TCR gene transfer with lentiviral vectors allows efficient redirection of tumor specificity in naïve and memory T-cells without prior stimulation of endogenous TCR

We investigated the possibility of introducing exogenous TCR genes into T-cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor-antigen specificity to recipient T-cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T-cells. Cytokine stimulation makes T-cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high affinity TCRs specific for self/tumor antigens. We selected a Mart-1 specific TCR from a vitiligo patient based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human PBMCs, preferential pairing of exogenous alpha and beta chain was observed, together with effective recognition of Mart-1+ melanoma cells. We tested transduction efficiency on different T-cell subsets prestimulated with IL-2, IL-7, IL-15 and IL-21 (alone or in combination). Both naïve and unfractionated CD8+ T-cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T-cell differentiation (34+/-5% Mart-1-TCR+ cells in naïve CD8+ and 16+/-6% in unfractionated CD8+). Cytokine prestimulated, Mart-1-redirected naïve and unfractionated CD8+ cells expanded better than CD3-CD28 prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart-1+ melanoma cells. This strategy allows the generation of tumor-specific T-cells encompassing truly naïve T-cells, endowed with an intact proliferative potential and a preserved differentiation stage.