Soluble divalent and multivalent heterodimeric analogs of proteins

Abstract

Specificity in immune responses is in part controlled by the selective interaction of T cell receptors with their cognate ligands, peptide/MHC molecules. The discriminating nature of this interaction makes these molecules, in soluble form, good candidates for selectively regulating immune responses. Attempts to exploit soluble analogs of these proteins has been hampered by the intrinsic low avidity of these molecules for their ligands. To increase the avidity of soluble analogs for their cognates to biologically relevant levels, divalent peptide/MHC complexes or T cell receptors (superdimers) were constructed. Using a recombinant DNA strategy, DNA encoding either the MHC class II/peptide or TCR heterodimers was ligated to DNA coding for murine Ig heavy and light chains. These constructs were subsequently expressed in a baculovirus expression system. Enzyme-linked immunosorbant assays (ELISA) specific for the Ig and polymorphic determinants of either the TCR or MHC fraction of the molecule indicated that infected insect cells secreted approximately 1 μg/ml of soluble, conformnationally intact chimeric superdimers. SDS PAGE gel analysis of purified protein showed that expected molecular weight species. The results of flow cytometry demonstrated that the TCR and class II chimeras bound specifically with high avidity to cells bearing their cognate receptors. These superdimers will be useful for studying TCR/MHC interactions, lymphocyte tracking, identifying new antigens, and have possible uses as specific regulators of immune responses.

We Claim:  
     
         1 . A soluble recombinant divalent protein composition comprising the extracellular domains of a heterodimeric protein operatively linked to immunoglobulin heavy and light chain polypeptides.  
     
     
         2 . The recombinant protein composition of  claim 1 , wherein said immunoglobulin is selected from the group consisting of IgM, IgD, IgG3, IgG1, IgG2 b , IgG2 a , IgE, and IgA.  
     
     
         3 . The recombinant protein composition of  claim 1 , wherein said extracellular domains of the heterodimeric protein minimally contain a binding site involved in immune recognition.  
     
     
         4 . The recombinant protein composition of  claim 1  further comprising a linker domain between said extracellular domains of the heterodimeric protein and said immunoglobulin polypeptides.  
     
     
         5 . The recombinant protein composition of  claim 3 , wherein said heterodimeric protein comprises an a polypeptide chain and β polypeptide chain.  
     
     
         6 . The recombinant protein composition of  claim 3 , wherein said heterodimeric protein comprises a γ polypeptide chain and δ polypeptide chain.  
     
     
         7 . The recombinant protein composition of  claim 5  wherein said heterodimeric protein is an MHC class II molecule.  
     
     
         8 . The recombinant protein composition of  claim 5  wherein said heterodimeric protein is a TcR molecule.  
     
     
         9 . The recombinant protein composition of  claim 7  additionally comprising an antigenic peptide in the peptide binding groove of said MHC class II molecule.  
     
     
         10 . A soluble recombinant multivalent protein composition comprising the extracellular domains of a heterodimeric protein operatively linked to immunoglobulin heavy and light chain polypeptides.  
     
     
         11 . The recombinant protein composition of  claim 10 , wherein said immunoglobulin is selected from the group consisting of IgM, IgD, IgG3, IgG1, IgG2 b , IgG2 a , IgE, and IgA.  
     
     
         12 . The recombinant protein composition of  claim 10 , wherein said extracellular domains of the heterodimeric protein minimally contain a binding site involved in immune recognition.  
     
     
         13 . The recombinant protein composition of  claim 10  further comprising a linker domain between said extracellular domains of a divalent heterodimeric protein and said immunoglobulin polypeptides.  
     
     
         14 . The recombinant protein composition of  claim 10 , wherein said heterodimeric protein comprises an α polypeptide chain and β polypeptide chain.  
     
     
         15 . The recombinant protein composition of claim I 0 , wherein said heterodimeric protein comprises a γ polypeptide chain and δ polypeptide chain.  
     
     
         16 . The recombinant protein composition of  claim 14  wherein said heterodimeric protein is an MHC class II molecule.  
     
     
         17 . The recombinant protein composition of  claim 14  wherein said heterodimeric protein is a TcR molecule.  
     
     
         18 . The recombinant protein composition of  claim 16  additionally comprising an antigenic peptide in the peptide binding groove of said MHC class II molecule.  
     
     
         19 . A method for producing an expression vector encoding the recombinant protein composition according to any one of claims  1 - 8  and  10 - 17 , comprising modifying an expression vector for immunoglobulin heavy and light chains by inserting DNA sequences which code for the extracellular domains of a heterodimeric integral membrane protein such that fusion proteins are produced which comprise at the amino terminus one of said extracellular domains operatively linked to said immunoglobulin heavy or light chain.  
     
     
         20 . The method of  claim 19  wherein said heavy and light chain fusion proteins are encoded on separate expression vectors.  
     
     
         21 . An expression vector comprising DNA sequences encoding a soluble heterodimeric protein each operatively linked to a DNA sequence encoding either an immunoglobulin heavy or light chain polypeptide, respectively.  
     
     
         22 . An expression vector comprising a DNA sequence encoding a soluble analog of one member of a heterodimeric protein operatively linked to a DNA sequence encoding either an immunoglobulin heavy or light chain polypeptide.  
     
     
         23 . The expression vector of  claim 21  further comprising linker domains between said DNA sequences encoding said soluble heterodimeric protein and said respective DNA sequences encoding said immunoglobulin heavy and light chain polypeptides.  
     
     
         24 . The expression vector of  claim 22  further comprising a linker domain between said DNA sequence encoding one member of a soluble heterodimeric protein and said DNA sequence encoding said immunoglobulin heavy or light chain polypeptide.  
     
     
         25 . A host cell comprising any one of the vectors of claims  21 - 24 .  
     
     
         26 . A host cell comprising two vectors according to either claims  22  or  24 , wherein one of said two vectors comprises a DNA sequence encoding one member of said heterodimeric protein operatively linked to a DNA sequence encoding an immunoglobulin heavy chain polypeptide, and the other of said two vectors comprises a DNA sequence encoding the other member of said heterodimeric protein operatively linked to a DNA sequence encoding an immunoglobulin light chain polypeptide.  
     
     
         27 . A pharmaceutical composition comprising the soluble recombinant divalent protein composition according to any one of claims  1 - 9  in a pharmaceutically-acceptable carrier.  
     
     
         28 . A pharmaceutical composition comprising a soluble recombinant multivalent protein composition according to any one of claims  10 - 18  in a pharmaceutically-acceptable carrier.  
     
     
         29 . A method for selectively inhibiting or decreasing an immune response, comprising administering to a patient an effective amount of a soluble recombinant divalent protein composition according to any one of claims  1 - 9  in a pharmaceutically-acceptable carrier such that said immune response is inhibited or decreased.  
     
     
         30 . A method according to  claim 29  wherein said immune response is directed to a foreign transplantation antigen.  
     
     
         31 . A method according to  claim 29  wherein said immune response results in an autoimmune disease.  
     
     
         32 . A method for inhibiting or decreasing an immune response, comprising administering to a patient an effective amount of a soluble recombinant multivalent protein composition according to any one of claims  10 - 18  in a pharmaceutically-acceptable carrier such that said immune response is inhibited or decreased.  
     
     
         33 . A method according to  claim 32  wherein said immune response is directed to a foreign transplantation antigen.  
     
     
         34 . A method according to  claim 32  wherein said immune response results in an autoimmune disease.  
     
     
         35 . A method for stimulating an antigen-specific T-cell response comprising immobilizing a soluble recombinant divalent protein composition according to  claim 9  on a substrate and exposing said immobilized protein composition to a population of T cells such that an antigen-specific T cell response is stimulated.  
     
     
         36 . A method for stimulating an antigen-specific T-cell response comprising immobilizing a soluble recombinant multivalent protein composition according to  claim 18  on a substrate and exposing said immobilized protein composition to a population of T cells such that an antigen-specific T cell response is stimulated.  
     
     
         37 . A method according to  claim 35  or  36  wherein said method is used for the identification and purification of specific T cell subsets.  
     
     
         38 . A method for identifying and purifying an unknown peptide/MHC complex comprising immobilizing a soluble recombinant divalent protein composition according to  claim 8  on a substrate and exposing said immobilized protein composition to a population of peptide/MHC complexes such that a particular peptide/MHCcomplex is identified.  
     
     
         39 . A method for identifying and purifying an unknown peptide/MHC complex comprising immobilizing a soluble recombinant multivalent protein composition according to  claim 17  on a substrate and exposing said immobilized protein composition to a population of peptide/MHC complexes such that a particular peptide/MHCcomplex is identified.  
     
     
         40 . A method according to  claim 38  or  39  wherein said peptide is a tumor or viral antigen.  
     
     
         41 . A soluble recombinant multivalent protein composition comprising the extracellular domains encoding a binding site operatively linked to both immunoglobulin heavy and light chain polypeptides.  
     
     
         42 . The protein composition of claim  42  wherein said binding site is encoded by a polypeptide, a carbohydrate or a glycoprotein.