Massively parallel interrogation and mining of natively-paired human TCRab repertoires

T cell receptors (TCRs) can be engineered into T cells as potent therapies for human disease; however, only a handful of TCRs have been thoroughly evaluated. Identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires, as well as the inherent challenges of working with primary T cells. We conduct extensive validation of a novel method for identification of high-avidity TCRs from diverse human T cell repertoires. The method uses massively parallel microfluidics to generate natively paired, full-length TCRab libraries, from millions of primary T cells, which are then transduced into Jurkat cells. These TCRab-Jurkat libraries are immortal and induce TCR-mediated cell signaling events, which enables repeated screening and panning for antigen-reactive TCRs using peptide:MHC binding and cellular activation. We captured over 2.7 million natively paired TCRab clonotypes from six virus-seropositive human donors and identified rare (<0.001% frequency) viral antigen-reactive TCRs. We also mined a clinical-grade tumor-infiltrating lymphocyte (TIL) sample and identified high-avidity, tumor-killing TCRs. This new approach improves the speed, sensitivity, and specificity for antigen-reactive therapeutic TCR mining. Additionally, it has the potential to scale, providing an opportunity to comprehensively profile the antigen reactivity of the human T cell repertoire.