Imaging the activity of extracellular protease in cells using mutant anthrax toxin protective antigens that are cleaved by specific extracellular proteases
First Claim
1. A method for imaging the activity of a specific extracellular protease expressed by a cell, the method comprising the steps of:
- (a) contacting a cell with a mutant anthrax protective antigen (μ
PrAg), under conditions where the μ
PrAg binds to a cell surface receptor expressed by the cell and is cleaved by a specific extracellular protease expressed by the cell, wherein the μ
PrAg comprises a domain for binding the cell surface receptor, and comprises a protease cleavage site that is cleaved by the specific extracellular protease and is in place of the furin cleavage site of the native anthrax protective antigen (PrAg);
(b) contacting the cell with a ligand linked to a detectable moiety, under conditions where the ligand specifically binds to the cleaved μ
PrAg of step (a), thereby, forming a ligand-μ
PrAg complex; and
(c) imaging the detectable moiety linked to the ligand bound in the ligand-μ
PrAg complex of step (b) and, thereby, generating an image of the detectable moiety, wherein the image of the detectable moiety is indicative of the activity of the specific extracellular protease.
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Abstract
This invention pertains to methods for imaging the activity of extracellular proteases in cells using the anthrax binary toxin-system to target cells expressing extracellular proteases with mutant anthrax toxin protective antigens (μPrAg) that bind to receptors on the cells and are cleaved by a specific extracellular protease expressed by the cells, and ligands that specifically bind to the cleaved μPrAg and are linked to a moiety that is detectable by an imaging procedure. The μPrAg proteins used in the methods comprise a protease cleavage site that is cleaved by a specific extracellular protease and is in place of the furin cleavage site of the native PrAg. The methods are useful for diagnosing and treating diseases and undesirable physiological conditions correlated with the activity of extracellular proteases, and for optimizing the therapeutic efficacy of drugs used to treat such diseases and conditions.
15 Citations
28 Claims
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1. A method for imaging the activity of a specific extracellular protease expressed by a cell, the method comprising the steps of:
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(a) contacting a cell with a mutant anthrax protective antigen (μ
PrAg), under conditions where the μ
PrAg binds to a cell surface receptor expressed by the cell and is cleaved by a specific extracellular protease expressed by the cell,wherein the μ
PrAg comprises a domain for binding the cell surface receptor, and comprises a protease cleavage site that is cleaved by the specific extracellular protease and is in place of the furin cleavage site of the native anthrax protective antigen (PrAg);
(b) contacting the cell with a ligand linked to a detectable moiety, under conditions where the ligand specifically binds to the cleaved μ
PrAg of step (a), thereby, forming a ligand-μ
PrAg complex; and
(c) imaging the detectable moiety linked to the ligand bound in the ligand-μ
PrAg complex of step (b) and, thereby, generating an image of the detectable moiety,wherein the image of the detectable moiety is indicative of the activity of the specific extracellular protease. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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28. A method for assaying for an inhibitor of an extracellular protease expressed by a cell, the method comprising the steps of:
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(a) contacting a cell with a potential inhibitory compound and a mutant anthrax protective antigen (μ
PrAg), under conditions where the μ
PrAg binds to a cell surface receptor expressed by the cell and is cleaved by a specific extracellular protease expressed by the cell,wherein the μ
PrAg comprises a domain for binding the cell surface receptor, and comprises a protease cleavage site that is cleaved by the specific extracellular protease and is in place of the furin cleavage site of the native anthrax protective antigen (PrAg);
(b) contacting the cell with a ligand linked to a detectable moiety, under conditions where the ligand specifically binds to the cleaved μ
PrAg of step (a), thereby, forming a ligand-μ
PrAg complex; and
(c) imaging the detectable moiety linked to the ligand bound in the ligand-μ
PrAg complex of step (b) and, thereby, generating an image of the detectable moiety,wherein the image of the detectable moiety is indicative of the activity of the specific extracellular protease, thereby identifying inhibitors of the extracellular protease.
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Specification