Modified ADAMTS4 molecules and method of use thereof

Abstract

The present invention relates to modified ADAMTS4 proteins having improved stability comparing to the corresponding native, unmodified proteins. The modified ADAMTS4 proteins can be expressed and isolated in large quantities, thus allowing further characterization of the proteins, such as crystallographic and enzyme kinetic studies. The purified, stable proteins would also facilitate the production of anti-ADAMTS antibodies and the development of inhibitors to ADAMTS enzymes.

1 - 8 . (canceled)  
     
     
         9 . An isolated, modified ADAMTS4 protein with improved stability compared to an ADAMTS4 protein having an amino acid sequence recited in SEQ ID NO:15, said modified ADAMTS4 protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:17, 19, 22, 24, 26, 27, and 46-49.  
     
     
         10 . An isolated polynucleotide, said polynucleotide comprising a nucleotide sequence encoding an isolated, modified ADAMTS4 protein with improved stability compared to a naturally-occurring, full-length ADAMTS4 protein, said modified ADAMTS4 protein differing from the naturally-occurring, full-length ADAMTS4 protein by at least one amino acid.  
     
     
         11 . An isolated polynucleotide, said polynucleotide comprising a nucleotide sequence encoding the modified ADAMTS4 protein of  claim 9 .  
     
     
         12 . A vector comprising the polynucleotide of  claim 10  in operative association with an expression control sequence.  
     
     
         13 . A vector comprising the polynucleotide of  claim 11  in operative association with an expression control sequence.  
     
     
         14 . A method for producing a modified ADAMTS4 protein, said method comprising: 
 introducing a polynucleotide comprising a nucleotide sequence encoding an isolated, modified ADAMTS4 protein with improved stability compared to a naturally-occurring, full-length ADAMTS4 protein, said modified ADAMTS4 protein differing from the naturally-occurring, full-length ADAMTS4 protein by at least one amino acid, into a host cell;    incubating said host cell under conditions that allow expression of the modified ADAMTS4 protein from the polynucleotide; and    purifying the modified ADAMTS4 protein from the host cell.    
     
     
         15 . A method for producing a modified ADAMTS4 protein, said method comprising: 
 introducing a polynucleotide comprising a nucleotide sequence encoding the modified ADAMTS4 protein of  claim 9  into a host cell;    incubating said host cell under conditions that allow expression of the modified ADAMTS4 protein from the polynucleotide; and    purifying the modified ADAMTS4 protein from the host cell.    
     
     
         16 . A method of identifying an inhibitor of an isolated, modified ADAMTS4 protein with improved stability compared to a naturally-occurring, full-length ADAMTS4 protein, said modified ADAMTS4 protein differing from the naturally-occurring, full-length ADAMTS4 protein by at least one amino acid, said method comprising the steps of: 
 determining the aggrecanase activity of the modified ADAMTS4 protein;    contacting the modified ADAMTS4 protein with a candidate agent;    determining the aggrecanase activity of the modified ADAMTS4 protein in the presence of said candidate agent; and    determining whether said candidate agent affects the activity of the modified ADAMTS4 protein.    
     
     
         17 . A pharmaceutical composition for treating an aggrecanase-related disease, comprising: 
 (a) an inhibitor of an isolated, modified ADAMTS4 protein with improved stability compared to a naturally-occurring, full-length ADAMTS4 protein, said modified ADAMTS4 protein differing from the naturally-occurring, full-length ADAMTS4 protein by at least one amino acid, or an antibody that binds specifically to the isolated, modified ADAMTS4 protein; and    (b) a pharmaceutically acceptable carrier.    
     
     
         18 . The pharmaceutical composition of  claim 17 , wherein the antibody inhibits the aggrecanase activity of the modified ADAMTS4 protein.  
     
     
         19 . A method for treating an aggrecanase-related disease in a mammal, said method comprising the step of: 
 introducing into the mammal an effective amount of the pharmaceutical composition of  claim 17 .    
     
     
         20 . The method of  claim 19 , wherein the aggrecanase-related disease is osteoarthritis.