Antibodies directed against particular T cell receptor epitopes can eliminate or reduce the severity of at least some T cell mediated diseases. Negative regulation of T cell activity has also recently been shown to be controlled by the T cell surface molecule CTLA-4. The long- range goals of the present study are to develop anti-T cell receptor antibody agents that modulate the activity of T cell subpopulations and to use these agents to further understand the basic mechanisms of T cell regulation. This project will explore the ability of these agents to suppress two different classes of diseases: 1) acute diseases such as SEB-mediated shock, that might be expected to be inhibited by simple competition for the TCR ligand SEB and 2) chronic diseases such as some autoimmune diseases, that might be expected to require complete inactivation of the T cells. The agents described below will be tested in vitro and in vivo for their effects on both types of T cell reactions. In order to explore the most effective strategies, anti-Vbeta8 agents that target only the Vbeta8+ population in mice will be developed. Continuing our efforts from the previous grant period, the scFv from the anti-Vbeta8 antibody KJ16 will be used to determine the effects of antibody affinity, kinetics, and valency on modulating T cell activity (Specific Aims 1 and 2). These aims are based on the premise that TCR: ligand binding properties dictate the fate of a T cell, yet the role of each of these parameters in anti-TCR antibody-mediated regulation has never been examined. A panel of KJ16 scFv variants that have a range of affinities and off-rates will be produced by mutagenesis, yeast display, and selection. Bivalent agents will be produced by F(ab) 2 anti-c-myc crosslinking (the scFv contains a ten residue c-myc tag). In selected cases, bivalent scFv2 will be produced using Fos and Jun scFv fusion proteins. In Specific Aim 3, the KJ16 scFv-Fos will be combined with the scFv-Jun of the anti-CTLA-4 antibody 4F10, which we have recently produced. This bispecific construction and bispecific, multivalent microspheres will be tested for their abilities to inactivate Vbeta8+ T cells by cross-linking TCR and CTLA-4. In Specific Aim 4, anti-Vbeta/anti-CTLA-4 agents that are effective in vitro will be examined in several animal models: C57B1/6 and 2C TCR transgenic mice; graft rejection in 2C TCR/RAG-/- mice; SEB-mediated shock in Vbeta8 transgenic mice; NOD mice. Finally, in specific Aim 5 a novel system for the discovery of scFv that inhibit T cells will be explored. This system involves the use of yeast that we have already engineered to display surface scFv-KJ16. Yeast that co-express scFv-KJ16 and scFv-4F10 will be tested for their ability to affect T cell activity in vitro.
NIH ExPorter
NIH ExPorter