398 Functional characterization of a class I-restricted CD8+ T cell receptor specific for the stem cell transcription factor Sox2

<h3>Background</h3> As relapse is a substantial cause of morbidity and mortality for cancer patients, novel therapies targeting mechanisms of relapse are urgently needed. Sox2 (Sex- determining region-box2) is a stem cell transcription factor whose expression confers drug resistance, enhanced proliferation, and survival advantage to residual cancer cells in several hematological and solid malignancies. Prior studies have suggested that T-cell immunity against SOX2 is associated with better clinical outcomes in patients treated with immunotherapies. Nonetheless, direct evidence of SOX2-specific T cell lytic activity against tumor cells is lacking. <h3>Methods</h3> We employed in silico predictions and immunopeptidomics to identify candidate HLA class I restricted SOX2-derived epitopes. SOX2-specific TCR sequences were identified from patients with plasma cell dyscrasias enrolled on an IRB-approved clinical studies at the University of Pennsylvania. Transgenic expression of synthetic TCRs was attained via lentiviral transduction of reporter cell lines or gene-edited (TCRabnull) primary CD8+ T cells. Antigen affinity was assessed via limiting peptide dilution. TCR reactivity to SOX2 expressing cell lines was assessed by FACS analysis and in vitro cytotoxicity assays (4h 51Cr-release, xCELLigence Real Time Cell Analysis). <h3>Results</h3> Two high-affinity peptides were identified, and their immunogenicity confirmed on peptide-expanded T cell cultures using peptide/MHC (p/MHC) multimers and in IFN-gamma ELISpot assays. In-vitro generated SOX2-specific CD8+ T cells provided a source of TCRs for further characterization and studies. Here, we described a SOX2 TCR with high-antigen affinity for p/MHC complexes as determined in peptide titration assays. This SOX2 TCR was efficiently expressed, as detected by p/MHC multimers, on TCR-null CD8+ T cells derived from healthy donors. Transgenic TCR expression confers T cells with in-vitro recognition and lytic activity against SOX2+ cancer cell lines of various histologies in short- and long-term cytotoxicity assays (figure 1). <h3>Conclusions</h3> Our findings demonstrate SOX2 is a novel immunological target that may enable the development of TCR engineered adoptive T-cell therapy for cancer patients whose tumor expresses this stem cell transcription factor. <h3>Ethics Approval</h3> The University of Pennsylvania Institutional Review Board approved this project, and informed consent was obtained from all patients.