Human insulin analogs, fusion proteins thereof and pharmaceutical uses

Abstract

Human insulin analogs, fusion proteins and pharmaceutical uses thereof. Human insulin analog-Fc fusion proteins have a very long in vivo pharmacokinetic profile and can be used to treat metabolic diseases (e.g., type 1 diabetes and type 2 diabetes).

Description

[Technical field]

[0001] This application claims priority to Chinese patent application No. 202210259967.6 filed on March 16, 2022.

[0002] The present disclosure relates to the biopharmaceutical field, specifically to human insulin analogs and fusion proteins thereof, and the use of such fusion proteins to treat metabolic diseases (e.g., diabetes). [Background technology]

[0003] Diabetes mellitus is a chronic metabolic disease that causes abnormalities in the metabolism of glucose, protein and lipids in the human body due to the lack of insulin secreted in vivo. It is mainly divided into insulin-dependent diabetes mellitus (type 1 diabetes) and non-insulin-dependent diabetes mellitus (type 2 diabetes). 90-95% of diabetic patients worldwide have type 2 diabetes, which is caused by pancreatic β-cell dysfunction and long-term insulin resistance, and is most notably characterized by lack of insulin levels in vivo, reduced insulin sensitivity and high blood glucose concentration in plasma. Research has shown that type 2 diabetes is associated with a variety of high-risk complications, which can lead to cardiovascular disease, renal failure, blindness, limb amputation and many other complications.

[0004] The diabetes drugs approved for commercial use mainly include chemically synthesized small molecule oral hypoglycemic agents, such as biguanides, sulfonyls, insulin sensitizers, α-glycosides and injectable peptide hypoglycemic agents such as glucagon-like peptide-1 (GLP-1) analogues. For all type 1 diabetes and some end-stage type 2 diabetes, only insulin injection-related drugs can control blood glucose levels relatively well.

[0005] At present, insulin analogue products that have been approved for sale are mainly divided into two types: fast-acting insulin and long-acting basal insulin. Fast-acting insulins that have been approved for sale include insulin lispro and insulin aspart. Fast-acting insulin is mainly administered by subcutaneous injection before a meal to patients, and inhibits the formation of natural insulin hexamers by modifying the insulin molecular structure, thereby having the pharmacokinetic characteristics of rapid onset of action and rapid clearance after subcutaneous administration. Among them, insulin lispro achieves the effect of inhibiting the formation of insulin hexamers by alternating the amino acid positions at positions 28 and 29 at the end of the natural insulin B-chain, while insulin aspart inhibits the formation of hexamers through charge-exclusive interaction by replacing proline with aspartic acid at position 28 of the insulin B-chain. Long-acting basal insulins that have been approved for sale include insulin glargine, insulin detemir, and insulin degludec. Among them, insulin glargine introduces positively charged arginine into the insulin molecular backbone to raise the isoelectric point of the molecule to neutral, so that the insulin analogue forms a precipitate at the administration site after subcutaneous injection and obtains significantly extended in vivo pharmacokinetic properties after being dissolved in a sustained release manner. Insulin detemir and insulin degludec extend their in vivo pharmacokinetic properties by introducing long-chain fatty acid modifications to the lysine side chain at the B-chain end, forming supramolecular multimers at the administration site, and reversibly binding to human serum albumin in plasma. Currently, insulin glargine and insulin degludec are administered once a day, and insulin detemir is administered once or twice a day.

[0006] However, fast-acting insulin products may cause side effects such as hypoglycemia and severe hypoglycemia, and patients need to inject insulin-like drugs once or even several times a day, which makes it inconvenient for patients to use, increases the burden of treatment, and reduces the quality of life. Meanwhile, traditional long-acting basal insulin products also require subcutaneous injection once a day, and the injection frequency is still high. Therefore, an ultra-long-acting basal insulin product with lower injection frequency and higher safety is required by clinical patients, which can significantly improve patient compliance and convenience, and the present disclosure provides such an ultra-long-acting basal insulin product. Summary of the Invention

[0007] The present disclosure provides fusion proteins of human insulin analogs and IgG Fc regions, methods for their preparation, and uses of said fusion proteins in the treatment of metabolic diseases (eg, type 1 diabetes or type 2 diabetes).

[0008] Insulin analogues The present disclosure provides insulin analogs, which include an insulin B-chain analog and an insulin A-chain analog, of which the insulin B-chain analog is X7 is selected from G, E, P, K, D, S, H, or is absent; X8 is selected from G, E, K, P, Q, D, H, or is absent; and X9 is selected from G, T, S, E, K, A, or is absent; Insulin A-chain analogues include The amino acid sequence is GIVEQCCZ1SICSLZ2QLENYCZ3Z4 (sequence number 44), in which Z1 is selected from T, H or E, Z2 is selected from Y, D, S or E, Z3 is selected from G or N, and Z4 is any naturally occurring amino acid or is absent, and when Z3 is N, Z4 is not G or N.

[0009] The present disclosure provides insulin analogs, which include an insulin B-chain analog and an insulin A-chain analog, of which the insulin B-chain analog is The amino acid sequence of FVX1QHLCGX2HLVEALX3X4VCGERGFX5Y (SEQ ID NO: 1) is included, wherein X1 is selected from N, G, or K, X2 is selected from S or E, X3 is selected from Y, H, or E, X4 is selected from H, R, L, or E, and X5 is selected from F or H; Insulin A-chain analogues include It comprises the amino acid sequence GIVEQCCZ1SICSLZ2QLENYCZ3 (SEQ ID NO: 2), in which Z1 is selected from T, H or E; Z2 is selected from Y, D, S or E; and Z3 is selected from G or N.

[0010] In some embodiments, the insulin B chain analog comprises (or is) the amino acid sequence set forth in SEQ ID NO:43 or SEQ ID NO:1, which has at least one, at least two, at least three, at least four, or five amino acid modifications in X1, X2, X3, X4, X5. In some specific embodiments, X1 is selected from N or K, X2 is selected from S or E, X3 is selected from Y or H, X4 is selected from L, and / or X5 is selected from H. In some specific embodiments, X2 is E and X5 is H. In some specific embodiments, X5 is H. In some specific embodiments, X3 is H and X5 is H. In some specific embodiments, X1 is K, X2 is E, and X5 is H.

[0011] In some embodiments, the insulin A-chain analog comprises (or is) the amino acid sequence set forth in SEQ ID NO:44 or SEQ ID NO:2, which has at least one, at least two, or three amino acid modifications in Z1, Z2, Z3. In some specific embodiments, Z1 is selected from T, H or E, Z2 is selected from E, and Z3 is selected from G or N. In some specific embodiments, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, Z1 is H, Z2 is E, and Z3 is G. In some specific embodiments, Z2 is E. In some specific embodiments, Z1 is E and Z2 is E.

[0012] In some embodiments, the insulin B-chain analog in the insulin analog comprises (or is) the sequence of SEQ ID NO: 43 or SEQ ID NO: 1, and the insulin A-chain analog comprises (or is) the amino acid sequence shown in SEQ ID NO: 44 or SEQ ID NO: 2. In some specific embodiments, X1 is selected from N or K, X2 is selected from S or E, X3 is selected from Y or H, X4 is selected from L, X5 is selected from H, and Z1 is selected from T, H or E, Z2 is selected from E, and Z3 is selected from G or N. In some specific embodiments, X2 is E, X5 is H, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, X5 is H, Z1 is H, Z2 is E, and Z3 is G. In some specific embodiments, X3 is H, X5 is H, and Z2 is E. In some specific embodiments, X1 is K, X2 is E, X5 is H, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, X2 is E, X5 is H, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, X2 is E, X5 is H, Z1 is E, and Z2 is E.

[0013] In some specific embodiments, X1 is N or K, X2 is E, X3 is Y, X4 is L, X5 is H, Z1 is E, Z2 is E, and Z3 is G; X1 is N, X2 is E, X3 is Y, X4 is L, X5 is H, Z1 is E, Z2 is E, and Z3 is N; X1 is N, X2 is S, X3 is Y, X4 is L, X5 is H, Z1 is H, Z2 is E, and Z3 is G; X1 is N, X2 is S, X3 is H, X4 is L, X5 is H, Z1 is T, Z2 is E and Z3 is G; or X1 is N, X2 is E, X3 is Y, X4 is L, X5 is H, Z1 is E, Z2 is E, and Z3 is G.

[0014] In the above embodiment, X6-X9 or X7-X9 may each be G, or X6-X9 or X7-X9 may be absent.

[0015] In the above embodiment, Z4 may be absent, or, if Z3 is N, then Z4 is an amino acid other than G, N, S, V, L or P.

[0016] In some embodiments, the insulin B chain analog in the insulin analog comprises (or is) any one of the following: FVNQHLCGEHLVEALYLVCGERGFHY (SEQ ID NO:31), FVNQHLCGSHLVEALYLVCGERGFHY (SEQ ID NO:32), FVNQHLCGSHLVEALHLVCGERGFHY (SEQ ID NO:33), FVKQHLCGEHLVEALYLVCGERGFHY (SEQ ID NO:34), FVNQHLCGEHLVEALYLVCGERGFHY (SEQ ID NO:35), FVNQHLCGEHLVEALYLVCGERGFHY (sequence number 36).

[0017] In some embodiments, the insulin A-chain analog in the insulin analog comprises (or is) any one of the following: GIVEQCCESICSLEQLENYCG (SEQ ID NO:37), GIVEQCCHSICSLEQLENYCG (SEQ ID NO:38), GIVEQCCTSICSLEQLENYCN (SEQ ID NO:39), GIVEQCCESICSLEQLENYCG (SEQ ID NO:40), GIVEQCCESICSLEQLENYCG (SEQ ID NO:41), GIVEQCCESICSLEQLENYCN (SEQ ID NO:42).

[0018] In some embodiments, the insulin B chain analog and the insulin A chain analog in the insulin analog comprise (or are) the amino acid sequences set forth in any one of SEQ ID NOs: 31 and 37, SEQ ID NOs: 32 and 38, SEQ ID NOs: 33 and 39, SEQ ID NOs: 34 and 40, SEQ ID NOs: 35 and 41, and SEQ ID NOs: 36 and 42.

[0019] In some embodiments, the insulin analogs have disulfide bonds, hi some specific embodiments, there are two disulfide bonds between the insulin A chain analog and the insulin B chain analog at CysA7-CysB7 and CysA20-CysB19, and / or an intrachain disulfide bond at CysA6-CysA11 in the insulin A chain analog.

[0020] Exemplarily, "CysA7-CysB7" indicates that an interchain disulfide bond is formed between the 7th Cys from the N-terminus in the insulin A-chain analog and the 7th Cys from the N-terminus in the insulin B-chain analog. "CysA20-CysB19" indicates that an interchain disulfide bond is formed between the 20th Cys from the N-terminus in the insulin A-chain analog and the 19th Cys from the N-terminus in the insulin B-chain analog.

[0021] In some embodiments, any one of the insulin analogs has insulin receptor agonist activity.

[0022] In some embodiments, the insulin analogue comprises an amino acid modification at any one or more (e.g., 2, 3, 4, 5, 6, 7, 8) positions in the insulin A chain analogue and insulin B chain analogue. In some embodiments, the modification is a chemical group modification to improve the chemical and / or physical stability of the insulin analogue, to modulate the efficacy of the insulin analogue, and / or to enhance expression.

[0023] In some embodiments, the modified insulin analog is a glucose-responsive insulin.

[0024] In some embodiments, the insulin analog is modified at the fatty acid chain. Fusion proteins The present disclosure provides fusion proteins of human insulin analogs.

[0025] In some embodiments, the fusion protein comprises an insulin analog, The insulin analogues have the general formula B1-L1-A1, Among them, B1 is an insulin B chain analogue, X7 is selected from G, E, P, K, D, S, H, or is absent; X8 is selected from G, E, K, P, Q, D, H, or is absent; and X9 is selected from G, T, S, E, K, A, or is absent; A1 is an insulin A-chain analogue, The amino acid sequence is GIVEQCCZ1SICSLZ2QLENYCZ3Z4 (sequence number 44), in which Z1 is selected from T, H or E, Z2 is selected from Y, D, S or E, Z3 is selected from G or N, and Z4 is any naturally occurring amino acid or is absent, and when Z3 is N, Z4 is not G or N.

[0026] L1 is a linker.

[0027] In some embodiments, the fusion protein comprises an insulin analog, The insulin analogues have the general formula B1-L1-A1, Among them, B1 is an insulin B chain analogue, The amino acid sequence of FVX1QHLCGX2HLVEALX3X4VCGERGFX5Y (SEQ ID NO: 1) is included, wherein X1 is selected from N, G, or K, X2 is selected from S or E, X3 is selected from Y, H, or E, X4 is selected from H, R, L, or E, and X5 is selected from F or H; A1 is an insulin A chain analogue, GIVEQCCZ1SICSLZ2QLENYCZ3 (SEQ ID NO:2), wherein Z1 is selected from T, H or E, Z2 is selected from Y, D, S or E, and Z3 is selected from G or N; L1 is a linker.

[0028] In some embodiments, B1 comprises (or is) SEQ ID NO:43 or the amino acid sequence set forth in SEQ ID NO:1, which has at least one, at least two, at least three, at least four, or five amino acid modifications in X1, X2, X3, X4, X5. In some specific embodiments, X1 is selected from N or K, X2 is selected from S or E, X3 is selected from Y or H, X4 is selected from L, and / or X5 is selected from H. In some specific embodiments, X2 is E and X5 is H. In some specific embodiments, X5 is H. In some specific embodiments, X3 is H and X5 is H. In some specific embodiments, X1 is K, X2 is E, and X5 is H.

[0029] In some embodiments, A1 comprises (or is) the amino acid sequence set forth in SEQ ID NO:44 or SEQ ID NO:2, which has at least one, at least two, or three amino acid modifications in Z1, Z2, Z3. In some specific embodiments, Z1 is selected from T, H or E, Z2 is selected from E, and Z3 is selected from G or N. In some specific embodiments, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, Z1 is H, Z2 is E, and Z3 is G. In some specific embodiments, Z2 is E. In some specific embodiments, Z1 is E and Z2 is E.

[0030] In some embodiments, B1 in the insulin analog comprises (or is) the sequence of SEQ ID NO: 43 or SEQ ID NO: 1, and A1 comprises (or is) the amino acid sequence shown in SEQ ID NO: 44 or SEQ ID NO: 2. In some specific embodiments, X1 is selected from N or K, X2 is selected from S or E, X3 is selected from Y or H, X4 is selected from L, X5 is selected from H, and Z1 is selected from T, H or E, Z2 is selected from E, and Z3 is selected from G or N. In some specific embodiments, X2 is E, X5 is H, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, X5 is H, Z1 is H, Z2 is E, and Z3 is G. In some specific embodiments, X3 is H, X5 is H, and Z2 is E. In some specific embodiments, X1 is K, X2 is E, X5 is H, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, X2 is E, X5 is H, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, X2 is E, X5 is H, Z1 is E, and Z2 is E.

[0031] In some specific embodiments, X1 is N or K, X2 is E, X3 is Y, X4 is L, X5 is H, Z1 is E, Z2 is E, and Z3 is G; X1 is N, X2 is E, X3 is Y, X4 is L, X5 is H, Z1 is E, Z2 is E, and Z3 is N; X1 is N, X2 is S, X3 is Y, X4 is L, X5 is H, Z1 is H, Z2 is E, and Z3 is G; X1 is N, X2 is S, X3 is H, X4 is L, X5 is H, Z1 is T, Z2 is E and Z3 is G; or X1 is N, X2 is E, X3 is Y, X4 is L, X5 is H, Z1 is E, Z2 is E, and Z3 is G.

[0032] In the above embodiment, X6-X9 or X7-X9 may each be G, or X6-X9 or X7-X9 may be absent.

[0033] In the above embodiment, Z4 may be absent, or, if Z3 is N, then Z4 is an amino acid other than G, N, S, V, L or P.

[0034] In some embodiments, B1 in the insulin analog comprises (or is) the amino acid sequence of any one of SEQ ID NOs: 31-36.

[0035] In some embodiments, A1 in the insulin analog comprises (or is) the amino acid sequence of any one of SEQ ID NOs: 37-42.

[0036] In some embodiments, B1 and A1 in the insulin analog include (or are) the amino acid sequences set forth in any one of the following groups: SEQ ID NOs: 31 and 37, SEQ ID NOs: 32 and 38, SEQ ID NOs: 33 and 39, SEQ ID NOs: 34 and 40, SEQ ID NOs: 35 and 41, and SEQ ID NOs: 36 and 42.

[0037] In some embodiments, the linker represented by L1 is a polypeptide capable of performing a linking function, e.g., a flexible polypeptide. In some specific embodiments, the linker represented by L1 comprises (or is) the following sequences, including, but not limited to, G4S (SEQ ID NO: 53), GS, GAP, ASGS (SEQ ID NO: 54), polyguanine (poly G). In some specific embodiments, the linker represented by L1 is (G m S p ) n wherein each m is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6), each n is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6), and each p is independently selected from 0 to 4. In some specific embodiments, the GS or poly G linker has at least 5 Gs.

[0038] In this disclosure, a GS linker covers any type of linker that includes the amino acids G and S in the linker.

[0039] In some specific embodiments, the linker depicted in L1 comprises (or is) GGSGGGG (SEQ ID NO:45), GSGGGG (SEQ ID NO:46), GGGGG (SEQ ID NO:47), GGGGGGSGGGG (SEQ ID NO:3) or GGGGGSGGGG (SEQ ID NO:52).

[0040] In some embodiments, the fusion protein further comprises a C1 domain, which is capable of extending the half-life of the insulin analog.

[0041] In some embodiments, the fusion protein further comprises a C1 domain, wherein the C1 is selected from, for example, an Fc region of an immunoglobulin (e.g., an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM), an HSA protein or polypeptide, an HSA binding domain (e.g., an anti-HSA antibody or an antigen-binding fragment thereof), an XTEN peptide, a glycine-rich high amino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.

[0042] In some specific embodiments, the XTEN peptides are unstructured hydrophilic long peptides that contain six amino acids, Ala, Glu, Gly, Ser, and Thr, in different percentages, e.g., XTEN TM Peptide (Amunix Operating Inc.) In some specific embodiments, the PAS polypeptide is a hydrophilic, uncharged polypeptide of at least about 100, 200, 300, 400, 500, or 600 amino acids in length consisting of Pro, Ala, and Ser residues.

[0043] In some embodiments, one fusion protein comprises one or more (eg, 2, 3, 4) insulin analogs and one C1.

[0044] In some specific embodiments, C1 is selected from the Fc region of human IgG1, IgG2, or IgG4. In some specific embodiments, the Fc region comprises a mutation, and the mutation in the Fc region is L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P or S228P / F234A / L235A on IgG4, N297A on IgG1, IgG2, IgG3, or IgG4, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358MV309L / A330S / P331S, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S on IgG4. A hybrid IgG2 / 4 Fc domain may also be used, for example an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4. In some specific embodiments, the human IgG4 Fc region has S228P, F234A, L235A and / or K447A mutations. In some specific embodiments, the human IgG1 Fc region has L234A / L235A or L234A / L235A / P329G mutations. In some embodiments, the mutation sites of the Fc region are numbered according to the EU numbering rules.

[0045] In some specific embodiments, C1 comprises (or is) an amino acid sequence set forth in any one of SEQ ID NOs: 17, 18, 48-51, or is selected from a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0046] >Human IgG1 Fc region 1 EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:48), >Human IgG1 Fc region 2 EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:49), >Human IgG4 Fc region 1 ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:50), >Human IgG4 Fc region 2 ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence number 51).

[0047] In some embodiments, the fusion protein further comprises L2, the linker represented by L2 being a polypeptide (e.g., a flexible polypeptide) capable of performing a linking function, and C1 forms a fusion protein with B1-L1-A1 via L2. In some specific embodiments, the linker represented by L2 comprises (or is) the following sequence: G4S, GS, GAP, ASGS, polyguanine (poly G), (G m Q) n , (G m A) n , (G m Q i ) n , (G m A i ) n or (PGPQ) s (SEQ ID NO:55), in which m is independently selected from an integer of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6), n is independently selected from an integer of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6), i is independently selected from an integer of 0 to 4 (e.g., 0, 1, 2, 3, 4), and s is selected from an integer of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6). In some specific embodiments, L2 comprises (or is) an amino acid sequence set forth in any one of SEQ ID NOs:10-16.

[0048] In some embodiments, C1 of the fusion protein is at the N-terminus of the insulin analog. In other embodiments, C1 of the fusion protein is at the C-terminus of the insulin analog. In some specific embodiments, the fusion protein is, from N-terminus to C-terminus, B1-L1-A1-L2-C1, C1-L2-B1-L1-A1, A1-L1-B1-L2-C1, or C1-L2-A1-L1-B1.

[0049] In some embodiments, the insulin analog is an insulin receptor agonist that has insulin receptor agonist activity.

[0050] In some embodiments, the fusion protein is a monomer, dimer, or multimer. In some specific embodiments, the dimer is a homodimer, in which the amino acid sequences of the two fusion proteins constituting the dimer are identical or essentially identical. In other specific embodiments, the dimer is a heterodimer, in which the amino acid sequences of the two fusion proteins constituting the dimer are different.

[0051] In some embodiments, the fusion protein comprises (or is) an amino acid sequence set forth in any one of SEQ ID NOs: 19-27, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0052] In some embodiments, a fusion protein of the present disclosure (e.g., any one of SEQ ID NOs: 19-27, further e.g., SEQ ID NOs: 19, 20, 21, or 27) has a reduced ability to promote cell proliferation, thereby reducing the risk of inducing tumorigenesis and / or tumor progression, such as in cells that express IGF-1R on their surface. In some embodiments, the reduction is due to the fusion protein having a reduced ability to promote cell (e.g., MCF-7) proliferation compared to native human insulin, such as in the EC 50 The value increases by 1-10 times, 1-100 times, 1-1000 times, 1-10000 times, 100-500 times, 200-400 times, e.g., EC 50The EC of cell proliferation is increased by about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold. In some specific embodiments, fusion protein 1 as set forth in SEQ ID NO: 19 has an about 200-fold reduction in its ability to promote cell proliferation compared to native human insulin. The EC of cell (e.g., MCF-7) proliferation was measured using the method of Example 5 of the present disclosure. 50 can be detected.

[0053] In some embodiments, the fusion protein of the present disclosure (e.g., any one of SEQ ID NOs: 19-27, further e.g., SEQ ID NO: 19 or 20) has a half-life of 1-10 days, 2-9 days, 2-8 days, or 3-7 days, e.g., about 1, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10 days. In some specific embodiments, the fusion protein 1 shown in SEQ ID NO: 19 has a half-life of about 4.5 days. The half-life of the protein can be detected using the method of Example 6 of the present disclosure.

[0054] Polynucleotides The present disclosure provides a polynucleotide encoding the fusion protein, insulin analog of the present disclosure. The polynucleotide of the present disclosure may be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the nucleic acid of the present disclosure is essentially an isolated nucleic acid.

[0055] "Essentially isolated" refers to removal from its naturally occurring or purified state, and in this case refers to the specified molecule being essentially free of other biological molecules or other materials (e.g., cellular debris or culture medium). "Essentially isolated" is not intended to imply the complete absence of such materials, or the absence of water, buffers or salts, unless those materials are present in amounts that would significantly interfere with therapeutic use.

[0056] The nucleic acid according to the present disclosure may be in the form of, present in and / or part of a vector, for example a plasmid, cosmid, YAC or viral vector. The vector may in particular be an expression vector, i.e. a vector that allows the expression of the fusion protein, insulin analogue in vitro and / or in vivo (i.e. in a suitable host cell, host organism and / or expression system). Such an expression vector generally comprises at least one nucleic acid according to the present disclosure, operably linked to one or more suitable expression control elements (e.g. promoters, enhancers, terminators, etc.). The selection of said elements and their sequences for expression in a particular host is within the knowledge of the skilled artisan. Regulatory elements and other elements useful or necessary for the expression of the fusion protein, insulin analogue according to the present disclosure are, for example, promoters, enhancers, terminators, integrons, selection markers, leader sequences, reporter genes.

[0057] The nucleic acids according to the present disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on information about the amino acid sequence of the polypeptide according to the present disclosure, and / or may be isolated from a suitable natural source.

[0058] host cell The present disclosure provides recombinant host cells that express one or more of the fusion proteins, insulin analogs, or contain the polynucleotides or vectors of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0059] Bacterial cells include, for example, cells of gram-negative strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0060] Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0061] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0062] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells and any other cells known in the art for expressing heterologous proteins.

[0063] The cells of the present disclosure are incapable of developing into complete plants or animal organisms.

[0064] Method of production or preparation The present disclosure provides a method for preparing a fusion protein, an insulin analog, of the present disclosure, the method generally comprising: - culturing a host cell of the present disclosure under conditions that allow expression of the fusion protein, insulin analog of the present disclosure; - recovering the fusion protein of interest, the insulin analog, expressed by said host cells from the culture; - optionally further purifying and / or modifying the fusion protein of interest according to the present disclosure, the insulin analogue; Includes.

[0065] The fusion proteins, insulin analogs of the present disclosure may be produced in an intracellular manner in cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies) and then isolated from the host cells and optionally further purified, or may be produced in an extracellular manner (e.g., in the medium in which the host cells are cultured) and then isolated from the medium and optionally further purified.

[0066] Methods and reagents for recombinantly producing polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc., are known in the art. Similarly, protein isolation and purification techniques applied to the disclosed protein production methods are known to those of skill in the art. As an example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can be stably transfected into CHO cells. Mammalian expression systems will cause glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in a bioreactor to produce antibodies. The culture medium into which the antibodies are secreted can be purified and collected by conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers may be removed by conventional methods such as molecular sieving, ion exchange, etc. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0067] However, the fusion proteins, insulin analogs of the present disclosure may also be obtained by other methods of protein production known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0068] Pharmaceutical Compositions The present disclosure provides a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of any one or combination of the above-mentioned fusion protein, insulin analog or its encoding polynucleotide, and one or more pharma- ceutically acceptable vectors, diluents, buffers or excipients.

[0069] In some specific embodiments, the unit dose of the pharmaceutical composition may contain 0.01-99% by weight of the fusion protein or insulin analog. In other specific embodiments, the amount of the fusion protein or insulin analog in the unit dose of the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0070] Reagent kit (or kit) The present disclosure provides a reagent kit or kit comprising one or more containers, each independently containing any one or combination of a fusion protein, insulin analog or its encoding polynucleotide according to the present disclosure.

[0071] In some embodiments, the device includes a syringe and a needle, hi some specific embodiments, the device is a pre-filled syringe.

[0072] Methods for preventing and treating diseases and pharmaceutical uses The present disclosure provides compositions, polynucleotides, pharmaceutical compositions comprising the fusion proteins or insulin analogs of the present disclosure, and methods of use in the prevention, treatment, or amelioration of a disease or condition.

[0073] In some embodiments, the method includes administering an effective amount of a fusion protein or insulin analog of the present disclosure to a subject in need thereof to prevent, treat, or alleviate a disease or condition.

[0074] In some embodiments, the disease or condition is selected from non-insulin dependent diabetes mellitus, insulin dependent diabetes mellitus, or other metabolic disease, and the subject in need thereof is untreated or has been treated with oral medications (e.g., sulfonylureas, metformin, thiazolidinediones such as pioglitazone, α-glucosidase inhibitors such as acarbose) and / or non-insulin injectables (including, e.g., incretin-based therapies such as DPP-4 inhibitors and GLP-1R agonists).

[0075] In some embodiments, the disease or condition is selected from diabetes, obesity, dyslipidemia and / or metabolic syndrome.

[0076] In some embodiments, the disease or condition is a diabetic complication, or diabetes-related heart disease, stroke, nephropathy, retinopathy, neuropathy, or kidney disease.

[0077] In some embodiments, the disease or condition is selected from hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, syndrome X, or dyslipidemia.

[0078] In some embodiments, a method is provided for administering to a subject in need thereof an effective amount of a fusion protein or insulin analog of the present disclosure to lower blood glucose.

[0079] The present disclosure further provides a pharmaceutical use of the fusion protein, insulin analogue of the present disclosure for the preparation of a medicament for preventing, treating or alleviating the above-mentioned diseases or symptoms.

[0080] definition In order to make the present disclosure more easily understandable, some technical and scientific terms are specifically defined below. Unless otherwise specifically defined in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those skilled in the art. The three-letter code and one-letter code of amino acids used in this disclosure are as described in J.Biol.Chem, 243, p3558 (1968).

[0081] "Insulin" includes naturally occurring insulin (e.g., human insulin), which is composed of two polypeptide chains, designated A and B chains, which contain 21 and 30 amino acid residues, respectively, and are linked by two cysteine ​​isulfide bonds. The A chain is, for example, set forth in SEQ ID NO:29 of the present disclosure, and the B chain is, for example, set forth in SEQ ID NO:30 of the present disclosure.

[0082] "Insulin analogues" include polypeptides, which have a molecular structure that may be derived formally from naturally occurring insulin (e.g., the human insulin structure) by removal and / or replacement (substitution) of one or more amino acid residues present in natural insulin and / or by addition of one or more amino acid residues. The added and / or substituted amino acid residues may be codable amino acid residues, or other naturally occurring amino acid residues, or purely synthetic amino acid residues.

[0083] An "insulin A-chain analog" refers to an A-chain analog that has one or more amino acid substitutions, deletions and / or extensions (additions) relative to the A-chain of human insulin.

[0084] An "insulin B chain analog" refers to one that has one or more amino acid substitutions, deletions and / or extensions (additions) of the B chain relative to the B chain of human insulin.

[0085] "Glucose-responsive insulin" (GRI) can reduce the incidence of hypoglycemia and make blood sugar control smoother by regulating the release and action of insulin according to blood sugar levels.

[0086] The term "insulin receptor agonist" refers to a protein that binds to and activates the insulin receptor, resulting in lowering of blood glucose levels and / or inhibition of hepatic glucose output, characterized by a property that can be tested and measured by known techniques (e.g., those shown in the studies described in the Examples of the present disclosure). In some embodiments, the insulin receptor agonist is formed by directly or indirectly linking (e.g., linking via a linker) an insulin A chain analog and an insulin B chain analog.

[0087] Unless otherwise limited in the present disclosure, "sequence" should generally be understood to include not only related amino acid sequences, but also nucleic acid or nucleotide sequences encoding said sequences.

[0088] "Homology" or "identity" refers to the similarity of sequences between two polynucleotide sequences or two polypeptides. If a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, if 6 out of 10 positions in two sequences are matched or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Generally, two sequences are compared when aligned to obtain the maximum percentage of homology.

[0089] "Nucleic acid molecule" may be used interchangeably with "polynucleotide" and refers to DNA and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, but preferably are double-stranded DNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

[0090] "Vector" refers to a construct capable of delivering and expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors bound to cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0091] A "host cell" includes a cell or cell culture that may be or has been a recipient of a vector. A host cell includes the progeny of a single host cell, and due to natural, accidental, or deliberate mutations, the progeny are not necessarily completely identical (in morphology or genomic DNA complement) to the original parent cell. A host cell includes cells transfected and / or transformed in vivo with a polynucleotide according to the present disclosure. "Cells," "cell lines," and "cell cultures" may be used interchangeably, and such designations include their progeny (without regard to the number of passages), including, for example, mutant progeny that have the same function or biological activity as the parent cell screened from the originally transformed cell.

[0092] A "pharmaceutical composition" refers to a mixture of one or more active ingredients described herein or their physiologically / pharmaceutical acceptable salts or prodrugs with other chemical components, and other components such as physiologically / pharmaceutical acceptable vectors and excipients. The pharmaceutical composition is intended to facilitate administration to a living body and contribute to the absorption of the active ingredients to further exert their biological activity.

[0093] A "pharmaceutical acceptable vector, diluent or excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and has no intended therapeutic purpose. Examples include, but are not limited to, any standard pharmaceutical vector, such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various wetting agents. In some embodiments, the diluent used for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such vectors are prepared by conventional methods well known in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and R Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0094] "Giving", "administration" and "treatment" refer to contact of an exogenous agent, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid, e.g., therapeutic, pharmacokinetic, diagnostic, research and experimental methods, when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid. Treatment of cells includes contact of a reagent with a cell, and contact of a reagent with a fluid, where the fluid contacts the cell. "Giving", "administration" and "treatment" also refer to in vitro and ex vivo treatment of, e.g., cells, with a reagent, diagnostic, binding composition, or through another type of cell. When applied to a human, veterinary or research subject, it refers to therapeutic treatment, preventative or prophylactic measures, research and diagnostic uses.

[0095] "Treatment" refers to administering to a subject a therapeutic agent, for example, comprising any one of the fusion proteins or insulin analogs of the present disclosure as a therapeutic agent, where the subject suffers from, is at risk of suffering from, or is prone to suffer from, one or more diabetes or hyperglycemia related diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, providing the subject or population being treated with a therapeutic agent in an amount that effectively relieves one or more disease symptoms is accomplished by preventing or delaying the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disease to any clinically measurable extent. The amount of therapeutic agent that effectively relieves any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on several factors, such as the disease state, age and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptom has been reduced can be evaluated by any clinical detection method commonly used by a physician or other professional health care provider to evaluate the severity or progression of the condition. An embodiment of the present disclosure (e.g., a method of treatment or a product) may be ineffective in alleviating a target disease symptom in a subject, but should alleviate a target disease symptom in a statistically significant number of subjects, as determined by any statistical testing method known in the art, such as, for example, Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test. The patient to be treated is a mammal, and preferably a human.

[0096] "Prevention" refers to reducing the risk or incidence of one or more medical conditions, symptoms, complications or conditions, or eliminating or alleviating the progression of one or more medical conditions, symptoms, complications or conditions.

[0097] "Optionally" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0098] By "subject" or "patient" is meant mammals, particularly primates, especially humans.

[0099] "About" or "approximately" refers to a numerical value being within an acceptable error range of the specific value as determined by one of ordinary skill in the art, as determined by how the numerical portion is measured (i.e., the limitations of the measurement system). For example, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" or "essentially including" may mean a variation of at most 20%, for example, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, between 0.5% and 1%, and in this disclosure, any instance where the term "about" precedes a number or range of values ​​includes the specified number of embodiments. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" or "essentially including" should be assumed to be within an acceptable error range of the specific value.

[0100] Unless the context clearly indicates otherwise, throughout the specification and claims, the words "comprise," "having," "containing," and the like, are to be understood to have an inclusive meaning, i.e., "including but not limited to," rather than an exclusive or exhaustive meaning. [Brief description of the drawings]

[0101] [Figure 1] FIG. 13 shows the blood glucose lowering effect after a single subcutaneous injection of fusion protein 1 in a rat model of type 1 diabetes induced by STZ. [Diagram 2] FIG. 13 shows the blood glucose lowering effect of LY3209590, fusion protein 2, and fusion protein 9 after a single subcutaneous injection in a rat model of type 1 diabetes induced by STZ. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0102] The present disclosure will be further described below with reference to examples, but these examples are not intended to limit the scope of the present disclosure.

[0103] Experimental methods for which specific conditions are not specified in the Examples or Test Examples of this disclosure generally follow conventional conditions or conditions recommended by the manufacturers of materials or products. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Modern Methods in Molecular Biology, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which specific sources are not specified are conventional reagents that are commercially available.

[0104] Example 1. Design of human insulin analogs and their fusion proteins with Fc This example provides human insulin analogs that have the general formula, from N-terminus to C-terminus, B1-L1-A1.

[0105] Of these, B1 is a human insulin B chain analogue, and the sequence is: FVX1QHLCGX2HLVEALX3X4VCGERGFX5Y (SEQ ID NO: 1), in which X1 is selected from the amino acid residues Asn, Gly, or Lys, X2 is selected from the amino acid residues Ser or Glu, X3 is selected from the amino acid residues Tyr, His, or Glu, X4 is selected from the amino acid residues His, Arg, Leu, or Glu, and X5 is selected from the amino acid residues Phe or His; A1 is an insulin A-chain analogue, the sequence of which is GIVEQCCZ1SICSLZ2QLENYCZ3 (SEQ ID NO: 2), in which Z1 is selected from the amino acid residues Thr, His or Glu, Z2 is selected from the amino acid residues Tyr, Asp, Ser or Glu, and Z3 is selected from the amino acid residues Gly or Asn; L1 is GGGGGGSGGGG (SEQ ID NO: 3) or GGGGGSGGGG (SEQ ID NO: 52).

[0106] The sequence of an exemplary human insulin analog is as follows: > Human insulin analogue 1 FVNQHLCGEHLVEALYLVCGERGFHY GGGGGGSGGGGGIVEQCCESICSLEQLENYCG (SEQ ID NO:4) > Human insulin analogue 2 FVNQHLCGSHLVEALYLVCGERGFHY GGGGGGSGGGG GIVEQCCHSICSLEQLENYCG (SEQ ID NO:5) > Human insulin analogue 3 FVNQHLCGSHLVEALHLVCGERGFHY GGGGGGSGGGG GIVEQCCTSICSLEQLENYCN (SEQ ID NO:6) > Human insulin analogue 4 FVKQHLCGEHLVEALYLVCGERGFHY GGGGGGSGGGG GIVEQCCESICSLEQLENYCG (SEQ ID NO:7) > Human insulin analogue 5 FVNQHLCGEHLVEALYLVCGERGFHY GGGGGSGGGG GIVEQCCESICSLEQLENYCG (SEQ ID NO:8) > Human insulin analogue 6 FVNQHLCGEHLVEALYLVCGERGFHY GGGGGGSGGGG GIVEQCCESICSLEQLENYCN (SEQ ID NO:9).

[0107] Furthermore, a fusion protein of the above-mentioned human insulin analog and Fc is provided, and the fusion of the two is achieved by a linker such as those shown in Table 1.

[0108] [Table 1] The above Fc is human IgG2 Fc and has the following amino acid sequence: > IgG2 Fc1 ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 17) > IgG2 Fc2 GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 18).

[0109] The amino acid sequence of an exemplary fusion protein is as follows: > Fusion protein 1 (insulin analog 1 + L2 + IgG2 Fc1) FVNQHLCGEHLVEALYLVCGERGFHY GGGGGGSGGGG GIVEQCCESICSLEQLENYCG GGGGQGGGGQGGGGQGGGGG ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 19) > Fusion protein 2 (insulin analog 2 + L2 + IgG2 Fc1) FVNQHLCGSHLVEALYLVCGERGFHY GGGGGGSGGGG GIVEQCCHSICSLEQLENYCG GGGGQGGGGQGGGGQGGGGG ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 20) > Fusion protein 3 (insulin analog 3 + L2 + IgG2 Fc1) FVNQHLCGSHLVEALHLVCGERGFHY GGGGGGSGGGG GIVEQCCTSICSLEQLENYCN GGGGQGGGGQGGGGQGGGGG ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21) > Fusion protein 4 (insulin analog 4 + L2 + IgG2 Fc1) FVKQHLCGEHLVEALYLVCGERGFHY GGGGGGSGGGG GIVEQCCESICSLEQLENYCG GGGGQGGGGQGGGGQGGGGG ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 22) > Fusion protein 5 (insulin analog 1 + L2 + IgG2 Fc1) FVNQHLCGEHLVEALYLVCGERGFHY GGGGGGSGGGG GIVEQCCESICSLEQLENYCG GGGGQGGGGQGGGGQGGGGQGGGGQ ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 23) > Fusion protein 6 (insulin analog 5 + L2 + IgG2 Fc1) FVNQHLCGEHLVEALYLVCGERGFHY GGGGGSGGGG GIVEQCCESICSLEQLENYCG GGGGQGGGGQGGGGQGGGGG ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 24) > Fusion protein 7 (insulin analog 1 + L2 + IgG2 Fc1) FVNQHLCGEHLVEALYLVCGERGFHY GGGGGGSGGGG GIVEQCCESICSLEQLENYCG PGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:25) > Fusion protein 8 (insulin analog 1 + L2 + IgG2 Fc1) FVNQHLCGEHLVEALYLVCGERGFHY GGGGGGSGGGG GIVEQCCESICSLEQLENYCG GGGGAGGGGAGGGGAGGGGG ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 26) > Fusion protein 9 (insulin analog 6 + L2 + IgG2 Fc1) FVNQHLCGEHLVEALYLVCGERGFHY GGGGGGSGGGG GIVEQCCESICSLEQLENYCN HGGGQGGGGQGGGGQGGGGG ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 27).

[0110] Furthermore, LY3209590 is a once-weekly ultra-long-acting insulin analogue developed by Eli Lilly and Company, USA, which is currently undergoing Phase III clinical trials and has the following amino acid sequence: FVNQHLCGSHLVEALELVCGERGFHY GGGGGGSGGGG GIVEQCCTSTCSLDQLENYCG GGGGQGGGGQGGGGQGGGGG ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 28) The underlined parts are all linker sequences.

[0111] The sequence of wild-type human insulin is as follows: >A chain GIVEQCCTSICSLYQLENYCN (SEQ ID NO:29) >B chain FVNQHLCGSHLVEALYLVCGERGFFYTPKT (sequence number 30).

[0112] Example 2. Preparation, characterization and identification of human insulin analog-Fc fusion proteins 1. Preparation 1) HEK-293 cell expression The correct plasmid was extracted using an endotoxin-removing medium extraction reagent kit (Promega, A2495), cloned, and sequenced correctly, and then the cells were prepared for transfection to produce protein. HEK293 suspension cells were passaged regularly using Freestyle 293 medium (invitrogen, cat no. 12338026) and grown at a density of 6 × 10 the day before transfection. 5The cells were passaged to 1.1 × 10 cells / mL, and cell activity and density were measured on the day of transfection to ensure cell activity >90% and a cell density of 1.1 × 10 6 The concentration was adjusted to cells / mL, and plasmid DNA was prepared at a volume of 1 μg DNA / mL cells. The DNA used and the corresponding 293-fectin (Invitrogen, cat No. 12347019) were diluted with opti-MEM (Invitrogen, cat No. 51985-034), mixed evenly by shaking gently, and left at room temperature for 5 min. The diluted DNA was mixed with liposomes by shaking gently, and left at room temperature for 20 min. Then, the diluted DNA was slowly added to the HEK293 cells to be transfected, and the cell culture flask was gently rotated. Then, the cells were placed in a shaker at 37 ° C for suspension shaking culture (5% CO2, 135 rpm, 75% humidity). Five days after transfection, the supernatant of the cell culture was collected and centrifuged at 4000 rpm for 20 min. The supernatant was collected and filtered through a 0.45 μm filter.

[0113] 2) Expression of ExpiCHO cells ExpiCHO suspension cells were passaged regularly using ExpiCHO expression medium (Invitrogen, Cat. No. A2910002) and transfected with the ExpiCHO expression system (Cat. no. A29133) at a rate of 1 μg DNA / mL of transfected cells. After transfection using the "High protocol" in the instruction manual, the cells were placed in a shaker at 32°C for suspension shaking culture (5% CO2, 110 rpm, 75% humidity). After transfection, the cell culture supernatant was collected on the 10th to 12th day, centrifuged at 4000 rpm for 20 min, and the supernatant was collected and filtered through a 0.45 μm filter.

[0114] 3) Detection of expression levels The human insulin analog-Fc fusion protein was purified by ultraviolet absorption method to measure the OD280 of the purified protein, and the expression amount of each fusion protein in different expression systems was estimated based on the results. The results show that fusion proteins 1 to 9 can all be expressed in the HEK293 expression system, and some fusion proteins have excellent yields. For example, fusion protein 1 was 119 mg / L, fusion protein 4 was 116 mg / L, fusion protein 6 was 132 mg / L, fusion protein 7 was 110 mg / L, and fusion protein 8 was 119 mg / L. In the ExpiCHO transient transfection expression system, the expression amount of fusion protein 1 is more than 400 mg / L.

[0115] 4) Purification Protein-A affinity chromatography: The cell culture supernatant expressing the Fc fusion protein was centrifuged at high speed to collect the supernatant, which was then filtered through a 0.22 μm filter membrane. The Protein-A (GE, Cat: 17-5474-99) affinity column was regenerated with 5 column volumes of 0.1 M NaOH aqueous solution (Sigma: Cat: 71687-500g), and then washed and equilibrated with 5 column volumes of 1× PBS buffer (pH 7.4) (Sangon Biotech (Shanghai) co., Ltd.: Cat. E607016-0500). The supernatant was applied to the column at a low flow rate for binding, and the flow rate was controlled so that the retention time was at least about 1 min. After completion of binding, the chromatography column was washed with 5 to 10 column volumes of 1× PBS buffer (pH 7.4) until the UV absorption returned to baseline. The sample was then eluted with 0.1 M glycine (pH 3.0) (Sigma: Cat. 410225-250G) buffer, the elution peak was collected by UV detection, and the elution product was quickly adjusted to pH 7-8 with 1 M Tris-HCl (pH 7.5) (Vetec, Cat. V900483) and stored for a while. The solution of the elution product can be replaced by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube, replacement of the solution with a desired buffer system, or replacement with a desired buffer system by molecular exclusion chromatography (e.g., G-25 desalting). The final storage system of the human insulin analog-Fc fusion protein was 1×PBS buffer, pH 7.4.

[0116] Anion Chromatography: In step 1, the sample was diluted with equilibration buffer 20 mM Tris pH 8.0 (Vetec, Cat. V900483) to a conductivity lower than 3 ms / cm, and equilibrated with a Q HP (GE, Cat: 17-1014-01) anion chromatography column by regenerating with 5 column volumes of 0.5 M aqueous NaOH (Sigma Cat: 71687-500g) and then washing with 15 column volumes of 20 mM Tris buffer pH 8.0 (Vetec, Cat. V900483). The supernatant was applied to the column at a low flow rate for binding, and the flow rate was controlled to ensure a retention time of at least 2 min. After binding was complete, the chromatography column was washed with 10 column volumes of 20 mM Tris buffer pH 8.0 (Vetec, Cat. V900483) until the UV absorption returned to baseline. Gradient elution was performed from 0 to 100% using the elution buffer 20 mM Tris pH 8.0 (Vetec, Cat. V900483) 0.5 M NaCl (Vetec, Cat. V900058), and the fraction was analyzed by SEC-HPLC (Column: TSKgel G3000SW XL The elution peak was detected and collected according to purity (Cat. 008541, 5 μm). The elution product can be subjected to solution replacement by methods well known to those skilled in the art, such as ultrafiltration concentration using ultrafiltration tubes, replacement of the solution with a desired buffer system, or exchange of the solution with a desired buffer system by molecular exclusion chromatography (e.g., G-25 desalting). The final storage buffer system of the human insulin analog-Fc fusion protein is 1×PBS buffer, pH 7.4.

[0117] 2. Characterization and Identification Size exclusion chromatography (SEC-HPLC): An Agilent 1260 infinity high performance liquid chromatography system was used, and the chromatography column was a Tosoh (TSGel-G3000SWXL, the size of the chromatography column was 7.8 mm x 300 mm). Before sample injection, the chromatography column and the system were equilibrated with 2 x PBS buffer containing 5% isopropanol, and then 15 μg of sample was injected and eluted with the same buffer, with a method duration of 20 min and a method flow rate of 1 mL / min.

[0118] Ultra-high performance liquid chromatography-mass spectrometry combined: Characterized by Agilent 1290-6530 ultra-high performance liquid chromatography-mass spectrometry combined system, the chromatography column used was Sepax-C4 (particle size 3 μm, size of the chromatography column: 2.1 mm × 50 mm), mobile phase A was an aqueous solution containing 0.1% formic acid, mobile phase B was acetonitrile containing 0.1% formic acid, the flow rate was 1 mL / min, the temperature of the chromatography column was maintained at 60 °C, and the chromatographic gradient used was as shown in Table 2. The relevant detection parameters setting mode of the mass spectrometer: 1 GHz, capillary voltage: 5000 V, desolvation gas temperature: 350 °C, gas flow rate: 12 L / min, Fragmentor voltage: 380 V, MS acquisition range: 100 m / z-4000 m / z.

[0119] [Table 2] According to the data in Table 3, fusion proteins 1 to 9 all have good purity and desired molecular weight after purification.

[0120] [Table 3] The following examples tested the biological activity of the fusion proteins of the present disclosure.

[0121] Example 3. Experiment to detect autophosphorylation of human insulin receptor B (hIR-B) The activities of different human insulin analog-Fc fusion proteins to promote autophosphorylation of human insulin receptor B subtype were examined and compared in CHO-K1 cells overexpressing the human insulin receptor B subtype. 50 The activity of each fusion protein to promote the autophosphorylation of human insulin receptor B subtype was evaluated by calculating the EC 50 A lower value means a higher in vitro activity.

[0122] Experimental method: 18 h before the start of the experiment, CHO-K1-hIRB cells (Shanghai Huiyuan, CHO-K1-hIRB-M-20170706) were cultured at 10 4 The cells were seeded at 1 / well in a 96-well plate, 100 μL of growth medium (F-12K medium, 10% FBS, 1× penicillin-streptomycin containing 4 μg / mL blasticidin) was added, and the cells were cultured overnight at 37°C and 5% CO2. On the morning of the experiment, the human insulin analog-Fc fusion protein was diluted with 50 μL of starvation medium (F-12K medium, 0.1% BSA, 1× penicillin-streptomycin containing 4 μg / mL blasticidin) as necessary, and after discarding the old medium, the cells were incubated in starvation medium containing the human insulin analog-Fc fusion protein. After incubation at 37°C and 5% CO2 for 20 min, the medium was discarded, and the cells were added with the reagent kit (IR-beta The cells were lysed using the cell lysis solution in the phospho-Y1150 / 1151 reagent kit (Cisbio, lot number: 63ADK016PEG) and incubated at 37°C for 30 min. After that, 16 μL of the lysis solution was aspirated and placed in a 96-well plate, and 4 μL of the prepared antibody mixture (containing Phospho InsuLin Receptor beta Cryptate antibody and Phospho InsuLin Receptor beta d2 antibody) was added and incubated at room temperature for 4 h.

[0123] Data processing method: Signals were read using a Tecan plate reader, with an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The signal ratio (665 nm / 620 nm × 10,000) was calculated, and the signal ratio and sample concentration were nonlinearly fitted with a four-parameter equation in GraphPad Prism 6 to obtain the EC 50 Values ​​were obtained and are shown in Table 4. Human insulin, which serves as a control in this and subsequent examples, was purchased from Sigma-Aldrich.

[0124] [Table 4]

[0125] According to the results in Table 4, all of the fusion proteins disclosed herein have some activity of activating the autophosphorylation of insulin receptor B subtype, and have EC 50 The values ​​show that the activity of all the fusion proteins is significantly weaker than that of natural human insulin, and the activity of some of the fusion proteins to activate the autophosphorylation of the insulin receptor B subtype is equal to or slightly stronger than that of LY3209590. This indicates that, although all of the disclosed fusion proteins are human insulin receptor agonists, they have weaker activity, a clinically safer administration window, and can reduce side effects related to hypoglycemia and the like in patients compared to natural human insulin.

[0126] Example 4. Detection experiment of AKT phosphorylation activity in C2C12 mouse myoblast cells When insulin analogs bind to the insulin receptor, they activate a series of downstream signaling pathways, where stimulation of AKT phosphorylation is one of the main signaling pathways through which insulin and its analogs produce hypoglycemic effects. Therefore, the AKT phosphorylation stimulating activities of different human insulin analog-Fc fusion proteins were examined and compared in C2C12 mouse myoblast cells. The EC 50The activity of each fusion protein to stimulate AKT phosphorylation in mouse myoblast cells was evaluated by calculating the EC 50 A lower value means a higher in vitro activity.

[0127] Experimental method: 18 h before the start of the experiment, C2C12 cells (Procell, CL-0044) were transfected with 10 4 The cells were seeded at 100 μL / well in a 96-well plate, 100 μL of growth medium (DMEM medium, 1× penicillin-streptomycin containing 10% FBS) was added, and the cells were cultured overnight at 37°C and 5% CO2. On the morning of the day of the experiment, the human insulin analog-Fc fusion protein was diluted with 50 μL of starvation medium (DMEM medium, 1× penicillin-streptomycin without FBS) as necessary, and the old medium was discarded. After incubating the cells in the starvation medium containing the human insulin analog-Fc fusion protein for 20 min at 37°C and 5% CO2 incubator, the medium was discarded, and the cells were lysed with the cell lysis solution in the reagent kit (AKT phospho-S473 kit, Cisbio, 64AKSPEG) and incubated for 30 min at 37°C. After that, 16 μL of the lysis solution was aspirated and placed in a 96-well plate, and the prepared antibody mixture (Phospho-AKT Eu Cryptate antibody and Phospho-AKT d2 The EC 50 The values ​​were obtained and are shown in Table 5.

[0128] [Table 5]

[0129] As a result, it was shown that all of the fusion proteins disclosed herein have some degree of activity in activating the phosphorylation of the insulin receptor AKT, and 50The values ​​show that the activity of all fusion proteins is significantly weaker than that of native human insulin, and some fusion proteins have activity equivalent to that of LY3209590. This indicates that all fusion proteins have significant activity in stimulating phosphorylation of AKT in mouse myoblasts, but the activity is weaker than that of native human insulin.

[0130] Example 5. Human MCF-7 (human breast cancer cell) proliferation promotion experiment The ability of some human insulin analog-Fc fusion proteins of the present disclosure to activate downstream signaling pathways and promote cell proliferation was examined in human MCF-7 breast cancer cells. Many studies have shown that insulin and insulin analogs have a series of metabolic activities such as blood glucose reduction that they induce, as well as the activity of activating other downstream signaling pathways to promote cell proliferation. The activity of promoting cell proliferation is closely linked to the phenomenon in which insulin and its analogs induce tumor formation. Among them, the late development of B10 Asp insulin, a fast-acting insulin analog developed by Novo Nordisk, was terminated due to the observation of significant tumor formation in a rat model. Therefore, this experiment was aimed at evaluating the ability of the fusion proteins of the present disclosure to induce cell proliferation using MCF-7 human breast cancer cells, and a significant decrease in the ability to promote cell proliferation means a decrease in the risk of inducing tumor formation at a later stage. Research has shown that the ability of insulin or its analogs to promote such cell proliferation is associated with the expression of IGF-1R on the surface of some cells, and when insulin or its analogs react with IGF-1R, they promote cell proliferation, thereby inducing the potential for the development of tumors.

[0131] Experimental procedure: Before starting the experiment, MCF-7 cells (Procell, CL-147) were resuspended in DMEM medium containing 0.5% FBS, counted, and 100 μL of the cell suspension was added to each well of a 96-well clear-bottom black cell culture plate at a concentration of 5 × 10 3The cells were added at a density of 100 cells / well and cultured overnight at 37°C, 5% CO2. On the morning of the day of the experiment, the human insulin analog-Fc fusion protein was diluted with 50 μL of starvation medium (DMEM medium, containing 0.5% FBS) as necessary. After discarding the medium, the cells were incubated with the starvation medium containing the human insulin analog-Fc fusion protein and incubated at 37°C, 5% CO2 for 3 days. After the incubation was completed, CellTiter-Glo detection solution (50 μL) was added to the 96-well plate, and the plate was shaken at room temperature for 2 minutes to mix evenly. After leaving the plate in the dark for 10 minutes, the fluorescence value was read using a Tecan plate reader and the EC 50 Values ​​were calculated and are shown in Table 6.

[0132] [Table 6]

[0133] As a result, multiple parallel experiments (Table 6 shows one of them) show that fusion protein 1 all have relatively weak activity to stimulate MCF-7 cell proliferation compared to native human insulin. Unexpectedly, compared to LY3209590, fusion protein 1 has a significantly reduced activity to stimulate proliferation of MCF-7 human breast cancer cells, suggesting that the fusion protein has a potentially lower risk of inducing tumor formation. At the same time, other fusion proteins 2 to 9 of the present disclosure also show a lower cell proliferation-promoting activity against MCF-7 compared to human insulin. For example, the EC 50 , EC of LY3209590 50 This is about twice the value.

[0134] Example 6. Determination of the pharmacokinetic properties of human insulin analog-Fc fusion proteins In this example, SD rats were used as an animal model to study the pharmacokinetic properties of fusion protein 1 after a single subcutaneous or intravenous injection into normal male SD rats.

[0135] Experimental method: Male SD rats (purchased from Weitong Lihua) weighing 170-200g and aged 5-7 weeks were selected. Fusion protein 1 was prepared in 1×PBS buffer and injected into the rats via tail vein or dorsal subcutaneous injection at a dose of 30nmol / kg. After administration, the rats were not fasted. Blood was collected from the orbit at 15min, 1h, 2h, 4h, 1st, 3rd, 5th, 8th, 11th, 14th, 21st and 25th days after administration, anticoagulated with EDTA, centrifuged, and plasma was obtained for analysis of in vivo pharmacokinetic properties. The pharmacokinetic properties were analyzed by enzyme-linked immunosorbent assay, in which a capture antibody, anti-human IgG-Fc (I2136, sigma), was coated on a multi-well plate, and insulin polyclonal antibody (337E2A20, invitrogen) was added to specifically bind with the molecule to be measured, and then the concentration of the molecule to be measured in plasma was quantified by a detection antibody, anti-rabbit IgG peroxidase (A0545, sigma). The calibration curve and samples tested during the study were both prepared in 1% rat plasma (EDTA). The results are shown in Tables 7 and 8.

[0136] [Table 7] [Table 8]

[0137] As a result, fusion protein 1 had very long in vivo pharmacokinetic properties after a single intravenous / subcutaneous injection into normal rats, with a half-life of about 4.5 days, which means that the fusion protein may have a lower administration frequency and longer administration interval in clinical practice.

[0138] Example 7. Pharmacodynamic study of human insulin analog-Fc fusion protein in a rat model of type 1 diabetes In this example, streptozotocin (STZ)-induced rats were used as a type 1 diabetes animal model to investigate the hypoglycemic effect and duration of some human insulin analog-Fc fusion proteins after a single subcutaneous injection, and to comprehensively evaluate their hypoglycemic activity and in vivo pharmacokinetic properties.

[0139] Experimental method: 8-week-old male SD rats (purchased from Weitong Lihua) weighing approximately 350g were selected and subjected to adaptation breeding for one week. A citric acid / sodium citrate buffer solution with a pH range between 4.2 and 4.5 was prepared, and STZ was dissolved in the buffer solution to a concentration of 1% (w / v). STZ was then intraperitoneally injected according to the rat's actual fasting body weight, with a dose of 60mg / kg per rat. On the third day after STZ injection, the rat's fasting blood glucose was measured, and when the actual blood glucose value was higher than 15mM, the modeling was judged to be successful.

[0140] The human insulin analog-Fc fusion protein was prepared to the corresponding concentration using 1× PBS buffer, pH 7.4. Before administration, the blood glucose of each type 1 diabetic rat was measured, and rats with blood glucose levels between 20-30 mM were selected, and randomly grouped so that the average blood glucose of each group (3 or 6 rats) was similar to the standard difference. After grouping was completed, the rats in each group were subcutaneously administered the corresponding human insulin analog-Fc fusion protein based on their body weight, and the rats were allowed to eat and drink water normally after administration, and the blood glucose levels of the animals were monitored 1 h, 2 h, 4 h, 24 h, 48 h, etc. after administration until the blood glucose returned to the blood glucose level before administration, and the corresponding blood glucose level time-course graphs were prepared.

[0141] In the STZ-induced type 1 diabetes rat model, the time course of blood glucose concentration after a single subcutaneous injection of fusion protein 1 (dosage: 90 nmol / kg) is shown in Figure 1, and the time course of blood glucose concentration after LY3209590, fusion protein 2, and fusion protein 9 (dosage: 30 nmol / kg each) is shown in Figure 2. As a result, it has been shown that fusion protein 1, fusion protein 2, and fusion protein 9 all have a relatively long-lasting blood glucose lowering effect, and among them, the blood glucose lowering effect of fusion protein 1 after a single subcutaneous injection can be sustained for at least 300 hours.

[0142] Example 8. Pharmacodynamic study of human insulin analog-Fc fusion protein in the db / db type 2 diabetes mouse model In this example, a db / db type 2 diabetes mouse model was used to investigate the improvement of glycated hemoglobin after multiple subcutaneous injections of some human insulin analog-Fc fusion proteins, and their hypoglycemic activity and in vivo pharmacokinetic properties were comprehensively evaluated.

[0143] Experimental method: 50 db / db mice were purchased and adapted to the environment for 5 days, and the animals with blood glucose levels higher than 20 mmol / L were randomly selected and divided into groups for the test. The test was divided into 4 groups, namely, model control group (solvent control group, components: 0.08 M histidine, 70 g / L sucrose, 0.4 g / L polysorbate 80), fusion protein 1-low dose group, fusion protein 1-medium dose group, and fusion protein 1-high dose group, with 10 mice per group, for a total of 40 mice. The drug was administered from the first day after administration, once a week for a total of 4 times, and the administration method was subcutaneous injection. At the end point of the test, glycated hemoglobin was detected in all mice, and the detection method was to collect 0.4 mL of blood from the abdominal main vein of the animals, inject it into an EDTA anticoagulant tube, and directly detect glycated hemoglobin with the instrument. Please refer to Table 9 for the results.

[0144] [Table 9]

[0145] According to the test results, the model control group had a glycated hemoglobin content of 11.27±0.87%, while the low, medium and high dose groups of fusion protein 1 had glycated hemoglobin contents of 11.01±0.86%, 10.58±0.44% (p=0.0481) and 10.41±0.45% (p=0.0171), respectively, and the medium and high dose groups were statistically different from the model control group, and there was a certain dose-effect relationship between the three dose groups. The above results suggest that the medium and high dose groups of fusion protein 1 can significantly reduce the glycated hemoglobin content in the bodies of db / db mice, while the low dose group had no obvious effect, and there was a certain dose-effect relationship between the three dose groups.

[0146] Example 9. In vivo pharmacokinetic characterization of human insulin analog-Fc fusion protein in pigs In this example, the pharmacokinetic properties of fusion protein 1 and LY3209590 after a single subcutaneous injection in Bama pigs were studied using Bama pigs as an animal model.

[0147] Experimental method: Male bama pigs (Yibin Hengzhi Biological Technology Co., Ltd.) weighing 13-18 kg and aged 4-6 months were selected. Fusion protein 1 and LY3209590 were prepared with solvent (composition: 80 mM histidine, 70 g / L sucrose, 0.4 g / L Tween 80) and 1×PBS buffer, respectively, and administered into the animal's body by subcutaneous injection at a dose of 5 nmol / kg and 2 nmol / kg, respectively. Approximately 1 mL of venous blood was collected at 1 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h after administration, and was not anticoagulated. The whole blood samples were temporarily placed in an ice box before centrifugation, and centrifuged at 2-8°C for 10 min at a centrifugal force of about 1800×g, and the serum was separated into two tubes (one of which was about 100 μL, and the remaining serum was stored in the other tube), and stored at -66°C or lower for analysis of in vivo pharmacokinetic properties. The analysis of pharmacokinetic properties was performed in a similar manner to Example 6, and the specific pharmacokinetic parameters are shown in Table 10.

[0148] [Table 10]

[0149] The results show that fusion protein 1 has a very long in vivo pharmacokinetic profile after a single subcutaneous injection into pigs. At the doses in Table 10, the drug half-life (t1 / 2) does not change significantly with dose. Compared with LY3209590, fusion protein 1 has a significantly longer half-life of about 120 h, suggesting that the fusion protein has a lower dosing frequency and a longer dosing interval in clinical practice.

[0150] The present disclosure provides a novel fusion protein of human insulin analog and IgG Fc region that can be used to treat metabolic diseases such as diabetes mellitus type 1 or type 2. The fusion protein has a very long in vivo pharmacokinetic profile, shows good and sustained hypoglycemic effect in STZ-induced type 1 diabetes rat model and db / db type 2 diabetes mouse model, has excellent physical / chemical stability and relatively high expression yield, and has significantly lower in vitro cell proliferation-inducing activity than native human insulin, suggesting that it can potentially meet the clinical demand for ultra-long-acting insulin products with good safety and relatively low dosage risk.

Claims

1. It is a fusion protein, From the N-terminus to the C-terminus, B １ -L １ -A １ It contains an insulin analog having the general formula, Eventually, B １ It is an insulin B chain analog, FVX １ QHLCGX ２ HLVEALX ３ X ４ VCGERGFX ５ comprises the amino acid sequence called Y (SEQ ID NO:1), among which, X １ is selected from N, G, or K, X ２ It is selected from S or E. X ３ is selected from Y, H, or E, X ４ is selected from H, R, L, or E. X ５ is selected from F or H, A １ It is an insulin A chain analog, GIVEQCCZ １ SICSLZ ２ QLENYCZ ３ It contains the amino acid sequence (SEQ ID NO: 2), of which, Z １ is selected from T, H, or E, Z ２ is selected from Y, D, S, or E, Z ３ is selected from G or N, L １ This is a linker, preferably a GS linker and / or a linker having at least five Gs, and more preferably a linker of the amino acid sequence shown in SEQ ID NO: 3 or 52. Fusion protein.

2. X １ is selected from N or K, X ２ is selected from S or E, X ３ is selected from Y or H, X ４ L is selected from X ５ He was selected from H. Z １ is selected from T, H, or E, Z ２ It was selected from E, Z ３ It is selected from G or N. The fusion protein according to claim 1.

3. The insulin B chain analog comprises or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 31 to 36. The insulin A chain analog comprises or is selected from any one of the amino acid sequences shown in SEQ ID NOs: 37 to 42. Preferably, the fusion protein contains the amino acid sequence shown in any one of SEQ ID NOs: 4 to 9. The fusion protein according to claim 1 or 2.

4. C １ Further including the C １ This is at least one selected from the Fc region of immunoglobulin, HSA, and HSA-binding domain. Preferably, the C １ It is selected from the Fc region of IgG, More preferably, C １ It is selected from the Fc region of human IgG1, IgG2, and IgG4. The fusion protein according to claim 1.

5. Said C １ The insulin analog is located at the N-terminus or C-terminus of the insulin analog, preferably at the C-terminus of the insulin analog. The fusion protein according to claim 4.

6. Said C １ This includes, or is, an amino acid sequence that is at least 90% identical to, the amino acid sequence shown in any one of sequence numbers 17, 18, 48-51. The fusion protein according to claim 4.

7. Linker L ２ It further includes C １ is L ２ via B １ -L １ -A １ It forms a fusion protein with, Preferably, the L ２ is, (G ｍ Q ｉ ) ｎ , (G ｍ A ｉ ) ｎ , (G ｍ Q) ｎ , (G ｍ A) ｎ (PGPQ) ｓ It is one of the following, and of which, m is independently chosen from integers between 1 and 10. n is independently chosen from integers between 1 and 10. Each i is independently selected from 0 to 4. s is selected from integers between 1 and 10. more preferably, L ２ It contains, or is, the amino acid sequence shown in any one of sequence numbers 10 to 16. The fusion protein according to claim 4.

8. It contains, or is, an amino acid sequence that has at least 90% sequence identity with any one of sequence numbers 19 to 27. The fusion protein according to claim 4.

9. It is a dimer, preferably a homodimer. The fusion protein according to claim 1.

10. The aforementioned insulin analog is an insulin receptor agonist having insulin receptor agonist activity. The fusion protein according to claim 1.

11. An insulin analog comprising an insulin B chain analog and an insulin A chain analog, Insulin B chain analogs are FVX １ QHLCGX ２ HLVEALX ３ X ４ VCGERGFX ５ It contains the amino acid sequence Y (Sequence ID 1), of which X １ is selected from N, G, or K, and X ２ is selected from S or E, X ３ is selected from Y, H, or E, X ４ is selected from H, R, L, or E, X ５ is selected from F or H, Insulin A chain analogs are GIVEQCCZ １ SICSLZ ２ QLENYCZ ３ It contains the amino acid sequence (SEQ ID NO: 2), of which Z １ is selected from T, H, or E, Z ２ is selected from Y, D, S, or E, Z ３ is selected from G or N, Preferably, X １ is selected from N or K, X ２ is selected from S or E, X ３ is selected from Y or H, X ４ L is selected from X ５ Selected from H, Z １ is selected from T, H, or E, Z ２ It was selected from E, Z ３ It is selected from G or N. Insulin analog.

12. The insulin B chain analog contains or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 31-36. Insulin A chain analogs contain, or are selected from, the amino acid sequence shown in any one of SEQ ID NOs: 37-42. Preferably, Insulin B chain analogs and insulin A chain analogs are, respectively, The amino acid sequences shown in Sequence IDs 31 and 37, The amino acid sequences shown in Sequence IDs 32 and 38, The amino acid sequences shown in Sequence IDs 33 and 39, The amino acid sequences shown in Sequence IDs 34 and 40, and The amino acid sequences shown in SEQ ID NOs: 36 and 42 It includes any one of the groups, or is selected from them. The insulin analog according to claim 11.

13. A disulfide bond is formed between the insulin A chain analog and the insulin B chain analog. The insulin analog according to claim 11 or 12.

14. The insulin analog described in claim 11, Glucose-responsive insulin.

15. Encoding the fusion protein of claim 1, or the insulin analog of claim 11, or the glucose-responsive insulin of claim 14, Polynucleotide.

16. A polynucleotide comprising the polynucleotide described in claim 15, host cell.

17. A method for preparing a fusion protein or insulin analog, Culture a host cell containing a polynucleotide encoding the fusion protein described in claim 1, or the insulin analog described in claim 11, To recover the aforementioned fusion protein or the aforementioned insulin analog, This includes optionally purifying and / or modifying the fusion protein, insulin analog, method.

18. A fusion protein according to claim 1, an insulin analog according to claim 11, or a glucose-responsive insulin according to claim 14, and one or more pharmaceutically acceptable carriers, diluents, or excipients. Pharmaceutical composition.

19. A pharmaceutical composition comprising the fusion protein according to claim 1, the insulin analog according to claim 11, or the glucose-responsive insulin according to claim 14, for treating diabetes and its complications or for lowering blood glucose, Preferably, the diabetes and its complications are selected from type 1 diabetes, type 2 diabetes and its complications. More preferably, the complications are selected from diabetes-related heart disease, stroke, retinopathy, neuropathy, or kidney disease. Pharmaceutical composition.

Images