4-1BBL costimulation enables CD4 T cells expressing a CD28/zeta chimeric antigen receptor to eliminate aggressive leukemia

Abstract Adoptive T-cell therapy using chimeric antigen receptor (CAR) engineered T cells is a promising cancer treatment. First generation CARs include an scFv targeting a surface antigen and the CD3ζ chain to direct tumor cell killing. CAR transduced T cells however need appropriate costimulation to proliferate and persist. Here we assessed in a B-cell leukemia xenograft model, the anti-tumor activities of human T cells transduced with an anti-CD19 CAR harboring the zeta chain alone (19z1+), fused to the CD28 or 4-1BB cytoplasmic domain (1928z+), (19BBz+), along with 4-1BBL (19z1-4-1BBL+), or with CD28 and 4-1BBL (1928z-4-1BBL+). We observed total tumor regression in NALM-6 bearing NSG mice treated with 1928z-4-1BBL+ T cells and no or partial regression in the other T-cell groups, as measured by bioluminescence imaging. 1928z-4-1BBL+ T cells showed the highest bone marrow accumulation and persisted the most. Experiments utilizing purified T cell subsets revealed that naïve and central memory 1928z-4-1BBL+ CD4+ T cells are sufficient to trigger leukemia eradication and are necessary to support CD8+ T-cell in vivo expansion. 1928z-4-1BBL+ CD4+ T cells displayed reduced apoptosis and increased cytotoxicity compared to 1928z+ CD4+ T cells. Thus, constitutive 4-1BBL expression and CAR-mediated CD28 signaling afforded the most efficient costimulatory combination to generate long-lived T cells capable of eradicating aggressive leukemia.