Abstract: The T cell receptor interaction with peptide-MHC is unique in that each receptor has the capacity to engage and differentially respond to functionally distinct ligands. Understanding the molecular basis for these properties is important not only for mechanistic insight but for clinical translation to antigen-specific immunotherapies. In this proposal we focus on the chemistry of the TCR/pMHC interface, its role in the specificity of both the recognition and triggering phases of TCR signal initiation, and the use of this information to advance immunotherapeutic strategies. In the prior term of this proposal, we developed a technology, yeast peptide-MHC display, which enabled us obtain a deep understanding of the extent of TCR specificity and cross-reactivity. We found that TCRs are highly specific for their cognate antigens despite exhibiting cross-reactivity. This property enabled us to recover the known peptide ligands of TCRs through un-biased screening of pMHC libraries. Now, in Aim #1, this finding has opened up many exciting possibilities: principally to use pMHC display technology to query the specificity of TCRs, and to identify peptide ligands for ‘orphan’ TCRs in the natural immune system (e.g. Treg) or from pathogenic systems (e.g. cancer, autoimmunity, infectious disease). In Aim #2 we wish to then then generate high-affinity “TCR mimic” antibodies, using a novel platform approach, that specifically recognize these newly discovered ligands and enables us to track tissue expression and inducing selective killing of cells expressing these antigens. In Aim #3, we show that pMHC library technology is also powerful for understanding the relationship between TCR recognition of pMHC and signaling through the capacity of this approach to generate large panels of peptides that can be characterized with respect to signaling and structure. Using this technology, we made the surprising finding that high-affinity, but non-stimulatory pMHC ligands exist in the natural human immune repertoire. We were able to ‘isolate’ the mechanism of TCR triggering to the formation of “catch bonds” in the TCR/pMHC interface, which can decouple TCR/pMHC binding strength from signaling. In Aim #3 of the current proposal we wish to expand on our studies of non-stimulatory TCRs to better understand the molecular mechanisms of triggering at the TCR/pMHC interface, and test a new idea, ‘catch bond engineering,’ for modifying TCRs to exhibit improved target killing potency in a way that bypasses the dangers of affinity-matured TCRs in adoptive cell therapy. Collectively, this proposal aims to exploit ‘first principles’ of TCR/pMHC binding chemistry along three different fronts that have direct translational impact.