Methods for determining dpp3 and therapeutic methods

Abstract

The present invention is directed to methods for determining active DPP3 in a bodily fluid sample, an assay or kit for determining active DPP3 in a bodily fluid sample, a method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes and methods of treating or preventing said disease.

1 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes comprising:
 determining the amount of total DPP3 and/or determining the amount of active DPP3 in a sample of bodily fluid of said subject,   comparing said determined amount of total DPP3 or active DPP3 to a predetermined threshold,   wherein said subject is diagnosed as having a disease or condition accompanied by or related to necrotic processes if said determined amount is above said predetermined threshold.   
     
     
         2 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to  claim 1  wherein the amount of total or active DPP3 is determined in the unit of concentration. 
     
     
         3 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to  claim 1  or  2 , wherein said sample is selected from the group comprising whole blood, serum and plasma. 
     
     
         4 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to any of  claims 1 - 3 , wherein said disease is selected from the group comprising heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burn injuries, traumatic injuries, severe infection (microbial, viral (e.g. AIDS), parasitic (e.g. Malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. seizures, neurodegenerative diseases), autoimmune diseases, vascular diseases (e.g. Kawasaki syndrome) and hypotension. 
     
     
         5 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to any of  claims 1 - 4 , wherein the amount of total DPP3 and/or the amount of active DPP3 is determined in a bodily fluid sample of said subject and comprises the steps:
 Contacting said sample with a capture-binder that binds specifically to full-length DPP3,   Separating DPP3 bound to said capture binder,   Adding substrate of DPP3 to said separated DPP3,   Quantifying of said active DPP3 by measuring and quantifying the conversion of a substrate of DPP3.   
     
     
         6 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to  claim 5 , wherein said capture-binder may be selected from the group of antibody, antibody fragment or non-IgG scaffold. 
     
     
         7 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to  claim 5  or  6 , wherein said capture-binder is an antibody. 
     
     
         8 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to any of  claims 5 - 7 , wherein said capture binder is immobilized on a surface. 
     
     
         9 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to any of  claims 5 - 8 , wherein said separation step is a washing step that removes ingredients of the sample that are not bound to said capture-binder from the captured DPP3. 
     
     
         10 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to any of  claims 5 - 9 , wherein DPP3 substrate conversion is detected by a method selected from the group comprising: fluorescence of fluorogenic substrates (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of chromogenic substrates, luminescence of substrates coupled to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC/FPLC (reversed phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (immobilized, active DPP3) or western blot (cleavage products). 
     
     
         11 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to any of  claims 5 - 10 , wherein said substrate may be selected from the group comprising: angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or di-peptide coupled to a fluorophore, a chromophore or aminoluciferin wherein the di-peptide is Arg-Arg. 
     
     
         12 . Method for diagnosing a disease or condition in a subject accompanied by or related to necrotic processes according to any of  claims 5 - 11 , wherein said substrate may be selected from the group comprising: a di-peptide coupled to a fluorophore, a chromophore or aminoluciferin wherein the di-peptide is Arg-Arg. 
     
     
         13 . Method for monitoring a disease or condition in a subject accompanied by or related to necrotic processes wherein the method of diagnosing according to any of  claims 1  to  12  is conducted at least twice. 
     
     
         14 . Inhibitor of the activity of DPP3 for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes. 
     
     
         15 . Inhibitor of the activity of DPP3 for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes according to  claim 14  wherein said inhibitor is selected from the group comprising an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold. 
     
     
         16 . Inhibitor of the activity of DPP3 for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes according to  claim 14  or  15  wherein said disease is selected from the group comprising heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burn injuries, traumatic injuries, severe infection (microbial, viral (e.g. AIDS), parasitic (e.g. Malaria)), SIRS or sepsis, cancer, acute kidney injury (AKI), CNS disorders (e.g. seizures, neurodegenerative diseases), autoimmune diseases, vascular diseases (e.g. Kawasaki syndrome) and hypotension. 
     
     
         17 . Inhibitor of the activity of DPP3 for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes according to any of  claims 14  to  16  wherein said inhibitor is an antibody that is mono-binding or at least two-binding. 
     
     
         18 . Inhibitor of the activity of DPP3 for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes according to any of  claims 14  to  17  wherein said inhibitor is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to SEQ ID No. 1, in particular that binds to SEQ ID No. 2. 
     
     
         19 . Inhibitor of the activity of DPP3 for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes according to according to any of  claims 14  to  18  wherein said inhibitor is an antibody or fragment or scaffold that exhibits a minimum binding affinity to DPP3 of equal or less than 10 −7  M. 
     
     
         20 . Inhibitor of the activity of DPP3 for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes according to any  claims 14  to  19  wherein said inhibitor is an antibody or fragment or scaffold that is monospecific. 
     
     
         21 . Inhibitor of the activity of DPP3 for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes according to any  claims 14  to  20  wherein said inhibitor is an antibody or fragment or scaffold binds to full-length DPP3 and inhibits activity of DPP3 of at least 10%, or at least 50%, more preferred at least 60%, even more preferred more than 70%, even more preferred more than 80%, even more preferred more than 90%, even more preferred more than 95%. 
     
     
         22 . Inhibitor of the activity of DPP3 for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes according to any  claims 14  to  21  wherein said inhibitor is selective and/or specific to DPP3 and not cross cell membranes and/or the blood brain barrier. 
     
     
         23 . Inhibitor of the activity of DPP3 for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes according to any  claims 14  to  22  wherein said subject has an amount of total DPP3 and/or an amount of active DPP3 in a sample of bodily fluid of said subject that is above a predetermined threshold. 
     
     
         24 . Pharmaceutical composition comprising an inhibitor of the activity of DPP3 according to any of the  claims 14  to  23  for use in prevention or therapy of a disease or condition in a subject accompanied by or related to necrotic processes. 
     
     
         25 . Use of an inhibitor of the activity of DPP3 according to any of the  claims 14  to  23  in a method of extracorporeal removal of DPP3 from plasma comprising apheresis and affinity chromatography. 
     
     
         26 . Method for determining active DPP3 in a bodily fluid sample of a subject comprising the steps:
 Contacting said sample with a capture-binder that binds specifically to full-length DPP3,   Separating DPP3 bound to said capture binder,   Adding substrate of DPP3 to said separated DPP3,   Quantifying of said active DPP3 by measuring and quantifying the conversion of a substrate of DPP3.   
     
     
         27 . Method for determining active DPP3 in a bodily fluid sample of a subject according to  claim 26  wherein said capture-binder may be selected from the group of antibody, antibody fragment or non-IgG scaffold. 
     
     
         28 . Method for determining active DPP3 in a bodily fluid sample of a subject according to  claim 26  or  27  wherein said capture-binder is an antibody. 
     
     
         29 . Method for determining active DPP3 in a bodily fluid sample of a subject according to any of  claims 26  to  28  wherein said capture binder is immobilized on a surface. 
     
     
         30 . Method for determining active DPP3 in a bodily fluid sample of a subject according to any of  claims 26  to  29  wherein said separation step is a washing step that removes ingredients of the sample that are not bound to said capture-binder from the captured DPP3. 
     
     
         31 . Method for determining active DPP3 in a bodily fluid sample of a subject according to any of  claims 26  to  30  wherein DPP3 substrate conversion is detected by a method selected from the group comprising: fluorescence of fluorogenic substrates (e.g. Arg-Arg-βNA, Arg-Arg-AMC), color change of chromogenic substrates, luminescence of substrates coupled to aminoluciferin (Promega Protease-Glo™ Assay), mass spectrometry, HPLC/FPLC (reversed phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining (immobilized, active DPP3) or western blot (cleavage products). 
     
     
         32 . Method for determining active DPP3 in a bodily fluid sample of a subject according to any of  claims 26  to  31 , wherein said substrate may be selected from the group comprising: A di-peptide coupled to a fluorophore, a chromophore or aminoluciferin wherein the di-peptide is Arg-Arg. 
     
     
         33 . Method for determining active DPP3 in a bodily fluid sample of a subject according to any of  claims 26  to  32 , wherein said sample is a blood sample selected from the group comprising whole blood, serum and plasma. 
     
     
         34 . Assay or kit for determining active DPP3 in a bodily fluid sample of a subject comprising:
 A capture-binder that binds specifically to full-length DPP3,   A substrate of DPP3.   
     
     
         35 . Assay or kit for determining active DPP3 in a bodily fluid sample of a subject according to  claim 34 , wherein said capture-binder may be selected from the group of antibody, antibody fragment or non-IgG scaffold. 
     
     
         36 . Assay or kit for determining active DPP3 in a bodily fluid sample of a subject according to  claim 34  or  35 , wherein said capture-binder inhibits less than 50% DPP3 activity in a liquid phase assay, preferably less than 40%, more preferably less than 30%. 
     
     
         37 . Assay or kit for determining active DPP3 in a bodily fluid sample of a subject according to any of  claims 34  to  36 , wherein the binding region of DPP3 for the capture-binder is not within the region of amino acids 316-669 of SEQ ID No. 1. 
     
     
         38 . Assay or kit for determining active DPP3 in a bodily fluid sample of a subject according to any of  claims 34  to  37  wherein said binder is an antibody. 
     
     
         39 . Assay or kit for determining active DPP3 in a bodily fluid sample of a subject according to any of  claims 34  to  38  wherein said capture-binder is immobilized on a surface. 
     
     
         40 . Assay or kit for determining active DPP3 in a bodily fluid sample of a subject according to any of  claims 34  to  39  wherein said substrate may be selected from the group comprising: angiotensin II, III and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or a di-peptide coupled to a fluorophore, a chromophore or aminoluciferin wherein the di-peptide is Arg-Arg. 
     
     
         41 . Use of a method for determining active DPP3 in a bodily fluid sample of a subject according to any of  claims 26 - 33  or use of an assay or kit according to any of  claims 34  to  40  in a method of for diagnosing or monitoring a disease or condition in a subject accompanied by or related to necrotic processes according to any of  claims 1 - 13 .