Engineering optimal CAR constructs for allogeneic mesothelin-directed CAR-NKT cells in solid tumor immunotherapy

• Various CAR designs were evaluated for allogeneic CAR-NKT cells in treating solid tumors to identify optimal antitumor efficacy. • The 4-1BB costimulatory domain was selected, as it conferred superior antitumor activity and robust in vivo persistence to CAR-NKT cells. Chimeric antigen receptor (CAR)–engineered cell therapies have revolutionized cancer immunotherapy and are expanding into other diseases, including autoimmune disorders. Optimizing CAR design, particularly the choice of costimulatory domains and the incorporation of immune-enhancing genes, has been critical to improving efficacy and safety in CAR-based cell therapy. Although the functional impact of CD28 and 4-1BB costimulatory domains, alone or in combination, has been extensively studied in CAR T cells, comparable insights into CAR-engineered invariant natural killer T (CAR-NKT) cells remain limited. CAR-NKT cells represent a promising platform for solid tumor therapy owing to their intrinsic tumor-homing and infiltration capacities. In this study, we systematically compared 4 CAR designs: CD28, 4-1BB, dual CD28/4-1BB, and an NK receptor (NKG2D/2B4)-based construct, in allogeneic hematopoietic stem and progenitor cell–engineered, mesothelin-targeting CAR-NKT cells. Among these, the 4-1BB–containing CAR conferred superior antitumor activity, demonstrating potent and sustained cytotoxicity against diverse solid tumors as well as enhanced in vivo persistence. Importantly, these CAR-NKT cells exhibited a favorable safety profile, with no risk of graft vs host response, minimal tissue toxicity, and no dysregulated growth. Collectively, this study provides the first systematic evaluation of costimulatory domain selection in allogeneic CAR-NKT cells, identifies 4-1BB as a leading design, and establishes a translational path toward clinical development of allogeneic CAR-NKT cell therapy for solid tumors.