Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy.

The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT ((U)CAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The (U)CAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These (U)CAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, (U)CAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of (U)CAR-NKT cell products and lay a foundation for their translational and clinical development.