Insulin analogs and uses thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an insulin analogue and application thereof. The present disclosure provides an insulin analogue selected from at least three mutations, relative to a parent insulin, from the group consisting of positions A2, A4, A8, A14, A18, B23, B26, B27, B31, B32, B34, B37, B38, B42, B44, B46, B47, and B50. According to the insulin analogue disclosed by the invention, by reducing the affinity with an insulin receptor, the timeliness can be improved, the compliance of a patient is improved, and the safety and the controllability are improved.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of Chinese Patent Application No. 2023116328496, filed on December 1, 2023, the entire text of which is incorporated herein by reference. Technical field

[0003] The present disclosure belongs to the field of biomedicine, and specifically relates to an insulin analogue and its uses. Background art

[0004] Diabetes is caused by hyperglycemia due to insulin deficiency or insulin resistance. Insulin therapy is an important method for diabetes management. By injecting insulin, it helps control blood glucose levels to prevent and reduce diabetes - related complications. Although the progression of diabetes may vary from person to person, insulin therapy is required in the later stage of the disease. Insulin lowers blood glucose through multiple pathways. First, it can directly bind to the insulin receptor, promoting the translocation of the glucose transporter GLUT4 from inside the cell to the cell membrane, increasing the glucose transporters on the cell membrane, enhancing the cell's ability to take up extracellular glucose, thus promoting glucose entry into the cell and enabling the cell to use glucose as an energy source. Second, insulin can inhibit glucose synthesis in the liver, reducing the release of glucose from the liver into the bloodstream. In addition, insulin can also promote the uptake and utilization of glucose by adipose tissue and muscle tissue, thereby further lowering blood glucose levels.

[0005] In diabetes treatment, insulin is widely used. For patients with type 1 diabetes, due to the loss of pancreatic β - cell function, they need exogenous insulin to replace the lack of insulin in the body. This insulin therapy can effectively control blood glucose levels and prevent and reduce diabetes - related complications. For patients with type 2 diabetes, insulin therapy is usually considered when oral drug treatment is ineffective or not applicable. Insulin can help improve the sensitivity of cells to insulin, promote the utilization of glucose, and control blood glucose levels. With the progress of technology, insulin formulations have also been continuously developed. From the first - generation insulin - animal insulin to the second - generation insulin - human insulin, and then to the third - generation insulin - insulin analogue, the research and development of insulin have been continuously pursuing a better simulation of the normal physiological blood - glucose - lowering mode in the human body. These improvements have made insulin therapy safer and more effective, providing better tools for diabetes patients to manage their blood glucose and being beneficial to improving the quality of life of patients.

[0006] Currently, insulin on the market (including recombinant insulin and its analogs) is mainly divided into long-acting, intermediate-acting, rapid-acting, and premixed insulin, etc. The market demand for long-acting insulin analogs mainly comes from type 2 diabetes patients. These patients need to use insulin for a long time to control blood glucose levels and avoid the occurrence of complications. The application prospect of long-acting insulin analogs is broad, and most doctors and patients expect an insulin treatment method that can well control blood glucose but does not require daily injection.

[0007] First of all, long-acting insulin can provide a more convenient treatment method. Traditional insulin treatment requires multiple injections, which brings inconvenience to patients. While long-acting insulin analogs can reduce the number of injections and improve patients' compliance and quality of life. Secondly, long-acting insulin analogs have a more stable drug effect. It can mimic the insulin secretion pattern, keep the blood glucose level stable within a certain range, and reduce the occurrence of hypoglycemia and hyperglycemia. In addition, long-acting insulin analogs also have lower risks and side effects, which can better meet the treatment needs of patients.

[0008] Most of the common insulins on the market currently have problems with treatment compliance and safety. The half-life of traditional insulin in the body is relatively short and it is easily cleared, resulting in insufficient long-term effectiveness and unable to provide long-lasting blood glucose control. This leads to patients needing to inject insulin frequently, increasing the burden and inconvenience of treatment and reducing patients' compliance. Traditional insulin has a relatively high affinity for binding to insulin receptors, resulting in too fast absorption rate of insulin in the body, causing a drastic fluctuation in blood glucose levels and possibly triggering a series of side effects such as hypoglycemia. These side effects not only affect patients' quality of life but also may pose potential risks to their physical health. Therefore, it is necessary to rationally reduce the affinity of insulin for receptors, slow down the receptor-mediated endocytosis and degradation process, design and develop new long-acting insulin analogs to improve the efficacy, compliance and safety of patients. Summary of the Invention

[0009] The present disclosure relates to a long-acting insulin analog and its use. This insulin analog has a reduced insulin receptor binding activity, thus having a reduced receptor-mediated clearance rate, and at the same time has a signal transduction activity that allows sufficient reduction of blood glucose levels in the body and can play a long-term role in patients.

[0010] In one aspect, the present disclosure provides an insulin analog that has at least three mutations selected from the following positions relative to the parental insulin: positions A2, A4, A8, A14, A18, B23, B26, B27, B31, B32, B34, B37, B38, B42, B44, B46, B47, and B50.

[0011] On the other hand, the present disclosure provides a nucleic acid molecule encoding the above insulin analogue.

[0012] On the other hand, the present disclosure provides a vector comprising the above nucleic acid molecule.

[0013] On the other hand, the present disclosure provides a host cell comprising the above vector.

[0014] On the other hand, the present disclosure provides a pharmaceutical composition comprising the above insulin analogue and a pharmaceutically acceptable carrier.

[0015] On the other hand, the present disclosure provides the use of the above insulin analogue in the preparation of a drug for treating, preventing or alleviating a disease or disorder, wherein the disease or disorder is related to diabetes or elevated blood glucose.

[0016] The insulin analogue provided by the present disclosure has the following beneficial effects:

[0017] 1. Improved timeliness: The novel long-acting insulin analogue extends the half-life of the drug in vivo by reducing its affinity for the insulin receptor, thereby prolonging the duration of the drug effect. This enables patients to maintain stable blood glucose levels with fewer injection times, reducing patient inconvenience and pain.

[0018] 2. Improved patient compliance: Traditional insulin therapy requires patients to inject insulin multiple times, either before or after meals, which causes great inconvenience to patients. The long-acting nature of the novel long-acting insulin analogue allows patients to inject once a day or once every two days, reducing the patient's burden and improving patient compliance.

[0019] 3. Improved safety: The novel long-acting insulin analogue has a longer duration of drug effect and smaller fluctuations in blood glucose levels, thereby reducing the risk of hypoglycemia and hyperglycemia.

[0020] 4. By introducing only site-directed mutations and not site-directed modification of fatty chains, the affinity of the insulin analogue for the insulin receptor is reduced, thereby achieving long-acting effects, which helps to reduce production costs and simplify the production process.

[0021] In summary, the novel long-acting insulin analogue provided by the present disclosure has advantages and characteristics such as improved timeliness, improved patient compliance, improved safety and strong controllability by reducing its affinity for the insulin receptor, providing a more convenient and effective treatment option for diabetic patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shows the half-life of the long-acting insulin analogue in Example 2.

[0023] Figure 2 Shows the time course of the long-acting insulin analogue in a mouse model in Example 3. Detailed implementation manners

[0024] I. Definitions

[0025] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory operation procedures used herein are all terms and conventional procedures widely used in the corresponding fields. Meanwhile, to better understand the present disclosure, definitions and explanations of related terms are provided below.

[0026] As used herein, the term "parent insulin" refers to naturally occurring insulin, that is, wild-type insulin without mutation. In some embodiments, the parent insulin is animal insulin, such as mammalian insulin. For example, the parent insulin can be human insulin, porcine insulin or bovine insulin. The term "human insulin" as used herein means the human insulin hormone, the structure and properties of which are well known. Human insulin has two polypeptide chains, named chain A and chain B. Chain A is a peptide of 21 amino acids, while chain B is a peptide of 30 amino acids, and these two chains are connected by the following disulfide bridges: the first bridge between cysteine at position 7 on chain A and cysteine at position 7 on chain B, and the second bridge between cysteine at position 20 on chain A and cysteine at position 19 on chain B. The third bridge exists between cysteines at positions 6 and 11 on chain A.

[0027] As used herein, the term "insulin analogue" means a modified human insulin, in which one or more amino acid residues of the insulin have been replaced by other amino acid residues, and / or in which one or more amino acid residues have been deleted from the insulin, and / or in which one or more amino acid residues have been added and / or inserted into the insulin.

[0028] As used herein, the term "amino acid" includes proteinogenic (or natural) amino acids (among which there are 20 standard amino acids) and non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are amino acids that are naturally incorporated into proteins. Standard amino acids are amino acids encoded by the genetic code. Non-proteinogenic amino acids either do not exist in proteins or are not produced by standard cellular mechanisms (for example, they may have undergone post-translational modifications).

[0029] As used herein, the terms "protein", "polypeptide", and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly encompasses variants with conservative modifications (conservative substitutions).

[0030] As used herein, the terms sequence "identity", "identity", or "homology" have the meanings recognized in the art, and the percentage of sequence identity between two nucleic acids or polypeptides or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods for measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art.

[0031] As used herein, the term "substitution-type" variant is a variant in which at least one amino acid residue in the native sequence is removed and a different amino acid is inserted in its place. The substitution can be single, where only one amino acid in the molecule is replaced; or it can be multiple, where two or more amino acids in the same molecule are replaced. Multiple substitutions can be at contiguous sites. Also, one amino acid can be replaced by multiple residues, where such variants include both substitutions and insertions. An "insertion-type" variant is a variant in which one or more amino acids are inserted adjacent to an amino acid at a particular position in a native sequence. Adjacent amino acid means linked to the α-carboxyl or α-amino functional group of the amino acid. A "deletion-type" variant is a variant in which one or more amino acids are removed from the native amino acid sequence. Typically, deletion-type variants have one or two amino acids deleted in a particular region of their molecule.

[0032] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to oligomers or polymers containing at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are typically linked together by phosphodiester bonds. As used herein, the terms "polynucleotide" and "nucleic acid molecule" are intended to include DNA molecules and RNA molecules, which can be single-stranded or double-stranded, and can be cDNA. The term also includes codon-optimized nucleic acid molecules.

[0033] As used herein, the terms "conservative substitution" or "conservative sequence modification" of a sequence refer to nucleotide and amino acid sequence modifications that do not eliminate the binding of an antibody encoded by or containing an amino acid sequence to an antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listings described herein by standard techniques known in the art (such as site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (such as lysine, arginine, histidine), amino acids having acidic side chains (such as aspartic acid, glutamic acid), amino acids having uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (such as threonine, valine, isoleucine), and amino acids having aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0034] As used herein, the term "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods can include, but are not limited to, quantifying the polypeptide in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0035] As used herein, the term "host cell" is a cell used to receive, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. A host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

[0036] As used herein, the term "vector" is a replicable nucleic acid that, when transformed into a suitable host cell, can express one or more heterologous proteins from the vector. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced by restriction enzyme digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplifying the nucleic acids or for expressing / displaying the polypeptides encoded by the nucleic acids. Vectors generally remain free, but can be designed to integrate a gene or part thereof into the chromosomes of the genome. Vectors of artificial chromosomes are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vectors are known to those skilled in the art.

[0037] As used herein, a vector also includes a "viral vector" or "vector of a virus". A vector of a virus is an engineered virus that is operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.

[0038] As used herein, the term "expression vector" includes a vector capable of expressing DNA that is operably linked to regulatory sequences capable of influencing the expression of such DNA fragments, such as a promoter region. Such additional fragments can include promoter and terminator sequences and optionally can include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors generally are derived from plasmid or viral DNA or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector that, when introduced into a suitable host cell, results in the expression of the cloned DNA. Suitable expression vectors are known to those skilled in the art and include expression vectors replicable in eukaryotic cells and / or prokaryotic cells and expression vectors that remain free or integrate into the genome of the host cell.

[0039] The term "pharmaceutical carrier" or "pharmaceutically acceptable formulation" refers to one or more non-toxic materials that are administered with a therapeutic agent and do not interfere with the biological activity of the active ingredient, including but not limited to buffers, preservatives, compatible carriers, diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), excipients, vehicles, and optionally other additives or encapsulating materials. Pharmaceutical carriers suitable for the present disclosure can be conventional pharmaceutical formulation excipients; and are suitable for delivering the compositions and formulations of the disclosed neutralizing antibodies.

[0040] As used herein, the term "treating" an individual having a disease or medical condition means that the symptoms of the individual are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing a potential disease and / or preventing the worsening of symptoms or the development of a disease. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided herein and any composition provided herein.

[0041] As used herein, the term "treating" an individual having a disease or medical condition means that the symptoms of the individual are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing a potential disease and / or preventing the worsening of symptoms or the development of a disease. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided herein and any composition provided herein.

[0042] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, is at least sufficient to produce a therapeutic effect. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or disorder.

[0043] As used herein, the term "preventively effective amount" or "preventively effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired preventive effect, e.g., preventing or delaying the onset or recurrence of a disease or symptoms, reducing the likelihood of the occurrence or recurrence of a disease or symptoms. A fully preventive effective dose need not occur by administration of a single dose and may occur only after administration of a series of doses. Thus, a preventively effective amount may be administered in one or more administrations.

[0044] As used herein, the term "patient" or "subject" refers to a mammal, such as a human.

[0045] II. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0046] In one aspect, the present disclosure provides an insulin analogue having at least three mutations relative to the parental insulin selected from the following positions: positions A2, A4, A8, A14, A18, B23, B26, B27, B31, B32, B34, B37, B38, B42, B44, B46, B47, and B50.

[0047] In some embodiments, the above parental insulin is human insulin, porcine insulin, or bovine insulin.

[0048] In some preferred embodiments, the above parental insulin is human insulin.

[0049] In some embodiments, the mutations in the above insulin analogs include:

[0050] (1) The amino acid at position A2 is replaced by aspartic acid (Asp);

[0051] (2) The amino acid at position A4 is replaced by lysine (Lys);

[0052] (3) The amino acid at position A8 is replaced by lysine (Lys);

[0053] (4) The amino acid at position A14 is replaced by an amino acid selected from methionine (Met), alanine (Ala), or threonine (Thr);

[0054] (5) The amino acid at position A18 is replaced by alanine (Ala);

[0055] (6) The amino acid at position B23 is replaced by alanine (Ala);

[0056] (7) The amino acid at position B26 is replaced by glutamine (Gln);

[0057] (8) The amino acid at position B27 is replaced by lysine (Lys);

[0058] (9) The amino acid at position B31 is replaced by an amino acid selected from glycine (Gly), glutamine (Gln), or serine (Ser);

[0059] (10) The amino acid at position B32 is replaced by histidine (His);

[0060] (11) The amino acid at position B34 is replaced by an amino acid selected from lysine (Lys), phenylalanine (Phe), serine (Ser), or alanine (Ala);

[0061] (12) The amino acid at position B37 is replaced by proline (Pro);

[0062] (13) The amino acid at position B38 is replaced by aspartic acid (Asp);

[0063] (14) The amino acid at position B42 is replaced by an amino acid selected from glutamine (Gln) or threonine (Thr);

[0064] (15) The amino acid at position B44 is replaced by glutamic acid (Glu);

[0065] (16) The amino acid at position B46 is replaced by glutamine (Gln);

[0066] The amino acid at position B47 is replaced by glycine (Gly); and / or

[0067] (18) The amino acid at position B50 is replaced by an amino acid selected from alanine (Ala) or asparagine (Asn). In some embodiments, the A chain of the insulin analog comprises a mutation or combination of mutations selected from the following:

[0068] (1) N18A;

[0069] (2) Y14M and N18A;

[0070] (3) T8K;

[0071] (4) E4K;

[0072] (5) E4K and Y14M;

[0073] (6) Y14M;

[0074] (7) Y14A;

[0075] (8) I2D and Y14T;

[0076] (9) Y14T;

[0077] (10) E4K and N18A;

[0078] (11) Y14A and N18A; or

[0079] (12) Y14M and N18A.

[0080] In some embodiments, the B chain of the insulin analog comprises a mutation or combination of mutations selected from the following:

[0081] (1) H31G, L32H and E34K;

[0082] (2) V23A, H31Q and E42Q;

[0083] (3) H31G and L38D;

[0084] (4) F46Q;

[0085] (5) L27K, E34F and G44E;

[0086] (6) E34S, Y47G and K50A;

[0087] (7) V23A, H31S and E42T;

[0088] (8) H31S and E34F;

[0089] (9) L38D, Y47G, and K50A;

[0090] (10) H31Q, E34K, and E42T;

[0091] (11) V23A, E34S, and Y37P;

[0092] (12) H31Q and Y37P;

[0093] (13) H31Q;

[0094] (14) H31G and K50A;

[0095] (15) H31G and K50N;

[0096] (16) H31S, Y37P, and Y47G;

[0097] (17) H31S and Y47G;

[0098] (18) E34S and K50N;

[0099] (19) H31S and E34A;

[0100] (20) V23A, H31G, and L38D;

[0101] (21) Y37P and E42T;

[0102] (22) H31S, E34A, and Y47G;

[0103] (23) H31Q, L38D, and Y47G;

[0104] (24) H31G, E34K, and L38D;

[0105] (25) V23A and E34A;

[0106] (26) H31G, Y37P, and L38D;

[0107] (27) H26Q and E34A;

[0108] (28) H31Q and E34A;

[0109] (29) H31G, E34A, and E42Q;

[0110] (30) H26Q;

[0111] (31) H26Q, E34S, and Y47G;

[0112] (32) Y37P;

[0113] (33)H31G and Y47G;

[0114] (34)E34F, E42T and Y47G;

[0115] (35)V23A, H31G and E34S;

[0116] (36)H31G, L32H and E34S;

[0117] (37)H31G and E34K;

[0118] (38)H31G and Y37P;

[0119] (39)H26Q, H31S and E34A;

[0120] (40)H26Q and H31Q; or

[0121] (41)H31S and E34S.

[0122] In some embodiments, the insulin analogs comprise a combination of mutations selected from the following:

[0123] (1) B-chain: H31G, L32H and E34K;

[0124] (2) B-chain: V23A, H31Q and E42Q;

[0125] (3) A-chain: N18A, and B-chain: H31G and L38D;

[0126] (4) A-chain: Y14M and N18A, and B-chain: F46Q;

[0127] (5) B-chain: L27K, E34F and G44E;

[0128] (6) B-chain: E34S, Y47G and K50A;

[0129] (7) B-chain: V23A, H31S and E42T;

[0130] (8) A-chain: T8K, and B-chain: H31S and E34F;

[0131] (9) B-chain: L38D, Y47G and K50A

[0132] (10) B-chain: H31Q, E34K and E42T

[0133] (11) B-chain: V23A, E34S and Y37P

[0134] (12) A-chain: E4K, and B-chain: H31Q and Y37P;

[0135] (13) Chain A: E4K and Y14M, and Chain B: H31Q;

[0136] (14) Chain A: Y14M, and Chain B: H31G and K50A;

[0137] (15) Chain A: N18A, and Chain B: H31G and K50N;

[0138] (16) Chain B: H31S, Y37P and Y47G;

[0139] (17) Chain A: Y14A, and Chain B: H31S and Y47G;

[0140] (18) Chain A: Y14M, and Chain B: E34S and K50N;

[0141] (19) Chain A: T8K, and Chain B: H31S and E34A;

[0142] (20) Chain B: V23A, H31G and L38D;

[0143] (21) Chain A: Y14A, and Chain B: Y37P and E42T;

[0144] (22) Chain B: H31S, E34A and Y47G;

[0145] (23) Chain B: H31Q, L38D and Y47G;

[0146] (24) Chain A: I2D and Y14T, and Chain B: H31Q;

[0147] (25) Chain B: H31G, E34K and L38D;

[0148] (26) Chain A: Y14A, and Chain B: V23A and E34A;

[0149] (27) Chain A: Y14T, and Chain B: E34S and K50N;

[0150] (28) Chain B: H31G, Y37P and L38D;

[0151] (29) Chain A: T8K, and Chain B: H26Q and E34A;

[0152] (30) Chain A: E4K, and Chain B: H31Q and E34A;

[0153] (31) Chain B: H31G, E34A and E42Q;

[0154] (32) Chain A: E4K and N18A, and Chain B: H26Q;

[0155] (33) Chain B: H26Q, E34S, and Y47G;

[0156] (34) Chain A: Y14A and N18A, and Chain B: Y37P;

[0157] (35) Chain A: Y14A, and Chain B: H31G and Y47G;

[0158] (36) Chain B: E34F, E42T, and Y47G;

[0159] (37) Chain B: V23A, H31G, and E34S;

[0160] (38) Chain A: Y14A, and Chain B: H31G and K50A;

[0161] (39) Chain A: Y14T, and Chain B: H31G and Y47G;

[0162] (40) Chain B: H31G, L32H, and E34S;

[0163] (41) Chain A: N18A, and Chain B: H31G and E34K;

[0164] (42) Chain A: Y14M and N18A, and Chain B: Y37P;

[0165] (43) Chain A: Y14A, and Chain B: H31G and Y37P;

[0166] (44) Chain B: H26Q, H31S, and E34A;

[0167] (45) Chain A: N18A, and Chain B: H31S and Y47G;

[0168] (46) Chain A: Y14A, and Chain B: H26Q and H31Q; or

[0169] (47) Chain A: Y14T, and Chain B: H31S and E34S.

[0170] In some embodiments, the A chain of the above insulin analogue comprises an amino acid sequence selected from those shown in SEQ ID NO: 1, 5, 7, 12, 17, 19, 21, 23, 26, 28, 30, 33, 37, 40, 42, 45, 47, 50, 53, 55, 59, 61, 64, 66, 68, 71, 73, and 75, or having an identity of 80% or more thereto; and / or, the B chain of the insulin analogue comprises an amino acid sequence selected from those shown in SEQ ID NO: 3, 4, 6, 8-11, 13-16, 18, 20, 22, 24, 25, 27, 29, 31, 32, 34-36, 38, 39, 41, 43, 44, 46, 48, 49, 51, 52, 54, 56-58, 60, 62, 63, 65, 67, 69, 70, 72, 74, and 76, or having an identity of 80% or more thereto.

[0171] In some embodiments, the above insulin analogue comprises an amino acid sequence combination selected from the following:

[0172] (1) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 3 or has an identity of 80% or more thereto;

[0173] (2) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 4 or has an identity of 80% or more thereto;

[0174] (3) The A chain is as shown in SEQ ID NO: 5 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 6 or has an identity of 80% or more thereto;

[0175] (4) The A chain is as shown in SEQ ID NO: 7 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 8 or has an identity of 80% or more thereto;

[0176] (5) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 9 or has an identity of 80% or more thereto;

[0177] (6) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 10 or has an identity of 80% or more thereto;

[0178] (7) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 11 or has an identity of 80% or more thereto;

[0179] (8) The A chain is as shown in SEQ ID NO: 12 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 13 or has an identity of 80% or more thereto;

[0180] (9) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 14 or has an identity of 80% or more thereto;

[0181] (10) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 15 or has an identity of 80% or more thereto;

[0182] (11) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 16 or has an identity of 80% or more thereto;

[0183] (12) The A chain is as shown in SEQ ID NO: 17 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 18 or has an identity of 80% or more thereto;

[0184] (13) The A chain is as shown in SEQ ID NO: 19 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 20 or has an identity of 80% or more thereto;

[0185] (14) The A chain is as shown in SEQ ID NO: 21 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 22 or has an identity of 80% or more thereto;

[0186] (15) The A chain is as shown in SEQ ID NO: 23 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 24 or has an identity of 80% or more thereto;

[0187] (16) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 25 or has an identity of 80% or more thereto;

[0188] (17) The A chain is as shown in SEQ ID NO: 26 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 27 or has an identity of 80% or more thereto;

[0189] (18) The A chain is as shown in SEQ ID NO: 28 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 29 or has an identity of 80% or more thereto;

[0190] (19) The A chain is as shown in SEQ ID NO: 30 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 31 or has an identity of 80% or more thereto;

[0191] (20) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 32 or has an identity of 80% or more thereto;

[0192] (21) The A chain is as shown in SEQ ID NO: 33 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 34 or has an identity of 80% or more thereto;

[0193] (22) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 35 or has an identity of 80% or more thereto;

[0194] (23) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 36 or has an identity of 80% or more thereto;

[0195] (24) The A chain is as shown in SEQ ID NO: 37 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 38 or has an identity of 80% or more thereto;

[0196] (25) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 39 or has an identity of 80% or more thereto;

[0197] (26) The A chain is as shown in SEQ ID NO: 40 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 41 or has an identity of 80% or more thereto;

[0198] (27) The A chain is as shown in SEQ ID NO: 42 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 43 or has an identity of 80% or more thereto;

[0199] (28) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 44 or has an identity of 80% or more thereto;

[0200] (29) The A chain is as shown in SEQ ID NO: 45 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 46 or has an identity of 80% or more thereto;

[0201] (30) The A chain is as shown in SEQ ID NO: 47 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 48 or has an identity of 80% or more thereto;

[0202] (31) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 49 or has an identity of 80% or more thereto;

[0203] (32) The A chain is as shown in SEQ ID NO: 50 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 51 or has an identity of 80% or more thereto;

[0204] (33) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 52 or has an identity of 80% or more thereto;

[0205] (34) The A chain is as shown in SEQ ID NO: 53 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 54 or has an identity of 80% or more thereto;

[0206] (35) The A chain is as shown in SEQ ID NO: 55 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 56 or has an identity of 80% or more thereto;

[0207] (36) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 57 or has an identity of 80% or more thereto;

[0208] (37) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 58 or has an identity of 80% or more thereto;

[0209] (38)The A chain is as shown in SEQ ID NO:59 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:60 or has an identity of 80% or more thereto;

[0210] (39)The A chain is as shown in SEQ ID NO:61 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:62 or has an identity of 80% or more thereto;

[0211] (40)The A chain is as shown in SEQ ID NO:1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:63 or has an identity of 80% or more thereto;

[0212] (41)The A chain is as shown in SEQ ID NO:64 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:65 or has an identity of 80% or more thereto;

[0213] (42)The A chain is as shown in SEQ ID NO:66 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:67 or has an identity of 80% or more thereto;

[0214] (43)The A chain is as shown in SEQ ID NO:68 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:69 or has an identity of 80% or more thereto;

[0215] (44)The A chain is as shown in SEQ ID NO:1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:70 or has an identity of 80% or more thereto;

[0216] (45)The A chain is as shown in SEQ ID NO:71 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:72 or has an identity of 80% or more thereto;

[0217] (46)The A chain is as shown in SEQ ID NO:73 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:74 or has an identity of 80% or more thereto; or

[0218] (47)The A chain is as shown in SEQ ID NO:75 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:76 or has an identity of 80% or more thereto.

[0219] On the other hand, the present disclosure provides a nucleic acid molecule encoding the above insulin analog.

[0220] On the other hand, the present disclosure provides a vector comprising the nucleic acid molecule as described above.

[0221] In some preferred embodiments, the above-mentioned vector is an expression vector.

[0222] On the other hand, the present disclosure provides a host cell comprising the above-mentioned vector.

[0223] In some preferred embodiments, the above-mentioned host cell is a prokaryotic cell or a eukaryotic cell.

[0224] In some preferred embodiments, the above-mentioned host cell is selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0225] In some preferred embodiments, the above-mentioned mammalian cells are selected from CHO cells, HEK293 cells or COS cells.

[0226] On the other hand, the present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned insulin analogue and a pharmaceutically acceptable carrier.

[0227] In some preferred embodiments, the above-mentioned pharmaceutical composition further comprises other therapeutic agents.

[0228] On the other hand, the present disclosure provides the use of the insulin analogue as described above in the preparation of a drug for treating, preventing or alleviating a disease or disorder, wherein the disease or disorder is related to diabetes or elevated blood glucose.

[0229] In some preferred embodiments, the above-mentioned diabetes is type I diabetes or type II diabetes.

[0230] On the other hand, the present disclosure provides a method for treating, preventing or alleviating a disease or disorder, wherein the disease or disorder is related to diabetes or elevated blood glucose, comprising administering a therapeutically / preventively effective amount of the insulin analogue according to any of the above aspects to a subject in need thereof in a pharmaceutically acceptable formulation.

[0231] In some preferred embodiments, the above-mentioned diabetes is type I diabetes or type II diabetes.

[0232] On the other hand, the present disclosure provides an insulin analogue for treating, preventing or alleviating a disease or disorder, the insulin analogue being as described in any of the above aspects, wherein the disease or disorder is related to diabetes or elevated blood glucose.

[0233] In some preferred embodiments, the above-mentioned diabetes is type I diabetes or type II diabetes.

[0234] The insulin analog can be prepared by any method considered appropriate. For example, the insulin analog can be prepared by recombinant methods or by solid-phase synthesis.

[0235] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments. However, it will be understood that the scope of the present disclosure may include embodiments having combinations of all or some of the described features.

[0236] Examples

[0237] Example 1: Preparation of a long-acting insulin analog

[0238] 1. Synthesis of the insulin analog

[0239] The parent insulin used in the present disclosure is human insulin, which comprises an A chain and a B chain. The amino acid sequence of the A chain is GIVEQCCTSICSLYQLENYCN (SEQ ID NO:1), and the amino acid sequence of the B chain is FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:2). The amino acid sequence of proinsulin is shown as SEQ ID NO:77 and is derived from the UniProt database (uniprot.org / uniprotkb / P01308).

[0240] (1) Prepare the raw materials. Synthesize the gene sequence of the proinsulin analog (His-B peptide-C peptide-A peptide) and insert it into the pet30a prokaryotic expression vector by recombinant DNA technology. After sequencing verification, transform the pet30a expression vector into Escherichia coli to obtain an Escherichia coli expression strain.

[0241] (2) Culture the Escherichia coli expression strain. Inoculate the transformed Escherichia coli strain into a culture flask containing an appropriate medium and culture it for a certain period of time under appropriate temperature, pH, and stirring conditions to promote the expression and accumulation of the proinsulin analog.

[0242] (3) Collect the cells. Centrifuge the Escherichia coli cells in the culture flask, then remove the supernatant and leave the cell pellet.

[0243] (4) Lyse the cells. Resuspend the cell pellet in an appropriate buffer and lyse the cells by sonication to release proinsulin analogs, including dissolving inclusion bodies in an appropriate buffer, usually a buffer containing a denaturing agent (such as urea or guanidinium salt) to fully unfold the proteins in the inclusion bodies. Currently, the more commonly used expression system is the Escherichia coli expression system. When foreign proteins are highly expressed in Escherichia coli, they often form insoluble and inactive aggregates, also known as inclusion bodies. Inclusion bodies must undergo in vitro refolding operations to form the correct higher-order structure to obtain the target protein with the expected biological activity.

[0244] (5) Refold the proinsulin analogs. Adjustment of the buffer: Gradually remove the denaturing agent while adjusting the pH and ionic strength of the buffer to promote the correct folding and formation of the higher-order structure of the protein. Addition of folding aids: Add some folding aids, such as redox agents (such as glutathione), coenzymes (such as NADH), etc. to the buffer to help the protein fold correctly. Optimization of temperature and time: Optimize the temperature and time during the refolding process to obtain the best refolding effect.

[0245] (6) Purify the proinsulin analogs. Use ion chromatography and size-exclusion chromatography to purify the refolded solution to remove impurities and other proteins, thereby obtaining a proinsulin analog with a higher purity.

[0246] (7) Enzymatic cleavage.

[0247] (8) Purify by size-exclusion chromatography using gel filtration: Use a gel column or a gel filtration membrane to separate and purify macromolecules according to the difference in molecular size through the pore size of the molecular sieve to obtain 47 purified insulin analogs.

[0248] (9) Structure identification and activity detection. Identify the structure of the purified insulin analogs by techniques such as mass spectrometry and nuclear magnetic resonance. Then, evaluate its activity using appropriate activity detection methods, such as insulin receptor binding assays, etc.

[0249] Results of affinity evaluation: Through surface plasmon resonance (SPR) experiments, evaluate the total binding strength / affinity between the insulin analogs and the insulin receptor. Compared with the parental insulin, the insulin analogs of the present disclosure have a lower binding affinity to the insulin receptor than the parental insulin, and the difference between the two is not less than 1 order of magnitude, that is, about a 10-fold difference. Compared with the reference insulin (i.e., insulin glargine), the insulin analogs of the present disclosure have a lower binding affinity to the insulin receptor than insulin glargine, and the difference between the two is not less than 0.5 order of magnitude, at least a 3.2-fold difference.

[0250] Cell activity level: The level of insulin receptor autophosphorylation was measured by Western Blot or flow cytometry (FCM) as a measure of insulin downstream signal transduction. The level of insulin receptor autophosphorylation after the insulin analogs of the present disclosure bind to the insulin receptor is not significantly different from the corresponding insulin receptor autophosphorylation level of insulin glargine. The insulin analogs of the present disclosure are designed to ensure that while the receptor affinity is reduced, insulin downstream signal transduction can still be ensured, the normal drug efficacy remains unchanged, or is improved.

[0251] The obtained insulin analogs with mutations are shown in Table 1 below.

[0252] The binding affinity of the insulin analogs of the present disclosure to the insulin receptor and the level of insulin receptor autophosphorylation are shown in Table 2.

[0253] Table 1: The produced human insulin analogs and their mutation sites

[0254]

[0255]

[0256] Table 2

[0257]

[0258]

[0259] Example 2: Plasma drug concentration of long-acting insulin analogs in animal models

[0260] A diabetic ICR mouse animal model was induced using streptozotocin. In the fasting state of male C57BL / 6J mice, it was intraperitoneally injected continuously for 3 days at a dose of 80 mg / kg STZ. Provide a suitable breeding environment, including temperature, humidity, lighting, and feed supply, etc., to ensure the health and welfare of the animals. After the model was established, the insulin analogs of the present disclosure were then intraperitoneally injected. According to the diagnostic criteria for diabetes, such as the increase in blood glucose level and the occurrence of insulin resistance, a diabetic ICR mouse model was successfully induced. The above 47 insulin analogs were administered in an appropriate insulin analog administration method and dose.

[0261] Blood was collected from the mouse tail vein at different time points after injecting the insulin analogs to detect blood glucose and plasma drug concentration. Use sterile blood collection instruments to avoid contaminating the blood samples. Place the collected blood in a blood collection tube and centrifuge to separate serum or plasma. Adopt a suitable analysis method, such as high performance liquid chromatography, to measure the concentration of insulin analogs in the blood samples. Perform statistics and analysis based on the measured plasma drug concentration data and draw a drug concentration-time curve, calculate the pharmacokinetic parameters of the drug, etc. As Figure 1As shown, 47 insulin analogs in the present disclosure all showed good performance in the pharmacokinetic experiments. Their half-lives were significantly higher than those of the parental insulin and insulin glargine, with a difference of no less than 24 hours, approximately twice or more the half-life of insulin glargine.

[0262] Example 3: Duration of action of long-acting insulin analogs in animal models

[0263] In this example, animal model experiments were conducted to evaluate the duration of action of 47 insulin analogs in the present disclosure. Before the start of the experiment, the experimental animals were subjected to feeding control to make them in a similar hungry state. The feeding time could be restricted or the same feed could be provided. The experimental animals were randomly divided into an experimental group and a control group. The experimental group was injected with the insulin analog sample, while the control group was injected with physiological saline as a control. After injecting the insulin analog or physiological saline, the blood glucose levels of the experimental animals were monitored regularly. A blood glucose measuring instrument could be used to measure the blood glucose levels. The values of each blood glucose measurement were recorded and correlated with the injection time. The data of the experimental group and the control group were compared and statistically analyzed to evaluate the duration of action of the insulin analog.

[0264] The specific experimental protocol is as follows:

[0265] (1) Forty 8-week-old male C57BL / 6J diabetic mice were randomly divided into 8 groups, with 5 mice in each group.

[0266] (2) The first group was intraperitoneally injected with PBS as a negative control.

[0267] (3) The second group was intraperitoneally injected with insulin glargine (25 nmol / kg, n = 5) as a positive control. The reason for choosing insulin glargine as the positive control is that insulin glargine is the insulin analog with the largest market share in the current long-acting insulin market. At the same time, insulin glargine is also the only insulin analog among the current mainstream insulin analogs on the market that only introduces missense mutations (without introducing fatty chain modifications).

[0268] (4) The third and fourth groups were injected with the long-acting insulin analogs of the present disclosure, with an injection concentration of 25 nmol / kg, n = 5.

[0269] (5) The initial blood glucose of each mouse was measured before injection. After injection, without interfering with the normal diet of the mice, at regular intervals, a blood routine instrument was used to record the blood glucose data and track the change of the concentration of the insulin analog in the blood over time.

[0270] The data analysis results are as Figure 2As shown, the time courses of 47 insulin analogs in the present disclosure all achieved good long-acting effects, and their in vivo pharmacodynamic durations were significantly higher than those of the parental insulin and insulin glargine, with a difference of no less than 48 hours, approximately twice or more than the in vivo pharmacodynamic duration of insulin glargine.

[0271] The sequence information involved in the present disclosure is shown in Table 3 below:

[0272] Table 3

[0273]

[0274]

Claims

1. An insulin analogue having at least three mutations relative to a parent insulin selected from the group consisting of positions A2, A4, A8, A14, A18, B23, B26, B27, B31, B32, B34, B37, B38, B42, B44, B46, B47 and B50.

2. The insulin analog according to claim 1, wherein the parent insulin is human insulin, porcine insulin or bovine insulin; Preferably, the parent insulin is human insulin.

3. The insulin analogue according to claim 1 or 2, wherein the mutation comprises: (1) The amino acid at position A2 is replaced by aspartic acid (Asp); (2) the amino acid at position A4 is replaced by lysine (Lys); (3) the amino acid at position A8 is substituted with lysine (Lys); (4) the amino acid at position A14 is substituted with an amino acid selected from methionine (Met), alanine (Ala) or threonine (Thr); (5) the amino acid at position A18 is substituted with alanine (Ala); (6) the amino acid at position B23 is substituted with alanine (Ala); (7) the amino acid at position B26 is replaced by glutamine (Gln); (8) the amino acid at position B27 is substituted with lysine (Lys); (9) the amino acid at position B31 is substituted with an amino acid selected from glycine (Gly), glutamine (Gln) or serine (Ser); (10) the amino acid at position B32 is replaced by histidine (His); (11) the amino acid at position B34 is substituted with an amino acid selected from lysine (Lys), phenylalanine (Phe), serine (Ser) or alanine (Ala); (12) the amino acid at position B37 is substituted with proline (Pro); (13) the amino acid at position B38 is substituted with aspartic acid (Asp); (14) the amino acid at position B42 is substituted with an amino acid selected from glutamine (Gln) or threonine (Thr); (15) the amino acid at position B44 is substituted with glutamic acid (Glu); (16) the amino acid at position B46 is substituted with glutamine (Gln); (17) the amino acid at position B47 is substituted with glycine (Gly); and / or (18) The amino acid at position B50 is substituted with an amino acid selected from alanine (Ala) or asparagine (Asn).

4. The insulin analogue according to any one of claims 1 to 3, wherein The A chain of the insulin analog comprises a mutation or a combination of mutations selected from the group consisting of: (1) N18A; (2) Y14M and N18A; (3) T8K; (4) E4K; (5) E4K and Y14M; (6)Y14M; (7)Y14A; (8) I2D and Y14T; (9)Y14T; (10) E4K and N18A; (11) Y14A and N18A; or (12)Y14M and N18A.

5. The insulin analogue according to any one of claims 1 to 4, wherein The B chain of the insulin analog comprises a mutation or a combination of mutations selected from the group consisting of: (1) H31G, L32H and E34K; (2) V23A, H31Q and E42Q; (3) H31G and L38D; (4) F46Q; (5) L27K, E34F and G44E; (6) E34S, Y47G and K50A; (7) V23A, H31S and E42T; (8) H31S and E34F; (9) L38D, Y47G and K50A; (10) H31Q, E34K and E42T; (11) V23A, E34S and Y37P; (12) H31Q and Y37P; (13)H31Q; (14) H31G and K50A; (15) H31G and K50N; (16) H31S, Y37P and Y47G; (17) H31S and Y47G; (18) E34S and K50N; (19) H31S and E34A; (20) V23A, H31G and L38D; (21) Y37P and E42T; (22) H31S, E34A and Y47G; (23) H31Q, L38D, and Y47G; (24) H31G, E34K, and L38D; (25) V23A and E34A; (26) H31G, Y37P, and L38D; (27) H26Q and E34A; (28) H31Q and E34A; (29) H31G, E34A, and E42Q; (30)H26Q; (31) H26Q, E34S and Y47G; (32)Y37P; (33) H31G and Y47G; (34) E34F, E42T and Y47G; (35) V23A, H31G and E34S; (36) H31G, L32H and E34S; (37) H31G and E34K; (38) H31G and Y37P; (39) H26Q, H31S and E34A; (40) H26Q and H31Q; or (41)H31S and E34S.

6. The insulin analogue according to any one of claims 1 to 5, wherein The insulin analog comprises a combination of mutations selected from the group consisting of: (1) B chain: H31G, L32H and E34K; (2) Chain B: V23A, H31Q, and E42Q; (3) A chain: N18A, and B chain: H31G and L38D; (4) Chain A: Y14M and N18A, and chain B: F46Q; (5) B chain: L27K, E34F and G44E; (6) Chain B: E34S, Y47G, and K50A; (7) Chain B: V23A, H31S and E42T; (8) Chain A: T8K, and chain B: H31S and E34F; (9) Chain B: L38D, Y47G and K50A (10) Chain B: H31Q, E34K and E42T (11) Chain B: V23A, E34S and Y37P (12) Chain A: E4K, and chain B: H31Q and Y37P; (13) Chain A: E4K and Y14M, and chain B: H31Q; (14) Chain A: Y14M, and chain B: H31G and K50A; (15) A chain: N18A, and B chain: H31G and K50N; (16) B chain: H31S, Y37P and Y47G; (17) Chain A: Y14A, and chain B: H31S and Y47G; (18) Chain A: Y14M, and chain B: E34S and K50N; (19) Chain A: T8K, and chain B: H31S and E34A; (20) B chain: V23A, H31G and L38D; (21) Chain A: Y14A, and chain B: Y37P and E42T; (22) Chain B: H31S, E34A, and Y47G; (23) B chain: H31Q, L38D and Y47G; (24) Chain A: I2D and Y14T, and chain B: H31Q; (25) Chain B: H31G, E34K, and L38D; (26) Chain A: Y14A, and chain B: V23A and E34A; (27) Chain A: Y14T, and chain B: E34S and K50N; (28) B chain: H31G, Y37P, and L38D; (29) Chain A: T8K, and chain B: H26Q and E34A; (30) Chain A: E4K, and chain B: H31Q and E34A; (31) Chain B: H31G, E34A, and E42Q; (32) Chain A: E4K and N18A, and chain B: H26Q; (33) Chain B: H26Q, E34S, and Y47G; (34) Chain A: Y14A and N18A, and chain B: Y37P; (35) Chain A: Y14A, and chain B: H31G and Y47G; (36) Chain B: E34F, E42T, and Y47G; (37) Chain B: V23A, H31G, and E34S; (38) Chain A: Y14A, and chain B: H31G and K50A; (39) Chain A: Y14T, and chain B: H31G and Y47G; (40) Chain B: H31G, L32H, and E34S; (41) A chain: N18A, and B chain: H31G and E34K; (42) Chain A: Y14M and N18A, and chain B: Y37P; (43) Chain A: Y14A, and chain B: H31G and Y37P; (44) Chain B: H26Q, H31S, and E34A; (45) A chain: N18A, and B chain: H31S and Y47G; (46) A chain: Y14A, and B chain: H26Q and H31Q; or (47) Chain A: Y14T, and chain B: H31S and E34S.

7. The insulin analogue according to any one of claims 1 to 6, wherein The A chain of the insulin analog comprises an amino acid sequence selected from SEQ ID NO: 1, 5, 7, 12, 17, 19, 21, 23, 26, 28, 30, 33, 37, 40, 42, 45, 47, 50, 53, 55, 59, 61, 64, 66, 68, 71, 73 and 75 or an amino acid sequence having 80% or more identity thereto; and / or The B chain of the insulin analog comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 6, 8-11, 13-16, 18, 20, 22, 24, 25, 27, 29, 31, 32, 34-36, 38, 39, 41, 43, 44, 46, 48, 49, 51, 52, 54, 56-58, 60, 62, 63, 65, 67, 69, 70, 72, 74 and 76 or an amino acid sequence with 80% or more identity thereto.

8. The insulin analogue according to any one of claims 1 to 7, wherein The insulin analog comprises an amino acid sequence combination selected from the following: (1) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 3 or has an identity of 80% or more thereto; (2) the A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 4 or has an identity of 80% or more thereto; (3) the A chain is as shown in SEQ ID NO: 5 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 6 or has an identity of 80% or more thereto; (4) the A chain is as shown in SEQ ID NO: 7 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 8 or has an identity of 80% or more thereto; (5) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 9 or has an identity of 80% or more thereto; (6) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 10 or has an identity of 80% or more thereto; (7) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 11 or has an identity of 80% or more thereto; (8) The A chain is as shown in SEQ ID NO: 12 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 13 or has an identity of 80% or more thereto; (9) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 14 or has an identity of 80% or more thereto; (10) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 15 or has an identity of 80% or more thereto; (11) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 16 or has an identity of 80% or more thereto; (12) the A chain is as shown in SEQ ID NO: 17 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 18 or has an identity of 80% or more thereto; (13) The A chain is as shown in SEQ ID NO: 19 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 20 or has an identity of 80% or more thereto; (14) The A chain is as shown in SEQ ID NO: 21 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 22 or has an identity of 80% or more thereto; (15) The A chain is as shown in SEQ ID NO: 23 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 24 or has an identity of 80% or more thereto; (16) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 25 or has an identity of 80% or more thereto; (17) The A chain is as shown in SEQ ID NO: 26 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 27 or has an identity of 80% or more thereto; (18) The A chain is as shown in SEQ ID NO: 28 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 29 or has an identity of 80% or more thereto; (19) The A chain is as shown in SEQ ID NO: 30 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 31 or has an identity of 80% or more thereto; (20) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 32 or has an identity of 80% or more thereto; (21) The A chain is as shown in SEQ ID NO: 33 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 34 or has an identity of 80% or more thereto; (22) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 35 or has an identity of 80% or more thereto; (23) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 36 or has an identity of 80% or more thereto; (24) The A chain is as shown in SEQ ID NO: 37 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 38 or has an identity of 80% or more thereto; (25) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 39 or has an identity of 80% or more thereto; (26) The A chain is as shown in SEQ ID NO:40 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:41 or has an identity of 80% or more thereto; (27) The A chain is as shown in SEQ ID NO:42 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:43 or has an identity of 80% or more thereto; (28) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 44 or has an identity of 80% or more thereto; (29) The A chain is as shown in SEQ ID NO:45 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:46 or has an identity of 80% or more thereto; (30) The A chain is as shown in SEQ ID NO:47 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:48 or has an identity of 80% or more thereto; (31) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 49 or has an identity of 80% or more thereto; (32) The A chain is as shown in SEQ ID NO:50 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:51 or has an identity of 80% or more thereto; (33) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 52 or has an identity of 80% or more thereto; (34) The A chain is as shown in SEQ ID NO:53 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:54 or has an identity of 80% or more thereto; (35) The A chain is as shown in SEQ ID NO:55 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:56 or has an identity of 80% or more thereto; (36) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 57 or has an identity of 80% or more thereto; (37) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 58 or has an identity of 80% or more thereto; (38) The A chain is as shown in SEQ ID NO:59 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO:60 or has an identity of 80% or more thereto; (39) The A chain is as shown in SEQ ID NO: 61 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 62 or has an identity of 80% or more thereto; (40) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 63 or has an identity of 80% or more thereto; (41) The A chain is as shown in SEQ ID NO: 64 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 65 or has an identity of 80% or more thereto; (42) The A chain is as shown in SEQ ID NO: 66 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 67 or has an identity of 80% or more thereto; (43) The A chain is as shown in SEQ ID NO: 68 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 69 or has an identity of 80% or more thereto; (44) The A chain is as shown in SEQ ID NO: 1 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 70 or has an identity of 80% or more thereto; (45) The A chain is as shown in SEQ ID NO: 71 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 72 or has an identity of 80% or more thereto; (46) the A chain is as shown in SEQ ID NO: 73 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 74 or has an identity of 80% or more thereto; or (47) The A chain is as shown in SEQ ID NO: 75 or has an identity of 80% or more thereto, and the B chain is as shown in SEQ ID NO: 76 or has an identity of 80% or more thereto.

9. A nucleic acid molecule encoding an insulin analogue according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid molecule according to claim 9; Preferably, the vector is an expression vector.

11. A host cell comprising the vector of claim 10; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; Preferably, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof; Preferably, the mammalian cell is selected from CHO cells, HEK293 cells or COS cells.

12. A pharmaceutical composition, wherein: The pharmaceutical composition comprises the insulin analogue according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises other therapeutic agents.

13. Use of the insulin analogue according to any one of claims 1 to 8 in the preparation of a medicament for treating, preventing or alleviating a disease or condition, wherein: The disease or condition is related to diabetes or elevated blood sugar; Preferably, the diabetes is type I diabetes or type II diabetes.

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