Physiologically active polypeptide conjugate having prolonged in vivo half-life

Abstract

A protein conjugate having a prolonged in vivo half-life of a physiological activity, comprising i) a physiologically active polypeptide, ii) a biocompatible non-peptidic polymer, and iii) an immunoglobulin, is useful for the development of a peptide drug due to the enhanced in vivo stability and prolonged half-life in blood, while reducing the possibility of inducing an immune response.

1 . A protein conjugate comprising i) a physiologically active polypeptide, ii) a non-peptidic polymer, and iii) an immunoglobulin, which are covalently linked to one another, and having a prolonged in vivo half-life of the physiologically active polypeptide.  
     
     
         2 . The protein conjugate according to  claim 1 , wherein the non-peptidic polymer has two reactive groups at both ends, through which the polymer is covalently linked to the physiologically active polypeptide and the immunoglobulin.  
     
     
         3 . The protein conjugate according to  claim 2 , wherein the immunoglobulin is covalently linked to at least two complexes of the physiologically active polypeptide and the non-peptidic polymer.  
     
     
         4 . The protein conjugate according to  claim 1 , wherein the immunoglobulin is selected from the group consisting of IgG, IgA, IgD, IgE, IgM and a mixture thereof.  
     
     
         5 . The protein conjugate according to  claim 4 , wherein the immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3, IgG4 and a mixture thereof.  
     
     
         6 . The protein conjugate according to  claim 4 , wherein the immunoglobulin is a human immunoglobulin.  
     
     
         7 . The protein conjugate according to  claim 2 , wherein the reactive group of the non-peptidic polymer is selected from the group consisting of aldehyde, propion aldehyde, maleimide and succinamide derivative.  
     
     
         8 . The protein conjugate according to  claim 7 , wherein the succinamide derivative is succinimidyl propionate, succinimidyl carboxymethyl, hydroxy succinimidyl or succinimidyl carbonate.  
     
     
         9 . The protein conjugate according to  claim 7 , wherein the non-peptidic polymer has aldehyde groups at both ends.  
     
     
         10 . The protein conjugate according to  claim 1 , wherein the non-peptidic polymer is covalently linked at the ends thereof to the amino terminal, lysine residue, histidine residue or cysteine residue of the immunoglobulin and the amino terminal, lysine residue, hisitidine residue or cysteine residue of the physiologically active polypeptide, respectively.  
     
     
         11 . The protein conjugate according to  claim 1 , wherein the non-peptidic polymer is selected form the group consisting of poly(ethylene glycol), poly(propylene glycol), ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, poly(lactic-glycolic acid), biodegradable polymer, lipid polymer, chitin, hyaluronic acids, and a mixture thereof.  
     
     
         12 . The protein conjugate according to  claim 11 , wherein the non-peptidic polymer is poly(ethylene glycol).  
     
     
         13 . The protein conjugate according to  claim 1 , wherein the physiologically active polypeptide is selected from the group consisting of hormone, cytokine, enzyme, antibody, growth hormone, transcription regulatory factor, blood factor, vaccine, structure protein, ligand protein and receptor.  
     
     
         14 . The protein conjugate according to  claim 13 , wherein the physiologically active polypeptide is selected from the group consisting of human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons, colony stimulating factor, interleukins, glucocerebrosidae, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, suppressive factor of allergy, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor inhibitory factor, transforming growth factor, alpha-1 antitrypsin, albumin, apolipoprotein-E, erythropoietin, hyper-glycosylated erythropoietin, factor VII, factor VIII, factor IX, plaminogen activator, urokinase, streptokinase, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, platelet derived growth factor, epidermal growth factor, osteogenic growth factor, osteogenesis stimulating protein, calcitonin, insulin, atriopeptin, cartilage inducing factor, connective tissue activator protein, follicle stimulating hormone, leutinizing hormone, FSH releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocorticotrophic hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotrophin releasing, thyroid stimulating hormone, monoclonal antibody, polyclonal antibody, antibody derivatives including [Fab]′, [Fab]′2 and scFv, and virus-derived vaccine antigen.  
     
     
         15 . The protein conjugate according to  claim 14 , wherein the physiologically active polypeptide is human growth hormone, interferon-alpha, granulocyte colony stimulating factor or erythropoietin.  
     
     
         16 . A method for preparing the protein conjugate of  claim 1 , comprising 
 (a) covalently linking at least one physiologically active polypeptide, at least one immunoglobulin with at least one non-peptidic polymer having reactive groups at both ends; and    (b) isolating a protein conjugate comprising essentially the active polypeptide, the immunoglobulin and the non-peptidic polymer, which are linked covalently.    
     
     
         17 . The method according to  claim 16 , wherein step (a) further comprises: 
 (a1) covalently coupling one end of the non-peptidic polymer with either an immunoglobulin or a physiologically active polypeptide;    (a2) isolating from the resulting reaction mixture a complex comprising the non-peptidic polymer coupled with the immunoglobulin or the physiologically active polypeptide; and    (a3) covalently coupling the free end of the non-peptidic polymer of the complex with the immunoglobulin or physiologically active polypeptide, to produce a protein conjugate comprising the physiologically active polypeptide, the non-peptidic polymer and the immunoglobulin, which are covalently interlinked.    
     
     
         18 . The method according to  claim 17 , wherein the molar ratio of the physiologically active polypeptide to the non-peptidic polymer in step (a1) ranges from 1:2.5 to 1:5.  
     
     
         19 . The method according to  claim 17 , wherein the molar ratio of the immunoglobulin to the non-peptidic polymer in step (a1) ranges from 1:5 to 1:10.  
     
     
         20 . The method according to  claim 17 , wherein the molar ratio of the complex obtained in step (a2) to physiologically active polypeptide or immunoglobulin in step(a3) ranges from 1:1 to 1:3.  
     
     
         21 . The method according to  claim 17 , wherein steps (a1) and (a3) are performed in the presence of a reducing agent.  
     
     
         22 . The method according to  claim 21 , wherein the reducing agent is sodium cyanoborohydride, sodium borohydride, dimethylamine borate or pyridine borate.  
     
     
         23 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         24 . A method for prolonging the in vivo half-life of a physiologically active polypeptide, which comprises the step of covalently linking a non-peptidic polymer having reactive groups at both ends with a physiologically active polypeptide and an immunoglobulin.  
     
     
         25 . The method according to  claim 24 , wherein the immunoglobulin is covalently linked to at least two complexes of the physiologically active polypeptide and the non-peptidic polymer.  
     
     
         26 . The method according to  claim 24 , wherein the immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM and a mixture thereof.  
     
     
         27 . The method according to  claim 26 , wherein the immunoglobulin is a human immunoglobulin.  
     
     
         28 . The method according to  claim 24 , wherein the reactive group of the non-peptidic polymer is selected from the group consisting of aldehyde, propion aldehyde, maleimide and succinamide derivative.  
     
     
         29 . The method according to  claim 24 , wherein the non-peptidic polymer is selected from the group consisting of poly(ethylene glycol), poly(propylene glycol), ethylene glycol-propylene glycol copolymer, polyoxyethylated polol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, poly(lactic-glycolic acid), biodegradable polymer, lipid polymer, chitin, hyaluronic acids, and a mixture thereof.  
     
     
         30 . The method according to  claim 29 , wherein the non-peptidic polymer is poly(ethylene glycol).  
     
     
         31 . The method according to  claim 24 , wherein the physiologically active polypeptide is selected from the group consisting of hormone, cytokine, enzyme, antibody, growth hormone, transcription regulatory factor, blood factor, vaccine, structural protein, ligand protein and receptor.  
     
     
         32 . The method according to  claim 31 , wherein the physiologically active polypeptide is selected from the group consisting of human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons, colony stimulating factor, interleukins, glucocerebrosidae, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, suppressive factor of allergy, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor inhibitory factor, transforming growth factor, alpha-1 antitrypsin, albumin, apolipoprotein-E, erythropoietin, hyper-glycosylated erythropoietin, factor VII, factor VIII, factor IX, plasminogen activator, urokinase, streptokinase, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, platelet derived growth factor, epidermal growth factor, osteogenic growth factor, osteogenesis stimulating protein, calcitonin, insulin, atriopeptin, cartilage inducing factor, connective tissue activator protein, follicle stimulating hormone, leutinizing hormone, FSH releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocorticotrophic hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, monoclonal antibody, polyclonal antibody, antibody derivatives including [Fab]′, [Fab]′2, and scFv, and virus-derived vaccine antigen.  
     
     
         33 . The method according to  claim 32 , wherein the physiologically active polypeptide is human growth hormone, interferon-alpha, granulocyte colony stimulating factor or erythropoietin.  
     
     
         34 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 2  and a pharmaceutically acceptable carrier.  
     
     
         35 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 3  and a pharmaceutically acceptable carrier.  
     
     
         36 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 4  and a pharmaceutically acceptable carrier.  
     
     
         37 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 5  and a pharmaceutically acceptable carrier.  
     
     
         38 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 6  and a pharmaceutically acceptable carrier.  
     
     
         39 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 7  and a pharmaceutically acceptable carrier.  
     
     
         40 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 8  and a pharmaceutically acceptable carrier.  
     
     
         41 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 9  and a pharmaceutically acceptable carrier.  
     
     
         42 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 10  and a pharmaceutically acceptable carrier.  
     
     
         43 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 11  and a pharmaceutically acceptable carrier.  
     
     
         44 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 12  and a pharmaceutically acceptable carrier.  
     
     
         45 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 13  and a pharmaceutically acceptable carrier.  
     
     
         46 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 14  and a pharmaceutically acceptable carrier.  
     
     
         47 . A pharmaceutical composition having a prolonged half-life of a physiologically active polypeptide, which comprises a protein conjugate of  claim 15  and a pharmaceutically acceptable carrier.