Insulin-like growth factor binding protein variants

Abstract

The invention relates to variants of native insulin-like growth factor binding protein 3 (IGFBP-3). Variant IGFBP-3s are disclosed which are modified to be resistant to hydrolysis. Also disclosed are variant IGFBP-3s where the nuclear localization signal (NLS) in native IGFBP-3 is altered. Additionally, amino-terminally extended IGFBP-3s are disclosed which include a variety of N-terminal extensions, including peptide and nucleotide binding domains, specific binding members such as ligand binding domains from receptors or antigen binding domains from immunoglobins, and peptide and protein hormones and growth factors. N-terminally extended IGFBP-3s may comprise hydrolysis-resistant or NLS variant IGFBP-3s.

1 . A stabilized IGFBP-3, comprising the sequence of FIG. 1, wherein position 116 is changed to an amino acid other than aspartate.  
     
     
         2 . The stabilized IGFBP-3 of  claim 1 , wherein position 116 is changed to glutamic acid.  
     
     
         3 . A stabilized IGFBP-3, comprising the sequence of FIG. 1, wherein position 135 is changed to an amino acid other than aspartate.  
     
     
         4 . The stabilized IGFBP-3 of  claim 3 , wherein position 135 is changed to glutamic acid.  
     
     
         5 . The stabilized IGFBP-3 of  claim 4 , wherein residue 116 is changed to an amino acid other than aspartate.  
     
     
         6 . The stabilized IGFBP-3 of  claim 5 , wherein residue 116 is changed to glutamic acid.  
     
     
         7 . A nuclear localization signal (NLS) variant IGFBP-3 wherein the NLS sequence is altered.  
     
     
         8 . The NLS variant IGFBP-3 of  claim 7  comprising the sequence of FIG. 1 wherein residue 228 is altered to an amino acid other than lysine.  
     
     
         9 . The NLS variant IGFBP-3 of  claim 8  wherein residue 228 is altered to glutamic acid.  
     
     
         10 . The NLS variant IGFBP-3 of  claim 7 , wherein residue 230 is altered to an amino acid other than arginine.  
     
     
         11 . The NLS variant IGFBP-3 of  claim 10 , wherein residue 230 is altered to glycine.  
     
     
         12 . The NLS variant IGFBP-3 of  claim 7 , wherein residue 228 is altered to an amino acid other than lysine and residue 230 is altered to a residue other than arginine.  
     
     
         13 . The NLS variant IGFBP-3 of  claim 12 , wherein residue 228 is altered to glutamic acid and residue 230 is altered to glycine.  
     
     
         14 . An N-terminally extended insulin-like growth factor binding protein 3 (IGFBP-3), comprising: 
 an N-terminal extension sequence selected from the group consisting of nucleotide-binding sequences, peptide binding sequences, antigen or ligand binding sequences, protein hormones, growth factors and enzymes; and    IGFBP-3, wherein said IGFBP-3 is linked to the carboxy terminus of said fusion partner, and wherein said N-terminal extension sequence is not DsbA, leaderless DsbA,  Vibro cholerae  TcpG, DsbC, or leaderless DsbC.    
     
     
         15 . The N-terminally extended IGFBP-3 of  claim 14 , wherein said IGFBP-3 comprises the sequence of FIG. 1.  
     
     
         16 . The N-terminally extended IGFBP-3 of  claim 14 , wherein said IGFBP-3 comprises a variant of the sequence of FIG. 1.  
     
     
         17 . The N-terminally extended IGFBP-3 of  claim 16 , wherein said variant comprises the sequence of FIG. 1 with position 5 changed to glycine.  
     
     
         18 . The N-terminally extended IGFBP-3 of  claim 16 , wherein said IGFBP-3 comprises an ND variant IGFBP-3.  
     
     
         19 . The N-terminally extended IGFBP-3 of  claim 14 , wherein said N-terminal extension is a nucleotide binding sequence.  
     
     
         20 . The N-terminally extended IGFBP-3 of  claim 14  wherein said N-terminal extension is an enzyme.  
     
     
         21 . The N-terminally extended IGFBP-3 of claim  20  wherein said enzyme is not glutathione-S-transferase or β-galactosidase.