Cleavage of VEGF and VEGF receptor by wildtype and mutant MT-SP1

Abstract

MT-SP1 mutein proteases with altered specificity for the target molecules they cleave can be used to treat human diseases, such as cancer. Cleaving VEGF or VEGFR at certain substrate sequences with wild-type and mutein MT-SP1 proteases can be used to treat pathologies associated with angiogenesis.

1 . A method of treating a subject having an angiogenesis-related disease or condition, comprising administering an MT-SP1 protease, wherein the protease cleaves vascular endothelial growth factor (VEGF) or a vascular endothelial growth factor receptor (VEGFR) thereby inactivating a VEGFR signaling pathway. 
     
     
         2 . The method of  claim 1 , wherein the MT-SP1 protease is set forth in SEQ ID NO:1, or is an allelic variant or a biologically active portion thereof. 
     
     
         3 . The method of  claim 1 , wherein the MT-SP1 protease is set forth in SEQ ID NO:2, or is an allelic variant or a biologically active portion thereof. 
     
     
         4 . The method of  claim 1 , wherein:
 the MT-SP1 protease is a mutein MT-SP1 protease containing N mutated residues relative to a wild-type MT-SP1 protease scaffold; and   the mutated residues modify the activity and/or target specificity of the MT-SP1 protease towards VEGF or a VEGFR.   
     
     
         5 . The method of  claim 4 , wherein the wild-type protease comprises a sequence of amino acids set forth in SEQ ID NO:1, or is an allelic variant or biologically active portion thereof. 
     
     
         6 . The method of  claim 4 , wherein the wild-type protease comprises a sequence of amino acids set forth in SEQ ID NO:2, or is an allelic variant or biologically active portion thereof. 
     
     
         7 . The method of  claim 4 , wherein the mutated residues modify the target specificity. 
     
     
         8 . The method of  claim 4 , wherein N mutated residues is between 1 and 4. 
     
     
         9 . The method of  claim 4 , wherein the mutein MT-SP1 protease comprises a polypeptide whose sequence is 95% identical to the amino acid sequence of wild-type MT-SP1 of SEQ ID NO:1 or SEQ ID NO:2, or to an allelic variant or biologically active portion thereof, wherein the polypeptide has at least one mutation at one or more positions which are selected from among 60b, 60c, 97, 99, 146, 172, 175, 180, 192, 215, 217 and 224, wherein the numbering is based on chymotrypsin. 
     
     
         10 . The method of  claim 4 , wherein the mutation is selected from among one or more mutations corresponding to D60bI, D60bF, D60bR, D60bA, R60cI, R60cF, R60cD, R60cA, R60cW, F97N, F97D, F97E, F97A, F97W, F97R, F99Y, F99W, F99N, F99D, F99E, F99A, F99V, F99R, Y146F, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R, L172N, L172D, L172E, L172A, L172V, L172F, L172R, Q175D, Q175E, Q175A, Q175V, Q175F, Q175R, M180E, M180Y, M180R, M180A, Q192A, Q192V, Q192D, Q192R, Q192F, W215F, W215Y, W215I, W215D, W215R, D217A, D217V, D217F, D217E, D217R, K224A, K224F, K224V, K224D, L172D/Q175D, F99V/L172D, F99V/L172D/Q175D, F99V/K224F, F99V/M180E, F99V/Y146D, Y146D/K224F, Y146D/M180E, Y146D/L172D/Q175D, F99V/Y146D/L172D/Q175D, F99A/L172D/Q175D, F99L/L172D/Q175D, F99T/L172D/Q175D, F99A/L172D/Q175D, F99I/K224F, F99L/K224F, F99T/K224F, F99V/Y146D/K224F, F991/Y146D/K224F, F99L/Y146D/K224F and F99T/Y146D/K224F, wherein the numbering is based on chymotrypsin. 
     
     
         11 . The method of  claim 4 , wherein the mutein MT-SP1 protease is more selective for a substrate recognition site of a VEGFR as compared to the wild-type MT-SP1 substrate recognition site. 
     
     
         12 . The method of  claim 11 , wherein the substrate recognition site of a VEGFR comprises an amino acid sequence of RRVR (SEQ ID NO:14). 
     
     
         13 . The method of  claim 12 , wherein the mutation is selected from among L172D, Y146F, Q175D, D217F, F99V, and K224F. 
     
     
         14 . The method of  claim 10 , wherein the mutation is K224F and F99V or K224F and F99I. 
     
     
         15 . The method of  claim 11 , wherein the substrate recognition site is located in the extracellular domain of a VEGFR. 
     
     
         16 . The method of  claim 15 , wherein the VEGFR is VEGF-R2/flk-1/KDR. 
     
     
         17 . The method of  claim 4 , wherein the mutein MT-SP1 protease target
 specificity is increased by at least 2-fold compared to the wild-type MT-SP1 protease.   
     
     
         18 . The method of  claim 1 , wherein the angiogenesis-related disease or condition is cancer, macular degeneration, inflammation or diabetes. 
     
     
         19 . The method of  claim 1 , wherein the angiogenesis-related disease or condition is tumor-associated angiogenesis. 
     
     
         20 . The method of  claim 1 , wherein the angiogenesis-related disease or condition is cancer. 
     
     
         21 . The method of  claim 1 , wherein the subject is a mammal. 
     
     
         22 . The method of  claim 1 , wherein the subject is a human. 
     
     
         23 . The method of  claim 1 , wherein the MT-SP1 protease is administered in combination with an anti-cancer agent. 
     
     
         24 . The method of  claim 23 , wherein the anti-cancer agent is the anti-VEGF antibody bevacizumab. 
     
     
         25 . The method of  claim 23 , wherein the anti-cancer agent is a chemotherapeutic agent. 
     
     
         26 . The method of  claim 23 , wherein the anti-cancer agent is selected from among a radioisotope, drug, cytokine and toxin. 
     
     
         27 . The method of  claim 23 , wherein the anti-cancer agent is a radioisotope, drug or toxin and the agent is conjugated to an antibody or antibody fragment that targets the agent to a cancer cell. 
     
     
         28 . The method of  claim 23 , wherein the anti-cancer agent and MT-SP1 protease are administered to the patient simultaneously or sequentially. 
     
     
         29 . The method of  claim 23 , wherein the MT-SP1 protease and anti-cancer agent are administered in the same or separate pharmaceutically acceptable carriers. 
     
     
         30 . The method of  claim 1 , wherein the MT-SP1 protease is formulated as a liquid, tablet, buccal tablets, troches, capsules, elixirs, suspensions, syrups or wafers. 
     
     
         31 . The method of  claim 1 , wherein the MT-SP1 protease is administered orally or by subcutaneous, intramuscular or intravenous injection.