Selective TRAF over-expression enhances CD19-targeted 41BB CAR T function by increasing NF-κB

Abstract

Abstract The prototypical second-generation CD19-targeted CAR (chimeric antigen receptor) features a costimulatory domain, either CD28 or 41BB, with both designs achieving comparable clinical outcomes against B cell malignancies. However, clinical application of CAR T cells has outpaced the understanding of their functions in vivo. We evaluated fully murine CD19-targeted CAR T cells containing CD28 or 41BB endodomain in an immune competent B-ALL (B cell acute lymphoblastic leukemia) mouse model. Mouse 41BB CAR T cells imparted inferior survival to mouse CD28 CAR T cells at a stress-test dose. Replacing mouse 41BB endodomain with human 41BB sequence significantly increased NF-κB signaling in 41BB CAR T cells and enhanced survival and persistence to match the mouse CD28 CAR T cells. TRAF binding and its subsequent NF-κB signaling are more important to 41BB than CD28 CAR T functions as evidenced by TRAF1 deficient 41BB but not CD28 CAR T cells showing inferior in vivo B cell killing and CAR T persistence to wild type CAR T cells. Also, 41BB CAR T cells with impaired TRAF binding showed remarkably decreased NF-κB signaling, viability and proliferation in vitro. Over-expressing TRAF2 or TRAF3 but not TRAF1 significantly enhanced CD19 targeted 41BB CAR T functions, such as cytotoxicity, viability and proliferation. Our study demonstrates how co-stimulatory domains impact CAR T functions and selective TRAF over-expression could be a new strategy to improve current CAR T therapy.