Insulin-fc fusion proteins

Abstract

The present invention relates to fusion proteins comprising an insulin receptor agonist fused to a human IgG2 Fc region, and the use of such fusion proteins in the treatment of diabetes. The fusion protein of the present invention has an extended time action profile and is useful for providing basal glucose control for an extended period of time.

Description

FUSION PROTEINS

[0001] The present invention relates to fusion proteins for use in the treatment of diabetes. More particularly, the invention relates to fusion proteins comprising an insulin receptor agonist fused to a human IgG2 Fc region, and the use of such proteins in the treatment of diabetes. The fusion proteins of the present invention have an extended time action profile and are useful for providing protracted basal glucose control and suppression of hepatic glucose output. BACKGROUND OF THE INVENTION

[0002] Diabetes mellitus is a chronic disorder characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Type 1 diabetes mellitus is characterized by little or no insulin secretory capacity, and patients with type 1 diabetes mellitus require insulin for survival. Insulin therapy for Type 1 diabetics generally involves the use of two types of exogenously administered insulin: a rapid-acting, mealtime insulin provided by bolus injections, and a 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 impaired insulin secretion, insulin resistance, excessive hepatic glucose output, and / or contributions from all of the above. Treatment of patients with Type 2 diabetes typically begins with prescribed weight loss, exercise, and a diabetic diet, but when these measures fail to control elevated blood sugars, then oral medications and incretin-based therapy, such as administration of glucagon-like peptide-1 (GLP-1) receptor agonists and / or dipeptidyl peptidase 4 (DPP-4) inhibitors that enable increased incretin levels, may be necessary. When these medications are still insufficient, treatment with insulin is considered. Type 2 diabetes patients whose disease has progressed to the point that insulin therapy is required are generally started on a single daily injection of a long- acting, basal insulin, although mealtime injections of rapid-acting insulins may be included, as necessary, in some cases.

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

[0004] Many diabetic patients are unwilling or unable to comply with treatment regimens involving daily injections, however, so research is being conducted to identify insulin products with longer duration of action; thus, requiring fewer injections than currently available insulin products to improve acceptance and compliance.

[0005] WO2016 / 178905 describes fusion proteins comprising insulin receptor agonists fused to human IgG Fc regions that have extended time action profiles for providing protracted basal glucose control.

[0006] Nevertheless, the need remains for additional options for basal insulin therapy, including therapies having improved chemical stability suitable for prolonged storage at room temperature. SUMMARY OF THE INVENTION

[0007] The present disclosure provides a fusion protein comprising an insulin receptor agonist fused to a human IgG2 Fc region, wherein the human IgG2 Fc region comprises 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] Also provided is a homodimer of two fusion proteins of the present disclosure.

[0009] Also provided is a method of treating a patient with diabetes mellitus comprising administering to a patient in need thereof a therapeutically effective amount of a fusion protein of the present disclosure.

[0010] Also provided is a fusion protein of the present disclosure for use in therapy.

[0011] Also provided is the use of a fusion protein of the present disclosure in the manufacture of a medicament.

[0012] Also provided are polynucleotides encoding a fusion protein of the present disclosure.

[0013] Also provided is a process for producing a fusion protein of the present disclosure, said process comprising the steps of: culturing a mammalian host cell comprising a polynucleotide encoding a fusion protein of the present disclosure under conditions such that said fusion protein is expressed; and recovering from said host cell a fusion protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1. Figure 1 provides pharmacodynamic data for exemplary fusion proteins of the present invention in a streptozotocin (STZ)-treated rat diabetes model. DETAILED DESCRIPTION OF THE INVENTION

[0015] The fusion proteins of the present invention comprise insulin receptor agonists fused to human IgG2 Fc regions. When used herein, the term “insulin receptor agonist” refers to a protein that binds to and activates the insulin receptor, resulting in a lowering of blood glucose levels and / or suppression of hepatic glucose output, characteristics which can be tested and measured using known techniques, such as those shown in the studies described below.

[0016] In certain embodiments, the insulin receptor agonist of fusion proteins of the present invention comprises an insulin-A chain, or analog thereof, and an insulin B-chain, or analog 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 No.11061-68-0), whose native wild type sequences are well-known. The human insulin A-chain consists of 21 amino acids, referred to in the art as A1-A21, having the following sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO:8).

[0017] The human insulin B-chain consists of 30 amino acids, referred to in the art as B1- B30, having the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:7).

[0018] In certain embodiments, the insulin receptor agonist portion of the fusion proteins of the present invention includes an analog of an insulin B-chain and an analog of an 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, when compared with the native human amino acid sequence.

[0019] In certain embodiments, the analog of the insulin B-chain includes one or more modifications to the amino acid sequence of the human insulin B-chain. In particular, in order to enhance chemical stability during in-use storage conditions, the insulin B-chain analog includes modification of the amino acid at position B3, to G, T, A, S, E, Q, R, K, H or D. In addition, in order to reduce the propensity of the insulin receptor agonistportions to self-associate, or dimerize, the insulin B-chain analog also includes one or more modifications from the B-chain of a molecule of human insulin at positions B16, B25 or B27-30,

[0020] In certain embodiments, the analog of the insulin B-chain comprises the amino acid sequence: X1X2X3QHLCGSHLVEALX16LVCGERGFX25YX27X28X29X30 wherein X1 is F, Q or A; X2 is V or G; X3 is G, T, A, S, E, Q, R, K, H or D; X16 is E, Y, Q, or H; X25 is H or F; X27 is G, T, S, H, V or is absent; X28 is G, E, P, K, D, S, H or is absent; X29 is G, E, K, P, Q, D, H or is absent; X30 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; X16 is E; X25 is H; X27 is G; X28 is G; X29 is G; and X30 is G.

[0023] In certain embodiments, the analog of the insulin A-chain may include one or more modifications to the amino acid sequence of the human insulin A-chain intended to improve chemical and physical stability, adjust potency, and / or enhance expression. In certain embodiments, the analog of the insulin A-chain comprises the amino acid sequence: GIVEQCCTSX10CSLX14QLENYCX21X22 wherein X10 is T or I; X14 is D, Y, Q or E; X21 is G, N, S or A; and X22 is any naturally occurring amino acid, or is absent, provided that if X21is N, then X22must be an amino acid other than G or N (SEQ ID NO:2).

[0024] In a preferred embodiment, the analog of the insulin A-chain comprises the amino acid sequence of SEQ ID NO:2, wherein X10 is T; X14 is D; X21 is G; and X22 is absent.

[0025] In a molecule of human insulin, the A- and B-chains are joined by two disulfide bonds, CysA7-CysB7 and CysA20-CysB19. The A-chain has an intra-chain disulfide bond at CysA6-CysA11. In certain embodiments, the insulin receptor agonist of fusionproteins of the present invention comprise insulin A- and B-chains, or analogs thereof, joined by disulfide bonds at the positions at which the A- and B-chains are joined in a molecule of human insulin. In certain embodiments, the insulin A-chain, or analogs thereof, comprises an intra-chain disulfide bond in the analog of the insulin A-chain at CysA6-CysA11.

[0026] In certain embodiments, the insulin receptor agonist of fusion proteins of the present invention comprise a single chain comprising 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 analog thereof, is fused to the N-terminal residue of a 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 analog thereof. The first peptide linker must provide sufficient flexibility for the analogs of the insulin A-chain and B-chain to achieve the structure necessary to bind to the insulin receptor, but must not be so long that it unduly interferes with that binding. The length and composition of the first peptide linker may be adjusted in order to adjust the potency and / or expression of the fusion proteins. In some embodiments, the first peptide linker is 5 to 10 amino acids in length, at least 5 of which are G residues.

[0027] In certain embodiments, the amino acid sequence of the first peptide linker comprises the following amino acid sequence: X1GX3GGGG

[0028] wherein X1 is G or is absent; and X3 is G, S or is absent (SEQ ID NO:3). In certain 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.

[0029] In certain embodiments, the insulin receptor agonist is fused directly to the human IgG2 Fc region. In other embodiments, the insulin receptor agonist is fused to the human IgG2 Fc region through the use of a linker. In certain embodiments, the linker is a “second peptide linker.” In certain of these embodiments, the C-terminal residue of the insulin receptor agonist portion of the fusion proteins of the present invention is fused to the N-terminal residue of a second peptide linker, and the C-terminal residue of the second peptide linker is fused directly to the N-terminal residue of the Fc portion.

[0030] It is preferred that the second peptide linker be glycine rich, to provide sufficient conformational flexibility. Preferably, the second peptide linker is less than 30 amino acids in length. In certain preferred embodiments, the second peptide linker is between 10 and 25 amino acids in length, with at least 50% of the amino acids being glycine residues. A preferred second peptide linker includes the sequence (GGGGX)n wherein X is Q, E or S and n = 2-5 (SEQ ID NO:4). In certain embodiments n is 4. In other embodiments n is 3.

[0031] In certain preferred embodiments, the linker comprises the sequence GGGGQGGGGQGGGGQGGGG (SEQ ID NO: 14).

[0032] When used herein, the term human IgG2 Fc region refers to an analog of the Fc region of a human IgG2 antibody that has been modified, elongated and / or truncated, for example, to alter properties or characteristics such as the complement and / or Fc receptor binding functions, effector functions, disulfide bond formation, glycosylation, antibody- dependent cell-mediated cytotoxicity (ADCC), manufacturability, and / or stability. In particular, the human IgG2 Fc regions in fusion proteins of the present invention comprise modification of the N residue at position 389 to an amino acid selected from the group consisting of S and Q.

[0033] It should be noted that when used herein “position 389” in an IgG2 Fc region refers to the amino acid at position 389 of an antibody according to the EU antibody numbering scheme. See, e.g., chart, available atEdelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); Kabat, E.A. 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 regions identified below by SEQ ID NO:5, this amino acid is located at X164.

[0034] The human IgG Fc regions of the fusion proteins of the present invention may also have some of the hinge region removed in order to simplify disulfide mediated Fc dimerization. Other examples of alterations include phosphorylation, sulfation, acylation, glycosylation, methylation, acetylation, amidation, and / or modifications to enable production of heterodimer molecules. Techniques for modifying the structures and properties of the human IgG Fc regions of the IgG subclasses are known in the art.

[0035] In certain preferred embodiments, the human IgG2 Fc region has the following amino acid sequence: ECPPCPAPPVAGPSVX16LX18PPKPKDTLMISRTPEVTCX37VX39DVSHEDPEVQFN WYVDGVEVHNAKTKPREEQFX72STFRVVSVLTVVHQDWLNGKEYKCKVSNKG LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPEX164NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGX222 wherein X16 is F, Q or E; X18 is F, Q or E; X37 is V or T; X39 is V or T; X72 is N, D or Q; X164 is S, Q, E, D or A; and X222 is K or absent (SEQ ID NO:5).

[0036] In certain preferred embodiments, the human IgG2 Fc region has the amino acid of SEQ ID NO: 5 wherein X164 is S or Q.

[0037] A preferred human IgG IgG2 Fc region has the sequence of SEQ ID NO:5 wherein X16 is F; X18 is F; X37 is V; X39 is V; X72 is N; X164 is Q; and X222 is absent.

[0038] Another preferred human IgG IgG2 Fc region has the sequence of SEQ ID NO:5 wherein X16 is F; X18 is F; X37 is V; X39 is V; X72 is N; X164 is S; and X222 is absent.

[0039] Although the amino acid sequences of the preferred human IgG Fc regions recited above have portions of the hinge regions removed to simplify disulfide mediated dimerization, those hinge regions may be present in certain embodiments. For example, a wild-type IgG2 Fc region includes the six amino acid sequence ERKCCV (SEQ ID NO:6) at its N-terminal end, and although these amino acids are not recited in the IgG2 Fc region sequence set forth in SEQ ID NO:5, it is contemplated that a human IgG Fc region which comprises the amino acid sequence set forth in SEQ ID NO:5 may further comprise some or all of the six amino acid sequence ERKCCV at its N-terminal end. Moreover, the precise delineation between which amino acid constitutes the C-terminal end of the second peptide linker and which amino acid constitutes the N-terminal end of the human IgG Fc region is not critical to the structure or function of the fusion protein of the present invention.

[0040] The fusion proteins of the present invention and their use share certain features with the fusion proteins and uses described in WO2016 / 178905, and may be produced using known techniques, such as those described in WO2016 / 178905. As described therein, human IgG2 Fc regions tend to self-associate to form dimers, typically associatedcovalently through disulfide bonds in the hinge region, and such dimers are formed from human IgG2 Fc regions of two fusion proteins of the present invention. When the amino acid sequences of the two fusion proteins that make up such a dimer are the same, the dimer is referred to herein as a “homodimer.” Expression of fusion proteins of the present invention in mammalian cells is preferred, and expression in such cells results in homodimers. When the amino acid sequences of two fusion proteins that make up a dimer are different, the dimer is referred to herein as a “heterodimer.”

[0041] References herein to pharmaceutical compositions comprising a fusion protein include pharmaceutical compositions which contain a homodimer of that fusion protein, and / or which contain a heterodimer, wherein one member of the heterodimer is that fusion protein. Similarly, references herein to methods comprising administering a fusion protein, include methods comprising administering a homodimer of that fusion protein and / or administering a heterodimer, wherein one member of the heterodimer is that fusion protein. Likewise, references to a fusion protein for use in therapy and / or a fusion protein for use in the manufacture of a medicament include a homodimer of that fusion protein, and / or a heterodimer wherein one member of the heterodimer is that fusion protein, for use in therapy and / or in the manufacture of a medicament,

[0042] As described above, the present invention also relates to polynucleotides that encode any of the fusion proteins of the present invention. The polynucleotides encoding the above-described fusion proteins may be in the form of RNA or DNA, which includes cDNA and synthetic DNA, and which may be double-stranded or single-stranded. The coding sequences that encode the proteins of the present invention may vary as a result of the redundancy or degeneracy of the genetic code.

[0043] The polynucleotides that encode for the fusion proteins of the present invention may include the following: only the coding sequence for the proteins, the coding sequence for the proteins and additional coding sequence, such as a leader or secretory sequence or a pro-protein sequence; the coding sequence for the proteins and non-coding sequence, such as introns or non-coding sequence 5' and / or 3' of the coding sequence for the proteins. Thus, the term “polynucleotide encoding a protein” encompasses a polynucleotide that may include not only coding sequence for the proteins but also a polynucleotide that includes additional coding and / or non-coding sequence.

[0044] The polynucleotides of the present invention will be expressed in a host cell after the sequences have been operably linked to an expression control sequence. The expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers to permit detection of those cells transformed with the desired DNA sequences.

[0045] The term “treatment” or “treating,” as used herein, refers to the management and care of a patient having diabetes or hyperglycemia, or other condition for which insulin administration is indicated for the purpose of combating or alleviating symptoms and complications of those conditions. Treating includes administering fusion proteins of the present invention to prevent or delay the onset of symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. The patient to be treated is a mammal, and preferably, a human being.

[0046] The term “prevent” or “preventing,” as used herein, refers to reducing the risk or incidence of, or eliminating or slowing the progression of, one or more conditions, symptoms, complications or disorders.

[0047] The fusion proteins of the present invention can be used to treat subjects with a wide variety of diseases and conditions. Included are subjects with hyperglycemia, insulin-dependent diabetes as well as subjects with non-insulin dependent diabetes, including treatment naïve subjects as well as subjects being treated with oral medications, such as a sulfonylurea, metformin, thiazolidinedione such as pioglitazone, α-glucosidase inhibitor such as acarbose, and / or noninsulin injectables, including incretin-based therapies, such as DPP-4 inhibitors and GLP-1R agonists. The fusion proteins of the present invention may be used to regulate blood glucose in such patients, and may treat conditions or complications that result from insufficient blood glucose control such as retinopathy, neuropathy or kidney disease.

[0048] In certain embodiments, the fusion protein of the present invention is administered every day, every other day, twice weekly, thrice weekly, once weekly, twice monthly or once monthly. In preferred embodiments, the duration of action is sufficiently extended to allow for once weekly dosing.

[0049] In certain embodiments, the fusion protein of the present invention is administered in combination with an additional active ingredient, such as insulin or an insulin analog, an incretin-based therapy, a DPP-4 inhibitor, an amylin receptor agonist, or an oral diabetes medication, such as a sulfonylurea, metformin, thiazolidinedione such as pioglitazone or an α-glucosidase inhibitor such as acarbose.

[0050] The term “incretin-based therapy” includes any treatment which comprises administration of, or promotes, enables, enhances and / or simulates the effects of, a group of metabolic hormones known as incretins, which are endogenous hormones secreted from the gut that stimulate insulin secretion in a nutrient-dependent manner by activation of the GLP-1 and / or GIP receptor. Incretins include any agonists (peptide or non-peptide) with activity on either the GLP-1 and / or GIP receptors. Incretins may contain additional activity at other receptors, such as the glucagon receptor (GCGR). Currently available incretin-based therapies include exenatide (Byetta® and Bydureon®), liraglutide (Victoza®), semaglutide (Ozempic®), albiglutide (Tanzeum®), dulaglutide (Trulicity®) and tirzepatide (Mounjaro®). Other incretin-based therapies in clinical development include mazdutide and retatrutide.

[0051] A “DPP-4 inhibitor” is a compound that blocks the DPP-4 enzyme, which is responsible for the degradation of incretins. Currently available DPP-4 inhibitors include sitagliptin (Januvia®), and linagliptin (Tradjenta®).

[0052] In embodiments wherein a fusion protein of the present invention is provided in combination with an additional active ingredient, the fusion protein and additional active ingredient may be administered simultaneously, sequentially or in a single, combined formulation.

[0053] The fusion proteins of the present invention are effective in treating such diseases and conditions by administering to a patient in need thereof a therapeutically effective amount of a fusion protein of the present invention. As used herein, the phrase “therapeutically effective amount” refers to that amount of a fusion protein of the present invention sufficient to regulate blood glucose in a patient without causing unacceptable side effects. A therapeutically effective amount of the fusion protein administered to a subject will depend on the type and severity of the disease and on the characteristics of the subject, such as general health, age, sex, body weight, and tolerance to drugs.

[0054] The fusion proteins of the present invention are administered parenterally, by nasal administration or pulmonary inhalation. Parenteral administration is preferred, and can include, for example, systemic administration, such as by intramuscular, intravenous, subcutaneous, or intraperitoneal injection.

[0055] The fusion proteins can be administered to the subject in a pharmaceutical composition, which comprises a fusion protein of the present invention and at least one pharmaceutically acceptable excipient. Such pharmaceutical compositions are typically, though not necessarily, parenteral in nature and may be prepared by any of a variety of techniques using conventional excipients for parenteral products, which are well known in the art.

[0056] As described above, the fusion protein of the present invention is a homodimer when expressed in mammalian cells. Thus, when used herein, the term “composition comprising a fusion protein” includes a composition, which contains a homodimer of a fusion protein.

[0057] The invention is further illustrated by the following examples, which are not to be construed as limiting.

[0058] Expression and Purification of Fusion Proteins

[0059] Fusion proteins of the present invention may be produced in a mammalian cell expression system using the CHO glutamine synthetase (GS) knockout (GSKO) cell line. The GS gene knockout enables tightened selection stringency by eliminating endogenous GS background activity which can allow survival of low- or non-productive cells under selection conditions. Genes coding for fusion proteins are sub-cloned into the glutamine synthetase (GS) containing expression plasmid. The cDNA sequence encoding the fusion proteins is fused in frame with the coding sequence of a signal peptide which enhances secretion of the fusion protein into the cell culture medium. The expression is driven by the cytomegalovirus (CMV) promoter. CHO GSKO cells are stably transfected using electroporation and the appropriate amount of recombinant expression plasmid.

[0060] Transfected cells undergo bulk selection in glutamine-free media. Transfected pools are plated at low density to allow for close-to-clonal outgrowth of stable expressing cells. The masterwells are screened for fusion protein expression and scaled-up in serum- free suspension cultures to be used for production.

[0061] Fusion proteins secreted into the media may be purified by Protein A affinity chromatography followed by size exclusion chromatography following standard chromatographic techniques. Briefly, fusion proteins from clarified media are captured by Mab Select Protein A (GE) that has been equilibrated with phosphate buffered saline pH 7.4. Following a wash step with phosphate buffered saline pH 7.4, bound fusion proteins are eluted with 10 mM citric acid pH 3.0. Fractions containing fusion protein are pooled and neutralized by adding 1 / 10 volume of 1M Tris pH 8.0. Soluble aggregates and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction or ion exchange chromatography. Fractions containing monomeric fusion protein (covalently linked homodimer), as determined by size exclusion chromatography, are pooled, sterile filtered, and stored.

[0062] Amino acid sequences of exemplary fusion proteins of the present invention are shown below:

[0063] Example 1 10 20 30 40 50 60 FVGQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGG 70 80 90 100 110 120 GGQGGGGQGGGGQGGGGGECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 130 140 150 160 170 180 HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG 190 200 210 220 230 240 LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 250 260 270 280 290 ESNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:9)

[0064] Example 2 10 20 30 40 50 60 FVGQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGG 70 80 90 100 110 120 GGQGGGGQGGGGQGGGGGECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 130 140 150 160 170 180 HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG 190 200 210 220 230 240 LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 250 260 270 280 290 EQNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:10)

[0065] Example 310 20 30 40 50 60 FVTQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGG 70 80 90 100 110 120 GGQGGGGQGGGGQGGGGGECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 130 140 150 160 170 180 HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG 190 200 210 220 230 240 LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 250 260 270 280 290 ESNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:11)

[0066] Example 4 10 20 30 40 50 60 FVTQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGG 70 80 90 100 110 120 GGQGGGGQGGGGQGGGGGECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 130 140 150 160 170 180 HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG 190 200 210 220 230 240 LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 250 260 270 280 290 EQNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:12)

[0067] Reference 10 20 30 40 50 60 FVGQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGG 70 80 90 100 110 120 GGQGGGGQGGGGQGGGGGECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 130 140 150 160 170 180 HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG 190 200 210 220 230 240 LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 250 260 270 280 290 ENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:13)

[0068] Stability

[0069] Example and reference fusion proteins are formulated at 10 mg / mL in 5 mM sodium phosphate with pH adjusted to 6.5, if necessary, using HCl. Formulation aliquots were stored at 5°C and 37°C for 8 days. After 8 days, percent acidic species are quantified by anion exchange chromatography, and species growth were determined bycomparing the percent of acidic species in 37°C sample to 5°C sample. Results are provided in Table 1 below:

[0070] Table 1. Change in acidic species for example and reference proteins.

[0071] As seen in Table 1, when stored at 37°C the change in acidic species is less for Examples 1-4 as compared to reference, demonstrating improved stability.

[0072] Studies in Streptozotocin (STZ)-Treated Rat Diabetes Model

[0073] Effects of Fusion Proteins are investigated in STZ-treated rat diabetes model. Male Sprague-Dawley rats, 400-425 gram body weight, are anesthetized with isoflurane and given a single injection of Zanosar®(STZ item # 89256, Teva Parenteral Medicines, 40 mg / kg IV). The rats are used in studies 3 days after injection of Zanosar®; only animals with non-fasted blood glucose between 400-550 mg / dL are used in these studies.

[0074] The rats are distributed into groups to provide comparable variance in blood glucose and body weight and then randomized. Blood glucose is measured using Accucheck Aviva glucometer (Roche). STZ-treated rats are given a single subcutaneous (SC) injection of 30 nmol / kg dose.

[0075] Blood samples for glucose measurements are collected by tail bleed. Animals have free access to food and water throughout the experiment. Blood glucose data are provided in Figure 1. Data shown are mean ± SEM (n=5). Blood glucose data for time points during the initial feeding period (between 0 and 24 hours) are collected, but are not included in Figure 1 for ease of visual representation. As shown in Figure 1, Example and Reference fusion proteins each provide glucose lowering for a prolonged period of time.

[0076] Sequences

[0077] SEQ ID NO:1 – Analog of insulin B-chain X1X2X3QHLCGSHLVEALX16LVCGERGFX25YX27X28X29X30 wherein X1 is F, Q or A; X2 is V or G; X3 is G, T, A, S, E, Q, R, K, H or D; X16 is E, Y, Q, or H; X25 is H or F; X27 is G, T, S, H, V or is absent; X28 is G, E, P, K, D, S, H or is absent; X29 is G, E, K, P, Q, D, H or is absent; X30 is G, T, S, E, K, A or is absent,

[0078] SEQ ID NO:2 – Analog of insulin A-chain GIVEQCCTSX10CSLX14QLENYCX21X22 wherein X10 is T or I; X14 is D, Y, Q or E; X21 is G, N, S or A; and X22 is any naturally occurring amino acid, or is absent, provided that if X21 is N, then X22 must be an amino acid other than G or N

[0079] SEQ ID NO:3 – First peptide linker X1GX3GGGG wherein X1 is G or is absent; and X3 is G, S or is absent

[0080] SEQ ID NO:4 – Second peptide linker GGGGXn wherein X is Q, E or S and n = 2-5

[0081] SEQ ID NO:5 – Human IgG2 Fc region ECPPCPAPPVAGPSVX16LX18PPKPKDTLMISRTPEVTCX37VX39DVSHEDPEVQFN WYVDGVEVHNAKTKPREEQFX72STFRVVSVLTVVHQDWLNGKEYKCKVSNKG LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPEX164NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGX222 wherein X16 is F, Q or E; X18 is F, Q or E; X37 is V or T; X39 is V or T; X72 is N, D or Q; X164 is S, Q, E, D and A; and X222 is K or absent.

[0082] SEQ ID NO:6 – Human IgG2 Fc region hinge ERKCCV

[0083] SEQ ID NO:7 – Human insulin B-chain FVNQHLCGSHLVEALYLVCGERGFFYTPKT

[0084] SEQ ID NO:8 – Human insulin A-chain GIVEQCCTSICSLYQLENYCN

[0085] SEQ ID NO:9 – Example fusion protein 10 20 30 40 50 60 FVGQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGG 70 80 90 100 110 120 GGQGGGGQGGGGQGGGGGECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 130 140 150 160 170 180 HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG 190 200 210 220 230 240 LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 250 260 270 280 290 ESNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0086] SEQ ID NO:10 – Example fusion protein 10 20 30 40 50 60 FVGQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGG 70 80 90 100 110 120 GGQGGGGQGGGGQGGGGGECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 130 140 150 160 170 180 HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG 190 200 210 220 230 240 LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 250 260 270 280 290 EQNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0087] SEQ ID NO:11 – Example fusion protein 10 20 30 40 50 60 FVTQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGG 70 80 90 100 110 120 GGQGGGGQGGGGQGGGGGECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 130 140 150 160 170 180 HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG 190 200 210 220 230 240 LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP250 260 270 280 290 ESNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0088] SEQ ID NO:12 – Example fusion protein 10 20 30 40 50 60 FVTQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGG 70 80 90 100 110 120 GGQGGGGQGGGGQGGGGGECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 130 140 150 160 170 180 HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG 190 200 210 220 230 240 LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 250 260 270 280 290 EQNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0089] SEQ ID NO:13 – Reference fusion protein 10 20 30 40 50 60 FVGQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGG 70 80 90 100 110 120 GGQGGGGQGGGGQGGGGGECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 130 140 150 160 170 180 HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG 190 200 210 220 230 240 LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 250 260 270 280 290 ENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0090] SEQ ID NO:14 – Second peptide linker GGGGQGGGGQGGGGQGGGG

Claims

WE CLAIM:

1. A fusion protein comprising an insulin receptor agonist fused to a human IgG2 Fc region, wherein the human IgG2 Fc region comprises 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 of claim 1 wherein the insulin receptor agonist comprising 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 amino acid sequence: X1X2X3QHLCGSHLVEALX16LVCGERGFX25YX27X28X29X30 wherein X1 is F, Q or A; X2 is V or G; X3 is G, T, A, S, E, Q, R, K, H or D; X16 is E, Y, Q, or H; X25 is H or F; X27 is G, T, S, H, V or is absent; X28 is G, E, P, K, D, S, H or is absent; X29 is G, E, K, P, Q, D, H or is absent; X30 is G, T, S, E, K, A or is absent, (SEQ ID NO:1).

4. The fusion protein of claim 3 wherein X1 is F; X2 is V; X3 is G or T; X16 is E; X25 is H; X27 is G; X28 is G; X29 is G; and X30 is 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 either of any of claims 2-6 wherein the insulin A-chain analog comprises the amino acid sequence: GIVEQCCTSX10CSLX14QLENYCX21X22 wherein X10 is T or I; X14 is D, Y, Q or E; X21 is G, N, S or A; and X22 is any naturally occurring amino acid, or is absent, provided that if X21 is N, then X22 must be an amino acid other than G or N (SEQ ID NO:2).

8. The fusion protein of claim 7 wherein X10 is T; X14 is D; X21 is G; and X22 is absent.

9. The fusion protein of any of claims 2-8 wherein the C-terminal residue of the insulin B-chain analog is fused to the N-terminal residue of a first peptide linker and 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 said amino acids are G residues.

11. The fusion protein of either of claims 9 or 10, wherein the first peptide linker comprises the following amino acid sequence: X1GX3GGGG wherein X1 is G or is absent; and X3 is G, S or is absent (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 of claims 2-12, wherein the C-terminal residue of the insulin A- chain analog is fused to the N-terminal residue of a second peptide linker, and 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 between 10 and 25 amino acids, wherein at least 50% of said amino acids are G residues.

15. The fusion protein of claim 14, wherein the second peptide linker comprises a peptide having the sequence [GGGGX]n wherein X is Q, E or S; and wherein 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 GGGGQGGGGQGGGGQGGGG (SEQ ID NO: 14).

18. The fusion protein of any of claims 1-17 wherein the human IgG2 Fc region comprises the amino acid sequence: ECPPCPAPPVAGPSVX16LX18PPKPKDTLMISRTPEVTCX37VX39DVSHEDPEVQFNWYVD GVEVHNAKTKPREEQFX72STFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEX164NYKTTPP MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX222 wherein X16 is F, Q or E; X18 is F, Q or E; X37 is V or T; X39 is V or T; X72 is N, D or Q; X164 is S, Q, E, D and A; and X222 is K or absent. (SEQ ID NO:5).

19. The fusion protein of claim 18 wherein X16 is F; X18 is F; X37 is V; X39 is V; X72 is N; X164 is Q or S; and X222 is absent.

20. The fusion protein of claim 19 wherein X164 is Q.

21. The fusion protein of claim 19 wherein X164 is S.

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 of any of claims 1-22.

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

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

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