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 .- 162 . (canceled) 
     
     
         163 . A method using a combination diagnostic test 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) utilizing the results of steps (1) and (2) in a diagnosis as to whether the first individual has a neoplastic disease such that the greater the extent to which the first individual's plasma fragment level exceeds the second individual's plasma fragment level the more likely that the diagnosis will be that a neoplastic disease is present in the first individual; and   (4) performing a test selected from the group consisting of an imaging test, a radiographic test, a magnetic resonance imaging test, and a nuclear medicine test on said first individual; wherein said fragment or fragments in both the first and second individuals are at least 6 continuous amino acyl residues in length but of a molecular weight less than 110 kDa; wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction.   
     
     
         164 . A method using a combination diagnostic test 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) utilizing the results of steps (1) and (2) in a diagnosis as to whether the first individual has a neoplastic disease such that the greater the extent to which the first individual's plasma fragment level exceeds the second individual's plasma fragment level the more likely that the diagnosis will be that a neoplastic disease is present in the first individual; and   (4) performing a test selected from the group consisting of an imaging test, a radiographic test, a magnetic resonance imaging test, and a nuclear medicine test on said first individual; wherein said fragment or fragments in both the first and second individuals either start between amino acyl residues I-165 and V-263, inclusive, and end between amino acyl residues R-792 and Y-982, inclusive, or is a portion of the range I-165 to Y-982, said portion being at least 150 amino acyl residues in size and wherein I-165, V-263, R-792 and Y-982 refer to residues 183, 281, 810, and 1000, respectively of SEQ ID NO:38.   
     
     
         165 . A method using a combination diagnostic test 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) utilizing the results of steps (1) and (2) in a diagnosis as to whether the first individual has a neoplastic disease such that the greater the extent to which the first individual's plasma fragment level exceeds the second individual's plasma fragment level the more likely that the diagnosis will be that a neoplastic disease is present in the first individual; and   (4) performing a test selected from the group consisting of an imaging test, a radiographic test, a magnetic resonance imaging test, and a nuclear medicine test on said first individual; wherein the molecular weight of said fragment or any of said fragments in both the first and second individuals are not more than 110 kDa, the molecular weight of said fragment or fragments in both the first and second individuals are at least 20 kDa, wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction, and wherein, in both the first and second individuals, 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.   
     
     
         166 . A method using a combination diagnostic test 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) utilizing the results of steps (1) and (2) in a diagnosis as to whether the first individual has a neoplastic disease such that the greater the extent to which the first individual's plasma fragment level exceeds the second individual's plasma fragment level the more likely that the diagnosis will be that a neoplastic disease is present in the first individual; and   (4) performing a test selected from the group consisting of an imaging test, a radiographic test, a magnetic resonance imaging test, and a nuclear medicine test on said first individual; wherein said fragment or fragments in both the first and second individuals are within a molecular weight range selected from the group consisting of 80 to 110 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.   
     
     
         167 . A method using a combination diagnostic test 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) utilizing the results of steps (1) and (2) in a diagnosis as to whether the first individual has a neoplastic disease such that the greater the extent to which the first individual's plasma fragment level exceeds the second individual's plasma fragment level the more likely that the diagnosis will be that a neoplastic disease is present in the first individual; and   (4) performing a test selected from the group consisting of an imaging test, a radiographic test, a magnetic resonance imaging test, and a nuclear medicine test on said first individual; wherein said plasma level is measured using a binding agent that is capable of binding to said fragment or fragments in both the first and second individuals 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, in both the first and second individuals, the molecular weight of each of the fragment or fragments is at least 20 kDa but not more than 110 kDa, wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction.   
     
     
         168 . A method using a combination diagnostic test 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) utilizing the results of steps (1) and (2) in a diagnosis as to whether the first individual has a neoplastic disease such that the greater the extent to which the first individual's plasma fragment level exceeds the second individual's plasma fragment level the more likely that the diagnosis will be that a neoplastic disease is present in the first individual; and   (4) performing a test selected from the group consisting of an imaging test, a radiographic test, a magnetic resonance imaging test, and a nuclear medicine test 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 110 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.   
     
     
         169 . A method using a combination diagnostic test 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; and   3) utilizing the difference between the quantitations obtained in steps (1) and (2) as a quantitation, for the plasma of said first individual, of the amount of thrombospondin fragment or fragments;   4) utilizing the first binding agent to obtain a quantification, for the plasma of a second individual, of a total, thrombospondin plus either the thrombospondin fragment or fragments, said second individual considered not to have neoplastic disease;   5) utilizing the second binding agent to obtain a quantification, for the plasma of said second individual, of thrombospondin only;   6) utilizing the difference between the quantifications obtained in steps (4) and (5) as a quantitation, for the plasma of said second individual, of the amount of thrombospondin fragment or fragments;   7) utilizing the results of steps (3) and (6) in a diagnosis as to whether the first individual has a neoplastic disease such that the greater the extent to which the first individual's plasma level of said thrombospondin fragment or fragments exceeds the second individual's plasma level of said thrombospondin fragment or fragments, the more likely that the diagnosis will be that a neoplastic disease is present in said first individual; and   8) performing a test selected from the group consisting of an imaging test, a radiographic test, a magnetic resonance imaging test, and a nuclear medicine test on said first individual; 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.