Long-acting conjugates of glucagon-like peptide-2 (GLP-2) derivatives

Abstract

The present invention relates to glucagon-like peptide-2 (GLP-2) derivatives, conjugates thereof and uses thereof. In addition, the present invention relates to a method for preparing glucagon-like peptide-2 (GLP-2) derivatives and conjugates thereof.

Description

Technical Field

[0001] The present invention relates to glucagon-like peptide-2 (GLP-2) derivatives, conjugates thereof and uses thereof. In addition, the present invention relates to methods for preparing glucagon-like peptide-2 (GLP-2) derivatives and conjugates thereof. Background Art

[0002] Glucagon-like peptide 2 (GLP-2) is a 33-amino acid peptide hormone produced by intestinal endocrine L cells after nutrient intake. GLP-2 stimulates mucosal growth in the small and large intestines and inhibits growth promotion and apoptosis of enterocytes and crypt cells. In addition, GLP-2 enhances the absorption of nutrients in the small intestine and reduces intestinal permeability. In addition, GLP-2 inhibits gastric emptying and gastric acid secretion, while increasing intestinal blood flow rate and relaxing intestinal smooth muscle. Because GLP-2 has the ability to absorb and conserve energy and activate intestinal cell function, it has shown high therapeutic potential in various in vivo models of intestinal diseases and injuries.

[0003] However, GLP-2 still has limitations in terms of being developed as a commercial drug. Peptides such as GLP-2 are easily denatured due to low stability, lose activity due to degradation by proteases in the body, and are easily removed by the kidneys due to their relatively small size. Therefore, in order to maintain optimal blood drug concentrations and titers of peptide drugs, peptide drugs need to be administered more frequently. However, most peptide drugs are administered with various types of injections, and frequent injections are required to maintain the blood drug concentration of peptide drugs, which causes severe pain in patients. In this regard, many attempts have been made to solve these problems, one of which has developed a method for increasing the membrane permeability of peptide drugs, which results in the delivery of peptide drugs to the body by oral or intranasal inhalation. However, compared with injecting it, this method has the limitation of low peptide drug delivery efficiency, and therefore it is still difficult to maintain sufficient biological activity of peptide drugs for therapeutic use.

[0004] Specifically, GLP-2 has an extremely short in vivo half-life (7 minutes or less) due to inactivation by dipeptidyl peptidase-IV (DPP IV), which cleaves between the amino acids at positions 2 (Ala) and 3 (Asp) of GLP-2 (Bolette H. et al., The Journal of Clinical Endocrinology & Metabolism. 85(8): 2884-2888, 2000). Attempts have been made to increase the in vivo half-life of GLP-2 mainly by amino acid substitution. Summary of the invention

[0005] Technical issues

[0006] The object of the present invention is to provide GLP-2 derivatives.

[0007] Another object of the present invention is to provide an isolated nucleic acid encoding a GLP-2 derivative, a recombinant expression vector comprising the same, and a transformant comprising the recombinant expression vector.

[0008] Still another object of the present invention is to provide a method for preparing a GLP-2 derivative.

[0009] Still another object of the present invention is to provide a GLP-2 conjugate, wherein a GLP-2 derivative is linked to a material capable of increasing its half-life in vivo.

[0010] Still another object of the present invention is to provide a method for preparing a GLP-2 conjugate.

[0011] Yet another object of the present invention is to provide a long-acting formulation of GLP-2 with increased in vivo durability and stability, wherein the long-acting formulation comprises a GLP-2 conjugate.

[0012] Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases, which pharmaceutical composition comprises a GLP-2 derivative and / or a GLP-2 conjugate.

[0013] Yet another object of the present invention is to provide a method for preventing or treating one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases, which method comprises administering a GLP-2 derivative, a GLP-2 conjugate or a pharmaceutical composition comprising the same as an active ingredient to a subject in need thereof.

[0014] Still another object of the present invention is to provide use of a GLP-2 derivative or a GLP-2 conjugate in preparing a medicament.

[0015] Still another object of the present invention is to provide use of a GLP-2 derivative or a GLP-2 conjugate in preventing or treating one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases.

[0016] Technical Solution

[0017] In one aspect, the present invention provides a glucagon-like peptide-2 (GLP-2) conjugate, wherein the GLP-2 derivative and the immunoglobulin Fc region are each covalently linked via a non-peptidyl polymer at both ends of the non-peptidyl polymer, and

[0018] The non-peptide polymer is selected from polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyethoxylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, lipid polymer, chitin, hyaluronic acid and combinations thereof.

[0019] In a specific embodiment, the present invention provides a GLP-2 conjugate, wherein the GLP-2 derivative comprises an amino acid sequence of the following general formula 1:

[0020] [General formula 1]

[0021] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO: 9),

[0022] Among them, in the above formula,

[0023] X1 is histidine, imidazolylacetyl dehistidine, deaminohistidine, β-hydroxyimidazolylpropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazolylpropionyl dehistidine;

[0024] X2 is alanine, glycine or 2-aminoisobutyric acid (Aib);

[0025] X 30 is lysine or arginine; and

[0026] X 34 does not exist, or is lysine, arginine, glutamine, histidine, 6-azido-lysine or cysteine; provided that any sequence in Formula 1 that is identical to the amino acid sequence of SEQ ID NO: 1 is excluded.

[0027] In another embodiment, the present invention provides a GLP-2 conjugate, wherein in the general formula 1 of the GLP-2 derivative, (1) X2 is glycine, (2) X 30 is arginine, or (3) X2 is glycine and X 30 It's arginine.

[0028] In yet another specific embodiment, the present invention provides a GLP-2 conjugate, wherein

[0029] (1) X1 is imidazolylacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It is cysteine;

[0030] (2) X1 is imidazolyl acetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It is lysine;

[0031] (3) X1 is imidazolylacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It is lysine;

[0032] (4) X1 is imidazolylacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 is 6-azido-lysine;

[0033] (5) X1 is imidazolylacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It is cysteine;

[0034] (6) X1 is imidazolylacetyl desyl histidine, X2 is Aib, X 30 is lysine, and X 34 is cysteine; or

[0035] (7) X1 is histidine, X2 is Aib, X 30 is lysine, and X 34 It's cysteine.

[0036] In yet another specific embodiment, the present invention provides a GLP-2 conjugate, wherein at least one residue of the GLP-2 derivative is cysteine, lysine, arginine, glutamine, histidine or 6-azido-lysine.

[0037] In yet another specific embodiment, the present invention provides a GLP-2 conjugate, wherein the GLP-2 derivative is an amino acid sequence selected from SEQ ID NOs: 2 to 8.

[0038] In yet another specific embodiment, the present invention provides a GLP-2 conjugate, wherein one end of the non-peptidyl polymer is conjugated to the immunoglobulin Fc region, and the other end thereof is conjugated to the hydroxyl group, thiol group, amino group or azide group of the GLP-2 derivative.

[0039] In yet another specific embodiment, the present invention provides a GLP-2 conjugate, wherein the immunoglobulin Fc region is aglycosylated.

[0040] In yet another specific embodiment, the present invention provides a GLP-2 conjugate, wherein the immunoglobulin Fc region further comprises a hinge region.

[0041] In yet another specific embodiment, the present invention provides a GLP-2 conjugate, wherein the immunoglobulin Fc region is an IgG4 Fc region.

[0042] In another aspect, the present invention provides a GLP-2 derivative comprising an amino acid sequence of the following general formula 1:

[0043] [General formula 1]

[0044] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO: 9),

[0045] Among them, in the above formula,

[0046] X1 is histidine, imidazolylacetyl dehistidine, deaminohistidine, β-hydroxyimidazolylpropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazolylpropionyl dehistidine;

[0047] X2 is alanine, glycine or 2-aminoisobutyric acid (Aib);

[0048] X 30 is lysine or arginine; and

[0049] X 34 does not exist, or is lysine, arginine, glutamine, histidine, 6-azido-lysine or cysteine; provided that any sequence in Formula 1 that is identical to the amino acid sequence of SEQ ID NO: 1 is excluded.

[0050] In yet another embodiment, the present invention provides a GLP-2 derivative, wherein in the general formula, (1) X2 is glycine, (2) X 30 is arginine, or (3) X2 is glycine and X 30 It's arginine.

[0051] In yet another specific embodiment, the present invention provides a GLP-2 derivative, wherein

[0052] (1) X1 is imidazolylacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It is cysteine;

[0053] (2) X1 is imidazolyl acetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It is lysine;

[0054] (3) X1 is imidazolylacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It is lysine;

[0055] (4) X1 is imidazolylacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 is 6-azido-lysine;

[0056] (5) X1 is imidazolylacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It is cysteine;

[0057] (6) X1 is imidazolylacetyl desyl histidine, X2 is Aib, X 30 is lysine, and X 34 is cysteine; or

[0058] (7) X1 is histidine, X2 is Aib, X 30 is lysine, and X 34 It's cysteine.

[0059] In yet another aspect, the present invention provides an isolated nucleic acid encoding a GLP-2 derivative.

[0060] In yet another aspect, the present invention provides a recombinant expression vector comprising a nucleic acid.

[0061] In yet another aspect, the present invention provides a transformant comprising the recombinant expression vector.

[0062] In yet another aspect, the present invention provides a method for preparing a GLP-2 derivative, comprising:

[0063] a) culturing a transformant comprising a nucleic acid encoding a GLP-2 derivative to express the GLP-2 derivative; and

[0064] b) Isolating and purifying the expressed GLP-2 derivative.

[0065] In yet another aspect, the present invention provides a method for preparing a GLP-2 conjugate, comprising:

[0066] (a) preparing a complex by reacting a non-peptidyl polymer having two or more terminal reactive groups with one of a GLP-2 derivative and an immunoglobulin Fc region, so that the complex has the GLP-2 derivative or the immunoglobulin Fc region attached to one terminal of the non-peptidyl polymer, and a reactive group at the other terminal; and

[0067] (b) preparing a conjugate by reacting the complex prepared in step (a) with one of an immunoglobulin Fc region and a GLP-2 derivative not attached to the complex, such that the GLP-2 derivative and the immunoglobulin Fc region are linked via a non-peptidyl polymer.

[0068] In yet another embodiment, the present invention provides a preparation method, wherein the non-peptidyl polymer comprises one or more reactive groups selected from the group consisting of an aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group, and a succinimide derivative.

[0069] In yet another specific embodiment, the present invention provides a preparation method, wherein the succinimide derivative is succinimidylcarboxymethyl, succinimidylvalerate, succinimidylbutyrate methyl, succinimidylpropionate methyl ester, succinimidylbutyrate, succinimidylpropionate, N-hydroxysuccinimide or succinimidyl carbonate.

[0070] In yet another aspect, the present invention provides a long-acting formulation of GLP-2 with increased in vivo durability and stability, wherein the long-acting formulation comprises a GLP-2 conjugate.

[0071] In yet another aspect, the present invention provides a pharmaceutical composition for preventing or treating one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases, the pharmaceutical composition comprising a GLP-2 conjugate or a GLP-2 derivative.

[0072] In yet another embodiment, the present invention provides a pharmaceutical composition, wherein the intestinal disease is short bowel syndrome, irritable bowel disease, inflammatory bowel disease, Crohn's disease, colitis, colitis, pancreatitis, ileitis, mucositis, or intestinal atrophy.

[0073] In yet another embodiment, the present invention provides the pharmaceutical composition, wherein the gastric disease is gastrospasm, gastritis, gastric ulcer, duodenitis or duodenal ulcer.

[0074] In yet another aspect, the present invention provides a method for preventing or treating one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases, which method comprises administering a GLP-2 conjugate, a GLP-2 derivative or a pharmaceutical composition comprising the same as an active ingredient to a subject in need thereof.

[0075] In yet another aspect, the present invention provides use of a GLP-2 derivative or a GLP-2 conjugate in the preparation of a medicament.

[0076] In another specific embodiment, the present invention provides the use of a GLP-2 derivative or a GLP-2 conjugate, wherein the medicament is used to prevent or treat one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases.

[0077] In yet another aspect, the present invention provides use of a GLP-2 derivative or a GLP-2 conjugate for preventing or treating one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases.

[0078] Beneficial Effects

[0079] Since the GLP-2 derivatives and long-acting conjugates thereof of the present invention have remarkably high activity and excellent sustained effects in vivo, these can be effectively used for preventing, improving and treating intestinal diseases, intestinal damage and gastric diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 The results of analyzing the purity of the long-acting conjugate of the GLP-2 derivative by a reverse phase column are shown.

[0081] Figure 2 It is a graph showing changes in the blood concentration of the long-acting conjugate of the GLP-2 derivative.

[0082] Figure 3 The present invention is a graph showing changes in the blood concentration of a long-acting conjugate of teduglutide and a GLP-2 derivative.

[0083] Figure 4 It is a graph showing the in vivo effect of the long-acting conjugate of teduglutide and a GLP-2 derivative (A: small intestine weight, B: small intestinal villus length). DETAILED DESCRIPTION

[0084] The specific details of the present invention will be described below. Specifically, the description and embodiments disclosed in the present invention can be applied to other descriptions and embodiments, respectively. That is, all combinations of the various elements disclosed in the present invention belong to the scope of the present invention. In addition, the scope of the present invention should not be limited by the specific disclosure provided below.

[0085] In addition, those skilled in the art will be able to identify or ascertain, based on routine experimentation, numerous equivalents to the specific embodiments of the invention described in this application, and such equivalents are intended to be encompassed by the present invention.

[0086] Throughout the specification, not only the conventional one-letter or three-letter codes for the naturally occurring amino acids are used, but also those three-letter codes commonly permitted for other amino acids, e.g., Aib (2-aminoisobutyric acid), AZ K (6-azidolysine) etc. In addition, the amino acids mentioned in the abbreviations herein are described as follows according to the IUPAC-IUB rules:

[0087]

[0088] In one aspect, the present invention provides GLP-2 derivatives.

[0089] In the present invention, "GLP-2 derivatives" include peptides having one or more amino acid sequence differences compared to natural GLP-2; peptides modified by modifying the natural GLP-2 sequence; and natural GLP-2 mimetics, which have the same functions as natural GLP-2 in preventing, treating and / or alleviating intestinal diseases, intestinal damage and gastric diseases. In addition, derivatives of GLP-2 also include derivatives with excellent in vitro and / or in vivo activity on GLP-2 receptors.

[0090] As used herein, the term "glucagon-like peptide-2 (GLP-2)" refers to a peptide that has the function of preventing, treating and / or alleviating intestinal diseases, intestinal damage and gastric diseases, and it includes not only the natural form of GLP-2, but also its agonists, fragments, variants, derivatives, etc.

[0091] As used herein, the term "GLP-2 agonist" refers to a substance that can bind to the GLP-2 receptor and induce the same or similar physiological activity as native GLP-2, regardless of its structural similarity to GLP-2.

[0092] As used herein, the term "GLP-2 fragment" refers to a peptide in which one or more amino acids are added or deleted at the N-terminus or C-terminus of GLP-2, wherein the added amino acids may be non-naturally occurring amino acids (eg, D-amino acids).

[0093] As used herein, the term "GLP-2 variant" refers to a peptide having one or more amino acids that differ from native GLP-2. For this purpose, substitutions with non-naturally occurring amino acids as well as naturally occurring amino acids can be induced.

[0094] In the present invention, modifications used to prepare agonists, fragments, variants and derivatives of natural GLP-2 may include all modifications using L- or D-type amino acids and / or non-natural amino acids; and / or modifications of the natural sequence or post-translational modifications (e.g., methylation, acylation, ubiquitination, intramolecular covalent binding, etc.).

[0095] Such agonists, fragments, variants and derivatives of natural GLP-2 can have the function of preventing, treating and alleviating intestinal diseases, intestinal damage and gastric diseases.

[0096] Agonists, fragments, variants and derivatives of native GLP-2 suitable for use in the present invention can be prepared by a combination of several methods for preparing agonists, fragments, variants and derivatives.

[0097] The GLP-2 used in the present invention can be synthesized by a solid phase synthesis method, and can also be produced by a recombinant method.

[0098] In a specific embodiment, the derivative of GLP-2 may be a derivative prepared by any one method or a combination of substitution, addition, deletion and modification of some amino acids of natural GLP-2.

[0099] The amino acid sequence of natural GLP-2 is as follows:

[0100] GLP-2(1-33)

[0101] HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 1)

[0102] Specifically, the GLP-2 derivatives include substitution of alanine at the second amino acid of native GLP-2 with glycine or Aib (2-aminoisobutyric acid), substitution of lysine at the 30th amino acid of native GLP-2 with arginine, or a combination thereof, but are not limited thereto.

[0103] Specifically, the GLP-2 derivative can be a derivative showing at least 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence homology in the amino acid sequence compared to native GLP-2, and / or a derivative in which some groups of the amino acid residues of GLP-2 are changed by chemical substitution (e.g., α-methylation, α-hydroxylation), deletion (e.g., deamination) or modification (e.g., N-methylation), but the sequence homology and form of the derivative are not limited thereto.

[0104] Specifically, the GLP-2 derivative may be in a form in which a thiol group, an amino group or an azide group is introduced, but is not limited thereto. Since the GLP-2 derivative has excellent in vitro and / or in vivo activity on the GLP-2 receptor, and since conjugation occurs in the introduced group when preparing a long-acting conjugate of the GLP-2 derivative, these can be used to prepare a GLP-2 conjugate whose binding site is selectively controlled.

[0105] Specifically, the hydroxyl group, thiol group, amino group or azido group of the GLP-2 derivative can be conjugated to one end of the non-peptidyl polymer, and a material capable of increasing the half-life in vivo (e.g., an immunoglobulin Fc region) can be conjugated to the other end of the non-peptidyl polymer. The thiol group, amino group or azido group can be introduced by adding amino acids to GLP-2, but is not limited thereto. The thiol group can be introduced by adding cysteine ​​(C) to GLP-2, the amino group can be introduced by adding lysine (K), arginine (R), glutamine (Q) or histidine (H) to GLP-2, and the 6-azido-lysine ( AZ K) to introduce an azide group, but these are not limited thereto.

[0106] Specifically, in the GLP-2 derivative, at least one residue may be cysteine, lysine, arginine, glutamine, histidine or 6-azido-lysine, but is not limited thereto.

[0107] In another specific embodiment, in the GLP-2 derivative, the N-terminal amino group may be substituted, removed or modified, but is not limited thereto. In order to prevent binding at the N-terminus, which is the in vivo active introduction site of the GLP-2 derivative, when preparing a long-acting conjugate, the GLP-2 derivative of the present invention may be prepared by the following methods: a method of removing the α-amino group of the N-terminal histidine, a method of replacing the N-terminal amino group with a hydroxyl group or a carboxyl group, a method of removing the α-carbon of the N-terminal histidine and the N-terminal amino group conjugated to the α-carbon so that only the imidazo-acetyl group remains, and a method of modifying the N-terminal amino group with two methyl groups.

[0108] Specifically, the GLP-2 derivative can be imidazolylacetyl-deshistidyl-GLP-2 (CA-GLP-2) prepared by removing the α-carbon of a histidine residue and the N-terminal amino group bonded thereto, which histidine residue is the first amino acid at the N-terminus of GLP-2; deamino-histidyl GLP-2 (DA-GLP-2) prepared by deleting the N-terminal amino group of GLP-2; β-hydroxyimidazolylpropionyl-deshistidyl GLP-2 (HY-GLP-2) prepared by replacing the N-terminal amino group of GLP-2 with a hydroxyl group; N-dimethyl-histidyl GLP-2 (DM-GLP-2) prepared by modifying the N-terminal amino group of GLP-2 with two dimethyl groups; or β-carboxyimidazolylpropionyl-deshistidyl-GLP-2 (CX-GLP-2) prepared by replacing the N-terminal amino group of GLP-2 with a carboxyl group; but are not limited thereto.

[0109] Specifically, the GLP-2 derivative of the present invention may include replacing alanine (which is the second amino acid of native GLP-2) with glycine and introducing a sulfhydryl group (e.g., cysteine) into the C-terminus of GLP-2; and more specifically, the GLP-2 derivative may include imidazolylacetyl dehistidine, in which the α-carbon of the histidine residue (which is the first amino acid at the N-terminus of GLP-2) and the N-terminal amino group bonded to the α-carbon are removed (e.g., it may have the amino acid sequence of SEQ ID NO: 2), but is not limited thereto.

[0110] Specifically, the GLP-2 derivative of the present invention may include replacing alanine (which is the second amino acid of native GLP-2) with glycine and introducing an amino group (e.g., lysine) into the C-terminus; and more specifically, the GLP-2 derivative may include imidazolylacetyl dehistidine, in which the α-carbon of the histidine residue (which is the first amino acid at the N-terminus of GLP-2) and the N-terminal amino group bonded to the α-carbon are removed (e.g., it may have an amino acid sequence of SEQ ID NO: 3), but is not limited thereto.

[0111] Specifically, the GLP-2 derivative of the present invention may include substitution of alanine (which is the second amino acid of natural GLP-2) with glycine, substitution of lysine (which is the 30th amino acid of natural GLP-2) with arginine, and introduction of an amino group (e.g., lysine) into the C-terminus; and more specifically, the GLP-2 derivative may include imidazolylacetyl dehistidine, in which the α-carbon of the histidine residue (which is the first amino acid at the N-terminus of GLP-2) and the N-terminal amino group bonded to the α-carbon are removed (e.g., it may have the amino acid sequence of SEQID NO: 4), but is not limited thereto.

[0112] Specifically, the GLP-2 derivative of the present invention may include substitution of alanine (which is the second amino acid of native GLP-2) with glycine and introduction of an azido group (e.g., 6-azido-lysine) into the C-terminus; and more specifically, the GLP-2 derivative may include imidazolylacetyl dehistidine, in which the α-carbon of the histidine residue (which is the first amino acid at the N-terminus of GLP-2) and the N-terminal amino group bonded to the α-carbon are removed (e.g., it may have the amino acid sequence of SEQ ID NO: 5), but is not limited thereto.

[0113] Specifically, the GLP-2 derivative of the present invention may include substitution of alanine (which is the second amino acid of native GLP-2) with glycine, substitution of lysine (which is the 30th amino acid of native GLP-2) with arginine, and introduction of a sulfhydryl group (e.g., cysteine) into the C-terminus; and more specifically, the GLP-2 derivative may include imidazolylacetyl dehistidine, in which the α-carbon of the histidine residue (which is the first amino acid at the N-terminus of GLP-2) and the N-terminal amino group bonded to the α-carbon are removed (e.g., it may have the amino acid sequence of SEQ ID NO: 6), but is not limited thereto.

[0114] Specifically, the GLP-2 derivative of the present invention may include substitution of alanine (which is the second amino acid of native GLP-2) with glycine and introduction of a sulfhydryl group (e.g., cysteine) into the C-terminus (e.g., it may have an amino acid sequence of SEQ ID NO: 8); and more specifically, the GLP-2 derivative may include imidazolylacetyl dehistidine, in which the α-carbon of the histidine residue (which is the first amino acid at the N-terminus of GLP-2) and the N-terminal amino group bonded to the α-carbon are removed (e.g., it may have an amino acid sequence of SEQ ID NO: 7), but is not limited thereto.

[0115] The GLP-2 derivatives of SEQ ID NOs: 2 to 8 are shown in Table 1 below.

[0116] [Table 1]

[0117] name sequence SEQ ID NO: CA GLP-2KC <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTKITDC]]> 2 CA GLP-2KK <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTKITDK]]> 3 CA GLP-2RK <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTRITDK]]> 4 <![CDATA[CA GLP-2K AZ K]]> <![CDATA[ ca HGDGFSDEMNTILDNLAARDFINWLIQTKITD AZ K]]> 5 CA GLP-2RC <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTRITDC]]> 6 CA GLP-2Aib <![CDATA[ ca HAibDGSFSDEMNTILDNLAARDFINWLIQTKITDC]]> 7 GLP-2Aib HAibDGSFSDEMNTILDNLAARDFINWLIQTKITDC 8

[0118] In Table 1 above, ca H represents a derivative in which histidine is replaced with an imidazolylacetyl group; Aib represents 2-aminoisobutyric acid; and AZ K represents 6-azido-L-lysine.

[0119] The GLP-2 derivative according to the present invention may be a peptide including the above specific sequence, or may be a peptide consisting (substantially) of the above specific sequence, but the GLP-2 derivative is not limited thereto.

[0120] Meanwhile, although it is described in the present invention that a peptide or a GLP-2 derivative "consists of a specific SEQ ID NO", such expression does not exclude mutations in the peptide or GLP-2 derivative that may occur by the addition of meaningless sequences upstream or downstream of the amino acid sequence of the corresponding SEQ ID NO, or mutations naturally occurring therein, or silent mutations therein, as long as the peptide or GLP-2 derivative having such mutations has the same or corresponding activity as the peptide or GLP-2 derivative consisting of the amino acid sequence of the corresponding SEQ ID NO. Even when there is a sequence addition or mutation, it obviously belongs to the scope of the present invention.

[0121] In yet another specific embodiment, the GLP-2 derivative may include the amino acid sequence of the following general formula 1, but is not limited thereto:

[0122] [General formula 1]

[0123] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO: 9)

[0124] Among them, in the above formula,

[0125] X1 is histidine, imidazolylacetyl dehistidine, deaminohistidine, β-hydroxyimidazolylpropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazolylpropionyl dehistidine;

[0126] X2 is alanine, glycine or 2-aminoisobutyric acid (Aib);

[0127] X 30 is lysine or arginine; and

[0128] X 34 does not exist, or is lysine, arginine, glutamine, histidine, 6-azido-lysine or cysteine; provided that any sequence in Formula 1 that is identical to the amino acid sequence of SEQ ID NO: 1 is excluded.

[0129] Specifically, in Formula 1, (1) X2 can be glycine, (2) X 30 can be arginine, or (3) X2 can be glycine and X 30 It may be arginine, but these are not limited thereto.

[0130] Specifically, in Formula 1,

[0131] (1) X1 can be imidazolylacetyl desyl histidine, X2 can be glycine, X 30 can be lysine, and X 34 It can be cysteine;

[0132] (2) X1 can be imidazolylacetyl desyl histidine, X2 can be glycine, X 30 can be lysine, and X 34 It can be lysine;

[0133] (3) X1 can be imidazolylacetyl desyl histidine, X2 can be glycine, X 30 can be arginine, and X 34 It can be lysine;

[0134] (4) X1 can be imidazolylacetyl desyl histidine, X2 can be glycine, X 30 can be lysine, and X 34 It may be 6-azido-lysine;

[0135] (5) X1 can be imidazolylacetyl desyl histidine, X2 can be glycine, X 30 can be arginine, and X 34 It can be cysteine;

[0136] (6) X1 can be imidazolylacetyl desyl histidine, X2 can be Aib, X 30 can be lysine, and X 34 may be cysteine; or

[0137] (7) X1 can be histidine, X2 can be Aib, X 30 can be lysine, and X 34 Cysteine ​​may be used, but these are not limited thereto.

[0138] Meanwhile, the GLP-2 derivative may include all those in the form of the peptide itself, a salt thereof (eg, a pharmaceutically acceptable salt of the peptide), or a solvate thereof.

[0139] Additionally, the peptide or GLP-2 derivative may be in any pharmaceutically acceptable form.

[0140] The kind of the salt is not particularly limited. However, the salt is preferably one that is safe and effective for a subject such as a mammal, but is not particularly limited thereto.

[0141] The term "pharmaceutically acceptable" refers to materials that are effective for their intended use within the scope of pharmaceutical medical determination without causing undue toxicity, irritation, allergic response, and the like.

[0142] As used herein, the term "pharmaceutically acceptable salt" refers to a salt derived from a pharmaceutically acceptable inorganic acid, organic acid or base. 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-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Examples of salts derived from suitable bases may include alkali metals such as sodium, potassium, and the like; alkaline earth metals such as magnesium; ammonium, and the like.

[0143] In addition, as used herein, the term "solvate" refers to a complex formed between the peptide or a salt thereof according to the present invention and solvent molecules.

[0144] The GLP-2 derivatives of the present invention can be synthesized by solid phase synthesis or prepared by recombinant methods, and can be prepared commercially.

[0145] In another aspect, the present invention provides an isolated nucleic acid encoding a GLP-2 derivative, a recombinant expression vector comprising the nucleic acid, and a transformant comprising the recombinant expression vector.

[0146] The GLP-2 derivatives are as described above.

[0147] As used herein, the term "nucleic acid" refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in a single-stranded or double-stranded form, including genomic DNA, cDNA and RNA transcribed therefrom, and nucleotides as basic constituent units include not only natural nucleotides but also analogs with modifications on sugars or bases (Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Uhlman and Peyman, Chemical Reviews, 90: 543-584, 1990). The nucleic acids of the present invention can be isolated or prepared using standard techniques in molecular biology. For example, nucleic acids can be amplified from natural GLP-2 gene sequences by PCR (polymerase chain reaction) using appropriate primer sequences, and nucleic acids can be prepared using standard synthesis techniques using an automated DNA synthesizer.

[0148] As used herein, the term "vector" refers to a recombinant vector capable of expressing a target protein in a suitable host cell, which is a nucleic acid construct comprising necessary regulatory factors operably linked to enable expression of a nucleic acid insert. In the present invention, a recombinant vector can be prepared, which includes a nucleic acid encoding a GLP-2 derivative. In addition, the GLP-2 derivative of the present invention can be obtained by transforming or transfecting the recombinant vector into a host cell.

[0149] The recombinant vector according to the present invention can generally be constructed as a cloning vector or an expression vector, and can also be constructed using prokaryotic or eukaryotic cells as host cells.

[0150] In the present invention, the nucleic acid encoding the GLP-2 derivative is operably linked to a promoter.

[0151] As used herein, the term "operably linked" refers to a functional connection between a regulatory sequence for nucleic acid expression (e.g., a promoter, a signal sequence, a ribosome binding site, a transcription termination sequence, etc.) and a different nucleic acid sequence, and the regulatory sequence can regulate the transcription and / or translation of the different nucleic acid sequence.

[0152] As used herein, the term "promoter" refers to an untranslated nucleic acid sequence located upstream of a coding region, which includes a polymerase binding site and has the activity of initiating transcription of a gene located downstream of the promoter into mRNA, i.e., a DNA domain that binds to a polymerase and initiates gene transcription, and it can be located in the 5' domain of the mRNA transcription start site.

[0153] For example, when the vector of the present invention is a recombinant vector and a prokaryotic cell is used as a host cell, it should include a strong promoter capable of performing transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for starting translation, and a transcription / translation termination sequence.

[0154] In addition, the vector used in the present invention can be prepared by manipulating plasmids commonly used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pPICZα series, pUC19, etc.), bacteriophages (e.g., λgt4·λB, λ-Charon, λΔz1, M13, etc.) or viruses (e.g., SV40, etc.).

[0155] Meanwhile, when the vector of the present invention is a recombinant vector and a eukaryotic cell is used as a host cell, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, 7.5K promoter of papillomavirus, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV) can be used, and a polyadenylation sequence (e.g., bovine growth hormone terminator and a polyadenylation sequence derived from SV40) is generally included as a transcription termination sequence.

[0156] In addition, the recombinant vector of the present invention includes an antibiotic resistance gene commonly used as a selection marker in the art, and may include, for example, genes resistant to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0157] The recombinant vector of the present invention may further include sequences to facilitate purification of the collected target protein, i.e., the GLP-2 derivative. The additionally included sequences may be tag sequences for protein purification, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), hexahistidine, etc., but the types of sequences required for purifying the target protein are not limited thereto.

[0158] The fusion protein expressed by the recombinant vector including the above tag sequence can be purified by affinity chromatography. For example, when fusion glutathione-S-transferase is used, glutathione as an enzyme substrate can be used, and when a 6-histidine tag is used, the target protein can be easily collected by a Ni-NTA column.

[0159] As used herein, the term "transformation" refers to a process in which DNA is introduced into a host cell and made replicable therein as a chromosomal factor or by completing chromosomal integration, which is a phenomenon of artificially causing genetic changes by introducing foreign DNA into cells.

[0160] The transformation method used in the present invention can be any transformation method, and can be easily carried out according to the conventional method used in the art. The example of commonly used transformation method can include CaCl precipitation method, the Hanahan method with improved efficiency using dimethyl sulfoxide (DMSO) as a reducing agent in the CaCl precipitation method, electroporation, CaPO precipitation method, protoplast fusion method, stirring method using silicon carbide fiber, Agrobacterium-mediated transformation, transformation using PEG, dextran sulfate-mediated transformation, liposome-mediated transformation and drying / inhibition-mediated transformation, etc.

[0161] The method for transforming a recombinant vector including a nucleic acid encoding a GLP-2 derivative according to the present invention is not limited to these methods, and any method commonly used in the art for transformation or transfection may be used, but is not limited thereto.

[0162] The transformant of the present invention can be obtained by introducing a recombinant vector including a target nucleic acid encoding a GLP-2 derivative into a host cell.

[0163] Suitable hosts for the present invention may not be particularly limited, but any host that can express the nucleic acid of the present invention may be used. Examples of suitable hosts may include bacteria such as Escherichia coli belonging to the genus Escherichia, bacteria such as Bacillus subtilis belonging to the genus Bacillus, bacteria such as Pseudomonas putida belonging to the genus Pichia, yeast such as Pichia pastoris, Saccharomyces cerevisiae and Schizosaccharomyces pombe, insect cells such as Spodoptera frugiperda (SF9), and animal cells such as CHO, COS and BSC. Specifically, Escherichia coli can be used as a host cell, but is not limited thereto.

[0164] In yet another aspect, the present invention provides a method for preparing a GLP-2 derivative using the transformant.

[0165] Specifically, the present invention provides a method for preparing a GLP-2 derivative, comprising:

[0166] a) culturing a transformant comprising a nucleic acid encoding a GLP-2 derivative to express the GLP-2 derivative; and

[0167] b) Isolating and purifying the expressed GLP-2 derivative.

[0168] In the present invention, the substratum for cultivating transformants must meet the requirements of host cell cultivation in a suitable manner. Can be determined by those skilled in the art, according to the type of transformant prepared by it, suitably select the carbon source contained in the substratum for growing host cells, and can select suitable culture conditions, so as to control the time and amount of cultivation.

[0169] Examples of sugar sources used in the culture medium may include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose; oils and fats such as soybean oil, sunflower 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 materials may be used alone or in combination.

[0170] Examples of the nitrogen source used may include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used alone or in combination.

[0171] Examples of phosphorus sources used may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts. In addition, the culture medium may contain metal salts necessary for growth, such as magnesium sulfate or iron sulfate.

[0172] Finally, essential growth substances such as amino acids and vitamins can be used. In addition, suitable precursors of culture medium can also be used. The above sources can be appropriately added to the culture during the cultivation process of batch culture or continuous culture. Basic compounds such as sodium hydroxide, potassium hydroxide and ammonia, or acidic compounds such as phosphoric acid or sulfuric acid can be used to appropriately adjust the pH of the culture. In addition, defoamers such as fatty acid polyethylene glycol esters can be added to prevent foam from generating. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas (such as air) can be injected into the culture.

[0173] The transformant of the present invention can be cultured at 20° C. to 45° C., and specifically at 25° C. to 40° C. In addition, the culture is continued until the maximum yield of the desired GLP-2 derivative is obtained, and in this regard, the culture can usually be continued for 10 to 160 hours.

[0174] As described above, when appropriate culture conditions are provided according to the host cells, the transformant of the present invention can produce GLP-2 derivatives, and the GLP-2 derivatives produced according to the vector composition and characteristics of the host cells can be secreted into the cytoplasm or into the periplasmic space of the host cells or into the outside of the cells.

[0175] Proteins expressed inside or outside the host cell can be purified by conventional methods. Examples of purification methods may include salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (e.g., protein fraction precipitation using acetone or ethanol, etc.), dialysis, gel filtration, ion exchange or chromatography such as reverse column chromatography, ultrafiltration, etc., and these methods may be used alone or in combination.

[0176] On the other hand, the present invention provides a preparation that increases the half-life and bioavailability of a GLP-2 derivative, or continuously maintains its activity. Specifically, the preparation refers to a preparation containing a carrier that is directly covalently bound to the GLP-2 derivative, or a preparation containing a component that can enhance the maintenance of the in vivo activity of the GLP-2 derivative even in the absence of a direct covalent bond.

[0177] In addition, in yet another aspect, the present invention provides a GLP-2 conjugate, wherein the GLP-2 derivative is connected to a material capable of increasing the in vivo half-life of the GLP-2 derivative. In addition, the GLP-2 derivative of the present invention has a higher activity than natural GLP-2, and its long-acting conjugate has a significantly improved blood half-life. Therefore, the GLP-2 conjugate of the present invention can be effectively used to prevent, treat and / or alleviate intestinal diseases, intestinal damage or gastric disease.

[0178] The GLP-2 derivatives are as described above.

[0179] In a specific embodiment, in the conjugate of the present invention, the GLP-2 derivative and the material capable of increasing the in vivo half-life of the GLP-2 derivative may be linked via a linker.

[0180] In the GLP-2 conjugate of the present invention, a covalent bond is formed between the linker and the thiol group, amino group or azide group introduced into the GLP-2 derivative, and thereby the binding site of the GLP-2 derivative and the linker can be selectively adjusted.

[0181] In addition, in the GLP-2 conjugate, the amino group at the N-terminus of the GLP-2 derivative is substituted, deleted or modified so that the binding of the linker to the N-terminus is prevented. The N-terminus is an important site for in vivo activity, and thus the binding site of the GLP-2 derivative and the linker can be selectively regulated, but is not limited to this.

[0182] As used herein, the term "material capable of increasing the half-life in vivo" refers to a substance that can be linked to a GLP-2 derivative to extend the half-life of the GLP-2 derivative. As used herein, the term "material capable of increasing the half-life in vivo" can be used interchangeably with the term "biocompatible material" or "carrier".

[0183] The biocompatible material or carrier may include any material as long as it can be linked to the GLP-2 derivative and prolong the half-life of the GLP-2 derivative, and the GLP-2 derivative is, for example, selected from polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin binding materials, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding materials, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, sugars and polymers, but is not limited thereto.

[0184] The biocompatible material or carrier can be linked to the GLP-2 derivative via a covalent bond or a non-covalent bond. In addition, the method of linking the GLP-2 derivative to the biocompatible material or carrier can include genetic recombination technology and in vitro connection using polymers or low molecular weight chemicals, but is not limited to any specific connection method.

[0185] In the present invention, when polyethylene glycol is used as a carrier, Ambrx, Inc.'s Recode technology (which enables position-specific attachment of polyethylene glycol) may be included, and Neose Technologies, Inc.'s glycopegylation technology (which enables specific attachment in the glycan region) may also be included. In addition, the method may include a releasable PEG technology that enables polyethylene glycol to be slowly released in vivo, but the method is not limited thereto, and may also include a technology that enables the use of PEG to improve in vivo bioavailability.

[0186] In addition, one or more polymers, such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyethoxylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipopolymers, chitin or hyaluronic acid can be linked to the GLP-2 derivatives by the above-mentioned techniques.

[0187] In the present invention, when albumin is used as a carrier, a technology capable of improving in vivo stability by direct covalent bonding between albumin or an albumin fragment and a GLP-2 derivative can be used. In addition, instead of directly linking albumin to a GLP-2 derivative, a technology for indirectly linking albumin to a GLP-2 derivative by linking a material capable of binding to albumin, such as an albumin-specific antibody or an antibody fragment thereof, to a GLP-2 derivative; a technology for linking a specific peptide / protein having a binding affinity to albumin (e.g., an albumin-binding peptide produced by an affinity body AB via the Albumod technology) to a GLP-2 derivative; and a technology for linking a fatty acid having a binding affinity to albumin, etc., but the method is not limited thereto, and any technology or linking method that uses albumin to improve in vivo stability can be used, but is not limited thereto.

[0188] In order to increase the half-life in vivo, the technology of using an antibody or its antibody fragment as a carrier to bind to the GLP-2 derivative can be included in the scope of the present invention. It can be an antibody or its antibody fragment including the FcRn-binding region, or any antibody fragment such as Fab, etc. that does not include the FcRn-binding region. The CovX-body technology using catalytic antibodies by CovX Research LLC can be included, and the use of immunoglobulin Fc region technology to increase the half-life in vivo can also be included in the scope of the present invention.

[0189] The FcRn-binding material may be an immunoglobulin Fc region.

[0190] As used herein, the term "immunoglobulin Fc region" refers to a portion excluding the variable regions of the heavy and light chains of an immunoglobulin, the heavy chain constant region 1 (CH1), and the light chain constant region (CL), and may further include the hinge region of the heavy chain constant region. Specifically, the immunoglobulin Fc region may be a fragment including part or all of the immunoglobulin Fc region, and thus may be used interchangeably with the terms "immunoglobulin fragment" or "immunoglobulin constant region".

[0191] Natural Fc has a sugar chain at Asn297 of the heavy chain constant region 1, but the recombinant Fc derived from E. coli is expressed in a glycosylated form. Removal of sugar chains from Fc results in a decrease in the binding affinity of Fcγ receptors 1, 2, 3 and complement (c1q) to the heavy chain constant region 1, resulting in a decrease or loss of antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity.

[0192] As used herein, the term "immunoglobulin constant region" refers to an immunoglobulin fragment that does not contain the variable regions of the light chain and heavy chain, the constant region 1 (CH1) of the heavy chain, and the constant region (CL) of the light chain, i.e., an Fc region consisting of the constant regions 2 and 3 (CH2 and CH3) of the heavy chain (or including the constant region 4 (CH4) of the heavy chain). Optionally, the immunoglobulin Fc region may further include a hinge region in the constant region of the heavy chain. In addition, the immunoglobulin constant region of the present invention may be an extended immunoglobulin Fc region, which includes only the constant region 1 of the heavy chain (CH1) excluding the variable regions of the heavy chain and light chain of the immunoglobulin and / or a part or the entirety of the constant region of the light chain (CL), as long as it shows substantially the same or better effects as those of the natural immunoglobulin constant region. Further, the immunoglobulin constant region of the present invention may lack a significant portion of the amino acid sequence corresponding to CH2 and / or CH3. Thus, the immunoglobulin constant region of the present invention may include (1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, (2) a CH1 domain and a CH2 domain, (3) a CH1 domain and a CH3 domain, (4) a CH2 domain and a CH3 domain, (5) a combination of one or more constant domains and an immunoglobulin hinge region (or a portion of a hinge region), or (6) a dimer of each constant domain of a heavy chain and the constant region of the light chain. The immunoglobulin constant region including the Fc region is a biodegradable polypeptide that can be metabolized in vivo, so that it can be safely used as a drug carrier. In addition, in terms of the production, purification, and productivity of the conjugate, the immunoglobulin Fc region is more advantageous than the entire immunoglobulin molecule due to its relatively low molecular weight. Further, because there is no Fab (Fab exhibits a high degree of heterogeneity due to the difference in amino acid sequence between one antibody and another antibody), the immunoglobulin Fc alone provides a conjugate with significantly enhanced homogeneity and reduces the possibility of inducing blood antigenicity.

[0193] On the other hand, immunoglobulin constant region can originate from humans or animals, such as cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., and can be specifically of human origin. In addition, immunoglobulin constant region can be selected from the constant region derived from IgG, IgA, IgD, IgE, IgM or its combination or hybrid. Specifically, constant region is derived from the most abundant IgG or IgM in blood, and most specifically from IgG known to improve the half-life of ligand-binding protein. In the present invention, immunoglobulin Fc region can be a dimer or polymer consisting of a single-chain immunoglobulin of a domain of the same origin.

[0194] As used herein, the term "combination" means that a polypeptide encoding a single-chain immunoglobulin constant region (preferably an Fc region) of the same origin is linked to a single-chain polypeptide of a different origin to form a dimer or multimer. That is, a dimer or multimer can be prepared from two or more fragments of the Fc fragment selected from IgGFc, IgA Fc, IgM Fc, IgD Fc and IgE Fc.

[0195] As used herein, the term "hybridization" means that the sequences encoding two or more immunoglobulin constant regions of different origins are present in a single chain of an immunoglobulin constant region (preferably, an Fc region). In the present invention, various hybridization forms are possible. For example, a hybridization domain can be composed of one to four domains of CH1, CH2, CH3 and CH4 selected from IgG Fc, IgMFc, IgA Fc, IgE Fc and IgD Fc, and can further include a hinge region.

[0196] IgG can be divided into IgG1, IgG2, IgG3 and IgG4 subclasses, and the present invention can include combinations or hybrids thereof. Preferred are IgG2 and IgG4 subclasses, and most preferably are the Fc regions of IgG4, which rarely have effector functions, such as complement dependent cytotoxicity (CDC).

[0197] In addition, the glycosylation form of the immunoglobulin constant region can be the same as the native form or larger or smaller, or can be a deglycosylated form. Increasing or reducing the glycosylation or deglycosylation of the immunoglobulin constant region can be achieved by typical methods, for example, by using a chemical method, an enzyme method or a genetic engineering method using a microorganism. Herein, when deglycosylated, the complement (C1q) bound to the immunoglobulin constant region becomes significantly reduced, and antibody-dependent cellular toxicity or complement-dependent cellular toxicity is reduced or removed, so as not to cause unnecessary immune responses in the body. In this context, the deglycosylated or glycosylated immunoglobulin constant region is more consistent with the purpose of the drug carrier. Accordingly, the immunoglobulin Fc region can be more specifically the glycosylated Fc region of humanized IgG4, that is, the glycosylated Fc region of humanized IgG4. Humanized Fc regions are more preferred relative to non-humanized Fc regions, and non-humanized Fc regions may act as antigens in the human body and cause adverse immune responses, such as producing new antibodies against antigens.

[0198] Further, the immunoglobulin constant region of the present invention includes not only the native amino acid sequence, but also its sequence derivatives (mutants). Amino acid sequence derivatives mean that due to the deletion, insertion, conservative or non-conservative substitution or its combination of one or more amino acid residues, it has an amino acid sequence different from the wild-type amino acid sequence. For example, it is known that the amino acid residues at positions 214 to 238, 297 to 299, 318 to 322 or 327 to 331 in the IgG Fc important for connection can be used as sites suitable for modification. Various derivatives can be used, such as those prepared by removing sites that can form disulfide bonds, removing several N-terminal amino acids from natural Fc, or adding methionine to the N-terminal of natural Fc. In addition, complement binding (fixation) sites, such as C1q binding sites or ADCC sites, can be eliminated to remove effector functions. The technology of preparing sequence derivatives of immunoglobulin constant regions is disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478.

[0199] Amino acid substitutions in protein or peptide molecules that do not alter the activity of the molecule are well known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common substitutions occur in both directions between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. Optionally, amino acids may be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0200] The above-mentioned immunoglobulin constant region derivatives can be such derivatives, which have biological activities comparable to the immunoglobulin constant region of the present invention, but have enhanced structural stability of the immunoglobulin constant region to heat, pH, etc. Further, the immunoglobulin constant region can be obtained from natural types isolated from humans or animals such as cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or can be their recombinants or derivatives obtained from transformed animal cells or microorganisms. In this article, they can be obtained from natural immunoglobulins by isolating complete immunoglobulins from human or animal organisms and treating them with proteolytic enzymes. Papain digests natural immunoglobulins into Fab and Fc regions, and pepsin treatment results in the production of pF'c and F(ab)2 fragments. These fragments can be subjected to, for example, size exclusion chromatography to separate Fc or pF'c.

[0201] Specifically, the human-derived immunoglobulin constant region may be a recombinant immunoglobulin constant region obtained from a microorganism.

[0202] In the present invention, the linker can bind to the N-terminus, C-terminus, thiol (e.g., cysteine), amino (e.g., lysine, arginine, glutamine or histidine) and / or hydroxyl of the material capable of increasing the in vivo half-life; and can bind to the N-terminus, C-terminus, thiol (e.g., cysteine), amino (e.g., lysine, arginine, glutamine or histidine), azido (e.g., 6-azido-lysine) and / or hydroxyl of the GLP-2 derivative, but these are not limited thereto.

[0203] The linker can be a peptidyl linker or a non-peptidyl linker.

[0204] By using a polymer resistant to proteases as a peptidyl linker, the blood half-life of the GLP-2 derivative can be maintained similarly in a material capable of increasing the in vivo half-life of the GLP-2 derivative. Therefore, the non-peptidyl linker that can be used in the present invention can be used without limitation as long as it is a non-peptidyl polymer resistant to in vivo proteases.

[0205] As used herein, the term "non-peptidyl polymer" includes a biocompatible polymer in which two or more repeating units are conjugated, and is used interchangeably with the term "non-peptidyl linker". The repeating units are linked to each other by any covalent bond other than a peptide bond. In the present invention, the non-peptidyl polymer includes a reactive group at its terminal, and thus can form a conjugate by reacting with other components constituting the conjugate. Such a non-peptidyl polymer may have two terminals or three terminals.

[0206] As used herein, the term "non-peptidyl polymer linking portion" refers to a constituent element in a conjugate, which is formed by linking a non-peptidyl polymer having reactive groups at both ends to an immunoglobulin Fc region and a GLP-2 derivative through each reactive group of the non-peptidyl polymer.

[0207] In a specific embodiment of the present invention, the GLP-2 conjugate can be a conjugate in which the immunoglobulin Fc region and the GLP-2 derivative are linked together by a non-peptidyl polymer, which includes reactive groups at both ends that can be linked to the immunoglobulin Fc region and the GLP-2 derivative.

[0208] Specifically, although not particularly limited thereto, the non-peptidyl polymer may be one selected from the following: polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyethoxylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ether, biodegradable polymers such as polylactic acid (PLA) and polylactic-glycolic acid (PLGA), lipopolymers, chitin, hyaluronic acid, oligonucleotides and combinations thereof. In a more specific embodiment, the non-peptidyl polymer may be polyethylene glycol, but is not limited thereto. In addition, derivatives of the above-mentioned materials known in the art and derivatives that can be easily produced at the technical level of the art also belong to the scope of the present invention.

[0209] The binding through the linker may be any chemical bond, such as a non-covalent chemical bond or a covalent chemical bond, but is not limited thereto.

[0210] The non-peptidyl polymer that can be used in the present invention can be used without limitation as long as it is a polymer resistant to in vivo proteases. Specifically, the molecular weight of the non-peptidyl polymer may be in the range of greater than 0 kDa to 200 kDa, specifically in the range of 1 kDa to 100 kDa, more specifically in the range of 1 kDa to 50 kDa, further more specifically in the range of 1 kDa to 20 kDa, still further more specifically in the range of 3.4 kDa to 10 kDa, and most specifically about 3.4 kDa, but is not limited thereto.

[0211] In addition, the carrier linked to the immunoglobulin Fc region, particularly the non-peptidyl polymer of the present invention, may be a single polymer or a combination of different polymers.

[0212] In a specific embodiment, both termini of the non-peptidyl polymer can be bound to the thiol, amino or hydroxyl group of the immunoglobulin Fc region, and can be bound to the thiol, amino, azide or hydroxyl group of the GLP-2 derivative.

[0213] Specifically, the non-peptidyl polymer may include reactive groups capable of binding to each of the immunoglobulin Fc and the GLP-2 derivative at both ends. Specifically, the reactive group may be bound to the thiol group of cysteine; the amino group at the N-terminus of lysine, arginine, glutamine and / or histidine; and / or the hydroxyl group at the C-terminus of the immunoglobulin Fc region and capable of binding to the thiol group; the amino group of lysine, arginine, glutamine and / or histidine; the azide group of azido-lysine; and / or the hydroxyl group of the GLP-2 derivative, but the reactive group is not limited thereto.

[0214] More specifically, the reactive group of the non-peptidyl polymer may be one or more selected from an aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.

[0215] In the above, examples of the aldehyde group may be a propionaldehyde group or a butyraldehyde group, but are not limited thereto.

[0216] In the above, examples of the succinimide derivative may be succinimidylcarboxymethyl, succinimidylvalerate, succinimidylbutyrate methyl, succinimidylpropionate methyl, succinimidylbutyrate, succinimidylpropionate, N-hydroxysuccinimide or succinimidyl carbonate, but are not limited thereto.

[0217] The non-peptidyl polymer can be linked to the immunoglobulin Fc and GLP-2 derivatives through a reactive group to be converted into a non-peptidyl polymer linker moiety.

[0218] In addition, the final product produced by reductive alkylation via an aldehyde bond is much more stable than the final product linked via an amide bond. The aldehyde reactive group reacts selectively with the N-terminus at low pH and can form a covalent bond with a lysine residue at high pH, ​​such as pH 9.0.

[0219] The terminal reactive groups of the non-peptidyl polymer of the present invention may be the same or different from each other. The non-peptidyl polymer may have an aldehyde reactive group at both ends. Alternatively, the non-peptidyl polymer may have an aldehyde group and a maleimide group at each end, or may have an aldehyde group and a succinimide reactive group at each end, but is not limited thereto.

[0220] For example, the non-peptidyl polymer may have a maleimide group at one end and an aldehyde group, a propionaldehyde group or a butyraldehyde group at the other end. As another example, the non-peptidyl polymer may have a succinimide group at one end and a propionaldehyde group or a butyraldehyde group at the other end.

[0221] When poly(ethylene glycol) having a hydroxyl group at the end of the propionaldehyde group is used as the non-peptidyl polymer, the hydroxyl group can be activated by various reactive groups by known chemical reactions, or poly(ethylene glycol) having a commercially available functional group for modifying reactivity can be used to prepare the conjugate of the present invention.

[0222] In a specific embodiment, the reactive group of the non-peptidyl polymer may be linked to a cysteine ​​residue of the GLP-2 derivative, specifically to a -SH group of cysteine, but not limited thereto.

[0223] In the case of using maleimide-PEG-aldehyde, the maleimide group can be linked to the -SH group of the GLP-2 derivative through a thioether bond, and the aldehyde group can be linked to the -NH2 group of the immunoglobulin Fc through a reductive alkylation reaction, but the present invention is not limited thereto and this corresponds to an example.

[0224] By this reductive alkylation, the N-terminal amine group of the immunoglobulin Fc region is linked to the oxygen atom located at one end of PEG through a linker functional group having a -CH2CH2CH2- structure to form a structure of -PEG-O-CH2CH2CH2NH-immunoglobulin Fc. In addition, a structure in which one end of PEG is linked to a sulfur atom located in cysteine ​​of the GLP-2 derivative through a thioether bond can be formed. The above-mentioned thioether bond may include structure.

[0225] However, the present invention is not particularly limited to the above-mentioned example, and this corresponds to one example.

[0226] In addition, in this conjugate, the reactive group of the non-peptidyl polymer may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, but this corresponds to one example.

[0227] In addition, in the conjugate, the reactive group of the GLP-2 derivative can be linked to the non-peptidyl polymer having a reactive group through the C-terminus of the GLP-2 derivative, and it corresponds to an example.

[0228] As used herein, for the purpose of the present invention, the term "C-terminus" refers to the carboxyl terminal of a peptide, and refers to a position capable of binding to a non-peptidyl polymer. For example, the C-terminus may include both the amino acid residue at the very end of the C-terminus and the amino acid residue near the C-terminus, and specifically may include the 1st to 20th amino acid residues from the terminal, but the C-terminus is not limited thereto.

[0229] In one embodiment, the linking of the material capable of extending the in vivo half-life of the GLP-2 derivative can be performed by genetic recombination.

[0230] In a specific embodiment, the GLP-2 conjugate of the present invention is a GLP-2 conjugate in which the GLP-2 derivative and the immunoglobulin Fc region are each covalently linked via a non-peptidyl polymer at both ends of the non-peptidyl polymer.

[0231] Herein, the non-peptidyl polymer may be selected from polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyethoxylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, lipopolymer, chitin, hyaluronic acid or a combination thereof.

[0232] Specifically, in the GLP-2 derivative of the present invention, a covalent bond can be formed between a linker as a non-peptidyl polymer and an introduced thiol group, an amino group or an azide group. Therefore, when the GLP-2 derivative of the present invention is used, a GLP-2 conjugate in which the binding site is selectively regulated can be obtained.

[0233] In addition, because the N-terminal amino group is substituted, deleted or modified in the GLP-2 derivatives of the present invention, the binding of the non-peptidyl polymer to the N-terminus (an important site for in vivo activity) is prevented, and thereby a GLP-2 conjugate in which the binding site is selectively regulated is obtained.

[0234] In the present invention, GLP-2 conjugate is used interchangeably with the terms "conjugate of a GLP-2 derivative", "long-acting conjugate of a GLP-2 derivative" or "long-acting GLP-2 derivative conjugate".

[0235] In yet another aspect, the present invention provides a method for preparing a GLP-2 conjugate, comprising linking a GLP-2 derivative to a material capable of increasing the in vivo half-life of the GLP-2 derivative.

[0236] The GLP-2 derivatives, materials capable of increasing the in vivo half-life of the GLP-2 derivatives, and GLP-2 conjugates are as described above.

[0237] Specifically, the method may include:

[0238] (a) preparing a complex by reacting a non-peptidyl polymer having two or more terminal reactive groups with one of a GLP-2 derivative and a carrier (e.g., an immunoglobulin Fc region) so that the complex has the GLP-2 derivative or the carrier attached to one terminal of the non-peptidyl polymer and a reactive terminal group at the other terminal; and

[0239] (b) preparing a conjugate by reacting the complex prepared in step (a) with one of a carrier and a GLP-2 derivative not attached to the complex, such that the GLP-2 derivative and the carrier are linked via a non-peptidyl polymer.

[0240] The above description is applicable to non-peptidyl polymers, carriers, GLP-2 derivatives and their linked structures.

[0241] As used herein, the term "complex" refers to an intermediate in which only one of the GLP-2 derivative and the carrier is linked to the non-peptidyl polymer via a covalent bond. The GLP-2 derivative or carrier not attached to the complex may be linked to the end of the non-peptidyl polymer of the complex, wherein the end is not linked to the GLP-2 derivative or the carrier.

[0242] In yet another aspect, the present invention provides a long-acting formulation of GLP-2 with increased in vivo durability and stability, which comprises a GLP-2 conjugate.

[0243] Meanwhile, formulations that can increase bioavailability or maintain long-acting activity may include sustained-release formulations using microparticles and nanoparticles using PLGA, hyaluronic acid, chitin, and the like.

[0244] Additionally, examples of other forms of formulations that may increase bioavailability or maintain prolonged activity may include implants, inhalants, nasal formulations, and patches.

[0245] The GLP-2 conjugate of the present invention can maintain the in vivo activity of conventional GLP-2, and can also increase the blood half-life of the GLP-2 derivative, and significantly increase the duration of the in vivo efficacy of the peptide, and therefore, the GLP-2 conjugate is used to treat intestinal diseases, intestinal damage and gastric diseases.

[0246] In yet another aspect, the present invention provides a composition, for example, a pharmaceutical composition including a GLP-2 derivative and / or a GLP-2 conjugate.

[0247] The pharmaceutical composition may be a pharmaceutical composition for preventing or treating one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases.

[0248] The GLP-2 derivatives and GLP-2 conjugates are as described above.

[0249] As used herein, the term "intestinal disease" may refer to short bowel syndrome, irritable bowel disease, inflammatory bowel disease, Crohn's disease, colitis, colitis, pancreatitis, ileitis, mucositis, or intestinal atrophy, but is not limited thereto.

[0250] As used herein, the term "gastric disease" may refer to gastric spasm, gastritis, gastric ulcer, duodenitis or duodenal ulcer, but is not limited thereto.

[0251] As used herein, the term "prevention" refers to any activity that inhibits or delays the onset of a disease by administering a pharmaceutical composition. The term "treatment" refers to all activities that ameliorate or favorably alter the symptoms of a disease by administering a pharmaceutical composition.

[0252] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier.

[0253] As used herein, the term "pharmaceutically acceptable" refers to a property of having a sufficient amount to exhibit a therapeutic effect without causing adverse effects, and this property can be easily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, age, weight, health status, sex, drug sensitivity of the patient, administration route, administration method, administration frequency, duration of treatment, simultaneously mixed or administered drug(s), etc.

[0254] Pharmaceutically acceptable carriers may include binders, glidants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc. for oral administration; buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injection, which may be used in combination; and bases, excipients, lubricants, preservatives, etc. for topical administration. Various types of pharmaceutical composition preparations of the present invention are prepared by combining with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the composition can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. For injection, the composition can be formulated into single-dose ampoules or multi-dose containers. In addition, the composition can also be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0255] Meanwhile, examples of suitable carriers, excipients and diluents 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, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, the composition may further include fillers, anticoagulants, lubricants, humectants, flavoring agents, preservatives, etc.

[0256] In addition, the GLP-2 derivative and / or GLP-2 conjugate of the present invention may be included in an amount of 0.001 wt % to 10 wt % based on the total weight of the composition of the present invention, but is not particularly limited.

[0257] In yet another aspect, the present invention provides a method for preventing or treating one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases, comprising administering a GLP-2 derivative, a GLP-2 conjugate and / or a pharmaceutical composition comprising the same as an active ingredient to a subject in need thereof.

[0258] The GLP-2 derivatives, GLP-2 conjugates, pharmaceutical compositions, intestinal diseases, intestinal damage, gastric diseases, prevention and treatment are as described above.

[0259] As used herein, the term "subject" refers to a subject suspected of having an intestinal disease, intestinal damage or gastric disease, and a subject suspected of having the disease refers to a mammal suffering from the disease or at risk of developing the disease, including humans, rats, cows, etc., but includes any subject that can be treated with the GLP-2 derivative, GLP-2 conjugate or composition comprising the same of the present invention, but is not limited thereto.

[0260] As used herein, the term "administer" refers to the introduction of a specific substance into a patient by any suitable method, and the route of administration may be any conventional route capable of delivering the drug to a target tissue. This may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc., but is not limited thereto. However, because the peptide is digested during oral administration, the active ingredient of the composition for oral administration is preferably coated or formulated to prevent degradation in the stomach, and specifically, may be administered in an injectable form. In addition, a certain device capable of transporting the active ingredient to the target cell may be used to administer the pharmaceutical composition.

[0261] In addition, the pharmaceutical composition of the present invention can be determined based on the type of drug used as an active ingredient, and various factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, the severity of the disease, etc. Since the pharmaceutical composition of the present invention has a significantly excellent in vivo duration, the number of administrations and the frequency of administration of the pharmaceutical composition of the present invention can be significantly reduced.

[0262] The total effective dose of the composition of the present invention can be administered to the patient in a single dose or can be administered in multiple doses for a long time according to a divided treatment regimen. In the pharmaceutical composition of the present invention, the content of the active ingredient can vary according to the severity of the disease. Specifically, the total daily dose of the GLP-2 derivative or GLP-2 conjugate of the present invention can be about 0.0001 mg to 500 mg per 1 kg of patient body weight.

[0263] However, in addition to the administration route and treatment frequency of the pharmaceutical composition, various factors including the patient's age, weight, health status, gender, disease severity, diet and excretion rate are considered to determine the effective dose of the GLP-2 derivative or GLP-2 conjugate. In this regard, those skilled in the art can easily determine the effective dose suitable for the specific use of the pharmaceutical composition of the present invention. The pharmaceutical composition according to the present invention has no particular limitation on the formulation, administration route and mode, as long as it shows the effect of the present invention.

[0264] In yet another aspect, the present invention provides use of a GLP-2 derivative or a GLP-2 conjugate in the preparation of a medicament.

[0265] In one aspect, the medicament can be used for preventing or treating one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases, but is not particularly limited thereto.

[0266] In yet another aspect, the present invention provides use of a GLP-2 derivative or a GLP-2 conjugate for preventing or treating one or more diseases selected from intestinal diseases, intestinal damage and gastric diseases.

[0267] The GLP-2 derivatives, GLP-2 conjugates, intestinal diseases, intestinal damage and gastric diseases are as described above.

[0268] Example

[0269] The present invention is further illustrated by the following examples. However, it should be understood that these examples are only used to specifically illustrate the present invention and should not be understood as limiting the present invention in any form.

[0270] Example 1: Preparation of CA-GLP-2KC-PEG (10K)-immunoglobulin Fc conjugate or CA-GLP-2RC-PEG (10K)-immunoglobulin Fc conjugate

[0271] The 34th cysteine ​​residue of CA-GLP-2KC or CA-GLP-2RC (CPC, Chinese Peptide Co, China) was PEGylated using 10 kDa MAL-ALD PEG (polyethylene glycol with a molecular weight of 10 kDa, in which the hydrogen at each terminal was modified with 3-[(3-N-maleimido) propionyl] aminopropyl and propionaldehyde, respectively; NOF Inc., Japan), and the reaction was carried out for 1 to 3 hours, the molar ratio of CA-GLP-2KC or CA-GLP-2RC to was 1:1 to 2, and the peptide concentration was 1 mg / mL to 3 mg / mL. The reaction was carried out in a mixed solvent of 50 mM Tris (pH 7.5) and isopropanol herein. The mono-PEGylated CA-GLP-2KC or mono-PEGylated CA-GLP-2RC was purified from the reaction solution using a SP Sepharose High Performance column (GE, USA) using a concentration gradient of potassium chloride and a buffer (pH 2.0) containing ethanol and sodium citrate.

[0272] Thereafter, the reaction is carried out at 2°C to 8°C for 12 to 20 hours, the molar ratio of the purified mono-PEGylated CA-GLP-2KC or mono-PEGylated CA-GLP-2RC and the immunoglobulin Fc fragment is 1:2 to 1:6, and the total protein concentration is 30 mg / mL to 35 mg / mL. Herein, the reaction solution contains 20 mM sodium cyanoborohydride (NaCNBH3), which is added as a reducing agent to 100 mM potassium phosphate buffer (pH 6.0) and isopropanol.

[0273] After the reaction was completed, a long-acting conjugate of a CA-GLP-2RC(10K PEG) derivative (CA-GLP-2KC-PEG(10K)-immunoglobulin Fc) and a long-acting conjugate of a CA-GLP-2RC(10K PEG) derivative (CA-GLP-2RC-PEG(10K)-immunoglobulin Fc), wherein CA-GLP-2KC or CA-GLP-2RC is covalently linked to immunoglobulin Fc via PEG, were purified from the reaction solution by using a concentration gradient of bis-Tris buffer (pH 6.5) and sodium chloride applied to Source 15Q column (GE, USA), and by using a concentration gradient of ammonium sulfate and sodium citrate (pH 5.0 to 5.2) applied to Source 15ISO column (GE, USA). As a result of HPLC reverse analysis, the purity of the conjugate was determined to be 92.9% and 95.6%, respectively, and the results are shown in Table 1. Figure 1 middle.

[0274] Example 2: Preparation of CA-GLP-2RK-PEG (3.4K or 10K)-immunoglobulin Fc conjugate

[0275] The 34th lysine residue of CA-GLP-2RK (CPC, Chinese Peptide Co., China) was PEGylated using 3.4 kDa or 10 kDa ALD(2)PEG (polyethylene glycol with a molecular weight of 3.4 kDa, wherein the hydrogen at each end was modified with a propionaldehyde group; NOF Inc., Japan), and the reaction was carried out at 2°C to 8°C for 4 to 16 hours, with a CA-GLP-2RK to PEG molar ratio of 1:5 to 1:20 and a peptide concentration of 5 mg / mL to 10 mg / mL. In this article, the reaction was carried out in 20 mM HEPES (pH 7.5) and ethanol, and was carried out by adding 20 mM sodium cyanoborohydride as a reducing agent. The mono-PEGylated CA-GLP-2RK was purified from the reaction solution by using a Source15S column (GE, USA) using a concentration gradient of potassium chloride and a buffer (pH 2.0) containing ethanol and sodium citrate.

[0276] Thereafter, the purified mono-PEGylated conjugate of CA-GLP-2RK and immunoglobulin Fc was prepared and purified according to the same reaction and purification conditions as in Example 1. As a result of HPLC reverse phase analysis, the purities of the long-acting conjugate of CA-GLP-2RK (3.4KPEG) derivative (CA-GLP-2RK-PEG (3.4K)-immunoglobulin Fc) and the long-acting conjugate of CA GLP-2RK (10KPEG) derivative (CA-GLP-2RK-PEG (10K)-immunoglobulin Fc) (in which CA-GLP-2RK is covalently linked to immunoglobulin Fc via PEG) were 94.3% and 92.6%, respectively, and the results are shown in Table 1. Figure 1 middle.

[0277] Example 3: Preparation of CA-GLP-2KK-PEG (10K)-immunoglobulin Fc conjugate and CA-GLP-2K AZ K-PEG (10K)-Immunoglobulin Fc Conjugate

[0278] By using CA-GLP-2KK and CA-GLP-2K according to the method of Example 2 AZ K, Preparation and purification of long-acting conjugates of CA GLP-2KK (10K PEG) derivatives (CA-GLP-2KK-PEG (10K)-immunoglobulin Fc) and CA GLP-2K AZA long-acting conjugate of a K(10K PEG) derivative, wherein CA-GLP-2KK or CA-GLP-2K AZ K is covalently linked to immunoglobulin Fc via PEG.

[0279] Example 4: Confirmation of in vitro activity of GLP-2 derivatives and their long-acting conjugates

[0280] In order to measure the activity of the GLP-2 derivatives and the long-acting conjugates of the GLP-2 derivatives obtained in the previous examples, the cell line in which the GLP-2 receptor was transformed was used to measure the in vitro cell activity. This cell line is a cell line that has been transformed to express the human GLP-2 receptor in Chinese hamster ovary (CHO)-K1 and is therefore suitable for measuring the activity of GLP-2 (DiscoverX, USA).

[0281] In order to measure the activity of GLP-2 derivatives and their long-acting conjugates, human GLP-2 and teduglutide (Shire) were diluted 3 times from 166.7nM to 0.0028nM; GLP-2 derivatives were diluted 3 times from 500nM to 0.0085nM; and the long-acting conjugates of GLP-2 derivatives were diluted 3 times from 3000nM to 0.0508nM. The culture medium was removed from the cultured CHO-K1 cells expressing the human GLP-2 receptor, and then each serially diluted material was added to the cells in an amount of 5μL, respectively. Thereafter, a buffer containing cAMP antibody (5μL) was added, and then cultured at room temperature for 15 minutes. Then, a detection mixture (10μL) containing a cell lysis buffer was added thereto to dissolve the cells, and reacted at room temperature for 60 minutes. After the reaction was completed, the cell lysate was applied to the LANCE cAMP kit (PerkinElmer, USA) to calculate the EC via the accumulated cAMP. 50 The values ​​were compared with each other. The relative titers compared with human GLP-2 are shown in Table 2.

[0282] [Table 2] Relative titer ratio of GLP-2 derivatives

[0283]

[0284] ND = Not Determined

[0285] It has been confirmed that the novel GLP-2 derivatives and their long-acting conjugates prepared as described above have the function of activating the GLP-2 receptor, and the relative titer of the GLP-2 derivatives is significantly better than that of human GLP-2. In addition, since it has been confirmed that the long-acting conjugates of the GLP-2 derivatives of SEQ ID NO: 4 (CA GLP-2RK) and the long-acting conjugates of the GLP-2 derivatives of SEQ ID NO: 6 (CA GLP-2RC) have higher activity than the long-acting conjugates of the GLP-2 derivatives of SEQ ID NO: 2 (CA GLP-2KC), these can be used as materials for treating desired diseases.

[0286] Example 5: Confirmation of the pharmacokinetics of long-acting conjugates of GLP-2 derivatives in SD rats

[0287] The pharmacokinetics of long-acting conjugates of GLP-2 derivatives were compared in normal rats.

[0288] 8-week-old normal rats were divided into the following groups: a group administered with a long-acting conjugate of a CA GLP-2KC (10K PEG) derivative (2.52 mg / kg), a group administered with a long-acting conjugate of a CA GLP-2RC (10K PEG) derivative (2.52 mg / kg), a group administered with a long-acting conjugate of a CA GLP-2RK (10K PEG) derivative (2.52 mg / kg), and a group administered with a long-acting conjugate of a CA GLP-2RK (3.4K PEG) (2.52 mg / kg). The test material was subcutaneously injected once into normal rats of each group (3 rats / group). Thereafter, for the group administered with a long-acting conjugate of a CA GLP-2KC (10K PEG) derivative, whole blood was obtained by collecting blood samples from the coccygeal vein at 1, 4, 8, 24, 48, 72, 96, 120, 144, and 168 hours. In addition, for other groups of administration of long-acting conjugates of CA GLP-2 derivatives, whole blood was obtained by collecting blood samples from the coccygeal vein at 1, 4, 8, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312 and 336 hours. The whole blood was placed in a 1.5 mL microtube, centrifuged at 5,000 rpm for 10 minutes at room temperature, and then the serum was separated and stored at -20°C. The concentration of the stored serum in each group was quantified using an ELISA analysis method. For the long-acting conjugates of CA GLP-2 derivatives, a biotin-labeled GLP-2 polyclonal antibody (Phoenix Pharmaceuticals, #B-028-14) was bound to a plate coated with Streptadivin (Roche, #11645692001) and then reacted with the serum for 1 hour. After washing, anti-human IgG4-HPR (Alpha Diagonosis, #10124) was added thereto and allowed to react at room temperature for 1 hour. Thereafter, the resultant was subjected to a colorimetric reaction using a TMB reagent, and the absorbance was measured at a wavelength of 450 nm. The pharmacokinetic parameters thereof were calculated using the serum concentration.

[0289] The results confirmed that similar AUC and half-life were exhibited in all long-acting conjugates of CA GLP-2 derivatives. Specifically, for the long-acting conjugate of CA GLP-2RK (3.4K PEG) derivative, its half-life was actually shortened due to the short PEG, but there was no significant difference in AUC. Figure 2 The results are shown in Table 3.

[0290] [Table 3] Pharmacokinetic parameters of long-acting conjugates of GLP-2 derivatives

[0291]

[0292] -AUC 持续 : PK parameter, represents the drug exposure level in vivo and is closely related to the drug's efficacy / toxicity (in order for a drug to be effective in vivo, a certain in vivo drug exposure level should be considered; excessive in vivo drug exposure levels may indicate toxic effects.

[0293] -MRT 持续 MRT: This represents the mean residence time of the drug, which is the average time until the drug disappears from the body. A higher MRT value indicates that the drug remains in the body for a long period of time and can therefore be assessed as having a longer duration compared to a drug with a lower MRT value.

[0294] Example 6: Comparison of the pharmacokinetics of long-acting conjugates of GLP-2 derivatives and teduglutide in SD rats

[0295] The pharmacokinetics of the long-acting conjugates of teduglutide and GLP-2 derivatives were compared. 8-week-old normal rats were divided into a group administered with teduglutide (2.5 mg / kg) and a group administered with a long-acting conjugate of a CA GLP-2RK (3.4K PEG) derivative (0.705 mg / kg). The test material was injected subcutaneously once into normal rats in each group (3 rats / group). Thereafter, for the group administered with teduglutide, whole blood was obtained by collecting blood samples from the coccygeal vein at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 24 hours; and for the group administered with the long-acting conjugate of the CA GLP-2RK derivative, whole blood was obtained by collecting blood samples from the coccygeal vein at 4, 8, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312 and 336 hours. Whole blood was placed in a 1.5 mL microtube, centrifuged at 5,000 rpm for 10 minutes at room temperature, and then the serum was separated and stored at -20 ° C. The concentration of the serum stored in each group was quantified using an ELISA analysis method. For teduglutide, a GLP-2 ELIS kit (Alpco, #48-GP2HU-E01.1) was used. For the long-acting conjugate of the CA GLP-2RK derivative, a biotin-labeled GLP-2 polyclonal antibody (Phoenix Pharmaceuticals, #B-028-14) was bound to a plate coated with streptavidin (Roche, #11645692001) and then reacted with the serum for 1 hour. After washing, anti-human IgG4-HPR (Alpha Diagnosis, #10124) was added thereto and allowed to react at room temperature for 1 hour. Thereafter, the resultant was subjected to a colorimetric reaction using a TMB reagent, and the absorbance was measured at a wavelength of 450 nm. Its pharmacokinetic parameters were calculated using serum concentrations.

[0296] The results confirmed that the AUC and half-life of the long-acting conjugate of CA GLP-2RK derivative were significantly increased compared with those of teduglutide. Figure 3 The results are shown in Table 4.

[0297] [Table 4] Pharmacokinetic parameters of long-acting conjugates of teduglutide and GLP-2 derivatives

[0298]

[0299] Example 7: Demonstration of the effect of a long-acting conjugate of a GLP-2 derivative on increasing intestinal weight in vivo in normal mice

[0300] The effect of a long-acting conjugate of teduglutide and a GLP-2 derivative on increasing intestinal weight in vivo was studied in normal mice.

[0301] 7-week-old C57BL / 6 mice were divided into a vehicle-administered group, a teduglutide-administered group (7.5 & 15 nmol / kg / BID) and a CA GLP-2RK (3.4K PEG) derivative-administered group (4.15, 7.5, 15, 30 nmol / kg / Q2D). Five mice were set in each group and autopsied 13 days after administration. The small intestine was perfused, and the weight of the small intestine and the length of the villi in the small intestine were measured.

[0302] As a result, in the long-acting conjugate of teduglutide and CA GLP-2RK derivative, the weight of the small intestine increased in a dose-dependent manner ( Figure 4 A), and based on the fact that the increase in intestinal weight is associated with the increase in villus length, it can be inferred that the increase in small intestine weight is due to the increase in villus length ( Figure 4 B). It is known that the high-dose administration group (15 nmol / kg / BID) showing the maximum efficacy of teduglutide is similar to the low-dose administration group (4.15 nmol / kg / Q2D) among the groups administered with the long-acting conjugate of the CA GLP-2RK derivative, and it is confirmed that the long-acting conjugate of the CA GLP-2RK derivative has an effect that exceeds the maximum efficacy of teduglutide in a dose-dependent manner. Figure 4 The results are shown in .

[0303] According to the foregoing, those skilled in the art to which the present invention belongs will be able to understand that, without modifying the technical concept or basic features of the present invention, the present invention can be implemented in other specific forms. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be interpreted as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only the exemplary embodiments, but also various alternatives, modifications, equivalents and other embodiments that may be included in the spirit and scope of the present invention as defined by the appended claims. <110> Hanmi Pharmaceutical Co., Ltd. <120> Long-acting conjugates of glucagon-like peptide-2 (GLP-2) derivatives <130> OPA18231 <150> KR 10-2017-0126577 <151> 2017-09-28 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 33 <212> PRT <213> Homo sapiens <400> 1 His Ala Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 2 <211> 34 <212> PRT <213> Artificial sequence <220> <223> GLP-2 derivatives <220> <221> MISC_FEATURE <222> (1) <223> Xaa is imidazolylacetyl desyl histidine <400> 2 Xaa Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Cys <210> 3 <211> 34 <212> PRT <213> Artificial sequence <220> <223> GLP-2 derivatives <220> <221> MISC_FEATURE <222> (1) <223> Xaa is imidazolylacetyl desyl histidine <400> 3 Xaa Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Lys <210> 4 <211> 34 <212> PRT <213> Artificial sequence <220> <223> GLP-2 derivatives <220> <221> MISC_FEATURE <222> (1) <223> Xaa is imidazolylacetyl desyl histidine <400> 4 Xaa Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Arg Ile Thr 20 25 30 Asp Lys <210> 5 <211> 34 <212> PRT <213> Artificial sequence <220> <223> GLP-2 derivatives <220> <221> MISC_FEATURE <222> (1) <223> Xaa is imidazolylacetyl desyl histidine <220> <221> MISC_FEATURE <222> (34) <223> Xaa is 6-azidolysine <400> 5 Xaa Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 6 <211> 34 <212> PRT <213> Artificial sequence <220> <223> GLP-2 derivatives <220> <221> MISC_FEATURE <222> (1) <223> Xaa is imidazolylacetyl desyl histidine <400> 6 Xaa Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Arg Ile Thr 20 25 30 Asp Cys <210> 7 <211> 34 <212> PRT <213> Artificial sequence <220> <223> GLP-2 derivatives <220> <221> MISC_FEATURE <222> (1) <223> Xaa is imidazolylacetyl desyl histidine <220> <221> MISC_FEATURE <222> (2) <223> Xaa is Aib (2-aminoisobutyric acid) <400> 7 Xaa Xaa Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Cys <210> 8 <211> 34 <212> PRT <213> Artificial sequence <220> <223> GLP-2 derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is Aib (2-aminoisobutyric acid) <400> 8 His Xaa Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Cys <210> 9 <211> 34 <212> PRT <213> Artificial sequence <220> <223> GLP-2 derivatives <220> <221> MISC_Features <222> (1) <223> Xaa = histidine, imidazolylacetyl des histidine, desamino histidine, β-hydroxyimidazolylpropionyl des histidine, N-dimethyl histidine, or β-carboxyimidazolylpropionyl des histidine <220> <221> MISC_Features <222> (2) <223> Xaa is alanine, glycine or Aib (2-aminoisobutyric acid) <220> <221> MISC_Features <222> (30) <223> Xaa is lysine or arginine <220> <221> MISC_Features <222> (34) <223> Xaa is absent or is lysine, arginine, glutamine, histidine, 6-azidolysine, or cysteine <400> 9 Xaa Xaa Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Xaa Ile Thr 20 25 30 Asp Xaa

Claims

1. A glucagon-like peptide-2 (GLP-2) conjugate, wherein the GLP-2 derivative and the immunoglobulin Fc region are each covalently linked via a non-peptidyl polymer at both ends of the non-peptidyl polymer, wherein the non-peptidyl polymer is polyethylene glycol, wherein the GLP-2 derivative is linked to the non-peptidyl polymer via the C-terminus of the GLP-2 derivative, and The GLP-2 derivative is composed of the following amino acid sequence of formula 1: [General formula 1] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9), Among them, in the above formula, X1 is imidazolylacetyl desyl histidine; X2 is glycine; X 30 is arginine; and X 34 It is lysine or cysteine.

2. The GLP-2 conjugate according to claim 1, wherein X1 is imidazolylacetyl des-histidine; X2 is glycine; X 30 is arginine; and X 34 It is lysine or cysteine. 3 . The GLP-2 conjugate according to claim 1 , wherein the GLP-2 derivative is an amino acid sequence of SEQ ID NO: 4 or 6.

4. The GLP-2 conjugate according to claim 1, wherein one end of the non-peptidyl polymer is conjugated to the immunoglobulin Fc region, and the other end thereof is conjugated to a hydroxyl group, a thiol group, an amino group or an azide group of the GLP-2 derivative.

5. The GLP-2 conjugate according to claim 1, wherein the immunoglobulin Fc region is non-glycosylated. The GLP-2 conjugate according to claim 1 , wherein the immunoglobulin Fc region includes a hinge region.

7. The GLP-2 conjugate according to claim 1, wherein the immunoglobulin Fc region is an IgG4 Fc region.

8. The GLP-2 conjugate according to claim 1, wherein the non-peptidyl polymer is polyethylene glycol, and The GLP-2 derivative consists of the amino acid sequence of SEQ ID NO:

4.

9. A GLP-2 derivative consisting of the amino acid sequence of the following general formula 1: [General formula 1] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9), Among them, in the above formula, (a) X1 is imidazolylacetyl desyl histidine; X2 is glycine; X 30 is lysine; and X 34 is lysine, or (b) X1 is imidazolylacetyl desyl histidine; X2 is glycine; X 30 is arginine; and X 34 It is lysine or cysteine.

10. The GLP-2 derivative according to claim 9, wherein X1 is imidazolylacetyl des-histidine; X2 is glycine; X 30 is lysine; and X 34 It's lysine.

11. The GLP-2 derivative according to claim 9, wherein X1 is imidazolylacetyl des-histidine; X2 is glycine; X 30 is arginine; and X 34 It is lysine or cysteine.

12. The GLP-2 derivative according to claim 9, The GLP-2 derivative is selected from the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:

6.

13. An isolated nucleic acid encoding the GLP-2 derivative according to any one of claims 9 to 12. A recombinant expression vector comprising the nucleic acid according to claim 13 . A transformant comprising the recombinant expression vector according to claim 14 .

16. A method for preparing a GLP-2 derivative according to any one of claims 9 to 12, the method comprising: a) culturing a transformant comprising a nucleic acid encoding a GLP-2 derivative according to any one of claims 9 to 12 to express the GLP-2 derivative; and b) isolating and purifying the expressed GLP-2 derivative.

17. A method for preparing a GLP-2 conjugate, the method comprising: (a) preparing a complex by reacting a non-peptidyl polymer having two or more terminal reactive groups with one of a GLP-2 derivative and an immunoglobulin Fc region, so that the complex has the GLP-2 derivative or the immunoglobulin Fc region attached to one terminal of the non-peptidyl polymer, and a reactive group at the other terminal; and (b) preparing a conjugate by reacting the complex prepared in step (a) with one of an immunoglobulin Fc region and a GLP-2 derivative that are not attached to the complex, such that the GLP-2 derivative and the immunoglobulin Fc region are linked via a non-peptidyl polymer, wherein the GLP-2 derivative is linked to the non-peptidyl polymer via the C-terminus of the GLP-2 derivative, The GLP-2 derivative is composed of the following amino acid sequence of formula 1: [General formula 1] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9), Among them, in the above formula, X1 is imidazole acetyl dehistidine; X2 is glycine; X 30 is arginine; and X 34 is lysine or cysteine, Wherein the non-peptidyl polymer is polyethylene glycol.

18. The method of claim 17, wherein the non-peptidyl polymer comprises one or more reactive groups selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative.

19. The method of claim 18, wherein the aldehyde group is a propionaldehyde group or a butyraldehyde group.

20. The method of claim 18, wherein the succinimide derivative is succinimidylcarboxymethyl, succinimidylvalerate, succinimidylbutyrate methyl, succinimidylpropionate methyl, succinimidylbutyrate, succinimidylpropionate, N-hydroxysuccinimide or succinimidyl carbonate.

21. Use of the GLP-2 conjugate or a composition comprising the GLP-2 conjugate according to any one of claims 1 to 8, or the GLP-2 derivative or a composition comprising the GLP-2 derivative according to any one of claims 9 to 12 for the preparation of a medicament for preventing or treating one or more diseases selected from short bowel syndrome, irritable bowel disease, inflammatory bowel disease, mucositis and intestinal atrophy.

22. Use of the GLP-2 conjugate or a composition comprising the GLP-2 conjugate according to any one of claims 1 to 8, or the GLP-2 derivative or a composition comprising the GLP-2 derivative according to any one of claims 9 to 12 for the preparation of a medicament for preventing or treating colitis or ileitis.

23. The use according to claim 21, wherein the inflammatory bowel disease is Crohn's disease.

24. A pharmaceutical composition for preventing or treating one or more diseases selected from pancreatitis, duodenitis and duodenal ulcer, comprising the GLP-2 conjugate according to any one of claims 1 to 8 or the GLP-2 derivative according to any one of claims 9 to 12.

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