The Johns Hopkins University

Sean O'Herrin

Jonathan Schneck

Michael S Lebowitz

Abdel Hamad

Drew M Pardoll

Tim Greten

Jill Slansky

Compositions comprising a cell in which a molecular complex with high affinity for its cognate ligand is bound to the surface of the cell are provided. To form the molecular complexes, extracellular domains of transmembrane heterodimeric proteins, particularly T cell receptor and major histocompatibility complex proteins, can be covalently linked to the heavy and light chains of immunoglobulin molecules. The molecular complexes can be used, inter alia, to detect and regulate antigen-specific T cells and as therapeutic agents for treating disorders involving immune system regulation, such as allergies, autoimmune diseases, tumors, infections, and transplant rejection. Optionally, identical antigenic peptides can be bound to each ligand binding site of a molecular complex.

Cell compositions comprising molecular complexes that modify immune responses

To increase the effective affinity of soluble analogs of peptide/MHC molecules for their cognate ligands, divalent peptide/MHC complexes were constructed. Using a recombinant DNA strategy, DNA encoding the MHC class I was ligated to DNA coding for murine Ig heavy chain. MHC/Ig complexes were exploited to homogeneously load with peptides of interest. The results of flow cytometry demonstrated that the MHC/Ig complexes bound specifically with high affinity to cells bearing their cognate receptors. MHC/Ig complexes are also useful in modulating effector functions of antigen-specific T cells. These MHC/Ig complexes are useful for studying TCR/MHC interactions and lymphocyte tracking and have uses as specific regulators of immune responses.

Use of multivalent chimeric peptide-loaded, MHC/Ig molecules to detect, activate or suppress antigen-specific T cell-dependent immune responses

Extracellular domains of transmembrane heterodimeric proteins, particularly T cell receptor and major histocompatibility complex proteins, can be covalently linked to the heavy and light chains of immunoglobulin molecules to provide soluble multivalent molecular complexes with high affinity for their cognate ligands. The molecular complexes can be used, inter alia, to detect and regulate antigen-specific T cells and as therapeutic agents for treating disorders involving immune system regulation, such as allergies, autoimmune diseases, tumors, infections, and transplant rejection.

Molecular complexes which modify immune responses

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 &mgr;g/ml of soluble, conformationally 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, liymphocyte tracking, identifying new antigens, and have possible uses as specific regulators of immune responses.

Soluble divalent and multivalent heterodimeric analogs of proteins

Use of multivalent chimeric peptide-loaded, MHC/ig molecules to detect, activate or suppress antigen-specific T cell-dependent immune responses

Polynucleotides encode soluble, multivalent molecular complexes which modify immune responses, and host cells comprise such polynucleotides. The molecular complexes comprise extracellular domains of transmembrane heterodimeric proteins, particularly T cell receptor and major histocompatibility complex proteins, which are covalently linked to the heavy and light chains of immunoglobulin molecules to provide soluble multivalent molecular complexes with high affinity for their cognate ligands. The molecular complexes can be used, inter alia, to detect and regulate antigen-specific T cells and as therapeutic agents for treating disorders involving immune system regulation, such as allergies, autoimmune diseases, tumors, infections, and transplant rejection.

Polynucleotides encoding molecular complexes which modify immune

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 .mu.g/ml of soluble, conformationally 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.

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Baltimore, MD, United States of America

Sean O'Herrin