Soluble tcr molecules and methods of use

Abstract

Disclosed are compositions and methods for detecting cells or tissue comprising a peptide antigen presented in the context of an MHC or HLA complex. The invention has a wide range of applications including providing a highly sensitive method for detecting cancer cells.

What is claimed is: 
     
         1 . A method for detecting cells or tissue comprising a peptide antigen presented on the cells or tissue in the context of an MHC complex, the method comprising:
 a) contacting the cells or tissue with at least one soluble TCR molecule or functional fragment thereof under conditions that form a specific binding complex between the presented peptide antigen and the soluble TCR or fragment,   b) washing the cells or tissue under conditions appropriate to remove any soluble TCR molecule or fragment not bound to the presented peptide antigen; and   c) detecting the specific binding complex as being indicative of cells or tissue comprising the presented peptide antigen.   
     
     
         2 . The method of  claim 1 , wherein the cells or tissue are further contacted with at least one blocking agent. 
     
     
         3 . The method of  claim 2 , wherein the method further comprises contacting the cells or tissue with the blocking agent before step a) to reduce non-specific binding between the soluble TCR or fragment and the cells. 
     
     
         4 . The method of  claim 2 , wherein the blocking agent is a peroxide, serum protein, antibody, or fragment thereof. 
     
     
         5 . The method of  claim 1 , wherein the method further comprises contacting the complex with a competing MHC molecule or fragment thereof under conditions that compete with and specifically bind the soluble TCR or fragment bound to the complex. 
     
     
         6 . The method of  claim 5 , wherein essentially all of the soluble TCR or fragment is bound to the competing MHC molecule or fragment thereof to form a competition complex. 
     
     
         7 . The method of  claim 6 , wherein the method further comprises detecting the competition complex and determining the binding specificity of the MHC molecule or the soluble TCR. 
     
     
         8 . The method of  claim 5 , wherein the MHC molecule or fragment is single-chain. 
     
     
         9 . The method of  claim 5 , wherein the MHC molecule or fragment is loaded with peptide antigen. 
     
     
         10 . The method of  claim 1 , wherein the method further comprises contacting the cells or tissue with at least one denaturing agent. 
     
     
         11 . The method of  claim 10 , wherein the method further comprises contacting the cells or tissue with the denaturing agent before step a) and denaturing (fixing) the cells or tissue. 
     
     
         12 . The method of  claim 1 , wherein the method further comprises placing a plurality of cells or tissue in an array. 
     
     
         13 . The method of  claim 12 , wherein the method is performed in each element of the array comprising cells or tissue. 
     
     
         14 . The method of  claim 12 , wherein the method is performed substantially simultaneously in each element of the array. 
     
     
         15 . The method of  claim 12 , wherein step c) further comprises scanning the array and generating image signals indicative of presence of the specific binding complex. 
     
     
         16 . The method of  claim 15 , wherein step c) further comprises outputting the signals in real-time to a user and optionally indexing stored images of the image signal. 
     
     
         17 . The method of  claim 1 , wherein the amount of any peptide antigen present on the cells is less than about 100,000 copies. 
     
     
         18 . The method of  claim 17 , wherein the amount of any peptide antigen present on the cells is less than about 400 copies. 
     
     
         19 . The method of  claim 1 , wherein the peptide antigen is a tumor-associated peptide antigen. 
     
     
         20 . The method of  claim 1 , wherein the cells or tissue are suspended. 
     
     
         21 . The method of  claim 1 , wherein the soluble TCR molecule or fragment comprises at least one single-chain TCR. 
     
     
         22 . The method of  claim 1 , wherein the soluble TCR molecule or fragment is detectably-labeled. 
     
     
         23 . The method of  claim 22 , wherein the detectable label is biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. 
     
     
         24 . The method of  claim 21 , wherein the single-chain TCR or fragment further comprises at least one covalently bound cytokine. 
     
     
         25 . The method of  claim 24 , wherein the soluble TCR molecule or fragment is a single-chain TCR comprising a covalently bound cytokine or fragment thereof. 
     
     
         26 . The method of  claim 25 , wherein the single-chain TCR or fragment comprises sequence encoding interleukin-2 (IL-2). 
     
     
         27 . The method of  claim 21 , wherein the single-chain TCR or fragment further comprises at least one covalently bound immunoglobin domain. 
     
     
         28 . The method of  claim 27 , wherein the soluble TCR molecule or fragment is a single-chain TCR comprising a covalently bound immunoglobin domain or fragment thereof. 
     
     
         29 . The method of  claim 28 , wherein the single-chain TCR or fragment comprises sequence encoding an IgG1 domain or fragment thereof. 
     
     
         30 . The method of  claim 1 , wherein the MHC complex is HLA-A2 restricted. 
     
     
         31 . The method of  claim 1 , wherein the method further comprises performing a control to detect any binding between the soluble TCR or fragment to cells that do not comprise the peptide antigen. 
     
     
         32 . The method of  claim 1 , wherein the presented peptide antigen is p53 (aa 149-157) or p53 (aa 264-272). 
     
     
         33 . The method of  claim 1 , wherein the method is performed in vivo, ex vivo, or in vitro. 
     
     
         34 . The method of  claim 1 , wherein the cell or tissue is a tumor cell or tissue. 
     
     
         35 . A method for detecting a tumor cell or tissue in a subject, wherein the cell or tissue comprises a tumor-associated peptide antigen presented on the cells or tissue in the context of an MHC complex, the method comprising:
 a) administering to the subject a soluble TCR molecule or functional fragment thereof under conditions that form a specific binding complex between the presented peptide antigen and the soluble TCR molecule or fragment; and   b) detecting the specific binding complex as being indicative of a tumor cell or tissue comprising the presented tumor-associated peptide antigen.   
     
     
         36 . The method of  claim 35 , wherein the soluble TCR molecule or fragment comprises at least one single-chain TCR. 
     
     
         37 . The method of  claim 35 , wherein the soluble TCR molecule or fragment is detectably-labeled. 
     
     
         38 . The method of  claim 37 , wherein the detectable label is biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule.