The Combination of 41BB and CD28 Costimulatory Signals Endows Lower Affinity CAR T Cells with the Most Optimal Properties for Selective Targeting of the Tumor Associated Antigen CD38

T cells equipped with Chimeric Antigen Receptors (CARs) have been emerged as a potent therapy for cancer. The scarcity of tumor-specific surface antigens in several cancer types, however, prompted the research towards improved CAR designs to optimally target tumor associated antigens (TAA), which are also expressed on normal cells at lower densities. To this end, we recently demonstrated the feasibility of targeting CD38, a multiple myeloma (MM) associated TAA, without on-target/off-tumor effects, by designing CARs with optimally lowered affinity for the antigen. It is, however, largely unknown whether lowering the antigen affinity affects the costimulatory requirements of CAR T cells for optimal expansion and in vivo persistence. Therefore we now evaluated the impact of different costimulation designs on CD38-CARs with lower affinity. Previous reports indicate that a CAR design including CD28 costimulation improves the cytotoxic potential of CAR T cells whereas a configuration involving the 4-1BB-costimulation confers more persistent responses. Therefore, we used CARs with 5 different affinities (Kd range 1.8 - >1915 nM), in combination with 3 different described costimulatory designs (28z, 4-1BBz and 28z + 4-1BBL). Lowering the affinity of CAR gradually diminished the cytolytic activity of CAR T cells with a 4-1BB costimulatory domain. Such an effect was however not observed with CARs containing the CD28 costimulatory domain, alone or expressed in combination with 4-1BBL. In vitro as well as in vivo experiments revealed that 28z + 4-1BBL expressing low affinity CD38-CAR T cells possessed a significantly better discriminative capacity between tumor and normal cells, displayed a more clear central memory phenotype, showed better in vitro expansion and improved in vivo persistence compared to the other two configurations tested. More importantly, the 28z + 4-1BBL design potentiated even CARs bearing scFvs with immeasurable affinity (>1915nM) and brought them to the functional level of 4-1BB- and 28z-based CARs of higher affinities. In conclusion, we show that the type of costimulatory domain is of importance when fine-tuning the CAR affinity. A combination of 4-1BB and CD28 signals provides the most optimal costimulation to affinity-tuned CAR T cells and suggests the careful construction of TAA-targeting CARs to enhance their clinical potential.