RU2779462C2 - Insulin analogue complex with reduced affinity to insulin receptor and its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to the production of insulin conjugates; it can be used in medicine for the treatment of diabetes. An insulin conjugate of the formula X-L₁-F (formula 1) is proposed, where X is an insulin analogue having the same sequence as native insulin, except for the fact that 14^th amino acid of A-chain is aspartic acid; L is polyethylene glycol as a linker; F is Fc-fragment of immunoglobulin. In the proposed conjugate, X, L, and F are bound to each other by means of a covalent bond in the order specified in the formula 1, and both ends of the specified linker are attached to N-end of B-chain of the specified insulin analogue and to N-end of the specified Fc-fragment of immunoglobulin.

EFFECT: resulting insulin conjugate of prolonged action of unnatural origin allows for improvement of compliance of patients, who need insulin administration.

6 cl, 4 dwg, 4 tbl, 9 ex

Description

FIELD OF TECHNOLOGY

This invention relates to an insulin analogue conjugate and its use.

PRIOR ART

Proteins are known to be eliminated from the body by various means, including cleavage by proteases in the blood, excretion by the kidneys, receptor-mediated elimination, and so on. In this regard, various attempts have been made to improve the therapeutic effects of proteins by increasing the half-life of physiological proteins by avoiding the mechanisms of protein clearance.

In general, insulin is a hormone secreted by the pancreas in the human body that regulates glucose levels and maintains normal blood glucose levels while allowing excess glucose from the blood to flow into the cells to provide energy to the cells. However, in diabetic patients, insulin does not function properly due to lack of insulin, insulin resistance, and dysfunction of beta cells. As a result, diabetic patients cannot utilize blood glucose as an energy source, but still show symptoms of hyperglycemia where there is high blood glucose and urinary glucose excretion, which causes various complications. Accordingly, insulin therapy is of fundamental importance for patients with abnormal insulin production (type I) or insulin resistance (type II), and normal regulation of glucose levels is carried out by administration of insulin.

However, like other proteins and peptide hormones, insulin has a very short in vivo half-life and therefore does not have a long therapeutic effect. Therefore, repeated administration of insulin is required to maintain this effect. Such frequent administration causes pain and discomfort to patients, and therefore, taking into account aspects such as patient compliance, safety and convenience, administration needs to be improved.

In this regard, research has focused on the development of various protein compositions, chemical conjugates (e.g. fatty acid conjugate), etc., which improve therapeutic effects as well as the quality of life of patients by reducing the frequency of administration due to the increase in in vivo half-life of such protein drugs like insulin.

According to a recent report, 50% or more of insulin is excreted via the kidneys, and the rest is excreted through the process of receptor-mediated clearance (ROC) at target sites such as muscle, fat, liver, etc.

According to publications (J Pharmacol Exp Ther (1998) 286: 959, Diabetes Care (1990) 13: 923, Diabetes (1990) 39: 1033 and others) in order to avoid insulin ROCK, reduce in vitro activity, thereby increasing insulin levels in blood. However, the insulin analogs proposed in J Pharmacol Exp Ther (1998) 286: 959, Diabetes Care (1990) 13: 923, which had at least two amino acid substitutions, did not lead to any specific result, while those described in Diabetes (1990) 39: 1033 insulin analogs showed no change in their receptor binding affinity or decreased activity as a result of substitution of amino acids directly involved in binding to insulin receptors. Thus, there is still a need to develop insulin analogues with extended duration of action.

SUMMARY OF THE INVENTION

Technical task

The object of the present invention is to provide an insulin analogue conjugate.

Another object of the present invention is to provide a polynucleotide encoding a conjugate, a vector comprising the polynucleotide, and a transformant comprising the polynucleotide or a vector comprising the polynucleotide.

Another object of this invention is to provide a method for producing a conjugate.

Another object of this invention is to provide a composition, for example a pharmaceutical composition, comprising an insulin analog conjugate.

Another object of the present invention is to provide a sustained release composition comprising an insulin analog conjugate with increased in vivo duration of action and/or stability compared to a native insulin conjugate.

Another object of the present invention is to provide a pharmaceutical composition comprising an insulin analog conjugate for the prevention or treatment of an insulin related disease such as diabetes.

Another object of the present invention is to provide a method for treating an insulin related disease, such as diabetes, comprising the step of administering an insulin analog conjugate or composition to a subject in need thereof.

Another object of the present invention is to provide the use of an insulin analog conjugate for the manufacture of a medicament.

Yet another object of the present invention is to provide the use of an insulin analogue conjugate in the treatment of an insulin related disease, in particular diabetes.

Technical solution

To solve the problems mentioned above, in one aspect of the present invention, an insulin analogue conjugate is provided.

More specifically, in an aspect of the present invention, there is provided a conjugate represented by Formula 1 below:

where in Formula 1

X is an insulin analogue having a modification of one or more amino acids selected from the group consisting of B chain amino acid 16, B chain amino acid 25, A chain amino acid 14, and amino acid A 19 - chains of native insulin;

L is a linker;

a is 0 or a natural number, provided that when a is 2 or more, each of L is independent of each other, and

F is a substance capable of increasing the half-life X.

In an exemplary embodiment, X has one or more modifications selected from the group consisting of a modification of the 16th amino acid of the native insulin B chain, tyrosine, to glutamic acid, serine, threonine, or aspartic acid; modifying the 25th amino acid of the native insulin B chain, phenylalanine, with aspartic acid or glutamic acid; modifying the 14th amino acid of the native insulin A chain, tyrosine, with histidine, lysine, alanine, or aspartic acid; and modifying the 19th amino acid of the native insulin A chain, tyrosine, with glutamic acid, serine, or threonine.

In another exemplary embodiment, X is an insulin analog comprising all combinations of an A chain with the sequence of SEQ ID NO: 55 as specified in General Formula 2 and a B chain with the sequence of SEQ ID NO: 56 as specified in General Formula 3 (except for native insulin, which means the exclusion of the peptide in which the A chain matches SEQ ID NO: 53, while the B chain matches SEQ ID NO: 54):

General Formula 2

Xaal-Ile-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21 (SEQ ID NO: 55), where in 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, seri, 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; and Xaa21 is asparagine, glycine, histidine or alanine; and General Formula 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), where in the general formula 3

Xaa16 is tyrosine, glutamic acid, sulfur, threonine or aspartic acid;

Xaa25 is phenylalanine, aspartic acid or glutamic acid;

Xaa27 is threonine or absent; and

Xaa28 is proline, glutamic acid, or aspartic acid, or is absent.

In yet another exemplary embodiment, X comprises an A chain with the sequence of SEQ ID NO: 55 as set forth in General Formula 2 and a B chain with the sequence of SEQ ID NO: 54.

In yet another exemplary embodiment, X comprises an A chain with the sequence of SEQ ID NO: 53 as specified in General Formula 2 and a B chain with the sequence of SEQ ID NO: 56 as specified in General Formula 3.

In yet another exemplary embodiment, in 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 general formula 3

Xaa16 is tyrosine, glutamic acid, serine, threonine, or aspartic acid; Xaa25 is phenylalanine, aspartic acid, or glutamic acid, Xa27 is threonine, and Xa28 is proline.

In yet another exemplary embodiment in 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 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, Xa27 is threonine, and Xa28 is proline.

In yet another exemplary embodiment, X is characterized in that

what:

(1) in general formula 2, Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is histidine, Xaa16 is leucine, Xaa19 is tyrosine, Xaa21 is asparagine; and in general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(2) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(3) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(4) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(5) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(6) in 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 general formula 3, Xaa16 is glutamic acid, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(7) in 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 general formula 3, Xaa16 is serine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(8) in 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 general formula 3, Xaa16 is threonine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(9) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(10) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(11) in 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 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 general formula 3, Xaa16 is tyrosine, Xaa25 is aspartic acid, Xaa27 is threonine, and Xaa28 is proline;

(13) in 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 general formula 3, Xaa16 is tyrosine, Xaa25 is glutamic acid, Xaa27 is threonine, and Xaa28 is proline.

In yet another exemplary embodiment, X is 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.

In the following exemplary embodiment in general formula 2, Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is glutamic acid, Xaa21 is asparagine; and in general formula 3, Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

In the following exemplary embodiment in general formula 2, Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is tyrosine, Xaa16 is leucine, Xaa19 is serine, Xaa21 is asparagine; and in general formula 3, Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

In the following exemplary embodiment in 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 general formula 3, Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

In the following exemplary embodiment, in 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 general formula 3, Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

In the following exemplary embodiment in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is aspartic acid, Xaa27 is threonine and Xaa28 is proline.

In the following exemplary embodiment in 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 general formula 3, Xaa16 is tyrosine, Xaa25 is glutamic acid, Xaa27 is threonine, and Xaa28 is proline.

In the following exemplary embodiment, F is selected from the group consisting of polymer, fatty acid, cholesterol, albumin and fragment thereof, albumin binder, polymer consisting of repeating blocks of a specific amino acid sequence, antibody, antibody fragment, FcRn binder , connective tissue in vivo, nucleotide, fibronectin, transferrin, saccharide, heparin and elastin.

In the following exemplary embodiment, the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and their combinations.

In yet another exemplary embodiment, F is an immunoglobulin Fc fragment.

In yet another exemplary embodiment, F is an IgG Fc fragment.

In yet another exemplary embodiment, L is selected from the group consisting of a peptide, a fatty acid, a saccharide, a polymer, a small molecule, a nucleotide, and combinations thereof.

In another exemplary embodiment, the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and their combinations.

In yet another exemplary embodiment, L is polyethylene glycol.

In yet another exemplary embodiment, the polymer has a molecular weight of 1 kDa to 100 kDa.

In yet another exemplary embodiment, the linker is attached to the N-terminus of the B chain of the insulin analog or to a lysine residue in the insulin analog.

In yet another exemplary embodiment, F is an immunoglobulin Fc fragment and the linker is attached to the N-terminus of the immunoglobulin Fc fragment.

In yet another exemplary embodiment, F is an Fc fragment and the conjugate has a structure in which both ends of the linker are attached to the N-terminus of the B chain of the insulin analogue and to the N-terminus of the Fc fragment of the immunoglobulin.

In yet another exemplary embodiment, the insulin analog is an analog in which the binding affinity for the native insulin receptor is reduced compared to native insulin.

In yet another exemplary embodiment, the binding affinity of the insulin analog to the native insulin receptor is from about 10% to about 90% compared to the affinity of native insulin.

In yet another exemplary embodiment, the conjugate is characterized in that F is an immunoglobulin Fc fragment; L is polyethylene glycol, and the binding affinity of the conjugate to the native insulin receptor is 0.1% to 50% compared to native insulin.

In yet another exemplary embodiment, the conjugate is characterized in that F is an immunoglobulin Fc fragment; L is polyethylene glycol and X is an insulin analogue having a sequence identical to that of native insulin, except that the 14th amino acid of the A chain is aspartic acid.

In another aspect of the invention, a polynucleotide encoding a conjugate is provided; a vector comprising a polynucleotide; and a transformant comprising a polynucleotide or a vector comprising a polynucleotide.

In yet another aspect of the present invention, a method for preparing a conjugate is provided.

In yet another aspect of the present invention, a composition is provided that contains a conjugate.

In an exemplary embodiment, the composition is a pharmaceutical composition.

In another exemplary embodiment, the composition is a pharmaceutical composition for the prevention or treatment of an insulin related disease.

In yet another exemplary embodiment, the composition is a pharmaceutical composition for the prevention or treatment of diabetes.

In yet another aspect of the present invention, there is provided a sustained release composition with increased in vivo duration of action and/or stability compared to a native insulin conjugate, comprising an insulin analog conjugate.

In yet another aspect of the present invention, there is provided a method of treating an insulin related disease comprising the step of administering an insulin analog conjugate or composition to a subject in need thereof.

In an exemplary embodiment, the insulin related disease is diabetes.

In yet another aspect of the present invention, the use of an insulin analog conjugate for the manufacture of a medicament is provided.

In an exemplary embodiment, the drug is for the treatment of an insulin related disease.

In another exemplary embodiment, the drug is for the prevention or treatment of diabetes.

In yet another aspect of the present invention, the use of an insulin analog conjugate in the treatment of an insulin-related disease, in particular diabetes, is provided.

Beneficial effects

The non-native insulin analog conjugate of the present invention may improve the compliance of patients requiring insulin administration.

BRIEF DESCRIPTION OF GRAPHICS

On FIG. 1 shows the results of analysis of the purity of insulin analogs by electrophoretic separation of proteins, and in particular the results for representative insulin analogs 9, 10, 11 and 12 (lane 1: fragment size marker; lane 2: native insulin; lane 3: insulin analog 9; lane 4: insulin analog 10; lane 5: insulin analog 11 and lane 6: insulin analog 12).

On FIG. 2a-2d show the results of high pressure chromatography purity analysis of insulin analogs, and in particular the results for representative insulin analogs 9, 10, 11 and 12 (FIGS. 2a-2d). On each of Figs. from top to bottom are the results of high performance liquid chromatography (HPLC) with reversed phase (C18), HPLC with reversed phase (C4) and size exclusion HPLC.

On FIG. 3a - 3c show the results of a comparison of the pharmacokinetic characteristics of the "10 analogue insulin-PEG-Fc immunoglobulin" conjugate, which is a representative insulin analogue conjugate of this invention, and the "native insulin-PEG-Fc immunoglobulin" conjugate, which is a conjugate of native insulin, in normal mice (Fig. 3a), SD rats (Fig. 3b) and beagle dogs (Fig. 3c).

On FIG. 4 shows the results of a comparison of the ability to lower blood glucose level of the "10 insulin-PEG-Fc immunoglobulin analog" conjugate, which is a representative insulin analog conjugate of this invention, and the "native insulin-PEG-Fc immunoglobulin" conjugate, which is a native insulin conjugate, in DIO/STZ rats.

Preferred embodiments of the invention

Next, exemplary embodiments of the present invention will be described in detail.

However, each of the explanations and embodiments given in this document as an example, may be true for other explanations and given as an example of embodiments. Thus, all combinations of the various factors described herein are within the scope of this invention. Moreover, the scope of the present invention is not limited to the specific description below.

In addition, a person skilled in the art can, based on routine techniques, detect or confirm many equivalents of the specific embodiments of this invention described in this application, and such equivalents are included in the scope of this invention.

In the description of the invention, standard 1-letter and 3-letter designations of amino acids are used. In addition, the amino acids mentioned in this document are abbreviated according to the IUPAC-IUB nomenclature rules.

In one aspect of the present invention, an insulin analog conjugate is provided.

In particular, the insulin analog conjugate may be in the form of a conjugate in which the insulin analog is linked to a biocompatible material to increase the in vivo half-life of the insulin analog. In the present invention, a biocompatible material can be used interchangeably with a carrier.

In the present invention, an insulin analog conjugate can retain efficacy for a longer time compared to an insulin analog that is not conjugated to a carrier, and such a conjugate is referred to as a "long-acting conjugate" in the present invention.

However, such a conjugate may be a conjugate not found in nature.

More specifically, the present invention provides a conjugate represented by Formula 1 below:

where in Formula 1

X is an insulin analogue having a modification of one or more amino acids selected from the group consisting of B chain amino acid 16, B chain amino acid 25, A chain amino acid 14, and amino acid A 19 - chains of native insulin;

L is a linker;

a is 0 or a natural number, provided that when a is 2 or more, each of L is independent of each other, and

F is a substance capable of increasing the half-life X.

As used herein, the term "insulin analog" refers to non-native insulin that is different from native insulin. The insulin analog includes non-native human insulin that is different from native human insulin. The insulin analogue is the moiety that forms the conjugate of this invention and corresponds to X in Formula 1.

Such an insulin analog includes analogs in which some of the native insulin amino acids are modified by insertion, deletion, or substitution.

In particular, compared to the sequence of native insulin, an insulin analog of the invention may have an identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. In addition, the insulin analog of this invention may have a lower binding affinity for native insulin receptor compared to native insulin, while still having the above sequence identity with native insulin. In addition, the insulin analog may have the ability to absorb glucose and/or the ability to lower blood glucose levels in vivo, as shown for native insulin.

In the present invention, the binding affinity of any insulin analog to the native insulin receptor refers to the percentage binding inhibition that the insulin analog and native insulin exhibit when determining binding inhibition levels under conditions of competitive receptor binding inhibition, where the binding of any detectable ligand to the native insulin receptor insulin is inhibited in the presence of native insulin or an appropriate insulin analogue. Binding inhibition can be indirectly determined by the values that are recorded when determining whether there is an effective change in the signals observed when the ligand binds to the insulin receptor, according to appropriate criteria. For example, binding affinity can be determined from a measured value, expressed as a percentage (eg, IC _{50 of} native insulin as a percentage relative to the IC _{50 of} the corresponding insulin analogue) obtained by determining how much the signals increase or decrease; which means that one measures the signal emitted when the radioactively labeled insulin binds to the insulin receptor and determines how much each signal increases or decreases after the addition of unlabeled native insulin or an insulin analog (for example, by measuring signal reduction IC ₅₀ values for native insulin or analogue of insulin). Such determination of binding affinity for receptors by competitive inhibition is well known in the art. In an exemplary embodiment of the present invention, a cell membrane expressing a native human insulin receptor (isoform A or isoform B, or both) can be used as the native insulin receptor necessary for such determination, for example, the cell membrane of cells that have been genetically modified to increase the expression of the native human insulin receptor. In addition, native insulin labeled with iodine-125 can be used as a detection ligand. In a more specific embodiment of the invention, a scintillation proximity assay (SPA) can be used to study competitive inhibition of receptor binding. In a further specific embodiment of the present invention, the binding affinity for the insulin receptor can be determined by the method described in Experimental Example 1.

More specifically, the insulin analog of this invention may exhibit an insulin receptor binding affinity 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, 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 compared to affinity with binding of native insulin to the insulin receptor (100%) (however, the binding affinity of the insulin analog of this invention to the insulin receptor is not 0%). In a specific embodiment of the present invention, the binding affinity of the insulin analog to the native insulin receptor is from about 10% to about 90%, but is not limited to the indicated values. In addition, if the binding affinity is weak compared to the affinity of native insulin, the insulin analog is within the scope of this invention. If the binding affinity is weaker than that of native insulin, the elimination of the insulin analog mediated by the insulin receptor will be reduced, and therefore an improvement in blood half-life and stability of effect can be expected.

In one embodiment of the invention, the linker in the insulin analog conjugate may be polyethylene glycol; a substance capable of increasing the half-life may be an Fc fragment of an immunoglobulin; and the binding affinity for the native insulin receptor can be 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, 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 compared to the binding affinity of native insulin (however, the binding affinity of con The insulin receptor jugate of this invention is not 0%. In a specific embodiment of the present invention, the binding affinity of the insulin analog conjugate to the native insulin receptor is preferably from 0.1% to 50%, but is not limited to the indicated values. In addition, if the binding affinity is weaker compared to the affinity of native insulin, it is within the scope of this invention. If the binding affinity is weaker than that of native insulin, the elimination of the insulin analog conjugate mediated by the insulin receptor will be reduced, and therefore an improvement in blood half-life and stability of effect can be expected.

As used herein, the term "about" refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc. and therefore includes all values in the range that are equivalent or close to the values specified after this term, without limitation.

In addition, the insulin analog of this invention may have the ability to mediate glucose uptake as shown for native insulin.

In particular, the insulin analog of this invention may have the ability to mediate glucose uptake of about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 55% or above, 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 native insulin's ability to mediate glucose uptake (100%).

The ability to mediate glucose uptake can be measured by various methods known in the art.

In particular, the insulin analog may be in the form of a single chain, in which both of the two sections of the sequences corresponding to the A chain and the B chain of its native form are linked into one polypeptide chain, or may be in the form of two polypeptide chains, where each of the two sections of the sequence is formed by individual polypeptide chains. Further, when reference is made herein to the A-chain or B-chain of an insulin analog, it should be interpreted depending on the context that it refers to the A-chain or B-chain of the corresponding insulin analog in the form of two polypeptide chains, and that it refers to the region sequence corresponding to the native A chain of the corresponding single chain polypeptide, or to a portion of the sequence corresponding to the native B chain of the corresponding single chain polypeptide, respectively, even when the analog is in single chain form. The insulin analogue of this invention may be in the form of a single chain or in the form of two polypeptide chains which are a combination of one chain selected from the native insulin A chain or its corresponding polypeptide and one chain selected from the native insulin B chain or the corresponding polypeptide. polypeptide. In particular, matching of the A-chain or B-chain of native insulin may, for example, refer to cases where any polypeptide chain of two polypeptide chains has an identity of at least 60%, at least 65%, at least 70%, according to at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% compared to the sequence of the A chain or B chain of native insulin, without particular limitation, and a person skilled in the art can easily determine this by comparing the sequence constituting the corresponding polypeptide and the sequence of the A chain or B chain of native insulin.

As indicated above, the following description applies to all descriptions relating to the sub-terms of insulin analog.

Native insulin is a hormone secreted by the pancreas that promotes intracellular glucose uptake and inhibits fat breakdown, thereby controlling blood glucose levels in vivo. Insulin, which is able to control blood glucose levels, is formed as a result of the processing of its precursor, proinsulin, which does not have the function of controlling blood glucose levels. Insulin consists of two polypeptide chains, i.e. A-chains and B-chains, which include 21 and 30 amino acids, respectively, and are linked by disulfide bridges. Each of the A chain and the B chain may include the amino acid sequences set forth in SEQ ID NOs: 53 and 54 below.

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)

In an exemplary embodiment, the insulin analogs described herein may be analogs with reduced receptor binding affinity while still performing the function of controlling blood glucose levels in vivo like native insulin. More specifically, the insulin analog may have the ability to lower blood glucose levels in vivo.

In addition, in the exemplary embodiment, the type and size of the insulin analogs is not limited to any particular, as long as they can exhibit reduced receptor-mediated internalization or receptor-mediated clearance. Accordingly, the insulin analogs of this invention may exhibit improved blood half-life compared to native insulin.

The insulin analogs of this invention include inverted insulin, native insulin derivatives, native insulin fragments, and the like. Insulin analogues can be prepared not only by recombinant methods, but also by solid phase synthesis, and the preparation methods are not limited to those.

As used herein, the term "native insulin derivatives" refers to a peptide whose amino acid sequence differs at least one from native insulin, a peptide resulting from a modification of the sequence of native insulin, and a native insulin mimetic that are capable of controlling blood glucose levels in vivo as native insulin. Such native insulin derivatives may be derivatives that perform the function of controlling blood glucose levels in vivo.

In particular, native insulin derivatives can be obtained by modifying a portion of the native insulin amino acids in any of a number of substitutions, insertions, deletions, modifications, or combinations of methods.

In particular, native insulin derivatives may have 80% or greater homology with each of the native insulin A chain and B chain amino acid sequences, and/or part of the amino acid residue groups may be modified by chemical substitution (e.g., alpha-methylation, alpha-hydroxylation ), deletions (eg, deaminations) or modifications (eg, N-methylations), etc., without limitation.

Derivatives of native insulin used in this invention can be obtained by a combination of various methods used to obtain derivatives.

In addition, such modifications to obtain derivatives of native insulin include modification using L-type or D-type amino acids and/or amino acids of artificial origin; and/or native sequence modification or post-translational modification (eg, methylation, acylation, ubiquitination, intermolecular covalent bonds, etc.).

In addition, all native insulin derivatives include derivatives in which one or more amino acids are added to the amino and/or carboxy terminus of native insulin.

When replacing or inserting amino acid(s), not only the 20 amino acids commonly found in human proteins, but also atypical or artificial amino acids can be used. Commercial sources of atypical amino acids may be Sigma-Aldrich, ChemPep Inc. and Genzyme Pharmaceuticals. Peptide sequences including these amino acids, as well as typical peptides, can be synthesized or purchased from commercial peptide synthesis companies such as American Peptide Company, Bachem (USA) or Anygen (Korea), without particular limitation.

As used herein, the term "native insulin fragments or native insulin derivative fragments" refers to a form of insulin that has at least one amino acid removed from the amino or carboxy terminus of the native insulin or native insulin derivative. Such an insulin fragment may function to control blood glucose levels in vivo.

In addition, the insulin analogs of this invention may be analogs obtained by methods for obtaining derivatives and fragments of native insulin, independently of each other or in combination.

In particular, the insulin analogs of this invention may include analogs having the native insulin A chain and B chain modifications described above, and in particular analogs in which specific amino acid residues of the native insulin A chain are modified and/or specific amino acids are modified. residues of the B-chain of native insulin.

In particular, insulin analogs may be analogs wherein at least one amino acid is selected from the group consisting of native insulin B chain amino acid 16, B chain amino acid 25, A chain amino acid 14, and The 19th amino acid of the native insulin A chain is substituted with another amino acid, and in particular, may be substituted with glutamic acid, serine, threonine, aspartic acid, histidine, lysine, or alanine, without limitation.

In particular, insulin analogs may be analogs in which at least one, at least two, at least three, or at least four amino acids of the above amino acids are substituted with other amino acids.

In particular, the modification may be a modification of the 16th amino acid of the insulin B chain (ie tyrosine) to glutamic acid, serine, threonine or aspartic acid; modification of the 25th amino acid of the insulin B chain (ie phenylalanine) to aspartic acid or glutamic acid; modifying the 14th amino acid of the insulin A chain (ie tyrosine) with histidine, lysine, alanine or aspartic acid; or modifying the 19th amino acid of the insulin A chain (ie tyrosine) with glutamic acid, serine or threonine.

Thus, an insulin analog may include modifying the 16th amino acid of the native insulin B chain (ie, tyrosine) to glutamic acid, serine, threonine, or aspartic acid; and/or modifying the 25th amino acid of the native insulin B chain (ie, phenylalanine) to aspartic acid or glutamic acid; and/or modifying the 14th amino acid of the native insulin A chain (i.e. tyrosine) with histidine, lysine, alanine or aspartic acid and/or modifying the 19th amino acid of the native insulin A chain (i.e. tyrosine) with glutamic acid, serine or threonine, without limitation.

More specifically, the insulin analogs may be analogs comprising the A chain of SEQ ID NO: 55 represented by General Formula 2 below and the B chain of SEQ ID NO: 56 represented by General Formula 3 below. These insulin analogs may be in form, where the A-chain and B-chain are linked by a disulfide bond, or in the form of proinsulin, but not limited to these.

General Formula 2

Xaal-Ile-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21 (SEQ ID NO: 55)

In 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; and

Xaa21 is asparagine, glycine, histidine or alanine.

General Formula 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)

in 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; and

Xaa28 is proline, glutamic acid, or aspartic acid, or is absent.

Meanwhile, peptides in which the A chain matches SEQ ID NO: 53 while the B chain matches SEQ ID NO: 54 may be excluded.

In addition, peptides that have a homology of 70% or more, in particular 80% or more, more specifically 90% or more, and even more specifically 95% or more, with the sequence of the corresponding A-chain insulin analog of the General Formula 2 above, and the B-chain of General Formula 3 above, while including characteristic modifications (i.e., amino acid residues not present in native insulin) of the 14th and/or 19th amino acids of the A-chain and/ or the 16th and/or 25th amino acids of the B chain described above and having a reduced binding affinity for receptors compared to native insulin are also within the scope of this invention.

As used herein, the term "homology" refers to the degree of similarity to the amino acid sequence of a wild-type protein or nucleotide sequence encoding it, and encompasses sequences that have the above degree of sequence similarity, expressed as a percentage, to the amino acid sequences or nucleotide sequences of this invention. Such homology can be determined by comparing two given sequences with the naked eye, or can be determined using a bioinformatics algorithm that analyzes homology by aligning the two sequences. Homology between two amino acid sequences may be indicated as a percentage. There are efficient automated algorithms that can be applied both in the GAP, BESTFIT and FASTA software modules of the Wisconsin Genetics (Genetics Computer Group, Madison, WI, USA) and TFASTA software packages. The automated alignment algorithms in these modules include the Needleman and Wunsch, Pearson and Lipman, and Smith and Waterman sequence alignment algorithms. Other automated algorithms that can be used for sequence alignment and homology determination include FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W.

In an exemplary embodiment, the insulin analog may be an insulin analog comprising the A chain of SEQ ID NO: 55 represented by the general Formula 2 above and the B chain of SEQ ID NO: 54; or an insulin analogue of insulin comprising the A chain of the sequence SEQ ID NO: 53 and the B chain of the sequence SEQ ID NO: 56 represented by the general Formula 3 above, without particular limitation.

More specifically, the insulin analog may be an insulin analog, where 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 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 Xa28 is proline, but not limited to.

More specifically, the insulin analog may be an insulin analog, where 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 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, but not limited to.

More specifically, the insulin analog may be an insulin analog, where 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 is proline, but not limited to.

In an exemplary embodiment, the insulin analog of this invention may correspond to the following insulin analogs:

(1) in general formula 2, Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is histidine, Xaa16 is leucine, Xaa19 is tyrosine, Xaa21 is asparagine; and in general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(2) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(3) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(4) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(5) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(6) in 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 general formula 3, Xaa16 is glutamic acid, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(7) in 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 general formula 3, Xaa16 is serine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(8) in 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 general formula 3, Xaa16 is threonine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(9) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(10) in 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 general formula 3 Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(11) in 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 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 general formula 3, Xaa16 is tyrosine, Xaa25 is aspartic acid, Xaa27 is threonine, and Xaa28 is proline; and

(13) in 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 general formula 3, Xaa16 is tyrosine, Xaa25 is glutamic acid, Xaa27 is threonine, and Xaa28 is proline.

In addition, in an exemplary embodiment, the insulin analogue may be 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, no limit.

The insulin analog of the present invention may be, but is not limited to, an analog comprising the defined sequence described above and an analog (essentially) consisting of the defined sequence described above.

In addition, although in this invention it is referred to as "a peptide consisting of a certain SEQ ID NO", it does not preclude any insertion of a nonsense sequence upstream or downstream of the amino acid sequence of the corresponding SEQ ID NO or its naturally occurring or silent mutations. , provided that the peptide has the same or equivalent activity as a peptide consisting of the amino acid sequence of the corresponding SEQ ID NO, and it is clear that a peptide comprising such a sequence insertion or mutation is also within the scope of this invention.

In this case, the insulin analog includes all of the following: the peptide itself, its salts (for example, a pharmaceutically acceptable salt of the peptide), or solvates.

In addition, the peptide may be in any pharmaceutically acceptable form.

The type of salt is not limited to any particular one. However, the salt is preferably in a form that is safe and effective for the subject (eg, mammals), but is not limited to specific salts.

As used herein, the term "pharmaceutically acceptable" refers to a substance that can be effectively used for its intended purpose within a medical-pharmaceutical solution without causing excessive toxicity, irritation, allergic response, etc.

As used herein, the term "pharmaceutically acceptable salt" includes a salt derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable salts may 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 para-sulfonic acid, tartaric acid , acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases may include alkali metals such as sodium, potassium, etc., alkaline earth metals such as magnesium, etc., ammonium, etc.

In addition, in this description, the term "solvate" refers to the complex formed between the peptide of this invention or its salt and a solvent molecule.

Insulin analogues can be readily prepared by one of skill in the art using methods for preparing peptides known in the art. For example, insulin analogues can be prepared by a process comprising the following steps:

a) expression of an insulin analogue by culturing a transformant comprising a nucleic acid encoding an insulin analogue; and

b) isolation and purification of the expressed insulin analog.

The medium used in the present invention for culturing transformants can meet the requirements for culturing host cells appropriately. The person skilled in the art can select the appropriate carbon sources to be contained in the growth medium of the host cell, depending on the obtained transformants, and can select the appropriate culture conditions to control the duration and extent of cultivation.

Examples of the sugar source used may include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose; oils and fats such as soybean oil, sunflower seed oil, castor oil and coconut oil; fatty acids such as palmitic acid, stearic acid and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used alone or in combination.

Examples of the nitrogen source used may include peptone, yeast extract, meat extract, maltose extract, corn extract, soy flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source can also be used alone or in combination.

Examples of a source of phosphorus used may include dipotassium phosphate or dipotassium phosphate or the corresponding sodium salt. In addition, the culture medium may contain a metal salt necessary for the growth of the transformant, such as magnesium sulfate or iron sulfate.

Finally, substances essential for growth, such as amino acids and vitamins, can be used. Additionally, appropriate culture medium precursors may also be used. The above sources can be appropriately added to the culture during batch culture or continuous culture. It is possible to properly adjust the pH of the culture with a basic compound such as sodium hydroxide, potassium and ammonium hydroxide, or an acidic compound such as phosphoric acid or sulfuric acid. In addition, an antifoam agent such as a polyglycol fatty acid ester may be added to prevent foaming. In addition, oxygen or an oxygen-containing gas (eg, air) can be pumped into the culture to maintain the aerobic state of the culture.

Transformants according to this invention can be cultivated at temperatures from 20°C to 45°C, and in particular from 25°C to 40°C. In addition, culturing is continued until the maximum amount of produced target insulin analogs is obtained, and in this regard, culturing can usually be continued from 10 hours to 160 hours.

As described above, the transformant of this invention can produce insulin analogs when appropriate culture conditions are provided to suit the host cells, and the produced insulin analogs can be secreted into the cytoplasm or periplasmic space of the host cell or extracellularly, depending on the composition of the vector and the characteristics of the cell. owner.

Expressed inside the host cell or outside the proteins can be purified in a standard way. Examples of the purification method may include salting out (for example, ammonium sulfate precipitation, calcium phosphate precipitation, etc.), solvent precipitation (for example, protein fraction precipitation using acetone or ethanol, etc.), dialysis, gel filtration, chromatography such as ion exchange chromatography or reverse phase column chromatography, ultrafiltration, etc. and these methods can be used alone or in combination.

In an exemplary embodiment, the invention may further include the following steps for isolating and purifying an insulin analog expressed as inclusion bodies from a transformant:

b-1) obtaining a transformant from the culture in step a) and grinding it;

b-2) isolating the expressed insulin analogue from the crushed cell lysate, followed by its refolding;

b-3) purifying the refolded insulin analog by cation exchange chromatography;

b-4) treating the purified insulin analogue with trypsin and carboxypeptidase B; and

b-5) sequentially purifying the treated insulin analog by cation exchange chromatography and anion exchange chromatography or reverse phase chromatography.

In the present invention, the nucleic acid encoding an insulin analog comprises the nucleotide sequences shown in SEQ ID NOS: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51. In the exemplary embodiment, the nucleic acid of the invention not only includes the nucleotide sequences represented by SEQ ID NOS: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51, but also includes all sequences that have at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% homology with said sequences, wherein the peptide encoded by the above nucleic acid shows reduced receptor binding affinity compared to native insulin, while essentially having the function of controlling blood glucose levels in vivo.

The recombinant vector of this invention can be constructed as a typical cloning or expression vector and can be designed as a vector for use in eukaryotic or prokaryotic host cells.

As used herein, the term "vector" refers to a recombinant vector capable of expressing a target protein in an appropriate host cell, which is a nucleic acid construct comprising essential regulatory factors operably linked to the inserted nucleic acid to allow its expression. In the present invention, a recombinant vector that includes a nucleic acid encoding an insulin analog can be produced, and the insulin analog of the present invention can be obtained by transforming or transfecting a host cell with the recombinant vector.

In the present invention, a nucleic acid encoding an insulin analog may be operably linked to a promoter.

The method of transformation with a recombinant vector comprising a nucleic acid encoding an insulin analogue of the present invention cannot be limited to these methods, on the contrary, any transformation or transfection method commonly used in the art can be used without limitation.

The transformant of this invention can be obtained by introducing a recombinant vector comprising a target nucleic acid that encodes an insulin analog into a host cell.

A suitable host for use in the present invention is not limited to any particular host, provided that it can express the nucleic acid of the present invention. Examples of suitable hosts may include bacteria belonging to the genus Escherichia such as E. coli; bacteria belonging to the genus Bacillus such as Bacillus subtilis; bacteria belonging to the genus Pseudomonas 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. In particular, E. coli can be used as the host cell, but hosts are not limited to them.

While F in the conjugate is a substance that can increase the half-life X; and corresponds to the grouping that is part of the conjugate according to this invention.

F and X may be bonded to each other via a covalent chemical bond or a non-covalent chemical bond, and alternatively F and X may be bonded to each other via a covalent chemical bond, a non-covalent chemical bond, or a combination thereof.

More specifically, X and L, as well as L and F, can be linked to each other via a covalent bond. Thus, a conjugate is a conjugate in which X, L and F are linked to each other via a covalent bond in the order shown in Formula 1.

The substance capable of increasing the half-life X may be a biocompatible material and, for example, may be selected from the group consisting of a polymer, a fatty acid, cholesterol, albumin and fragment thereof, an albumin-binding agent, a polymer consisting of repeating units of a particular amino acid sequence, antibody, antibody fragment, FcRn binding agent, in vivo connective tissue, nucleotide, fibronectin, transferrin, saccharide, heparin, and elastin, but not limited to.

With regard to elastin, it may be human tropoelastin, which is a water-soluble precursor, and may be a polymer of a portion of a sequence or a portion of its repeat units. For example, elastin may include, but is not limited to, an elastin-like peptide.

Examples of the polymer include a polymer selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and their combinations, but not limited to those listed.

Polyethylene glycol is a general term including, but not limited to, all forms of ethylene glycol homopolymers, PEG copolymer, or monomethyl-substituted PEG (mPEG) polymer.

In addition, the biocompatible material includes, but is not limited to, poly-amino acids such as poly-lysine, poly-aspartic acid, and poly-glutamic acid.

In addition, the fatty acid may be, but is not limited to, a fatty acid capable of binding to albumin in the body.

As a more specific example, the FcRn binding agent can be, but is not limited to, an immunoglobulin Fc fragment, and more specifically, an IgG Fc fragment.

To increase in vivo solubility and/or improve half-life and/or bioavailability of the insulin analog, a biocompatible material may be attached to the side chain of one or more amino acids within the insulin analog of this invention. Such modifications may also reduce the clearance of therapeutic proteins and peptides.

The biocompatible material described above may be water-soluble (amphipatic or hydrophilic) and/or non-toxic and/or pharmaceutically acceptable.

F may be directly linked to X (ie, a is 0 in Formula 1) or may be linked to X via a linker (L).

In particular, L may be a peptide linker or a non-peptide linker.

When L is a peptide linker, it may include one or more amino acids, such as 1 to 1000 amino acids, without limitation. In the present invention, various known peptide linkers can be used to link F to X. For example, [GS] _x linker, [GGGS] _x linker, [GGGGS] _x linker, etc. may be included, and x in the above linkers may be a natural number greater than or equal to 1. However, the linkers are not limited to the following. above examples.

In the present invention, a "non-peptide linker" includes a biocompatible polymer having two or more repeating units linked to each other. The repeating blocks are linked by any covalent bond, with the exception of the peptide bond. The non-peptidic linker may be a moiety constituting a part of the conjugate of this invention and corresponds to L in Formula 1. In the present invention, when L is a biocompatible polymer as the non-peptidic linker, the distal end of the non-peptidic linker may further include a linking element for attaching the linker L to an insulin analog X and/or F via a covalent bond. Such a linking element is not limited to any specific ones, provided that it is an atom or a group of atoms that is not a repeating block of the biocompatible polymer, and is suitable for attaching the biocompatible polymer to X and/or F via a covalent bond.

In L _a , a can be 1 or more, and each L can be independent when a is 2 or more.

In the present invention, a non-peptide linker can be used interchangeably with a non-peptide polymer.

In addition, in an exemplary embodiment, the conjugate may be a conjugate in which F and X are covalently linked to each other via a non-peptidic linker containing at both ends a reactive group capable of conjugation with F, in particular with the Fc fragment of an immunoglobulin, and X.

In particular, the non-peptide linker may be selected from the group consisting of fatty acids, saccharides, polymers, small molecules, nucleotides, and combinations thereof.

The polymer of this invention can be in the range of 0 kDa to about 100 kDa or more, in particular, in the range of about 1 kDa to about 100 kDa, and more specifically, in the range of about 1 kDa to about 20 kDa, without particular limitation. .

The non-peptidic linker may be selected from the group consisting of a biodegradable polymer such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA), and polylactide-polyglycolide (PLGA), lipid polymer, chitin, hyaluronic acid, oligonucleotide and combinations thereof, but not limited to.

In an exemplary embodiment, the non-peptide polymer may be, but is not limited to, polyethylene glycol. In addition, the scope of this invention includes its derivatives known in the technical field, and derivatives easily obtained by a method known in the technical field.

Any non-peptide linker can be used in the present invention without limitation, as long as it is a polymer that is resistant to the proteolytic enzyme in vivo. The molecular weight of the non-peptide polymer of this invention is in the range of 0 kDa to about 100 kDa or more, in particular, in the range of about 1 kDa to about 100 kDa, and more specifically, in the range of from about 1 kDa to about 20 kDa, without specific limitation.

As used herein, the term "about" refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc. and therefore may include all values in the range that are equivalent or close to the values specified after this term, without limitation.

In addition, as the non-peptide linker of the present invention to be linked to the F-corresponding polypeptide, not one type of polymer but also a combination of different types of polymers can be used.

As indicated above, in the exemplary embodiment, each of the two ends of the non-peptidic linker may be attached to F and X indirectly via a linker. For example, in a specific embodiment of the present invention, the insulin analog conjugate of the present invention is prepared by a reaction (addition reaction) between an Fc fragment of an immunoglobulin and/or an insulin analog and a non-peptide polymer (non-peptide polymer having a reactive group) having a reactive group capable of forming a covalent bond (i.e., the reactive group is converted into a linking element of the conjugate) with the amino group or thiol group of the Fc fragment of the immunoglobulin and the amino group or thiol group of X as a result of a chemical reaction at its end. Thus, a non-peptide polymer having a reactive group becomes a non-peptide linker forming part of the conjugate as a result of the addition reaction. Thus, the non-peptide linker in the conjugate consists of repeating blocks and a connecting element of a non-peptide polymer with a reactive group.

In particular, a non-peptide polymer having a reactive group that can be used to prepare a conjugate can carry a reactive group at both of its ends that can bind to F (for example, an immunoglobulin Fc fragment) and X, respectively, and in particular, a reactive group , which can bind to an amino group located at the N-terminus, or a lysine residue or a thiol group of a cysteine X residue; or with an amino group located at the N-terminus, or a lysine residue or a thiol group of a cysteine residue F (for example, an immunoglobulin Fc fragment), but the reactive group is not limited to these.

In addition, a reactive group of a non-peptide polymer capable of forming a covalent bond with F, i. e. An immunoglobulin Fc fragment, and X, may be selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but are not limited to.

In the above, examples of the aldehyde group may include a propionaldehyde group or a butyraldehyde group, however, the aldehyde group is not limited thereto.

The succinimidyl derivative may include, but is not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidylcarboxymethyl, or succinimidyl carbonate.

A non-peptidic linker can be attached to X and F through such a linking element by converting a reactive group, but is not limited to the above.

In addition, the end product obtained by reductive amination with an aldehyde bond is more stable than that attached with an amide bond. The aldehyde reactive group selectively reacts with the N-terminus at low pH and is able to form a covalent bond with lysine residues at high pH, eg pH 9.0.

In addition, the reactive groups at both ends of a non-peptide polymer having a reactive group may be the same or different, for example, it may have a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group at the other end. However, reactive groups are not necessarily limited, as long as F (ie, an immunoglobulin Fc fragment) and X can be attached to both ends of a non-peptide polymer having a reactive group.

For example, as a reactive group, a maleimide group may be included at one end of the non-peptide polymer having a reactive group, and an aldehyde group, a propionaldehyde group, or a butyraldehyde group, etc. may be included at the other end.

When polyethylene glycol having a reactive hydroxy group at both of its ends is used as the non-peptide polymer, the hydroxy group can be activated by known chemical reactions known in the art to form various reactive groups as described above, or a long-acting protein conjugate can be obtained. according to this invention using commercially available polyethylene glycol having modified reactive groups.

In an exemplary embodiment, the non-peptidic polymer may be a polymer attached to an X, such as, but not limited to, an N-terminal region or a lysine residue of an insulin analog. In particular, a non-peptide polymer, such as PEG, may be attached to the N-terminal region of an insulin analog, such as, but not limited to, the amino group of a terminal amino acid or the amino group of a lysine present in the insulin analog.

Additionally, in an exemplary embodiment, a non-peptidic polymer may be attached to the N-terminal F region, such as, but not limited to, an Fc fragment of an immunoglobulin. In particular, the non-peptide polymer can be attached to the N-terminal region of the Fc of the immunoglobulin, in particular, to the amino group of the terminal amino acid, but is not limited thereto.

As used herein, the term "N-terminal region" refers to the amino-terminal region of an insulin analog or immunoglobulin Fc, and also refers to the position at which a linker can be attached to practice the present invention. For example, the N-terminal region may include not only the terminal amino acid residues of the N-terminal region, but also amino acid residues near the N-terminal amino acid residues, and in particular, may include amino acid residues 1 to 20 of the distal end, without limitation.

If an aldehyde group is present at one end of a non-peptide polymer having a reactive group and is attached to an amino group located at the N-terminus of an insulin analog, the aldehyde group can be attached to the amino group by a reductive amination reaction. In addition, if an aldehyde group is present at the other end of the non-peptide polymer having a reactive group and is attached to the amino group located at the N-terminus of the immunoglobulin Fc, the aldehyde group can also be attached to the amino group by a reductive amination reaction.

In an embodiment of the present invention, the non-peptide polymer having a reactive group that was used to prepare the conjugate is a PEG having a reactive group at both ends, and therefore the conjugate is formed by adding the non-peptide polymer to the amino group X and F by reductive amination. Thus, the conjugate has the following structure:

X-NHCH ₂ -W-group PEG-Z-CH ₂ NH-F,

where -CH ₂ W- represents a connecting element connecting the non-peptide polymer with X; and -Z-CH ₂ - is a linking element connecting a non-peptide polymer to F. In addition, W and Z are absent or are divalent functional groups such as alkylene, etc. Methylenes -CH ₂ W- and -Z-CH ₂ - come from the aldehyde reactive group, and the nitrogen atoms X-NH and NH-F come from the amino groups of the insulin analogue and F, respectively, which were used in the addition reaction.

In the above structure, the PEG moiety can contain not only the -(CH ₂ CH ₂ O) _n - structure, but also an oxygen atom located between the linking element and -(CH ₂ CH ₂ O) _n -.

As used herein, the term “Immunoglobulin Fc” refers to a region including a heavy chain constant region 2 ( _CH 2 ) and/or a heavy chain constant region 3 ( _CH 3 ) excluding immunoglobulin heavy chain and light chain variable regions. The Fc fragment of an immunoglobulin may be one element constituting the grouping of the conjugate of this invention.

An immunoglobulin Fc fragment may include, but is not limited to, a hinge region in the heavy chain constant region. In addition, the Fc fragment of an immunoglobulin of the present invention may be an extended Fc fragment comprising all or part of the heavy chain constant region 1 (CH ₁ ) and/or light chain constant region 1 (CL), excluding the heavy chain variable regions and light chain immunoglobulin, provided that it has essentially the same or improved effect compared to the native type. In addition, the Fc fragment of the immunoglobulin according to this invention may be a fragment in which a rather large part of the amino acid sequence corresponding to CH ₂ and/or CH ₃ has been deleted.

For example, an Fc fragment of an immunoglobulin of this invention may be 1) CH ₁ domain, CH ₂ domain, CH ₃ domain and CH ₄ domain, 2) CH ₁ domain and CH ₂ domain, 3) CH ₁ domain and CH ₃ domain, 4) CH ₂ domain and CH ₃ domain, 5) a combination of one or more of: CH ₁ domain, CH ₂ domain, CH ₃ domain and CH ₄ domain and immunoglobulin hinge region (or part of the hinge region) and 6) dimer all heavy chain constant region domains and light chain constant region domains, without limitation.

In addition, in an exemplary embodiment, the immunoglobulin Fc may be in dimeric form, and one X molecule may be covalently linked to one Fc in dimeric form, where the immunoglobulin Fc and X may be linked to each other via a non-peptide polymer . It is also possible that both X molecules are symmetrically attached to the Fc fragment in dimeric form. In particular, the Fc and X of an immunoglobulin may be linked to each other via a non-peptidic linker, without being limited to the examples above.

In addition, the Fc fragment of the immunoglobulin of the present invention not only includes a native amino acid sequence, but also derived sequences. An amino acid sequence derivative refers to an amino acid sequence that differs in at least one amino acid as a result of a deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

For example, amino acid residues at positions 214-238, 297-299, 318-322, or 327-331, which are known to be conjugated to an immunoglobulin Fc, may be suitable sites for modification.

In addition, various kinds of other derivatives can be obtained by deleting sites involved in the formation of a disulfide bond, deleting several amino acids at the N-terminus of native Fc, inserting a methionine residue at the N-terminus of native Fc, etc. In addition, a complement binding site, such as the C1q binding site or an antibody-dependent cell-mediated cytotoxicity (ADCC) site, can be deleted to eliminate effector functions. Methods for obtaining such derivatives of the immunoglobulin Fc fragment sequence are described in international publications W097/34631, WO 96/32478 and others.

Amino acid substitutions in proteins and peptides are known in the art that do not completely alter the activity of the proteins or peptides (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most common substitutions are 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 both directions. Depending on the situation, the Fc fragment can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

The Fc derivatives described above exhibit identical biological activity to that of the Fc fragment of this invention and may have improved structural stability, eg to heat, pH, etc.

In addition, the immunoglobulin Fc fragment can be obtained from native forms isolated in vivo from humans and animals such as cows, sheep, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or can be recombinant forms or derivatives obtained from transformed cells of animals or microorganisms. Thus, an Fc fragment can be obtained from native immunoglobulin by isolating whole immunoglobulins from a living human or animal and treating the isolated immunoglobulin with a protease. When the whole immunoglobulin is treated with papain, it is cleaved into Fab and Fc fragments, while when the whole immunoglobulin is treated with pepsin, it is cleaved into pF'c and F(ab) ₂ fragments. Fc or pF'c can be isolated by size exclusion chromatography, etc. In a more specific embodiment, the Fc fragment of human origin is an Fc fragment of a recombinant immunoglobulin derived from a microorganism.

In addition, the immunoglobulin Fc fragment may be a form having native glycans, an increased or decreased amount of glycans compared to the native form, or may be a deglycosylated form. Increasing, decreasing, or removing immunoglobulin Fc glycans can be achieved by standard methods such as a chemical method, an enzymatic method, and a genetically engineered method that uses microorganisms. An immunoglobulin Fc fragment obtained by removing Fc glycans shows a significant reduction in C1q binding affinity and a reduction or loss of antibody-dependent cytotoxicity or complement-dependent cytotoxicity, and therefore does not elicit unnecessary immune responses in vivo. In this regard, an immunoglobulin Fc fragment in deglycosylated or aglycosylated form may be more suitable for the purposes of this invention as a drug carrier.

As used herein, the term "deglycosylation" refers to the enzymatic removal of the carbohydrate moieties of an Fc fragment, and the term "aglycosylation" means that the Fc fragment is produced in a non-glycosylated form by prokaryotes, more specifically E. coli.

While the Fc fragment of the immunoglobulin may originate from humans or other animals, including cattle, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs. In a more specific embodiment, it is of human origin.

Also, an immunoglobulin (Ig) Fc fragment may be derived from IgG, IgA, IgD, IgE, and IgM, or be a combination or hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which are the most abundant proteins in human blood and, in an even more specific embodiment, it is derived from IgG, which is known to increase the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc is an IgG4 Fc, and in a most specific embodiment, the IgG4 Fc is, but is not limited to, an aglycosylated Fc derived from human IgG4.

In particular, the term "combination" as used herein means that polypeptides encoding an Fc fragment of single chain immunoglobulins of one origin are linked to a single chain polypeptide of another origin to form a dimer or multimer. This means that a dimer or multimer can be formed from two or more fragments selected from the group consisting of IgG Fc fragments, IgA Fc fragments, IgM Fc fragments, IgD Fc fragments and IgE Fc fragments.

In another aspect of the invention, a polynucleotide encoding a conjugate is provided; a vector comprising a polynucleotide; and a transformant comprising a polynucleotide or a vector comprising a polynucleotide.

The conjugate is the same as described above.

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

In addition, within the scope of this invention, the isolated polynucleotide encoding the conjugate includes a polynucleotide sequence identical to the corresponding sequence by 75% or more, in particular, 85% or more, more specifically, 90% or more, and even more specifically, by 95% or more.

The above description of the vector and transformant also applies to this embodiment.

In yet another aspect of the present invention, a method for preparing a conjugate is provided.

The conjugate is as described above.

In particular, the method may include the following steps:

(a) reacting a non-peptide polymer having two or more reactive end groups with one of an insulin analog and a carrier to produce a non-peptide polymer having an insulin analog or polymer attached to one end and a reactive end group to the other end; and

(b) reacting the non-peptide polymer obtained in step (a) having an insulin analog or polymer attached at one end and a reactive end group at the other end with the second of: the insulin carrier or insulin analog to obtain a conjugate wherein the insulin analog and the carrier are connected through a non-peptide polymer.

The non-peptide polymer, carrier, and insulin analog and formations formed therefrom are as described above.

In yet another aspect of the present invention, a composition is provided comprising a conjugate.

The conjugate is as described above.

In this invention, the composition may be a pharmaceutical composition. In particular, the composition may be a pharmaceutical composition for the prevention or treatment of an insulin related disease. More specifically, the composition may be a pharmaceutical composition for the prevention or treatment of diabetes.

In this description, the term "disease associated with insulin" refers to a disease that occurs or progresses due to low or absent biological activity of insulin; and an example of an insulin related disease may include, but is not limited to, diabetes.

As used herein, the term "prevention" refers to all activities that suppress or delay the onset of an insulin-related disease by administering the aforementioned conjugate or composition containing the conjugate, and the term "treatment" refers to all activities that improve or beneficially alter symptoms of an insulin related disease due to the administration of the above conjugate or composition containing the conjugate.

The pharmaceutical composition containing the insulin analog conjugate of the present invention may include a pharmaceutically acceptable carrier. Such a carrier may be of artificial origin.

As used herein, the term "pharmaceutically acceptable" refers to the properties of exhibiting a therapeutic effect in sufficient quantity to do so without causing side effects, which can be readily ascertained by one of ordinary skill in the art based on factors well known in the medical field, such as the type of disease, age, body weight, health status, gender, patient drug sensitivity, route of administration, route of administration, frequency of administration, duration of treatment, drug(s) to be mixed or administered simultaneously, etc.

A pharmaceutically acceptable carrier for oral administration may include a binder, lubricant, disintegrant, excipient, solvent, dispersing agent, stabilizer, suspending agent, coloring agent, flavoring agent, and the like. In injectable preparations, the pharmaceutically acceptable carrier may include a buffering agent, a preservative, an analgesic, a diluent, an isotonic agent, and a stabilizer. In topical preparations, a pharmaceutically acceptable carrier may include a base, an excipient, a lubricant, a preservative, and the like. The pharmaceutical composition of this invention may be presented in various dosage forms in combination with the above-mentioned pharmaceutically acceptable carriers. For example, a pharmaceutical composition for oral administration may be in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, or cachets. The pharmaceutical composition for injection may be in single dose ampoules or in a multi-dose container. The pharmaceutical composition may also be presented as solutions, suspensions, tablets, pills, capsules and sustained release preparations.

While examples of carriers, excipients and diluents suitable for formulating the composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, the pharmaceutical compositions may further contain a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

As used herein, the term "administration" refers to administering a particular substance to a patient by any appropriate route, and the conjugate of this invention may be administered by any conventional route, provided that the drug is delivered to the target tissue. For example, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or rectal administration may be performed, but the route of administration is not limited to the above. However, since peptides are digested upon oral administration, the active ingredients of the oral composition must be coated or protected from degradation in the stomach. In particular, the composition may be administered in injectable form. In addition, the pharmaceutical composition can be administered using certain devices capable of delivering the active ingredients to the target cell.

In addition, the pharmaceutical composition of the present invention may be determined by the type of drug active ingredient, as well as by a number of factors, including the type of disease being treated, the route of administration, the age, sex and body weight of the patient, and the severity of the disease.

The total effective dose of the composition of this invention may be administered to a patient as a single dose, or may be administered over an extended period of time in multiple doses according to a split-dose treatment protocol. The amount of active(s) ingredient(s) contained in the pharmaceutical composition of this invention may vary depending on the severity of the disease. In particular, the total daily dose of the conjugate of this invention may be from about 0.0001 mg to 500 mg per 1 kg of patient body weight. At the same time, the effective dose of the conjugate is determined taking into account various factors, including, in addition to the route of administration and frequency of administration of the pharmaceutical composition, age, body weight, health status, gender of the patient, severity of the disease, diet, and excretion rate. In this regard, those skilled in the art can easily determine the effective dose appropriate for a particular use of the pharmaceutical composition of this invention. The pharmaceutical composition of this invention is not limited to a specific composition and method and route of administration, provided that it exhibits the effects described in this invention.

To carry out the present invention, in yet another aspect of the present invention, there is provided a sustained release composition comprising an insulin analogue conjugate with increased in vivo duration of action and/or stability compared to a native insulin conjugate.

The insulin analog and conjugate are as described above.

To carry out the present invention, in yet another aspect of the present invention, there is provided a method for treating an insulin related disease comprising administering an insulin analog conjugate or composition to a subject in need thereof.

The insulin analog and conjugate are as described above.

In one embodiment, the insulin-related disease is, but is not limited to, diabetes.

To carry out the present invention, in another aspect of the present invention, the use of an insulin analog conjugate for the manufacture of a medicament is provided.

In one embodiment, the drug is for the prevention or treatment of an insulin related disease, without particular limitation.

In another embodiment, the drug is for the prevention or treatment of diabetes, but is not limited to said disease.

To carry out the present invention, in another aspect of the present invention, the use of an insulin analog conjugate in the treatment of an insulin-related disease, in particular diabetes, is provided.

The insulin analog, conjugate and insulin related disease are as described above.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and do not limit the scope of the invention in any way.

Example 1 Preparation of a Vector Expressing a Single Chain Insulin Analog

To obtain insulin analogs having a single amino acid modification in the A chain or B chain, respectively, using a native insulin expression vector as a template, forward and reverse oligonucleotides were synthesized (Table 2) and then PCR was performed, thereby amplifying the analog genes.

Altered A chain or B chain amino acid sequences and analog names are shown in Table 1 below. In Table 1, analog 1 is an analog in which the 14th amino acid of the A chain (i.e., tyrosine, Y) is replaced by histidine (H), and analog 6 is an analog in which the 16th amino acid of the B chain (t ie tyrosine, Y) is replaced by glutamic acid (E).

Primers for the amplification of insulin analogues are presented in Table 2 below.

The PCR reaction for the amplification of insulin analogues was carried out under the conditions: 95°C for 30 seconds, 55°C for 30 seconds and 68°C for 6 minutes, 18 repeated cycles. Fragments of insulin analogs obtained under these conditions were inserted into the pET22b vector for expression as intracellular inclusion bodies, and the expression vectors thus obtained were designated pET22b insulin analogs 1 to 13. The expression vectors included nucleic acids encoding the amino acid sequences of insulin analogs c 1 to 13 under the control of the T7 promoter; and insulin analog proteins were expressed in host cells in the form of inclusion bodies containing expression vectors.

The DNA sequences and protein sequences of insulin analogues 1 to 13 are shown in Table 3 below.

Each sequence modification was studied by DNA sequencing, and as a result, it was confirmed that the sequence of each insulin analog was modified in accordance with the intended purpose.

Example 2 Expression of Fusion Peptides of Recombinant Insulin Analogs

The expression of recombinant insulin analogs was under the control of the T7 promoter. E. coli BL21-DE3 (E. coli B F-dcm ompT hsdS(rB-mB-) gal λDE3; Novagen) was transformed with each of the vectors expressing the recombinant insulin analogue. Transformation was performed according to the recommended protocol (Novagen). For inoculation of 2X Luria (LB) medium containing ampicillin (50 μg/ml), single colonies transformed with each of the recombinant expression vectors were taken and cultured at 37°C for 15 hours. The culture broth of the recombinant strain and 2X LB medium containing 30% glycerol were mixed in a 1:1 (v/v) ratio, each mixture was poured into 1 ml cryovials and stored at -140°C in a cell bank to obtain a recombinant fusion protein.

For the expression of recombinant insulin analogues, one ampoule from the cell bank was thawed and inoculated with 500 ml of 2X Luria medium and cultured with stirring at 37°C for 14-16 hours. The cultivation was stopped when the OD600 reached 5.0 or higher, and the culture broth was used as a seed culture. The seed culture was used to inoculate 17 L of fermentation medium using a 50 L fermenter (MSJ-U2, B.E. MARUBISHI, Japan) and the first batch of fermentation was started. Culture conditions: temperature 37° C., aeration rate 20 l/min (rpm), stirring speed 500 rpm, pH value 6.70 was maintained with 30% aqueous ammonia solution. Fermentation was carried out in a fed-batch mode, adding a nutrient solution when the nutrients in the culture broth were depleted. The growth of the strain was followed by the value of optical density (OD). When an OD value of 100 or higher was reached, IPTG was administered at a final concentration of 500 μM. After the introduction continued further cultivation for 23 - 25 hours. After completion of cultivation, the recombinant strains were collected by centrifugation and stored at -80°C until use.

Example 3 Isolation and Refolding of Recombinant Insulin Analogs

To convert the recombinant insulin analogs expressed in Example 2 into a soluble form, they were refolded after cell disruption. The cell pellet (wet weight 100 g) 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). Cells were destroyed using a microfluidizer (Microfluidic Inc. Model M-110EN-30) at an operating pressure of 15,000 psi. inch (103421 kPa). The lysate of cells thus destroyed was centrifuged at 7000 rpm at 4°C to 8°C for 20 minutes. The supernatant was discarded and the pellet was resuspended in 3 L of wash buffer (0.5% Triton X-100 and 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl and 1 mM EDTA. After centrifugation at 7000 rpm at a temperature of 4°C to 8°C for 20 minutes, the cell pellet was resuspended in distilled water, then centrifuged in the same way.The pellet thus obtained was resuspended in buffer (1 M L-glycine, 3.78 g L-cysteine-HCl , pH 10.6) and stirred at room temperature for 1 hour. To isolate the thus resuspended recombinant insulin analog, 8 M urea was added to it and stirred for 3-5 hours. To refold the dissolved recombinant proinsulin analogs, centrifugation was performed at 7000 rpm /min at a temperature of 4°C to 8°C for 30 minutes, the supernatant was collected and treated with 15 mm L-cysteine-HCl, i.e. reducing agent, for 1 hour.A predetermined volume of distilled water was added with peristaltic pump and stirred at a temperature of from 4°C to 8°C for at least 12 hours.

Example 4 Purification by Cation Exchange Chromatography

After refolding was completed, the sample was loaded onto a balanced SP FF column (GE healthcare) equilibrated with 20 mM sodium citrate buffer (pH 2.0) containing 45% ethanol, and then insulin analog proteins were eluted with a linear gradient (0-100%) of 20 mM sodium citrate buffer solution (pH 2.0) containing 0.5 M potassium chloride and 45% ethanol (10 column volumes).

Example 5 Trypsin and Carboxypeptidase B Treatment

The eluted samples were desalted using an ultrafiltration membrane followed by a buffer change (10 mM Tris-HCl, pH 8.0). A sample of the thus obtained protein was treated with trypsin in a molar ratio to the amount of protein in the sample, corresponding to approximately 30,000, and carboxypeptidase B in a molar ratio to the amount of protein in the sample, corresponding to approximately 3000, and stirred at a temperature of from 4°C to 8°C in for approximately 16 hours.

Example 6 Purification by Cation Exchange Chromatography

After completion of the reaction, the sample was reloaded onto an equilibrated SP HP column (GE healthcare) equilibrated with 20 mM sodium citrate buffer (pH 2.0) containing 45% ethanol, and then insulin analog proteins were eluted with a linear gradient (0-100%) 20 mM sodium citrate buffer (pH 2.0) containing 0.5 M potassium chloride and 45% ethanol (10 column volumes).

Example 7 Purification by Reverse Phase Chromatography

To cleanly isolate the pure insulin analog from the sample prepared in Example 6, it was loaded onto a Source30RPC reverse phase chromatography column (GE healthcare, USA) equilibrated with sodium phosphate and isopropanol and the insulin analog proteins were eluted with a linear gradient using a buffer containing phosphate sodium and isopropanol.

Thus purified insulin analogs were analyzed by protein electrophoresis (SDS-PAGE, Fig. 1) and HPLC, purity data for representative insulin analogs 9, 10, 11 and 12 are shown in FIG. 2.

Example 8 Preparation of a long-acting insulin analog conjugate

The insulin analog conjugates prepared in Examples 1-7 were prepared as follows.

Extended release conjugate analog 10 was prepared by the following representative method.

For PEGylation of the N-terminus of the B-chain 10 of the insulin analog, a reaction was performed between 10 insulin analog at a concentration of 5 mg/ml and 3.4K PEG-propion-ALD(2) (PEG (3.4 kDa) bearing a propionaldehyde group at both ends; NOF Inc., USA) in a 1:4 molar ratio to PEG, at 4°C for approximately 2 hours. Here, the reaction proceeded by adding the reducing agent 3 mM sodium cyanoborohydride (NaCNBH ₃ ) in a mixed solvent containing 50 mM sodium citrate buffer (pH 5.0) and 45% isopropanol. The reaction solution was purified on SP-HP columns (GE Healthcare) using a concentration gradient of KCl and a buffer containing sodium citrate (pH 3.0) and 45% EtOH.

To attach the PEG to which the insulin analog is attached to the N-terminus of the Fc fragment of the immunoglobulin, the molar ratio of the purified mono-PEGylated insulin analog and the Fc fragment of the immunoglobulin should be 1:1.2, the reaction was carried out at 25°C for 15 hours, setting total protein concentration 20 mg/ml. At the same time, 100 mM HEPES buffer solution (pH 8.2) was used as the reaction solution, to which 20 mM sodium cyanoborohydride was added as a reducing agent and sodium chloride.

After completion of the reaction, the reaction solution was applied to a Q-HP column (GE, USA) using Tris-HCl buffer (pH 7.5) and a NaCl concentration gradient, and applied to Source 15ISO resin (GE, USA) using ammonium sulfate and a concentration gradient Tris-HCl (pH 7.5), thus obtaining a purified conjugate: insulin analog 10-3.4K PEG-Fc immunoglobulin.

Comparative example: preparation of a long-acting native insulin conjugate

For PEGylation of the N-terminus of the native insulin B chain (Biocon Inc., India), a PEGylation reaction was performed with 3.4K PEG-nponnoH-ALD(2) (PEG (3.4 kDa) bearing a propionaldehyde group at both ends; NOF Inc. , USA) and purified mono-PEGylated native insulin on a column as in Example 8.

To attach the PEG to which native insulin is attached to the N-terminus of the immunoglobulin Fc fragment, the reaction conditions and purification column described in Example 8 and purified mono-PEGylated native insulin and immunoglobulin Fc fragment were used, thus obtaining a conjugate: native insulin- 3.4K PEG-Fc immunoglobulin.

Experimental Example 1: Comparison of Binding Affinity of Insulin Analogs to Insulin Receptors

Scintillation proximity assay (SPA) was used to determine the binding affinity of insulin analogs to insulin receptors. First, to obtain a cell membrane expressing the insulin receptor isoform B, a CHO cell line overexpressing the insulin receptor isoform B was obtained by transfecting CHO cells with lipofectamine (Life Technology, USA). The resulting cells were then homogenized with a homogenizer (Wheaton, USA) and the cell membrane was separated using a sucrose cushion. The resulting cell membrane of CHO cells expressing the B isoform of the native human insulin receptor and PVT SPA microparticles were introduced into 96-well Pico plates (PerkinElmer, Model No. 6005162, USA). As a competitor, human insulin or each of the insulin analogs was added at more than 10 different concentrations (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 0.1% BSA) along with radioisotope I ¹²⁵ labeled native human insulin (PerkinElmer , Model No. NEX-420, USA) which was used as a detection ligand and then allowed to compete at room temperature for 4 hours. Four hours later, binding affinity to insulin receptors was determined using a beta particle counter (PerkinElmer, Model No. C9904V1, USA). Based on the values obtained for each substance using a beta counter, IC ₅₀ was calculated using GraphPad Prism 6 software and the relative binding affinity of insulin analogs to the insulin receptor relative to human insulin was expressed as a percentage of IC ₅₀ values.

As a result, 1 insulin analogue had an insulin receptor binding affinity of 90% compared to human insulin, 2 insulin analogues 95%, 3 insulin analogues 1.0%, 4 insulin analogues less than 0.1%, 6 insulin analogues 20%, 7 insulin analogues 8.5%, 9 insulin analogues 79%, 10 insulin analogues 79%; 11 insulin analogues 24%; 12 insulin analogues had less than 0.1% and less than 0.1% for 13 insulin analogues (Table 4). Therefore, the insulin analogs of this invention have a reduced binding affinity for insulin receptors compared to native insulin.

Experimental Example 2 Confirmation of Receptor Binding Affinity for a Long Release Insulin Analogue Conjugate

The binding affinity of the long acting insulin analogue to the insulin receptor and the IGF-1 receptor was confirmed by the method described above in Experimental Example 1.

As a result, compared with native human insulin, the binding affinity for the insulin receptor was 2.4±0.4% for the "native insulin-PEG-Fc immunoglobulin" conjugate and 1.3±0.2% for the "10 insulin analog-PEG" conjugate. -Fc immunoglobulin". In addition, compared to native human IGF-1, the binding affinity for the IGF-1 receptor was less than 1% for the native insulin-PEG-Fc immunoglobulin conjugate and for the insulin-PEG-Fc analogue 10 immunoglobulin conjugate. Accordingly, as a result of conjugation with PEG and immunoglobulin Fc, the insulin-PEG-Fc analogue 10 immunoglobulin conjugate was confirmed to have a reduced binding affinity for the insulin receptor compared to the native insulin-PEG-Ig Fc conjugate, and both substances had a reduced binding affinity for the IGF-1 receptor.

1) The exact value was not determined, since at the highest concentration the dose-response curve did not reach the plateau level.

Experimental Example 3: PK Study of a Long-Release Insulin Analogue Conjugate

To confirm the pharmacokinetics of the extended release insulin analog conjugate, a comparative study of blood concentration levels over time was performed in normal rats (male SD rats at 6 weeks of age), normal mice and dogs that were allowed to adapt to laboratory conditions over a period of time.

Native insulin-PEG-Fc immunoglobulin conjugate and insulin-PEG-Fc analogue 10 immunoglobulin conjugate were administered subcutaneously to mice at a concentration of 43.05 nmol/kg and to rats at a concentration of 65.1 nmol/kg in a single dose. Blood samples were then taken at 0, 1, 4, 8, 24, 48, 72, 96, 120, 144 and 168 hours. In addition, the "native insulin-PEG-Fc immunoglobulin" conjugate and the "10 analogue insulin-PEG-Fc immunoglobulin" conjugate were administered subcutaneously to dogs at a concentration of 6.3 nmol/kg in a single dose. Blood samples were then taken at 0, 1, 4, 8, 24, 48, 72, 96, 120, 144, 168, 192, 216 and 240 hours.

The blood concentrations of the "native insulin-PEG-Fc immunoglobulin" conjugate and the "10 analog insulin-PEG-Fc immunoglobulin" conjugate at each time point were determined by enzyme-linked immunosorbent assay (ELISA), and the ELISA kit manufactured by ALPCO was used as a kit for determining insulin, USA. In this case, a mouse antibody to human IgG4 conjugated with HRP (Alpha Diagnostic Intl, Inc, USA) was used as a detecting antibody.

As a result, compared with the conjugate "native insulin-PEG-Fc immunoglobulin", the half-life of the conjugate "10 analog insulin-PEG-Fc immunoglobulin" increased by 1.9 times, 1.2 times and 1.6 times in mice, rats and dogs , respectively, and the average retention time of the drug in the body increased by 2.5 times, 1.9 times and 2.0 times, respectively. Accordingly, it was confirmed that the blood duration of the "Insulin-PEG-Fc Immunoglobulin Analogue" conjugate was longer than that of the "Native Insulin-PEG-Ig Fc" conjugate.

Experimental Example 4 Determination of the Glucose-Lowering Effect of a Long-Release Insulin Analogue Conjugate

To confirm the pharmacodynamics of a long-acting insulin analog conjugate in a model of type 2 diabetes, normal rats (male SD rats at 7 weeks of age) were fed a high-fat diet (60% fat) for 2 weeks and injected with streptozotocin (STZ), which can degrade beta- pancreatic cells at a dose of 30 mg/kg twice a week to create a model of type 2 diabetes (hereinafter referred to as DIO/STZ rats). To maintain the established diabetes model, mice were continuously maintained on a high fat diet.

DIO/STZ rats were injected subcutaneously with 12.1 nmol/kg of the "native insulin-PEG-Fc immunoglobulin" conjugate and 8.09 nmol/kg of the "10 analogue of insulin-PEG-Fc immunoglobulin" conjugate and the administration was repeated with an interval of 3 days. To determine the blood glucose-lowering effect in vivo with the introduction of the "native insulin-PEG-Fc immunoglobulin" conjugate and the "10 analog insulin-PEG-Fc immunoglobulin" conjugate, blood samples were taken from the tail vein at 0, 1, 3, 6, 7 , 9, 10, 12, 13, 15, 16, 21, 22, 24, 25, 27 and 28 days after administration and in vivo blood glucose levels were determined using a OneTouch® Ultra® glucometer (LifeScan, Inc., USA) .

As a result, it was confirmed that the blood glucose lowering effect in vivo was equivalent between the 10 insulin-PEG-Fc immunoglobulin analog conjugate and the native insulin-PEG-Fc immunoglobulin conjugate, even when the DIO/STZ conjugate was administered to rats at a dose of 50 %. Based on the above results, it was confirmed that due to the extended blood lifespan in vivo, the superior effect of the 10-PEG-Fc Immunoglobulin Insulin Analogue conjugate in lowering blood glucose levels was maintained even at lower doses compared to the conjugate. native insulin-PEG-Fc immunoglobulin".

Based on the foregoing, those skilled in the art will appreciate that other embodiments of the present invention are possible without changing the technical concept or essential features of the present invention. In this regard, the exemplary embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. On the contrary, the present invention covers not only the exemplary embodiments, but also various alternatives, modifications, equivalents, and other embodiments which may be within the spirit of the present invention and fall within its scope as defined by the appended claims.

--->

SEQUENCE LIST

<110> HANMI PHARM. CO., LTD.

<120> A conjugate of insulin analog with reduced affinity to insulin

receptor and use thereof

<130>OPA18124

<150> KR 10-2017-0037101

<151> 2017-03-23

<160> 56

<170> KoPatentIn 3.0

<210> 1

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 1

cagcatctgc tccctccatc agctggagaa ctac 34

<210> 2

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 2

gtagttctcc agctgatgga gggagcagat gctg 34

<210> 3

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 3

cagcatctgc tccctcaagc agctggagaa ctac 34

<210> 4

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 4

gtagttctcc agctgcttga gggagcagat gctg 34

<210> 5

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 5

ctaccagctg gagaacgagt gcaactgagg atcc 34

<210> 6

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 6

ggatcctcag ttgcactcgt tctccagctg gtag 34

<210> 7

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 7

ctaccagctg gagaactcct gcaactgagg atcc 34

<210> 8

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 8

ggatcctcag ttgcaggagt tctccagctg gtag 34

<210> 9

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 9

ctaccagctg gagaacacct gcaactgagg atcc 34

<210> 10

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 10

ggatcctcag ttgcaggtgt tctccagctg gtag 34

<210> 11

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 11

ctggtggaag ctctcgagct agtgtgcggg gaac 34

<210> 12

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 12

gttccccgca cactagctcg aggcttcca ccag 34

<210> 13

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 13

ctggtggaag ctctctccct agtgtgcggg gaac 34

<210> 14

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 14

gttccccgca cactagggag agagcttcca ccag 34

<210> 15

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 15

ctggtggaag ctctcaccct agtgtgcggg gaac 34

<210> 16

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 16

gttccccgca cactagggtg agagcttcca ccag 34

<210> 17

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 17

cagcatctgc tccctcgcc agctggagaa ctac 34

<210> 18

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 18

gtagttctcc agctggggcga gggagcagat gctg 34

<210> 19

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 19

cagcatctgc tccctcgacc agctggagaa ctac 34

<210> 20

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 20

gtagttctcc agctggtcga gggagcagat gctg 34

<210> 21

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 21

ctggtggaag ctctcgacct agtgtgcggg gaac 34

<210> 22

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 22

gttccccgca cactaggtcg agagcttcca ccag 34

<210> 23

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 23

ggggaacgag gcttcgacta cacacccaag acc 33

<210> 24

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 24

ggtcttgggt gtgtagtcga agcctcgttc ccc 33

<210> 25

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 25

ggggaacgag gcttcgagta cacacccaag acc 33

<210> 26

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 26

ggtcttgggt gtgtactcga agcctcgttc ccc 33

<210> 27

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 1

<400> 27

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctccatcag 240

ctggagaact actgcaac 258

<210> 28

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 1

<400> 28

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu His Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 29

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 2

<400> 29

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcaagcag 240

ctggagaact actgcaac 258

<210> 30

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analogue 2

<400> 30

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Lys Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 31

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 3

<400> 31

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaacg agtgcaac 258

<210> 32

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 3

<400> 32

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Glu Cys Asn

85

<210> 33

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 4

<400> 33

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact cctgcaac 258

<210> 34

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 4

<400> 34

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Ser Cys Asn

85

<210> 35

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 5

<400> 35

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaaca cctgcaac 258

<210> 36

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 5

<400> 36

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Thr Cys Asn

85

<210> 37

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 6

<400> 37

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcgagct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 38

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 6

<400> 38

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Glu

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 39

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 7

<400> 39

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctccct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 40

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 7

<400> 40

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ser

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 41

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 8

<400> 41

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcaccct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 42

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 8

<400> 42

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Thr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 43

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 9

<400> 43

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcgcccag 240

ctggagaact actgcaac 258

<210> 44

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 9

<400> 44

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Ala Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 45

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 10

<400> 45

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcgaccag 240

ctggagaact actgcaac 258

<210> 46

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 10

<400> 46

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Asp Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 47

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 11

<400> 47

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcgacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 48

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 11

<400> 48

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Asp

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 49

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 12

<400> 49

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcgactacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 50

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 12

<400> 50

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Asp Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 51

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 13

<400> 51

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcgagtacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 52

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> Analog 13

<400> 52

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Glu Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210> 53

<211> 21

<212> PRT

<213> Homo sapiens

<400> 53

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

twenty

<210> 54

<211> 30

<212> PRT

<213> Homo sapiens

<400> 54

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

20 25 30

<210> 55

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Insulin analogue, A-chain

<220>

<221> MISC_FEATURE

<222> (1)

<223> Xaa is alanine, glycine, glutamine, histidine, glutamine

acid or asparagine

<220>

<221> MISC_FEATURE

<222> (5)

<223> Xaa is alanine, glutamic acid, glutamine, histidine, or

asparagine

<220>

<221> MISC_FEATURE

<222> (12)

<223> Xaa is alanine, serine, glutamine, glutamic acid, histidine, or

asparagine

<220>

<221> MISC_FEATURE

<222> (14)

<223> Xaa is tyrosine, histidine, lysine, alanine or aspartic acid

<220>

<221> MISC_FEATURE

<222> (16)

<223> Xaa is alanine, leucine, tyrosine, histidine, glutamic acid, or

asparagine

<220>

<221> MISC_FEATURE

<222> (19)

<223> Xaa is tyrosine, glutamic acid, serine or threonine

<220>

<221> MISC_FEATURE

<222> (21)

<223> Xaa is asparagine, glycine, histidine or alanine

<400> 55

Xaa Ile Val Glu Xaa Cys Cys Thr Ser Ile Cys Xaa Leu Xaa Gln Xaa

1 5 10 15

Glu Asn Xaa Cys Xaa

twenty

<210> 56

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> Insulin analogue, B-chain

<220>

<221> MISC_FEATURE

<222> (16)

<223> Xaa is tyrosine, glutamic acid, serine,

threonine or aspartic acid

<220>

<221> MISC_FEATURE

<222> (25)

<223> Xaa is phenylalanine, aspartic acid, or

glutamic acid

<220>

<221> MISC_FEATURE

<222> (27)

<223> Xaa is threonine or absent

<220>

<221> MISC_FEATURE

<222> (28)

<223> Xaa is proline, glutamic acid, or

aspartic acid or absent

<400> 56

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Xaa

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Xaa Tyr Xaa Xaa Lys Thr

20 25 30

<---

Claims (15)

1. Conjugate for the treatment of diabetes, represented by Formula 1: [Formula 1] X-La-F, where in Formula 1 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; L is a linker, where the linker is a polyethylene glycol; a is 1; F is an Fc fragment of an immunoglobulin; and X, L and F are linked to each other via a covalent bond in the order shown in Formula 1; and both ends of said linker are attached to the N-terminus of the B chain of said insulin analog and to the N-terminus of said immunoglobulin Fc fragment. 2. The conjugate of claim 1 wherein F is an IgG Fc fragment. 3. The conjugate according to claim 1, wherein L has a molecular weight of 1 kDa to 100 kDa. 4. The conjugate according to any one of paragraphs. 1-3, where the binding affinity of the conjugate to the native insulin receptor is from 0.1% to 50% compared to the affinity of native insulin. 5. Pharmaceutical composition for the prevention or treatment of diabetes, containing an effective dose of the conjugate according to any one of paragraphs. 1-4 and a pharmaceutically acceptable carrier. 6. Method for the treatment of diabetes, including the introduction of an effective dose of the conjugate according to any one of paragraphs. 1-4 or compositions according to claim 5 to a subject in need thereof.

Images