Engineering Intracellular Signaling Domains to Drive CAR-T Cell Differentiation from Pluripotent Stem Cell 2865

Description Immunotherapy has revolutionized cancer treatment by harnessing the immune system to target malignant cells, with chimeric antigen receptor (CAR) T cell therapy showing notable efficacy against B cell lymphomas. While conventional CAR-T therapy relies on patient-derived cells, induced pluripotent stem cell (iPSC)-derived CAR T cells offer a scalable, off-the-shelf alternative, mitigating issues of cost and patient variability. However, differentiating iPSCs into CAR T cells is challenged by a tendency of cells to skew toward innate-like phenotypes due to tonic signaling from the CAR during early development. To overcome this, we are engineering CAR intracellular signaling domains (ICDs) to modulate tonic signaling and direct iPSC differentiation toward functional CD4+ and CD8+ CAR T cells. Our CAR signaling domain library screen revealed that a novel ICD combination demonstrated a 2.5-fold reduction in tonic signaling index compared to the conventional CD28 ICD. Additionally, these CAR-transduced iPSC-derived hematopoietic stem progenitor cells (HSPCs) showed a fivefold increase in CD4+CD8+ T cells, which are precursors to mature CD4+ and CD8+ T cells, suggesting reduced innate skewing. This work enhances our understanding of lymphoid cell development with CAR signaling, supporting the identification of optimal ICD combinations to promote iPSC differentiation into CAR T cells under feeder- and serum-free conditions, and advancing standardized CAR-T cell therapy options.