Detailed knowledge at the molecular level concerning the interaction of T cell receptors (TCRs) with cognate peptide-MHC complexes would facilitate the development of immunotherapy for the treatment of, inter alia, autoimmune disease and cancer. The initial aim of this study is to express TCRs in soluble form as either single Valpha, Vbeta domains, Fv-like Valpha-Vbeta heterodimers, Fab-like ValphaCalpha:Vbeta-C beta heterodimers or as Fab-like Valpha-CH1:Vbeta-Ckappa immunoglobulin-chimeras, using Escherichia coli as a host. Using murine experimental allergic encephalomyelitis (EAE) as a model system, TCR genes will be isolated from a well characterized encephalitogenic T call hybridoma (1 934.4), which is associated with EAE induction in the H-2u mouse. This system has been chosen as the TCRs, restricting MHC molecules and peptide antigen have been defined, and murine disease models are available for the testing of the efficacy of immunotherapeutic reagents. The recombinant TCRs will be expressed and secreted into the E. coli culture supernatant, and the functional activity of the recombinant TCR fragments will be analyzed using direct and competition binding assays. The latter will be designed to assess the ability of the TCR fragments to interfere with the interaction of the 1934.4 hybridoma with cognate peptide-MHC. This E. coli expression system could allow the production of functional Fv-like or Fab-like TCRs in sufficient yields to allow crystallographic analyses to be carried out. In addition, site-directed mutagenesis experiments will be focused towards the identification of the TCR residues which are functionally important in binding peptide-MHC. This, together with the high resolution structural work, will be followed by random mutagenesis of regions of the TCR involved in binding, with the aim for generating TCRs of higher affinity. Such TCRs could be of considerable utility in the blocking of pathogenic TCRs in EAE, and will be used in passive immunization to assess their efficacy in both preventing and reversing this disease in the H-2u mouse. Thus, the ultimate aim of this project is to use data from the proposed structure-function studies of recombinant, soluble TCRs to develop immunotherapeutic strategies, using murine EAE as the model. These strategies could in the long term be applicable to the therapy of autoimmune disease in humans.
| Radu, C G; Ober, B T; Colantonio, L et al. (1998) Expression and characterization of recombinant soluble peptide: I-A complexes associated with murine experimental autoimmune diseases. J Immunol 160:5915-21 |
| Popov, S; Hubbard, J G; Ward, E S (1996) A novel and efficient route for the isolation of antibodies that recognise T cell receptor V alpha(s). Mol Immunol 33:493-502 |
