Gamma-delta T cell CARs; a combinatorial approach to immunotherapy

Abstract

Background: The ability of Vgamma9+ gammagamma T cells to target cancer cells via recognition of phosphoantigens has resulted in treatments such as ImmuniCell, which is currently being evaluated in an adaptive phase II/III study. In addition, we have developed gammagamma T cells expressing chimeric antigen receptors (CARs) to maximise the therapeutic potential of both gammagamma T cell and CAR-T therapy. We rationally designed a CAR construct that takes advantage of the defined antigen specificity of Vgamma9+ gammagamma T cells resulting in a potential cellular therapy with enhanced effector functions whilst minimising 'on-target, off-tumour' side effects. Methods: A CD19 targeting CAR was designed comprising costimulatory domains from CD28 and CD137 ('signal-2'). This design was tested against a classical CAR, comprising the aforementioned costimulatory domains plus a 'signal-1' providing CD3zeta activation domain. Vgamma9+ gammagamma T cells from healthy individuals were expanded in culture, and CAR expression achieved by lentiviral transduction. Ability of transduced gammagamma T cells to target CD19+ cancer cell lines, RAMOS and DAUDI, was assessed using a flow cytometry based cytotoxicity assay. Results: gammagamma T cells were successfully transduced with both CAR constructs, these were discriminated by qPCR using two primer sets. gammagamma T cells transduced with either construct exhibited increased cytotoxicity against the CD19+ DAUDI and RAMOS target cells. Strikingly, a 3-fold increase in cytotoxicity was measured against RAMOS cells, which usually display low sensitivity to unmodified gammagamma T cell-mediated killing. Conclusions:We have demonstrated a novel gammagamma specific CAR design that does not require incorporation of a CD3zeta signalling domain to elicit effector function. 'Signal 1' is provided by the gammagamma T cell receptor (TCR) resulting in a TCR-tuneable CAR construct. As healthy cells do not accumulate phosphoantigens, this CAR design should only elicit effector function against cancer cells, reducing 'on-target, off tumour' side effects - a major safety concern. Moreover, CAR expressing gammagamma T cells may result in a potent targeted treatment, as both phosphoantigens and the CAR target contribute to identification and killing of cancerous cells in vivo.