Thrombospondin Fragments and Uses Thereof In Clinical Assays for Cancer and Generation of Antibodies and Other Binding Agents

Abstract

The invention relates to thrombospondin fragments found in plasma, their use or use of portions thereof in diagnostic methods, as method calibrators, method indicators, and as immunogens, and as analytes for methods with substantial clinical utility; and their detection in plasma or other bodily fluids for purpose of diagnostic methods, especially for cancer.

1 - 242 . (canceled) 
     
     
         243 . A method to detect the presence and/or clinical course of a neoplastic disease in an individual, wherein the method comprises the steps of:
 (1) measuring a first individual's plasma level of a thrombospondin fragment or fragments, the plasma level of said fragment or fragments in the first individual being the first individual's plasma fragment level;   (2) measuring, in a second individual, the plasma level of the same thrombospondin fragment or fragments measured for the first individual, said second individual considered to not have neoplastic disease, the plasma level of said fragment or fragments in the second individual being the second individual's plasma fragment level; and   (3) performing a biopsy on said first individual; wherein said fragment or fragments being at least 6 continuous amino acyl residues in length but of a molecular weight less than 140 kDa; wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction.   
     
     
         244 . A method to detect the presence and/or clinical course of a neoplastic disease in an individual, wherein the method comprises the steps of:
 (1) measuring a first individual's plasma level of a thrombospondin fragment or fragments, the plasma level of said fragment or fragments in the first individual being the first individual's plasma fragment level;   (2) measuring, in a second individual, the plasma level of the same thrombospondin fragment or fragments measured for the first individual, said second individual considered to not have neoplastic disease, the plasma level of said fragment or fragments in the second individual being the second individual's plasma fragment level;   (3) performing a biopsy on said first individual; wherein said fragment or fragments either start between amino acyl residues 1-165 and V-263, inclusive, and end between amino acyl residues R-792 and Y-982, inclusive, or is a portion of the range 1-165 to Y-982, said portion being at least 150 amino acyl residues in size and wherein 1-165, V-263, R-792 and Y-982 refer to residues 183, 281, 810, and 1000, respectively of SEQ ID NO:38.   
     
     
         245 . A method to detect the presence and/or clinical course of a neoplastic disease in an individual, wherein the method comprises the steps of:
 (1) measuring a first individual's plasma level of a thrombospondin fragment or fragments, the plasma level of said fragment or fragments in the first individual being the first individual's plasma fragment level;   (2) measuring, in a second individual, the plasma level of the same thrombospondin fragment or fragments measured for the first individual, said second individual considered to not have neoplastic disease, the plasma level of said fragment or fragments in the second individual being the second individual's plasma fragment level;   (3) performing a biopsy on said first individual; wherein the molecular weight of said fragment or any of said fragments less than 140 kDa, the molecular weight of said fragment or fragments being at least 20 kDa, wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction, and wherein the fragment or each of said fragments comprises a portion of thromobospondin selected from the group consisting of   a collagen type V binding domain, and   a domain or a part thereof within the protease-resistant core of thrombospondin, said domain being selected from the group consisting of a domain of inter-chain disulfide bonds, an oligomerization domain, a procollagen-like domain, a type 1 repeat, a type 2 repeat, and a type 3 repeat.   
     
     
         246 . A method to detect the presence and/or clinical course of a neoplastic disease in an individual, wherein the method comprises the steps of:
 (1) measuring a first individual's plasma level of a thrombospondin fragment or fragments, the plasma level of said fragment or fragments in the first individual being the first individual's plasma fragment level;   (2) measuring, in a second individual, the plasma level of the same thrombospondin fragment or fragments measured for the first individual, said second individual considered to not have neoplastic disease, the plasma level of said fragment or fragments in the second individual being the second individual's plasma fragment level;   (3) performing a biopsy on said first individual; wherein said fragment or fragments being within a molecular weight range selected from the group consisting of 80 to 140 kDa, 40 to 60 kDa, and 20 to 35 kDa such that the greater the plasma level of said thrombospondin fragment or fragments, the more likely that the diagnosis will be that a neoplastic disease is present in said individual, wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction.   
     
     
         247 . A method to detect the presence and/or clinical course of a neoplastic disease in an individual, wherein the method comprises the steps of:
 (1)) measuring a first individual's plasma level of a thrombospondin fragment or fragments, the plasma level of said fragment or fragments in the first individual being the first individual's plasma fragment level;   (2) measuring, in a second individual, the plasma level of the same thrombospondin fragment or fragments measured for the first individual, said second individual considered to not have neoplastic disease, the plasma level of said fragment or fragments in the second individual being the second individual's plasma fragment level; and   (3) performing a biopsy on said first individual; wherein said plasma level is measured using a binding agent that is capable of binding to said fragment or fragments provided that said binding agent does not bind a region selected from the group consisting of the fibrinogen-binding region in the amino-terminal domain of thrombospondin, and a heparin-binding sequence in the amino-terminal domain of thrombospondin; wherein the molecular weight of each of the fragment or fragments is at least 20 kDa but not more than 140 kDa, wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction.   
     
     
         248 . A method to detect the presence and/or clinical course of a neoplastic disease in an individual, wherein the method comprises the steps of:
 (1) measuring a first individual's plasma level of a thrombospondin fragment or fragments, the plasma level of said fragment or fragments in the first individual being the first individual's plasma fragment level;   (2) measuring, in a second individual, the plasma level of the same thrombospondin fragment or fragments measured for the first individual, said second individual considered to not have neoplastic disease, the plasma level of said fragment or fragments in the second individual being the second individual's plasma fragment level;   (3) performing a biopsy on said first individual; wherein said method comprises the use of a binding agent that binds to an epitope within a plasma fragment in the molecular weight range selected from the group consisting of 80 to 140 kDa, 40 to 60 kDa, and 20 to 35 kDa, wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction.   
     
     
         249 . A method to detect the presence and/or clinical course of a neoplastic disease in an individual, wherein the method comprises the steps of:
 1) utilizing a first binding agent to obtain a quantification, for the plasma of a first individual, of a total, thrombospondin plus either the thrombospondin fragment or fragments;   2) utilizing a second binding agent to obtain a quantification, for the plasma of said first individual, of thrombospondin only; wherein the first binding agent binds to an epitope shared by thrombospondin and the thrombospondin fragment or fragments, and wherein the second binding agent binds to an epitope present in thrombospondin but not present in the fragment or fragments.