Optimization of Murine 4-1BB Signaling Results in Enhanced CD19-Targeted CAR T Cell Function in Immune Competent Mice

Adding a co-stimulatory domain to chimeric antigen receptor (CAR) design is the most critical innovation that allowed this therapy to be translated from the laboratory to the bedside. Early-stage clinical trials of CD19-targeted CAR T cell therapy have shown its potential for B cell malignancies with up to 90% complete response (CR) rates that are durable for patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Clinical benefit is also seen in patients with chemotherapy-resistant non-Hodgkin lymphoma (NHL). Despite these successes, the clinical application of this technology has far exceeded the understanding of its function in vivo. CD28 and 4-1BB are the two co-stimulatory domains that have been most widely evaluated and used in patients. Previous studies have suggested that 4-1BB has advantages over CD28 by reversing CAR T cell exhaustion and driving CAR T cells towards a central memory phenotype. In contrast, CD28 appears to provide marked expansion and IL2 production. Despite accumulating studies comparing 4-1BB CAR and CD28 CAR T cells, there has been no side-by-side comparison in immune competent pre-clinical animal models. Here we compared mouse CD19 targeting CARs containing CD28 or 4-1BB co-stimulatory domains in terms of in vitro proliferation, viability, cytokine production, as well as in vivo persistence in blood and bone marrow, and survival from leukemia. We found that, while mouse 4-1BB CAR and mouse CD28 CAR were functional in vitro, the 4-1BB containing CAR T cells offered much less survival and persistence than the CD28 containing CAR T cells. We have dissected the molecular mechanisms for the differences in vivo and with only a few amino acid substitutions are able to significantly enhance 4-1BB CAR T cell function. Our study sheds light on critical sequences that support optimal 4-1BB co-stimulation in CAR design and provides a platform for enhanced 3rd generation CARs.