Abstract
Despite success in treating some hematological malignancies, CAR-T cells have not yet produced similar outcomes in solid tumors due, in part, to the tumor microenvironment, poor persistence, and a paucity of suitable target antigens. Importantly, the impact of the CAR components on these challenges remains focused on the intracellular signaling and antigen-binding domains. In contrast, the flexible hinge and transmembrane domains have been commoditized and are the least studied components of the CAR. Here, we compared the hinge and transmembrane domains derived from either the CD8ɑ or CD28 molecule in identical GUCY2C-targeted third-generation designs for colorectal cancer. While these structural domains do not contribute to differences in antigen-independent contexts, such as CAR expression and differentiation and exhaustion phenotypes, the CD8ɑ structural domain CAR has a greater affinity for GUCY2C. This results in increased production of inflammatory cytokines and granzyme B, improved cytolytic effector function with low antigen-expressing tumor cells, and robust anti-tumor efficacy in vivo compared with the CD28 structural domain CAR. This suggests that CD8α structural domains should be considered in the design of all CARs for the generation of high-affinity CARs and optimally effective CAR-T cells in solid tumor immunotherapy.