Generation of Functional iPSC-Derived CAR-T Cells for Cancer Immunotherapy Via G9a/GLP Inhibition

Cancer immunotherapy using patient-derived T cells modified with chimeric antigen receptors (CAR) has shown exceptional success against lymphoid malignancies. However, these autologous cell therapies involve complex and labor-intensive cell processing. Utilizing human induced Pluripotent Stem Cells (iPSCs) to produce CAR T cells offers significant potential for developing allogeneic, off-the-shelf cancer immunotherapies. The main challenge is generating fully mature T cells from iPSCs, as iPSC-derived T cells often display characteristics of innate-like gamma-delta T cells, lacking the robust functionality of mature alpha-beta T cells derived from peripheral blood. Previously, we developed a stroma-free culture system for differentiating iPSCs into T cells that entailed genetic knockdown of the EZH1 histone methyltransferase, which functions as an epigenetic barrier to hematopoietic maturation (Jing et al, Cell Stem Cell, 2022). To facilitate cell manufacture for clinical translation, we conducted small molecule screens to identify compounds that phenocopy EZH1 deficiency in affecting lymphoid development. We discovered that inhibiting G9a/GLP similarly promotes the generation of mature T cells from iPSC-derived hemogenic endothelial cells (HECs). ATAC-seq and RNA-seq analyses revealed that G9a/GLP inhibition regulates chromatin accessibility and gene expression patterns associated with lymphoid differentiation, and influences the fate choice between myeloid and lymphoid lineages. We also showed that inhibiting G9a/GLP enhances lymphopoiesis in zebrafish, demonstrating the evolutionary conservation of G9a/GLP’s role in T cell development. Most importantly, chemically-induced epigenetic reprogramming via G9a/GLP inhibition enables the generation of highly functional iPSC-derived T cells with a molecular profile similar to that of mature alpha-beta T cells from peripheral blood. Single-cell RNA-seq analysis further revealed that iPSC-derived T cells give rise to both effector and memory-like T cell subpopulations upon activation. When these epigenetically reprogrammed iPSC-derived T cells were engineered to express an anti-CD19 CAR, they showed potent effector responses and antitumor activity in vitro and in a xenograft lymphoma mouse model. We also demonstrated that mice treated with epigenetically modulated iPSC-CAR-T cells were resistant to tumor cell rechallenge, further demonstrating that iPSC-CAR-T cells derived through G9a/GLP inhibition can persist and induce sustained remission. These findings support the efficient production of clinically relevant iPSC-derived T cells for adoptive cell therapies.