A Regulated Cytokine Engineering Strategy to Augment <i>in Vitro</i> and <i>In Vivo</i> persistence of Allogeneic iPSC Derived CD8 T-Cells

Large-scale clinical production of induced pluripotent stem cell (iPSC)-derived CAR-T cells represents a remarkable opportunity in allogeneic cell therapies. Despite the growing advantages offered by iPSCs such as multiplex gene editing and unlimited starting material, most iPSC derived CAR-T cells demonstrate limited clinical proliferation and persistence. This has been partially attributed to existing iPSC differentiation protocols that yield only cytotoxic CD8 T-cells, whereas conventional autologous and donor-derived CAR-T therapies contain a mixture of CD8 T-cells and supportive CD4 T-cells. It has been well established that cytokine signaling via the JAK/STAT pathway plays an important role in promoting CAR-T cell function. Herein we describe novel engineered receptors that can induce JAK/STAT signaling in iPSC derived CD8 T-cells. We demonstrate that iPSC derived CD8 T-cells have a unique JAK/STAT signaling requirement for long-term cytotoxicity and proliferation in our in vitro assays. We show that our engineered receptors can induce JAK/STAT signaling in the absence of exogenous cytokines in culture media. When fully differentiated CD8 T-cells are transduced with engineered cytokine receptors, they show long-term persistence in the absence of exogenous cytokines in vitro. We observed that premature activation of JAK/STAT signaling pathways can skew cells toward a myeloid or innate lymphoid cell lineage. To circumvent this, we designed iPSC clones with an engineered cytokine receptor integrated into a specific locus under the control of context-specific regulatory sequences. These designs exhibited a stringent off-state during differentiation and CAR-dependent transcriptional responsiveness when exposed to target antigen. This data reveals that a regulated strategy can control cytokine receptor expression during differentiation, to preserve phenotype and improve efficacy of differentiated cells. Further, constitutive activation of JAK/STAT signaling may drive adverse side-effects, and we demonstrate that our regulated designs show complete lack of activity in the absence of antigen stimulation. Finally, we evaluated iPSC derived CD8 T-cells harboring the regulated cytokine engineering strategy in vivo. Engineered iPSC derived CD8 T-cells showed tumor growth inhibition and persistence in the absence of exogenous cytokines at comparable levels to unmodified iPSC derived CD8 T-cells receiving exogenous cytokine support. Engineering of appropriate cytokine support and strategies to regulate expression during differentiation is an important first step to enhance the persistence of iPSC derived CD8 T-cells. One of the major advantages of using iPSC derived CD8 T-cells is their potential for drug readministration without toxic lymphodepletion and exogenous cytokine support. We believe that cell-intrinsic cytokine support is critical to develop durable and safe allogeneic T-cell therapies for patients.