Tumor- and neoantigen-reactive T-cell receptors can be identified based on their frequency in fresh tumor

AdoptivetransferofTcellswithengineeredT-cellreceptor(TCR) genes that target tumor-specific antigens can mediate cancer regression.Accumulatingevidencesuggeststhattheclinicalsuccess ofmanyimmunotherapiesismediatedbyTcellstargetingmutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8PD-1 T-cell subset, matching of TCRA-TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity.