Bispecific T-cells expressing polyclonal repertoire of endogenous γδ T-cell receptors and introduced CD19-specific chimeric antigen receptor

Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR⁺ γδ T cells were expanded on CD19⁺ artificial antigen-presenting cells (aAPC), which resulted in >10 9 CAR⁺ γδ T cells from <10 6 total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR⁺ γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19⁺ tumor cell lines compared with CAR neg γδ T cells. CD19⁺ leukemia xenografts in mice were reduced with CAR⁺ γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.