions that allow the fusion protein to be expressed, and recovering the fusion protein from the host cells. [Brief explanation of the drawing]

[0014] [Figure 1] This document provides pharmacodynamic data for an exemplary fusion protein of the present invention in a streptozotocin (STZ)-treated rat diabetic model. [Modes for carrying out the invention]

[0015] The fusion protein of the present invention comprises an insulin receptor agonist fused to the human IgG2 Fc region. As used herein, the term “insulin receptor agonist” refers to a protein that binds to the insulin receptor and activates it, thereby resulting in a reduction in blood glucose levels and / or suppression of glucose production in the liver, which can be tested and measured using known techniques such as those shown in the studies below.

[0016] In certain embodiments, the insulin receptor agonist of the fusion protein of the present invention comprises an insulin A chain or analogue thereof, and an insulin B chain or analogue thereof. As used herein, the terms “insulin A chain” and “insulin B chain” refer to the A and B chains of the human insulin molecule (CAS number 11061-68-0), whose natural wild-type sequence is well known. The human insulin A chain has the following sequences, known in the art as A1-A 21 It is composed of 21 amino acids, which are known as [the amino acid]. GIVEQCCTSICSLYQLENYCN (Sequence ID 8).

[0017] Human insulin B chains have the following sequences, in the art, B1-B 30 It is composed of 30 amino acids, which are referred to as [a specific amino acid]. FVNQHLCGSHLVEALYLVCGERGFFYTPKT(Sequence ID 7).

[0018] In certain embodiments, the insulin receptor agonist portion of the fusion protein of the present invention includes an analogue of the insulin B chain and an analogue of the insulin A chain. As used herein, the term “analog” refers to a structure having one or more modifications, including amino acid substitutions, deletions, inversions, or additions, compared to the natural human amino acid sequence.

[0019] In certain embodiments, insulin B-chain analogs include one or more modifications to the amino acid sequence of the human insulin B-chain. In particular, to enhance chemical stability during storage conditions in use, insulin B-chain analogs include modifications to the amino acid at position B3 to G, T, A, S, E, Q, R, K, H, or D. Furthermore, to reduce the tendency of the insulin receptor agonist moiety to self-associate or dimerize, insulin B-chain analogs also include one or more modifications from the B-chain of the human insulin molecule to B 16 B 25 or B 27~30 Included in the rank.

[0020] In certain embodiments, an analogue of the insulin B chain has the following amino acid sequence: X1X2X3QHLCGSHLVEALX 16 LVCGERGFX 25 YX 27 X 28 X 29 X 30 comprising, X1 is F, Q or A, X2 is V or G, X3 is G, T, A, S, E, Q, R, K, H or D, X 16 is E, Y, Q, or H, X 25 is H or F, X 27 is G, T, S, H, V, or is absent, X 28 is G, E, P, K, D, S, H, or is absent, X 29 is G, E, K, P, Q, D, H, or is absent, X 30 is G, T, S, E, K, A, or is absent (SEQ ID NO: 1).

[0021] In certain preferred embodiments, the analog of the insulin B chain comprises the amino acid sequence of SEQ ID NO: 1, wherein X3 is G, T, A or S.

[0022] In certain preferred embodiments, the analog of the insulin B chain comprises the amino acid sequence of SEQ ID NO: 1, wherein X3 is G or T. In certain preferred embodiments, the analog of the insulin B chain comprises the amino acid sequence of SEQ ID NO: 1, wherein X1 is F, X2 is V, X3 is G or T, X 16 is E, X 25 is H, X 27 is G, X 28 is G, X 29 is G, X 30 is G.

[0023] In certain embodiments, the analog of the insulin A chain may comprise one or more modifications to the amino acid sequence of the human insulin A chain, aimed at improving chemical and physical stability, adjusting potency, and / or enhancing expression. In certain embodiments, the analog of the insulin A chain has the amino acid sequence: GIVEQCCTSX10 CSLX 14 QLENYCX 21 X 22 Includes, X 10 is T or I, and X 14 is D, Y, Q, or E, and X 21 is G, N, S or A, and X 22 is any naturally occurring amino acid, or is not present, however X 21 If X is N, 22 It must be an amino acid other than G or N (Sequence ID 2).

[0024] In a preferred embodiment, an analogue of the insulin A chain comprises the amino acid sequence of SEQ ID NO: 2, and X 10 is T, and X 14 D is X 21 G is X 22 It does not exist.

[0025] In the human insulin molecule, the A and B chains are joined by two disulfide bonds: CysA7-CysB7 and CysA20-CysB19. The A chain has intrachain disulfide bonds between CysA6-CysA11. In certain embodiments, the insulin receptor agonist of the fusion protein of the present invention comprises insulin A and B chains, or analogs thereof, joined by disulfide bonds at the position where the A and B chains are joined in the human insulin molecule. In certain embodiments, the insulin A chain or analog contains intrachain disulfide bonds between CysA6-CysA11 in the insulin A chain analog.

[0026] In certain embodiments, the insulin receptor agonist of the fusion protein of the present invention comprises a single chain containing human insulin A and B chains, or analogs thereof. In certain embodiments, the human insulin A and B chains, or analogs thereof, are joined by a peptide linker referred to herein as the “first peptide linker.” In certain embodiments, the C-terminal residue of the insulin B chain, or an analog thereof, is fused to the N-terminal residue of the first peptide linker, and the C-terminal residue of the first peptide linker is fused to the N-terminal residue of the insulin A chain, or analog thereof. The first peptide linker needs to provide sufficient flexibility to the insulin A and B chain analogs to achieve the structure necessary for binding to the insulin receptor, but it should not be so long as to excessively interfere with the binding. The length and composition of the first peptide linker may be adjusted to modulate the potency and / or expression of the fusion protein. In some embodiments, the first peptide linker has an amino acid length of 5 to 10, of which at least 5 are G residues.

[0027] In a particular embodiment, the amino acid sequence of the first peptide linker is: X1GX3GGGG Includes, X1 is either G or absent, and X3 is either G, S, or absent (SEQ ID NO: 3). In a particular preferred embodiment, the first peptide linker comprises the sequence of SEQ ID NO: 3, where X1 and X3 of SEQ ID NO: 3 are G and S, respectively.

[0028] In certain embodiments, the insulin receptor agonist is directly fused to the human IgG2 Fc region. In other embodiments, the insulin receptor agonist is fused to the human IgG2 Fc region via the use of a linker. In certain embodiments, the linker is a "second peptide linker". In these particular embodiments, the C-terminal residue of the insulin receptor agonist portion of the fusion protein of the present invention is fused to the N-terminal residue of the second peptide linker, and the C-terminal residue of the second peptide linker is directly fused to the N-terminal residue of the Fc portion.

[0029] To provide sufficient conformational flexibility, the second peptide linker is preferably glycine-rich. Preferably, the second peptide linker has an amino acid length of less than 30. In certain preferred embodiments, the second peptide linker has an amino acid length of 10 to 25, and at least 50% of these amino acids are glycine residues. A preferred second peptide linker has the sequence (GGGGX) n (X is Q, E, or S, and n=2 to 5) (Sequence ID 4) is included. In certain embodiments, n In other embodiments, n The answer is 3.

[0030] In a particular preferred embodiment, the linker is arranged GGGGQGGGGQGGGGQGGGG (Sequence 14) Includes.

[0031] As used herein, the term "human IgG2 Fc region" refers to analogues of the Fc region of a human IgG2 antibody that have been modified, extended, and / or cleaved to alter properties or characteristics such as complement and / or Fc receptor binding function, effector function, disulfide bond formation, glycosylation, antibody-dependent cell-mediated cytotoxicity (ADCC), manufacturability, and / or stability. In particular, the human IgG2 Fc region in the fusion protein of the present invention includes modification of the N residue at position 389 to an amino acid selected from the group consisting of S and Q.

[0032] It should be noted that, as used herein, “position 389” in the IgG2 Fc region refers to the amino acid at position 389 of the antibody according to the EU antibody numbering scheme. See, for example, the IMGT Scientific chart, available at https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html, Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969), and Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n°91-3242, pp 662, 680, 689 (1991). In the preferred IgG2 Fc region identified below by Sequence ID No. 5, this amino acid is X 164 It is located in [location].

[0033] The human IgG Fc region of the fusion protein of the present invention may also have a portion of the hinge region removed to simplify disulfide-mediated Fc dimerization. Other examples of modifications include modifications that enable phosphorylation, sulfation, acylation, glycosylation, methylation, acetylation, amidation, and / or the production of heterodimer molecules. Techniques for modifying the structure and properties of human IgG Fc regions of IgG subclasses are known in the art.

[0034] In certain preferred embodiments, the human IgG2 Fc region has the amino acid sequence: ECPPCPAPPVAGPSVX 16 LX 18 PPKPKDTLMISRTPEVTCX 37 VX 39 DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFX 72STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEX 164 NYKTTPPMLDSDGGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX 222 It has, X 16 is F, Q, or E, and X 18 is F, Q, or E, and X 37 is V or T, and X 39 However, V or T, X 72 is N, D, or Q, and X 164 is S, Q, E, D or A, and X 222 It is either K or does not exist (SEQ ID NO: 5).

[0035] In certain preferred embodiments, the human IgG2 Fc region has the amino acid of SEQ ID NO: 5, and X 164 It is either S or Q.

[0036] The preferred human IgG IgG2 Fc region has the sequence of Sequence ID No. 5, X 16 is F, and X 18 is F, and X 37 However, V is X 39 However, V is X 72 N is X 164 Q is X 222 It does not exist.

[0037] Another preferred human IgG IgG2 Fc region has the sequence of Sequence ID No. 5, X 16 is F, and X 18 However, F is X 37 However, V is X 39 However, V is X 72 N is X 164 S is X 222 It does not exist.

[0038] The preferred human IgG Fc region amino acid sequences listed above have portions of the hinge region removed to simplify disulfide-mediated dimerization, although these hinge regions may be present in certain embodiments. For example, the wild-type IgG2 Fc region contains a six-amino acid sequence ERKCCV (SEQ ID NO: 6) at its N-terminus, and these amino acids are not described in the IgG2 Fc region sequence shown in SEQ ID NO: 5. However, a human IgG Fc region containing the amino acid sequence shown in SEQ ID NO: 5 may further contain some or all of the six amino acid sequence ERKCCV at its N-terminus. Furthermore, a precise description between the amino acids constituting the C-terminus of the second peptide linker and the amino acids constituting the N-terminus of the human IgG Fc region is not essential to the structure or function of the fusion protein of the present invention.

[0039] The fusion proteins of the present invention and their uses share certain characteristics with the fusion proteins and uses described in International Publication No. 2016 / 178905 and can be produced using known techniques such as those described in International Publication No. 2016 / 178905. As described therein, the human IgG2 Fc region has a tendency to self-associate to form a dimer, typically covalently associating via a disulfide bond in the hinge region, and such a dimer is formed from the human IgG2 Fc regions of two fusion proteins of the present invention. When the amino acid sequences of the two fusion proteins constituting such a dimer are the same, the dimer is referred to herein as a “homodimer”. Expression of the fusion proteins of the present invention in mammalian cells is preferred, and expression in such cells results in a homodimer. When the amino acid sequences of the two fusion proteins constituting the dimer are different, the dimer is referred to herein as a “heterodimer”.

[0040] References herein to a pharmaceutical composition containing a fusion protein include a pharmaceutical composition containing a homodimer of the fusion protein and / or a heterodimer in which one of the heterodimers is the fusion protein. Similarly, references herein to a method involving the administration of a fusion protein include the administration of a homodimer of the fusion protein and / or the administration of a heterodimer in which one of the heterodimers is the fusion protein. Likewise, references herein to a fusion protein for use in therapy and / or for use in the manufacture of a pharmaceutical include a homodimer of the fusion protein and / or a heterodimer in which one of the heterodimers is the fusion protein for use in therapy and / or the manufacture of a pharmaceutical.

[0041] As described above, the present invention also relates to polynucleotides encoding any of the fusion proteins of the present invention. The polynucleotides encoding the above fusion proteins may be in the form of RNA or DNA, comprising cDNA and synthetic DNA, and may be double-stranded or single-stranded. The coding sequence encoding the proteins of the present invention may change as a result of genetic coding duplication or degeneracy.

[0042] The polynucleotides encoding the fusion protein of the present invention may include only the protein coding sequence, the protein coding sequence and further coding sequences, e.g., a leader sequence or secretory sequence or a proprotein sequence; or the protein coding sequence and non-coding sequences, e.g., introns or non-coding sequences 5' and / or 3' of the protein coding sequence. Therefore, the term "protein-coding polynucleotide" encompasses polynucleotides that may include not only the coding sequence for a protein but also further coding and / or non-coding sequences.

[0043] The polynucleotides of the present invention are expressed in host cells after their sequences have been operably ligated to an expression control sequence. The expression vector is typically replicable in the host organism, either as an episome or as an integrated portion of host chromosomal DNA. Generally, the expression vector will contain a selection marker to enable detection of cells converted with the desired DNA sequence.

[0044] The terms “treatment” or “to treat” as used herein refer to the management and care of a patient having diabetes mellitus or hyperglycemia, or other conditions (in which insulin administration is indicated for the purpose of counteracting or alleviating the symptoms and complications of these conditions). To treat includes administering the fusion protein of the present invention to prevent or delay the onset of symptoms or complications, to alleviate symptoms or complications, or to eliminate a disease, condition, or disorder. The patient to be treated is a mammal, preferably a human.

[0045] As used herein, the terms “prevent” or “prevent” refer to reducing the risk or incidence of one or more conditions, symptoms, complications, or diseases, or eliminating or delaying their progression.

[0046] The fusion protein of the present invention can be used to treat subjects suffering from a wide range of diseases and conditions. This includes subjects with hyperglycemia, insulin-dependent diabetes mellitus, and non-insulin-dependent diabetes mellitus, including untreated subjects and subjects receiving oral medications. Oral medications include thiazolidinediones such as sulfonylurea, metformin, and pioglitazone, α-glucosidase inhibitors such as acarbose, and / or non-insulin injectable therapies including incretin therapies such as DPP-4 inhibitors and GLP-1R agonists. The fusion protein of the present invention may be used to regulate blood glucose in such patients and can treat conditions or complications arising from inadequate blood glucose control, such as retinopathy, neuropathy, or renal disease.

[0047] In certain embodiments, the fusion protein of the present invention is administered daily, every other day, twice a week, three times a week, once a week, twice a month, or once a month. In preferred embodiments, the duration of action is significantly extended, allowing for weekly administration.

[0048] In certain embodiments, the fusion protein of the present invention is administered in combination with additional active ingredients, such as insulin or insulin analogues, incretin therapy, DPP-4 inhibitors, amyrin receptor agonists, or oral antidiabetic drugs, such as thiazolidinediones like sulfonylurea, metformin, or pioglitazone, or α-glucosidase inhibitors like acarbose.

[0049] The term "incretin therapy" includes, or any treatment that promotes, enables, enhances and / or stimulates, the effects of, a group of metabolic hormones known as incretins, which are endogenous hormones secreted from the intestines that stimulate insulin secretion in a nutrient-dependent manner by activating GLP-1 and / or GIP receptors. Incretins include any agonist (peptide or non-peptide) that has activity against either the GLP-1 and / or GIP receptor. Incretins may contain further activity at other receptors, such as the glucagon receptor (GCGR). Currently available incretin therapies include exenatide (Byetta® and Bydureon®), liraglutide (Victoza®), semaglutide (Ozempic®), albiglutide (Tanzeum®), dulaglutide (Trulicity®), and tilzepatide (Mounjaro®). Other incretin therapies currently in clinical development include mazdutide and letatoltide.

[0050] DPP-4 inhibitors are compounds that interfere with the DPP-4 enzyme, which is involved in incretin degradation. Currently available DPP-4 inhibitors include sitagliptin (Januvia®) and linagliptin (Tradjenta®).

[0051] In embodiments in which the fusion protein of the present invention is provided in combination with further active ingredients, the fusion protein and the further active ingredients may be administered simultaneously, sequentially, or as a single combined formulation.

[0052] The fusion protein of the present invention is effective in treating such diseases and conditions by administering a therapeutically effective dose of the fusion protein to patients in need. As used herein, the term "therapeutic dose" refers to an amount of the fusion protein of the present invention sufficient to regulate blood glucose in a patient without causing unacceptable side effects. The therapeutically effective dose of the fusion protein administered to a subject will depend on the type and severity of the disease, as well as the subject's characteristics, such as overall health, age, sex, weight, and drug tolerance.

[0053] The fusion protein of the present invention is administered parenterally, intranasally, or by pulmonary inhalation. Parenteral administration is preferred and may include systemic administration, such as by intramuscular, intravenous, subcutaneous, or intraperitoneal injection.

[0054] The fusion protein can be administered to a subject in a pharmaceutical composition comprising the fusion protein of the present invention and at least one pharmaceutically acceptable excipient. Such a pharmaceutical composition can be prepared, though not necessarily, typically by any of various techniques using conventional excipients for parenteral products that are originally parenteral and are well known in the art.

[0055] As described above, the fusion protein of the present invention is homodimer when expressed in mammalian cells. Therefore, as used herein, the term "composition comprising the fusion protein" includes compositions containing homodimers of the fusion protein.

[0056] The present invention will be further illustrated by the following embodiments, but these should not be construed as limiting.

[0057] Expression and purification of fusion proteins The fusion protein of the present invention can be produced in a mammalian cell expression system using a CHO glutamine synthetase (GS) knockout (GSKO) cell line. GS gene knockout allows for strict selectivity by eliminating endogenous GS background activity, which can allow for the survival of low-productivity or non-productivity cells under secretion conditions. The gene encoding the fusion protein is subcloned into glutamine synthetase (GS) containing an expression plasmid. The cDNA sequence encoding the fusion protein is fused in-frame with the encoding sequence of a signal peptide that enhances the secretion of the fusion protein into the cell medium. Expression is driven by a cytomegalovirus (CMV) promoter. CHO GSKO cells are stably transfected using electroporation and an appropriate amount of recombinant expression plasmid.

[0058] Transfected cells are subjected to bulk selection in glutamine-free culture medium. The transfected pool is plated at a low density to allow for near-clonal growth of stable expression cells. Master wells are screened for fusion protein expression and then scaled up in serum-free suspension culture for production.

[0059] The fusion protein secreted into the culture medium can be purified by protein A affinity chromatography, followed by size exclusion chromatography, and then by standard chromatography techniques. In short, the fusion protein from the clarified medium is captured with Mab Select Protein A(GE) equilibrated with pH 7.4 phosphate-buffered saline. After a washing step with pH 7.4 phosphate-buffered saline, the bound fusion protein is eluted with 10 mM citrate at pH 3.0. The fraction containing the fusion protein is pooled and neutralized by adding 1 / 10 volume of 1 M Tris at pH 8.0. Soluble aggregates and polymers can be efficiently removed by common techniques including size exclusion, hydrophobic interaction, or ion exchange chromatography. The fraction containing monomeric fusion protein (covalently bound homodimer) as determined by size exclusion chromatography was pooled, filtered sterile, and stored.

[0060] An example amino acid sequence of the fusion protein of the present invention is shown below.

[0061] [Table 1]

[0062] [Table 2]

[0063] [Table 3]

[0064] [Table 4]

[0065] [Table 5]

[0066] stability The example and reference fusion proteins were formulated at a concentration of 10 mg / mL in 5 mM sodium phosphate, with the pH adjusted to 6.5 using HCl as needed. Aliquot formulations were stored at 5°C and 37°C for 8 days. After 8 days, the proportion of acidic species was quantified by anion exchange chromatography, and species growth was determined by comparing the proportion of acidic species in the 37°C and 5°C samples. The results are shown in Table 1 below.

[0067] [Table 6]

[0068] As shown in Table 1, when stored at 37°C, Examples 1-4 showed less change in acidic species compared to the reference, demonstrating improved stability.

[0069] A study in a rat model of diabetes treated with streptozotocin (STZ) The effects of the fusion protein will be investigated in a rat diabetic model treated with STZ. Male Sprague-Dawley rats weighing 400-425 grams will be anesthetized with isoflurane and given a single injection of Zanosar® (STZ catalog number 89256, Teva Parenteral Medicines, 40 mg / kg IV). Rats will be used in the study 3 days after the Zanosar® injection. Only animals with non-fasting blood glucose levels of 400-550 mg / dL will be used in these studies.

[0070] Rats were divided into groups to ensure similar variance in blood glucose and body weight, and then randomized. Blood glucose was measured using an Accucheck Aviva blood glucose meter (Roche). STZ-treated rats were given a single subcutaneous (SC) injection of 30 nmol / kg.

[0071] Blood samples for glucose measurement are collected by tail amputation. Animals are allowed free access to food and water throughout the experiment. Blood glucose data are provided in Figure 1. The data shown are mean ± SEM (n=5). Blood glucose data for the time points (0 to 24 hours) during the initial feeding period are collected, but not included in Figure 1 for ease of visual presentation. As shown in Figure 1, the Example and reference fusion proteins each produce glucose lowering over an extended period of time.

[0072] Sequence SEQ ID NO: 1 - Analogue of insulin B chain X1X2X3QHLCGSHLVEALX 16 LVCGERGFX 25 YX 27 X 28 X 29 X 30 X1 is F, Q or A, X2 is V or G, X3 is G, T, A, S, E, Q, R, K, H or D, X 16 is E, Y, Q, or H, X 25 is H or F, X 27 is G, T, S, H, V, or is absent, X 28 is G, E, P, K, D, S, H, or is absent, X 29 is G, E, K, P, Q, D, H, or is absent, X 30 is G, T, S, E, K, A, or is absent.

[0073] SEQ ID NO: 2 - Analogue of insulin A chain GIVEQCCTSX 10 CSLX 14 QLENYCX 21 X 22 X 10 is T or I, X 14 is D, Y, Q or E, X 21 is G, N, S or A, X 22 is any naturally occurring amino acid, or is absent, provided that when X 21 is N, X 22must be an amino acid other than G or N

[0074] SEQ ID NO: 3 - First peptide linker X1GX3GGGG X1 is G or is absent, and X3 is G, S or is absent

[0075] SEQ ID NO: 4 - Second peptide linker GGGGX n , X is Q, E or S, and n=2~5

[0076] SEQ ID NO: 5 - Human IgG2 Fc region ECPPCPAPPVAGPSVX 16 LX 18 PPKPKDTLMISRTPEVTCX 37 VX 39 DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFX 72 STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEX 164 NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX 222 X 16 is F, Q or E, and X 18 is F, Q or E, and X 37 is V or T, and X 39 is V or T, and X 72 is N, D or Q, and X 164 is S, Q, E, D, and A, and X 222 is K or is absent.

[0077] SEQ ID NO: 6 - Human IgG2 Fc region hinge ERKCCV

[0078] SEQ ID NO: 7 - Human insulin B chain FVNQHLCGSHLVEALYLVCGERGFFYTPKT

[0079] Sequence ID 8 - Human insulin A chain GIVEQCCTSICSLYQLENYCN

[0080] [Table 7]

[0081] [Table 8]

[0082] [Table 9]

[0083] [Table 10]

[0084] [Table 11]

[0085] Sequence ID No. 14 - Second Peptide Linker GGGGQGGGGQGGGGGGGGGG

Claims

1. A fusion protein comprising an insulin receptor agonist fused to a human IgG2 Fc region, wherein the human IgG2 Fc region includes a modification of the N residue at position 389 to an amino acid selected from the group consisting of S, Q, E, D, and A.

2. The fusion protein according to claim 1, wherein the insulin receptor agonist comprises an insulin B chain analog and an insulin A chain analog.

3. The aforementioned insulin B chain analog has the amino acid sequence: X １ X ２ X ３ QHLCGSHLVEALX １６ LVCGERGFX ２５ YX ２７ X ２８ X ２９ X ３０ Includes, X １ is F, Q, or A, and X ２ is V or G, X ３ is G, T, A, S, E, Q, R, K, H or D, X １６ is E, Y, Q, or H, and X ２５ is H or F, and X ２７ is G, T, S, H, V, or does not exist, X ２８ is G, E, P, K, D, S, H, or does not exist, X ２９ is G, E, K, P, Q, D, H, or does not exist, X ３０ The fusion protein according to claim 2, wherein is G, T, S, E, K, A, or absent (SEQ ID NO: 1).

4. X １ However, F is X ２ However, V is X ３ However, it is G or T, and X １６ However, E is X ２５ However, H is X ２７ However, G is X ２８ However, G is X ２９ However, G is X ３０ However, the fusion protein according to claim 3 is G.

5. X ３ However, the fusion protein according to claim 4 is G.

6. X ３ The fusion protein according to claim 4, wherein T is present.

7. The aforementioned insulin A chain analog has the amino acid sequence: GIVEQCCTSX １０ CSLX １４ QLENYCX ２１ X ２２ Includes, X １０ is T or I, X １４ is D, Y, Q, or E, and X ２１ is G, N, S, or A, X ２２ is any naturally occurring amino acid, or is not present, however X ２１ If X is N, ２２ The fusion protein according to any one of claims 2 to 6, wherein the amino acid is one other than G or N (Sequence ID 2).

8. X １０ However, T is X １４ However, D is X ２１ However, G is X ２２ The fusion protein according to claim 7, wherein the element is absent.

9. The fusion protein according to any one of claims 2 to 8, wherein the C-terminal residue of the insulin B-chain analog is fused to the N-terminal residue of a first peptide linker, and the C-terminal residue of the first peptide linker is fused to the N-terminal residue of the insulin A-chain analog.

10. The fusion protein according to claim 9, wherein the first peptide linker comprises 5 to 10 amino acids, and at least 5 of the amino acids are G residues.

11. The first peptide linker described above has the following amino acid sequence: X １ GX ３ GGGG Includes, X １ is G, or does not exist, X ３ The fusion protein according to claim 9 or 10, wherein is G, S, or absent (SEQ ID NO: 3).

12. X of sequence number 3 １ and X ３ The fusion protein according to claim 11, wherein the components are G and S, respectively.

13. The fusion protein according to any one of claims 2 to 12, wherein the C-terminal residue of the insulin A chain analog is fused to the N-terminal residue of a second peptide linker, and the C-terminal residue of the second peptide linker is fused to the N-terminal residue of the human IgG2 Fc region.

14. The fusion protein according to claim 13, wherein the second peptide linker is a peptide having 10 to 25 amino acids, and at least 50% of the amino acids are G residues.

15. The second peptide linker is sequence [GGGGX] ｎ It contains a peptide having X is Q, E, or S, ｎ The fusion protein according to claim 14, wherein the coefficient is 2 to 5 (SEQ ID NO: 4).

16. ｎ The fusion protein according to claim 15, wherein the value is 3.

17. The fusion protein according to claim 16, wherein the second peptide linker comprises a peptide having the sequence GGGGQGGGGQGGGGQGGGG (SEQ ID NO: 14).

18. The aforementioned human IgG2 Fc region has the following amino acid sequence: ECPPCPAPPVAGPSVX １６ LX １８ PPKPKDTLMISRTPEVTCX ３７ VX ３９ DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFX ７２ STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEX １６４ NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX ２２２ Includes, X １６ is F, Q, or E, and X １８ is F, Q, or E, and X ３７ is V or T, X ３９ is V or T, X ７２ is N, D, or Q, and X １６４ These are S, Q, E, D, and A, and X ２２２ The fusion protein according to any one of claims 1 to 17, wherein is K or is absent (SEQ ID NO: 5).

19. X １６ However, F is X １８ However, F is X ３７ However, V is X ３９ However, V is X ７２ However, N is X １６４ However, Q or S, X ２２２ The fusion protein according to claim 18, wherein the element is absent.

20. X １６４ However, Q is the fusion protein according to claim 19.

21. X １６４ The fusion protein according to claim 19, wherein S is present.

22. A fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:

12.

23. A homodimer of two fusion proteins according to any one of claims 1 to 22.

24. A pharmaceutical composition comprising the fusion protein described in any one of claims 1 to 22.

25. The pharmaceutical composition according to claim 24, wherein the composition is sufficiently stable to allow an increase of 1% or less per week in the acidic species of the fusion protein when stored at 30°C.

26. A method for treating a patient with diabetes mellitus, comprising administering a therapeutically effective amount of the fusion protein described in any one of claims 1 to 22 to the patient in need of the treatment.

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