CAR T cells recognizing CD276 and Dual-CAR T cells against CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models

Abstract

AbstractBackgroundRhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, whose prognosis is still poor especially for metastatic, high-grade, and relapsed RMS. New treatments are urgently needed, especially systemic therapies. Chimeric Antigen Receptor T cells (CAR Ts) are very effective against hematological malignancies, but their efficacy against solid tumors needs to be improved. CD276 is a target upregulated in RMS and detected at low levels in normal tissues. FGFR4 is a very specific target for RMS. Here, we optimized CAR Ts for these two targets, alone or in combination, and tested their anti-tumor activityin vitroandin vivo.MethodsFour different single-domain antibodies were used to select the most specific FGFR4-CAR construct. RMS cell killing and cytokine production by CD276- and FGFR4-CAR Ts expressing CD8α or CD28 HD/TM domains in combination with 4-1BB and/or CD28 co-stimulatory domains were testedin vitro.The most effective CD276- and FGFR4-CAR Ts were used to generate Dual-CAR Ts. Tumor killing was evaluatedin vivoin three orthotopic RMS mouse models.ResultsCD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3z) showed the strongest killing of RMS cells, and the highest release of IFN-γ and Granzyme Bin vitro. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3z) showed the most specific killing. CD276-CAR Ts successfully eradicated RD- and Rh4-derived RMS tumorsin vivo, achieving complete remission in 3/5 and 5/5 mice, respectively. In CD276lowJR-tumors, however, they achieved complete remission in only 1/5 mice. FGFR4 CAR Ts instead delayed of Rh4 tumor growth. Dual-CAR Ts promoted Rh4-tumors clearance in 5/5 mice.ConclusionsCD276- and CD276/FGFR4-directed CAR Ts showed effective RMS cell killingin vitroand eradication of CD276highRMS tumorsin vivo. CD276lowtumors escaped the therapy showing a correlation of antigen density and effectiveness. FGFR4-CAR Ts showed specific killingin vitrobut could only delay RMS growthin vivo. Our results show that combined expression of CD276-CAR with other CAR does not reduce its benefit. Introducing immunotherapy with CD276-CAR Ts in RMS seems to be feasible and promising, although CAR constructs design and target combinations have to be further improved to eradicate tumors with low target expression.