Derivatives of Growth Hormone and Related Proteins, and Methods of Use Thereof

Abstract

The growth hormone supergene family comprises greater than 20 structurally related cytokines and growth factors. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine residues to non-essential regions of the proteins or substituting cysteine residues for non-essential amino acids in the proteins using site-directed mutagenesis and then covalently coupling a cysteine-reactive polymer or other type of cysteine-reactive moiety to the proteins via the added cysteine residue. Disclosed herein are preferred sites for adding cysteine residues or introducing cysteine substitutions into the proteins, and the proteins and protein derivatives produced thereby. Also disclosed are therapeutic methods for using the cysteine variants of the invention.

1 . A method to protect an animal from a disease or condition that can be treated by granulocyte colony-stimulating factor (G-CSF), comprising administering to an animal having said disease or condition a composition comprising a cysteine variant of G-CSF of SEQ ID NO: 6, wherein said G-CSF cysteine variant comprises at least one cysteine residue substituted for at least one amino acid located in at least one region of G-CSF selected from the group consisting of: the A-B loop, the B-C loop, the C-D loop, the first three or last three amino acids in helix B, the first three or last three amino acids in helix C, the first three or last three amino acids in helix D, and the amino acids following helix D.  
     
     
         2 . The method of  claim 1 , wherein said cysteine variant comprises at least one cysteine residue substituted for at least one amino acid selected from the group consisting of: K40, S53, G55, W58, A59, P60, S62, S63, P65, S66, Q67, A68, Q70, A72, Q90, A91, E93, G94, S96, E98, G100, G125, M126, A127, A129, Q131, T133, Q134, G135, A136, A139, A141, S142, A143, Q145, Q173 and P174.  
     
     
         3 - 59 . (canceled)  
     
     
         60 . An isolated, PEGylated granulocyte colony stimulating factor (G-CSF) variant of SEQ ID NO:6, wherein a polyethylene glycol is attached to the protein through an amino acid position, with respect to SEQ ID NO:6, selected from the group consisting of: 2, 6, 7, 58, 68, 93, 129, 131, 133, 134, 136, 139, 141, and 173, and wherein said variant has biological activity in vitro as measured by proliferation of a cell line that proliferates in response to G-CSF.  
     
     
         61 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to the protein through amino acid 2 of said G-CSF variant.  
     
     
         62 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 6 of said G-CSF variant.  
     
     
         63 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 7 of said G-CSF variant.  
     
     
         64 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 58 of said G-CSF variant.  
     
     
         65 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 68 of said G-CSF variant.  
     
     
         66 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 93 of said G-CSF variant.  
     
     
         67 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 129 of said G-CSF variant.  
     
     
         68 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 131 of said G-CSF variant.  
     
     
         69 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 133 of said G-CSF variant.  
     
     
         70 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 134 of said G-CSF variant.  
     
     
         71 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 136 of said G-CSF variant.  
     
     
         72 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 139 of said G-CSF variant.  
     
     
         73 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 141 of said G-CSF variant.  
     
     
         74 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to amino acid 173 of said G-CSF variant.  
     
     
         75 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein the variant is monoPEGylated.  
     
     
         76 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a non-cysteine residue is substituted for cysteine-17 of said G-CSF variant.  
     
     
         77 . The isolated, PEGylated G-CSF variant of  claim 76 , wherein said non-cysteine amino acid is selected from the group consisting of serine and alanine.  
     
     
         78 . The isolated, PEGylated G-CSF variant of  claim 60 , wherein a polyethylene glycol is attached to a cysteine residue substituted for the amino acid at said amino acid position.  
     
     
         79 . A composition comprising at least one PEGylated granulocyte colony stimulating factor (G-CSF) variant according to  claim 60 .  
     
     
         80 . A method to stimulate proliferation and differentiation of hematopoietic cells in an animal, comprising administering to said animal an isolated PEGylated granulocyte colony stimulating factor (G-CSF) variant of  claim 60 .  
     
     
         81 . The method of  claim 80 , wherein said composition is administered by intravenous administration or subcutaneous administration.  
     
     
         82 . The method of  claim 80 , wherein said animal has a deficiency of hematopoietic cells.  
     
     
         83 . The method of  claim 80 , wherein said deficiency of hematopoietic cells is associated with myelosuppressive chemotherapy, bone marrow transplantation, infection with the human immunodeficiency virus, or chronic neutropenia.