Mouse CD19-targeted CAR T cells with an optimized 4-1BB domain enhance protection against CD19+ leukemia and retain proliferation potential after long-term rest in vivo.

Abstract Chimeric antigen receptor (CAR) T cells have mediated exciting results for patients. The CAR is a novel antigen-receptor that includes an antigen-binding domain, derived from an antibody, linked to an intracellular T cell activation domain. This 1st generation CAR design worked well in vitro but mediated poor results in vivo. Modification of the CAR to include a co-stimulatory domain, classified as a 2nd generation CAR design, achieved optimal T cell persistence and tumor killing in mice, which served as the rationale for evaluation of CAR T cells in patients. The most common clinically evaluated CARs target CD19 and include CD28 or 4-1BB co-stimulatory domains. However, there has been no extensive comparison of 2nd generation CAR T cells in mice, which could serve as a model for how these domains impact CAR T cell function in patients. Therefore, we evaluated and compared the function of mouse CD19 targeted CARs modified with a CD28 or 4-1BB domain. In contrast to clinical results, mouse CD28 CAR T cells imparted better leukemia-killing and persistence than mouse 4-1BB CAR T cells. We identified a defect in CAR T cell signaling mediated by mouse 4-1BB and mutation of this domain restored CAR signaling and in vivo leukemia killing. Also, we determined that CAR T cells with the mutation-optimized 4-1BB co-stimulatory domain proliferated in response to antigen in the bone marrow (BM) after a > 1-month period of rest, while CAR T cells with the CD28 co-stimulatory domain did not. We determined that this is likely due to the acquisition of an exhausted phenotype in the CD28 CAR T cells. Our results illustrate how co-stimulation modulates CAR T cell function in mice and suggest mechanisms for its impact on persistence and cancer-killing in patients.