Bispecific T-cells Expressing Polyclonal Repertoire of Endogenous γδ T-cell Receptors and Introduced CD19-specific Chimeric Antigen Receptor

Even though other gamma delta T-cell subsets exhibit antitumor activity, adoptive transfer of gamma delta Tcells is currently limited to one subset (expressing V gamma 9V82 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating gamma delta T cells. Therefore, we developed an approach to propagate polyclonal gamma delta T cells and rendered them bispecific through expression of a CD1 9-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 gamma delta T-cell subsets. CAR(+)gamma delta T cells were expanded on CD19(+) artificial antigen-presenting cells (aAPC), which resulted in >10(9) CAR(+)gamma delta T cells from <10(6) total cells. Digital multiplex assay detected TCR mRNA coding for V delta 1, V delta 2, and V delta 3 with V gamma 2, V gamma 7, V gamma 8, V gamma 9, and V gamma 10 alleles. Polyclonal CAR(+)gamma delta T cells were functional when TCR gamma delta and CAR were stimulated and displayed enhanced killing of CD19(+) tumor cell lines compared with CAR(neg)gamma delta T cells. CD19(+) leukemia xenografts in mice were reduced with CAR(+)gamma delta 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 gamma delta T cells.