Fusion proteins having mutated immunoglobulin hinge region

Abstract

A fusion protein having a non-immunoglobulin polypeptide having a cysteine residue proximal to the C terminal thereof, and an immunoglobulin component with a mutated hinge region is provided. The mutation comprises a point mutated site corresponding in position to the position in a native hinge region of the cysteine residue located nearest the cysteine residue of the non-Ig component. The distance from the cysteine residue of the non-immunoglobulin polypeptide and any remaining cysteine residues of the mutated hinge region is sufficient to prevent the formation of a disulphide bond therebetween.

1 . A fusion protein comprising:
 a non-immunoglobulin polypeptide comprising a cysteine residue proximal to the C terminal thereof; and   an immunoglobulin component comprising a mutated hinge region, the mutation comprising a point mutated site corresponding in position to the position in a native hinge region of the cysteine residue located nearest the cysteine residue of the non-Ig component, whereby the distance from the cysteine residue of the non-immunoglobulin polypeptide and any remaining cysteine residues of the mutated hinge region is sufficient to prevent the formation of a disulphide bond therebetween.   
     
     
         2 . A fusion protein according to  claim 1 , wherein the non-immunoglobulin polypeptide is selected from the group consisting of a cytokine, a ligand-binding protein, a hormone, a neurotrophin, a neutrophin receptor, a body-weight regulator, a serum protein, a clotting factor, a protease, an extracellular matrix component, an angiogenic factor, an anti-angiogenic factor, an immunoglobulin receptor, a blood factor, a cancer antigen, a statin, a growth factor, a therapeutic peptide, a non-human protein, a non-mammalian protein and a protein toxin. 
     
     
         3 . A fusion protein according to  claim 2 , wherein the cytokine is selected from the group consisting of hematopoietic factor, interferon, interleukin and tumor necrosis factor. 
     
     
         4 . A fusion protein according to  claim 3 , wherein the hematopoietic factor is selected from the group consisting of erythropoietin, granulocyte-macrophage colony stimulating factor and granulocyte colony stimulating factor. 
     
     
         5 . A fusion protein according to  claim 2 , wherein the ligand-binding protein is selected from the group consisting of a CD molecule, CTLA-4, TNF receptor, and interleukin receptor. 
     
     
         6 . A fusion protein according to  claim 1 , wherein the human immunoglobulin component comprises an Fc fragment. 
     
     
         7 . A fusion protein according to  claim 6 , wherein the Fc fragment is derived from an IgG. 
     
     
         8 . A fusion protein according to  claim 7 , wherein the IgG is selected from the group consisting of IgG 1, IgG 2, IgG 3 and IgG 4. 
     
     
         9 . A fusion protein according to  claim 8 , wherein the Fc fragment is a IgG Fc fragment comprising the mutated hinge region and CH2 and CH3 domains. 
     
     
         10 . A fusion protein according to  claim 9 , wherein the IgG Fc fragment derived from IgG 1. 
     
     
         11 . A fusion protein according to  claim 1  wherein the C-terminal of the non-immunoglobulin polypeptide is directly linked to the N-terminal of the mutated hinge region. 
     
     
         12 . A fusion protein according to  claim 1 , wherein the mutated hinge region comprises at least 9 amino acids. 
     
     
         13 . A fusion protein according to  claim 12 , wherein the mutated hinge region comprises between 10 and 20 amino acids. 
     
     
         14 . A fusion protein according to  claim 1 , wherein the point mutated site comprises a non-cysteine amino acid. 
     
     
         15 . A fusion protein according to  claim 1 , wherein the point mutated site is the sixth amino acid position measured from the N-terminal of the mutated hinge region and comprises a non-cysteine amino acid. 
     
     
         16 . A fusion protein according to  claim 14 , wherein the non-cysteine amino acid is a neutral amino acid. 
     
     
         17 . A fusion protein according to  claim 14 , wherein the non-cysteine amino acid is glycine. 
     
     
         18 . A fusion protein according to  claim 14 , wherein the non-cysteine amino acid is alanine. 
     
     
         19 . A fusion protein according to  claim 1 , wherein the non-immunoglobulin polypeptide is a human granulocyte-macrophage colony stimulating factor or a variant thereof 
     
     
         20 . A multimeric protein comprising a plurality of fusion proteins according to  claim 1 . 
     
     
         21 . A multimeric protein according to  claim 20  wherein the multimeric protein is a dimer. 
     
     
         22 . A method of producing a fusion protein as defined in  claim 1  comprising culturing a cell line transfected with a DNA molecule that encodes the sequence of the fusion protein and purifying the protein encoded thereby. 
     
     
         23 . A cell line as defined in  claim 22 , wherein the cell line is a CHO cell line. 
     
     
         24 . A method of stimulating white blood cell production in a mammal comprising administering to the mammal a fusion protein according to  claim 19 . 
     
     
         25 . A method according to  claim 24  wherein the mammal is a human. 
     
     
         26 . A pharmaceutical composition comprising a fusion protein according to  claim 19  and a pharmaceutically acceptable carrier, adjuvant or diluent. 
     
     
         27 . A method of stimulating white blood cell production in a mammal comprising administering to the mammal a pharmaceutical composition according to  claim 26 . 
     
     
         28 . A method according to  claim 27  wherein the mammal is a human. 
     
     
         29 . A fusion protein comprising:
 a non-immunoglobulin polypeptide; and   an immunoglobulin component comprising a mutated hinge region, the mutation comprising a point mutated site in a hinge region of said component promixate to said polypeptide, whereby a cysteine residue of said hinge region is substituted by a non-cysteine residue.   
     
     
         30 . A fusion protein comprising a non-immunoglobulin polypeptide and a human immunoglobulin component, wherein the fusion protein has a prolonged half-life in vivo in comparison to naturally occurring or recombinant native non-immunoglobulin polypeptide. 
     
     
         31 . A fusion protein according to  claim 30  wherein the non-immunoglobulin polypeptide is directly linked to the human immunoglobulin component. 
     
     
         32 . A fusion protein according to  claim 30  wherein the non-immunoglobulin polypeptide is linked to the human immunoglobulin component by a synthetic linker. 
     
     
         33 . A fusion protein according to  claim 30 , wherein the half-life of the fusion protein is at least three fold higher than the native non-immunoglobulin polypeptide. 
     
     
         34 . A fusion protein according to  claim 30 , wherein the half-life of the fusion protein is at least four fold higher than the native non-immunoglobulin polypeptide. 
     
     
         35 . A fusion protein according to  claim 30 , wherein the fusion protein has enhanced biological activity in comparison to the native non-immunoglobulin polypeptide. 
     
     
         36 . A fusion protein according to  claim 30 , wherein the non-immunoglobulin polypeptide is selected from the group consisting of a cytokine, a ligand-binding protein, a hormone, a neurotrophin, a neutrophin receptor, a body-weight regulator, a serum protein, a clotting factor, a protease, an extracellular matrix component, an angiogenic factor, an anti-angiogenic factor, an immunoglobulin receptor, a blood factor, a cancer antigen, a statin, a growth factor, a therapeutic peptide, a non-human protein, a non-mammalian protein and a protein toxin. 
     
     
         37 . A fusion protein according to  claim 36 , wherein the cytokine is selected from the group consisting of hematopoietic factor, interferon, interleukin and tumor necrosis factor. 
     
     
         38 . A fusion protein according to  claim 37 , wherein the hematopoietic factor is selected from the group consisting of erythropoietin, granulocyte-macrophage colony stimulating factor and granulocyte colony stimulating factor. 
     
     
         39 . A fusion protein according to  claim 37 , wherein the ligand-binding protein is selected from the group consisting of a CD molecule, CTLA-4, TNF receptor, and interleukin receptor. 
     
     
         40 . A fusion protein according to  claim 30 , wherein the human immunoglobulin component comprises an Fc fragment. 
     
     
         41 . A fusion protein according to  claim 40 , wherein the Fc fragment is derived from an IgG. 
     
     
         42 . A fusion protein according to  claim 41 , wherein the IgG is selected from the group consisting of IgG 1, IgG 2, IgG 3 and IgG 4. 
     
     
         43 . A fusion protein according to  claim 40 , wherein the Fc fragment comprises a hinge region and CH2 and CH3 domains. 
     
     
         44 . A fusion protein according to  claim 43 , wherein the hinge region comprises at least 9 amino acids. 
     
     
         45 . A fusion protein according to  claim 44 , wherein the hinge region comprises between 10 and 20 amino acids. 
     
     
         46 . A fusion protein according to  claim 45 , wherein the hinge region is mutated. 
     
     
         47 . A fusion protein according to  claim 46 , wherein the hinge region is point-mutated. 
     
     
         48 . A fusion protein according to  claim 47 , wherein the point-mutated site corresponds to the position of the first cysteine from the N-terminal of a native hinge region. 
     
     
         49 . A fusion protein according to  claim 48 , wherein the first cysteine is substituted by a non-cysteine amino acid. 
     
     
         50 . A fusion protein according to  claim 49 , wherein the non-cysteine amino acid is a neutral amino acid. 
     
     
         51 . A fusion protein according to  claim 50 , wherein the non-cysteine amino acid is glycine. 
     
     
         52 . A fusion protein according to  claim 50 , wherein the non-cysteine amino acid is alanine. 
     
     
         53 . A fusion protein according to  claim 30 , wherein the non-immunoglobulin polypeptide is a human granulocyte-macrophage colony stimulating factor or a variant thereof 
     
     
         54 . A multimeric protein comprising a plurality of fusion proteins according to  claim 30 . 
     
     
         55 . A multimeric protein according to  claim 54  wherein the multimeric protein is a dimer. 
     
     
         56 . A method of producing a fusion protein as defined in  claim 30  comprising culturing a cell line transfected with a DNA molecule that encodes the sequence of the fusion protein and purifying the protein encoded thereby. 
     
     
         57 . A cell line as defined in  claim 56 , wherein the cell line is a CHO cell line. 
     
     
         58 . A method of stimulating white blood cell production in a mammal comprising administering to the mammal a fusion protein according to  claim 53 . 
     
     
         59 . A method according to  claim 58  wherein the mammal is a human. 
     
     
         60 . A pharmaceutical composition comprising a fusion protein according to  claim 53  and a pharmaceutically acceptable carrier, adjuvant or diluent. 
     
     
         61 . A method of stimulating white blood cell production in a mammal comprising administering to the mammal a pharmaceutical composition according to  claim 60 . 
     
     
         62 . A method according to  claim 61  wherein the mammal is a human. 
     
     
         63 . A fusion protein comprising the amino acid sequence of SEQ ID NO:2 or a sequence substantially homologous thereto. 
     
     
         64 . A recombinant DNA molecule comprising the nucleic acid sequence of SEQ ID NO:1 or a sequence substantially homologous thereto. 
     
     
         65 . A fusion protein comprising the amino acid sequence of SEQ ID NO:6 or a sequence substantially homologous thereto. 
     
     
         66 . A recombinant DNA molecule comprising the nucleic acid sequence of SEQ ID NO:5 or a sequence substantially homologous thereto.