Dual function proteins comprising fgf21 mutant protein and pharmaceutical composition comprising same

Abstract

A dual function protein is disclosed. The dual function protein may be prepared by linking a biologically active protein and an FGF mutant protein to an Fc region of an immunoglobulin. The dual function protein has improved pharmacological efficacy, in vivo duration and protein stability. The dual function protein exhibits improved pharmacological efficacy, in vivo duration and protein stability. A pharmaceutical composition containing the dual function protein as an active ingredient may be effectively used as a therapeutic agent for diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver diseases, non-alcoholic steatohepatitis or cardiovascular diseases.

1 . A dual function protein comprising a fibroblast growth factor 21 (FGF21) mutant protein; a biologically active protein, or a mutant or fragment thereof; and an Fc region of an immunoglobulin,
 wherein the FGF21 mutant protein comprises the following mutation:   (i) a substitution of the amino acid at position 180 from the N-terminus of a wild-type FGF21 protein with the amino acid E.   
     
     
         2 . The dual function protein of  claim 1 , wherein the FGF21 mutant protein further comprises the following mutation:
 (ii) a substitution of the amino acids at positions 170 to 174 from the N-terminus of a wild-type FGF21 protein with the amino acid sequence of TGLEAN (SEQ ID NO: 44).   
     
     
         3 . The dual function protein of  claim 1 , wherein the FGF21 mutant protein further comprises the following mutation:
 (iii) a substitution of the amino acid at position 170 from the N-terminus of a wild-type FGF21 protein with the amino acid N.   
     
     
         4 . The dual function protein of  claim 1 , wherein the FGF21 mutant protein further comprises the following mutation:
 (iv) a substitution of the amino acids at positions 98 to 101 from the N-terminus of a wild-type FGF21 protein with the amino acid sequence of EIRP (SEQ ID NO: 42);   (v) a substitution of the amino acids at positions 170 to 174 from the N-terminus of a wild-type FGF21 protein with the amino acid sequence of TGLEAV (SEQ ID NO: 43);   (vi) a substitution of the amino acid at position 174 from the N-terminus of a wild-type FGF21 protein with the amino acid N; or   (vii) a combination of the substitution (iv) and the substitution (v), or a combination of the substitution (iv) and the substitution (v).   
     
     
         5 . The dual function protein of  claim 1 , wherein the amino acid residue N of the FGF21 mutant protein introduced by a mutation is glycosylated. 
     
     
         6 . The dual function protein of  claim 1 , wherein the biologically active protein is one selected from the group consisting of insulin, C-peptide, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6), bone morphogenetic protein-9 (BMP-9), oxyntomodulin, oxytocin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), irisin, fibronectin type III domain-containing protein 5 (FNDC5), apelin, adiponectin, C1q and tumor necrosis factor related protein (CTRP family), resistin, visfatin, omentin, retinol binding protein-4 (RBP-4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4, growth hormone, and a combination thereof. 
     
     
         7 . The dual function protein of  claim 6 , wherein the biologically active protein is one selected from GLP-1, a mutant thereof, and exendin-4. 
     
     
         8 . The dual function protein of  claim 7 , wherein the mutant of GLP-1 comprises an amino acid sequence selected from the group consisting SEQ ID NOs: 43 to 46. 
     
     
         9 . The dual function protein of  claim 1 , wherein the wild-type FGF21 protein comprises the amino acid sequence represented by SEQ ID NO: 1. 
     
     
         10 . The dual function protein of  claim 1 , wherein the FGF21 mutant protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 23. 
     
     
         11 . The dual function protein of  claim 1 , wherein the dual function protein further comprises a linker. 
     
     
         12 . The dual function protein of  claim 11 , wherein the linker connects the FGF21 mutant protein to the Fc region of the immunoglobulin. 
     
     
         13 . The dual function protein of  claim 12 , wherein the linker is connected to the C-terminus of the Fc region of the immunoglobulin and the N-terminus of the FGF21 mutant protein. 
     
     
         14 . The dual function protein of  claim 12 , wherein the linker is a peptide consisting of 10 to 30 amino acid residues. 
     
     
         13 . The dual function protein of  claim 14 , wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5. 
     
     
         14 . The dual function protein of  claim 1 , wherein the Fc region of the immunoglobulin is any one of the Fc region of IgG1, IgG2, IgG3, IgG4 and IgD, or a hybrid Fc containing a combination thereof. 
     
     
         15 . The dual function protein of  claim 14 , wherein the hybrid Fc comprises an IgG4 region and an IgD region. 
     
     
         16 . The dual function protein of  claim 1 , wherein the dual function protein comprises the biologically active protein, the Fc region of the immunoglobulin and the FGF21 mutant protein, connected in this order from the N-terminus to the C-terminus. 
     
     
         17 . The dual function protein of  claim 16 , wherein a linker is additionally connected between the Fc region of the immunoglobulin and the FGF21 mutant protein. 
     
     
         18 . The dual function protein of  claim 17 , wherein the linker is connected to the C-terminus of the Fc region of the immunoglobulin and the N-terminus of the FGF21 mutant protein. 
     
     
         19 . The dual function protein of  claim 17 , wherein the linker is a peptide consisting of 10 to 30 amino acid residues. 
     
     
         20 . The dual function protein of  claim 17 , wherein the linker comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5. 
     
     
         21 . The dual function protein of  claim 1 , wherein the dual function protein comprise the amino acid sequence of SEQ ID NO: 65. 
     
     
         22 . The dual function protein of  claim 1 , wherein the dual function protein comprises the amino acid sequence of SEQ ID NO: 66. 
     
     
         23 . The dual function protein of  claim 1 , wherein the dual function protein has an amino acid sequence represented by SEQ ID NO: 67. 
     
     
         24 . A pharmaceutical composition comprising the dual function protein according to  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         25 . An isolated nucleic acid molecule encoding the fusion protein according to  claim 1 . 
     
     
         26 . An expression vector comprising the nucleic acid molecule of  claim 25 . 
     
     
         27 . A host cell comprising the nucleic acid molecule of  claim 25 . 
     
     
         28 . A method for treating diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or cardiovascular diseases in a subject in need thereof, comprising administering the pharmaceutical composition of  claim 24  to the subject. 
     
     
         29 . A method selected from the group consisting of:
 reducing blood glucose level in a subject;   reducing body weight in a subject;   reducing triglyceride or low-density lipoprotein levels in a subject; and   improving insulin sensitivity in a subject   wherein the method comprising administering the pharmaceutical composition of  claim 24  to the subject.   
     
     
         30 . The method of  claim 29 , wherein the subject is obese. 
     
     
         31 . The method of  claim 30 , wherein the subject has diabetes. 
     
     
         32 . A method for inhibiting cAMP production in a subject in need thereof, comprising administering the composition of  claim 24  to the subject.