Fusion proteins and medical uses thereof

Abstract

Fusion proteins and medical uses thereof are provided. In particular, a fusion protein comprising insulin or an analog thereof in combination with a serum albumin binding domain, and medical uses thereof for the treatment of diseases are provided.

Description

Fusion protein and its medical use

[0001] This disclosure claims priority to Chinese patent application 2023114825456 filed on November 8, 2023. Technical Field

[0002] The present disclosure relates to the field of biomedicine, and specifically to a class of human insulin or its analogs and fusion proteins thereof with a serum albumin binding domain, as well as the use of such fusion protein molecules in treating metabolic diseases such as type I diabetes or type II diabetes. Background Art

[0003] Diabetes is a chronic metabolic disease characterized by dysregulation of glucose, protein, and lipid metabolism in the human body due to insufficient insulin secretion. It is primarily divided into insulin-dependent diabetes mellitus (Type I diabetes) and non-insulin-dependent diabetes mellitus (Type II diabetes). Globally, 90-95% of diabetic patients suffer from Type II diabetes, which is caused by impaired pancreatic beta cell function and long-term insulin resistance. Its most prominent characteristics are a lack of insulin levels, decreased insulin sensitivity, and high plasma glucose concentrations. Studies have shown that Type II diabetes is associated with a variety of high-risk complications, leading to cardiovascular disease, kidney failure, blindness, amputation, and numerous other complications.

[0004] Approved diabetes medications primarily include chemically synthesized small-molecule oral glucose-lowering drugs, such as biguanides, sulfonyl agonists, insulin sensitizers, α-glycosides, and injectable peptide glucose-lowering drugs like glucagon-like peptide-1 (GLP-1) analogs. For all patients with type 1 diabetes and some patients with advanced type 2 diabetes, only insulin injections can effectively control blood sugar levels.

[0005] Currently, approved insulin analogs are primarily categorized into two main categories: rapid-acting insulin and long-acting basal insulin. Approved rapid-acting insulins include insulin lispro and insulin aspart. Rapid-acting insulins are primarily administered subcutaneously before meals. By modifying the insulin molecule's structure, they inhibit the formation of native insulin hexamers, resulting in rapid onset and clearance after subcutaneous administration. Insulin lispro inhibits hexamer formation by alternating amino acids at positions 28 and 29 at the end of native insulin's B-chain, while insulin aspart replaces proline with aspartic acid at position 28 of the insulin B-chain, inhibiting hexamer formation through charge repulsion. Approved long-acting basal insulins include insulin glargine, insulin detemir, and insulin degludec. Insulin glargine introduces a positively charged arginine amino acid into the insulin backbone, raising its isoelectric point to neutral. This allows the insulin analog to precipitate at the site of administration after subcutaneous injection, resulting in significantly prolonged in vivo pharmacokinetic properties after sustained-release dissolution. Insulin detemir and insulin degludec are modified by introducing a long-chain fatty acid into the terminal lysine side chain of the B-chain, forming a supramolecular polymer at the administration site, and reversibly binding to human serum albumin in plasma to prolong their in vivo pharmacokinetic properties. Currently, insulin glargine and insulin degludec are administered once daily, while insulin detemir is administered once to twice daily.

[0006] However, rapid-acting insulin products may cause adverse reactions such as hypoglycemia and severe hypoglycemic symptoms, and patients need to inject insulin once or even multiple times daily, which causes inconvenience, a heavy treatment burden, and a reduced quality of life. Existing long-acting basal insulin products also require subcutaneous injection once a day, and the injection frequency is still high. Therefore, the development of ultra-long-acting basal insulin products to further reduce the injection frequency, meet the needs of clinical patients, and improve patient compliance and convenience is an important issue that needs to be addressed in this field.

[0007] Summary of the Invention

[0008] The present disclosure provides a fusion protein of human insulin or an analog thereof and a serum albumin binding domain, a preparation method thereof, and use of the fusion protein in treating metabolic diseases (such as diabetes, obesity, etc.).

[0009] serum albumin binding molecules

[0010] The present disclosure provides serum albumin binding molecules.

[0011] In some embodiments, the serum albumin binding molecule comprises an immunoglobulin single variable domain comprising the CDR1, CDR2, and CDR3 of any one of (1) to (3):

[0012] (1) CDR1, CDR2, and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 1, 8-17

[0013] (2) CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 2-4, respectively;

[0014] (3) Any one, two or three CDRs among CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 2-4, respectively.

[0015] The above CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, for example, the CDRs are defined according to the Kabat numbering system.

[0016] In some embodiments, the serum albumin binding molecule comprises an immunoglobulin single variable domain comprising a CDR1 having 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned CDR1s; and / or, the immunoglobulin single variable domain comprises a CDR2 having 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned CDR2s; and / or, the immunoglobulin single variable domain comprises a CDR3 having 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned CDR3s. In some specific embodiments, the amino acid mutations in CDR1, CDR2, and / or CDR3 are conservative substitutions, replacements, or modifications, and / or deletions and / or additions that do not affect function.

[0017] In some embodiments, the serum albumin binding molecule comprises an immunoglobulin single variable domain comprising any one or a combination of any several (eg, 2 or 3) of the aforementioned CDR1, CDR2, and CDR3.

[0018] In some embodiments, the aforementioned immunoglobulin single variable domain is of camelid origin.

[0019] In some embodiments, the aforementioned immunoglobulin single variable domain is modified by any of the following: humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerization, or a combination thereof.

[0020] In some embodiments, the framework regions in the aforementioned humanized immunoglobulin single variable domains are derived from the human heavy chain variable region germline genes IGHV3-23 or IGVH3-66.

[0021] In some specific embodiments, the aforementioned humanized immunoglobulin single variable domain comprises an amino acid mutation at at least one of the following positions: positions 20, 23, 27, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83, and 84 (positions based on the EU numbering system). It should be emphasized that although the present disclosure limits amino acid mutations at specific positions, it should not be understood as: when determining whether an amino acid sequence falls within the scope of a claim, whether the amino acid residue at the corresponding position is mutated. In any case, the amino acid residue before the mutation does not limit the scope of protection.

[0022] In some specific embodiments, in the aforementioned humanized immunoglobulin single variable domain, the amino acid residue at a position selected from the group consisting of: positions 20, 23, 27, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83, and 84 (positions based on the EU numbering system) is a mutated amino acid residue;

[0023] As an example, the mutation comprises at least one amino acid mutation selected from the group consisting of: 20V, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 74A, 78V, 83Q or 84P (numbers indicate positions, and capital letters indicate the amino acid residue at the position after mutation);

[0024] As an example, the mutation comprises a combination of amino acid mutations selected from any one of the following:

[0025] 27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0026] 20V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0027] 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0028] 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 74A;

[0029] 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 78V;

[0030] 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A;

[0031] 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 83Q, 84P;

[0032] 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 84P;

[0033] 30L, 37F, 44E, 45R, 47G, 84P; or

[0034] 30L, 37F, 44E, 45R, 47G.

[0035] As an example, the mutation comprises a combination of amino acid mutations selected from any one of the following:

[0036] F27N, F29Y, S30L, V37F, G44E, L45R, W47G;

[0037] L20V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G;

[0038] A23V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G;

[0039] F27N, F29Y, S30L, V37F, G44E, L45R, W47G, S74A;

[0040] F27N, F29Y, S30L, V37F, G44E, L45R, W47G, L78V;

[0041] F27N, F29Y, S30L, V37F, G44E, L45R, W47G, S49A;

[0042] F27N, F29Y, S30L, V37F, G44E, L45R, W47G, R83Q, A84P;

[0043] F27N, F29Y, S30L, V37F, G44E, L45R, W47G, A84P;

[0044] S30L, V37F, G44E, L45R, W47G, A84P; or

[0045] S30L, V37F, G44E, L45R, W47G.

[0046] In some embodiments, the serum albumin binding molecules provided by the present disclosure comprise an immunoglobulin single variable domain comprising or being any one of SEQ ID NOs: 1, 8-17, or an amino acid sequence having at least 80%, at least 90% sequence identity thereto.

[0047] In the present disclosure, "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity; "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity, and ranges between any two of the foregoing values, including integers and decimals.

[0048] In some embodiments, the immunoglobulin single variable domain in the serum albumin binding molecules provided herein comprises three complementarity determining regions CDR1, CDR2 and CDR3 and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0049] In some embodiments, the serum albumin binding molecules provided by the present disclosure are antibodies or antigen-binding fragments thereof that bind to serum albumin, or conjugates or fusion proteins comprising the antibodies or antigen-binding fragments thereof.

[0050] In some embodiments, the aforementioned antibody is a camel antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0051] In some embodiments, the aforementioned antigen-binding fragment is an sdAb, a bispecific antibody, or a multispecific antibody.

[0052] In some embodiments, the aforementioned immunoglobulin single variable domain is a VHH.

[0053] In some embodiments, the aforementioned immunoglobulin single variable domain is a humanized VHH.

[0054] In some embodiments, the aforementioned immunoglobulin single variable domain is an affinity-matured VHH.

[0055] In some embodiments, a serum albumin binding molecule is provided, which comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) the aforementioned immunoglobulin single variable domains, wherein the immunoglobulin single variable domains can be the same or different, and any two immunoglobulin single variable domains can be directly connected or connected through a linker.

[0056] In some embodiments, serum albumin binding molecules are provided that bind to or compete for binding to the same epitope as the aforementioned immunoglobulin single variable domains of the present disclosure.

[0057] In some embodiments, serum albumin binding molecules are provided that block the binding of the aforementioned immunoglobulin single variable domains of the present disclosure to serum albumin.

[0058] In some embodiments, serum albumin binding molecules are provided, the binding of which to serum albumin is blocked by the immunoglobulin single variable domains disclosed above.

[0059] In some embodiments, the serum albumin binding molecules provided by the present disclosure further comprise an immunoglobulin Fc region, such as an Fc region selected from human IgG1, IgG2, IgG3, and IgG4; or a histidine tag, such as a (His)6 tag or a (His)8 tag.

[0060] The Fc region useful in the present disclosure can be derived from immunoglobulins of different subtypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM. In some embodiments, the Fc region includes the hinge region or a portion of the hinge region, the CH2 region, and the CH3 region of the constant region. The Fc region may or may not contain mutations.

[0061] In some embodiments, the aforementioned immunoglobulin single variable domain and the Fc region or histidine tag may be connected by a linker. The linker may be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure. For example, the linker is a flexible linker, such as G4S (SEQ ID NO: 122), GS, GAP, (G4S) 2( SEQ ID NO: 123), (G4S) 3( SEQ ID NO: 124), (G4S) 4( SEQ ID NO: 125), (G4S) 5(SEQ ID NO: 126), ASGS (SEQ ID NO: 127), G3S (SEQ ID NO: 128), or a combination thereof. In some embodiments, the linker is GGGGSGGGS (SEQ ID NO: 129).

[0062] In some embodiments, the serum albumin binding molecules provided by the present disclosure comprise or are set forth in any one of SEQ ID NOs: 6, 18-19, or an amino acid sequence having at least 80%, at least 90% sequence identity thereto.

[0063] In some embodiments, the immunoglobulin single variable domain in the aforementioned serum albumin binding molecule specifically binds to serum albumin, which can be rodent serum albumin (e.g., mouse serum albumin (mSA)), or primate serum albumin (e.g., macaque serum albumin (cySA), human serum albumin (HSA)). In some specific embodiments, the serum albumin is HSA.

[0064] In some alternatives, the serum albumin binding molecules of the present disclosure may comprise any of the aforementioned complete immunoglobulin single variable domains; or may comprise a functional portion of any of the aforementioned immunoglobulin single variable domains or variants thereof, such as CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0065] In some embodiments, the variant of the functional portion of the immunoglobulin single variable domain may be a polypeptide that retains the serum albumin binding function of CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, or FR1-CDR1-FR2-CDR2-FR3-CDR3 and has at least 80% or at least 90% sequence homology therewith. For example, it may be a polypeptide that retains the serum albumin binding function of CDR3 and has a certain sequence homology therewith, for example, a polypeptide that has at least 80% or at least 90% sequence homology with any of the above-mentioned CDR3s.

[0066] In some embodiments, in the serum albumin binding molecules provided by the present disclosure, the immunoglobulin single variable domain can be covalently or non-covalently linked to any other macromolecular or small molecule compound as a carrier for extending half-life.

[0067] In some embodiments, the serum albumin binding molecules of the present disclosure comprise one or more therapeutic or diagnostic agents, wherein the therapeutic or diagnostic agent is covalently or non-covalently linked to the immunoglobulin single variable domain.

[0068] In some embodiments, the therapeutic or diagnostic agent is selected from the group consisting of a therapeutic or diagnostic protein, a nucleic acid, and a small molecule compound.

[0069] In some embodiments, the serum albumin binding molecules provided herein or the immunoglobulin single variable domains therein are present in an amount of ≤1×10 -7 M, for example, ≤2×10 -8 M, ≤1×10 -8 M, ≤9×10 -9 M, ≤8×10 -9 M, ≤7×10 -9 M, ≤6×10 -9 M, ≤5×10 -9 M, ≤4×10 -9 M, ≤3×10 -9 M, ≤2×10 -9 M, or ≤1×10 -9 M, or ≤9×10 -10 M, ≤8×10 -10 M, ≤7×10 -10 M, ≤6×10 -10 M, ≤5×10 -10 M, ≤4×10 -10 M, ≤3×10 -10 M, ≤2×10 -10 M, ≤1×10 -10 M's K D The value binds to serum albumin, such as human serum albumin.

[0070] In some embodiments, the affinity K of serum albumin binding molecules provided by the present disclosure for human serum albumin is D The value is 10nM-100nM, for example 10nM-50nM. D The method for detecting the value is commonly used in the art, such as that provided in Example 1 of the present disclosure.

[0071] In some embodiments, the serum albumin binding molecules provided herein have at least one activity selected from the group consisting of:

[0072] (a) ≤1×10 -7 M, for example, ≤1×10 -8 M's K D Value combined with HSA;

[0073] (b) does not detectably block the binding of serum albumin, such as HSA, to FcRn, such as human FcRn, in vivo and / or in vitro;

[0074] (c) when comprising a therapeutic or diagnostic agent, extending the plasma half-life of the therapeutic or diagnostic agent.

[0075] In some embodiments, the serum albumin binding molecules provided by the present disclosure retain the favorable properties of single domain antibodies (e.g., VHHs) and have an extended lifespan in the individual's circulation. Thus, such molecules can circulate in the serum of a subject for several days, thereby reducing the frequency of treatment, the inconvenience to the subject, and the cost of treatment.

[0076] In some embodiments, the half-life of the serum albumin binding molecules of the present disclosure can be controlled by the number of single variable domains that specifically bind to serum proteins present in the molecule.

[0077] In some embodiments, the serum albumin binding molecules of the present disclosure promote the binding of serum albumin to FcRn in vivo and / or in vitro.

[0078] Insulin or its analogs

[0079] The present disclosure provides insulin or its analog.In some embodiments, insulin or its analog is selected from natural insulin, for example, human insulin.In some embodiments, insulin or its analog is selected from insulin analog.

[0080] In some embodiments, insulin or its analogs comprise a B chain and an A chain; wherein the A chain comprises the following amino acid sequence:

[0081] FVX1QHLCGX2HLVEALX3X4VCGEX5GFX6X7X8X9X 10 T (SEQ ID NO: 120), wherein X1 is selected from N or S; X2 is selected from S, E or R; X3 is selected from Y, H, E or R; X4 is selected from L, H, R, E or K; X5 is selected from R or K; X6 is selected from F or H; X7 is selected from Y, G, T, S, H, V or is absent; X8 is selected from G, E, P, K, D, S, T, H or is absent; X9 is selected from G, E, K, P, Q, D, H or is absent; X 10 Selected from G, T, S, E, K, A or absent;

[0082] The insulin A chain analog contains the following amino acid sequence:

[0083] GIVEQCCZ1Z2Z3CSLZ4QLENYCZ5Z6( SEQ ID NO:121), wherein Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I, K or T, Z4 is selected from Y, E, S, K or D, and Z5 is selected from G or N; Z6 is any natural amino acid or does not exist. When Z5 is N, Z6 is not G or N.

[0084] In some embodiments, X1 is selected from N or S. In some embodiments, X1 is N. In some embodiments, X1 is S.

[0085] In some embodiments, X2 is selected from S, E, or R. In some embodiments, X2 is S. In some embodiments, X2 is E. In some embodiments, X2 is R.

[0086] In some embodiments, X3 is selected from Y, H, E, or R. In some embodiments, X3 is selected from Y, H, or E. In some embodiments, X3 is Y. In some embodiments, X3 is H. In some embodiments, X3 is E.

[0087] In some embodiments, X4 is selected from L, H, R, E, or K. In some embodiments, X4 is L. In some embodiments, X4 is H. In some embodiments, X4 is R. In some embodiments, X4 is E. In some embodiments, X4 is K.

[0088] In some embodiments, X5 is selected from R or K. In some embodiments, X5 is R. In some embodiments, X5 is K.

[0089] In some embodiments, X6 is selected from F or H. In some embodiments, X6 is F. In some embodiments, X6 is H.

[0090] In some embodiments, X7 is selected from Y, G, T, S, H, V or is absent. In some embodiments, X7 is Y. In some embodiments, X7 is absent.

[0091] In some embodiments, X8 is selected from G, E, P, K, D, S, T, H, or is absent. In some embodiments, X8 is T. In some embodiments, X8 is absent.

[0092] In some embodiments, X9 is selected from G, E, K, P, Q, D, H or is absent. In some embodiments, X9 is P. In some embodiments, X9 is absent.

[0093] In some embodiments, X 10 is selected from G, T, S, E, K, A or is absent. In some embodiments, X 10 is K. In some embodiments, X10 Does not exist.

[0094] In some embodiments, Z1 is selected from T, H, or E. In some embodiments, Z1 is T. In some embodiments, Z1 is H. In some embodiments, Z1 is E.

[0095] In some embodiments, Z2 is selected from S or K. In some embodiments, Z2 is S. In some embodiments, Z2 is K.

[0096] In some embodiments, Z3 is selected from I, K, or T. In some embodiments, Z3 is selected from I or K. In some embodiments, Z3 is I. In some embodiments, Z3 is K. In some embodiments, Z3 is T.

[0097] In some embodiments, Z4 is selected from Y, E, S, K, or D. In some embodiments, Z4 is selected from Y, E, S, or K. In some embodiments, Z4 is Y. In some embodiments, Z4 is E. In some embodiments, Z4 is S. In some embodiments, Z4 is K. In some embodiments, Z4 is D.

[0098] In some embodiments, Z5 is selected from G or N. In some embodiments, Z5 is G. In some embodiments, Z5 is N.

[0099] In some embodiments, Z6 is any natural amino acid or is absent, and when Z5 is N, Z6 is not G or N. In some embodiments, Z6 is absent.

[0100] The present disclosure provides insulin or an analog thereof, which comprises a B chain and an A chain, wherein the B chain comprises the following amino acid sequence:

[0101] FVX1QHLCGX2HLVEALX3X4VCGEX5GFX6YTPKT (SEQ ID NO: 21), wherein X1 is selected from N or S, X2 is selected from S, E or R, X3 is selected from Y, H, E or R, X4 is selected from L, H, R, E or K, X5 is selected from R or K, and X6 is selected from F or H;

[0102] The A chain contains the following amino acid sequence:

[0103] GIVEQCCZ1Z2Z3CSLZ4QLENYCZ5 (SEQ ID NO: 22), wherein Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I, K or T, Z4 is selected from Y, E, S, K or D, and Z5 is selected from N or G.

[0104] In some embodiments, the present disclosure provides insulin or an analog thereof comprising a B chain and an A chain, wherein the B chain comprises the amino acid sequence shown in SEQ ID NO: 21, X1 is N, X2 is selected from S, E or R, X3 is selected from Y, H or E, X4 is selected from L, H, R, E or K, X5 is selected from R or K, and X6 is selected from H.

[0105] The A chain comprises the amino acid sequence shown in SEQ ID NO: 22, Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I or K, Z4 is selected from Y, E, S or K, and Z5 is G.

[0106] In some embodiments, the B chain comprises (or is) an amino acid sequence as shown in SEQ ID NO: 21, wherein X1, X2, X3, X4, X5, and X6 are selected from any one of the following:

[0107] (a1) X1 is N, X2 is selected from S, E or R, X3 is selected from Y, H or E, X4 is selected from L, H, R, E or K, X5 is selected from R or K, and X6 is selected from F or H;

[0108] (a2) X1 is N, X2 is selected from S, E or R, X3 is selected from Y, H or E, X4 is selected from L, H, R, E or K, X5 is R, and X6 is H;

[0109] (a3) X1 is N, X2 is S, X3 is selected from Y, H or E, X4 is selected from L, H, R or E, X5 is R, and X6 is H;

[0110] (a4) X1 is N, X2 is S, X3 is selected from Y or H, X4 is selected from L, H or E, X5 is R, and X6 is H;

[0111] (a5) X1 is N, X2 is E, X3 is selected from Y or E, X4 is selected from L, H, E or K, X5 is R; X6 is selected from F or H, preferably H;

[0112] (a6) X1 is N, X2 is E, X3 is selected from Y or E, X4 is selected from L, H, E or K, X5 is K, and X6 is H;

[0113] (a7) X1 is selected from N, X2 is R, X3 is selected from Y, H or E, X4 is selected from L, H, R or E, X5 is R, and X6 is H;

[0114] (a8) X1 is S, X2 is S, X3 is R, X4 is selected from L, H, R or E, X5 is R, and X6 is H.

[0115] In some embodiments, the B chain comprises (or is) an amino acid sequence as shown in SEQ ID NO: 21, wherein X1, X2, X3, X4, X5, and X6 are selected from any one of the following:

[0116] (b1) X1 is selected from N or S, X2 is selected from S, E or R, X3X4 is selected from HL, YH, YL, YR, EL, YE, YK or RL, X5 is selected from R or K, and X6 is selected from F or H;

[0117] (b2) X1 is N, X2 is S, X3 and X4 are selected from HL, YH, YL, YR, YE or EL, X5 is selected from R, and X6 is H;

[0118] (b3) X1 is N, X2 is E, X3X4 is selected from YE, YH, YL, EL or YK, X5 is selected from R, and X6 is H;

[0119] (b4) X1 is N, X2 is E, X3X4 is YL, X5 is selected from K or R, and X6 is H;

[0120] (b5) X1 is N, X2 is E, X3X4 is YL, X5 is selected from K or R, and X6 is F;

[0121] (b6) X1 is N, X2 is R, X3 and X4 are YH, X5 is R, and X6 is H;

[0122] (b7) X1 is S, X2 is S, X3 and X4 are RL, X5 is R, and X6 is H.

[0123] (b8) X1 is N, X2 is S, X3 and X4 are selected from HL, YH or YE, X5 is R, and X6 is H.

[0124] In some embodiments, the A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22, wherein Z1, Z2, Z3, Z4, and Z5 are selected from any one of the following:

[0125] (c1) Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I, K or T, Z4 is selected from Y, E, S, K or D, and Z5 is G;

[0126] (c2) Z1 is T, Z2 is S, Z3 is selected from I or T, Z4 is selected from E, S or D, and Z5 is G;

[0127] (c3) Z1 is H, Z2 is S, Z3 is I, Z4 is selected from E, S or Y, and Z5 is G;

[0128] (c4) Z1 is E, Z2 is selected from S or K, Z3 is selected from I or K, Z4 is selected from E, Y or K, and Z5 is G.

[0129] In some embodiments, the A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22, wherein Z1, Z2, Z3, Z4, and Z5 are selected from any one of the following:

[0130] (d1) Z1 is T, Z2Z3 is SI, Z4 is selected from E, S or D, and Z5 is G;

[0131] (d2) Z1 is T, Z2 and Z3 are ST, Z4 is selected from E, S or D, and Z5 is G;

[0132] (d3) Z1 is H, Z2Z3 is SI, Z4 is selected from E, S or Y, and Z5 is G;

[0133] (d4) Z1 is E, Z2Z3 is SI, Z4 is selected from E, Y or K, and Z5 is G;

[0134] (d5) Z1 is E, Z2 and Z3 are selected from KI or SK, Z4 is E, and Z5 is G.

[0135] In some embodiments, the B chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 21, and the A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22; wherein:

[0136] 1) X1 is N, X2 is S, X3 is H, Y or E, X4 is L, H or R, X5 is R, X6 is H; Z1 is T, Z2 is S, Z3 is I or T, Z4 is E, S or D, and Z5 is G; or,

[0137] 2) X1 is N, X2 is S, X3 is Y, X4 is H, L or R, X5 is R, X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is S, E or Y, Z5 is G; or,

[0138] 3) X1 is N, X2 is S, X3 is Y or E, X4 is R, H, E or L, X5 is R, X6 is H; Z1 is E, Z2 is S or K, Z3 is I, Z4 is E, Y or K, Z5 is G; or,

[0139] 4) X1 is N, X2 is S, X3 is Y, X4 is H or R, X5 is R, X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is S, Z5 is G; or,

[0140] 5) X1 is N, X2 is E, X3 is Y or E, X4 is E, H, K or L, X5 is R or K, X6 is H or F; Z1 is E, Z2 is S, Z3 is I or K, Z4 is E or K, Z5 is G; or,

[0141] 6) X1 is N, X2 is R, X3 is Y, X4 is H, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is E, Z5 is G; or,

[0142] 7) X1 is S, X2 is S, X3 is R, X4 is L, X5 is R, X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is Y, and Z5 is G.

[0143] In some embodiments, the B chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 21, and the A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22, wherein:

[0144] (1) X1 is N, X2 is S, X3 and X4 are HL, YH or YR, X5 is R, and X6 is H; Z1 is T, Z2 is S, Z3 is I, Z4 is E, and Z5 is G; or

[0145] (2) X1 is N, X2 is S, X3X4 is YH or YR, X5 is R, X6 is H; Z1 is T, Z2 is S, Z3 is I, Z4 is S, Z5 is G; or (3) X1 is N, X2 is S, X3X4 is EL, X5 is R, X6 is H; Z1 is T, Z2 is S, Z3 is T, Z4 is D, Z5 is G; or

[0146] (4) X1 is N, X2 is S, X3 and X4 are YH, YL or YR, X5 is R, and X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is E, and Z5 is G; or

[0147] (5) X1 is N, X2 is S, X3 and X4 are YH, YL or YR, X5 is R, and X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is Y, and Z5 is G; or

[0148] (6) X1 is N, X2 is S, X3 and X4 are YR, YH or YE, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is E, and Z5 is G; or

[0149] (7) X1 is N, X2 is S, X3 and X4 are YH or YE, X5 is R, X6 is H; Z1 is E, Z2 is K, Z3 is I, Z4 is E, and Z5 is G; or

[0150] (8) X1 is N, X2 is S, X3 and X4 are YH or EL, X5 is R, and X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is Y or K, and Z5 is G; or

[0151] (9) X1 is N, X2 is S, X3 and X4 are YH or YR, X5 is R, and X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is S, and Z5 is G; or

[0152] (10) X1 is N, X2 is E, X3 and X4 are YE, YH or YK, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is E, and Z5 is G; or

[0153] (11) X1 is N, X2 is E, X3 and X4 are YL, X5 is R or K, X6 is H or F; Z1 is E, Z2 is S, Z3 is I, Z4 is E, and Z5 is G; or

[0154] (12) X1 is N, X2 is E, X3 and X4 are YL, X5 is R, and X6 is H; Z1 is E, Z2 is S, Z3 is I or K, Z4 is K or E, and Z5 is G; or

[0155] (13) X1 is N, X2 is E, X3X4 is EL, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is K, Z5 is G.

[0156] In some embodiments, the present disclosure provides insulin analogs comprising a B chain and an A chain;

[0157] The B chain comprises (or is) the amino acid sequence of SEQ ID NO: 21, wherein: X1 is selected from N or S, X2 is selected from S, E or R, X3 is selected from Y, H, E or R, X4 is selected from L, H, R, E or K, X5 is selected from R or K, and X6 is selected from F or H;

[0158] The A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22, wherein: Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I, K or T, Z4 is selected from Y, E, S, K or D, and Z5 is G.

[0159] In some embodiments, the present disclosure provides insulin analogs comprising a B chain and an A chain;

[0160] The B chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 21, wherein: X1 is N, X2 is selected from S, E or R, X3 is selected from Y, H or E, X4 is selected from L, H, R, E or K, X5 is selected from R or K, and X6 is selected from F or H;

[0161] The A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22, wherein: Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I or K, Z4 is selected from Y, E, S or K, and Z5 is G.

[0162] In some embodiments, the present disclosure provides an insulin analog comprising a B chain and an A chain; wherein:

[0163] X1 is N, X2 is S, X3 is selected from Y, H or E, X4 is selected from E, L, H or R, X5 is R, X6 is H; Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is I, Z4 is Y, E, S or K, and Z5 is G;

[0164] X1 is N, X2 is E, X3 is selected from Y or E, X4 is selected from E, L, H or K, X5 is R or K, X6 is H or F; Z1 is E, Z2 is S, Z3 is selected from I or K, Z4 is E or K, and Z5 is G; or

[0165] X1 is N, X2 is R, X3 is selected from Y, X4 is selected from H, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is E, and Z5 is G.

[0166] In some embodiments, the B chain comprises the amino acid sequence of any one of SEQ ID NOs: 23-37, or a sequence at least 80% or at least 90% identical thereto.

[0167] The amino acid sequence is shown below:

[0168] In some embodiments, the A chain comprises the amino acid sequence of any one of SEQ ID NOs: 38-48, or a sequence at least 80% or at least 90% identical thereto.

[0169] The amino acid sequence is shown below:

[0170] The present disclosure provides insulin or an analog thereof, which contains a B chain and an A chain, wherein the B chain and the A chain comprise or are selected from any one of the following groups:

[0171] The amino acid sequences shown in SEQ ID NO: 23 and SEQ ID NO: 38,

[0172] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 39,

[0173] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 40,

[0174] The amino acid sequences shown in SEQ ID NO: 25 and SEQ ID NO: 40,

[0175] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 41,

[0176] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 38,

[0177] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 42,

[0178] The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 41,

[0179] The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 42,

[0180] The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 43,

[0181] The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 40,

[0182] The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 38,

[0183] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 44,

[0184] The amino acid sequences shown in SEQ ID NO: 27 and SEQ ID NO: 43,

[0185] The amino acid sequences shown in SEQ ID NO: 28 and SEQ ID NO: 43,

[0186] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 45,

[0187] The amino acid sequences shown in SEQ ID NO: 29 and SEQ ID NO: 45,

[0188] The amino acid sequences shown in SEQ ID NO: 30 and SEQ ID NO: 43,

[0189] The amino acid sequences shown in SEQ ID NO: 31 and SEQ ID NO: 43,

[0190] The amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 46,

[0191] The amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 47,

[0192] The amino acid sequences shown in SEQ ID NO: 33 and SEQ ID NO: 47,

[0193] The amino acid sequences shown in SEQ ID NO: 34 and SEQ ID NO: 47,

[0194] The amino acid sequences shown in SEQ ID NO: 35 and SEQ ID NO: 43,

[0195] The amino acid sequences shown in SEQ ID NO: 36 and SEQ ID NO: 43,

[0196] The amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 43,

[0197] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 43,

[0198] The amino acid sequences shown in SEQ ID NO: 29 and SEQ ID NO: 43,

[0199] The amino acid sequences shown in SEQ ID NO: 37 and SEQ ID NO: 39, or

[0200] The amino acid sequences shown in SEQ ID NO: 34 and SEQ ID NO: 48.

[0201] In some embodiments, disulfide bonds are present in the insulin analog. In some specific embodiments, two disulfide bonds are present at CysA7-CysB7 and CysA20-CysB19 joining the A chain of the insulin analog and the B chain of the insulin analog; and / or an intrachain disulfide bond is present at CysA6-CysA11 in the A chain of the insulin analog.

[0202] For example, "CysA7-CysB7" indicates that an interchain disulfide bond is formed between the Cys at position 7 from the N-terminus in the A chain of the insulin analog and the Cys at position 7 from the N-terminus in the B chain of the insulin analog. "CysA20-CysB19" indicates that an interchain disulfide bond is formed between the Cys at position 20 from the N-terminus in the A chain of the insulin analog and the Cys at position 19 from the N-terminus in the B chain of the insulin analog.

[0203] In some embodiments, the insulin analog comprises the following structure (from N-terminus to C-terminus): B-L1-A or A-L1-B, wherein A is the A chain of the insulin analog, and B is the B chain of the insulin analog. L1 is a linker, which may be present or absent.

[0204] In some embodiments, the L1 is a polypeptide having a linking function, such as a flexible linker. In some embodiments, the linker represented by L1 comprises (or is) the following sequence: including but not limited to G4S, GS, GAP, ASGS, polyguanine (poly G). In some specific embodiments, the linker represented by L1 comprises (or is) (G m S p ) n , wherein each m is independently selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6), each n is independently selected from an integer of 1-10 (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.

[0205] In some specific embodiments, L1 is selected from GS linkers and / or comprises a linker having at least 4 Gs. In the present disclosure, GS linkers encompass any type of linker comprising amino acids G and S.

[0206] In some specific embodiments, the linker represented by L1 comprises (or is) GGSGGGG (SEQ ID NO: 130), GSGGGG (SEQ ID NO: 131), GGGGG (SEQ ID NO: 132), GGGGGG (SEQ ID NO: 49), GGGGGGSGGGG (SEQ ID NO: 50) or GGGGGSGGGG (SEQ ID NO: 51).

[0207] In some specific embodiments, the insulin analog comprises the amino acid sequence shown in any one of SEQ ID NOs: 55-84, or a sequence having at least 80% or 90% identity thereto.

[0208] In some embodiments, any of the above insulin analogs have insulin receptor agonist activity.

[0209] In some embodiments, the above insulin analogs have amino acid modifications at any one or more (2, 3, 4, 5, 6, 7, 8, etc.) positions in the insulin A chain analog and the insulin B chain analog. In some embodiments, the modifications are chemical group modifications to improve the chemical and / or physical stability of the insulin analog, regulate the efficacy of the insulin analog, and / or increase expression.

[0210] In some embodiments, the insulin analog is selected from any type disclosed in WO2021022149 and WO2023174370, the entire contents of which are incorporated herein by reference.

[0211] In some embodiments, the above insulin analogs are modified with a fatty acid chain.

[0212] Fusion protein

[0213] The present disclosure provides a fusion protein comprising insulin or an analog thereof, and a C1 domain, wherein the C1 domain is capable of extending the half-life of insulin or an analog thereof.

[0214] In some embodiments, the C1 is selected from, for example, an Fc region of an immunoglobulin (e.g., IgG (e.g., 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 antigen-binding fragment thereof), an XTEN, a peptide 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.

[0215] Illustratively, XTEN peptides are unstructured, hydrophilic long peptides that contain varying percentages of the six amino acids: Ala, Glu, Gly, Ser, and Thr, e.g., TM Peptide (Amunix Operating Inc.) In some embodiments, the PAS polypeptide is a hydrophilic, uncharged polypeptide consisting of Pro, Ala, and Ser residues and is at least about 100, 200, 300, 400, 500, or 600 amino acids in length.

[0216] In some embodiments, one fusion protein contains one or more (e.g., 2, 3, 4, 5, etc.) insulins or their analogs, and one C1. In some embodiments, one fusion protein contains one or more (e.g., 2, 3, 4, 5, etc.) insulins or their analogs, and two or more C1s (e.g., 2, 3, etc.).

[0217] In some embodiments, the C1 domain is any immunoglobulin single variable domain defined in the aforementioned “serum albumin binding molecule” of this disclosure.

[0218] Exemplarily, the immunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3 described in any one of (1) to (3):

[0219] (1) CDR1, CDR2, and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 1, 8-17

[0220] (2) CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 2-4, respectively;

[0221] (3) Any one, two or three CDRs among CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 2-4, respectively.

[0222] The above CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, for example, the CDRs are defined according to the Kabat numbering system.

[0223] Illustratively, the immunoglobulin single variable domain comprises a CDR1 having 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned CDR1s; and / or, the immunoglobulin single variable domain comprises a CDR2 having 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned CDR2s; and / or, the immunoglobulin single variable domain comprises a CDR3 having 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned CDR3s. In some specific embodiments, the amino acid mutations in CDR1, CDR2, and / or CDR3 are conservative substitutions, replacements, or modifications, and / or deletions and / or additions that do not affect function.

[0224] Exemplarily, the immunoglobulin single variable domain comprises any one or a combination of any several (eg, 2 or 3) of the aforementioned CDR1, CDR2, and CDR3.

[0225] Illustratively, the immunoglobulin single variable domain is of camelid origin.

[0226] Exemplarily, the immunoglobulin single variable domain is engineered by any of the following: humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerization, or a combination thereof.

[0227] Illustratively, the framework regions in the immunoglobulin single variable domain are derived from the human heavy chain variable region germline genes IGHV3-23 or IGVH3-66.

[0228] Exemplarily, the humanized immunoglobulin single variable domain comprises an amino acid mutation selected from the group consisting of amino acid residues at positions 20, 23, 27, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83, and 84 (positions based on the EU numbering system);

[0229] For example, the mutation comprises at least one amino acid mutation selected from the group consisting of 20V, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 74A, 78V, 83Q, or 84P;

[0230] For example, the mutation comprises a combination of amino acid mutations selected from any one of the following:

[0231] 27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0232] 20V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0233] 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0234] 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 74A;

[0235] 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 78V;

[0236] 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A;

[0237] 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 83Q, 84P;

[0238] 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 84P;

[0239] 30L, 37F, 44E, 45R, 47G, 84P; or

[0240] 30L, 37F, 44E, 45R, 47G.

[0241] For example, comprising a combination of amino acid mutations selected from any of the following:

[0242] F27N, F29Y, S30L, V37F, G44E, L45R, W47G;

[0243] L20V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G;

[0244] A23V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G;

[0245] F27N, F29Y, S30L, V37F, G44E, L45R, W47G, S74A;

[0246] F27N, F29Y, S30L, V37F, G44E, L45R, W47G, L78V;

[0247] F27N, F29Y, S30L, V37F, G44E, L45R, W47G, S49A;

[0248] F27N, F29Y, S30L, V37F, G44E, L45R, W47G, R83Q, A84P;

[0249] F27N, F29Y, S30L, V37F, G44E, L45R, W47G, A84P;

[0250] S30L, V37F, G44E, L45R, W47G, A84P; or

[0251] S30L, V37F, G44E, L45R, W47G.

[0252] Exemplarily, the immunoglobulin single variable domain comprises or is any one of SEQ ID NOs: 1, 8-17, or an amino acid sequence having at least 80% or at least 90% sequence identity therewith.

[0253] Exemplarily, the aforementioned immunoglobulin single variable domain is a VHH. In some embodiments, the aforementioned immunoglobulin single variable domain is a humanized VHH. In some embodiments, the aforementioned immunoglobulin single variable domain is an affinity-matured VHH.

[0254] In some embodiments, the fusion protein comprises insulin or its analog. In some embodiments, insulin or its analog is selected from natural insulin, for example, human insulin. In some embodiments, insulin or its analog is selected from insulin analogs.

[0255] In some embodiments, insulin or an analog thereof comprises a B chain and an A chain; wherein the A chain comprises the following amino acid sequence:

[0256] FVX1QHLCGX2HLVEALX3X4VCGEX5GFX6X7X8X9X 10 T (SEQ ID NO: 120), wherein X1 is selected from N or S; X2 is selected from S, E or R; X3 is selected from Y, H, E or R; X4 is selected from L, H, R, E or K; X5 is selected from R or K; X6 is selected from F or H; X7 is selected from Y, G, T, S, H, V or is absent; X8 is selected from G, E, P, K, D, S, T, H or is absent; X9 is selected from G, E, K, P, Q, D, H or is absent; X 10 Selected from G, T, S, E, K, A or absent;

[0257] The insulin A chain analog contains the following amino acid sequence:

[0258] GIVEQCCZ1Z2Z3CSLZ4QLENYCZ5Z 6( SEQ ID NO:121), wherein Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I, K or T, Z4 is selected from Y, E, S, K or D, and Z5 is selected from G or N; Z6 is any natural amino acid or does not exist. When Z5 is N, Z6 is not G or N.

[0259] In some embodiments, X1 is selected from N or S. In some embodiments, X1 is N. In some embodiments, X1 is S.

[0260] In some embodiments, X2 is selected from S, E, or R. In some embodiments, X2 is S. In some embodiments, X2 is E. In some embodiments, X2 is R.

[0261] In some embodiments, X3 is selected from Y, H, E, or R. In some embodiments, X3 is selected from Y, H, or E. In some embodiments, X3 is Y. In some embodiments, X3 is H. In some embodiments, X3 is E.

[0262] In some embodiments, X4 is selected from L, H, R, E, or K. In some embodiments, X4 is L. In some embodiments, X4 is H. In some embodiments, X4 is R. In some embodiments, X4 is E. In some embodiments, X4 is K.

[0263] In some embodiments, X5 is selected from R or K. In some embodiments, X5 is R. In some embodiments, X5 is K.

[0264] In some embodiments, X6 is selected from F or H. In some embodiments, X6 is F. In some embodiments, X6 is H.

[0265] In some embodiments, X7 is selected from Y, G, T, S, H, V or is absent. In some embodiments, X7 is Y. In some embodiments, X7 is absent.

[0266] In some embodiments, X8 is selected from G, E, P, K, D, S, T, H, or is absent. In some embodiments, X8 is T. In some embodiments, X8 is absent.

[0267] In some embodiments, X9 is selected from G, E, K, P, Q, D, H or is absent. In some embodiments, X9 is P. In some embodiments, X9 is absent.

[0268] In some embodiments, X 10is selected from G, T, S, E, K, A or is absent. In some embodiments, X 10 is K. In some embodiments, X 10 Does not exist.

[0269] In some embodiments, Z1 is selected from T, H, or E. In some embodiments, Z1 is T. In some embodiments, Z1 is H. In some embodiments, Z1 is E.

[0270] In some embodiments, Z2 is selected from S or K. In some embodiments, Z2 is S. In some embodiments, Z2 is K.

[0271] In some embodiments, Z3 is selected from I, K, or T. In some embodiments, Z3 is selected from I or K. In some embodiments, Z3 is I. In some embodiments, Z3 is K. In some embodiments, Z3 is T.

[0272] In some embodiments, Z4 is selected from Y, E, S, Y, K, or D. In some embodiments, Z4 is selected from Y, E, S, or K. In some embodiments, Z4 is Y. In some embodiments, Z4 is E. In some embodiments, Z4 is S. In some embodiments, Z4 is Y. In some embodiments, Z4 is K. In some embodiments, Z4 is D.

[0273] In some embodiments, Z5 is selected from G or N. In some embodiments, Z5 is G. In some embodiments, Z5 is N.

[0274] In some embodiments, Z6 is any natural amino acid or is absent, and when Z5 is N, Z6 is not G or N. In some embodiments, Z6 is absent.

[0275] In some embodiments, the insulin or its analogue comprises a B chain and an A chain, wherein the B chain comprises the following amino acid sequence:

[0276] FVX1QHLCGX2HLVEALX3X4VCGEX5GFX6YTPKT (SEQ ID NO: 21), wherein X1 is selected from N or S, X2 is selected from S, E or R, X3 is selected from Y, H, E or R, X4 is selected from L, H, R, E or K, X5 is selected from R or K, and X6 is selected from F or H;

[0277] The A chain contains the following amino acid sequence:

[0278] GIVEQCCZ1Z2Z3CSLZ4QLENYCZ5 (SEQ ID NO: 22), wherein Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I, K or T, Z4 is selected from Y, E, S, K or D, and Z5 is selected from N or G.

[0279] In some embodiments, the present disclosure provides insulin or an analog thereof comprising a B chain and an A chain, wherein the B chain comprises the amino acid sequence shown in SEQ ID NO: 21, X1 is N, X2 is selected from S, E or R, X3 is selected from Y, H or E, X4 is selected from L, H, R, E or K, X5 is selected from R or K, and X6 is H.

[0280] The A chain comprises the amino acid sequence shown in SEQ ID NO: 22, Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I or K, Z4 is selected from Y, E, S or K, and Z5 is G.

[0281] In some embodiments, the B chain comprises (or is) an amino acid sequence as shown in SEQ ID NO: 21, wherein X1, X2, X3, X4, X5, and X6 are selected from any one of the following:

[0282] (a1) X1 is N, X2 is selected from S, E or R, X3 is selected from Y, H or E, X4 is selected from L, H, R, E or K, X5 is selected from R or K, and X6 is selected from F or H;

[0283] (a2) X1 is N, X2 is selected from S, E or R, X3 is selected from Y, H or E, X4 is selected from L, H, R, E or K, X5 is R, and X6 is H;

[0284] (a3) X1 is N, X2 is S, X3 is selected from Y, H or E, X4 is selected from L, H, R or E, X5 is R, and X6 is H;

[0285] (a4) X1 is N, X2 is S, X3 is selected from Y or H, X4 is selected from L, H or E, X5 is R, and X6 is H;

[0286] (a5) X1 is N, X2 is E, X3 is selected from Y or E, X4 is selected from L, H, E or K, X5 is R; X6 is selected from F or H, for example, H;

[0287] (a6) X1 is N, X2 is E, X3 is selected from Y or E, X4 is selected from L, H, E or K, X5 is K, and X6 is H;

[0288] (a7) X1 is N, X2 is R, X3 is selected from Y, H or E, X4 is selected from L, H, R or E, X5 is R, and X6 is H;

[0289] (a8) X1 is S, X2 is S, X3 is R, X4 is selected from L, H, R or E, X5 is R, and X6 is H.

[0290] In some embodiments, the B chain comprises (or is) an amino acid sequence as shown in SEQ ID NO: 21, wherein X1, X2, X3, X4, X5, and X6 are selected from any one of the following:

[0291] (b1) X1 is selected from N or S, X2 is selected from S, E or R, X3X4 is selected from HL, YH, YL, YR, EL, YE, YK or RL, X5 is selected from R or K, and X6 is selected from F or H;

[0292] (b2) X1 is N, X2 is S, X3X4 is selected from HL, YH, YL, YR, YE or EL, X5 is R, and X6 is H;

[0293] (b3) X1 is N, X2 is E, X3X4 is selected from YE, YH, YL, EL or YK, X5 is R, and X6 is H;

[0294] (b4) X1 is N, X2 is E, X3X4 is YL, X5 is selected from K or R, and X6 is H;

[0295] (b5) X1 is N, X2 is E, X3X4 is YL, X5 is selected from K or R, and X6 is F;

[0296] (b6) X1 is N, X2 is R, X3 and X4 are YH, X5 is R, and X6 is H;

[0297] (b7) X1 is S, X2 is S, X3 and X4 are RL, X5 is R, and X6 is H.

[0298] (b8) X1 is N, X2 is S, X3 and X4 are selected from HL, YH or YE, X5 is R, and X6 is H.

[0299] In some embodiments, the A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22, wherein Z1, Z2, Z3, Z4, and Z5 are selected from any one of the following:

[0300] (c1) Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I, K or T, Z4 is selected from Y, E, S, K or D, and Z5 is G;

[0301] (c2) Z1 is T, Z2 is S, Z3 is selected from I or T, Z4 is selected from E, S or D, and Z5 is G;

[0302] (c3) Z1 is H, Z2 is S, Z3 is I, Z4 is selected from E, S or Y, and Z5 is G;

[0303] (c4) Z1 is E, Z2 is selected from S or K, Z3 is selected from I or K, Z4 is selected from E, Y or K, and Z5 is G.

[0304] In some embodiments, the A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22, wherein Z1, Z2, Z3, Z4, and Z5 are selected from any one of the following:

[0305] (d1) Z1 is T, Z2Z3 is SI, Z4 is selected from E, S or D, and Z5 is G;

[0306] (d2) Z1 is T, Z2 and Z3 are ST, Z4 is selected from E, S or D, and Z5 is G;

[0307] (d3) Z1 is H, Z2Z3 is SI, Z4 is selected from E, S or Y, and Z5 is G;

[0308] (d4) Z1 is E, Z2Z3 is SI, Z4 is selected from E, Y or K, and Z5 is G;

[0309] (d5) Z1 is E, Z2 and Z3 are selected from KI or SK, Z4 is E, and Z5 is G.

[0310] In some embodiments, the B chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 21, and the A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22; wherein:

[0311] 1) X1 is N, X2 is S, X3 is H, Y or E, X4 is L, H or R, X5 is R, X6 is H; Z1 is T, Z2 is S, Z3 is I or T, Z4 is E, S or D, and Z5 is G; or,

[0312] 2) X1 is N, X2 is S, X3 is Y, X4 is H, L or R, X5 is R, X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is S, E or Y, Z5 is G; or,

[0313] 3) X1 is N, X2 is S, X3 is Y or E, X4 is R, H, E or L, X5 is R, X6 is H; Z1 is E, Z2 is S or K, Z3 is I, Z4 is E, Y or K, Z5 is G; or,

[0314] 4) X1 is N, X2 is S, X3 is Y, X4 is H or R, X5 is R, X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is S, Z5 is G; or,

[0315] 5) X1 is N, X2 is E, X3 is Y or E, X4 is E, H, K or L, X5 is R or K, X6 is H or F; Z1 is E, Z2 is S, Z3 is I or K, Z4 is E or K, Z5 is G; or,

[0316] 6) X1 is N, X2 is R, X3 is Y, X4 is H, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is E, Z5 is G; or,

[0317] 7) X1 is S, X2 is S, X3 is R, X4 is L, X5 is R, X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is Y, and Z5 is G.

[0318] In some embodiments, the B chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 21, and the A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22, wherein:

[0319] (1) X1 is N, X2 is S, X3 and X4 are HL, YH or YR, X5 is R, and X6 is H; Z1 is T, Z2 is S, Z3 is I, Z4 is E, and Z5 is G; or

[0320] (2) X1 is N, X2 is S, X3 and X4 are YH or YR, X5 is R, and X6 is H; Z1 is T, Z2 is S, Z3 is I, Z4 is S, and Z5 is G; or

[0321] (3) X1 is N, X2 is S, X3 and X4 are EL, X5 is R, and X6 is H; Z1 is T, Z2 is S, Z3 is T, Z4 is D, and Z5 is G; or

[0322] (4) X1 is N, X2 is S, X3 and X4 are YH, YL or YR, X5 is R, and X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is E, and Z5 is G; or

[0323] (5) X1 is N, X2 is S, X3 and X4 are YH, YL or YR, X5 is R, and X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is Y, and Z5 is G; or

[0324] (6) X1 is N, X2 is S, X3 and X4 are YR, YH or YE, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is E, and Z5 is G; or

[0325] (7) X1 is N, X2 is S, X3 and X4 are YH or YE, X5 is R, and X6 is H; Z1 is E, Z2 is K, Z3 is I, Z4 is E, and Z5 is G; or

[0326] (8) X1 is N, X2 is S, X3 and X4 are YH or EL, X5 is R, and X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is Y or K, and Z5 is G; or

[0327] (9) X1 is N, X2 is S, X3 and X4 are YH or YR, X5 is R, and X6 is H; Z1 is H, Z2 is S, Z3 is I, Z4 is S, and Z5 is G; or

[0328] (10) X1 is N, X2 is E, X3 and X4 are YE, YH or YK, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is E, and Z5 is G; or

[0329] (11) X1 is N, X2 is E, X3 and X4 are YL, X5 is R or K, X6 is H or F; Z1 is E, Z2 is S, Z3 is I, Z4 is E, and Z5 is G; or

[0330] (12) X1 is N, X2 is E, X3 and X4 are YL, X5 is R, and X6 is H; Z1 is E, Z2 is S, Z3 is I or K, Z4 is K or E, and Z5 is G; or

[0331] (13) X1 is N, X2 is E, X3X4 is EL, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is K, Z5 is G.

[0332] In some embodiments, the fusion protein comprises an insulin analog comprising a B chain and an A chain;

[0333] The B chain comprises (or is) the amino acid sequence of SEQ ID NO: 21, wherein: X1 is selected from N or S, X2 is selected from S, E or R, X3 is selected from Y, H, E or R, X4 is selected from L, H, R, E or K, X5 is selected from R or K, and X6 is selected from F or H;

[0334] The A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22, wherein: Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I, K or T, Z4 is selected from Y, E, S, K or D, and Z5 is G.

[0335] In some embodiments, the fusion protein comprises an insulin analog comprising a B chain and an A chain;

[0336] The B chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 21, wherein: X1 is N, X2 is selected from S, E or R, X3 is selected from Y, H or E, X4 is selected from L, H, R, E or K, X5 is selected from R or K, and X6 is selected from F or H;

[0337] The A chain comprises (or is) the amino acid sequence shown in SEQ ID NO: 22, wherein: Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I or K, Z4 is selected from Y, E, S or K, and Z5 is G.

[0338] In some embodiments, the fusion protein comprises an insulin analog comprising a B chain and an A chain; wherein:

[0339] X1 is N, X2 is S, X3 is selected from Y, H or E, X4 is selected from E, L, H or R, X5 is R, X6 is H; Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is I, Z4 is Y, E, S or K, and Z5 is G;

[0340] X1 is N, X2 is E, X3 is selected from Y or E, X4 is selected from E, L, H or K, X5 is R or K, X6 is H or F; Z1 is E, Z2 is S, Z3 is selected from I or K, Z4 is E or K, and Z5 is G; or

[0341] X1 is N, X2 is R, X3 is Y, X4 is H, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is E, Z5 is G.

[0342] In some embodiments, the B chain comprises the amino acid sequence of any one of SEQ ID NOs: 23-37, or a sequence at least 80% or at least 90% identical thereto.

[0343] The amino acid sequence is shown below:

[0344] In some embodiments, the A chain comprises the amino acid sequence of any one of SEQ ID NOs: 38-48, or a sequence at least 80% or at least 90% identical thereto.

[0345] The amino acid sequence is shown below:

[0346] In some embodiments, the B chain and A chain of the insulin analog include or are selected from any one of the following groups:

[0347] The amino acid sequences shown in SEQ ID NO: 23 and SEQ ID NO: 38,

[0348] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 39,

[0349] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 40,

[0350] The amino acid sequences shown in SEQ ID NO: 25 and SEQ ID NO: 40,

[0351] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 41,

[0352] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 38,

[0353] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 42,

[0354] The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 41,

[0355] The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 42,

[0356] The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 43,

[0357] The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 40,

[0358] The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 38,

[0359] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 44,

[0360] The amino acid sequences shown in SEQ ID NO: 27 and SEQ ID NO: 43,

[0361] The amino acid sequences shown in SEQ ID NO: 28 and SEQ ID NO: 43,

[0362] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 45,

[0363] The amino acid sequences shown in SEQ ID NO: 29 and SEQ ID NO: 45,

[0364] The amino acid sequences shown in SEQ ID NO: 30 and SEQ ID NO: 43,

[0365] The amino acid sequences shown in SEQ ID NO: 31 and SEQ ID NO: 43,

[0366] The amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 46,

[0367] The amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 47,

[0368] The amino acid sequences shown in SEQ ID NO: 33 and SEQ ID NO: 47,

[0369] The amino acid sequences shown in SEQ ID NO: 34 and SEQ ID NO: 47,

[0370] The amino acid sequences shown in SEQ ID NO: 35 and SEQ ID NO: 43,

[0371] The amino acid sequences shown in SEQ ID NO: 36 and SEQ ID NO: 43,

[0372] The amino acid sequences shown in SEQ ID NO: 32 and SEQ ID NO: 43,

[0373] The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 43,

[0374] The amino acid sequences shown in SEQ ID NO: 29 and SEQ ID NO: 43,

[0375] The amino acid sequences shown in SEQ ID NO: 37 and SEQ ID NO: 39, or

[0376] The amino acid sequences shown in SEQ ID NO: 34 and SEQ ID NO: 48.

[0377] In some embodiments, the insulin analog is selected from any type disclosed in WO2021022149 and WO2023174370, which are incorporated herein by reference.

[0378] In some embodiments, the fusion protein comprises the structure shown in any one of the following from the N-terminus to the C-terminus:

[0379] B-L1-A-L2-VHH Formula (I)

[0380] A-L1-B-L2-VHH Formula (II)

[0381] VHH-L2-B-L1-A Formula (III)

[0382] VHH-L2-A-L1-B Formula (IV)

[0383] in,

[0384] A is the A chain of any of the aforementioned insulins or their analogs,

[0385] B is the B chain of any of the aforementioned insulins or their analogs,

[0386] VHH is any of the aforementioned VHHs;

[0387] L1 and L2 are linkers,

[0388] L1 and L2 are independently present or absent;

[0389] Optionally, - represents a chemical bond.

[0390] In some embodiments, the L1 is a polypeptide having a linking function, such as a flexible linker. In some embodiments, the linker represented by L1 comprises (or is) the following sequence: including but not limited to G4S, GS, GAP, ASGS, polyguanine (poly G). In some specific embodiments, the linker represented by L1 comprises (or is) (G m S p ) n , wherein each m is independently selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6), each n is independently selected from an integer of 1-10 (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.

[0391] In some specific embodiments, L1 is selected from GS linkers and / or comprises a linker having at least 4 Gs. In the present disclosure, GS linkers encompass any type of linker comprising amino acids G and S.

[0392] In some specific embodiments, the linker represented by L1 comprises (or is) GGSGGGG (SEQ ID NO: 130), GSGGGG (SEQ ID NO: 131), GGGGG (SEQ ID NO: 132), GGGGGG (SEQ ID NO: 49), GGGGGGSGGGG (SEQ ID NO: 50), or GGGGGSGGGG (SEQ ID NO: 51). In some specific embodiments, the linker represented by L1 comprises (or is) the amino acid sequence shown in any one of SEQ ID NOs: 49-51.

[0393] In some embodiments, the linker represented by L2 is a polypeptide capable of achieving a connection function (e.g., a flexible polypeptide), and VHH forms a fusion protein with B1-L1-A1 through L2. In some specific embodiments, the linker represented by L2 comprises (or is) the following sequences: including but not limited to 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 、(PGPQ) s , (PGPA) s , wherein each m is independently selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), each n is independently selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), each i is independently selected from 0-4 (e.g., 0, 1, 2, 3, 4), and s is selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some specific embodiments, L2 comprises (or is) the amino acid sequence of any one of SEQ ID NOs: 52-54.

[0394] In some embodiments, the fusion protein is a single-chain protein, a dimeric protein, or a multimeric protein. In some embodiments, the fusion protein is a single-chain protein.

[0395] In some embodiments, the fusion protein comprises (or is) an amino acid sequence as set forth in any one of SEQ ID NOs: 85-117, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some specific embodiments, the fusion protein comprises (or is) an amino acid sequence as set forth in any one of SEQ ID NOs: 85-116, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some specific embodiments, the fusion protein comprises (or is) an amino acid sequence as set forth in any one of SEQ ID NOs: 85-112, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0396] In some embodiments, the fusion proteins of the present disclosure are capable of binding to human serum albumin molecules. For example, at a concentration of less than 1×10 -7 M, ≤1×10 -8 M, ≤1×10 -9 M, ≤1×10 -10 M, ≤1×10 -11 M's K D Values ​​bind to human serum albumin.

[0397] In some embodiments, the fusion protein of the present disclosure has the activity of binding to human insulin α receptor (IR-A) and / or promoting the phosphorylation of human insulin β receptor (IR-B).

[0398] In some embodiments, the fusion proteins of the present disclosure (e.g., any of SEQ ID NOs: 85-117, 85-116, or 85-112) have a half-life that is prolonged compared to native insulin. For example, the half-life is extended from 1 day to 25 days, from 1 day to 15 days, from 1 day to 10 days, from 2 days to 9 days, from 2 days to 8 days, from 3 days to 7 days, for example, by 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.

[0399] In some embodiments, the fusion protein of the present disclosure, when administered to a subject in need thereof, can reduce the risk of hypoglycemia in the subject and has a safe dosing window.

[0400] In some embodiments, the present disclosure fuses novel human insulin analogs with albumin-binding domains. In humans, serum albumin has a relatively long half-life of approximately 19-21 days, making it an ideal target for extending the half-life of other drugs. Therefore, fusion proteins can leverage albumin's inherently large molecular weight and long half-life in humans to effectively inhibit the renal clearance of certain low-molecular-weight drugs, thereby significantly prolonging the drug's pharmacokinetic properties in vivo.

[0401] In some embodiments, the present disclosure provides novel fusion proteins of human insulin analogs and human HSA-binding domains, which can be used to treat metabolic diseases such as type I diabetes or type II diabetes. These fusion proteins exhibit exceptionally long in vivo pharmacokinetic properties, demonstrating a robust and sustained hypoglycemic effect in STZ-induced type I diabetes rat models and db / db type II diabetes mouse models. They also possess excellent physical and chemical stability and high expression yields, potentially meeting clinical demands for ultra-long-acting insulin products.

[0402] polynucleotides

[0403] The present disclosure provides polynucleotides encoding the fusion proteins, insulin or analogs thereof of the present disclosure. The polynucleotides of the present disclosure may be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the nucleic acids of the present disclosure are substantially isolated nucleic acids.

[0404] Nucleic acid of the present disclosure can also be in the form of a vector, can be present in a vector and / or can be a part of a vector, such as a plasmid, a cosmid, a YAC or a viral vector. The vector can be an expression vector in particular, can provide a vector for expressing fusion protein, insulin or its analog in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector usually comprises at least one nucleic acid of the present disclosure, which is operably connected to one or more suitable expression control elements (such as promoters, enhancers, terminators, etc.). It is common sense for those skilled in the art to select the elements and their sequences for the expression in a specific host. Fusion protein of the present disclosure, insulin or its analog are expressed useful or necessary control elements and other elements such as promoters, enhancers, terminators, integrons, selection markers, leader sequences, reporter genes.

[0405] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0406] host cells

[0407] The present disclosure provides recombinant host cells that express one or more fusion proteins, insulin or its analogs of the present disclosure, or contain polynucleotides or vectors of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0408] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0409] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0410] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0411] 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.

[0412] The cells of the present disclosure are incapable of developing into complete plants or animals.

[0413] Production or preparation method

[0414] The present disclosure provides a method for preparing the fusion protein, insulin or its analogs of the present disclosure, which generally comprises the following steps:

[0415] - culturing the host cell of the present disclosure under conditions that allow expression of the fusion protein, insulin or its analogs of the present disclosure; and

[0416] - recovering the target fusion protein, insulin or its analogue expressed by the host cell from the culture; and

[0417] - Optionally, further purification and / or modification of the target fusion protein, insulin or analog thereof of the present disclosure is included.

[0418] The fusion proteins, insulin or analogs thereof of the present disclosure can be produced intracellularly in the cells as described above (e.g., in the cytoplasm, in the periplasm or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0419] Methods and reagents for recombinant production of polypeptides, such as specific expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, protein isolation and purification techniques suitable for use in methods for producing the proteins disclosed herein are well known to those skilled in the art. As an example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems result in glycosylation of antibodies, particularly 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 bioreactors to produce antibodies. The culture medium containing the secreted antibodies can be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, for example, at -70°C, or lyophilized.

[0420] However, the fusion protein, insulin or analogs thereof of the present disclosure can also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0421] Pharmaceutical composition

[0422] The present disclosure provides a pharmaceutical composition containing a preventively or therapeutically effective amount of any one or a combination of the following: the fusion protein, insulin or its analogues or their encoding polynucleotides as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

[0423] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the fusion protein or insulin or its analog in a unit dose. In other embodiments, the amount of the fusion protein or insulin analog in a unit dose is 0.1-2000 mg; in some embodiments, 1-1000 mg.

[0424] Reagent kit (or medicine box)

[0425] The present disclosure provides a kit or a pharmaceutical kit comprising one or more containers, each of which independently contains any one or a combination thereof selected from the following: the fusion protein of the present disclosure, insulin or an analog thereof, or a polynucleotide encoding the same.

[0426] Methods for preventing and treating diseases and pharmaceutical uses

[0427] The present disclosure provides uses and methods of pharmaceutical compositions comprising the fusion protein of the present disclosure, the insulin or its analogs of the present disclosure, or polynucleotides encoding the same in preventing, treating, or alleviating diseases or symptoms.

[0428] In some embodiments, the method comprises administering to a subject in need thereof an effective amount of the fusion protein of the present disclosure, the insulin or its analogs of the present disclosure, or a polynucleotide encoding the same, for preventing, treating, or ameliorating a disease or symptom.

[0429] In some embodiments, the disease or condition is selected from a metabolic disease, condition, and / or disorder.

[0430] In some embodiments, the disease or condition is selected from diabetes or its complications, obesity or its complications, dyslipidemia, and / or metabolic syndrome.

[0431] In some embodiments, the disease or condition is diabetes or its complications, obesity or its complications, dyslipidemia, and / or metabolic syndrome.

[0432] In some embodiments, the diabetes or its complications is selected from the group consisting of type I diabetes, type II diabetes, and complications thereof.

[0433] In some embodiments, the fusion protein, insulin or its analogs, and pharmaceutical compositions of the present disclosure are administered to subjects with metabolic diseases, conditions, and / or disorders to reduce the risk of hypoglycemia in subjects after receiving drug treatment.

[0434] In some embodiments, methods are provided for administering an effective amount of a fusion protein of the present disclosure, insulin or an analog thereof, or a polynucleotide encoding the same to a subject in need thereof for lowering blood glucose.

[0435] In some embodiments, a method is provided for reducing the risk of hypoglycemia in a subject in need thereof while receiving drug therapy, comprising administering an effective amount of the disclosed fusion protein, insulin or an analog thereof, or a polynucleotide encoding the same.

[0436] The present disclosure also provides pharmaceutical uses of the fusion protein, insulin or its analogs, or polynucleotides encoding the same, for preparing drugs for preventing, treating or alleviating the above-mentioned diseases or symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0437] FIG1A and FIG1B show the experimental results of the effect of HSA antibodies on the binding between FcRn and HSA.

[0438] Figure 2 shows the concentration-time curve of HSA antibody in plasma after intravenous administration in the pharmacokinetic study of mice.

[0439] Figures 3A and 3B show the results of the pharmacokinetic study of HSA antibodies in mice, wherein Figure 3A shows the T of HSA antibodies in plasma calculated using a non-compartmental model. 1 / 2 Parameter comparison results,Figure 3B is the Cl_obs (mL / (min*kg)) parameter comparison results.

[0440] FIG4 is a schematic diagram of the structure of the fusion protein.

[0441] FIG5A and FIG5B show the detection results of the fusion protein promoting the phosphorylation level of hIRB in CHO-K1-hIRB cells overexpressing hIRB receptor. DETAILED DESCRIPTION

[0442] definition

[0443] To facilitate understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure pertains. The three-letter and one-letter amino acid codes used herein are as described in J. Biol. Chem, 243, p3558 (1968).

[0444] "Insulin" includes naturally occurring insulin, such as human insulin, which consists of two polypeptide chains, referred to as the A and B chains, comprising 21 and 30 amino acid residues, respectively, and which are linked by two cystine disulfide bonds.

[0445] "Insulin analogs" include polypeptides that have a molecular structure that can be formally derived from the structure of naturally occurring insulin, such as human insulin, by removing and / or replacing (displacing) one or more amino acid residues present in natural insulin and / or by adding one or more amino acid residues. The added and / or substituted amino acid residues can be codable amino acid residues or other naturally occurring amino acid residues or purely synthetic amino acid residues.

[0446] "Insulin A chain analogue", "insulin A chain analogue", "insulin B chain analogue", "insulin B chain analogue" respectively include the A and B chains of human insulin, which have one or more substitutions, deletions and / or extensions (additions) of the A and B amino acid chains, respectively, relative to the A and B chains of human insulin, respectively.

[0447] "Sequence" should generally be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless the present disclosure requires further limited explanation.

[0448] An "immunoglobulin variable domain" refers to a domain essentially consisting of "Framework Region 1" (FR1), "Framework Region 2" (FR2), "Framework Region 3" (FR3), and "Framework Region 4" (FR4), as well as "Complementarity Determining Region 1" (CDR1), "Complementarity Determining Region 2" (CDR2), and "Complementarity Determining Region 3" (CDR3). Therefore, the general structure (or sequence) of a variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domain confers specificity for an antigen by having an antigen-binding site.

[0449] "Antibody framework (FR)" refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain.

[0450] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the VH / VL interactions required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, e.g., camelized human VH), IgNAR, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs), and antibodies that are VH domains or derived from VH domains. TM ) and (single domain) antibodies (such as dAbs) as the VL domain or derived from the VL domain TM ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0451] "VHH domain", also known as heavy chain single domain antibody, VHH, V HHDomain, VHH antibody fragment, VHH antibody, nanobody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e., "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the VH and VL present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to the epitope without the need for other antigen-binding domains (for the VH or VL domain in conventional tetrapeptide chain structure antibodies, the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH", "V HH domain", "VHH antibody fragment", "VHH antibody", as well as" "VHH domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) can be used interchangeably. "VHH domain" includes but is not limited to natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained by phage display technology. The total number of amino acid residues in a VHH domain will generally be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17 June, 2009 and WO94 / 04678.

[0452] As is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, and AbM.

[0453] "Humanized antibodies", also known as CDR-grafted antibodies, refer to antibodies produced by transplanting non-human CDR sequences into the variable region framework of a human antibody. This can overcome the strong immune response induced by chimeric antibodies due to the large amount of non-human protein components they carry. In order to avoid a decrease in activity while also reducing immunogenicity, the fully human antibody variable region can be subjected to minimal reverse mutations to maintain activity. Examples of "humanization" include the humanization of a VHH domain derived from Camelidae by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in the VH domain of a human conventional tetrapeptide chain structure antibody. The humanized VHH domain may contain one or more fully human framework region sequences, and in some specific embodiments, may contain the human framework region sequence of IGHV3. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), i.e., CDR "grafting" onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies that are further subjected to affinity maturation of CDRs by phage display. In addition, in order to avoid a decrease in immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequences can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0454] One skilled in the art can determine the amino acid sequence boundaries of an antibody CDR using any of a number of known numbering schemes, including those described in Kabat et al., supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefran et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Plückthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme); each of which is incorporated by reference in its entirety.

[0455] In general, the term "specificity" refers to the number of different types of antigens or epitopes to which a particular antigen binding molecule or antigen binding protein (e.g., a serum albumin binding molecule or fusion protein of the present disclosure) can bind. The specificity of an antigen binding protein can be determined based on its affinity and / or avidity. The dissociation equilibrium constant (K) between the antigen and the antigen binding protein is determined by the affinity and / or avidity of the antigen binding protein. D ) is a measure of the binding strength between an epitope and the antigen binding site on an antigen binding protein: K D The smaller the value, the stronger the binding strength between the epitope and the antigen binding protein (or, affinity can also be expressed as a binding constant (K a ), which is 1 / K D As will be appreciated by those skilled in the art, affinity can be determined in a known manner, depending on the specific antigen of interest. Avidity is a measure of the strength of binding between an antigen binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and the relevant antigen. Avidity is related to both the affinity of the antigen binding site on the antigen binding protein and the number of relevant binding sites present on the antigen binding protein.

[0456] "Serum albumin binding domain" means any polypeptide that can specifically bind to serum albumin or its epitope. "Serum albumin binding molecule" means any molecule that can specifically bind to serum albumin or its epitope, including but not limited to proteins and polypeptides. Serum albumin binding molecules may include antibodies or antigen-binding fragments thereof as defined in the present disclosure that are directed against serum albumin or its epitope, or conjugates or fusion proteins comprising the antibodies or antigen-binding fragments thereof. Antigen-binding fragments are, for example, sdAbs or bispecific antibodies or multispecific antibodies. The serum albumin binding molecules of the present disclosure may comprise at least one (e.g., 2, 3, 4 or more) immunoglobulin single variable domains (e.g., VHH) that bind to serum albumin. In addition to the immunoglobulin single variable domains, the serum albumin binding molecules of the present disclosure may also comprise a linker and / or a portion having the function of an effector molecule, the effector molecule including but not limited to a diagnostic agent or therapeutic agent, for example, an anti-tumor agent, an immunomodulator, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof.

[0457] An "affinity matured" serum albumin antibody (eg, VHH) has one or more changes in one or more CDRs which result in an increase in affinity for serum albumin compared to its parent anti-serum albumin antibody. Affinity matured anti-serum albumin antibodies can be prepared by methods known in the art, for example, as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci. USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3):889-896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0458] "Back mutation" refers to the mutation of the amino acid residues in the FR region of a human antibody to the amino acid residues at the corresponding position in the original source antibody. This is usually done to avoid the decrease in immunogenicity and activity caused by the humanized antibody. The humanized antibody variable region can be subjected to a minimum of back mutations to maintain the activity of the antibody.

[0459] Typically, the serum albumin binding molecules of the present disclosure will be expressed as preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (K D ), and / or with at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The binding constant (K a ) binds to the antigen to be bound (ie serum albumin). Any -4 M's K D Values ​​are generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined by any suitable means known in the art, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (ELISA), and sandwich competitive assays as described herein.

[0460] The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking.

[0461] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent 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 × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0462] "Nucleic acid molecule" and "polynucleotide" are used interchangeably to refer to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0463] "Vector" means a construct capable of delivering and, in some embodiments, 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 conjugated to a cationic condensing agent, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0464] "Host cell" includes individual cells or cell cultures that can be or have been recipients of vectors for incorporating polynucleotide inserts. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutations. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny thereof; for example, including mutant progeny that have the same function or biological activity as the parent cell selected in the originally transformed cell.

[0465] A "pharmaceutical composition" refers to a mixture containing one or more active ingredients described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredients and thereby exerting their biological activity.

[0466] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any carrier, diluent, buffer, or excipient, such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th edition Mack Publishing, 2000).

[0467] "Administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0468] "Treatment" means administering a therapeutic agent, such as a fusion protein or insulin analog comprising any of the present disclosure, to a subject who has, is suspected of having, or is predisposed to having one or more diabetes or hyperglycemia-related diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a treated subject or population, by preventing or delaying the onset of symptoms or complications, alleviating symptoms or complications, or eliminating the disease, condition, or disorder to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of 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 symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although the embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating the symptoms of the target disease in a subject, they should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test. The patient to be treated is a mammal, and preferably a human.

[0469] "Prevent" or "prevent" means reducing the risk or incidence, or eliminating or slowing the progression of one or more conditions, symptoms, complications, or disorders.

[0470] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0471] "Subject" or "patient" refers to a mammal, particularly a primate, and especially a human.

[0472] "About" and "approximately" refer to values ​​within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as 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, each instance of a number or numerical range preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0473] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be construed to have an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0474] The ordinal numbers "first", "second", "L1", "L2", "C1", etc. in the present disclosure are not intended to limit the quantity, level, grade, or sequence, and their purpose is only to distinguish different elements, steps, and technical features.

[0475] Example 1. Screening and preparation of anti-HSA antibodies

[0476] 1.1 Screening of antibodies that specifically bind to human serum albumin (HSA)

[0477] (1) Camel Immunization

[0478] A native Bactrian camel from Inner Mongolia was immunized with commercially available serum albumin extracted from human blood (Sigma, Cat. No. 126658). Five milliliters of pre-immune camel serum was collected and separated. Freund's complete or incomplete adjuvant was mixed with the antigen at a 1:1 ratio by volume, and the camel was immunized subcutaneously at multiple sites (100 μg protein per animal per immunization). Booster immunizations were performed every two weeks, and titers were measured after four immunizations. Plates were coated with 5 μg / mL human serum albumin (HSA) (Costar, Cat. No. 9018) at 100 μL / well and incubated at 4°C overnight. The next day, the plates were washed three times with PBST (0.05% Tween 20), at 300 μL / well, followed by blocking with 4% skim milk powder and incubation at 37°C for 2 hours. After washing, serial dilutions of the immunized camel serum were added and incubated at 37°C for 1 hour. Negative controls consisted of the same serial dilutions of pre-immune serum and blank PBS solution. After incubation, the cells were washed three times with PBST and then incubated at 37°C for 1 hour with a horseradish peroxidase-conjugated goat anti-camel Fc polyclonal antibody (Thermo, Cat. No. A16060, 1:5000 dilution). The cells were then washed again and developed with TMB colorimetric buffer. The color was terminated with 1M sulfuric acid and the absorbance was read at OD 450 nm using a SpectraMax M5 microplate reader. The titer was determined at a 1:51200 dilution. If the titer met the criteria, peripheral blood was collected from the camels for library preparation.

[0479] (2) Phage library establishment

[0480] 100-200 mL of camel peripheral blood was collected and peripheral blood lymphocytes (PBMCs) were separated using the Ficoll method. The cell count was 1.2 × 10 8 Add Trizol reagent and resuspend (1×10 7 cells / mL Trizol) to lyse the cells and place on ice for 5 minutes; centrifuge at 13000 rpm for 3 minutes, remove the supernatant, and discard the precipitate; add 1 / 5 volume of chloroform, shake vigorously for 30-60 seconds, and let it stand in an ice bath for 2 minutes; centrifuge at 13000 rpm for 10 minutes, aspirate the upper aqueous phase into a new 1.5 mL tube; add an equal volume of isopropanol, mix well, and let it stand at -20°C for 30 minutes; centrifuge at 13000 rpm for 10 minutes, remove the supernatant, and retain the precipitate; wash the precipitate with pre-cooled 75% ethanol and let it stand at room temperature for 5-10 minutes; add 600 μL of RNase-removed deionized water, redissolve it, obtain RNA, reverse transcribe it to obtain cDNA, and construct a phage library.

[0481] (3) Screening of phage libraries

[0482] An HSA protein lacking some amino acids in the C-terminal third domain (also known as truncated HSA) was expressed and purified in HEK293 cells, and this protein was used to screen a phage library to obtain antibodies with affinity for HSA.

[0483] 10 μg of the truncated HSA-avi-biotin protein was bound to 1 mg of Dynabeads M-280 streptavidin (Invitrogen, Cat No. 11206D) and allowed to stand at room temperature for one hour. The beads were then washed three times with 1× PBS and blocked with 2% skim milk for two hours at room temperature. The camel heavy chain single domain antibody phage display library was then added and allowed to react at room temperature for one hour. Unbound phage was removed by washing nine times with PBST (0.05% Tween-20). Phage specifically binding to the truncated HSA was eluted with 1 mg / mL trypsin and infected with Escherichia coli TG1 in logarithmic phase growth to generate and purify phage for the next round of screening. After repeating the same screening process for 1-2 rounds, positive clone sequences were enriched.

[0484] (4) Phage ELISA identification

[0485] From the clones obtained in the last round of screening, monoclonal colonies were picked and packaged into phage single-chain antibodies for phage ELISA testing. The ELISA plates were coated with 2 μg / mL of human, monkey, and mouse serum albumin (HSA, CySA, mSA) overnight, blocked with 4% skim milk at 37°C for 1 hour, and then the phage supernatant blocked with 2% skim milk was added. The plates were allowed to react for 1 hour at room temperature, washed 3 times with PBST (0.05% Tween 20), and anti-M13 HRP was added for detection. The clones with OD450 values ​​greater than 0.5 for the three albumins in the ELISA binding test were sequenced to obtain 50 specific sequences. Only some antibodies that showed high OD450 values ​​for both human and monkey serum albumin were sequenced to obtain another 7 new specific sequences. A total of 57 new specific sequences were obtained from phage screening.

[0486] 1.2 Construction of Antibody Mammalian Cell Expression Vector

[0487] The 57 specific sequences obtained by phage library screening in 1.1 were classified and sorted, 19 of which were cloned, and the phage supernatant ELISA binding of these 19 HSA antibodies was tested. The OD of clone #7 was 450 The values ​​are shown in Table 1.

[0488] Table 1. Results of ELISA test of HSA antibody phage supernatant

[0489] Some of the complete VHH sequences are as follows:

[0490] >#7aHSA

[0491] The above sequence SEQ ID NO: 1 comprises the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the FR sequences are in italics and the CDR1, CDR2, and CDR3 sequences are underlined. The CDR sequences of the HSA antibodies provided herein, as encoded by Kabat, are summarized in Table 2.

[0492] Table 2. CDR sequences of anti-HSA antibodies (Kabat numbering system)

[0493] The VHH sequence was fused to human IgG1-Fc (CH2-CH3) and constructed into the PTT5 expression vector. The sequence of the linked human IgG1-Fc is shown below:

[0494] >Human IgG1-Fc

[0495] The following is the complete protein sequence of the VHH sequence fused to the human IgG1-Fc (CH2-CH3) segment. The single underline represents the human IgG1-Fc (CH2-CH3) segment sequence (shown in SEQ ID NO: 5), and the double underline represents the linker sequence. The complete protein sequence is as follows:

[0496] >#7aHSA-IgG1Fc:

[0497] 1.3 Expression, screening, and detection of anti-HSA antibodies

[0498] The #7aHSA antibody fused to human IgG1-Fc was transiently expressed in HEK293 cells and affinity purified using Protein A. The protein was then tested using ELISA binding assays and Biacore protein interaction assays.

[0499] The in vitro binding ability of the #7aHSA antibody to human, monkey, and mouse serum albumin was tested by ELISA binding assays using human serum albumin (Sigma, Cat No. 126658), monkey serum albumin (Abcam, Cat No. ab184894), and mouse serum albumin (Abcam, Cat No. ab183228). The negative control was PBS, and the positive control was ALB8 fused to human IgG1-Fc (the variable region sequence was derived from the ALB8 sequence in Ablynx patent publication WO 2008 / 028977). The sequence is as follows:

[0500] >ALB8-IgG1Fc

[0501] (Note: The single underline is the Fc region of IgG1, and the double underline is the linker region)

[0502] Human, monkey, and mouse albumin were diluted to 1 μg / mL in PBS buffer (pH 7.4) and added to a 96-well microtiter plate (Corning, Cat No. 9018) at a volume of 100 μL / well. The plate was incubated at 4°C overnight for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (pH 7.4, 0.05% Tween-20) buffer. A blocking buffer of 2% skim milk powder diluted in PBS buffer was added (300 μL / well) and incubated at 37°C for 1 hour. After blocking, the blocking buffer was discarded, and the plate was washed three times with PBST buffer. HSA antibody was added at an initial concentration of 2 μg / mL and diluted threefold in 11 steps with PBS buffer. The plate was incubated at 25°C for 1 hour. After incubation, the reaction solution in the plate was discarded, the plate was washed three times with PBST, and 100 μL of HRP-labeled goat anti-human IgG Fc secondary antibody (Jackson immu, 109-035-190) (1:5000 dilution) was added to each well and incubated at 25°C for 1 hour. After washing the plate three times with PBST, 100 μL of TMB colorimetric substrate was added for color development. The reaction was terminated with 1 M sulfuric acid and the absorbance was read at OD450 nm using a SpectraMax M5 microplate reader. The absorbance and antibody concentration were plotted using a 4-parameter fitting method using GraphPad prism to calculate the EC value of antibody-antigen binding. 50 The results are shown in Table 3.

[0503] Table 3. ELISA binding EC values ​​of anti-HSA antibodies to human, monkey, and mouse albumin 50( μg / mL)

[0504] Note: “ / ” means not detected

[0505] In addition, the affinity constants of human IgG1Fc-fused HSA antibodies for human, monkey, and mouse albumins (HSA, CySA, and mSA) were determined using a Biacore T200 (GE Healthcare) instrument. Protein A was first covalently coupled to a CM5 S series chip (GE, Cat. 29-1049-88) using an amino coupling kit (GE, Cat. BR-1000-50). The HSA antibody was then affinity-captured onto the protein A-coupled chip. Different concentrations of human, monkey, and mouse albumin were then passed over the chip surface. The reaction signals were detected in real time using a Biacore instrument to generate binding and dissociation curves. Affinity constants were obtained by fitting the curves to a Langmuir 1:1 binding model using BIAevaluation version 4.1, GE software. The buffer used in the experiment was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4, GE, Cat.# BR-1006-69). After each cycle, the chip was regenerated using glycine-HCl (pH 1.5) (GE, Cat.# BR-1003-54). Affinity results are shown in Table 4.

[0506] Table 4. Affinity results of anti-HSA antibodies to human, monkey and mouse albumin

[0507] 1.4 Humanization of anti-HSA antibodies

[0508] By performing three-dimensional homology modeling on the selected specific anti-HSA antibody molecule #7aHSA and comparing the results with the V-base human germline sequence database and the IMGT human antibody heavy chain variable region germline gene database, the highly homologous heavy chain variable region germline gene IGHV3-23 was selected as a template. The CDRs of the camelid single-domain antibody were transplanted into the corresponding human template, resulting in a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The transplanted single-domain antibody was subjected to further three-dimensional structural modeling and analysis. Specific sites within the FR region that affect CDR morphology were backmutated. The resulting modified antibody exhibits enhanced stability. Amino acid residues were assigned and annotated using the Kabat numbering system. The FRs of #7_Hu-9 and #7_Hu-10 retain more human germline sequences, resulting in lower immunogenic mutations.

[0509] The design of back mutation sites and mutation methods are shown in Table 5.

[0510] Table 5. Reversion mutation sites and mutation patterns

[0511] The specific humanized sequences obtained are as follows, with the CDR regions underlined and the numbering system being the Kabat code:

[0512] >#7_Hu_1

[0513] >#7_Hu_2

[0514] >#7_Hu_3

[0515] >#7_Hu_4

[0516] >#7_Hu_5

[0517] >#7_Hu_6

[0518] >#7_Hu_7

[0519] >#7_Hu_8

[0520] >#7_Hu_9

[0521] >#7_Hu_10

[0522] The method described in 1.4 was used to construct a humanized anti-HSA antibody fusion protein with the Fc (CH2-CH3) region of hIgG1 or a protein fused with a 6His tag. The single underline is the hIgG1-Fc (CH2-CH3) region sequence (shown in SEQ ID NO: 5), the italicized 6His tag sequence, and the double underlined linker sequence. The protein sequences are as follows (using #7_hu_1-hIgG1Fc and #7_hu_10-6His as examples, and the same applies to other humanized HSA antibodies). For the antibody numbering system, #7_hu_10-6His is the C-terminal fusion of #7_Hu_10 (SEQ ID NO: 17). The same is true for other antibodies.

[0523] >#7_hu_1-hIgG1Fc:

[0524] >#7_Hu_10-6his

[0525] >ALB8-6his

[0526] The plasmid was transiently transfected into HEK293 cells. After 5 days of cell culture, the expression supernatant was collected and centrifuged at 4000 rpm for 15 minutes to remove cells. The supernatant was harvested and filtered through a 0.45 μM filter. The supernatant was then purified using a Protein A column or nickel column, or other conventional methods in the art, to obtain the HSA antibody disclosed herein.

[0527] 1.5 Affinity determination of humanized anti-HSA antibodies for human, monkey, and mouse serum albumin

[0528] The affinity constants of a humanized anti-HSA antibody fused with a 6His tag for human, monkey, and mouse albumin were determined using a Biacore T200 (GE Healthcare) instrument. First, amino coupling was performed on a CM5 Sseries chip (GE, Cat. 29-1049-88) using an amino coupling kit (GE, Cat. BR-1000-50) and a His capture kit (GE, Cat. 28-9950-56) according to the kit instructions, achieving a surface response of approximately 10,000 RU. The chip was then blocked with ethanolamine and ready for use. The humanized HSA antibody fused with a 6His tag was then affinity captured onto the anti-His chip. Different concentrations of human, monkey, and mouse albumin were then passed over the chip surface. The reaction signals were monitored in real time using the Biacore instrument to generate binding and dissociation curves. Affinity constants were obtained by fitting the curves to a Langmuir 1:1 binding model using BIAevaluation version 4.1, GE software. The buffer used in the experiment was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4, GE, Cat.# BR-1006-69). After each cycle, the chip was regenerated using pH 1.5 glycine-HCl (GE, Cat.# BR-1003-54). The affinity results of some humanized antibodies are shown in Table 6. The results show that the affinity of the antibodies obtained in the present disclosure for HSA is comparable to or slightly better than that of the positive control.

[0529] Table 6. Affinity of humanized HSA antibodies to human, monkey, and mouse albumin

[0530] 1.6 Effect of anti-HSA antibodies on FcRn and biotin-HSA binding in cells stably expressing hFcRn

[0531] The blocking effect of anti-HSA antibodies on the binding of HSA to FcRn was detected by measuring the fluorescence change of randomly biotinylated HSA bound to the surface of HEK293 cells stably expressing hFcRn mutants (293-hFcRn-mut cells). This experiment used HEK293 cells stably expressing hFcRn mutants (L320A, L321A), which are well localized and maintained on the cell membrane surface. Cells expressing hFcRn mutants were incubated with different concentrations of HSA antibodies and biotin-HSA, followed by incubation with SA-FITC secondary antibody. The degree of reduction in the secondary antibody fluorescence signal (MFI) was used as a measure of the blocking effect of HSA antibodies on the binding of biotin-HSA to FcRn. The specific experimental method is as follows:

[0532] After 293-hFcRn-mut cells were washed twice with pH 5.5 PBS, 5 × 10 5 cells. After centrifugation and removal of the supernatant, 90 μL of HSA antibody diluted in PBS (pH 5.5) and 10 μL of biotin-HSA (final concentration 250 μg / mL) were added to each well and incubated on ice for 40 minutes. After washing twice with 200 μL PBS (pH 5.5), 100 μL of streptavidin-FITC secondary antibody (eBioscience, Cat No. 11-4317-87) diluted in PBS (pH 5.5) was added to each well at a dilution ratio of 1:200 and incubated on ice for 40 minutes. After washing twice with 200 μL PBS (pH 5.5), 200 μL PBS (pH 5.5) was added to each well. Fluorescence values ​​were measured using a flow cytometer (BD Biosciences, BD Accuri C6) using the FL1 channel. Data were analyzed using Flowjo software, and the analysis results were plotted using Prism6 software. The inhibitory test results of some antibodies are shown in Figures 1A and 1B.

[0533] Result analysis: The fluorescence signal value (MFI) was used to determine whether the binding of HSA antibody to HSA blocked the binding of HSA to FcRn. If the fluorescence signal decreased, it indicated that the binding was blocked.

[0534] As shown in Figure 1A, the biotin-HSA + FcRn stably transfected cells + SA-FITC group (non-blocking group) served as a baseline; the biotin-HSA + HSA + FcRn stably transfected cells + SA-FITC group (HSA self-competing group) served as a positive control; Nc is an isotype control antibody. The results showed that the disclosed HSA antibodies #7_Hu_6-6his and #7_Hu_7-6his did not block the binding of HSA to FcRn.

[0535] As shown in FIG1B , #7_Hu_10-6his and #7_Hu_10 did not block the binding of HSA to FcRn (tested at a concentration of 1 mg / mL).

[0536] This indicates that the anti-HSA antibody disclosed herein does not affect the pH-dependent binding of FcRn to HSA and can well maintain the FcRn-mediated serum albumin recycling mechanism.

[0537] 1.7 Pharmacokinetics of anti-HSA antibodies in mice

[0538] The pharmacokinetic characteristics of the anti-HSA antibodies of the present disclosure (#7_Hu_6-6his; #7_Hu_7-6his; #7_Hu_10-6his), a positive control antibody (ALB8-6his), and an isotype control antibody (isotype, a non-HSA-binding VHH molecule) were studied in CD-1 male mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.). Three mice were administered via tail vein injection at a dose of 4 mg / kg on day 0. Blood samples were collected before administration and at 5 minutes, 1 hour, 7 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, and 144 hours after administration. Plasma was prepared using EDTA anticoagulation and centrifugation for pharmacokinetic analysis.

[0539] PK assay: Coated with 5 μg / mL anti-his antibody (Thermo, MAI-21315-1mg), the plate was added to a 96-well ELISA plate (Corning, Cat No. 9018) at a volume of 100 μL / well and placed at 4°C overnight for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (pH 7.4, 0.05% Tween-20) buffer, and then Casin Blocker (Thermo, 37528) blocking solution (300 μL / well) was added and incubated in a 37°C incubator for 2 hours for blocking. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST buffer. Mouse plasma at different time points was added and appropriately diluted using diluent (Casin Blocker containing 1% CD1 mouse plasma) and incubated at 37°C for 1.5 hours. After incubation, the reaction solution was discarded from the plate, and the plate was washed three times with PBST. 100 μL of HRP-labeled rabbit anti-camelid cocktail VHH secondary antibody (Genescript, A02016, BSA-free) (1:5000 dilution) was added to each well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, 100 μL of TMB chromogenic substrate was added for color development. The reaction was terminated with 1 M sulfuric acid, and the absorbance was read at OD450 nm using a SpectraMax M5 microplate reader. The VHH antibody concentration in the plasma sample was calculated based on the standard curve, and the results are shown in Figure 2. The calculated concentration was used to calculate the half-life using a PK solver, and the results are shown in Figures 3A and 3B and Table 7.

[0540] Table 7. Mouse plasma half-life of HSA antibodies

[0541] (Note: *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ****, p < 0.00001; all compared with the positive control ALB8-6his)

[0542] The results showed that the HSA antibodies #7_Hu_6-6his, #7_Hu_7-6his, and #7_Hu_10-6his disclosed herein had similar pK curves, half-lives within the half-life range of mouse serum albumin, and were superior to the positive control ALB8-6his antibody, with clearance rates also lower than that of the ALB8-6his antibody.

[0543] Example 2. Design and preparation of insulin analogs and HSAVHH fusion proteins

[0544] This embodiment provides an insulin analog, which comprises the following structure from the N-terminus to the C-terminus: B1-L1-A1.

[0545] Among them, B1 is a human insulin B chain analogue with the sequence:

[0546] FVX1QHLCGX2HLVEALX3X4VCGEX5GFX6YTPKT (SEQ ID NO: 21), wherein X1 is an amino acid residue selected from Asn or Ser, X2 is an amino acid residue selected from Ser, Glu or Arg, X3 is an amino acid residue selected from Tyr, His, Glu or Arg, X4 is an amino acid residue selected from Leu, His, Arg, Glu or Lys, X5 is an amino acid residue selected from Arg or Lys, and X6 is an amino acid residue selected from Phe or His;

[0547] A1 is an insulin A chain analogue with the sequence:

[0548] GIVEQCCZ1Z2Z3CSLZ4QLENYCZ5 (SEQ ID NO: 22), wherein Z1 is an amino acid residue selected from Thr, His, or Glu, Z2 is an amino acid residue selected from Ser or Lys, Z3 is an amino acid residue selected from Ile, Lys, or Thr, Z4 is an amino acid residue selected from Tyr, Glu, Ser, Lys, or Asp; and Z5 is an amino acid residue of Gly;

[0549] L1 is selected from GGGGGG (SEQ ID NO: 49), GGGGGGSGGGG (SEQ ID NO: 50), GGGGGSGGGG (SEQ ID NO: 51), specifically GGGGGG (SEQ ID NO: 49).

[0550] The sequence of an exemplary human insulin analog is as follows:

[0551] >Human insulin analog 1

[0552] >Human insulin analogue 2

[0553] >Human insulin analogue 3

[0554] >Human insulin analogue 4

[0555] >Human insulin analogue 5

[0556] >Human insulin analog 6

[0557] >Human insulin analog 7

[0558] >Human insulin analogue 8

[0559] >Human insulin analogue 9

[0560] >Human insulin analog 10

[0561] >Human insulin analog 11

[0562] >Human insulin analog 12

[0563] >Human insulin analog 13

[0564] >Human insulin analog 14

[0565] >Human insulin analog 15

[0566] >Human insulin analog 16

[0567] >Human insulin analog 17

[0568] >Human insulin analog 18

[0569] >Human insulin analog 19

[0570] >Human insulin analogue 20

[0571] >Human insulin analogue 21

[0572] >Human insulin analogue 22

[0573] >Human insulin analogue 23

[0574] >Human insulin analogue 24

[0575] >Human insulin analogue 25

[0576] >Human insulin analogue 26

[0577] >Human insulin analogue 27

[0578] >Human insulin analog 28

[0579] >Human insulin analogue 29

[0580] >Human insulin analogue 32

[0581] Furthermore, the above-mentioned single-chain insulin analog was further fused with the anti-HSA single-domain antibody to form a fusion protein. The fusion of the two was achieved through a linker as shown in Table 8. The structural diagram is shown in Figure 4.

[0582] Table 8. Linker sequences of single-chain insulin analogs fused to single-domain antibodies against HSA

[0583] The amino acid sequence of the anti-HSA single domain antibody is as follows:

[0584] >#7_Hu_10

[0585] The amino acid sequence of an exemplary fusion protein is as follows:

[0586] >SA0001

[0587] >SA0003

[0588] >SA0004

[0589] >SA0005

[0590] >SA0010

[0591] >SA0011

[0592] >SA0012

[0593] >SA0013

[0594] >SA0014

[0595] >SA0015

[0596] >SA0016

[0597] >SA0017

[0598] >SA0019

[0599] >SA0020

[0600] >SA0022

[0601] >SA0027

[0602] >SA0028

[0603] >SA0031

[0604] >SA0032

[0605] >SA0034

[0606] >SA0035

[0607] >SA0036

[0608] >SA0037

[0609] >SA0038

[0610] >SA0039

[0611] >SA0041

[0612] >SA0046

[0613] >SA0047

[0614] >1015

[0615] >1017

[0616] >1018

[0617] >1019

[0618] Among them, the italicized ones are anti-HSA single domain antibodies; the underlined ones are linkers.

[0619] In addition, this example uses fusion protein 1016 as a positive control, and fusion protein 1016 comes from Example 29 in WO2021022149.

[0620] >1016

[0621] Note: The underlined sequence is the linker sequence.

[0622] The wild-type human insulin sequence is as follows:

[0623] >A Chain

[0624] GIVEQCCTSICSLYQLENYCN(SEQ ID NO: 118)

[0625] >B Chain

[0626] FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 119).

[0627] The preparation and purification of insulin analog-HSAVHH fusion protein were performed as follows:

[0628] 1. Preparation of fusion protein:

[0629] The DNA sequence encoding the fusion protein was codon-optimized, synthesized, and cloned into the pTT5 expression vector. The expression plasmid was transiently transfected into expiCHO (ThermoFisher, Cat#.A29127) cells using the ExpiFectamine CHO Transfection Kit (ThermoFisher, Cat#.A29133) according to the kit's instructions. The "High" protocol was used. The day after transfection, the cells were rehydrated and incubated in a 32°C shaker (5% CO2, 110 rpm, 75% humidity) in suspension. On days 10-12 after transfection, the cell culture supernatant was harvested, centrifuged at 4000 rpm for 20 minutes, and filtered through a 0.45 μM filter.

[0630] 2. Purification of fusion protein:

[0631] A3 affinity chromatography to capture the fusion protein: The supernatant of cell culture expressing the fusion protein was collected by high-speed centrifugation and filtered through a 0.22 μm filter. An A3 (JSR Life Sciences, Cat#. BP-AMS-A3-0100) affinity column was regenerated with 5 column volumes of 0.1 M NaOH (Sigma, Cat#. 71687) and then washed and equilibrated with 5 column volumes of 1× PBS (pH 7.4) (Sangon Biotech (Shanghai) Co., Ltd., Cat#. E607016-0500). The filtered supernatant was loaded at a low flow rate to facilitate binding to the affinity column. The flow rate was controlled to maintain a retention time of approximately 1 min or longer. After binding, the column was rinsed with 5 column volumes of 1× PBS (pH 7.4), 5 column volumes of 1× PBS (pH 7.4) 0.8 M NaCl (Vetec, Cat#. V900058), and 5 column volumes of 1× PBS (pH 7.4) until UV absorbance returned to baseline. The sample was eluted with 0.1 M Glycine (pH 3.0) (Sigma, Cat#. 410225) buffer. The eluted peak was collected for UV detection. The eluted product was quickly adjusted to pH 7-8 with 1 M Tris-HCl (pH 7.5) (Vetec, Cat#. V900483) and stored temporarily. For the eluted product, solution replacement can be performed using methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube, and solution replacement to the desired buffer system, or replacement to the desired buffer system using molecular exclusion (such as G-25 desalting).

[0632] Example 3. Affinity determination of fusion protein and human serum albumin HSA

[0633] The affinity of the insulin analog-HSAVHH fusion protein to human serum albumin (HSA) was determined using a Biacore T200 (GE Healthcare) instrument, with a positive control of 1016. Human albumin (HSA) (Sigma, Cat# 126658) was used for the experiment.

[0634] Human serum albumin (HSA) was conjugated to an S-series CM5 sensor chip (GE, Cat. 29-1049-88) using an amino coupling kit (GE, Cat. BR-1000-50). HSA was diluted to 2 μg / mL using 10 mM sodium acetate, pH 4.5 (GE, Cat. BR-1003-50). After EDC / NHS activation, the CM5 chip was passed through the diluted albumin to a surface response of approximately 200 (180 to 230) RU, followed by blocking with ethanolamine. A blank control channel also required EDC / NHS activation and ethanolamine blocking.

[0635] A series of serial dilutions of the single-chain insulin analog fusion protein were prepared using 1× HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20): 222.22, 74.074, 24.69, 8.23, 2.74, 0.91, and 0.305 nM. Fusion protein samples of varying concentrations were passed through the HSA-coupled channel and a blank channel for 3 minutes at a flow rate of 50 μL / min, with a dissociation time of 4 minutes. After each cycle, the chip was regenerated using 10 mM glycine-HCl, pH 1.5 (GE, Cat.# BR-1003-54). Binding and dissociation curves were generated using a Biacore T200 (GE) system for real-time signal monitoring. The data were fitted to a Langmuir 1:1 binding model using BIA evaluation version 4.1 software at appropriate concentrations. The resulting affinity values ​​are shown in Table 9.

[0636] Table 9. Affinity for human serum albumin HSA

[0637] The results showed that the binding affinity of the fusion protein of the present disclosure to human serum albumin HSA was higher than that of the positive control 1016 molecules (Table 9).

[0638] Example 4. Affinity determination of fusion protein and human IR-A

[0639] The affinity of the insulin analog-HSAVHH fusion protein to human IR-A (Cat#.INR-H52Ha, Acro) was determined using a Biacore T200 (GE Healthcare) instrument.

[0640] Anti-His tag antibodies were coupled to the CM5 sensor chip (Cat. 29-1049-88, GE) using an amino coupling kit (Cat#. BR-1000-50, GE) and a His capture kit (Cat#. 28-9950-56, GE) according to the kit instructions, so that the surface response value reached approximately 10,000 RU, and then blocked with ethanolamine for use.

[0641] Human IR-A was diluted to 5 μg / ml in 1X HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20) and passed through the FC2 channel of an anti-his CM5 chip at a flow rate of 10 μL / min for antigen capture, resulting in a capture response of approximately 400-500 RU. A serial dilution series of 1X HBS-EP dilutions was performed: 100, 50, 25, 12.5, 6.25, and 3.125 μM. Samples of varying concentrations of the single-chain insulin analog fusion protein were then passed through the human IR-A capture channel and a blank channel for 1 minute at a flow rate of 50 μL / min, with a dissociation time of 1 minute. After each cycle, the chip was regenerated with 10 mM glycine-HCl, pH 1.5 (GE, Cat.# BR-1003-54). Biacore T200 (GE) was used to detect the reaction signal in real time to obtain the binding and dissociation curves. The obtained data were fitted with the Langmuir 1:1 binding model or steady-state affinity using BIAevaluation version 4.1 software at appropriate concentration points to obtain the affinity values ​​(Table 10).

[0642] Table 10. Affinity for human IR-A

[0643] Example 5. Biological activity of the fusion protein in CHO-K1 cells overexpressing hIRB receptors

[0644] Insulin binds to the receptor on CHO-K1-hIRB cells and promotes hIR phosphorylation. Therefore, this example examined the in vitro activity of different human insulin analog-HSAVHH fusion proteins by examining their ability to promote autophosphorylation of the human insulin receptor B subtype in CHO-K1 cells overexpressing the human insulin receptor B subtype.

[0645] This example used a homogeneous time-resolved fluorescence (HTRF) IR beta phospho-Y1150 / 1151 kit (Cisbio, Cat# 63ADK016PEG) according to the manufacturer's protocol. CHO-K1-hIRB (Shanghai Huiyuan, CHO-K1-hIRB-M-20170706) cells were cultured at 37°C, 5% CO₂ in growth medium consisting of F12K mixture, 10% FBS, 1% penicillin-streptomycin, and 4 μg / mL blasticidin. Separately, experimental medium 1 (EM1) consisted of DMEM without glucose, 1 mM sodium pyruvate solution, and 25 mM HEPES solution. Experimental medium 2 (EM2) consisted of EM1 supplemented with 1% HSA. On the first day of the experiment, cells were plated at 1E-04 cells / well in a 96-well plate. 100 μL of growth medium was used and the cells were incubated overnight at 37°C, 5% CO₂. The next day, serially dilute the test compound using EM1 or EM2. A maximum recommended concentration of 100 μM (final concentration) is recommended. After two 5-fold serial dilutions, perform five 10-fold dilutions, for a total of eight concentrations. Discard the supernatant from each well, wash the cells twice with 200 μL PBS, and immediately add 50 μL of the test compound dilution mixture. Incubate at 37°C, 5% CO₂ for 20 minutes. Discard the supernatant and add 40 μL of 1× lysis buffer (Cisbio, Cat#. 63ADK016PEG). Place the plate in a thermostatted shaker at 37°C, 300 rpm, for 30 minutes. Transfer 16 μL of the lysate to a low-volume 96-well plate (Cisbio, Cat#. 66PL96025). Add 4 μL of the prepared antibody mixture (d2-antibody (acceptor fluorophore) and cryptate-antibody (donor fluorophore)) and incubate at room temperature for 4 hours. The readings were then taken on a Tecan (Infinite 200PRO) at wavelengths of 620 nm and 665 nm. The signal intensity was expressed as the ratio of fluorescence intensity at 665 nm / fluorescence intensity at 620 nm × 10,000. Graphpad Prism was used for nonlinear fitting to plot the cAMP concentration curve (limiting the maximum activity of human insulin to 100%), and the EC values ​​of the test samples and controls for promoting hIRB phosphorylation in CHO-K1-hIRB cells were obtained. 50 The results are shown in Table 11, Figures 5A and 5B.

[0646] Table 11. In vitro activity of fusion proteins (CHO-K1-hIRB cell assay)

[0647] The results showed that the in vitro activity of the fusion protein of the present disclosure was comparable to or slightly stronger than that of the positive control molecule in the absence of HSA, indicating that the insulin analogs of the present disclosure all have agonist activity at the human insulin receptor.

[0648] In the presence of HSA, the in vitro activity of the fusion protein disclosed herein is generally weaker than that of the positive molecule, indicating that the fusion protein disclosed herein has a lower risk of inducing hypoglycemia in subjects during clinical use and has a safer dosing window.

[0649] Example 6. Pharmacokinetics of single-chain insulin analog fusion protein in rats

[0650] Three healthy male Sprague-Dawley rats, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., were used in this study. They were 7-8 weeks old and weighed 200-250 g. Upon arrival, the animals were acclimated for at least 7 days. During this period, they were housed on a 12 / 12 hour light / dark cycle, maintained at a temperature of 16-26°C and a relative humidity of 40-70%. On the day of the experiment, each of the three animals was intravenously injected with the fusion protein disclosed herein.

[0651] PK Sample Collection: Blood samples were collected from the orbital canthal vein at the following time points: before dosing and 15 minutes, 1 hour, 2 hours, and 4 hours after dosing, as well as on days 1, 3, 7, 11, 14, 21, and 28 after dosing. Blood samples were collected from the canthal vein of the SD rats via direct canthal vein puncture. 0.03 mL of whole blood was collected from each animal, anticoagulated with EDTA-K2, and immediately centrifuged at 4500 rpm for 5 minutes. Serum was collected in EP tubes and stored at -20°C. After the experiment, the serum was transferred to -80°C for analysis.

[0652] Blood glucose was measured simultaneously during the trial: before administration (T0), and on days 1, 3, 7, 11, 14, 21, and 28 after administration. Plasma drug concentrations were measured using ELISA.

[0653] Example 7. Effect of fusion protein on lowering blood sugar in STZ rats

[0654] For STZ rat (T1D) modeling, male SD rats weighing approximately 350 g and approximately 8 weeks old were used. Adaptive feeding was performed for one week. STZ was prepared in a citrate buffer solution at a dose of 60 mg / kg. Following STZ administration, the animals were fed normally. Blood glucose was measured 3 days after STZ injection. A blood glucose level of 15 mM or higher was considered successful modeling.

[0655] STZ rat (T1D) dosing experiment uses STZ male rats, weighing about 400g each. No fasting is required on the day of the experiment. Blood glucose is measured at T0 before dosing in the afternoon. Rats with blood glucose values ​​between 20 and 30mM are included in the group. Groups are divided according to blood glucose, and after grouping, the average blood glucose and standard deviation of each group of rats are ensured to be similar. If conditions permit, it is best that the average body weight and standard deviation of each group are also similar. In the afternoon, each group of STZ rats is given a single subcutaneous dose of the positive drug and the test drug. No fasting is required after dosing, and blood is collected from the tail vein at 4h, 24h, and on the second to Nth day (if the blood glucose value is lower than the T0 point, it needs to be tested again the next day) to monitor blood glucose levels and draw a curve chart of blood glucose changes over time. After dosing, the animals are fed normally until the end of the experiment.

Claims

1. A fusion protein comprising: serum albumin binding domain, and Insulin or its analogs, in, The serum albumin binding domain comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 selected from (1) or (2): (1) CDR1, CDR2 and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 1, 8-17, (2) CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 2-4, respectively, Wherein, the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

2. The fusion protein according to claim 1, wherein the immunoglobulin single variable domain is modified by any one of the following: humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerization, or a combination thereof; Preferably, the framework region template of the human germline gene used in the humanization modification is derived from IGHV3-23 or IGVH3-66.

3. The fusion protein according to claim 1 or 2, wherein the immunoglobulin single variable domain comprises an amino acid mutation, wherein the amino acid mutation is selected from the group consisting of amino acid residues at positions 20, 23, 27, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83, 84, and combinations thereof; The positions stated are based on the EU numbering system; Preferably, the mutation is selected from the group consisting of 20V, 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A, 74A, 78V, 83Q, 84P and combinations thereof; More preferably, the mutation is selected from the following combinations: 27N, 29Y, 30L, 37F, 44E, 45R, 47G; 20V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G; 23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G; 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 74A; 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 78V; 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A; 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 83Q, 84P; 27N, 29Y, 30L, 37F, 44E, 45R, 47G, 84P; 30L, 37F, 44E, 45R, 47G, 84P; or 30L, 37F, 44E, 45R, 47G.

4. The fusion protein according to any one of claims 1 to 3, wherein the immunoglobulin single variable domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1, 8 to 17 or having at least 90% sequence identity thereto.

5. The fusion protein according to any one of claims 1 to 4, wherein the immunoglobulin single variable domain is VHH.

6. The fusion protein according to any one of claims 1 to 5, wherein The insulin or its analogues comprises an A chain and a B chain, wherein: The B chain comprises the following amino acid sequence: FVX1QHLCGX2HLVEALX3X4VCGEX5GFX6YTPKT (SEQ ID NO: 21), wherein X1 is selected from N or S, X2 is selected from S, E or R, X3 is selected from Y, H, E or R, X4 is selected from L, H, R, E or K, X5 is selected from R or K, X6 is selected from F or H; The A chain comprises the following amino acid sequence: GIVEQCCZ1Z2Z3CSLZ4QLENYCZ5 (SEQ ID NO: 22), wherein Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I, K or T, Z4 is selected from Y, E, S, K or D, Z5 is selected from N or G; Preferably, X1 is N, X2 is selected from S, E or R, X3 is selected from Y, H or E, X4 is selected from L, H, R, E or K, X5 is selected from R or K, X6 is selected from F or H; and, Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I or K, Z4 is selected from Y, E, S or K, and Z5 is G.

7. The fusion protein according to claim 6, wherein The B chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 23-37, or a sequence having at least 90% identity thereto; and / or, The A chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 38-48, or a sequence having at least 90% identity thereto. Preferably, the B chain and the A chain comprise or are selected from any one of the following groups: The amino acid sequences shown in SEQ ID NO: 23 and SEQ ID NO: 38, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 41, The amino acid sequences shown in SEQ ID NO: 29 and SEQ ID NO: 45, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 39, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 40, The amino acid sequences shown in SEQ ID NO: 25 and SEQ ID NO: 40, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 38, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 42, The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 41, The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 42, The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 43, The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 40, The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 38, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 44, The amino acid sequences shown in SEQ ID NO: 27 and SEQ ID NO: 43, The amino acid sequences shown in SEQ ID NO: 28 and SEQ ID NO: 43, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 45, The amino acid sequences shown in SEQ ID NO:30 and SEQ ID NO:43, The amino acid sequences shown in SEQ ID NO:31 and SEQ ID NO:43, The amino acid sequences shown in SEQ ID NO:32 and SEQ ID NO:46, The amino acid sequences shown in SEQ ID NO:32 and SEQ ID NO:47, The amino acid sequences shown in SEQ ID NO:33 and SEQ ID NO:47, The amino acid sequences shown in SEQ ID NO:34 and SEQ ID NO:47, The amino acid sequences shown in SEQ ID NO:35 and SEQ ID NO:43, The amino acid sequences shown in SEQ ID NO:36 and SEQ ID NO:43, The amino acid sequences shown in SEQ ID NO:32 and SEQ ID NO:43, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 43, The amino acid sequences shown in SEQ ID NO: 29 and SEQ ID NO: 43, The amino acid sequences shown in SEQ ID NO:37 and SEQ ID NO:39, or The amino acid sequences shown in SEQ ID NO:34 and SEQ ID NO:

48.

8. The fusion protein according to any one of claims 1 to 7, wherein The fusion protein comprises a structure selected from any one of the following from the N-terminus to the C-terminus: B-L1-A-L2-VHH Formula (I) A-L1-B-L2-VHH Formula (II) VHH-L2-B-L1-A Formula (III) VHH-L2-A-L1-B Formula (IV) in, The A is the A chain defined in claim 6 or 7, Said B is the B chain defined in claim 6 or 7, The VHH is the VHH defined in claim 5; L1 and L2 are linkers. L1 and L2 are each independently present or absent.

9. The fusion protein according to claim 8, wherein The L1 is selected from a GS linker and / or comprises a linker having at least 4 Gs; preferably, the L1 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 49-51; The L2 is selected from any one of the following: m Q i ) n , (G m A i ) n , (G m Q) n , (G m A) n 、(PGPQ) s , and (PGPA) s , wherein m is independently selected from an integer of 1-10, n is independently selected from an integer of 1-10, i is independently selected from an integer of 0-4; s is selected from an integer of 1-10; More preferably, said L2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 52-54.

10. The fusion protein according to any one of claims 1 to 9, wherein The insulin analog comprises an amino acid sequence shown in any one of SEQ ID NOs: 55-84, or a sequence having at least 90% identity thereto.

11. The fusion protein according to any one of claims 1 to 10, wherein The fusion protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 85-116, or a sequence having at least 90% identity thereto.

12. The fusion protein according to any one of claims 1 to 11, wherein The fusion protein is a single-chain protein, a dimer protein or a multimer protein; preferably a single-chain protein.

13. The fusion protein according to any one of claims 1 to 12, having at least one property selected from the group consisting of: (a)≤1×10 -7 M, preferably ≤1×10 -9 M of K D Values ​​bind to human serum albumin; (b) agonist activity at the insulin receptor; (c) Binds to human insulin alpha receptor (IR-A); (d) Promotes phosphorylation of human insulin β receptor (IR-B); (e) Compared with natural insulin, it has a prolonged half-life.

14. An insulin analogue comprising an A chain and a B chain, wherein The B chain comprises the following amino acid sequence: FVX1QHLCGX2HLVEALX3X4VCGEX5GFX6YTPKT (SEQ ID NO: 21), wherein X1 is selected from N or S, X2 is selected from S, E or R, X3 is selected from Y, H, E or R, X4 is selected from L, H, R, E or K, X5 is selected from R or K, X6 is selected from F or H; The A chain comprises the following amino acid sequence: GIVEQCCZ1Z2Z3CSLZ4QLENYCZ5 (SEQ ID NO: 22), wherein Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I, K or T, Z4 is selected from Y, E, S, K or D, Z5 is G; Preferably, X1 is N, X2 is selected from S, E or R, X3 is selected from Y, H or E, X4 is selected from L, H, R, E or K, X5 is selected from R or K, X6 is selected from F or H; and, Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is selected from I or K, Z4 is selected from Y, E, S or K, and Z5 is G.

15. The insulin analogue according to claim 14, wherein: 1) X1 is N, X2 is S, X3 is selected from Y, H or E, X4 is selected from E, L, H or R, X5 is R, X6 is H; Z1 is selected from T, H or E, Z2 is selected from S or K, Z3 is I, Z4 is Y, E, S or K, and Z5 is G; or 2) X1 is N, X2 is E, X3 is selected from Y or E, X4 is selected from E, L, H or K, X5 is R or K, X6 is H or F; Z1 is E, Z2 is S, Z3 is selected from I or K, Z4 is E or K, and Z5 is G; or, 3) X1 is N, X2 is R, X3 is selected from Y, X4 is selected from H, X5 is R, X6 is H; Z1 is E, Z2 is S, Z3 is I, Z4 is E, and Z5 is G.

16. The insulin analogue according to claim 14 or 15, wherein: The B chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 23-37, or a sequence having at least 90% identity thereto; and / or, The A chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 38-48, or a sequence having at least 90% identity thereto; Preferably, the B chain and the A chain comprise or are selected from any one of the following groups: The amino acid sequences shown in SEQ ID NO: 23 and SEQ ID NO: 38, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 41, The amino acid sequences shown in SEQ ID NO: 29 and SEQ ID NO: 45, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 39, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 40, The amino acid sequences shown in SEQ ID NO: 25 and SEQ ID NO: 40, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 38, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 42, The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 41, The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 42, The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 43, The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 40, The amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 38, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 44, The amino acid sequences shown in SEQ ID NO: 27 and SEQ ID NO: 43, The amino acid sequences shown in SEQ ID NO: 28 and SEQ ID NO: 43, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 45, The amino acid sequences shown in SEQ ID NO:30 and SEQ ID NO:43, The amino acid sequences shown in SEQ ID NO:31 and SEQ ID NO:43, The amino acid sequences shown in SEQ ID NO:32 and SEQ ID NO:46, The amino acid sequences shown in SEQ ID NO:32 and SEQ ID NO:47, The amino acid sequences shown in SEQ ID NO:33 and SEQ ID NO:47, The amino acid sequences shown in SEQ ID NO:34 and SEQ ID NO:47, The amino acid sequences shown in SEQ ID NO:35 and SEQ ID NO:43, The amino acid sequences shown in SEQ ID NO:36 and SEQ ID NO:43, The amino acid sequences shown in SEQ ID NO:32 and SEQ ID NO:43, The amino acid sequences shown in SEQ ID NO: 24 and SEQ ID NO: 43, The amino acid sequences shown in SEQ ID NO: 29 and SEQ ID NO: 43, The amino acid sequences shown in SEQ ID NO:37 and SEQ ID NO:39, or The amino acid sequences shown in SEQ ID NO:34 and SEQ ID NO:

48.

17. The insulin analogue according to any one of claims 14 to 16, wherein A disulfide bond is formed between the A chain and the B chain; or, the A chain and the B chain are connected directly or through a linker; Preferably, the linker is selected from a GS linker and / or a linker comprising at least 4 Gs, and more preferably comprises an amino acid sequence shown in any one of SEQ ID NOs: 49-51.

18. A polynucleotide encoding: The fusion protein according to any one of claims 1 to 13, or The insulin analogue according to any one of claims 14 to 17.

19. A vector comprising or expressing the polynucleotide of claim 18.

20. A host cell comprising or expressing the vector of claim 19.

21. A method for producing or preparing the fusion protein according to any one of claims 1 to 13 or the insulin analogue according to any one of claims 14 to 17, comprising the steps of: Cultivating the host cell of claim 20; isolating the fusion protein or the insulin analog, and Optionally, the fusion protein or insulin analog is purified and / or modified.

22. A pharmaceutical composition comprising: The fusion protein of any one of claims 1 to 13, the insulin analog of any one of claims 14 to 17, the polynucleotide of claim 18 or the vector of claim 19; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

23. Use of the fusion protein according to any one of claims 1 to 13, the insulin analogue according to any one of claims 14 to 17, the polynucleotide according to claim 18, the vector according to claim 19 or the pharmaceutical composition according to claim 22 in any of the following: (1) Use in the preparation of a medicament for treating or preventing metabolic diseases, disorders and / or conditions; (2) for the treatment or prevention of metabolic diseases, conditions and / or disorders; (3) To reduce the risk of hypoglycemia in subjects with metabolic diseases, conditions and / or disorders after receiving drug treatment; Preferably, the metabolic disease, condition and / or disorder is selected from diabetes or its complications, obesity or its complications, dyslipidemia and / or metabolic syndrome; Preferably, the diabetes or its complications are selected from: type I diabetes, type II diabetes and their complications.

Images