Bispecific CD33/CD123 targeted chimeric antigen receptor T cells for the treatment of acute myeloid leukemia

Abstract

CD33 and CD123 are expressed on the surface of human acute myeloid leukemia blasts and other noncancerous tissues such as hemopoietic stem cells. On-target off-tumor toxicities may limit chimeric antigen receptor T cell therapies that target both CD33 and CD123. To overcome this limitation we developed bispecific human CD33/CD123 CAR T cells with an "AND" Logic gate. We produced novel CD33 and CD123 scFvs from monoclonal antibodies that bound CD33 and CD123 and activated T cells. Screening of CD33 and CD123 CAR T cells for cytotoxicity, cytokine production, and proliferation was performed and we selected scFvs for CD33/CD123 bispecific CARs. The bispecific CARs split 4-1BB costimulation on one scFv and CD3ζ on the other. In vitro testing of cytokine secretion and cytotoxicity resulted in selecting bispecific CAR 1 construct for in vivo analysis. The CD33/CD123 bispecific CAR T cells were able to control AML in a xenograft AML mouse model similar to monospecific CD33 and CD123 CAR T cells while showing no on-target off-tumor effects. Based on our findings, human CD33/CD123 bispecific CAR T cells are a promising cell-based approach to prevent AML and support clinical investigation.