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 - 164 . (canceled) 
     
     
         165 . A kit for the determination of the presence of, and/or the amount of, and/or the concentration of, a thrombospondin fragment or fragments in a material taken or gathered from an individual, said kit comprising a binding agent capable of binding a plasma thrombospondin fragment or plasma thrombospondin fragments and/or a portion of said plasma thrombospondin fragment or plasma thrombospondin fragments, each of said plasma fragment or plasma thrombospondin fragments being one that starts between amino acyl residues 1-165 and V-263, inclusive, and ends between amino acyl residues R-792 and Y-982, inclusive, each of said plasma thrombospondin fragment, fragments or portion being at least 20 kDa in size wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction, said kit further comprising a reference molecule, said reference molecule being selected from the group consisting of a thrombospondin fragment and a peptide derived from a thrombospondin fragment, said fragment or peptide corresponding to or within the region of thrombospondin extending from amino acyl residues I-165 to Y-982, said reference molecule capable of binding to the binding agent, 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. 
     
     
         166 . A kit for the determination of the presence of, and/or the amount of, and/or the concentration of, a thrombospondin fragment or fragments in a material taken or gathered from an individual, said kit comprising a binding agent capable of binding a plasma thrombospondin fragment or plasma thrombospondin fragments, each of said plasma thrombospondin fragment or plasma thrombospondin fragments having a molecular weight not exceeding 110 kDa, wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction, and wherein the plasma thrombospondin fragment or each of the plasma thrombospondin fragments comprises a region of thrombospondin, said region selected from the group of regions consisting of:
 a domain or a portion thereof within the protease-resistant core of thrombospondin, said domain being selected from the group consisting of a domain of inter-chain disulfide bonds, a procollagen-like domain, a type 1 repeat, a type 2 repeat, and a type 3 repeat; and   a collagen type V binding domain a or portion thereof,   and wherein the binding agent specifically binds to an epitope within said region, said kit further comprising a reference molecule, said reference molecule being selected from the group consisting of a thrombospondin fragment and a peptide derived from a thrombospondin fragment, said fragment or peptide corresponding to all or a portion of one or more regions selected from said group of regions, said reference molecule capable of binding to the binding agent.   
     
     
         167 . A kit of  claim 166  wherein the region of thrombospondin is a domain or a portion thereof within the protease-resistant core of thrombospondin, said domain being selected from the group of regions consisting of a domain of inter-chain disulfide bonds, a procollagen-like domain, a type 1 repeat, a type 2 repeat, and a type 3 repeat, the fragment or peptide corresponding to all or a portion of one or more regions selected from said group, said reference molecule capable of binding to the binding agent. 
     
     
         170 . A kit of  claim 166  wherein the region of thrombospondin is a collagen type V binding domain. 
     
     
         171 . A kit of  claim 165 ,  179 ,  167  or  168  wherein the sequence of the reference molecule comprises the sequence TEENKE (SEQ ID NO:1). 
     
     
         172 . A kit of  claim 171  wherein the binding agent capable of binding the plasma thrombospondin fragment or plasma thrombospondin fragments is a first binding agent and said kit further comprises a second binding agent, said second binding agent capable of binding thrombospondin but not the plasma thrombospondin fragment or plasma thrombospondin fragments. 
     
     
         173 . A kit comprising a first binding agent and a second binding agent, said first binding agent capable of a binding thrombospondin fragment or fragments in the plasma of an individual or in a material taken from the plasma of an individual, said second binding agent capable of binding thrombospondin but not the thrombospondin fragment or fragments in the plasma of the individual. 
     
     
         174 . A kit of  claim 173  wherein the first binding agent is capable of binding to a thrombospondin fragment, in the plasma of an individual, that is 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. 
     
     
         175 . A kit of  claim 173  wherein the first binding agent is capable of binding to a thrombospondin fragment, in the plasma of an individual, that is at least 20 kDa in size but does not exceed 110 kDa in size; wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction. 
     
     
         176 . A kit of  claim 173  wherein the first binding agent is capable of binding to a thrombospondin fragment, in the plasma of an individual, that is at least 20 kDa in size but does not exceed 35 kDa in size; wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction. 
     
     
         177 . A kit of  claim 173  wherein the first binding agent is capable of binding to a thrombospondin fragment, in the plasma of an individual, that starts between amino acyl residues I-165 and V-263, inclusive, and ends between amino acyl residues R-792 and Y-982, inclusive, said plasma thrombospondin fragment being at least 20 kDa in size wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction, 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. 
     
     
         178 . A kit of  claim 173  wherein the first binding agent is capable of binding to a thrombospondin fragment, in the plasma of an individual, that has a molecular weight not exceeding 110 kDa, wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction, and wherein the plasma thrombospondin fragment comprises a region of thrombospondin, said region selected from the group of regions consisting of:
 a domain or a portion thereof within the protease-resistant core of thrombospondin, said domain being selected from the group consisting of a domain of inter-chain disulfide bonds, a procollagen-like domain, a type 1 repeat, a type 2 repeat, and a type 3 repeat; and 
 a collagen type V binding domain a or portion thereof, 
 and wherein the binding agent specifically binds to an epitope within said region. 
 
     
     
         179 . A kit of  claim 173  wherein the kit further comprises a reference molecule selected from the group consisting of a reference molecule capable of binding to the first binding agent and a reference molecule capable of binding to the second binding agent. 
     
     
         180 . A kit of  claim 177  said kit further comprising a reference molecule, said reference molecule comprising a thrombospondin fragment corresponding to or within the region of thrombospondin extending from amino acyl residues I-165 to Y-982, said reference molecule capable of binding to the binding agent. 
     
     
         181 . A kit of  claim 178  said kit further comprising a reference molecule, said reference molecule comprising a thrombospondin fragment consisting of all or a portion of one or more regions selected from said group of regions, said reference molecule capable of binding to the binding agent. 
     
     
         182 . A kit of  claim 165 .  166 ,  167 , or  168  wherein said kit further comprise a means for minimizing platelet activation and/or protease activity said means selected from the group consisting of heparin, a heparin fragment, a protease inhibitor, a platelet inhibitor, and a clotting inhibitor. 
     
     
         183 . A kit of  claim 165 ,  166 ,  167 , or  168  wherein said kit further comprises a device for separation of plasma. 
     
     
         184 . A kit of  claim 165 ,  166 .  167 ,  168 ,  171 ,  182  or  183  wherein the binding agent is an antibody. 
     
     
         185 . A kit of  claim 172 ,  173 ,  174 ,  175 ,  176 ,  177 .  178 ,  179 ,  180  or  181  wherein the first binding agent is an antibody and the second binding agent is an antibody.