Adoptive T cell therapy can be an effective and highly specific approach to the treatment of cancer. Introduction of receptors that recognize cancer antigens provides sufficient numbers of T cells with the appropriate specificity to destroy large, established tumors. The receptors used to date include either T cell receptors (TCRs) against pepMHC or antibody (scFv)-directed chimeric antigen receptors (CARs) against conventional cell surface cancer antigens. The purpose of this proposal is to improve upon current therapies and to develop a robust strategy that combines the advantages of each of these targeting approaches. The strategy involves a single-chain TCR (V-linker-V, called scTv) fused to signaling domains of CD28 (or 4-1BB) and CD3?. When endowed with a high-affinity scTv, this novel signaling receptor, which we call TCR-SCS (TCR-Single-Chain Signaling fusions), redirects activity of both CD4 and CD8 T cells. The TCR-SCS can be used without some of the risks associated with conventional TCRs. For example, TCR-SCS receptors limit self-peptide, off-target reactivities and they avoid mis-pairing with endogenous TCR chains. The reduced levels of off-target reactivities arise from the inability of the single-chain signaling protein to synergize with CD8, a process that enhances sensitivity in CD8 T cells but also increases the level of stimulation by other pepMHC. To enable the rapid use of these TCR-SCS receptors in human therapies, we will also use structure-guided design to engineer high-affinity TCRs against specific pep/HLA-A2 antigens. Our central hypotheses are that TCR-SCS (TCR-Single-Chain Signaling fusions) can be rapidly engineered against many different pepMHC target antigens, and that the TCR-SCS will mediate effective CD4 and CD8 T cell activity without off-target cross-reactivity. Accordingly, the specific aims are: Aim 1. To isolate human T cell receptors against diverse peptide/HLA-A2 antigens using a single TCR scaffold. The approach will involve a combination of structure-based design, taking advantage of computational analyses for library construction and advanced techniques of yeast display for the rapid isolation and evolution of the TCRs. Aim 2. To determine if a TCR-SCS (TCR-Single-Chain Signaling fusion) influences T cell persistence and function in mice. Because the TCR-SCS format is a completely novel approach, we will use the model system involving the peptide SIY, and the high-affinity m33 TCR-SCS against SIY/Kb, to further examine in vivo properties of transduced CD4 and CD8 T cells (collaboration with Dr. Hans Schreiber). Tumor models will include an inducible B-Raf/PTEN-/- melanoma that expresses SIY/Kb (collaboration with Dr. Tom Gajewski). Aim 3. To use TCR-SCS (TCR-Single-Chain Signaling fusions) with high-affinity, human TCRs against WT1 to target tumors. Established, transplanted WT1/HLA-A2 human tumors will be used to examine effectiveness of CD4 and CD8 T cells, transduced with WT1-specific TCR-SCS receptors (collaboration with Dr. Philip Greenberg). Preliminary studies with TCRs isolated in Aim 1 will also extend results to other human tumor antigens.