Fusion proteins

Abstract

This invention relates to fusion proteins comprising an insulin receptor agonist fused to the Fc region of human IgG2, and the use of such fusion proteins in the treatment of diabetes. The fusion proteins of this invention have a prolonged duration of action and can be used to provide extended periods of basal glucose control.

Description

[0001] This invention relates to fusion proteins for the treatment of diabetes. More specifically, this invention relates to fusion proteins comprising an insulin receptor agonist fused to the Fc region of human IgG2, and the use of such proteins in the treatment of diabetes. The fusion proteins of this invention have a prolonged duration of action and can be used to provide prolonged basal glucose control and inhibition of hepatic glucose output. Background of the Invention Diabetes is a chronic condition characterized by high blood sugar due to defects in insulin secretion, insulin action, or both. Type 1 diabetes is characterized by little or no insulin secretion, and patients with type 1 diabetes require insulin to survive. Insulin therapy for patients with type 1 diabetes generally involves the use of two types of exogenously administered insulin: rapid-acting mealtime insulin delivered via high-dose injections, and long-acting basal insulin administered once or twice daily to control blood sugar levels between meals. Type 2 diabetes is characterized by elevated blood sugar levels due to impaired insulin secretion, insulin resistance, excessive hepatic glucose output, and / or contributing factors from all of the above. Treatment for patients with type 2 diabetes typically begins with prescribed weight loss, exercise, and a diabetic diet; however, when these measures fail to control elevated blood sugar, oral medications and incretin-based therapies, such as the administration of glucagon-like peptide-1 (GLP-1) receptor agonists and / or dipeptidyl peptidase-4 (DPP-4) inhibitors to achieve elevated incretin levels, may be necessary. When these medications are still insufficient, insulin therapy is considered. Type 2 diabetes patients whose disease has progressed to the point where insulin therapy is required generally begin treatment with a single daily injection of long-acting basal insulin, although in some cases, rapid-acting insulin may be included as needed during mealtimes.

[0003] Several types of basal insulin are currently indicated for once-daily administration, including insulin glargine sold under the brand name LANTUS®, insulin detemir sold under the brand name LEVEMIR®, and insulin degludec sold under the brand name TRESIBA®. All of these insulins are indicated for once-daily administration.

[0004] However, many people with diabetes are unwilling or unable to adhere to treatment regimens involving daily injections, so research is underway to identify insulin products with a longer duration of action; therefore, fewer injections are needed than with currently available insulin products to improve acceptability and adherence.

[0005] WO2016 / 178905 describes a fusion protein comprising an insulin receptor agonist fused to the Fc region of human IgG, the fusion protein having a prolonged action profile for providing prolonged basal glucose control.

[0006] However, additional options are still needed for basal insulin therapy, including therapies with improved chemical stability suitable for long-term storage at room temperature. Invention Overview This disclosure provides a fusion protein comprising an insulin receptor agonist fused to a human IgG2 Fc region, wherein the human IgG2 Fc region comprises an N residue at position 389 modified with an amino acid selected from S, Q, E, D, and A.

[0008] Homodimers of the two fusion proteins disclosed herein are also provided.

[0009] Methods for treating patients with diabetes are also provided, which include administering a therapeutically effective amount of the disclosed fusion protein to the patient in need.

[0010] The present disclosure also provides fusion proteins for use in therapeutics.

[0011] The use of the fusion protein disclosed herein in the manufacture of pharmaceuticals is also provided.

[0012] It also provides polynucleotides encoding the fusion protein disclosed herein.

[0013] A method for generating the fusion protein of this disclosure is also provided, the method comprising the following steps: Under conditions that enable expression of the fusion protein, mammalian host cells containing a polynucleotide encoding the fusion protein disclosed herein are cultured; and The fusion protein was recovered from the host cell. Brief description of the attached diagram Figure 1 . Figure 1 Exemplary pharmacokinetic data of the fusion protein of the present invention in a rat model of diabetes treated with streptozotocin (STZ) are provided. Invention Details The fusion protein of the present invention comprises an insulin receptor agonist fused to the Fc region of human IgG2. When used herein, the term "insulin receptor agonist" refers to a protein that binds to and activates the insulin receptor, resulting in a decrease in blood glucose levels and / or inhibition of hepatic glucose output, characterized by being detectable and measurable using known techniques, such as those shown in the studies described below.

[0016] In some embodiments, the insulin receptor agonist of the fusion protein of the present invention comprises an insulin A chain or an analogue thereof, and an insulin B chain or an analogue thereof. When 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 sequences are well known. The human insulin A chain consists of 21 amino acids, which are referred to in the art as A1-A. 21 It has the following sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO:8).

[0017] The human insulin B chain consists of 30 amino acids and is referred to in this field as B1-B. 30 It has the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:7).

[0018] In some embodiments, the insulin receptor agonist portion of the fusion protein of the present invention comprises analogs of the insulin B chain and analogs of the insulin A chain. When used herein, the term "analog" refers to a structure having one or more modifications (including amino acid substitutions, deletions, inversions, or additions) compared to a natural human amino acid sequence.

[0019] In some embodiments, insulin B-chain analogs include one or more modifications to the amino acid sequence of the human insulin B-chain. Specifically, to enhance chemical stability under storage conditions, insulin B-chain analogs include modifying the amino acid at position B3 to G, T, A, S, E, Q, R, K, H, or D. Furthermore, to reduce the tendency for self-association or dimerization of the insulin receptor agonist moiety, insulin B-chain analogs also include modifications to the position B3 of the human insulin molecule. 16 B 25 Or B 27-30 One or more modifications at the location, In some implementations, the insulin B-chain analog comprises the following amino acid sequence: X1X2X3QHLCGSHLVEALX 16 LVCGERGFX 25 YX 27 X 28 X 29 X 30 Where 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 It is E, Y, Q, or H; X 25 It is H or F; X27 It is G, T, S, H, V or does not exist; X 28 It is G, E, P, K, D, S, H or does not exist; X 29 It is G, E, K, P, Q, D, H or does not exist; X 30 It is G, T, S, E, K, A or does not exist (SEQ ID NO:1).

[0020] In some preferred embodiments, the insulin B-chain analog comprises the amino acid sequence of SEQ ID NO:1, wherein X3 is G, T, A, or S.

[0021] In some preferred embodiments, the insulin B-chain analog comprises the amino acid sequence of SEQ ID NO:1, wherein X3 is G or T. In some preferred embodiments, the insulin B-chain analog comprises the amino acid sequence of SEQ ID NO:1, wherein X1 is F; X2 is V; X3 is G or T; X 16 It is E; X 25 It is H; X 27 It is G; X 28 It is G; X 29 It is G; and X 30 It's G.

[0022] In some embodiments, insulin A-chain analogs may include one or more modifications to the amino acid sequence of the human insulin A-chain, said modifications intended to improve chemical and physical stability, regulatory efficacy, and / or enhance expression. In some embodiments, insulin A-chain analogs comprise the following amino acid sequence: GIVEQCCTSX 10 CSLX 14 QLENYCX 21 X 22 Where X 10 Is it T or I; X 14 It is D, Y, Q, or E; X 21 It is G, N, S, or A; and X 22 It is any naturally occurring amino acid or it does not exist, provided that X 21 If N, then X 22 It must be an amino acid other than G or N (SEQ ID NO:2).

[0023] In a preferred embodiment, the insulin A chain analog comprises the amino acid sequence of SEQ ID NO:2, wherein X 10 It is T; X 14 It is D; X 21 It is G; and X 22 It does not exist.

[0024] In the human insulin molecule, chains A and B are linked by two disulfide bonds, CysA7-CysB7 and CysA20-CysB19. Chain A has intrachain disulfide bonds at CysA6-CysA11. In some embodiments, the insulin receptor agonist of the fusion protein of the present invention comprises insulin chains A and B, or analogues thereof, which are linked by disulfide bonds at the sites where chains A and B are linked in the human insulin molecule. In some embodiments, the insulin chain A, or analogues thereof, contains intrachain disulfide bonds at CysA6-CysA11 within the insulin chain analogue.

[0025] In some 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 analogues thereof. In some embodiments, the human insulin A and B chains or analogues thereof are conjugated via a peptide linker referred to herein as a “first peptide linker.” In some embodiments, the C-terminal residue of the insulin B chain or analogue is fused to the N-terminal residue of the first peptide linker, and wherein the C-terminal residue of the first peptide linker is fused to the N-terminal residue of the insulin A chain or analogue. The first peptide linker must provide sufficient flexibility for the insulin A chain and the analogue of the insulin B chain to achieve the structure necessary for binding to the insulin receptor, but not so long as to excessively interfere with the binding. The length and composition of the first peptide linker can be tuned to modulate the efficacy and / or expression of the fusion protein. In some embodiments, the first peptide linker is 5 to 10 amino acids long, at least 5 of which are G residues.

[0026] In some embodiments, the amino acid sequence of the first peptide linker comprises the following amino acid sequence: X1GX3GGGG Where X1 is G or absent; and X3 is G, S or absent (SEQ ID NO:3). In some preferred embodiments, the first peptide linker comprises the sequence of SEQ ID NO:3, wherein X1 and X3 of SEQ ID NO:3 are G and S, respectively.

[0027] In some 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 using a linker. In some embodiments, the linker is a "second peptide linker". In some of these 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.

[0028] Preferably, the second peptide linker is glycine-rich to provide sufficient conformational flexibility. Preferably, the second peptide linker is less than 30 amino acids in length. In some preferred embodiments, the second peptide linker is 10 to 25 amino acids in length, wherein at least 50% of said amino acids are glycine residues. Preferred second peptide linkers include the sequence (GGGGX). n Where X is Q, E, or S and n = 2-5 (SEQ ID NO: 4). In some embodiments, n is 4. In other embodiments, n is 3.

[0029] In some preferred embodiments, the connector contains the sequence GGGGQGGGGGQGGGGGG (SEQ ID NO: 14).

[0030] When used herein, the term human IgG2 Fc region refers to an analogue of the Fc region of a modified, extended, and / or truncated human IgG2 antibody, for example, 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 comprises modifying the N residue at position 389 with an amino acid selected from S and Q.

[0031] It should be noted that, when 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); 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 SEQ ID NO:5, this amino acid is located at X 164 .

[0032] The human IgG Fc region of the fusion protein of the present invention may also have some hinge regions removed to simplify disulfide-mediated Fc dimerization. Other examples of alterations include phosphorylation, sulfation, acylation, glycosylation, methylation, acetylation, amidation, and / or modifications capable of producing heterodimeric molecules. Techniques for altering the structure and properties of the human IgG Fc region of IgG subclasses are known in the art.

[0033] In some preferred embodiments, the human IgG2 Fc region has the following amino acid sequence: ECPPCPAPPVAGPSVX 16 LX 18 PPKPKDTLMISRTPEVTCX 37 VX 39 DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFX 72 STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEX 164 NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX 222 Where X 16 It is F, Q, or E; X 18 It is F, Q, or E; X 37 It is V or T; X 39 It is V or T; X 72 It is N, D, or Q; X 164 It is S, Q, E, D, or A; and X 222 It is K or does not exist (SEQ ID NO:5).

[0034] In some preferred embodiments, the human IgG2 Fc region has the amino acid SEQ ID NO:5, wherein X 164 It is either S or Q.

[0035] A preferred human IgG2 Fc region has the sequence of SEQ ID NO:5, wherein X 16 It is F; X 18 It is F; X 37 It is V; X 39 It is V; X 72 It is N; X 164 It is Q; and X 222 It does not exist.

[0036] Another preferred human IgG IgG2 Fc region has the sequence of SEQ ID NO:5, wherein X 16 It is F; X 18 It is F; X 37 It is V; X 39 It is V; X 72 It is N; X 164 It is S; and X 222 It does not exist.

[0037] Although the hinge region portion of the preferred human IgG Fc region amino acid sequence described above is removed to simplify disulfide-mediated dimerization, those hinge regions may be present in some embodiments. For example, the wild-type IgG2 Fc region includes the six-amino acid sequence ERKCCV (SEQ ID NO:6) at its N-terminus, and although these amino acids are not described in the IgG2 Fc region sequence given in SEQ ID NO:5, it is contemplated that the human IgG Fc region containing the amino acid sequence given in SEQ ID NO:5 may further include some or all of the six-amino acid sequence ERKCCV at its N-terminus. Furthermore, the precise delineation of the boundary between the C-terminal amino acids constituting the second peptide linker and the N-terminal amino acids constituting the human IgG Fc region is not critical to the structure or function of the fusion protein of the present invention.

[0038] The fusion proteins and their uses of the present invention share certain characteristics with the fusion proteins and uses described in WO2016 / 178905 and can be produced using known techniques, such as those described in WO2016 / 178905. As described therein, the human IgG2 Fc region tends to self-associate to form dimers, typically covalently associated via disulfide bonds in the hinge region, and such dimers are formed from the human IgG2 Fc regions of two fusion proteins of the present invention. When the two fusion proteins constituting such a dimer have the same amino acid sequence, the dimer is referred to herein as a "homogeneous dimer". The fusion proteins of the present invention are preferably expressed in mammalian cells, and expression in such cells produces homodimers. When the two fusion proteins constituting a dimer have different amino acid sequences, the dimer is referred to herein as a "heterogeneous dimer".

[0039] Pharmaceutical compositions comprising fusion proteins as mentioned herein include: pharmaceutical compositions containing homodimers and / or heterodimers of the fusion protein, wherein one member of the heterodimer is the fusion protein. Similarly, methods of administering a fusion protein as mentioned herein include: administering a homodimer of the fusion protein and / or administering a heterodimer, wherein one member of the heterodimer is the fusion protein. Likewise, fusion proteins used for therapeutic purposes and / or for manufacturing pharmaceuticals include: homodimers and / or heterodimers of the fusion protein, wherein one member of the heterodimer is the fusion protein, for use in therapeutic purposes and / or for manufacturing pharmaceuticals.

[0040] As described above, the present invention also relates to polynucleotides encoding any of the fusion proteins of the present invention. The polynucleotides encoding the aforementioned fusion proteins may be in the form of RNA or DNA, including cDNA and synthetic DNA, and may be double-stranded or single-stranded. The coding sequence of the protein of the present invention may vary due to redundancy or degeneracy of the genetic code.

[0041] The polynucleotide encoding the fusion protein of the present invention may include: a protein-coding sequence only; a protein-coding sequence and additional coding sequences (such as leader or secretory sequences or preprotein sequences); and a protein-coding sequence and non-coding sequences (such as introns or non-coding sequences at the 5' and / or 3' ends of the protein-coding sequence). Therefore, the term "protein-coding polynucleotide" encompasses not only polynucleotides that may include the protein-coding sequence but also polynucleotides that include additional coding and / or non-coding sequences.

[0042] The polynucleotides of this invention will be expressed in host cells after the sequence has been operatively ligated to an expression control sequence. Expression vectors generally replicate in the host organism as episomes or as part of the host chromosomal DNA. Typically, expression vectors will contain selection markers to allow detection of cells transformed with the desired DNA sequence.

[0043] As used herein, the term "treatment" or "treating" refers to the management and care of a patient with diabetes or hyperglycemia or other conditions, for which insulin is administered to combat or alleviate symptoms and complications. Treatment includes administering the fusion protein of the present invention to prevent or delay the onset of symptoms or complications, alleviate symptoms or complications, or eliminate the disease, condition, or symptom. The patient to be treated is a mammal, and preferably a human.

[0044] As used herein, “prevent” or “preventing” means reducing the risk or incidence of one or more conditions, symptoms, complications or ailments, or eliminating or slowing the progression of one or more conditions, symptoms, complications or ailments.

[0045] The fusion protein of this invention can be used to treat subjects with a variety of diseases and conditions, including subjects with hyperglycemia, insulin-dependent diabetes mellitus, and non-insulin-dependent diabetes mellitus, including untreated subjects and subjects treated with oral medications (such as sulfonylureas, metformin, thiazolidinediones such as pioglitazone, alpha-glucosidase inhibitors such as acarbose) and / or non-insulin injectable agents (including glucagon-based therapies such as DPP-4 inhibitors and GLP-1R agonists). The fusion protein of this invention can be used to regulate blood glucose in such patients and can treat conditions or complications resulting from inadequate glycemic control, such as retinopathy, neuropathy, or nephropathy.

[0046] In some 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 a preferred embodiment, the duration of action is sufficiently prolonged to allow for weekly administration.

[0047] In some embodiments, the fusion protein of the present invention is administered in combination with additional active ingredients such as insulin or insulin analogs, glucagon-based therapies, DPP-4 inhibitors, amylin receptor agonists, or oral diabetes medications such as sulfonylureas, metformin, thiazolidinediones such as pioglitazone, or α-glucosidase inhibitors such as acarbose.

[0048] The term "glucagon-based therapy" includes any treatment that involves administering a group of metabolic hormones called enterotropins, or promoting, achieving, enhancing, and / or mimicking the effects of such a group of metabolic hormones, which are endogenous hormones secreted from the intestine by stimulating insulin secretion in a nutrient-dependent manner through activation of GLP-1 and / or GIP receptors. Enterotropins include any agonist (peptide or non-peptide) active at GLP-1 and / or GIP receptors. Enterotropins may contain additional activity at other receptors, such as the glucagon receptor (GCGR). Currently available enterotropin-based therapies include exenatide (Byetta® and Bydureon®), liraglutide (Victoza®), smegglutide (Ozempic®), abiglutide (Tanzeum®), duraglutide (Trulicity®), and telpoglutide (Mounjaro®). Other enterotropin-based therapies in clinical development include mashidotide and retatrutide.

[0049] "DPP-4 inhibitors" are compounds that block the DPP-4 enzyme, which is responsible for degrading glucagon. Currently available DPP-4 inhibitors include sitagliptin (Januvia®) and linagliptin (Tradjenta®).

[0050] In embodiments in which the fusion protein of the present invention is provided in combination with additional active ingredients, the fusion protein and the additional active ingredients may be administered simultaneously, sequentially, or in single or combined formulations.

[0051] The fusion protein of the present invention can effectively treat such diseases and conditions by administering a therapeutically effective amount of the fusion protein of the present invention to patients in need. As used herein, the term "therapeutically effective amount" means 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 amount of the fusion protein administered to a subject will depend on the type and severity of the disease and the characteristics of the subject, such as general health, age, sex, weight, and tolerance to the drug.

[0052] The fusion protein of the present invention can be administered parenterally, via nasal administration, or by pulmonary inhalation. Parenterally administration is preferred and may include, for example, systemic administration, such as by intramuscular injection, intravenous injection, subcutaneous injection, or intraperitoneal injection.

[0053] Fusion proteins 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 pharmaceutical compositions are typically (but not necessarily) parenteral in nature and can be prepared using conventional excipients for parenteral products via any of a variety of techniques well known in the art.

[0054] As described above, the fusion protein of the present invention is a homodimer when expressed in mammalian cells. Therefore, when used herein, the term "composition comprising a fusion protein" includes compositions containing a homodimer of the fusion protein.

[0055] The present invention is further illustrated by the following embodiments, which should not be considered limiting.

[0056] Expression and purification of fusion proteins The fusion protein of this invention can be produced in a mammalian cell expression system using a CHO glutamine synthase (GS) knockout (GSKO) cell line. GS gene knockout achieves tighter selection stringency by eliminating endogenous GS background activity that allows low-productivity or non-productivity cells to survive under selected conditions. The gene encoding the fusion protein is subcloned into an expression plasmid containing glutamine synthase (GS). The cDNA sequence encoding the fusion protein is fused in cascade with the coding sequence of a signal peptide, which enhances the secretion of the fusion protein into the cell culture 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.

[0057] Transfected cells underwent batch selection in glutamine-free medium. The transfected pools were plated at low density to allow for near-clonal growth of stably expressing cells. Fusion protein expression was screened in master wells and scaled up in serum-free suspension cultures for production.

[0058] Fusion proteins secreted into the culture medium can be purified using standard chromatographic techniques, specifically protein A affinity chromatography followed by size exclusion chromatography. In short, fusion proteins from clarified culture medium are captured using Mab Select Protein A (GE), which has been equilibrated with phosphate-buffered saline at pH 7.4. After washing with phosphate-buffered saline at pH 7.4, the bound fusion protein is eluted with 10 mM citrate at pH 3.0. Fractions containing the fusion protein are combined and neutralized by adding 1 / 10 volume of 1 M Tris at pH 8.0. Soluble aggregates and polymers can be efficiently removed using common techniques, including size exclusion, hydrophobic interaction, or ion exchange chromatography. Fractions containing monomeric fusion proteins (covalently linked homodimers) as determined by size exclusion chromatography are combined, sterilely filtered, and stored.

[0059] The amino acid sequence of an exemplary fusion protein of the present invention is shown below: Example 1 Example 2 Example 3 Example 4 refer to stability The example and reference fusion proteins were prepared at 10 mg / mL in 5 mM sodium phosphate, with the pH adjusted to 6.5 using HCl if necessary. Aliquots were prepared and stored at 5°C and 37°C for 8 days. After 8 days, the percentage of acidic species was quantified by anion-exchange chromatography, and species growth was determined by comparing the percentage of acidic species in the 37°C and 5°C samples. The results are presented in Table 1 below: name Δ% acidic species refer to 6.82 Example 1 3.13 Example 2 2.76 Example 3 2.60 Example 4 3.55 Table 1. Changes in acidic species for the examples and reference proteins.

[0060] As shown in Table 1, when stored at 37°C, the acid species in Examples 1-4 showed less change compared to the reference, indicating improved stability.

[0061] Studies in a rat model of diabetes treated with streptozotocin (STZ) The role of the fusion protein was investigated in a STZ-treated rat model of diabetes. Male Sprague-Dawley rats weighing 400–425 g were anesthetized with isoflurane and given a single injection of Zanosar® (STZ item # 89256, Teva Parenteral Medicines, 40 mg / kg IV). Rats were used in the study 3 days after the Zanosar® injection; only animals with non-fasting blood glucose levels between 400–550 mg / dL were used in these studies.

[0062] The rats were grouped to provide comparable variances 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 administered a single subcutaneous (SC) injection of 30 nmol / kg.

[0063] Blood samples were collected for glucose measurement via tail sampling. Animals had free access to food and water throughout the experiment. Blood glucose data were provided... Figure 1 The data shown is mean ± SEM (n=5). Blood glucose data were collected at time points during the initial feeding period (between 0 and 24 hours), but for ease of visual representation, this blood glucose data is not included in the table. Figure 1 In the middle. For example Figure 1 As shown, the examples and reference fusion proteins each provide a prolonged period of glucose reduction.

[0064] sequence SEQ ID NO:1—An analogue of the insulin B chain X1X2X3QHLCGSHLVEALX 16 LVCGERGFX 25 YX 27 X 28 X 29 X 30 Where 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 It is E, Y, Q, or H; X 25 It is H or F; X 27 It is G, T, S, H, V or does not exist; X 28 It is G, E, P, K, D, S, H or does not exist; X 29 It is G, E, K, P, Q, D, H or does not exist; X 30 It is G, T, S, E, K, A or does not exist. SEQ ID NO:2—An analogue of the insulin A chain GIVEQCCTSX 10 CSLX14 QLENYCX 21 X 22 Where X 10 Is it T or I; X 14 It is D, Y, Q, or E; X 21 It is G, N, S, or A; and X 22 It is any naturally occurring amino acid or it does not exist, provided that X 21 If N, then X 22 It must be an amino acid other than G or N. SEQ ID NO:3—First peptide linker X1GX3GGGG Where X1 is G or does not exist; and X3 is G, S or does not exist. SEQ ID NO:4—Second peptide linker GGGGX n Where X is Q, E, or S and n = 2-5 SEQ ID NO:5—Human IgG2 Fc region ECPPCPAPPVAGPSVX 16 LX 18 PPKPKDTLMISRTPEVTCX 37 VX 39 DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFX 72 STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEX 164 NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX 222 Where X 16 It is F, Q, or E; X 18 It is F, Q, or E; X 37 It is V or T; X 39 It is V or T; X 72 It is N, D, or Q; X 164 The numbers are S, Q, E, D, and A; and X 222 It is K or it does not exist.

[0065] SEQ ID NO:6—Human IgG2 Fc region hinge ERKCCV SEQ ID NO:7—Human Insulin B Chain fvnqhlcgshlvealylvcgergffytpkt SEQ ID NO:8—Human Insulin A Chain giveqcctsicslyqlenycn SEQ ID NO:9—Example: Fusion Protein SEQ ID NO:10—Example: Fusion Protein SEQ ID NO:11—Example: Fusion Protein SEQ ID NO:12—Example: Fusion Protein SEQ ID NO:13—Reference fusion protein SEQ ID NO:14—Second peptide linker GGGGQGGGGQGGGGQGGGG

Claims

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

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

3. The fusion protein of claim 2, wherein the insulin B-chain analog comprises the following amino acid sequence: X1X2X3QHLCGSHLVEALX 16 LVCGERGFX 25 YX 27 X 28 X 29 X 30 Where 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 It is E, Y, Q, or H; X 25 It is H or F; X 27 It is G, T, S, H, V or does not exist; X 28 It is G, E, P, K, D, S, H or does not exist; X 29 It is G, E, K, P, Q, D, H or does not exist; X 30 It is G, T, S, E, K, A or does not exist (SEQ ID NO:1).

4. The fusion protein of claim 3, wherein X1 is F; X2 is V; X3 is G or T; X 16 It is E; X 25 It is H; X 27 It is G; X 28 It is G; X 29 It is G; and X 30 It's G.

5. The fusion protein of claim 4, wherein X3 is G.

6. The fusion protein of claim 4, wherein X3 is T.

7. The fusion protein of any one of claims 2-6, wherein the insulin A chain analog comprises the following amino acid sequence: GIVEQCCTSX 10 CSLX 14 QLENYCX 21 X 22 Where X 10 Is it T or I; X 14 It is D, Y, Q, or E; X 21 It is G, N, S, or A; and X 22 It is any naturally occurring amino acid or it does not exist, provided that X 21 If N, then X 22 It must be an amino acid other than G or N (SEQ ID NO:2).

8. The fusion protein of claim 7, wherein X 10 It is T; X 14 It is D; X 21 It is G; and X 22 It does not exist.

9. The fusion protein of any one of claims 2-8, wherein the C-terminal residue of the insulin B chain analog is fused to the N-terminal residue of the first peptide linker, and wherein 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 of claim 9, wherein the first peptide linker comprises 5 to 10 amino acids, wherein at least 5 of the amino acids are G residues.

11. The fusion protein of claim 9 or 10, wherein the first peptide linker comprises the following amino acid sequence: X1GX3GGGG Where X1 is G or does not exist; and X3 is G, S or does not exist (SEQ ID NO:3).

12. The fusion protein of claim 11, wherein X1 and X3 of SEQ ID NO:3 are G and S, respectively.

13. The fusion protein of any one of claims 2-12, wherein the C-terminal residue of the insulin A chain analog is fused to the N-terminal residue of the second peptide linker, and wherein 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 of claim 13, wherein the second peptide linker is a peptide having 10 to 25 amino acids, wherein at least 50% of the amino acids are G residues.

15. The fusion protein of claim 14, wherein the second peptide linker comprises the sequence [GGGGX]. n peptides Where X is Q, E or S; and where n is 2-5 (SEQ ID NO:4).

16. The fusion protein of claim 15, wherein n is 3.

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

18. The fusion protein of any one of claims 1-17, wherein the human IgG2 Fc region comprises an amino acid sequence: ECPPCPAPPVAGPSVX 16 LX 18 PPKPKDTLMISRTPEVTCX 37 VX 39 DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFX 72 STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEX 164 NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX 222 Where X 16 It is F, Q, or E; X 18 It is F, Q, or E; X 37 It is V or T; X 39 It is V or T; X 72 It is N, D, or Q; X 164 The numbers are S, Q, E, D, and A; and X 222 It is K or does not exist (SEQ ID NO:5).

19. The fusion protein of claim 18, wherein X 16 It is F; X 18 It is F; X 37 It is V; X 39 It is V; X 72 It is N; X 164 It is Q or S; and X 222 It does not exist.

20. The fusion protein of claim 19, wherein X 164 It's Q.

21. The fusion protein of claim 19, wherein X 164 It is S.

22. A fusion protein comprising an amino acid sequence selected from 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 1-22.

24. A pharmaceutical composition comprising the fusion protein of any one of claims 1-22.

25. The pharmaceutical composition of claim 24, wherein the composition is sufficiently stable to allow the increase in acidic species of the fusion protein to not exceed 1% / week when the composition is stored at 30°C.

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

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