Compositions and methods for detecting proteolytic activity

Abstract

The invention provides compositions and methods for non-invasive imaging of enzyme (e.g., protease) activity in cells, tissues and organs and entire bodies in vitro, in vivo and in situ. The invention provides a chimeric polypeptide having a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and at least one silencing moiety, and a protease cleavage motif positioned between the first and second domains. The imaging can be by computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), or bioluminescence imaging (BLI).

What is claimed is:  
     
         1 . A chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains.  
     
     
         2 . The chimeric polypeptide of  claim 1 , wherein the chemiluminescent polypeptide comprises luciferase.  
     
     
         3 . The chimeric polypeptide of  claim 1 , wherein the bioluminescent or chemiluminescent polypeptide comprises an aequorin, an obelin, a ninemiopsin or a berovin.  
     
     
         4 . The chimeric polypeptide of  claim 1 , wherein the bioluminescent or chemiluminescent polypeptide comprises a green fluorescent protein, an alpha-galactosidase or a chloramphenicol acetyltransferase.  
     
     
         5 . The chimeric polypeptide of  claim 1 , wherein the heterologous kinase comprises a herpes simplex virus-1 thymidine kinase (HSV-1 TK).  
     
     
         6 . The chimeric polypeptide of  claim 1 , wherein the silencing moiety comprises a ligand binding domain.  
     
     
         7 . The chimeric polypeptide of  claim 6 , wherein the ligand binding domain comprises a steroid hormone receptor ligand binding domain.  
     
     
         8 . The chimeric polypeptide of  claim 6 , wherein the steroid hormone receptor ligand binding domain comprises an estrogen receptor ligand binding domain.  
     
     
         9 . The chimeric polypeptide of  claim 6 , wherein the hormone receptor is selected from the group consisting of glucocorticoid receptor, progesterone receptor, androgen receptor, mineralcorticoid receptor, thyroid hormone receptor, retinoic acid receptor and RXR receptor.  
     
     
         10 . The chimeric polypeptide of  claim 1 , wherein the silencing moiety comprises a sequence of a transcription factor.  
     
     
         11 . The chimeric polypeptide of  claim 10 , wherein the transcription factor comprises an estrogen receptor ligand binding domain polypeptide as set forth in SEQ ID NO:1.  
     
     
         12 . The chimeric polypeptide of  claim 1 , wherein the endogenous protease cleavage motif is specifically cleaved by an endogenous cellular protease.  
     
     
         13 . The chimeric polypeptide of  claim 1 , wherein the activity of the endogenous cellular protease is increased or decreased during apoptosis.  
     
     
         14 . The chimeric polypeptide of  claim 1 , wherein the endogenous cellular protease cleavage motif comprises a caspase recognition motif.  
     
     
         15 . The chimeric polypeptide of  claim 14 , wherein the caspase recognition motif comprises an amino acid sequence selected from group consisting of DEVD (SEQ ID NO:1), IETD (SEQ ID NO:2) and LEHD (SEQ ID NO:3).  
     
     
         16 . The chimeric polypeptide of  claim 12 , wherein the endogenous cellular protease comprises caspase 3, caspase 6, caspase 7, procaspase 8, caspase 8, caspase 9, caspase 10, matrix metalloproteinase (MMP) or gamma-secretase.  
     
     
         17 . The chimeric polypeptide of  claim 1 , wherein the endogenous protease cleavage recognition motif comprises a PACE/furin cleavage recognition motif.  
     
     
         18 . The chimeric polypeptide of  claim 1 , wherein the endogenous protease cleavage motif comprises a metalloprotease cleavage recognition motif, a serine protease cleavage recognition motif, or a gamma-secretase cleavage recognition motif.  
     
     
         19 . The chimeric polypeptide of  claim 1 , wherein the endogenous protease recognition motif further comprises at least one glycine residue flanking the carboxy or amino terminal amino acid of the motif.  
     
     
         20 . The chimeric polypeptide of  claim 1 , further comprising a cellular transport sequence.  
     
     
         21 . The chimeric polypeptide of  claim 20 , wherein the cellular transport sequence comprises a herpes simplex virus (HSV) VP-22 sequence.  
     
     
         22 . The chimeric polypeptide of  claim 20 , wherein the cellular transport sequence is located between the endogenous cleavage protease motif and the first domain or between the endogenous protease cleavage motif and the second domain.  
     
     
         23 . The chimeric polypeptide of  claim 1 , wherein the polypeptide comprises at least about 30 amino acids, at least about 50 amino acids, at least about 100 amino acids or at least about 200 amino acids.  
     
     
         24 . The chimeric polypeptide of  claim 1 , wherein the polypeptide is a recombinant fusion protein.  
     
     
         25 . The chimeric polypeptide of  claim 1 , wherein the bioluminescent or cbemiluminescent polypeptide, or the heterologous kinase, can directly or by enzymatic reaction with a reagent generate a molecule that can be imaged by computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), bioluminescence image (BLI) or equivalent.  
     
     
         26 . A chimeric polypeptide comprising a bioluminescent or chemiluminescent polypeptide domain flanked on both sides by a silencing moiety and an endogenous protease cleavage motif positioned between the bioluminescent or chemiluminescent polypeptide domain and each silencing moiety.  
     
     
         27 . The chimeric polypeptide of  claim 26 , wherein the chemiluminescent polypeptide is luciferase and the silencing domain is an estrogen receptor ligand binding domain.  
     
     
         28 . A chimeric polypeptide comprising a luciferase flanked on both sides by an estrogen receptor ligand binding domain and an endogenous protease cleavage motif positioned between the luciferase and the estrogen receptor ligand binding domain.  
     
     
         29 . A nucleic acid encoding a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains.  
     
     
         30 . An expression cassette comprising a nucleic acid encoding a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains.  
     
     
         31 . A transformed host cell comprising a nucleic acid encoding a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains.  
     
     
         32 . A non-human transgenic animal that expresses a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains.  
     
     
         33 . A non-human transgenic animal comprising a nucleic acid encoding a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains.  
     
     
         34 . The non-human transgenic animal of  claim 33 , wherein the animal is a mouse or a rat.  
     
     
         35 . A kit comprising a chimeric polypeptide or a nucleic acid and instructions for use 
 wherein the chimeric polypeptide comprises a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains    wherein the nucleic acid encodes a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains.    
     
     
         36 . The kit of  claim 35 , further comprising a substrate for the bioluminescent or chemiluminescent polypeptide or the heterologous kinase.  
     
     
         37 . The kit of  claim 35 , wherein the instructions are for measuring apoptosis in a living subject.  
     
     
         38 . A pharmaceutical formulation comprising a chimeric polypeptide and a pharmaceutically acceptable excipient suitable, 
 wherein the chimeric polypeptide comprises a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains.    
     
     
         39 . The pharmaceutical composition of  claim 38 , further comprising a substrate for the bioluminescent or the chemiluminescent polypeptide or the heterologous kinase.  
     
     
         40 . The pharmaceutical composition of  claim 39 , wherein the chemiluminescent polypeptide is luciferase and the substrate is luciferin.  
     
     
         41 . The pharmaceutical composition of  claim 39 , wherein the heterologous kinase is 8-[18F] fluoroganciclovir (FGCV) and the substrate is a herpes simplex virus-1 thymidine kinase (HSV-1 TK).  
     
     
         42 . The pharmaceutical composition of  claim 38 , further comprising a liposome.  
     
     
         43 . A method for detecting a protease activity in a sample comprising 
 (a) contacting a polypeptide as set forth in  claim 1  with a sample containing or suspected of containing a protease under conditions allowing cleavage of the endogenous protease cleavage motif, wherein the protease is capable of cleaving the endogenous protease cleavage motif; and    (b) detecting the amount of bioluminescent or a chemiluminescent signal in the sample, thereby detecting a protease activity in the sample.    
     
     
         44 . The method of  claim 43 , wherein the contacting is in solution, in solid phase or is in a cell in vitro, in situ, or in vivo.  
     
     
         45 . The method of  claim 43 , wherein the endogenous protease specifically cleaves the endogenous protease cleavage motif.  
     
     
         46 . The method of  claim 45 , wherein the endogenous cellular protease comprises a caspase.  
     
     
         47 . The method of  claim 43 , wherein the endogenous cellular protease comprises a caspase 3, a caspase 6, a caspase 7, a procaspase 8, a caspase 8, a caspase 9, a caspase 10, a matrix metalloproteinase (MMP) or a gamma-secretase.  
     
     
         48 . The method of  claim 43 , further comprising providing a substrate for the bioluminescent or chemiluminescent polypeptide, or the heterologous kinase, and administering the substrate before, with or after administration of the bioluminescent or chemiluminescent polypeptide or the heterologous kinase.  
     
     
         49 . The method of  claim 48 , wherein the chemiluminescent polypeptide is luciferase and the substrate is luciferin.  
     
     
         50 . The method of  claim 48 , wherein the heterologous kinase is 8-[18F] fluoroganciclovir (FGCV) and the substrate is a herpes simplex virus-1 thymidine kinase (HSV-1 TK).  
     
     
         51 . A method for identifying the presence of a caspase in a sample comprising 
 (a) contacting a polypeptide with a sample under conditions allowing cleavage of the endogenous protease cleavage motif,    wherein the polypeptide comprises a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains, and, the endogenous protease cleavage motif is a cleavage motif specific for the caspase; and    (b) detecting the amount of bioluminescent or a chemiluminescent signal in the sample, thereby identifying the presence of the caspase.    
     
     
         52 . A method for detecting apoptosis in a cell comprising 
 (a) contacting a polypeptide with the cell under conditions allowing cleavage of the endogenous protease cleavage motif by a cellular enzyme,    wherein the polypeptide comprises a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains, and, the endogenous protease cleavage motif is a cleavage motif specific for an enzyme associated with apoptosis; and    (b) detecting the amount of bioluminescent or chemiluminescent signal in the cell, thereby identifying the presence and activity of the apoptosis-associated enzyme and detecting apoptosis.    
     
     
         53 . The method of  claim 52 , wherein the contacting is in a cell, a tissue, an organ or an entire body in vitro, in situ, or in vivo.  
     
     
         54 . The method of  claim 53 , wherein the cell, tissue or organ is undergoing aberrant proliferation or aberrant degeneration.  
     
     
         55 . The method of  claim 53 , wherein the aberrant proliferation comprises hyperproliferation.  
     
     
         56 . The method of  claim 53 , wherein the cell comprises a benign or a metastatic tumor.  
     
     
         57 . The method of  claim 53 , wherein the cell comprises a solid tumor.  
     
     
         58 . The method of  claim 53 , wherein the aberrant proliferation comprises deficient proliferation.  
     
     
         59 . The method of  claim 53 , wherein the aberrant degeneration comprises decreased cell death or increased cell death.  
     
     
         60 . The method of  claim 53 , wherein the amount of bioluminescent or chemiluminescent signal in the cell can be imaged by computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), bioluminescence image (BLI) or equivalent.  
     
     
         61 . A method for detecting changes in apoptosis in a cell comprising: 
 a) contacting a polypeptide with the cell under conditions allowing cleavage of the endogenous protease cleavage motif by a cellular enzyme,    wherein the polypeptide comprises a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains, and, the endogenous protease cleavage motif is a cleavage motif specific for an enzyme associated with apoptosis; and    b) detecting any change in an amount of bioluminescent or chemiluminescent signal in the cell, thereby identifying the presence and activity of the apoptosis-associated enzyme and detecting a change in cell apoptosis.    
     
     
         62 . The method of  claim 61 , wherein the apoptosis-associated enzyme comprises a caspase.  
     
     
         63 . The method of  claim 61 , wherein the contacting is in a cell, a tissue, an organ or an entire body in vitro, in situ, or in vivo.  
     
     
         64 . The method of  claim 63 , wherein the tissue comprises breast, brain, head or neck, eye, nasopharynx, lung, liver, pancreas, kidney, esophagus, stomach, small or large intestine, bladder, rectum, prostate, testicle, ovary, uterus, bone, muscle, skin or blood.  
     
     
         65 . The method of  claim 63 , wherein the cell comprises a subject undergoing a therapy that increases or decreases cell proliferation or cell death.  
     
     
         66 . The method of  claim 65 , wherein the subject has or is at risk of having a cell proliferative disorder or a cell degenerative disorder.  
     
     
         67 . The method of  claim 66 , wherein the cell proliferative disorder comprises cell hyperplasia.  
     
     
         68 . The method of  claim 67 , wherein the cell hyperplasia comprises a cancer.  
     
     
         69 . The method of  claim 68 , wherein the cancer is a lymphoma, a myeloma or a leukemia.  
     
     
         70 . The method of  claim 68 , wherein the cancer is a solid tumor.  
     
     
         71 . The method of  claim 68 , wherein the cancer is a metastatic tumor.  
     
     
         72 . The method of  claim 68 , wherein the cancer comprises a sarcoma or fibrosarcoma.  
     
     
         73 . The method of  claim 68 , wherein the cancer is a glioma or neuroblastoma.  
     
     
         74 . The method of  claim 66 , wherein the cell degenerative disorder is produced by a stroke, a heart attack or a partial or complete arterial obstruction.  
     
     
         75 . The method of  claim 66 , wherein the cell degenerative disorder afflicts neural tissue, muscular tissue, cardiac tissue or bone marrow cells.  
     
     
         76 . The method of  claim 66 , wherein the cell degenerative disorder is dementia, Alzheimer's disease, Parkinson's disease, ALS, Huntington's disease, MachadoJoseph disease, spino-cerebellar ataxias, Kennedy's disease, muscular dystrophy, multiple sclerosis, beta-thalasemia, sickle cell anemia, aplastic anemia, ischemia/reperfusion injury, rheumatoid arthritis or graft versus host disease.  
     
     
         77 . A method for monitoring the effectiveness of a therapy that modulates cell proliferation or cell survival in a subject comprising: 
 a) contacting a cell, a tissue or an organ in the subject, before therapy, with a polypeptide, under conditions allowing cleavage of the polypeptide by an endogenous cell protease associated with cell proliferation or cell survival,    wherein the polypeptide comprises a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains, and, the endogenous protease cleavage motif is a cleavage motif specific for an enzyme associated with cell proliferation or cell survival;    b) detecting the amount of bioluminescent or chemiluminescent signal in the cell, tissue or organ, thereby identifying the presence and activity of the cell proliferation- or cell survival-associated enzyme;    c) administering a therapy to the subject; and    d) detecting any change in the amount of bioluminescent or chemiluminescent signal in the cell, tissue or organ after the therapy,    wherein a change in the amount of bioluminescent or chemiluminescent signal after the therapy indicates the amount of cell proliferation or cell survival, thereby indicating the effectiveness of the therapy.    
     
     
         78 . The method of  claim 77 , further comprising re-contacting the cell, tissue or organ with the polypeptide as set forth in  claim 1  after administering the therapy to the subject.  
     
     
         79 . The method of  claim 77 , wherein effectiveness of the therapy is indicated by an increase in the bioluminescent or chemiluminescent signal after the therapy.  
     
     
         80 . The method of  claim 77 , wherein effectiveness of the therapy is indicated by a decrease in the bioluminescent or chemiluminescent signal after the therapy.  
     
     
         81 . The method of  claim 77 , wherein the therapy inhibits cell proliferation.  
     
     
         82 . The method of  claim 77 , wherein the therapy stimulates cell death.  
     
     
         83 . The method of  claim 77 , wherein the therapy stimulates cell proliferation.  
     
     
         84 . The method of  claim 77 , wherein the therapy inhibits cell death.  
     
     
         85 . The method of  claim 77 , wherein the therapy comprises anti-cancer therapy.  
     
     
         86 . The method of  claim 77 , wherein the therapy comprises chemotherapy or radiation therapy.  
     
     
         87 . The method of  claim 77 , wherein the therapy comprises a treatment for a cell degenerative disorder.  
     
     
         88 . The method of  claim 87 , wherein the cell degenerative disorder afflicts neural tissue, muscle tissue, cardiac tissue or bone marrow cells.  
     
     
         89 . The method of  claim 77 , wherein the therapy comprises a treatment for ischemia.  
     
     
         90 . The method of  claim 77 , wherein the therapy comprises a treatment for stroke, a heart attack or a partial or complete arterial obstruction.  
     
     
         91 . The method of  claim 77 , wherein the therapy comprises a treatment for dementia, Alzheimer's disease, Parkinson's disease, ALS, Huntington's disease, Machado-Joseph disease, spino-cerebellar ataxias, Kennedy's disease, muscular dystrophy, multiple sclerosis, beta-thalasemia, sickle cell anemia, aplastic anemia, ischemia/reperfusion injury, rheumatoid arthritis and graft versus host disease.  
     
     
         92 . A computer-implemented method for monitoring relative effectiveness of a therapy that modulates cell proliferation or cell survival in a subject comprising: 
 (a) providing an imaging device in operable association with a computer, wherein the imaging device is a computer assisted tomography (CAT) device, a magnetic resonance spectroscopy (MRS) device, a magnetic resonance imaging (MRI) device, a positron emission tomography (PET) device, a single-photon emission computed tomography (SPECT) device, a bioluminescence imaging (BLI) device or equivalent;    (b) taking an image of a defined area of the subject before or during therapy in which the area has been contacted with a polypeptide to image the amount of bioluminescent or chemiluminescent signal, wherein the endogenous protease cleavage motif is an endogenous cleavage motif specific for an enzyme associated with cell proliferation or cell survival, and the polypeptide comprises a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains;    (c) outputting the image data to the computer;    (d) administering a therapy to the subject and again imaging the defined area;    (e) comparing the image data obtained in step (b) with the image data obtained in step (d) with the computer to generate a differential histogram; and    (f) analyzing the differential histogram to quantitate any change in bioluminescent or chemiluminescent signal after therapy,    wherein a change in the amount or activity of the bioluminescent or cbemiluminescent signal after the therapy indicates a relative effectiveness of the therapy in modulating cell proliferation or cell survival.    
     
     
         93 . A method for identifying an agent that modulates an enzyme activity comprising: 
 (a) contacting a sample comprising the enzyme with a polypeptide in the presence and absence of a test agent, wherein the endogenous cleavage motif is an endogenous cleavage motif specific for the enzyme,    wherein the polypeptide comprises a chimeric polypeptide comprising a first domain comprising a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and a second domain comprising at least one silencing moiety, and an endogenous protease cleavage motif positioned between the first and second domains, and;    (b) measuring the amount of bioluminescent or chemiluminescent signal in the sample after adding the polypeptide and in the presence and absence of the test agent,    wherein an increase or decrease in the amount or activity of the bioluminescent or chemiluminescent signal in the presence of the test agent identifies the test agent as a modulator of the enzyme's activity.    
     
     
         94 . The method of claim  93 , wherein the contacting occurs in solution or in solid phase.  
     
     
         95 . The method of claim  93 , wherein the contacting occurs in a cell.  
     
     
         96 . The method of claim  93 , wherein the cell comprises a tissue, an organ or an entire body.  
     
     
         97 . The method of claim  95 , wherein the cell comprises a non-human transgenic animal.  
     
     
         98 . The method of claim  95 , wherein the non-human transgenic animal is a mouse or a rat.