Methods And Compositions For Noninvasive Detection Of Organ Transplant Rejection

Abstract

An activity-based nanosensor composition for detecting protease activity comprising a cleavable detectable substrate and methods of use are disclosed.

1 . An activity-based nanosensor comprising:
 a scaffold;   a linker coupled to the scaffold;   a peptide substrate coupled to the linker; and   a detectable reporter coupled to the peptide substrate.   
     
     
         2 . The nanosensor of  claim 1 , wherein the scaffold is selected from the group consisting of a nanoparticle, a nanostructure, a microparticle, a protein, a sugar, a nucleic acid-based scaffold, an imaging contrast agent, and a polymer. 
     
     
         3 . The nanosensor of  claim 1 , wherein the scaffold is configured to prevent renal clearance of the peptide substrate. 
     
     
         4 . The nanosensor of  claim 1  wherein the scaffold comprises a nanoparticle having a diameter from about 3 nm to about 2 microns. 
     
     
         5 . The nanosensor of  claim 1 , wherein the scaffold comprises an iron oxide nanoparticle. 
     
     
         6 . (canceled) 
     
     
         7 . The nanosensor of  claim 1 , wherein the linker comprises a cysteine residue. 
     
     
         8 . The nanosensor of  claim 1 , wherein the peptide substrate comprises a target protease cleavage sequence. 
     
     
         9 . The nanosensor of  claim 1 , wherein the peptide substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 2-136. 
     
     
         10 . The nanosensor of  claim 1 , wherein the peptide substrate comprises a target protease selected from the group consisting of T cell proteases, complement proteases, fibrosis proteases, and inflammation-related proteases. 
     
     
         11 . The nanosensor of  claim 1 , wherein the peptide substrate comprises a target protease selected from the group consisting of Granzyme B, Granzyme A, MALT1, Caspase 8, Calpain 2, Cathepsin X, Cls, Clr, MASP2, Factor I, Factor D, ADAMTS1, MMP2, MMP9, elastase, cathepsin G, PR-3, thrombin, kallikrein 1, kallikrein 6, tryptase, and chymase. 
     
     
         12 . The nanosensor of  claim 1 , wherein the detectable reporter is selected from the group consisting of a fluorophore, a luminescent reporter, a ligand encoded reporter, a mass spectrometry tag, a contrast agent for imaging, a PET-detectable domain, and a nucleic acid tag. 
     
     
         13 .- 16 . (canceled) 
     
     
         17 . The nanosensor of  claim 1 , wherein the linker is coupled to the scaffold via a first spacer;
 wherein the peptide substrate is coupled to the linker via a second spacer;   wherein the detectable reporter is coupled to the substrate via a third spacer; and   wherein at least one of the first, second, and third spacers comprise a GGS amino acid sequence.   
     
     
         18 . A method of diagnosing tissue rejection comprising:
 administering a nanosensor to a subject, the nanosensor comprising:   a scaffold;   a linker coupled to the scaffold;   a target protease coupled to the linker; and   a detectable reporter coupled to the target protease;   obtaining a sample of a bodily fluid from the subject;   detecting a level of the detectable reporter in the sample;   determining an activity of the target protease based at least in part on the level of the detectable reporter in the sample;   comparing the activity of the target protease in the sample to a reference activity of the target protease; and   diagnosing tissue rejection in the subject if the activity of the target protease in the sample is greater than the reference activity of the target protease.   
     
     
         19 . The method of  claim 18 , wherein administering the nanosensor comprises intravenously administering the nanosensor to the subject. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 18 , wherein the bodily fluid is urine. 
     
     
         22 . The method of  claim 18 , wherein the method is for diagnosing acute organ rejection in a transplant recipient subject; and
 wherein the reference activity of the target protease is determined by one or both of the activity of the target protease in a sample taken from the subject before receiving the transplanted tissue and the activity of the target protease in a sample taken from a control subject.   
     
     
         23 . The method of  18 , wherein the method is for diagnosing acute organ rejection in a transplant recipient subject and further comprises treating the acute organ rejection by administering a therapeutic agent configured to treat acute organ rejection when acute organ rejection is diagnosed. 
     
     
         24 . The method of  claim 18 , wherein the method is for diagnosing acute organ rejection in a transplant recipient subject;
 wherein the scaffold is selected from the group consisting of a nanoparticle, a nanostructure, a microparticle, a protein, a sugar, a nucleic acid-based scaffold, an imaging contrast agent, and a polymer;   wherein the target protease is selected from the group consisting of T cell proteases, complement proteases, fibrosis proteases, and inflammation-related proteases; and   wherein the detectable reporter is selected from the group consisting of a fluorophore, a luminescent reporter, a ligand encoded reporter, a mass spectrometry tag, a contrast agent for imaging, a PET-detectable domain, and a nucleic acid tag.   
     
     
         25 . An activity-based nanosensor comprising:
 a scaffold selected from the group consisting of a nanoparticle, a nanostructure, a microparticle, a protein, a sugar, a nucleic acid-based scaffold, an imaging contrast agent, and a polymer;   a linker coupled to the scaffold via a first spacer;   a target protease coupled to the linker via a second spacer, the target protease selected from the group consisting of Granzyme B, Granzyme A, MALT1, Caspase 8, Calpain 2, Cathepsin X, Cls, Clr, MASP2, Factor I, Factor D, ADAMTS1, MMP2, MMP9, elastase, cathepsin G, PR-3, thrombin, kallikrein 1, kallikrein 6, tryptase, and chymase; and   a detectable reporter coupled to the target protease via a third spacer, the detectable reporter selected from the group consisting of a fluorophore, a luminescent reporter, a ligand encoded reporter, a mass spectrometry tag, a contrast agent for imaging, a PET-detectable domain, and a nucleic acid tag;   wherein the scaffold is configured to prevent renal clearance of the target protease; and   wherein at least one of the first, second, and third spacers comprises a GGS amino acid sequence.   
     
     
         26 . The nanosensor of  claim 25 , wherein the linker comprises a thiol group.