Abstract The success of T cell therapy for treatment of leukemia and melanoma provides rationale to pursue similar approaches for other cancers, particularly solid tumors. Merkel cell carcinoma, a highly aggressive skin cancer, is a model tumor for developing immunotherapies, as it is immune sensitive and most cases require obligate expression of conserved viral antigens. Like many solid tumor antigens, Merkel cell polyomavirus (MCPyV) oncoproteins are intracellular and thus not accessible to CAR-T cells, but can be targeted with T cell receptor (TCR) based therapy. TCRs for therapeutic translation for cellular immunotherapy must have sufficient affinity to bind the limited number of peptide-MHC molecules commonly expressed on tumor cells. Isolating high affinity natural TCRs offers a safety advantage to mutating lower affinity TCRs since the former TCRs have undergone selection in the thymus. However, identifying such TCRs by limited dilution cloning has been constrained not only by the time and effort required but also by the rarity of the highest affinity clones. Single cell RNA sequencing techniques allow for TCR identification from tiny, clonally diverse samples. We hypothesized this technique would be useful for isolating high-affinity TCRs from the peripheral repertoire of healthy donors or tumor bearing patients and allow for rapid clinical translation. We derived TCRs specific for the HLA-A*0201 restricted MCPyV viral epitope KLLEIAPNC from two sources: 1) the peripheral repertoire of 6 healthy HLA-A*02:01 donors stimulated 3 times in vitro with KLL peptide, and 2) polyclonal MCPyV-specific T cells that were adoptively transferred in a previous clinical trial and shown to localize to tumor and mediate a durable complete remission in a 60-year-old man with metastatic MCC. A total of 20,000 T cells were loaded onto the 10x Genomics 5' V(D)J scRNAseq platform; 14,022 cells (70.1%) were recovered, of which 11,417 (81% of recovered cells) yielded productive TCR alpha-beta pairing. Recovery was similar between healthy peripheral repertoire and patient TIL sources. TCR gene segments were codon optimized and assembled into a PRRLSIN lentiviral vector that is currently in clinical use. Initial screening in TCR-transduced cells revealed 5 TCRs that strongly bind KLL-pMHC tetramer independent of the CD8 co-receptor, including TCRs identified from both healthy donors and the immunotherapy responder. The candidate TCRs selected for further pre-clinical analysis produced inflammatory cytokines and specifically lysed HLA matched fibroblasts expressing physiologic levels of endogenously presented MCPyV oncoproteins. Safety analyses are ongoing. In summary, single cell RNA sequencing allows for the rapid identification from small, polyclonal human samples of functional highly avid paired TCRs, despite their presence at low frequency, thus facilitating rapid therapeutic translation. Citation Format: Megan S. McAfee, Kelly Paulson, Thomas Schmitt, Natalie Miller, Daniel Hunter, Jason Bielas, David Koelle, Valentin Voillet, Raphael Gottardo, Phillip Greenberg, Paul Nghiem, Aude Chapuis. Novel approaches to high-affinity TCR isolation for clinical translation enabled by single cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3567.