Non-signaling Chimeric Antigen Receptors Enhance Antigen-Directed Killing by γδ T Cells in Contrast to αβ T Cells

Chimeric antigen receptor (CAR)-modified T cells have demonstrated efficacy against B cell leukemias/lymphomas. However, redirecting CAR T cells to malignant T cells is more challenging due to product-specific cis- and trans-activation causing fratricide. Other challenges include the potential for product contamination and T cell aplasia. We expressed non-signaling CARs (NSCARs) in γδ T cells since donor-derived γδ T cells can be used to prevent product contamination, and NSCARs lack signaling/activation domains, but retain antigen-specific tumor cell-targeting capability. As a result, NSCAR targeting requires an alternative cytotoxic mechanism, which can be achieved through utilization of γδ T cells that possess major histocompatibility complex (MHC)-independent cytotoxicity. We designed two distinct NSCARs and demonstrated that they do not enhance tumor-killing by αβ T cells, as predicted. However, both CD5-NSCAR- and CD19-NSCAR-modified γδ T cells enhanced cytotoxicity against T and B cell acute lymphoblastic leukemia (T-ALL and B-ALL) cell lines, respectively. CD5-NSCAR expression in γδ T cells resulted in a 60% increase in cytotoxicity of CD5-expressing T-ALL cell lines. CD19-NSCAR-modified γδ T cells exhibited a 350% increase in cytotoxicity against a CD19-expressing B-ALL cell line compared to the cytotoxicity of naive cells. NSCARs may provide a mechanism to enhance antigen-directed anti-tumor cytotoxicity of γδ T cells through the introduction of a high-affinity interaction while avoiding self-activation.