Specific recognition by a T cell is dictated by a heterodimeric alpha-beta receptor (TCR). Although T cells undergo selection so that reactions with """"""""self"""""""" antigens should not occur, some autoimmune diseases are caused by aberrant T cells that recognize """"""""self"""""""" antigens. Various pathologic conditions have also been associated with the massive T cell stimulation that accompanies infection with superantigens such as bacterial enterotoxins. The inactivation or elimination of only detrimental T cells in these diseases will require the development of reagents that can be used to target specific T cells. Although anti-TCR antibodies have been used in a number of cases to prevent T cell mediated diseases in mice, there are many aspects of such treatments that could be optimized. The ideal reagents will specifically inactivate or eliminate T cell populations at low doses and with no adverse side effects. There is evidence that some side effects may be due to TCR cross-linking by bivalent antibodies and that monovalent antibodies may be more useful in this regard. With this in mind, the proposed studies will engineer monovalent single-chain anti-receptor antibodies (scFv) that will be expressed in E. coli. The scFv proteins will be used in vivo to determine if they can inactivate and/or delete specific T cell populations. The results should provide information on the effect of antibody size and binding affinity in targeting T cells and on the mechanisms involved in T cell inactivation. The system to be used in this proposal involves the alpha-beta TCR from the BALB.B derived CTL clone 2C. The 2C alpha-beta has been expressed in trangenic mice and there is a syngeneic clonotypic monoclonal antibody, 1B2, specific for the 2C alpha-beta and an allogeneic antibody, F23.1, specific for the V-beta region. CTL 2C and T cells from the transgenic mice recognize the Ld alloantigen and SEB/class II complexes. Our lab has engineered and characterized an active 29 kD scFv of the 1B2 antibody that can be purified in large quantities from E. coli inclusion body pellets. This scFv-1B2 will be used in the proposed study, which has three aims: 1) To construct and characterize the scFv of the anti-V-beta8 antibody F23.1. 2) To engineer a higher affinity anti-receptor antibody by linking the scFv-1B2 and scFv-F23.1 genes. As the 1B2 and F23.1 antibodies bind to different epitopes on the same TCR, we will thus test the hypothesis that a high affinity bivalent antibody might be generated by covalent linkage of the two binding sites. 3) To compare the in vivo effectiveness of the scFv-1B2, scFv-F23.1, and bivalent scFv2. In these studies, scFv proteins, Fab fragments, and intact antibodies will be administered to 2C transgenic mice or normal BALB/c mice (F23. I reagents only). Treated animals will be examined for inactivation or deletion of the targeted T cells.
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