Protocol to generate allorejection-resistant universal CAR-NKT cells and evaluate their efficacy, mechanism of action, safety, and immunogenicity.

Universal chimeric antigen receptor-engineered invariant natural killer T ((U)CAR-NKT) cells overcome the limitations of allogeneic chimeric antigen receptor (CAR)-T cell therapies, such as graft-versus-host disease and allorejection. Here, we present a protocol for generating (U)CAR-NKT cells from hematopoietic stem and progenitor cells (HSPCs), followed by feeder-free ex vivo differentiation. We also outline assays to assess antitumor efficacy, safety, and allorejection resistance. For complete details on the use and execution of this protocol, please refer to Li et al.(1).