Stem Cell Engineering for the Generation of Allogeneic CAR-Directed Natural Killer T Cells Targeting Endometrial Carcinoma

Aggressive subtypes of uterine endometrial carcinoma (UEC) often result in mortality due to recurrence of disease with chemoresistant tumor cells surrounded by an immune suppressive microenvironment. Current CAR-T cell therapies have shown limited efficacy in solid tumors, largely constrained by poor tumor infiltration, immune suppression, and the logistical limitations of autologous cell production, which hinder broad patient access. In this study, we conducted comprehensive immunophenotyping of primary UEC patient samples and identified a therapeutic opportunity for CAR-engineered invariant natural killer T (CAR-NKT) cells capable of targeting both tumor cells and the immunosuppressive TME. Using a hematopoietic stem and progenitor cell (HSPC) engineering platform coupled with ex vivo differentiation culture, we generated allogeneic mesothelin-targeting CAR-NKT cells (AlloMCAR-NKT) with high purity and yield. AlloMCAR-NKT cells exhibited potent cytotoxic activity against UEC tumor cells and CD1d⁺ tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Importantly, compared to conventional CAR-T cells, AlloMCAR-NKT cells demonstrated an improved safety profile, showing no evidence of graft-versus-host disease (GvHD) and minimal cytokine release syndrome (CRS)-related toxicity. These findings highlight the potential of AlloMCAR-NKT cells as a safe and effective off-the-shelf cellular immunotherapy for the treatment of UEC and potentially other solid tumors characterized by an immunosuppressive microenvironment.