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Background Tumor-associated antigen-specific cytotoxic T cells (TAA-Ts) are antigen-specific T cell products that have demonstrated therapeutic potential for high-risk malignancies. However, TAA-Ts face challenges from the tumor microenvironment (TME) in vivo, including immunosuppressive cytokine signaling by tumor cells. Transforming growth factor beta (TGFb) is a potent cytokine identified in the TME of high-risk tumors known to suppress T cell proliferation and differentiation. Thus, mitigation of TGFb signaling through introduction of a non-functional receptor to TAA-Ts may improve persistence and efficacy in vivo while maintaining antigen specificity. We hypothesize that TAA-T products targeting preferentially expressed antigen in melanoma (PRAME) can be transduced with a TGFbRII dominant negative receptor (DNR) while maintaining antigen specificity and phenotype.Methods Peripheral blood mononuclear cells (PBMCs) were pulsed with PRAME peptides and anti-CD28 antibody. After 16-24 hours, activated cells were isolated by magnetic bead selection for CD137 (4-1BB) and cultured with irradiated CD137-negative feeder cells in the presence of cytokines. Cells were restimulated on day 11 with PRAME-pulsed, irradiated phytohaemagglutinin (PHA) blasts and transduced with the TGFb DNR on days 14 and/or 15 using one of 4 protocols to optimize transduction. On day 18, specificity for PRAME was measured via interferon gamma ELISpot while phenotype, exhaustion, and transduction efficiency were assessed via flow cytometry.Results We generated 17 distinct transduced products from 13 donors. Transduced products were CD3 enriched with variable CD4+ (0.08 – 97.7%) and CD8+ (0.1 – 81.2%) T cell proportions and a median transduction efficiency of 46.7% (9.37—94.5%). Adding a second day of transduction improved median efficiency (19.9 versus 61.1%) as did a spinoculation of viral supernatant (14.7 versus 63.1%). Untransduced and transduced products showed median specificity of 510 and 338.5 spot forming units/1e5 cells above background, respectively; a difference that was not statistically significant. However, some transduced products demonstrated increased expression of exhaustion markers LAG3, PD1, TIM3 and CTLA4, and most transduced products showed reduced expansion. CD39, a checkpoint associated with T cell exhaustion, has also been observed on TAA-T products (range 28-56% on CD3+ cells) and remained highly expressed amongst both transduced and untransduced products.Conclusions We successfully transduced TAA-Ts with a TGFb DNR while maintaining specificity for PRAME but observed increased expression of T cell exhaustion markers and reduced expansion in some donors. Future studies will further optimize this protocol by testing the use of immune checkpoint inhibitors to improve expansion, specificity, and tumor-directed killing of transduced TAA-Ts in vitro and in murine models.Acknowledgements Thank you to the Center for Cancer and Immunology Research at Children’s National Hospital; Cancer Grand Challenges (award NCI/CRUK OT2CA278700); and to Brett Laufer for cell passaging