Immunoglobulin fusion proteins

Abstract

The present invention relates to novel methods for making fusion proteins comprising a cytokine or growth factor fused to an immunoglobulin domain. The growth factor/cytokine can be fused directly to an immunoglobulin domain or through a peptide linker. The purified growth factor/cytokine-IgG fusion proteins produced by the novel methods are biologically active and can be used to treat diseases for which the non-fused growth factor/cytokine are useful.

1 .- 37 . (canceled) 
     
     
         38 . A fusion protein comprising a granulocyte colony-stimulating factor protein joined without an intervening peptide linker to a immunoglobulin (Ig) domain that does not contain a variable region. 
     
     
         39 . The fusion protein of  claim 38 , wherein the Ig domain is selected from the group consisting of IgG-Fc, IgG-C H  and IgG-C L . 
     
     
         40 . A pharmaceutical composition comprising the fusion protein of  claim 38  in a pharmaceutically acceptable carrier. 
     
     
         41 . A composition comprising the fusion protein of  claim 38 , wherein said fusion protein is dimeric and wherein said composition is essentially free of monomeric fusion protein. 
     
     
         42 . A nucleic acid encoding the fusion protein of  claim 38 . 
     
     
         43 . An isolated host cell transfected or transformed with the nucleic acid of  claim 42 , enabling the host cell to express the fusion protein. 
     
     
         44 . The isolated host cell of  claim 43 , wherein the host cell is a eukaryotic cell. 
     
     
         45 . The isolated host cell of  claim 44 , wherein the eukaryotic cell is a mammalian cell. 
     
     
         46 . A method of producing a fusion protein of  claim 38 , comprising:
 a) transfecting or transforming a host cell with an expression vector comprising at least one nucleic acid encoding the fusion protein of  claim 38 ;   b) culturing the host cell under conditions effective to express said fusion protein; and   c) harvesting the fusion protein expressed by the host cell.   
     
     
         47 . A method of purifying the fusion protein of  claim 38 , comprising:
 a) obtaining a composition comprising the fusion protein; and   b) isolating the fusion protein from contaminants by column chromatography.   
     
     
         48 . The method of  claim 47 , wherein the fusion protein is isolated from contaminants by size-exclusion chromatography. 
     
     
         49 . The fusion protein of  claim 38 , wherein the granulocyte colony-stimulating factor is a full-length human granulocyte colony-stimulating factor. 
     
     
         50 . The fusion protein of  claim 38  wherein the fusion protein has an EC 50  of less than about 300 ng/ml in a granulocyte colony-stimulating factor-dependent in vitro bioassay using a murine NFS60 cell line that proliferates in response to granulocyte colony-stimulating factor. 
     
     
         51 . The fusion protein of  claim 38 , wherein said fusion protein has an EC 50  of less than 30 ng/ml in a granulocyte colony-stimulating factor-dependent in vitro bioassay using a murine NFS60 cell line that proliferates in response to granulocyte colony-stimulating factor. 
     
     
         52 . The fusion protein of  claim 38 , wherein said fusion protein has an EC 50  within 4 fold of the EC 50  of non-fused granulocyte colony-stimulating factor, on a molar basis, in a granulocyte colony-stimulating factor-dependent in vitro bioassay using a murine NFS60 cell line that proliferates in response to granulocyte colony-stimulating factor. 
     
     
         53 . A fusion protein comprising a granulocyte colony-stimulating factor protein joined without an intervening peptide linked to a immunoglobulin (Ig) domain that does not contain a variable region wherein the Ig domain is selected from the group consisting of full-length IgG-Fc, IgG-C H  and IgG-C L , wherein the fusion protein comprises the natural granulocyte colony-stimulating factor amino acid sequence and the natural immunoglobulin domain amino acid sequence at the junction of the fusion protein, and wherein the fusion protein has an EC 50  of less than about 1000 ng/ml in a granulocyte colony-stimulating factor-dependent in vitro bioassay using a murine NFS60 cell line that proliferates in response to granulocyte colony-stimulating factor. 
     
     
         54 . The fusion protein of  claim 38 , wherein a serine is substituted for cysteine-17 of granulocyte colony-stimulating factor. 
     
     
         55 . A method of treating a condition treatable with granulocyte colony-stimulating factor, comprising administering an effective amount of the fusion protein of  claim 38  to a patient in need thereof. 
     
     
         56 . The method of  claim 55 , wherein the condition is a deficient white blood cell count, and wherein administration of the fusion protein increases the number of white blood cells of the patient. 
     
     
         57 . A fusion protein comprising granulocyte colony-stimulating factor joined at its carboxy-terminus by a peptide linker to the amino terminus of an immunoglobulin domain that does not contain a variable region, wherein the peptide linker is selected from the group consisting of SerGly, SerGlyGlySer (SEQ ID NO:1) and Ser(GlyGlySer) 2  (SEQ ID NO:3).