Key Points
Evaluate various CAR designs for allogeneic CAR-NKT cells in treating solid tumors with best antitumor efficacy.
Select 4-1BB as the CAR co-stimulatory domain rendering CAR-NKT cells with best antitumor capacity and potent in vivo persistence.
Abstract
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 co-stimulatory domains and the incorporation of immune-enhancing genes, has been critical to improving efficacy and safety in CAR-based cell therapy. While the functional impact of CD28 and 4-1BB co-stimulatory domains, alone or in combination, has been extensively studied in CAR-T cells, comparable insights in 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 four CAR designs: CD28, 4-1BB, dual CD28/4-1BB, and a natural killer receptor (NKG2D/2B4)-based construct, in allogeneic hematopoietic stem and progenitor cell (HSPC)-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-versus-host response, minimal tissue toxicity, and no dysregulated growth. Collectively, this study provides the first systematic evaluation of co-stimulatory 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 therapy for solid tumors.
Author notes
These authors contribute equally
