Long-acting conjugates of glp-2 derivatives

Abstract

A glucagon-like peptide-2 (GLP-2) derivative, a conjugate thereof, and a use thereof are disclosed. The GLP-2 derivative or a conjugate thereof are useful in preventing or treating one or more diseases selected from intestinal disease, intestinal injury, or gastrosia. Additionally, a method for preparing a glucagon-like peptide-2 (GLP-2) derivative and a conjugate thereof is disclosed.

1 . A GLP-2 derivative comprising an amino acid sequence of the following Formula 1: 
       
         
           
                 
                 
               
                     
                   [Formula 1] 
                 
                     
                   (SEQ ID NO: 9) 
                 
                     
                   X 1 X 2 DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 , 
                 
             
                
                
                
               
            
           
         
         wherein, in the above formula, 
         X 1  is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; 
         X 2  is glycine; and 
         (i) X 30  is lysine and X 34  is lysine or 6-azido-lysine, or (ii) X 30  is arginine and X 34  is cysteine. 
       
     
     
         2 . The GLP-2 derivative according to  claim 1 , wherein
 (1) X 1  is imidazoacetyldeshistidine, X 2  is glycine, X 30  is lysine, and X 34  is lysine;   (2) X 1  is imidazoacetyldeshistidine, X 2  is glycine, X 30  is lysine, and X 34  is 6-azido-lysine; or   (3) X 1  is imidazoacetyldeshistidine, X 2  is glycine, X 30  is arginine, and X 34  is cysteine.   
     
     
         3 . The GLP-2 derivative according to  claim 1 , wherein the GLP-2 derivative comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 5, and 6. 
     
     
         4 . A glucagon-like peptide-2 (GLP-2) conjugate, wherein the GLP-2 derivative according to  claim 1  and an immunoglobulin Fc region are each covalently linked via a non-peptidyl polymer at both termini of the non-peptidyl polymer, and
 wherein the non-peptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, lipid polymer, chitin, hyaluronic acid, and a combination thereof. 
 
     
     
         5 . The GLP-2 conjugate according to  claim 4 , wherein
 (1) X 1  is imidazoacetyldeshistidine, X 2  is glycine, X 30  is lysine, and X 34  is lysine;   (2) X 1  is imidazoacetyldeshistidine, X 2  is glycine, X 30  is lysine, and X 34  is 6-azido-lysine; or   (3) X 1  is imidazoacetyldeshistidine, X 2  is glycine, X 30  is arginine, and X 34  is cysteine.   
     
     
         6 . The GLP-2 conjugate according to  claim 4 , wherein the GLP-2 derivative comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 5, and 6. 
     
     
         7 . The GLP-2 conjugate according to  claim 4 , 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. 
     
     
         8 . The GLP-2 conjugate according to  claim 4 , wherein the immunoglobulin Fc region is non-glycosylated. 
     
     
         9 . The GLP-2 conjugate according to  claim 4 , wherein the immunoglobulin Fc region comprises a hinge region. 
     
     
         10 . The GLP-2 conjugate according to  claim 4 , wherein the immunoglobulin Fc region is an IgG4 Fc region. 
     
     
         11 . An isolated nucleic acid encoding the GLP-2 derivative according to  claim 1 . 
     
     
         12 . A recombinant expression vector comprising the nucleic acid according to  claim 11 . 
     
     
         13 . A transformant comprising the recombinant expression vector according to  claim 12 . 
     
     
         14 . A method for preparing the GLP-2 derivative according to  claim 1 , comprising:
 a) culturing a transformant comprising a nucleic acid encoding the GLP-2 derivative according to  claim 1  to express the GLP-2 derivative; and   b) isolating and purifying the expressed GLP-2 derivative.   
     
     
         15 . A method for preparing a GLP-2 conjugate, comprising:
 (a) preparing a complex by reacting a non-peptidyl polymer having two or more terminal reactive groups with one of the GLP-2 derivative according to  claim 1  and an immunoglobulin Fc region such that the complex has the GLP-2 derivative or the immunoglobulin Fc region attached to one terminal end of the non-peptidyl polymer, and a reactive group at the other terminal end; and   (b) preparing a conjugate by reacting the complex prepared in Step (a) with one of the immunoglobulin Fc region and the 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.   
     
     
         16 . The method according to  claim 15 , 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. 
     
     
         17 . The method according to  claim 16 , wherein the succinimide derivative is succinimidyl carboxymethyl, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, or succinimidyl carbonate. 
     
     
         18 . A method for preventing or treating one or more diseases selected from intestinal disease, intestinal injury, or gastrosia, comprising administering an effective amount of a composition comprising
 the GLP-2 derivative according to  claim 1 ; or   a GLP-2 conjugate, wherein the GLP-2 derivative according to  claim 1  and an immunoglobulin Fc region are each covalently linked via a non-peptidyl polymer at both termini of the non-peptidyl polymer, and wherein the non-peptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, lipid polymer, chitin, hyaluronic acid, and a combination thereof to a subject in need thereof.   
     
     
         19 . The method according to  claim 18 , wherein the intestinal disease is short-bowel syndrome, hypersensitive intestinal disease, inflammatory intestinal disease, Crohn's disease, colonitis, colitis, pancreatitis, ileitis, mucositis, or intestine atrophy. 
     
     
         20 . The method according to  claim 18 , wherein the gastrosia is stomach cramps, gastritis, gastric ulcer, duodenitis, or duodenal ulcer.