Long-acting insulin-Fc fusion protein

Abstract

The present invention provides long-acting insulin-Fc fusion proteins, which have reduced insulin receptor affinity, increased growth factor receptor selectivity, and a prolonged half-life in the body, which can extend the duration of blood glucose reduction in the body, shorten the interval between insulin doses, and reduce side effects such as the risk of hypoglycemia.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein for treating diabetes. More specifically, the present invention relates to a fusion protein in which an insulin analog is fused to a human IgG Fc region via a peptide linker, a method for producing the same, and its use in treating diabetes. [Background technology]

[0002] Diabetes mellitus (DM) is a chronic disease characterized by hyperglycemia due to insufficient insulin secretion and / or insulin resistance. Type 1 diabetes is caused by an absolute deficiency of insulin secretion, primarily due to the destruction of pancreatic islet β cells, and can only be treated with insulin. Type 2 diabetes, which is primarily caused by insulin resistance and a compensatory deficiency of insulin secretion, accounts for approximately 90-95% of all cases, and as the disease progresses to later stages, insulin therapy is still required.

[0003] Outpatient insulin supplementation is one of the important therapeutic measures to improve the quality of life of diabetic patients and slow the progression of the disease. Insulin has a short half-life in the blood, and current conventional treatments involve large doses of insulin injected during meals and exogenous insulin given orally or by injection one or more times a day.

[0004] Frequent insulin injections and self-monitoring of glucose control cause considerable inconvenience and distress to diabetic patients, leading to poor treatment compliance. Currently available insulin products are prone to hypoglycemia and weight gain, making many patients unwilling to start insulin therapy, resulting in poor overall glycemic control. Even those already using insulin are unable to strictly follow insulin instructions to precisely control blood glucose. Poor glycemic control can lead to a variety of serious complications, including atherosclerotic cardiovascular disease, diabetes-related nephropathy and liver disease, cancer, and infection. To date, no medications have been approved for basal insulin injections less frequently than once daily. To improve patient compliance, insulin products with longer duration of hypoglycemic effect and fewer injections than current products are needed.

[0005] Currently, basal insulin-Fc fusion proteins with extended half-lives have been published. CN103509118 describes a protein in which the human insulin B chain and human insulin A chain are linked via a 4-50 amino acid C-peptide linking sequence, and the insulin A chain is directly linked to an immunoglobulin Fc fragment. Mouse experiments have shown that this protein has an in vivo half-life of approximately 3 days. KR1020150087130 describes a protein in which a proinsulin analog is linked to the immunoglobulin Fc region via a non-peptide linker. This protein has demonstrated a serum half-life longer than conventional therapies. US20180177851 describes a protein in which an insulin receptor agonist with reduced insulin receptor affinity is linked to a human Fc region via a second peptide linker, and the human insulin B chain and human insulin A chain are linked via a first peptide linker of 5 to 10 amino acids. In a two-phase clinical trial, this once-weekly insulin fusion protein injection, LY3209590, demonstrated similar therapeutic efficacy and safety compared to once-daily basal insulin (Frias JP, et al. J Endocr Soc. 2021;5(Suppl 1):A448-A449.).

[0006] Despite these and / or other disclosures, there remains a need in the art for insulin products that have an extended duration of hypoglycemic action. Summary of the Invention

[0007] The present invention meets clinical needs, allowing for less frequent injections and improving patient compliance, with an insulin analog, a fusion protein comprising a peptide linker between the human insulin B chain and the human insulin A chain, and a human IgG Fc fragment.

[0008] The present invention employs the following design strategies to extend the duration of insulin action: First, the affinity of insulin analogs for the insulin receptor is reduced by protein engineering, reducing insulin receptor-mediated insulin clearance while still retaining sufficient biological potency to activate the receptor and downstream signaling pathways. Second, to avoid renal clearance, insulin analogs are fused to a human immunoglobulin Fc region, increasing hydrodynamic size, and the Fc region is engineered to reduce unwanted insulin aggregation to avoid loss of biological activity due to insulin self-aggregation caused by Fc fragment dimerization.

[0009] The fusion proteins of the present invention have a prolonged duration of hypoglycemic action and require less frequent injections than conventional once-daily insulin therapy.

[0010] The fusion proteins according to the present invention have a flat pharmacokinetic profile and can provide a lower incidence of hypoglycemia.

[0011] In one aspect, the present invention provides a fusion protein comprising: a) an insulin analog having a structure represented by the general formula Z1-Z2-Z3; Among them, Z1 is an insulin B chain analogue comprising the following amino acid sequence: FVNQX1LCGSHLVEALX2LVCGERGFX3YX4X5X6X7 (SEQ ID NO: 1) Among them X1 is H or A, X2 is Y, A or E; X3 is T, I, F or H; X4 is E, D, T, or absent, X5 is P, E, or does not exist, X6 is K, E, or not present, X7 is T or absent; Z2 is a first peptide linker, preferably comprising an amino acid sequence selected from the group consisting of GGVGGG (SEQ ID NO: 16), GGGSGG (SEQ ID NO: 17) or GGGGGV (SEQ ID NO: 18); Z3 is an insulin A chain analogue comprising the following amino acid sequence: GIX8EX9CCX 10 SICSLYQLENYCX 11 (SEQ ID NO: 2) Among them X8 is A, L or V; X9 is Q or A; X 10 is T, A, F or K; X 11 is G, S or N; b) a second peptide linker; and c) a human IgG Fc region or a variant thereof; Including, wherein the C-terminal residue of the insulin analog is directly 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 human IgG Fc region or variant thereof; A fusion protein is provided.

[0012] In one embodiment, the insulin B chain analog comprises a modification to the amino acid sequence of the B chain of a human insulin molecule at X1 of SEQ ID NO:1.

[0013] In one embodiment, in Z1, X1 is A; X2 is Y; X3 is F; X4 is T; X5 is P; X6 is K; and X7 is T.

[0014] In one embodiment, the insulin B chain analogue comprises a modification to the amino acid sequence of the B chain of a human insulin molecule at X4.

[0015] In one embodiment, in Z1, X1 is H; X2 is A; X3 is F; X4 is T; X5 is P; X6 is K; and X7 is T.

[0016] In one embodiment, the insulin B chain analogue comprises modifications to the amino acid sequence of the B chain of a human insulin molecule at X5 and X6.

[0017] In one embodiment, in Z1, X1 is H; X2 is Y; X3 is T or I; X4 is E; X5 is P; X6 is K; and X7 is T.

[0018] In one embodiment, the insulin B chain analog comprises a modification to the amino acid sequence of the B chain of a human insulin molecule at X6 of SEQ ID NO:1.

[0019] In one embodiment, in Z1, X1 is H; X2 is Y; X3 is F; X4 is E or D; X5 is P; X6 is K; and X7 is T.

[0020] In one embodiment, the insulin B chain analogue comprises modifications to the amino acid sequence of the B chain of a human insulin molecule at X4, X5, X6, X7.

[0021] In one embodiment, in Z1, X4 to X7 are absent.

[0022] In one embodiment, the insulin A chain analogue is X or X of SEQ ID NO:2. 11 and contains at least one modification to the amino acid sequence of human insulin A chain.

[0023] In one embodiment, the insulin A chain analogue comprises X8 or X9 and X of SEQ ID NO:2. 11 and contains at least one modification to the amino acid sequence of the A chain of the human insulin molecule.

[0024] In one embodiment, the insulin A chain analogue comprises X8, X9 and X of SEQ ID NO:2. 11 and contains modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0025] In one embodiment, in Z3, X8 is A or L; X9 is A; and X 10 is T; and X 11 is G.

[0026] In one embodiment, the insulin A chain analogue is X of SEQ ID NO:2. 11 and contains modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0027] In one embodiment, in Z3, X8 is V; X9 is Q; and X 10 is T; and X 11 is G.

[0028] In one embodiment, the insulin A chain analogue is 10 and X 11 and contains modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0029] In one embodiment, in Z3, X8 is V; X9 is Q; and X 10 is A, K, or F; and X 11 is G.

[0030] In one embodiment, the insulin A chain analogue comprises X8 and X 10 and X 11 and contains modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0031] In one embodiment, in Z3, X8 is A or L; X9 is Q; and X 10 is A or K; and X 11 is G.

[0032] In one embodiment, the insulin B chain analog comprises a modification to the amino acid sequence of the B chain of a human insulin molecule at X6 of SEQ ID NO:1; and the insulin A chain analog comprises a modification to the amino acid sequence of the B chain of a human insulin molecule at X8 and X9 of SEQ ID NO:1. 11 Both contain modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0033] In one embodiment, in Z1, X1 is H; X2 is Y; X3 is F; X4 is E or D; X5 is P; X6 is K; X7 is T; and the insulin A-chain analog is selected from X8 and X9 of SEQ ID NO:2. 11 both of which contain modifications to the amino acid sequence of the A chain of the human insulin molecule, and in Z3, X8 is A; 11 is G.

[0034] In one embodiment, the insulin B chain analog comprises the B chain amino acid sequence of a human insulin molecule; and the insulin A chain analog comprises X8 and X9 of SEQ ID NO:2. 11 Both contain modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0035] In one embodiment, in Z1, X1 is H; X2 is Y; X3 is F; X4 is T; X5 is P; X6 is K; X7 is T; and the insulin A-chain analogue is 11 and a modification to the amino acid sequence of the A chain of the human insulin molecule; wherein X8 is A or L; X 11 is G.

[0036] In one embodiment, the insulin B chain analog comprises the amino acid sequence of the B chain of a human insulin molecule; and the insulin A chain analog comprises X and X 11 Both contain modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0037] In one embodiment, in Z1, X1 is H; X2 is Y; X3 is F; X4 is T; X5 is P; X6 is K; X7 is T; and the insulin A-chain analogue is 11 and a modification to the amino acid sequence of the A chain of the human insulin molecule, wherein X9 is A; X 11 is G.

[0038] In one embodiment, the insulin B chain analog comprises the amino acid sequence of the B chain of a human insulin molecule; and the insulin A chain analog comprises X 10 and X 11 Both contain modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0039] In one embodiment, in Z1, X1 is H; X2 is Y; X3 is F; X4 is T; X5 is P; X6 is K; and X7 is T; and the insulin A-chain analog is 10 and X 11 and containing modifications to the amino acid sequence of the A chain of the human insulin molecule, wherein X 10 is A, K or F;X 11 is G.

[0040] In one embodiment, the insulin B chain analog comprises a modification to the amino acid sequence of the B chain of a human insulin molecule at X1; and the insulin A chain analog comprises a modification to the amino acid sequence of the B chain of a human insulin molecule at X 11 and contains modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0041] In one embodiment, in Z1, X1 is A; X2 is Y; X3 is F; X4 is T; X5 is P; X6 is K; and X7 is T; and the insulin A-chain analog is 11 and contains modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0042] In one embodiment, the insulin B chain analog comprises a modification to the amino acid sequence of the B chain of a human insulin molecule at X2; and the insulin A chain analog comprises a modification to the amino acid sequence of the B chain of a human insulin molecule at X8 and X9. 11 Both contain modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0043] In one embodiment, in Z1, X1 is H; X2 is A; X3 is F; X4 is T; X5 is P; X6 is K; X7 is T; and the insulin A-chain analogue is 11 Both of these contain modifications to the amino acid sequence of the A chain of the human insulin molecule, wherein X8 is A; X 11 is G.

[0044] In one embodiment, the insulin B chain analog comprises modifications to the amino acid sequence of the B chain of a human insulin molecule at X3 and X4 of SEQ ID NO: 1; and the insulin A chain analog comprises modifications to the amino acid sequence of the B chain of a human insulin molecule at X8 and X9 of SEQ ID NO: 1. 11 Both contain modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0045] In one embodiment, in Z1, X1 is H; X2 is Y; X3 is T or I; X4 is E; X5 is P; X6 is K; X7 is T; and the insulin A-chain analogue is 11 Both contain modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0046] In one embodiment, the insulin B chain analog comprises modifications to the amino acid sequence of the B chain of a human insulin molecule at X4, X5, X6, X7; and the insulin A chain analog comprises modifications to the amino acid sequence of the B chain of a human insulin molecule at X8 and X9. 11 and contains modifications to the amino acid sequence of the A chain of the human insulin molecule.

[0047] In one embodiment, in Z1, X4 to X7 are absent; and the insulin A-chain analogue comprises X8 and X 11and a modification to the amino acid sequence of the A chain of the human insulin molecule, wherein X8 is A; X 11 is G.

[0048] In one embodiment, the first peptide linker Z2 comprises an amino acid sequence selected from the group consisting of: GGVGGG (SEQ ID NO: 16), GGGSGG (SEQ ID NO: 17), GGGGGV (SEQ ID NO: 18).

[0049] In one embodiment, the first peptide linker Z2 comprises the following amino acid sequence: GGVGGG (SEQ ID NO: 16).

[0050] In one embodiment, as the insulin receptor agonist, Z1-Z2-Z3 in the fusion protein comprises the following amino acid sequence: FVNQX1LCGSHLVEALX2LVCGERGFX3YX4X5X6X7GGVGGGGIX8EX9CCX 10 SICSLYQLENYCX 11 (SEQ ID NO: 19) Among them, X1 is H or A; X2 is Y, A or E; X3 is T, I, F or H; X4 is E, D, T or absent; X5 is P, E, or absent; X6 is K, E, or absent; X7 is T or absent; X8 is A, L or V; X9 is Q or A; X 10 is T, A, F or K; X 11 is G, S or N.

[0051] In one embodiment, as the insulin receptor agonist, Z1-Z2-Z3 in the fusion protein has the following amino acid sequence: FVNQHLCGSHLVEALYLVCGERGFFYEPKTGGVGGGGIAEQCCTSICSLYQLENYCG (SEQ ID NO: 3).

[0052] In one embodiment, the second peptide linker comprises a peptide having the sequence [GGGGX]n, where X is S or T; and n is 2, 3, 4, or 5.

[0053] In another embodiment, the second peptide linker comprises the following amino acid sequence: GGGGX 12 GGGGX 13 GGGGX 14 GGGGX 15 (SEQ ID NO: 20) Among them, X 12 is S or T;X 13 is S or T;X 14 is S or T;X 15 is S or T.

[0054] In another embodiment, the second peptide linker has the following amino acid sequence: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4) In one embodiment, the human IgG Fc region is an Fc region from an IgG2 or IgG4 antibody.

[0055] In one embodiment, the human IgG Fc region is an Fc region from an IgG2 antibody and comprises the following amino acid sequence: ERKX 16 X 17 VEX 18 PPX 19 PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSX 20 EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVX 21 HQDWLNGKEYKCKVSNKGLPX 22 X 23IEKTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX 24 GX 25 (SEQ ID NO: 5) Among them, X 16 is S or C; X 17 is S or C; X 18 is S or C; X 19 is S or C; X 20 is Q or H; X 21 is L or V; X 22 is S or A; X 23 is S or P; X 24 is L or P; X 25 is K or does not exist.

[0056] In one embodiment, the human IgG Fc region is an Fc region from an IgG4 antibody and comprises the following amino acid sequence: ESKYGPPSPPSPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 6) In one embodiment, the fusion protein of the invention comprises the following amino acid sequence: FVNQX1LCGSHLVEALX2LVCGERGFX3YX4X5X6X7GGVGGGGIX8EX9CCX 10SICSLYQLENYCX 11 GGGGX 12 GGGGX 13 GGGGX 14 GGGGX 15 ERKX 16 X 17 VEX 18 PPX 19 PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSX 20 EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVX 21 HQDWLNGKEYKCKVSNKGLPX 22 X 23 IEKTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX 24 GX 25 (SEQ ID NO: 7) Among them, X1 is H or A; X2 is Y, A or E; X3 is T, I, F or H; X4 is E, D, T or absent; X5 is P, E, or absent; X6 is K, E, or absent; X7 is T or absent; X8 is A, L or V; X9 is Q or A; X 10 is T, A, F or K; X 11 is G, S or N; X 12 is S or T; X 13 is S or T; X 14 is S or T; X 15 is S or T; X 16 is S or C; X 17 is S or C; X 18 is S or C; X 19 is S or C; X 20 is Q or H; X 21 is L or V; X 22 is S or A; X 23 is S or P; X 24 is L or P; X 25 is K or does not exist.

[0057] In one embodiment, the IgG Fc region comprises the amino acid sequence of SEQ ID NO: 7, and further comprises some or all of the amino acids found in the wild-type IgG2 Fc sequence N-terminal to the E residue at position 1 in SEQ ID NO: 7. In one preferred embodiment, the human IgG Fc region comprises the amino acid sequence of SEQ ID NO: 7, wherein X 16 is S;X 17 is S;X 18 is S;X 19 is S;X 20 is Q;X 21 is L;X 22 is S;X 23 is S;X 24 is L;X 25 does not exist.

[0058] In one embodiment, the fusion protein of the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

[0059] In one embodiment, the fusion protein of the invention comprises the following amino acid sequence: FVNQHLCGSHLVEALYLVCGERGFFYEPKTGGVGGGGIAEQCCTSICSLYQLENYCGGGGGSGGGGSGGGGSGGGGSERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 8). In one embodiment, the fusion proteins of the present invention exist as dimers. In some embodiments, the dimers are homodimers, in which the amino acid sequences of the two fusion proteins comprising the dimer are the same. In some embodiments, the dimers are heterodimers, in which the amino acid sequences of the two fusion proteins comprising the dimer are different.

[0060] Therefore, the present invention provides a dimer comprising the fusion protein.

[0061] In another aspect, the present invention provides a pharmaceutical composition comprising the fusion protein of the present invention or a dimer thereof, and optionally at least one pharmaceutically acceptable excipient.

[0062] In another aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein of the invention.

[0063] In another aspect, the present invention provides a recombinant vector comprising the above-described nucleic acid molecule.

[0064] In another aspect, the present invention provides a recombinant cell comprising the above-described nucleic acid molecule or recombinant vector.

[0065] In another aspect, the present invention provides the use of the fusion protein or dimer or pharmaceutical composition of the present invention in the manufacture of a medicament for treating diabetes, obesity or metabolic syndrome.

[0066] In another aspect, the present invention provides a method for treating a patient suffering from diabetes, obesity or metabolic syndrome, comprising administering to a patient in need thereof a therapeutically effective amount of a fusion protein or dimer or pharmaceutical composition of the present invention. [Brief explanation of the drawings]

[0067] [Figure 1] 1 shows pharmacokinetic data of an exemplary fusion protein of the invention in a streptozotocin (STZ)-treated mouse diabetes model. DETAILED DESCRIPTION OF THE INVENTION

[0068] term As used herein, the term "insulin analog" refers to a protein that has insulin activity and can bind to and activate the insulin receptor, thereby causing a decrease in blood glucose levels and / or suppression of hepatic glucose output, characteristics that can be measured and detected by conventional techniques (e.g., those shown in the studies described below).

[0069] 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 No. 11061-68-0), the native wild-type sequences of which are known. The human insulin A chain consists of 21 amino acids, known in the art as A1-A2. 21 and has the following sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 21) The human insulin B chain consists of 30 amino acids and is known in the art as B1-B 30 and has the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 22) As used herein, the terms "insulin analog" and "insulin receptor agonist" can be used interchangeably herein.

[0070] As used herein, the terms "peptide linker," "linking sequence," and "linker" can be used interchangeably. In some embodiments, this refers to a structure that links one or more amino acids of an insulin B chain analog and an insulin A chain analog and provides sufficient flexibility to bind to the insulin receptor. In some embodiments, this refers to one or more amino acids that link an insulin analog and an immunoglobulin Fc structural region and provides sufficient flexibility. Examples of suitable linkers include monoglycine (Gly), serine (Ser), or valine (Val) residues, and the sequence and arrangement of amino acid residues in the linker can vary depending on the type of secondary structure element desired to be achieved by the linker.

[0071] As used herein, EC 50 means the concentration of insulin-Fc fusion protein that is half the maximal response observed in in vitro insulin receptor binding (eg, the concentration at which the insulin receptor is half-bound).

[0072] As used herein, IC 50 IC refers to the concentration of insulin-Fc fusion protein at which a given biological function or biochemical process (e.g., binding) is inhibited by half. In some embodiments, IC 50 means the concentration of insulin-Fc fusion protein at which the binding of insulin to the human insulin receptor was reduced by half.

[0073] As used herein, the term "fusion protein," e.g., an "insulin-Fc fusion" protein, refers to a protein comprising one or more structural regions, e.g., structural regions generally of different origin (e.g., different proteins, polypeptides, cells, etc.), joined by a polypeptide linker. In some embodiments, the fusion protein is produced recombinantly. In some embodiments, the structural regions of the fusion protein are linked by linking the genetic sequences encoding each structural region onto a single nucleic acid molecule. In some embodiments, the insulin-Fc fusion protein is a protein, such as a single polypeptide, comprising an insulin polypeptide and an Fc fragment polypeptide, where the insulin and Fc fragment polypeptides are linked by a peptide linker to form a single polypeptide.

[0074] insulin analogues In the fusion proteins of the present invention, the analog of the insulin B chain in the insulin analog contains one or more modifications relative to the human insulin B chain. Specifically, to reduce the tendency of the insulin analog moiety to dimerize, the insulin B chain analog contains one or more modifications at positions B5, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, B16, B17, B18, B19, B20, B21, B22, B23, B24, B25, B26, B27, B28, B30, B31, B32, B33, B44, B45, B46, B47, B50, B51, B52, B53, B54, B55, B56, B57, B58, B59, B60, B61, B62, B63, B64, B65, B66, 16 , B 25 or B 27-30 and wherein said positions are positions X1, X2, X3 and X4 in SEQ ID NO: 1, respectively. 4-7 For example, X1 (corresponding to B5 in the B chain of the human insulin molecule) may be modified to A; X5 (corresponding to B5 in the B chain of the human insulin molecule) may be modified to A; 16 X3 (corresponding to B in the B chain of the human insulin molecule) may be modified to A; 25 X4 (corresponding to B in the B chain of the human insulin molecule) may be modified to T, I, or F; 27 X5 (corresponding to B in the B chain of the human insulin molecule) may be deleted or modified to E or D; 28 X6 (corresponding to B in the B chain of the human insulin molecule) may be deleted or modified to E; 29X7 (corresponding to B in the B chain of the human insulin molecule) may be deleted or modified to E, 30 ) may be missing.

[0075] In some embodiments, the analog of insulin B chain in the insulin partial agonist comprises one or more modifications to human insulin B chain, and is selected from the group consisting of positions X1, X2, X3 and X4 of SEQ ID NO:1. 4-7 In one preferred embodiment, X2 is E.

[0076] In some embodiments, the insulin A chain analog in the insulin partial agonist contains one or more modifications to the amino acid sequence of human insulin A chain to improve chemical and physical stability, adjust efficacy, eliminate deamidation, and / or enhance expression. 10 and X 11 The inclusion of the modification at is to modify X8 (corresponding to A3 in the A chain of the human insulin molecule) to A; X9 (corresponding to A5 in the A chain of the human insulin molecule) to A; 10 (corresponding to A8 in the A chain of the human insulin molecule) is modified to A, K, or F; X 11 (A in the A chain of the human insulin molecule 21 (corresponding to) is modified to G, S or A. In one preferred embodiment, X8 is A and X4 is G.

[0077] In the fusion proteins of the present invention, the C-terminal residue of the insulin B chain analog is fused directly to the N-terminal residue of the first peptide linker, and the C-terminal residue of the first peptide linker is fused directly to the N-terminal residue of the insulin B chain analog. The first peptide linker must provide sufficient flexibility to the insulin B chain and insulin A chain analogs, the structure required for binding to the insulin receptor. In some embodiments, the amino acid sequence of the first peptide linker is selected from the group consisting of GGVGGG (SEQ ID NO: 16), GGGSGG (SEQ ID NO: 17), or GGGGGV (SEQ ID NO: 18). Most preferably, the sequence of the first peptide linker is GGVGGG (SEQ ID NO: 16).

[0078] As described above, the C-terminal residue of the insulin receptor agonist portion of the fusion protein of the invention is fused to the N-terminal residue of the second peptide linker, and the C-terminal residue of this second peptide linker is fused directly to the N-terminal residue of the Fc portion. 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 15 to 25 amino acids in length, wherein at least 50% of the amino acids are glycine residues. A preferred second peptide linker comprises the sequence (GGGGX)n, wherein X is S or T and n=2, 3, 4, or 5; the most preferred second peptide linker has the amino acid sequence of SEQ ID NO:4.

[0079] Human IgG Fc region As used herein, the term "human IgG Fc region" has the definition commonly provided in the field of immunology. In particular, this term refers to a human IgG antibody fragment obtained by removing the two antigen-binding regions (Fab fragments) from an antibody. Specifically, the Fc region includes the CH2 and CH3 constant structural regions of the antibody, and may also include part or all of the hinge region.

[0080] As described above, in some embodiments of the fusion proteins of the present invention, the human IgG Fc region comprises a fragment derived from the constant region of one heavy chain of an IgG antibody, and in other embodiments, the human IgG Fc region comprises fragments derived from the constant regions of two heavy chains of an IgG antibody, in which the constant regions of these two heavy chains are associated with each other by non-covalent interactions.

[0081] Human IgG isotypes (subclasses of mature gamma globulin G antibodies; IgG1, IgG2, IgG3, and IgG4) have distinct structures and exhibit distinct biological functions, referred to as "effector / substance functions." These include antibody-dependent cellular cytotoxicity (ADCC, e.g., IgG1 and IgG3), antibody-dependent cellular phagocytosis (ADCP, e.g., IgG1, IgG2, IgG3, and IgG4), and complement-dependent cytotoxicity (CDC, e.g., IgG1 and IgG3). Among the various subclasses, IgG1 and IgG3 have very high relative affinity for Fcγ receptors (e.g., FcγRI, FcγRIIa / b / c, FcγRIIIa / b) and significantly lower affinity for IgG2 (limited to the FcγRIIa 131H polymorphism), while IgG4 only has measurable affinity for FcγRI. The fusion proteins of the present invention can comprise an Fc region from any IgG subclass, with IgG2 and IgG4 being more preferred as they have lower receptor binding and effector function activities compared to IgG1 and IgG3 antibodies.

[0082] As used herein, the term human IgG Fc region also includes antibody fragments that have been modified, extended, and / or truncated to alter properties or characteristics, such as Fc receptor binding function, effector function, disulfide bond formation, glycosylation, antibody-dependent cell-mediated cytotoxicity (ADCC), manufacturability, and / or stability. For example, the human IgG Fc region of the fusion proteins of the invention can be modified to reduce or eliminate N-linked glycosylation sites, decrease Clq binding affinity and cytotoxicity, affect conformational stability and bioclearance rate, and / or alter effector function.

[0083] The human IgG Fc region of the fusion protein of the present invention can further be modified by removing disulfide bonds in the hinge region to simplify disulfide-mediated Fc dimerization. Other examples include phosphorylation, sulfation, acylation, glycosylation, methylation, acetylation, amidation, and / or modifications that can generate heterodimeric molecules. Techniques for modifying the structure and properties of human IgG Fc regions of IgG subclasses are known in the art.

[0084] Regardless of the final structure of the fusion protein, the human IgG Fc region must extend the plasma half-life of the insulin analog in vivo and minimize any Fc effector functions. The fused insulin receptor agonist must also maintain its ability to bind and activate the insulin receptor and reduce blood glucose levels and / or suppress hepatic glucose output.

[0085] A preferred human IgG Fc region is an IgG2 Fc region that has been modified to further reduce effector function, for example, the H268Q, V309L, A330S, and P330S modifications (EU numbering) described in US20070148167, and has four pairs of disulfide bonds missing in the hinge region and C219S, C220S, C223S, and C226S modifications, for example, as shown in SEQ ID NO: 5, in which X 16 is S;X 17 is C;X 18 is S;X 19 is S;X 20 is Q;X 21 is L;X 22 is S;X 23 is S;X 24 is L;X 25 is K.

[0086] Another preferred human IgG Fc region is an IgG4 Fc region, modified to further reduce effector function and promote homodimer formation, for example, as set forth in SEQ ID NO:6.

[0087] The present invention provides any polynucleotide of the fusion protein disclosed herein. The recombinant nucleic acid sequences disclosed herein and engineered eukaryotic cells containing these vectors can be transfected with recombinant nucleic acid sequences (e.g., mRNA, cDNA, DNA) encoding the insulin-Fc fusion proteins described herein.

[0088] Fusion protein expression and purification The fusion proteins of the present invention are produced in a mammalian cell expression system using a CHO glutamine synthetase (GS) knockout (GSKO) cell line. The GS gene knockout allows for increased selection stringency by eliminating endogenous GS background activity, allowing low- or non-producing cells to survive under selection conditions. The gene encoding the fusion protein is subcloned into a glutamine synthetase (GS)-containing expression plasmid. The cDNA sequence encoding the fusion protein is fused in frame with a coding sequence for a signal peptide that enhances secretion of the fusion protein into the cell culture medium. Expression is driven by the cytomegalovirus (CMV) promoter. CHO GSKO cells are stably transfected using electroporation and an appropriate amount of the recombinant expression plasmid.

[0089] Transfected cells undergo bulk selection in glutamine-free medium. Transfected fusions are plated at low density to initiate close-to-clonal outgrowth of stable expressing cells. Primary wells are screened for fusion protein expression and proportionally expanded in serum-free suspension cultures for production use.

[0090] The fusion protein secreted into the medium can be purified by protein A affinity chromatography using standard chromatography techniques. Briefly, the fusion protein from the clarified medium is captured with fusion protein selection protein A (ge) balanced with phosphate-buffered saline, pH 7.4. After a wash step with phosphate-buffered saline, pH 7.4, the bound fusion protein is eluted with glycine, pH 3.0. Fractions containing the fusion protein are combined and neutralized by adding 1 / 10 of their volume of 1 M Tris, pH 9.0. Soluble aggregates and multimers can be efficiently removed by common techniques such as size exclusion, hydrophobic interaction, and ion exchange chromatography. Fractions containing the monomeric fusion protein (non-covalently bound homodimer), as determined by size exclusion chromatography, are combined, sterile filtered, and saved. [Example]

[0091] The present invention will now be further described with reference to examples and drawings. The experimental methods used in the following examples are conventional unless otherwise noted. Unless otherwise specified, all materials, reagents, etc. used in the following examples are commercially available.

[0092] Example 1: (B27E+A3-A+A21G) The amino acid sequence of the fusion protein of the present invention is as follows: FVNQHLCGSHLVEALYLVCGERGFFYEPKTGGVGGGGIAEQCCTSICSLYQLENYCGGGGGSGGGGSGGGGSGGGGSERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 8) Example 2: (A3-A+A21G) The amino acid sequence of the fusion protein of the present invention is as follows: FVNQHLCGSHLVEALYLVCGERGFFYTPKTGGVGGGGIAEQCCTSICSLYQLENYCGGGGGSGGGGSGGGGSGGGGSERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 9) Example 3: (A5-A+A21G) The amino acid sequence of the fusion protein of the present invention is as follows: FVNQHLCGSHLVEALYLVCGERGFFYTPKTGGVGGGGIVEACCTSICSLYQLENYCGGGGGSGGGGSGGGGSGGGGSERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 10) Example 4: (A8-A+A21G) The amino acid sequence of the fusion protein of the present invention is as follows: FVNQHLCGSHLVEALYLVCGERGFFYTPKTGGVGGGGIVEQCCASICSLYQLENYCGGGGGSGGGGSGGGGSGGGGSERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 11) Example 5: (B5-A+A21G) The amino acid sequence of the fusion protein of the present invention is as follows: FVNQALCGSHLVEALYLVCGERGFFYTPKTGGVGGGGIVEQCCTSICSLYQLENYCGGGGGSGGGGSGGGGSGGGGSERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 12) Example 6: (B16-A+A3-A+A21G) The amino acid sequence of the fusion protein of the present invention is as follows: FVNQHLCGSHLVEALALVCGERGFFYTPKTGGVGGGGIAEQCCTSICSLYQLENYCGGGGGSGGGGSGGGGSGGGGSERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 13) Example 7: (B25-T + B27-E A3-A + A21G) The amino acid sequence of the fusion protein of the present invention is as follows: FVNQHLCGSHLVEALYLVCGERGFTYEPKTGGVGGGGIAEQCCTSICSLYQLENYCGGGGGSGGGGSGGGGSGGGGSERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 14) Example 8: (desB27-30+A3-A+A21G) The amino acid sequence of the fusion protein of the present invention is as follows: FVNQHLCGSHLVEALYLVCGERGFFYGGVGGGGIAEQCCTSICSLYQLENYCGGGGGSGGGGSGGGGSGGGGSERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 15) On the other hand, the amino acid sequence of the unmodified Insulin-Fc fusion protein used in the present invention is as follows: FVNQHLCGSHLVEALYLVCGERGFFYEPKTGGVGGGGIVEQCCTSICSLYQLENYCNGGGGSGGGGSGGGGSGGGGSERKSSVESPPSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 23)

[0093] Experimental Example Experimental Example 1: Purity and thermal stability testing of fusion proteins The purity of the fusion protein monomer was determined using high-performance liquid size-exclusion chromatography (SEC-HPLC). Specifically, the protein solution was diluted to 1 mg / mL and a 10 μL sample volume was used for analysis using a high-performance liquid size-exclusion chromatography column, MAbPac™ SEC1 (7.8 mm x 300 mm, Thermo Fisher Scientific, Waltham, MA, USA). Analytical SEC was performed for 20 minutes on an Agilent 1100 (Agilent Technologies, Inc., Santa Clara, CA, USA) system using 20 mM phosphate buffer (pH 7.0, Sinopharm Co., Ltd., Shanghai, China) containing 150 mM sodium chloride at a flow rate of 0.6 mL / min. Signals were obtained using a 280 nm UV detector.

[0094] This accelerated test investigated the purity and aggregation changes of the fusion protein at high temperatures. The incubation conditions were 60°C for 1 hour, and the change in purity after heating was measured using SEC-HPLC. Table 1 summarizes the change in monomer purity of the fusion protein at time zero and after heating at 60°C for 1 hour. Unmodified Insulin-Fc is an Fc fusion protein (SEQ ID NO: 23) with the same structure as the example protein but with unmodified insulin amino acids. After heating at 60°C for 1 hour, the fusion protein of the example of the present invention showed a change in monomer purity from time zero of 2.9% to 8.8%, demonstrating the high thermal stability of the fusion protein of the example of the present invention at 60°C.

[0095] Table 1. Monomer purity and thermal stability of insulin-Fc fusion proteins

[0096] [Table 1]

[0097] Experimental Example 2: Detection of the binding between insulin fusion protein and receptor by enzyme-linked immunosorbent assay (ELISA) The assay procedure was as follows: Human insulin receptor extracellular domain protein (hIRB-ECD, Sino Biological Inc., Beijing, China) was diluted to 5 μg / mL in PBS (pH 7.2), 50 μL / well was applied to a 96-well high-binding U-plate (Greiner Bio-One North America Inc., Monroe, NC, USA) and incubated overnight at 4°C. The plate was then washed three times with PBS. The 96-well plate was then blocked with blocking solution (1% casein, Thermo Fisher Scientific, Waltham, MA, USA) at 200 μL / well and left at room temperature for 1.5 hours. The plate was then washed three times with 0.1% PBST. Each well was diluted 1:3 with blocking solution (example concentrations: 10 μM, 3.3 μM, 1.1 μM, 0.37 μM, 0.12 μM, 0.041 μM, 0.014 μM, 0 μM). The samples contained insulin-Fc fusion protein or Fc isotype control antibody (Thermo Fisher Scientific (Cat: 31154)). 50 μL / well was added in duplicate and incubated at room temperature for 1 hour. The wells were then washed three times with 0.1% PBST. HRP Goat Anti-Human IgG (H+L) (ABclonal Technology Co., Ltd., Wuhan, China) diluted 1:2000 with blocking solution was added and incubated at room temperature for 1 hour. The wells were then washed three times with 0.1% PBST. TMB solution (Thermo Fisher Scientific) was added at 50 μL / well and incubated at room temperature for 5 minutes. The reaction was stopped by adding 50 μL / well of 2 M sulfuric acid. Absorbance values ​​were read at 450 nm using a SpectraMax M5e plate reader (Molecular Devices, LLC, San Jose, CA, USA). Data analysis was performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA).

[0098] Table 2 shows the EC values ​​of the insulin-Fc fusion proteins binding to the insulin receptor. 50 The values ​​were compiled and the results showed that the fusion proteins of the present invention had reduced hIRB binding ability compared to unmodified Insulin-Fc.

[0099] Table 2. EC of insulin-Fc fusion proteins with insulin receptor 50 value

[0100] [Table 2]

[0101] Experimental Example 3: Detection of the affinity between insulin-Fc fusion protein and human insulin-like growth factor 1 receptor (IGF-1R) using biofilm interference technology ForteBio Octet (登録商標) The affinity of each fusion protein with IGF-1R-His tag (Sino Biological) was analyzed using a Pall ForteBio LLC (Fremont, CA, USA) instrument.

[0102] His-tagged IGF-1R protein (10 μg / mL) was captured with a Ni-NTA probe (Pall ForteBio LLC), and the interaction activity of insulin glargine (Lantus, Sanofi), unmodified Insulin-Fc, and the fusion proteins of Examples 1 and 2 was measured at concentrations of 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.6 nM, 7.8 nM, and 0 nM in 0.1% PBST general-purpose buffer. The affinity constants between IGF-1R and each insulin fusion protein were calculated by fitting calculations using the 1:1 affinity and kinetics binding mode in Octet Analysis software.

[0103] Table 3 shows the binding and dissociation kinetics of insulin-Fc fusion proteins with IGF-1R, as well as the average K on , K.off The apparent KD values ​​were summarized. Research has shown that IGF-1R is highly expressed in various cancer tissues, and insulin exerts its growth-promoting effect by binding to IGF-1R. An ideal insulin analog should have IGF-1R binding affinity similar to or reduced to that of unmodified insulin. The results in Table 3 show that the fusion proteins of Examples 1 and 2 of the present invention have reduced affinity for IGF-1R compared to insulin glargine (Lantus, Sanofi) and unmodified Insulin-Fc, indicating that the fusion proteins of the Examples of the present invention do not have the potential risk of promoting growth.

[0104] Table 3. KD values ​​of insulin-Fc fusion proteins and insulin-like growth factor 1 receptor (IGF-1R)

[0105] [Table 3]

[0106] Experimental Example 4: Measurement of the binding affinity of insulin-Fc fusion protein to IRA / IRB and IGF-1R by scintillation pulse assay (SPA) Cell membranes prepared from two CHO cell lines overexpressing hIRA and hIRB, and H19-7 cells overexpressing human IGF-1R, were used to measure IRA, IRB, and IGF-1R binding during SPA assays in a 96-well plate. Cell membranes from hIRA-, hIRB-, and IGF-1R-overexpressing cell lines diluted with 25 μl of 1 M Trizma hydrochloride buffer (Sigma-Aldrich) were added to each well of a 96-well conical plate (Agilent). The fusion protein of the example and native recombinant human insulin (RHI, CAS No.: 11061-68-0, Sigma-Aldrich) were diluted 1:4 with 1 M Trizma buffer and added at 25 μl per well to the 96-well plate containing the cell membranes, with two duplicate wells for each. During IRA / IRB binding assays, 50 μl of a final concentration of 100 pM was added to the above system. [125I]Similarly, during IGF-1R binding assays, 50 μl of 100 μM final concentration of insulin (PerkinElmer, Inc., Waltham, MA) was added. [125I] 100 μL of scintillation fluid (PerkinElmer, Inc.) was added to each well, and the cells were then sealed with a plate sealing membrane and incubated at room temperature on a shaker for 1 hour. The cells were then harvested from the above system using a cell harvester onto a 96-well GF / C filter plate (PerkinElmer, Inc.) that had been presoaked with 0.5% BSA solution. The plate was washed six times with 1 M Trizma buffer and then dried in an oven at 50°C for 1 hour. 50 μL of scintillation fluid (PerkinElmer, Inc.) was then added to each well, and the cells were then incubated with MicroBeta 2 (PerkinElmer, Inc.) instrument. A four-parameter fitting was performed using a nonlinear model in GraphPad Prism 5.0 (San Diego, CA) to calculate the IC. 50 Values ​​were calculated and quantified by the percentage (%) of maximal receptor binding activity relative to native recombinant human insulin (RHI).

[0107] Table 4 shows the IC values ​​of insulin-Fc fusion proteins and native recombinant human insulin with hIRA, hIRB, and hIGF-1R. 50 The values ​​and percentage of maximum receptor binding activity relative to native insulin were summarized. The results showed that the insulin-Fc fusion proteins of Examples 1 and 2 of the present invention had significantly reduced binding affinities for IRA, IRB, and IGF-1R compared with native recombinant human insulin and unmodified insulin-Fc fusion protein, and the maximum receptor binding activities of Examples 1 and 2 for IRA, IRB, and IGF-1R were all significantly lower than those of recombinant human insulin.

[0108] Table 4. IC of insulin-Fc fusion protein with IRA, IRB, and IGF-1R 50 Values ​​and percentage of maximum receptor binding activity for recombinant human insulin

[0109] [Table 4]

[0110] Experimental Example 5: Study of insulin-Fc fusion protein in STZ-treated diabetic mouse model Normal C57BL / 6 mice (SCXK(Kyoto) 2019-0008, weighing 18-22 g) were acclimated for approximately one week, fasted overnight for 12 hours, and then intraperitoneally injected with 60 mg / kg streptozotocin (STZ, Sigma-Aldrich). After refeeding, the mice were injected with 60 mg / kg streptozotocin (STZ, Sigma-Aldrich) at the same time each day for six consecutive days. After seven consecutive days of feeding, fasting blood glucose levels were measured using a blood glucose meter (Roche, Basel, Switzerland). If blood glucose levels exceeded 12 mmol / L in both cases, the model was considered successful. The mice were then divided into groups according to their final weight and blood glucose levels and used for the following pharmacological evaluation.

[0111] Mice that were successfully modeled were randomly divided into groups and fine-tuned by measuring blood glucose one hour before administration, and then subcutaneously injected slightly behind the neck with 300 nMol / kg of the insulin-Fc fusion protein of Example 1 and / or Example 2 prepared in sterile PBS. Blood glucose levels were measured 2, 4, 6, 8, and 24 hours after injection, and then every 24 hours until they returned to modeling levels.

[0112] Figure 1 shows the collected blood glucose data (mean ± SEM, n = 6). For a more intuitive presentation, data from 0 to 24 hours are not included. The results show that the fusion protein of Example 1 maintained blood glucose fluctuations at normal levels within 240 hours after administration, with the mice showing little hypoglycemia. The blood glucose-lowering effect lasted for approximately 14 days, demonstrating superior blood glucose-lowering efficacy and duration compared with the fusion protein of Example 2. Injection of 300 nMol / kg of unmodified Insulin-Fc fusion protein caused obvious hypoglycemia in mice within 48 hours of administration, which is not shown in the figure.

[0113] Experimental Example 6: Pharmacokinetics of insulin-Fc fusion protein Four 4-week-old C57BL / 6 mice (weight 18-22 g) were subcutaneously injected (sc) with 300 nMol / kg of Example 1, and blood was collected intravenously at 1, 2, 4, 8, 12, 24, 48, 96, 144, 196, and 240 hours after injection (approximately 100 μL of blood was collected from each mouse). 10 μL of 100% EDTA was added to the blood and mixed uniformly. The mixture was centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected.

[0114] The concentration of insulin fusion protein in plasma was measured by ELISA, using the human insulin receptor extracellular domain (hIRB, aa28-956) as the capture probe. 50 μL of 10 μg / mL hIRB (aa28-956) was coated onto a 96-well high-binding U-plate at 50 μL / well and incubated overnight at 4°C. The plate was washed three times with PBS. The 96-well plate was blocked with 200 μL / well of 1% casein blocking solution and incubated at room temperature for 1.5 hours. The plate was washed three times with 0.1% PBST. A standard curve was prepared by gradient dilution of the fusion protein at a 1:2 ratio using 5% mouse plasma in blocking solution (1% casein, Thermo Scientific Fisher Scientific). Plasma samples (n=4) from different time points were diluted 20-fold with 1% casein blocking solution and incubated at room temperature for 1.5 hours at 50 μL / well. The plate was washed three times with 0.1% PBST. The absorbance at 450 nm was measured using HRP Goat Anti-Human IgG (H+L) (ABclonal Technology Co., Ltd.) diluted 1:2000 with 1% casein blocking solution.

[0115] Table 5 shows the plasma concentrations of the fusion protein of Example 1 at different time points, and Table 6 shows the pharmacokinetic parameters of the fusion protein of Example 1 obtained by non-compartmental analysis of the data in Table 4. The data show that the fusion protein of Example 1 has a long half-life in the body of about 38 hours, and has increased bioavailability and a prolonged duration of action in the body compared to conventional insulins that have been approved.

[0116] Table 5. Blood drug concentrations of the fusion protein of Example 1 in mice.

[0117] [Table 5]

[0118] Table 6. PK parameters of the fusion protein of Example 1 in mice

[0119] [Table 6]

[0120] Abbreviation: T 1 / 2 - Half-life, Cmax - maximum concentration, Tmax - time at maximum concentration, AUC last - area under the curve from 0 to the administration point, CL_F - elimination rate, MRT last Mean time spent between -0 and the administration point.

Claims

1. A fusion protein comprising: a) General formula Z １ -Z ２ -Z ３ and an insulin analog having the structure Among them, Z １ is an insulin B chain analogue comprising the following amino acid sequence: FVNQX １ LCGSHLVEALX ２ LVCGERGFX ３ YX ４ X ５ X ６ X ７ (SEQ ID NO: 1) Among them, X １ is H or A, and X ２ is Y, A or E, and X ３ is T, I, F or H, and X ４ is E, D, T, or does not exist, and X ５ is P, E, or does not exist, and X ６ is K, E, or does not exist, and X ７ is T or absent; Z ２ is the first peptide linker; Z ３ is an insulin A chain analogue comprising the following amino acid sequence: GIX ８ EX ９ CCX １０ SICSLYQLENYCX １１ (SEQ ID NO: 2) Among them, X ８ is A, L or V; X ９ is Q or A; X １０ is T, A, F or K; X １１ is G, S or N, b) a second peptide linker; and c) a human IgG Fc region or a variant thereof; Including, wherein the C-terminal residue of the insulin analog is directly 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 human IgG Fc region or variant thereof; Fusion proteins.

2. In the insulin analogue, Z １ In this case, X １ is A and X ２ is Y and X ３ is F and X ４ is T and X ５ is P and X ６ is K and X ７ is T; or Z １ In this case, X １ is H and X ２ is A and X ３ is F and X ４ is T and X ５ is P and X ６ is K and X ７ is T; or Z １ In the formula, χ is H and X ２ is Y and X ３ is T or I, and X ４ is E and X ５ is P and X ６ is K and X ７ is T; or Z １ In this case, X １ is H; X ２ is Y; X ３ is F; X ４ is E or D; X ５ is P; X ６ is K; X ７ is T; or Z １ In this case, X ４ -X ７ does not exist, The fusion protein of claim 1.

3. In the insulin analogue, Z ３ In this case, X ８ is A or L, and X ９ is A and X １０ is T and X １１ is G; or Z ３ In this case, X ８ is V and X ９ is Q and X １０ is T and X １１ is G; or Z ３ In this case, X ８ is V and X ９ is Q and X １０ is A, K or F, and X １１ is G; or Z ３ In this case, X ８ is A or L, and X ９ is Q and X １０ is A or K, and X １１ is G, The fusion protein of claim 1.

4. In the insulin analogue, Z １ In this case, X １ is H and X ２ is Y and X ３ is F and X ４ is E or D, and X ５ is P and X ６ is K and X ７ is T; and Z ３ In this case, X ,８ is A; X １１ is G; or Z １ In this case, X １ is H and X ２ is Y and X ３ is F and X ４ is T and X ５ is P and X ６ is K and X ７ is T; and Z ３ In this case, X ８ is A or L, and X １１ is G; or Z １ In this case, X １ is H and X ２ is Y and X ３ is F and X ４ is T and X ５ is P and X ６ is K and X ７ is T; and Z ３ In this case, X ９ is A and X １１ is G; or Z １ In this case, X １ is H and X ２ is Y and X ３ is F and X ４ is T and X ５ is P and X ６ is K and X ７ is T; and Z ３ In this case, X １０ is A, K or F, and X １１ is G; or Z １ In this case, X １ is H and X ２ is A and X ３ is F and X ４ is T and X ５ is P and X ６ is K and X ７ is T; and Z ３ In this case, X ８ is A and X １１ is G; or Z １ In this case, X ４ -X ７ is not present; and Z ３ In this case, X ８ is A and X １１ is G, The fusion protein of claim 1.

5. Z １ -Z ２ -Z ３ comprises the following amino acid sequence: FVNQX １ LCGSHLVEALX ２ LVCGERGFX ３ YX ４ X ５ X ６ X ７ GGVGGGGIX ８ EX ９ CCX １０ SICSLYQLENYCX １１ (SEQ ID NO: 19) Among them, X １ is H or A; X ２ is Y, A or E; X ３ is T, I, F or H; X ４ is E, D, T or absent; X ５ is P, E, or absent; X ６ is K, E, or absent; X ７ is T or absent; X ８ is A, L or V; X ９ is Q or A; X １０ is T, A, F or K; X １１ is G, S or N, Preferably, Z １ -Z ２ -Z ３ has the following amino acid sequence: FVNQHLCGSHLVEALYLVCGERGFFYEPKTGGVGGGGGIAEQCCTSICSLYQLENYCG (SEQ ID NO: 3) The fusion protein of claim 1.

6. the first peptide linker comprises an amino acid sequence selected from the group consisting of GGVGGG (SEQ ID NO: 16), GGGSGG (SEQ ID NO: 17), GGGGGV (SEQ ID NO: 18), and / or the second peptide linker comprises a peptide having the sequence [GGGGX]n, wherein X is S or T; and wherein n is 2, 3, 4, or 5; Preferably, the second peptide linker comprises the following amino acid sequence: GGGGX １２ GGGGX １３ GGGGX １４ GGGGX １５ (SEQ ID NO: 20) Among them, X １２ is S or T; １３ is S or T; １４ is S or T; X １５ is S or T, More preferably, the second peptide linker has the following amino acid sequence: GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 4) The fusion protein of claim 1.

7. The human IgG Fc region is an Fc region derived from an IgG2 or IgG4 antibody, preferably an Fc region derived from an IgG2 antibody, and more preferably comprises the following amino acid sequence: ERKX １６ X １７ VEX １８ PPX １９ PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCCVVVDVSX ２０ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSLTVX ２１ HQDWLNGKEYKCKVSNKGLPX ２２ X s ２３ IEKTISKTKGQPREPQVYTLPPSREEMTKNPQVSLTCLVKGFYPSDIAVEWESNGQPENNYYKTTPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX ２４ GX ２５ (SEQ ID NO: 5) Among them, X １６ is S or C; X １７ is S or C; X １８ is S or C; X １９ is S or C; X ２０ is Q or H; X ２１ is L or V; X ２２ is S or A; X ２３ is S or P; X ２４ is L or P; X ２５ is K or does not exist, Even more preferably, the human IgG Fc region comprises the following amino acid sequence: ESKYGPPSPPSPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 6) The fusion protein of claim 1.

8. The fusion protein comprises the following amino acid sequence: FVNQX １ LCGSHLVEALX ２ LVCGERGFX ３ YX ４ X ５ X ６ X ７ GGVGGGGIX ８ EX ９ CCX １０ SICSLYQLENYCX １１ GGGGX １２ GGGGX １３ GGGGX １４ GGGGX １５ ERKX １６ X １７ VEX １８ PPX １９ PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCCVVVDVSX ２０ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSLTVX ２１ HQDWLNGKEYKCKVSNKGLPX ２２ X ２３ IEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYYTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLLSX ２４ GX ２５ (SEQ ID NO: 7) Among them X １ is H or A; X ２ is Y, A or E; X ３ is T, I, F or H; X ４ is E, D, T or absent; X ５ is P, E or absent; X ６ is K, E, or absent; X ７ is T or absent; X ８ is A, L or V; X ９ is Q or A; X １０ is T, A, F or K; X １１ is G, S or N; X １２ is S or T; X １３ is S or T; X １４ is S or T; X １５ is S or T; X １６ is S or C; X １７ is S or C; X １８ is S or C; X １９ is S or C; X ２０ is Q or H; X ２１ is L or V; X ２２ is S or A; X ２３ is S or P; X ２４ is L or P; X ２５ is K or does not exist, The fusion protein of claim 1.

9. The fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:

15. The fusion protein of claim 1.

10. A protein dimer comprising the fusion protein of any one of claims 1 to 9.

11. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 9 or the protein dimer of claim 10, and optionally at least one pharmaceutically acceptable excipient.

12. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 9 or the protein dimer of claim 10.

13. A recombinant vector comprising the nucleic acid molecule of claim 12.

14. A recombinant cell comprising the nucleic acid molecule of claim 12 or the recombinant vector of claim 13.

15. Use of the fusion protein of any one of claims 1 to 9, the protein dimer of claim 10, or the pharmaceutical composition of claim 11 in the manufacture of a medicament for treating diabetes, obesity, or metabolic syndrome.

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