Conjugate of insulin analogue with reduced binding ability to insulin receptor and use thereof

Abstract

The present invention relates to an insulin analogue conjugate and its use. [Selected Figure] Figure 1

Description

[Technical field]

[0001] The present invention relates to an insulin analogue conjugate and uses thereof. [Background technology]

[0002] It is known that proteins in the body are eliminated through several pathways, such as degradation by proteolytic enzymes in the blood, excretion via the kidney, or elimination by receptors, etc. Therefore, various attempts have been made to circumvent such protein elimination mechanisms and increase the half-life of physiologically active proteins to enhance their therapeutic efficacy.

[0003] Meanwhile, insulin is a blood sugar regulating hormone secreted by the pancreas of the human body, which transfers excess glucose in the blood to cells to supply them with an energy source while maintaining blood sugar at a normal level. However, in the case of diabetic patients, insulin does not function normally due to a lack of insulin or insulin resistance and loss of beta cell function. As a result, diabetic patients do not use glucose in the blood as an energy source, and instead show symptoms of hyperglycemia, which is a high level of glucose in the blood, and ultimately excrete sugar in the urine, which causes various complications. Therefore, diabetes patients with abnormalities in insulin production (Type I) or insulin resistance (Type II) require insulin treatment, and blood sugar levels can be regulated to normal levels by administering insulin.

[0004] However, insulin has a drawback in that it does not show a sustained therapeutic effect because of its extremely short half-life in the body, unlike other protein or peptide hormones, and must be administered repeatedly and continuously to be effective. Since such frequent administration causes great pain and inconvenience to patients, there is a need for improvements in terms of patient compliance, safety, and convenience.

[0005] In order to increase the half-life of protein drugs such as insulin in the body and reduce the frequency of administration, research is being conducted into various protein formulations and chemical conjugates (e.g., fatty acid conjugates) to improve the quality of life and therapeutic effects of patients. According to existing reports, more than 50% of insulin is eliminated by the kidney, and the remaining insulin is eliminated from target sites such as muscle, fat, and liver via the receptor mediated clearance (RMC) process.

[0006] In this regard, there are several reports in non-patent literature 1 and 2, etc., of increasing blood concentrations by weakening in vitro activity in order to avoid insulin RMC; however, in the case of non-patent literature 1, the presented insulin analogues have substituted two or more amino acids, and no specific results are presented, and in the case of non-patent literature 2, it is reported that the insulin analogues have no change in receptor binding ability, or that activity has been reduced by substituting amino acids directly involved in the insulin receptor, so there is still a need for the development of insulin analogues with increased durability. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Patent Publication No. WO 97 / 34631 [Patent Document 2] International Patent Publication No. 96 / 32478 [Non-patent literature]

[0008] [Non-Patent Document 1] J Pharmacol Exp Ther(1998)286:959, Diabetes Care(1990)13:923 [Non-Patent Document 2] Diabetes(1990)39:1033 [Non-Patent Document 3] H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979 Summary of the Invention [Problem to be solved by the invention]

[0009] One object of the present invention is to provide conjugates of insulin analogues. Another object of the present invention is to provide a polynucleotide encoding the conjugate, a vector containing the polynucleotide, and a transformant containing the polynucleotide or the vector containing the same. Another object of the present invention is to provide a method for producing the conjugate. Another object of the present invention is to provide a composition, e.g., a pharmaceutical composition, comprising said insulin analogue conjugate.

[0010] Another object of the present invention is to provide a long-acting preparation having increased in vivo duration and / or stability compared to conjugates of natural insulin, including conjugates of the insulin analogue. Another object of the present invention is to provide a pharmaceutical composition for preventing or treating insulin-related diseases, such as diabetes, comprising the conjugate of said insulin analogue. Another object of the present invention is to provide a method for treating insulin-related disorders, such as diabetes, comprising administering said insulin analogue conjugate or composition thereof to an individual in need thereof. Another object of the present invention is to provide the use of said conjugates of insulin analogues in the manufacture of a medicament. Another object of the present invention is to provide the use of said insulin analogue conjugates in the treatment of insulin-related diseases, particularly diabetes. [Means for solving the problem]

[0011] One embodiment of the present invention for solving the above problems is a conjugate of an insulin analogue.

[0012] More specifically, one embodiment of the present invention is a conjugate represented by the following general formula (1): X-La-F (1) wherein X is an insulin analogue comprising one or more amino acid mutations selected from the group consisting of amino acid 16 of the native insulin B chain, amino acid 25 of the B chain, amino acid 14 of the A chain, and amino acid 19 of the A chain; L is a linker; a is 0 or a natural number, provided that when a is 2 or more, each L is independent of the others; F is a substance that can increase the half-life of X.

[0013] In one specific example, X is characterized by including one or more mutations selected from the group consisting of a mutation of the 16th amino acid, tyrosine, of the native insulin B chain to glutamic acid, serine, threonine, or aspartic acid; a mutation of the 25th amino acid, phenylalanine, of the native insulin B chain to aspartic acid or glutamic acid; a mutation of the 14th amino acid, tyrosine, of the native insulin A chain to histidine, lysine, alanine, or aspartic acid; and a mutation of the 19th amino acid, tyrosine, of the native insulin A chain to glutamic acid, serine, or threonine.

[0014] As another specific example, X is an insulin analogue comprising any combination of an A-chain of SEQ ID NO: 55, represented by the following general formula (2), and a B-chain of SEQ ID NO: 56, represented by the following general formula (3), characterized in that it excludes native insulin, i.e., it excludes peptides in which the A-chain corresponds to SEQ ID NO: 53 and the B-chain corresponds to SEQ ID NO: 54.

[0015] Xaa1-Ile-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21 (SEQ ID NO: 55) (2) In the general formula (2), Xaa1 is alanine, glycine, glutamine, histidine, glutamic acid, or asparagine; Xaa5 is alanine, glutamic acid, glutamine, histidine, or asparagine; Xaa12 is alanine, serine, glutamine, glutamic acid, histidine, or asparagine; Xaa14 is tyrosine, histidine, lysine, alanine, or aspartic acid; Xaa16 is alanine, leucine, tyrosine, histidine, glutamic acid, or asparagine; Xaa19 is tyrosine, glutamic acid, serine, or threonine; Xaa21 is asparagine, glycine, histidine, or alanine.

[0016] Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Xaa16-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Xaa25-Tyr-Xaa27-Xaa28-Lys-Thr (SEQ ID NO: 56) (3) In the general formula (3), Xaa16 is tyrosine, glutamic acid, serine, threonine, or aspartic acid; Xaa25 is phenylalanine, aspartic acid, or glutamic acid; Xaa27 is threonine or absent; Xaa28 is proline, glutamic acid, aspartic acid, or absent.

[0017] In another specific example, X is an insulin analogue comprising an A chain of SEQ ID NO: 55 and a B chain of SEQ ID NO: 54, as represented by the general formula (2).

[0018] In another specific example, X is an insulin analogue comprising an A chain of SEQ ID NO: 53 and a B chain of SEQ ID NO: 56 represented by general formula (3).

[0019] As another specific example, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, histidine, lysine, alanine, or aspartic acid, Xaa16 is leucine, Xaa19 is tyrosine, glutamic acid, serine, or threonine, and Xaa21 is asparagine; In the general formula (3), Xaa16 is tyrosine, glutamic acid, serine, threonine, or aspartic acid, Xaa25 is phenylalanine, aspartic acid, or glutamic acid, Xaa27 is threonine, and Xaa28 is proline.

[0020] As another specific example, In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, glutamic acid, or serine, and Xaa21 is asparagine; In the general formula (3), Xaa16 is tyrosine, glutamic acid, serine, or aspartic acid, Xaa25 is phenylalanine, aspartic acid, or glutamic acid, Xaa27 is threonine, and Xaa28 is proline.

[0021] As another specific example, The X is (1) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is histidine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (2) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is lysine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (3) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is glutamic acid, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (4) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is serine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (5) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is threonine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (6) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is glutamic acid, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (7) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is serine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (8) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is threonine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (9) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is alanine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (10) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is aspartic acid, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (11) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is aspartic acid, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; (12) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is aspartic acid, Xaa27 is threonine, and Xaa28 is proline; (13) In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is glutamic acid, Xaa27 is threonine, and Xaa28 is proline.

[0022] In another embodiment, X is an insulin analogue comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52. As another specific example, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is glutamic acid, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0023] As another specific example, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is serine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0024] As another specific example, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is threonine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0025] As another specific example, In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is aspartic acid, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0026] As another specific example, In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is aspartic acid, Xaa27 is threonine, and Xaa28 is proline.

[0027] As another specific example, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is tyrosine, Xaa25 is glutamic acid, Xaa27 is threonine, and Xaa28 is proline.

[0028] In another specific example, F is selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin.

[0029] In another specific example, the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

[0030] In another embodiment, F is an immunoglobulin Fc region. In another specific example, F is an IgG Fc region. In another specific example, L is selected from the group consisting of peptides, fatty acids, saccharides, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof. In another specific example, the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

[0031] In another specific example, L is polyethylene glycol. As another specific example, the high molecular weight polymer is characterized by having a molecular weight of 1 to 100 kDa. In another specific example, a linker is linked to the N-terminus of the B chain of the insulin analogue or to a lysine residue in the insulin analogue. In another specific example, F is an immunoglobulin Fc region, and is characterized in that a linker is linked to the N-terminus of the immunoglobulin Fc region.

[0032] In another specific example, F is an immunoglobulin Fc region, and the conjugate is characterized in that both ends of the linker are linked to the N-terminus of the B chain of the insulin analogue and the N-terminus of the immunoglobulin Fc, respectively. As another specific example, the insulin analogue is characterized by having a reduced binding affinity to the native insulin receptor compared to native insulin. As another specific example, the binding strength of the insulin analogue to a native insulin receptor is about 10% to about 90% of that of native insulin. As another specific example, the conjugate is characterized in that F is an immunoglobulin Fc region, L is polyethylene glycol, and the binding strength to a native insulin receptor is 0.1% to 50% of that of native insulin.

[0033] In another embodiment, the conjugate is characterized in that F is an immunoglobulin Fc region, L is polyethylene glycol, and X is an insulin analogue having the same sequence as native insulin, except that the 14th amino acid of the A chain is aspartic acid. Other embodiments of the present invention are a polynucleotide encoding the conjugate, a vector containing the polynucleotide, and a transformant containing the polynucleotide or the vector containing the same. Another aspect of the present invention is a method for producing the conjugate. Another aspect of the present invention is a composition comprising the conjugate. In one embodiment, the composition is a pharmaceutical composition. In another embodiment, the composition is a pharmaceutical composition for preventing or treating an insulin-related disease.

[0034] In another embodiment, the composition is a pharmaceutical composition for preventing or treating diabetes. Another embodiment of the present invention is a long-acting formulation comprising a conjugate of an insulin analogue, which has increased in vivo persistence and / or stability compared to a conjugate of natural insulin.

[0035] Another embodiment of the present invention is a method for treating an insulin-related disorder comprising administering the insulin analogue conjugate or composition thereof to an individual in need thereof. In one embodiment, the insulin-related disease is diabetes. Another aspect of the present invention is the use of the conjugate of the insulin analogue in the manufacture of a medicament. In one embodiment, the drug is for preventing or treating an insulin-related disease.

[0036] In another embodiment, the drug is for preventing or treating diabetes. Another aspect of the present invention is the use of the conjugates of insulin analogues in the treatment of insulin-related disorders, particularly diabetes. Effect of the Invention

[0037] The conjugate of the non-natural insulin analogue according to the present invention can improve the convenience of patients who require insulin administration. [Brief description of the drawings]

[0038] [Figure 1] This figure shows the results of analyzing the purity of insulin analogs by protein electrophoresis. As representatives, the results are shown for insulin analogs 9, 10, 11, and 12. Lane 1: size marker, lane 2: native insulin, lane 3: insulin analog 9, lane 4: insulin analog 10, lane 5: insulin analog 11, lane 6: insulin analog 12. [Figures 2a-2d] The purity of insulin analogs was analyzed by high pressure chromatography. As representatives, the results of insulin analogs 9, 10, 11, and 12 are shown in order (Figures 2a to 2d). In each figure, the results of RP-HPLC (C18), RP-HPLC (C4), and SE-HPLC are shown from the top to the bottom. [Figure 3a-3c] FIG. 3 shows the results of comparing the pharmacokinetic properties between "insulin analog 10-PEG-immunoglobulin Fc," a representative insulin analog conjugate according to the present invention, and "native insulin-PEG-immunoglobulin Fc" conjugate, a native insulin conjugate, in normal mice (FIG. 3a), SD rats (FIG. 3b), and beagle dogs (FIG. 3c). [Figure 4]FIG. 1 shows the results of confirming the blood glucose lowering ability between "insulin analog 10-PEG-immunoglobulin Fc," a representative insulin analog conjugate according to the present invention, and "native insulin-PEG-immunoglobulin Fc" conjugate, a native insulin conjugate, in DIO / STZ rats. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention will now be described in further detail. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. In other words, all combinations of various elements disclosed in the present application belong to the category of the present invention. In addition, the category of the present invention is not limited by the specific descriptions described below.

[0040] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the present invention.

[0041] Throughout this specification, however, the conventional one-letter and three-letter codes for amino acids are used, and the amino acids referred to herein by abbreviations are written according to the IUPAC-IUB nomenclature system.

[0042] One embodiment of the present invention provides a conjugate of an insulin analogue.

[0043] Specifically, the conjugate of the insulin analogue may be in the form of a conjugate in which a biocompatible substance is conjugated to the insulin analogue to increase its half-life in vivo. In the present specification, the biocompatible substance may be mixed with a carrier.

[0044] In the present invention, conjugates of the insulin analogues can exhibit increased duration of effect compared to the insulin analogues not conjugated to a carrier, and these conjugates are referred to as "long-acting conjugates" in the present invention.

[0045] Alternatively, such conjugates may be non-naturally occurring. More specifically, the present invention provides a conjugate represented by the following general formula (1): X-La-F (1) wherein X is an insulin analogue comprising one or more amino acid mutations selected from the group consisting of amino acid 16 of the native insulin B chain, amino acid 25 of the B chain, amino acid 14 of the A chain, and amino acid 19 of the A chain; L is a linker; a is 0 or a natural number, provided that when a is 2 or more, each L is independent of the others; F is a substance that can increase the half-life of X.

[0046] In the present invention, the term "insulin analog" refers to a non-natural insulin that is different from natural insulin. The insulin analog includes a non-natural human insulin that is different from natural human insulin. The insulin analog is a moiety that constitutes the conjugate of the present invention, and corresponds to X in the general formula (1). These insulin analogues include analogues in which some amino acids of natural insulin have been modified by addition, deletion or substitution.

[0047] Specifically, the insulin analog of the present invention may have a sequence identity of at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more when compared with the sequence identity of the natural insulin sequence. The insulin analog of the present invention may have the above sequence identity with the natural insulin sequence but have a lower binding ability to the natural insulin receptor compared to natural insulin. The insulin analog may / may have glucose absorption ability like natural insulin, and may have the ability to lower blood glucose in vivo. In the present invention, the binding ability of any insulin analog to the natural insulin receptor means the percentage ratio of the binding inhibition degree of the insulin analog to the binding inhibition degree of natural insulin when the binding inhibition degree is measured under competitive inhibition of receptor binding conditions in which the binding of any detector ligand to the natural insulin receptor is inhibited in the presence of natural insulin or the corresponding insulin analog. Here, the degree of binding inhibition may be indirectly defined as a value that measures whether the signal expressed by the ligand binding of the insulin receptor is effectively changed based on an appropriate criterion. For example, when insulin labeled with a label such as a radioisotope binds to the insulin receptor, the degree to which the signal generated from this label decreases or increases after the addition of unlabeled natural insulin or an insulin analog is measured (e.g., IC for the decrease in the signal of natural insulin or an insulin analog). 50 The percentage of the measured value (e.g., IC value of the corresponding insulin analogue) obtained by 50 IC of natural insulin against 50The binding strength can be defined as a percentage of the binding strength of the receptor. Measurement of receptor binding strength via these competitive inhibition methods is well known in the art. In a specific embodiment of the present invention, the native insulin receptor required for these measurements may be a cell membrane expressing the human native insulin receptor (A isoform or B isoform or both), for example, a cell membrane of a cell engineered to overexpress the human native insulin receptor via genetic engineering methods, and the detector ligand may be native insulin labeled with iodine-125. In a more specific embodiment of the present invention, the competitive inhibition of receptor binding may be measured using a scintillation proximity assay (SPA). In a more specific embodiment, the binding strength to the insulin receptor may be measured using the method of Experimental Example 1.

[0048] More specifically, the insulin analogues of the present invention have an insulin receptor binding ability of about 99% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, or about 50% or less of the insulin receptor binding ability of natural insulin (100%). It may exhibit a binding strength to the insulin receptor of about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.1% or less (however, the insulin analog of the present invention does not have a binding strength to the insulin receptor of 0%). In one specific embodiment of the present invention, the binding strength of the insulin analog to the native insulin receptor is suitably about 10% to about 90% of that of native insulin, but the numerical value is not limited, and if the binding strength is weaker than that of native insulin, it belongs to the category of the present invention. When the binding strength is weaker than that of native insulin, the removal of the insulin analog by the insulin receptor is weakened, the half-life in blood is improved, and the duration of efficacy can be expected.

[0049] In one embodiment of the invention, the insulin analogue conjugate has a linker that is polyethylene glycol, a substance that can increase the half-life is an immunoglobulin Fc region, and has a binding ability to the native insulin receptor that is about 99% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about The insulin analog conjugate may exhibit a binding strength to the insulin receptor of 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.1% or less (however, the insulin analog conjugate of the present invention does not have a binding strength to the insulin receptor of 0%). In one specific embodiment of the present invention, the binding strength of the insulin analog conjugate to the native insulin receptor is suitably 0.1 to 50% of that of native insulin, but the numerical value is not limited, and if the binding strength is weaker than that of native insulin, it belongs to the category of the present invention. When the binding strength is weaker than that of native insulin, the removal of the insulin analog conjugate by the insulin receptor is weakened, the half-life in blood is improved, and the duration of efficacy can be expected.

[0050] In the present invention, the term "about" refers to a range that includes, but is not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., including all numerical values ​​in a range that is equal to or similar to the numerical value that follows the term about. Furthermore, the insulin analogue of the present invention may retain the glucose absorption ability like natural insulin. Specifically, the insulin analogue of the present invention may have a glucose absorption capacity of about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or more, about 110% or more, about 120% or more, about 130% or more, about 140% or more, about 150% or more, about 160% or more, about 170% or more, about 180% or more, about 190% or more, or about 200% or more compared to the glucose absorption capacity of natural insulin (100%).

[0051] Measurement of glucose absorption capacity may be accomplished using a variety of methods for measuring glucose absorption capacity known in the art. Specifically, the insulin analogue may be in the form of a short chain in which two sequence sites corresponding to the natural A chain and B chain are linked in one polypeptide chain, or in the form of two polypeptide chains in which these sequence sites are each independent polypeptide chains. Hereinafter, when the term A chain or B chain of an insulin analogue is used, it should be interpreted accordingly to mean not only the constituent A chain or B chain when the insulin analogue is in the form of two polypeptide chains, but also the sequence site corresponding to the natural A chain or the sequence site corresponding to the natural B chain in the short chain polypeptide when the analogue is in the form of a short chain. The insulin analogue of the present invention may be in the form of a short chain or two polypeptide chains in which one selected from the A chain of natural insulin or a polypeptide corresponding thereto and one selected from the B chain of natural insulin or a polypeptide corresponding thereto are combined. Here, "corresponding to the A chain or B chain of native insulin" means, for example, that when any one of the polypeptides has a sequence identity of at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more when comparing the sequence identity of the polypeptide with that of the A chain or B chain of native insulin, but is not particularly limited thereto, and can be easily grasped by a person skilled in the art by comparing the sequence constituting the polypeptide with the sequence of the A chain or B chain of native insulin.

[0052] As mentioned above, everything described herein is intended to apply to the description of the lower concepts of insulin analogues as well. Native insulin is a hormone secreted from the pancreas, and generally plays a role in regulating blood glucose in the body by promoting intracellular glucose absorption and inhibiting fat decomposition. Insulin is processed from a proinsulin precursor that does not have a blood glucose regulating function to insulin with blood glucose regulating function. Insulin is composed of two polypeptide chains, namely, A chain and B chain, which contain 21 and 30 amino acid residues, respectively, and are interconnected by two disulfide bridges. The A chain and B chain of native insulin contain the amino acid sequences shown in the following SEQ ID NOs: 53 and 54, respectively.

[0053] A chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 53) B chain: Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Lys-Thr (SEQ ID NO: 54) According to one embodiment of the present invention, the insulin analogues described herein may have reduced receptor binding ability while retaining the blood glucose regulating function in vivo like natural insulin. More specifically, the insulin analogues may have the ability to lower blood glucose in vivo.

[0054] In one embodiment of the present invention, the insulin analogue may not be limited to a particular type or size as long as it exhibits low receptor-mediated internalization or receptor-mediated clearance, and may therefore exhibit an increased half-life in blood compared to native insulin.

[0055] The insulin analogues of the present invention include reverse insulin, derivatives of natural insulin, fragments of natural insulin, etc. These insulin analogues can be produced not only by recombinant methods but also by solid phase methods, but are not limited thereto.

[0056] In the present invention, the term "derivative of natural insulin" includes peptides having one or more differences in the amino acid sequence compared to natural insulin, peptides in which the natural insulin sequence has been altered through modification, and mimetics of natural insulin that regulate blood glucose regulation in vivo like natural insulin. These derivatives of natural insulin may have blood glucose regulation function in vivo. Specifically, a derivative of natural insulin may be obtained by modifying some amino acids from natural insulin through any one of substitution, addition, deletion and modification, or a combination of these methods.

[0057] Specifically, it may / may have at least 80% or more amino acid sequence homology with the A-chain and B-chain of natural insulin, respectively, or it may have a form in which a partial group of single amino acid residues of insulin has been chemically substituted (e.g., alpha-methylation, alpha-hydroxylation), deleted (e.g., deamination) or modified (e.g., N-methylation), but is not limited to these. The derivatives of natural insulin that are applicable to the present invention may be produced by a combination of several methods for producing derivatives.

[0058] Furthermore, these modifications for producing derivatives of natural insulin include modifications using L- or D-amino acids and / or non-natural amino acids; and / or modifications by modifying the natural sequence or post-translational modifications (e.g., methylation, acylation, ubiquitination, intramolecular covalent bonds, etc.). Furthermore, derivatives of native insulin include all those in which one or more amino acids have been added to the amino and / or carboxy termini of native insulin.

[0059] The substituted or added amino acids may be atypical or non-naturally occurring amino acids, as well as the 20 amino acids commonly found in human proteins. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep and Genzyme pharmaceuticals. Peptides containing these amino acids and typical peptide sequences can be synthesized and purchased through commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea, but are not limited thereto.

[0060] In the present invention, the term "fragment of native insulin or a derivative of native insulin" refers to a form in which one or more amino acids have been removed from the amino or carboxy terminus of native insulin or a derivative of native insulin. These insulin fragments may retain blood glucose regulating function in the body.

[0061] The insulin analogues of the present invention may be produced by using the respective production methods used for producing the above-mentioned natural insulin derivatives and fragments independently or in combination.

[0062] Specifically, the insulin analogue of the present invention comprises modifications of specific amino acid residues in the A-chain and B-chain of natural insulin as described above, specifically, the specific amino acid residues in the A-chain of natural insulin may be modified / altered, or the specific amino acid residues in the B-chain may be modified.

[0063] Specifically, the insulin analogue may be one in which one or more amino acids selected from the group consisting of amino acid 16 of the natural insulin B chain, amino acid 25 of the B chain, amino acid 14 of the A chain, and amino acid 19 of the A chain are substituted with other amino acids, such as, but not limited to, glutamic acid, serine, threonine, aspartic acid, histidine, lysine, or alanine.

[0064] Specifically, one or more, two or more, three or more, or four of the amino acids described above may be substituted with other amino acids rather than the original amino acids.

[0065] Specifically, the mutation may be a mutation of the 16th amino acid of the insulin B chain, tyrosine, to glutamic acid, serine, threonine, or aspartic acid; a mutation of the 25th amino acid of the insulin B chain, phenylalanine, to aspartic acid or glutamic acid; a mutation of the 14th amino acid of the insulin A chain, tyrosine, to histidine, lysine, alanine, or aspartic acid; or a mutation of the 19th amino acid of the insulin A chain, tyrosine, to glutamic acid, serine, or threonine.

[0066] Thus, said insulin analogues may comprise, but are not limited to, a mutation of amino acid tyrosine 16 of the native insulin B chain to glutamic acid, serine, threonine or aspartic acid, and / or a mutation of amino acid phenylalanine 25 of the native insulin B chain to aspartic acid or glutamic acid, and / or a mutation of amino acid tyrosine 14 of the native insulin A chain to histidine, lysine, alanine or aspartic acid, and / or a mutation of amino acid tyrosine 19 of the native insulin A chain to glutamic acid, serine or threonine.

[0067] More specifically, the insulin analogue may be an insulin analogue comprising an A chain of SEQ ID NO: 55, represented by the following general formula (2), and a B chain of SEQ ID NO: 56, represented by the following general formula (3). These insulin analogues may be in a form in which the A and B chain sequences are interconnected via a disulfide bond, or in the form of proinsulin, but are not limited thereto.

[0068] Xaa1-Ile-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21 (SEQ ID NO: 55) (2) In the general formula (2), Xaa1 is alanine, glycine, glutamine, histidine, glutamic acid, or asparagine; Xaa5 is alanine, glutamic acid, glutamine, histidine, or asparagine; Xaa12 is alanine, serine, glutamine, glutamic acid, histidine, or asparagine; Xaa14 is tyrosine, histidine, lysine, alanine, or aspartic acid; Xaa16 is alanine, leucine, tyrosine, histidine, glutamic acid, or asparagine; Xaa19 is tyrosine, glutamic acid, serine, or threonine; Xaa21 is asparagine, glycine, histidine, or alanine.

[0069] Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Xaa16-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Xaa25- Tyr-Xaa27-Xaa28-Lys-Thr (SEQ ID NO: 56) (3) In the general formula (3), Xaa16 is tyrosine, glutamic acid, serine, threonine, or aspartic acid; Xaa25 is phenylalanine, aspartic acid, or glutamic acid; Xaa27 is threonine or absent; Xaa28 is proline, glutamic acid, aspartic acid, or absent.

[0070] Here, peptides in which the A chain corresponds to SEQ ID NO: 53 and the B chain corresponds to the B chain of SEQ ID NO: 54 may be excluded. Also included in the category of the present invention are peptides which contain the characteristic amino acid residues described above, in particular the characteristic mutations at amino acid 14 and / or amino acid 19 of the A chain and / or amino acid 16 and / or amino acid 25 of the B chain (i.e., amino acid residues not present in native insulin), and which have a homology of 70% or more, specifically 80% or more, more specifically 90% or more, and even more specifically 95% or more with an insulin analogue comprising the A chain of general formula (2) and the B chain of general formula (3), and which have a reduced receptor binding ability compared to native insulin.

[0071] In the present invention, the term "homology" refers to the degree of similarity between the amino acid sequence of a wild type protein or the polynucleotide sequence encoding the same, and includes sequences having the above percent identity with the amino acid sequence or polynucleotide sequence of the present invention. The homology may be determined by visually comparing two sequences, or by using a bioinformatic algorithm that aligns the sequences to be compared and analyzes the degree of homology. The homology between the two amino acid sequences may be expressed as a percentage. Convenient automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA). Automated sequence algorithms in the modules include the Needleman & Wunsch, Pearson & Lipman and Smith & Waterman sequence algorithms. Other useful sequence algorithms and homology determinations are automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.

[0072] In one specific embodiment, the insulin analog may comprise an A chain of SEQ ID NO: 55 represented by general formula (2) and a B chain of SEQ ID NO: 54, or an insulin analog may comprise an A chain of SEQ ID NO: 53 and a B chain of SEQ ID NO: 56 represented by general formula (3), but is not limited thereto.

[0073] More specifically, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, histidine, lysine, alanine, or aspartic acid, Xaa16 is leucine, Xaa19 is tyrosine, glutamic acid, serine, or threonine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, glutamic acid, serine, threonine, or aspartic acid, Xaa25 is phenylalanine, aspartic acid, or glutamic acid, Xaa27 is threonine, and Xaa28 is proline, but is not limited thereto.

[0074] More specifically, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, glutamic acid, or serine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, glutamic acid, serine, or aspartic acid, Xaa25 is phenylalanine, aspartic acid, or glutamic acid, Xaa27 is threonine, and Xaa28 may be proline, but is not limited thereto.

[0075] More specifically, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine or aspartic acid, Xaa16 is leucine, Xaa19 is tyrosine, glutamic acid, serine, or threonine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, aspartic acid, or glutamic acid, Xaa27 is threonine, and Xaa28 may be proline, but is not limited thereto.

[0076] According to a specific embodiment, the insulin analogue according to the invention may be:

[0077] (1) An insulin analogue, wherein in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is histidine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0078] (2) An insulin analogue in which, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is lysine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and, in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0079] (3) An insulin analogue in which, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is glutamic acid, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0080] (4) An insulin analogue in which, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is serine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0081] (5) An insulin analogue in which, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is threonine, and Xaa21 is asparagine; and, in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0082] (6) An insulin analogue in which, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and, in the general formula (3), Xaa16 is glutamic acid, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0083] (7) An insulin analogue in which, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is serine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0084] (8) An insulin analogue in which, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is threonine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0085] (9) An insulin analogue in which, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is alanine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and, in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0086] (10) An insulin analogue, wherein in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is aspartic acid, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0087] (11) An insulin analogue, wherein in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine, and in the general formula (3), Xaa16 is aspartic acid, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

[0088] (12) An insulin analogue in which, in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and, in the general formula (3), Xaa16 is tyrosine, Xaa25 is aspartic acid, Xaa27 is threonine, and Xaa28 is proline.

[0089] (13) An insulin analogue, wherein in the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is glutamic acid, Xaa27 is threonine, and Xaa28 is proline.

[0090] Also, according to a specific embodiment, the insulin analog may be an insulin analog comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52, but is not limited thereto.

[0091] The insulin analogue of the present invention may comprise the specific sequence described above or may consist (essentially) of the specific sequence described above, but is not limited thereto.

[0092] In addition, even if the present application describes a "peptide composed of a specific sequence number," if the peptide has the same or corresponding activity as a peptide consisting of the amino acid sequence of the sequence number, this does not exclude meaningless additions of sequences before or after the amino acid sequence of the sequence number, or naturally occurring mutations, or silent mutations thereof, and it is self-evident that even if such additions of sequences or mutations are present, they fall within the scope of the present application.

[0093] On the other hand, the insulin analogues include the peptide itself, a salt thereof (eg, a pharma- ceutically acceptable salt of the peptide), or any of the solvate forms thereof. Furthermore, the peptide may be in any pharma- ceutically acceptable form. The type of the salt is not particularly limited, provided that it is in a form that is safe and effective for an individual, for example, a mammal, but is not particularly limited thereto. The term "pharmacologically acceptable" means a substance that can be effectively used for its intended purpose without causing excessive toxicity, irritation, or allergic reactions, within the scope of medical judgment.

[0094] In the present invention, the term "pharmaceutically acceptable salts" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, potassium, alkaline earth metals such as magnesium, and ammonium, and the like.

[0095] Furthermore, the term "solvate" used in the present invention refers to a complex formed between the peptide of the present invention or a salt thereof and a solvent molecule.

[0096] The insulin analogues can be easily produced by those skilled in the art using methods for producing peptides known in the art, for example, by a method including the following steps:

[0097] a) culturing a transformant containing a nucleic acid encoding the insulin analog to express the insulin analog; and b) isolating and purifying the expressed insulin analogue.

[0098] In the present invention, the medium used for culturing the transformant must meet the requirements for culturing the host cell in an appropriate manner. The carbon source that can be contained in the medium for the growth of the host cell can be appropriately selected by those skilled in the art depending on the type of the transformant produced, and suitable culture conditions can be adopted to adjust the culture time and amount.

[0099] Sugar sources that may be used include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid, etc. These materials may be used individually or in mixtures.

[0100] Nitrogen sources that may be used include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may also be used individually or as mixtures.

[0101] Phosphorus sources which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium may also contain metal salts such as magnesium sulfate or ferrous sulfate required for growth.

[0102] Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins may be used. Also, suitable precursors may be used in the culture medium. The above mentioned raw materials may be added to the culture in a suitable manner during the cultivation in a batch or continuous manner. The pH of the culture may be adjusted using a suitable manner with basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid. Also, foam formation may be suppressed using antifoaming agents such as fatty acid polyglycol esters. Oxygen or oxygen-containing gas (e.g. air) may be injected into the culture to maintain aerobic conditions.

[0103] The transformant according to the present invention is cultured usually at a temperature of 20° C. to 45° C., specifically, 25° C. to 40° C. The culture is continued until the desired amount of insulin analogue produced is maximized, and for such purpose, the culture may be continued usually for 10 to 160 hours.

[0104] As described above, when appropriate culture conditions are created depending on the host cell, the transformant of the present invention will produce an insulin analogue, and depending on the vector configuration and the characteristics of the host cell, the insulin analogue produced may be secreted into the cytoplasm, periplasmic space or extracellularly of the host cell.

[0105] The protein expressed inside or outside a host cell may be purified by a conventional method, such as salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (e.g., protein fraction precipitation using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, chromatography such as reverse-phase column chromatography, and ultrafiltration, which may be used alone or in combination.

[0106] In a specific embodiment of the present invention, the method may further comprise the steps of: separating and purifying the insulin analogue expressed in the form of inclusion bodies from the transformant: b-1) harvesting and disrupting the transformant from the culture medium of step a); b-2) recovering and refolding the expressed insulin analogue from the disrupted cell lysate; b-3) purifying the refolded insulin analogue by cation exchange chromatography; b-4) treating the purified insulin analogue with trypsin and carboxypeptidase B; b-5) Purifying the treated insulin analogue sequentially by cation exchange chromatography and anion exchange chromatography or reverse phase chromatography.

[0107] In the present invention, the nucleic acid encoding the insulin analogue specifically includes the nucleotide sequences set forth in SEQ ID NOs: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, and 51. In one embodiment, the present invention includes not only the nucleotide sequences set forth in SEQ ID NOs: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, and 51, but also all sequences showing 70% or more homology with the above sequences, specifically 80% or more homology, more specifically 90% or more homology, even more specifically 95% or more homology, and most specifically 98% or more homology, and wherein the peptide substantially encoded by the nucleic acid has a reduced receptor binding ability compared to natural insulin while retaining the blood glucose regulating function in the body.

[0108] The recombinant vector according to the present invention may typically be constructed as a vector for cloning or as a vector for expression, and may be constructed in a prokaryotic or eukaryotic host cell.

[0109] In the present invention, the term "vector" refers to a recombinant vector capable of expressing a protein of interest in a suitable host cell, and a nucleic acid construct containing essential regulatory elements operably linked to allow a nucleic acid insert to be expressed. The present invention can produce a recombinant vector containing a nucleic acid encoding an insulin analogue, and the insulin analogue of the present invention can be obtained by transformation or transfection of the recombinant vector into a host cell.

[0110] The nucleic acid encoding the insulin analogue of the present invention may be operably linked to a promoter.

[0111] The method for transforming a recombinant vector containing a nucleic acid encoding the insulin analogue of the present invention is not limited to the above examples, and any transformation or transfection method commonly used in the art may be used without limitation.

[0112] The transformant of the present invention can be obtained by introducing a recombinant vector containing a nucleic acid encoding an insulin analogue, which is a target nucleic acid, into a host cell.

[0113] The host suitable for the present invention is not particularly limited as long as it expresses the nucleic acid of the present invention. Specific examples of hosts that may be used in the present invention include Escherichia bacteria such as E. coli; Bacillus bacteria such as Bacillus subtilis; Pseudomonas bacteria such as Pseudomonas putida; yeasts such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe; insect cells such as Spodoptera frugiperda (SF9); and animal cells such as CHO, COS, and BSC. Specifically, E. coli may be used as a host cell, but is not limited thereto.

[0114] Meanwhile, in the conjugate, F is a substance capable of increasing the half-life of X, and corresponds to one of the components constituting the conjugate of the present invention.

[0115] The F may be bonded to X via a covalent or non-covalent chemical bond, or F and X may be bonded to each other via L via a covalent chemical bond, a non-covalent chemical bond, or a combination thereof.

[0116] More specifically, X and L, and L and F may be linked to each other via a covalent bond. In this case, the conjugate is a conjugate in which X, L, and F are linked to each other via a covalent bond in the order of general formula (1).

[0117] The substance capable of increasing the half-life of X may be a biocompatible substance, for example, selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin, but is not particularly limited thereto.

[0118] In the case of elastin, it may be human tropoelastin, which is a water-soluble precursor, or it may be a polymer of a partial sequence or a partial repeating unit of these, including, for example, all cases of elastin-like polypeptides, but is not particularly limited thereto.

[0119] Examples of the polymer include, but are not limited to, polymers selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

[0120] The polyethylene glycol is a term that encompasses all forms of ethylene glycol homopolymer, PEG copolymer, and monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto.

[0121] The biocompatible material also includes, but is not limited to, polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid.

[0122] Furthermore, the fatty acid may be one that has a binding ability to albumin in vivo, but is not particularly limited thereto.

[0123] As a more specific example, the FcRn-binding substance may be an immunoglobulin Fc region, more specifically, an IgG Fc region, but is not particularly limited thereto. One or more amino acid side chains in the insulin analogues of the invention may be conjugated to biocompatible substances to increase the solubility and / or half-life of the insulin analogue in vivo or to increase its bioavailability. Such modifications may decrease the clearance of therapeutic proteins and peptides. The biocompatible materials mentioned above may be water-soluble (amphiphilic or hydrophilic) and / or non-toxic and / or pharma- ceutical acceptable.

[0124] The F may be directly linked to X (ie, a is 0 in the general formula (1)) or may be linked via a linker (L). Specifically, the L may be a peptidic linker or a non-peptidic linker. When L is a peptidic linker, it may contain one or more amino acids, for example, 1 to 1000 amino acids, but is not particularly limited thereto. In the present invention, various known peptide linkers for linking F and X may be used in the present invention, examples of which include [GS]x linker, [GGGS]x linker, and [GGGGS]x linker, where x may be a natural number of 1 or more. However, it is not limited to the above examples.

[0125] In the present invention, the "non-peptidic linker" includes a biocompatible polymer having two or more repeating units bonded thereto. The repeating units are linked to each other via any covalent bond, not a peptide bond. The non-peptidic linker may be a part of the conjugate of the present invention, and corresponds to L in the general formula (1). In the present invention, when L is a non-peptidic linker and a biocompatible polymer, the non-peptidic linker may further include a linking element at the end for covalently linking the linker L to the insulin analogue X and / or F. There is no particular restriction on the linking element, so long as it is not a repeating unit of the biocompatible polymer, but is an atom or atomic group suitable for covalently linking the biocompatible polymer to X and / or F.

[0126] In the above La, a may be 1 or more, and when a is 2 or more, each L may be independent.

[0127] In the present invention, the non-peptidyl linker may be used in combination with a non-peptidyl polymer. In a specific embodiment, the conjugate may be one in which F and X are covalently linked to each other via a non-peptide linker that contains reactive groups at both ends that can be coupled to F, specifically to an immunoglobulin Fc region, and to X.

[0128] Specifically, the non-peptide linker may be selected from the group consisting of fatty acids, saccharides, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof. Although not particularly limited thereto, in the present invention, the high molecular weight polymer may be in the range of more than 0 to about 100 kDa, specifically in the range of about 1 to about 100 kDa, and more specifically in the range of about 1 to about 20 kDa, but is not limited thereto.

[0129] The non-peptide linker may be selected from the group consisting of, but is not limited to, polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers such as PLA (polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, oligonucleotides, and combinations thereof.

[0130] In a more specific embodiment, the non-peptidic polymer may be, but is not limited to, polyethylene glycol. In addition, derivatives thereof already known in the art and derivatives that can be easily produced at the technical level in the art are also included within the scope of the present invention.

[0131] The non-peptidyl linker that may be used in the present invention may be any polymer that is resistant to in vivo protease, without limitation. The molecular weight of the non-peptidyl polymer is greater than 0 and in the range of about 100 kDa, specifically, in the range of about 1 to about 100 kDa, more specifically, in the range of about 1 to about 20 kDa, but is not limited thereto.

[0132] In the present invention, the term "about" refers to a range including, but not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., including all numerical values ​​in a range equivalent or similar to the numerical value appearing after the term about. Furthermore, the non-peptide linker of the present invention to be bonded to the polypeptide corresponding to F may be made of not only one type of polymer, but also a combination of different types of polymers.

[0133] As described above, in one specific embodiment, both ends of the non-peptidic linker can be linked to F and X, respectively, via a linking element. For example, in this specific embodiment, the insulin analogue conjugate of the present invention is produced by reacting (linking) an immunoglobulin Fc region and / or an insulin analog with a non-peptidic polymer (reactive group-bearing non-peptidic polymer) having at its end a reactive group that can form a covalent bond (i.e., be converted into a linking element of the conjugate) by chemically reacting an amine group or thiol group of the immunoglobulin Fc region with an amine group or thiol group of X. That is, the reactive group-bearing non-peptidic polymer becomes a non-peptidic linker that constitutes a part of the conjugate through a linking reaction, and in this case, the non-peptidic linker in the conjugate is composed of a repeating unit of the reactive group-bearing non-peptidic polymer and a linking element.

[0134] Specifically, the reactive group-containing nonpeptidyl polymer that may be used to prepare the conjugate may be, but is not limited to, a nonpeptidyl polymer having reactive groups at both ends that can be bonded to F (e.g., an immunoglobulin Fc region) and X, respectively, specifically, a reactive group that can be bonded to X or the N-terminus or an amine group located at a lysine of F (e.g., an immunoglobulin Fc region), or a thiol group of cysteine.

[0135] Furthermore, the reactive group of the non-peptidyl polymer capable of forming a covalent bond with F, e.g., an immunoglobulin Fc region, and X, may be selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto. In the above, the aldehyde may be, for example, a propionaldehyde group or a butyraldehyde group, but is not limited thereto.

[0136] The succinimide derivative may be, but is not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate.

[0137] The non-peptide linker may be connected to X and F via a linking element through conversion of these reactive groups, but is not particularly limited thereto.

[0138] Furthermore, the final product generated by reductive amination with an aldehyde bond is much more stable than that with an amide bond. The aldehyde reactive group selectively reacts with the N-terminus at low pH and can form a covalent bond with lysine residues at high pH, ​​e.g., pH 9.0.

[0139] Furthermore, the reactive groups at both ends of the reactive group-containing nonpeptidic polymer may be the same or different from each other, and may, for example, have a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group at the other end. However, there is no particular limitation thereto, so long as F, specifically, the immunoglobulin Fc region and X can be bound to each end of the reactive group-containing nonpeptidic polymer.

[0140] For example, the reactive group-containing nonpeptidyl polymer may contain a maleimide group as a reactive group at one end and an aldehyde group, a propionaldehyde group, a butyraldehyde group or the like at the other end.

[0141] When polyethylene glycol having hydroxy reactive groups at both ends is used as the non-peptidic polymer, the hydroxy groups may be activated with various reactive groups by known chemical reactions, or the long-acting protein conjugate of the present invention may be produced using commercially available polyethylene glycol having modified reactive groups.

[0142] In one specific embodiment, the non-peptidyl polymer may be linked to X, i.e., the N-terminal region or lysine residue of the insulin analogue, but is not particularly limited thereto. Specifically, the non-peptidyl polymer, specifically PEG, may be linked to the N-terminal region of the insulin analogue, specifically the amine group of the most terminal amino acid, or the amine group of a lysine present within the insulin analogue, but is not particularly limited thereto.

[0143] Furthermore, in a specific embodiment, the non-peptidyl polymer may be linked to an N-terminal region of F, for example, immunoglobulin Fc, but is not particularly limited thereto. Specifically, the non-peptidyl polymer may be linked to the N-terminal region of the immunoglobulin Fc, specifically, the amine group of the most terminal amino acid, but is not particularly limited thereto. The term "N-terminal region" refers to the amino-terminal region of an insulin analog or immunoglobulin Fc, and for the purposes of the present invention, refers to a position that can be bound to a linker. Examples of this include, but are not limited to, not only the most terminal amino acid residue of the N-terminal region, but also all amino acid residues surrounding the N-terminal amino acid residue, and specifically, the first to 20th amino acid residues from the most end. However, this is not particularly limited.

[0144] If an aldehyde group is present at one end of the reactive group-containing nonpeptidyl polymer and this aldehyde group is linked to an amine group located at the N-terminus of an insulin analogue, the linkage may be carried out via a reductive amination reaction. If an aldehyde group is present at the other end of the reactive group-containing nonpeptidyl polymer and this aldehyde group is linked to an amine group located at the N-terminus of an immunoglobulin Fc, the linkage may also be carried out via a reductive amination reaction.

[0145] In one specific embodiment of the conjugate of the present invention, the reactive group-bearing nonpeptidyl polymer used in the preparation is PEG having aldehyde reactive groups at both ends, which is linked to the amino groups of X and F via reductive amination to form a conjugate, and the conjugate has a structure of the following form:

[0146] X-NHCH2-W-PEG moiety-Z-CH2NH-F Here, -CHW- is a linking element connecting the nonpeptidyl polymer to X, and -Z-CH- is a linking element connecting the nonpeptidyl polymer to F, and W and Z are absent or are a divalent functional group such as alkylene. The methylenes in -CHW- and -Z-CH- are derived from the respective aldehyde reactive groups, and the nitrogen atoms in X-NH and NH-F are derived from the insulin analog used in the linking reaction and the amino group of F, respectively.

[0147] The PEG moiety in the above structure may include not only the -(CH2CH2O)n- structure, but also the intervening oxygen atom between the linking element and the -(CH2CH2O)n- structure.

[0148] In the present invention, the "immunoglobulin Fc region" refers to a portion of the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3) excluding the heavy and light chain variable regions of an immunoglobulin. The immunoglobulin Fc region may be a component that forms a part of the conjugate of the present invention.

[0149] These immunoglobulin Fc regions may include a hinge portion in the heavy chain constant region, but are not limited thereto. Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes a part or the entire heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1) excluding only the heavy and light chain variable regions of the immunoglobulin, so long as it has an effect substantially equivalent to or improved from that of the natural type. Alternatively, it may be a region in which a very long part of the amino acid sequence corresponding to CH2 and / or CH3 has been deleted.

[0150] For example, the immunoglobulin Fc region of the present invention may be a combination of one or more of the following domains with an immunoglobulin hinge region (or a part of a hinge region): 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; 5) a combination of one or more of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a part of a hinge region); or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region. However, the present invention is not limited thereto.

[0151] In addition, as a specific example, the immunoglobulin Fc region may be in a dimeric form, and one molecule of X may be covalently linked to one Fc region in the dimeric form, and in this case, the immunoglobulin Fc and X may be linked to each other via a non-peptide polymer. Meanwhile, two molecules of X may be symmetrically linked to one Fc region in the dimeric form. In this case, the immunoglobulin Fc and X may be linked to each other via a non-peptide linker. However, the present invention is not limited to the above-described examples.

[0152] Furthermore, the immunoglobulin Fc region of the present invention includes not only naturally occurring amino acid sequences but also sequence derivatives thereof, which means that one or more amino acid residues in the naturally occurring amino acid sequence have a different sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

[0153] For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, may be used as suitable sites for modification.

[0154] Various types of derivatives are possible, such as those in which sites capable of forming disulfide bonds are removed, those in which several amino acids at the N-terminus of native Fc are removed, or those in which a methionine residue is added to the N-terminus of native Fc. In addition, in order to eliminate effector functions, complement binding sites, such as C1q binding sites, and ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed. Techniques for producing sequence derivatives of these immunoglobulin Fc regions are disclosed in Patent Documents 1 and 2, etc.

[0155] Amino acid exchanges in proteins and peptides that do not change the overall activity of the molecule are known in the art (Non-Patent Document 3). The most commonly occurring exchanges are between the amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, the amino acid residues may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.

[0156] The above-mentioned Fc derivatives may exhibit biological activity equivalent to that of the Fc region of the present invention, and may have increased structural stability against heat, pH, and the like of the Fc region.

[0157] Furthermore, these Fc regions may be obtained from natural forms isolated in the living body of an animal such as a human, cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, or may be recombinant forms or derivatives thereof obtained from transformed animal cells or microorganisms. Here, the method of obtaining from natural forms may be a method of obtaining by separating the whole immunoglobulin from the living body of a human or animal and then treating it with a protease. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated using size-exclusion chromatography or the like. In a more specific embodiment, it is a recombinant immunoglobulin Fc region obtained by obtaining a human-derived Fc region from a microorganism.

[0158] The immunoglobulin Fc region may also be in a form having a native sugar chain, an increased sugar chain compared to the native type, a decreased sugar chain compared to the native type, or a form in which the sugar chain has been removed. To increase, decrease, or remove these immunoglobulin Fc sugar chains, conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used. Here, an immunoglobulin Fc region from which the sugar chains have been removed from Fc has a significantly reduced binding ability to complement (C1q) and antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity is reduced or removed, so that it does not induce unnecessary immune reactions in vivo. In this respect, a form that is more suitable for the original purpose as a drug carrier would be an immunoglobulin Fc region from which the sugar chains have been removed or which has been deglycosylated.

[0159] In the present invention, "deglycosylation" refers to an Fc region from which sugars have been removed using an enzyme, and "non-glycosylated" refers to an Fc region that is produced in a prokaryote, or in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0160] On the other hand, the immunoglobulin Fc region may be of human or animal origin, such as cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc., and in a more specific embodiment, is of human origin.

[0161] The immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid of these. In a more specific embodiment, it is derived from IgG or IgM, which are the most abundant in human blood, and in a more specific embodiment, it is derived from IgG, which is known to improve the half-life of ligand-binding proteins. In a further specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0162] Meanwhile, in the present invention, the term "combination" means that a polypeptide encoding a short-chain immunoglobulin Fc region of the same origin forms a bond with a short-chain polypeptide of a different origin when forming a dimer or multimer. In other words, a dimer or multimer can be produced from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and Fc fragments of IgE.

[0163] Another embodiment of the present invention provides a polynucleotide encoding the conjugate, a vector containing the polynucleotide, and a transformant containing the polynucleotide or the vector containing the same.

[0164] The conjugate is as described above.

[0165] The polynucleotide may encode the conjugate in the form of a fusion protein.

[0166] In addition, isolated polynucleotides encoding the conjugates are included in the category of polynucleotide sequences having 75% or more sequence identity with the sequence of interest, specifically 85% or more, more specifically 90% or more, and even more specifically 95% or more sequence identity. The above-mentioned explanations regarding the vector and the transformant also apply to this embodiment.

[0167] Another aspect of the present invention provides a method for producing the conjugate. The conjugate is as described above. Specifically, the method comprises: (a) reacting a non-peptidyl polymer having two or more terminal reactive groups with either an insulin analog or a carrier to produce a non-peptidyl polymer having an insulin analog or a carrier attached at one end and a reactive end group at the other end; and (b) reacting the insulin analogue or carrier produced in step (a) with a non-peptidyl polymer having an insulin analogue or carrier attached at one end and a terminal reactive group at the other end with the other of the carrier or insulin analogue to produce a conjugate in which the insulin analogue and the carrier are linked via the non-peptidyl polymer.

[0168] All of the above-mentioned descriptions are applicable to the non-peptidyl polymer, carrier, insulin analogue and the linking structure thereof. Another aspect of the present invention provides a composition comprising the conjugate. The conjugate is as described above. In the present invention, the composition may be a pharmaceutical composition. More specifically, the composition may be a pharmaceutical composition for preventing or treating an insulin-related disease. More specifically, the composition may be a pharmaceutical composition for preventing or treating diabetes. In the present invention, the term "insulin-related disease" refers to a disease that occurs or progresses when the physiological activity of insulin is absent or low, and examples thereof include, but are not limited to, diabetes.

[0169] In the present invention, the term "prevention" means any action of suppressing or delaying the onset of insulin-related diseases by administering the conjugate or a composition containing the same, and "treatment" means any action of improving or favoring the symptoms of insulin-related diseases by administering the conjugate or a composition containing the same.

[0170] Pharmaceutical compositions containing insulin analogue conjugates according to the invention may include pharma- ceutically acceptable carriers, which may be non-naturally occurring.

[0171] In the present invention, the term "pharmaceutical acceptable" means a sufficient amount capable of exhibiting a therapeutic effect and causing no side effects, and may be easily determined by a person skilled in the art depending on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, sensitivity of the patient to the drug, administration route, administration method, number of doses, treatment period, and drugs used in combination or concomitantly.

[0172] The pharma- ceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a dye, and a flavoring agent when administered orally, a buffer, a preservative, a soothing agent, a solubilizer, an isotonic agent, and a stabilizer when administered in an injection, and a base, an excipient, a lubricant, and a preservative when administered locally. The dosage form of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharma-ceutically acceptable carrier as described above. For example, the pharmaceutical composition may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, and wafers when administered orally, and in the form of unit dose ampoules or multiple doses when administered in an injection. The pharmaceutical composition may also be prepared in the form of solutions, suspensions, tablets, pills, capsules, and sustained release preparations.

[0173] On the other hand, examples of carriers, excipients and diluents suitable for formulation may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, etc. Also, fillers, anti-agglomerating agents, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc. may be further included.

[0174] In the present invention, "administration" means introducing a given substance into a patient by any suitable method, and the administration route of the conjugate may be any common route as long as the drug can reach the target tissue. It may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc., but is not limited thereto. However, since peptides are digested when administered orally, it is desirable to formulate oral compositions so that the active agent is coated or protected from degradation in the stomach. Specifically, it may be administered in the form of an injection. In addition, the pharmaceutical composition may be administered by any device that can transfer the active ingredient to the target cell.

[0175] In addition, the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, together with several related factors such as the disease to be treated, the route of administration, the age, sex, weight, and severity of the disease of the patient.

[0176] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have a different content of active ingredient depending on the severity of the disease. Specifically, the desired total dose of the conjugate of the present invention may be about 0.0001 mg to 500 mg per kg of patient body weight per day. However, the effective dose of the conjugate is determined in consideration of various factors such as the age, body weight, health condition, sex, severity of disease, diet, and excretion rate of the patient as well as the administration route and number of treatments of the pharmaceutical composition. Taking these points into consideration, a person having ordinary skill in the art can determine an appropriate effective dose according to a specific use of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route, and administration method as long as it exhibits the effects of the present invention.

[0177] Another aspect of the present invention provides a long-acting formulation comprising a conjugate of an insulin analogue, which has increased in vivo persistence and / or stability compared to a conjugate of natural insulin.

[0178] The insulin analogues and conjugates are as described above.

[0179] Another embodiment of the present invention provides a method for treating an insulin-related disorder, comprising administering the insulin analogue conjugate or a composition thereof to an individual in need thereof.

[0180] The insulin analogues and conjugates are as described above.

[0181] In one embodiment, the insulin-related disease is diabetes, but is not limited thereto. Another aspect of the present invention provides the use of the conjugate of the insulin analogue in the manufacture of a medicament.

[0182] In one embodiment, the medicament is for preventing or treating an insulin-related disease, but is not particularly limited thereto.

[0183] Another embodiment is for the prevention or treatment of diabetes, but is not particularly limited thereto.

[0184] Another embodiment of the present invention provides the use of the conjugate of insulin analogue in the treatment of insulin-related diseases, particularly diabetes.

[0185] The insulin analogues, conjugates, and insulin-related diseases are as described above. The present invention will be described in more detail with reference to the following examples, however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0186] Example 1: Construction of short insulin analogue expression vectors To produce insulin analogs in which one amino acid in the A or B chain was modified, forward and reverse oligonucleotides were synthesized using the natural insulin expression vector as a template (Table 2), and then PCR was performed to amplify each analog gene.

[0187] Table 1 shows the altered amino acid sequence and the analog name of each of the A and B chains. In other words, in the case of Analog 1, tyrosine 14 of A chain is replaced with histidine (H), and in the case of Analog 6, tyrosine 16 of B chain is replaced with glutamic acid (E).

[0188] [Table 1] Primers for amplifying insulin analogues are shown in Table 2 below.

[0189] [Table 2] TIFF0007604098000003.tif93168 The PCR conditions for amplifying insulin analogs were 95°C for 30 seconds, 55°C for 30 seconds, and 68°C for 6 minutes, and this process was repeated 18 times. The insulin analog fragments obtained under these conditions were inserted into a pET22b vector to be expressed in the form of inclusion bodies within cells, and the expression vectors thus obtained were named pET22b insulin analogs 1-13. The expression vectors contain nucleic acids encoding the amino acid sequences of insulin analogs 1-13 under the control of a T7 promoter. The insulin analog proteins were expressed in the form of inclusion bodies in a host containing the expression vector.

[0190] Table 3 shows the DNA and protein sequences of each of the insulin analogues 1 to 13.

[0191] Each sequence change was confirmed via DNA sequence analysis, and the results confirmed that each insulin analog had the desired sequence changes.

[0192] [Table 3] TIFF0007604098000005.tif155162 TIFF0007604098000006.tif155162 TIFF0007604098000007.tif155162 TIFF0007604098000008.tif155162 TIFF0007604098000009.tif155162 TIFF0007604098000010.tif81162

[0193] Example 2: Expression of fusion peptides of recombinant insulin analogues Expression of recombinant insulin analogs under the control of the T7 promoter was carried out. Each recombinant insulin analog expression vector was transformed into E. coli BL21-DE3 (E. coli B F-dcm ompT hsdS(rB-mB-)galλDE3; Novagen). The transformation method was according to the method recommended by Novagen. Individual single colonies transformed with each recombinant expression vector were picked and inoculated into 2x Luria Broth (LB) medium containing ampicillin (50 μg / ml) and cultured at 37°C for 15 hours. The culture medium of the recombinant strain and 2x LB medium containing 30% glycerol were mixed at a 1:1 (v / v) ratio, and 1 ml of each was dispensed into cryotubes and stored at -140°C. These were used as cell stocks for the production of recombinant fusion proteins.

[0194] For the expression of recombinant insulin analogs, one vial of each cell stock was dissolved and inoculated into 500 ml of 2x Luria broth and cultured at 37°C for 14-16 hours with shaking. When the OD600 value was 5.0 or more, the culture was terminated and used as the seed culture. The seed culture was inoculated into 17 L of fermentation medium using a 50 L fermenter (MSJ-U2, BEMARUBISHI, Japan) to start initial bath fermentation. The culture conditions were a temperature of 37°C, an air volume of 20 L / min (1 vvm), an agitation speed of 500 rpm, and a pH of 6.70 maintained using 30% ammonia water. The progress of fermentation was carried out by adding a feeding solution when nutrients in the culture became limiting. The growth of the strain was monitored by the OD value, and IPTG was introduced at a final concentration of 500 μM when the OD value was 100 or more. The culture was allowed to proceed for about 23 to 25 hours after the introduction, and after the culture was completed, the recombinant strain was harvested using a centrifuge and stored at -80°C until use.

[0195] Example 3: Recovery and refolding of recombinant insulin analogues To convert the recombinant insulin analogue expressed in Example 2 into a soluble form, the cells were disrupted and refolded. 100 g (wet weight) of cell pellet was resuspended in 1 L of lysis buffer (50 mM Tris-HCl (pH 9.0), 1 mM EDTA (pH 8.0), 0.2 M NaCl, and 0.5% Triton X-100). The cells were disrupted using a fine lysis processor Microfluidizer (Microfluidic Corp. Model M-110EH-30) operated at a pressure of 15,000 psi. The disrupted cell lysate was centrifuged at 7,000 rpm at 4-8°C for 20 minutes, the supernatant was discarded, and the cells were resuspended in 3 L of washing buffer (0.5% Triton X-100 and 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, and 1 mM EDTA). The mixture was centrifuged at 7,000 rpm for 20 minutes at 4-8°C, and the pellet was resuspended in distilled water and centrifuged in the same manner. The pellet was taken and resuspended in a buffer (1M L-glycine, 3.78 g L-cysteine-HCl, pH 10.6) and stirred at room temperature for 1 hour. To recover the resuspended recombinant insulin analog, 8M urea was added and stirred for 3-5 hours. To refold the solubilized recombinant proinsulin analog, the mixture was centrifuged at 7,000 rpm for 30 minutes at 4-8°C, and the supernatant was taken and treated with a reducing agent (15 mM L-cysteine-HCl) for 1 hour. A certain amount of distilled water was added using a peristaltic pump and stirred at 4-8°C for more than 12 hours.

[0196] Example 4: Cation exchange chromatography purification The refolded sample was bound to an SP FF (GE Healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol, and the insulin analog protein was then eluted with a linear concentration gradient of 10 column volumes from 0% to 100% using 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol.

[0197] Example 5: Treatment with Trypsin and Carboxypeptidase B The eluted sample was subjected to ultrafiltration to remove salts, and then replaced with a buffer solution (10 mM Tris-HCl, pH 8.0). Trypsin equivalent to about 30,000 molar ratio of the obtained sample protein and carboxypeptidase B equivalent to about 3,000 molar ratio were added, and the mixture was stirred at 4 to 8°C for 16 hours or more.

[0198] Example 6: Cation exchange chromatography purification After the reaction, the sample was bound again to an SP HP (GE Healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol, and the insulin analog protein was eluted with a linear concentration gradient of 10 column volumes from 0% to 100% using 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol.

[0199] Example 7: Purification by reversed phase chromatography To separate pure insulin analog from the sample obtained in Example 6, the sample was bound to a reversed-phase chromatography Source30RPC (GE Healthcare, USA) equilibrated with a buffer containing sodium phosphate and isopropanol, and the insulin analog protein was then eluted with a linear concentration gradient using a buffer containing sodium phosphate and isopropanol.

[0200] The insulin analogs purified in this manner were analyzed using protein electrophoresis (SDS-PAGE, Figure 1) and high pressure chromatography (HPLC). The results of the purity analysis of insulin analogs 9, 10, 11, and 12 are shown as representative examples (Figure 2).

[0201] Example 8: Preparation of long-acting insulin analogue conjugates Conjugates of the insulin analogs prepared in Examples 1 to 7 were prepared as follows. Typically, a long-acting conjugate of Analog 10 was prepared as follows.

[0202] In order to PEGylate the N-terminus of the B chain of insulin analogue 10 with 3.4K propion-ALD(2)PEG (3.4 kDa PEG with one propionaldehyde group at each end, NOF, USA), the insulin analogue 10 was reacted at a molar ratio of 1:4 with a concentration of 5 mg / ml at 4°C for about 2 hours. The reaction was carried out in a mixed solvent of 50 mM sodium citrate buffer (pH 5.0) and 45% isopropanol, with the addition of a 3 mM sodium cyanoborohydride (NaCNBH3) reducing agent. The reaction solution was purified using a SP-HP (GE Healthcare) column using a buffer containing sodium citrate (pH 3.0), 45% EtOH, and a KCl concentration gradient.

[0203] Next, in order to link the PEG to which the insulin analogue was attached to the N-terminus of the immunoglobulin Fc fragment, the purified mono-PEGylated insulin analogue and the immunoglobulin Fc fragment were reacted at a molar ratio of 1:1.2, a total protein concentration of 20 mg / ml, and 15 hours at 25° C. The reaction solution was 100 mM HEPES buffer (pH 8.2) and sodium chloride, with 20 mM sodium cyanoborohydride added as a reducing agent.

[0204] After the reaction was completed, the reaction solution was applied to a Q-HP (GE, USA) column using a Tris-HCl (pH 7.5) buffer and a NaCl gradient, and then applied to a Source 15ISO (GE, USA) using an ammonium sulfate and Tris-HCl (pH 7.5) gradient to purify the insulin analog 10-3.4K PEG-immunoglobulin Fc conjugate.

[0205] Comparative Example: Preparation of Long-acting Native Insulin Conjugate To PEGylate the N-terminus of the B chain of native insulin (Biocon, India) with 3.4K propion-ALD(2) PEG (3.4 kDa PEG having one propionaldehyde group at each end, NOF, USA), the PEGylation reaction and purification column were applied as in Example 8 to purify mono-PEGylated native insulin.

[0206] Next, in order to link the PEG to which the native insulin was attached to the N-terminus of the immunoglobulin Fc fragment, the purified mono-PEGylated native insulin and the immunoglobulin Fc fragment were subjected to the same reaction conditions and purification column as in Example 8 to prepare a native insulin-3.4K PEG-immunoglobulin Fc conjugate.

[0207] Experimental Example 1: Comparison of the binding ability of insulin analogues to the insulin receptor The binding strength of insulin analogues to insulin receptors was measured using SPA (Scintillation Proximity Assay). First, to obtain cell membranes expressing insulin receptor B isoform, CHO cell lines were transfected using Lipofectamine (Life Technologies, USA) to produce CHO cell lines overexpressing insulin receptor B isoform. The cells were homogenized using a homogenizer (Wheaton, USA) and the cell membranes were isolated using the sucrose cushion method. The obtained cell membrane of the CHO cell line expressing the human native insulin receptor B isoform and PVT SPA beads were placed together in a 96-well picoplate (PerkinElmer, model number 6005162, USA), and human insulin or each insulin analog diluted in reaction solution (50 mM Tris-HCl pH 7.8, 150 mM, 0.1% BSA) at 10 or more concentrations as competitors, and human native insulin labeled with the radioisotope iodine-125 (PerkinElmer, model number NEX-420, USA) as a detector ligand were added together, and the competitive reaction was carried out at room temperature for 4 hours. After 4 hours, the binding strength of the insulin receptor was measured using a beta counter (PerkinElmer, model number C9904V1, USA). The values ​​measured by the beta counter were used to calculate the IC of each substance in the experiment using GraphPad Prism 6 software. 50 The insulin receptor IC of the insulin analogue was calculated relative to the insulin receptor binding affinity of human insulin. 50 The percentage was expressed as a binding strength.

[0208] As a result, the receptor binding strength was confirmed to be 90% for insulin analog (No. 1), 95% for insulin analog (No. 2), 1.0% for insulin analog (No. 3), <0.1% for insulin analog (No. 4), 20% for insulin analog (No. 6), 8.5% for insulin analog (No. 7), 79% for insulin analog (No. 9), 79% for insulin analog (No. 10), 24% for insulin analog (No. 11), <0.1% for insulin analog (No. 12), and <0.1% for insulin analog (No. 13) compared to human insulin (Table 4). Thus, it was observed that the insulin analog of the present invention had a decreased insulin receptor binding strength compared to natural insulin.

[0209] [Table 4] Experimental Example 2: Confirmation of receptor binding ability of long-acting insulin analogue conjugate The binding ability of the long-acting insulin analogues to the insulin receptor and IGF-1 receptor was examined by the method described in Experimental Example 1 above.

[0210] As a result, it was confirmed that the binding strength to the insulin receptor was 2.4±0.4% for the native insulin-PEG-immunoglobulin Fc conjugate and 1.3±0.2% for the insulin analog 10-PEG-immunoglobulin Fc conjugate compared to native human insulin, and that the binding strength to the IGF-1 receptor was less than 1% for both the native insulin-PEG-immunoglobulin Fc conjugate and the insulin analog 10-PEG-immunoglobulin Fc conjugate compared to native human IGF-1. Thus, it was confirmed that the insulin analog 10-PEG-immunoglobulin Fc conjugate of the present invention had a reduced insulin receptor binding strength compared to the native insulin-PEG-immunoglobulin Fc conjugate, and that both of the two substances had low IGF-1 receptor binding strength via the PEG-immunoglobulin Fc bond.

[0211] [Table 5] 1) The dose-response curve did not reach saturation even at the highest concentration, so the exact value was not determined. Experimental Example 3: Pharmacokinetic analysis of long-acting insulin analogue conjugates To confirm the pharmacokinetics of the long-acting insulin analogue conjugate, a comparative study of blood concentrations over time was conducted in normal rats (SD rats, male, 6 weeks old), normal mice, and dogs that had been adapted to the laboratory for a certain period of time.

[0212] Native insulin-PEG-immunoglobulin Fc conjugates and insulin analog 10-PEG-immunoglobulin Fc conjugates were administered subcutaneously in a single dose of 43.05 nmol / kg to mice and 65.1 nmol / kg to rats, respectively, and blood samples were taken at 0, 1, 4, 8, 24, 48, 72, 96, 120, 144, and 168 hours after administration. Native insulin-PEG-immunoglobulin Fc conjugates and insulin analog 10-PEG-immunoglobulin Fc conjugates were administered subcutaneously in a single dose of 6.3 nmol / kg to dogs, and blood samples were taken at 0, 1, 4, 8, 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 hours after administration.

[0213] The remaining concentrations of native insulin-PEG-immunoglobulin Fc conjugate and insulin analog 10-PEG-immunoglobulin Fc conjugate in the blood at each time point were measured by enzyme-linked immunosorbent assay (ELISA) using an Insulin ELISA kit (ALPCO, USA) with mouse anti-human IgG4 HRP conjugate (Alpha Diagnostic Intl, Inc., USA) as the detection antibody.

[0214] As a result, the half-life of the insulin analog 10-PEG-immunoglobulin Fc conjugate in mice, rats, and dogs was increased by 1.9-fold, 1.2-fold, and 1.6-fold, respectively, and the MRT was increased by 2.5-fold, 1.9-fold, and 2.0-fold, respectively, compared with that of the native insulin-PEG-immunoglobulin Fc conjugate. Thus, it was confirmed that the insulin analog 10-PEG-immunoglobulin Fc conjugate of the present invention has a longer duration in blood compared with that of the native insulin-PEG-immunoglobulin Fc conjugate.

[0215] [Table 6] Experimental Example 4: Confirmation of the glucose lowering effect of long-acting insulin analogue conjugate To confirm the pharmacodynamics of the long-acting insulin analogue conjugate in a type 2 diabetes model, normal rats (SD rats, male, 7 weeks old) were fed a high-fat diet (60% Fat) for 2 weeks, and then STZ, which can destroy pancreatic beta cells, was administered at a dose of 30 mg / kg twice at a one-week interval to create a type 2 diabetes model (hereinafter referred to as DIO / STZ rats). The model was continued to consume a high-fat diet to maintain diabetes.

[0216] Native insulin-PEG-immunoglobulin Fc conjugates and insulin analog 10-PEG-immunoglobulin Fc conjugates were subcutaneously administered at 12.1 nmol / kg and 8.09 nmol / kg, respectively, to DIO / STZ rats, and repeated administration was performed at 3-day intervals. To confirm the blood glucose lowering effect of native insulin-PEG-immunoglobulin Fc conjugates and insulin analog 10-PEG-immunoglobulin Fc conjugates, blood was collected from a microvein on days 0, 1, 3, 6, 7, 9, 10, 12, 13, 15, 16, 21, 22, 24, 25, 27, and 28 after administration, and blood glucose values ​​were confirmed using a blood glucose meter (OneTouch Ultra, LifeScan, Inc., USA).

[0217] As a result, it was confirmed that the insulin analog 10-PEG-immunoglobulin Fc conjugate had the same blood glucose lowering effect as the native insulin-PEG-immunoglobulin Fc conjugate even when administered at a 50% lower dose in DIO / STZ rats. This confirmed that the improved blood persistence of the insulin analog 10-PEG-immunoglobulin Fc conjugate of the present invention allows it to continuously maintain an excellent blood glucose lowering effect even at a lower dose than the native insulin-PEG-immunoglobulin Fc conjugate.

[0218] From the above description, a person skilled in the art to which the present invention belongs can understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims below and their equivalent concepts from the above detailed description.

Claims

1. A conjugate represented by the following general formula (1): [Chemical formula 1] X-La-F (1) In the formula (1), X is an insulin analogue comprising an A chain of SEQ ID NO: 55 represented by the following general formula (2) and a B chain of SEQ ID NO: 56 represented by the following general formula (3), L is a linker; a is 0 or a natural number, provided that when a is 2 or more, each L is independent of the others; F is a substance capable of increasing the half-life of X; The conjugate, in which X, L and F are covalently linked to each other in the order of general formula (1) (excluding a peptide in which the A chain corresponds to SEQ ID NO: 53 and the B chain also corresponds to the B chain of SEQ ID NO: 54). [Chemical 2] Xaa1-Ile-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21 (SEQ ID NO: 55) (2) [Chemical 3] Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Xaa16-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Xaa25- Tyr-Xaa27-Xaa28-Lys-Thr (SEQ ID NO: 56) (3) (In the general formula (2), Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is aspartic acid, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in the general formula (3), Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.)

2. 2. The conjugate of claim 1, wherein X is an insulin analog comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

46.

3. 3. The conjugate according to claim 1 or 2, wherein F is selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin.

4. The conjugate according to any one of claims 1 to 3, wherein the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

5. The conjugate according to any one of claims 1 to 4, wherein F is an immunoglobulin Fc region.

6. The conjugate of any one of claims 1 to 5, wherein F is an IgG Fc region.

7. The conjugate according to any one of claims 1 to 6, wherein L is selected from the group consisting of a peptide, a fatty acid, a saccharide, a high molecular weight polymer, a low molecular weight compound, a nucleotide, and combinations thereof.

8. The conjugate according to claim 7, wherein the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

9. The conjugate of any one of claims 1 to 8, wherein L is polyethylene glycol.

10. The conjugate according to claim 7, wherein the polymer has a molecular weight of 1 to 100 kDa.

11. The conjugate according to any one of claims 1 to 10, wherein the linker is linked to the N-terminus of the B chain of the insulin analogue or to a lysine residue of the insulin analogue.

12. The conjugate according to any one of claims 1 to 11, wherein F is an immunoglobulin Fc region and a linker is attached to the N-terminus of the immunoglobulin Fc region.

13. The conjugate according to any one of claims 1 to 12, wherein F is an immunoglobulin Fc region, and the conjugate has a structure in which both ends of a linker are linked to the N-terminus of the B chain of an insulin analogue and the N-terminus of the immunoglobulin Fc, respectively.

14. The conjugate according to any one of claims 1 to 13, wherein the insulin analogue has reduced binding affinity to a native insulin receptor compared to native insulin.

15. 15. The conjugate of any one of claims 1 to 14, wherein the conjugate is an insulin analogue having the same sequence as native insulin, except that F is an immunoglobulin Fc region, L is polyethylene glycol, and X is an aspartic acid at amino acid 14 of the A chain.

16. A pharmaceutical composition for preventing or treating diabetes, comprising the conjugate according to any one of claims 1 to 15.

17. 16. A method for treating diabetes, comprising administering a conjugate according to any one of claims 1 to 15 or a composition comprising the same to a non-human individual in need of such treatment.

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