Background Adoptive cellular immunotherapy as a new paradigm to treat tumors is exemplified by the FDA approval of six different chimeric antigen receptor (CAR)-T cell therapies targeting hematological malignancies in recent years. Conventional alpha/beta (ab) T cells applied in these therapies have proven efficacy but with hitherto disappointing outcomes when applied to solid tumors, largely due to poor survival and reduced efficacy of CAR-T cells in the immunosuppressive solid tumor microenvironment (TME). Moreover, these therapies are confined exclusively to autologous use since, upon infusion, they elicit devastating graft-versus-host disease (GvHD) in human leukocyte antigen (HLA)-mismatched patients. One way to overcome these challenges is to use allogeneic immune cell types, in particular gamma/delta (gd) T cells, which occupy the interface between innate and adaptive immune cells and recognize a wide variety of ligands expressed on transformed cells. Importantly, specific gd T cell subsets such as Vd1 T cells, have a natural propensity to home to solid tissues.

Methods We propose to harness these characteristics of gd T cells by generating CAR-T cells using cord blood (CB)- compared with peripheral blood (PB)-derived gd T cells. We expanded CB- and PB-gd T cells using a feeder cell-based protocol and transduced them with a HER2-targeting CAR bearing CD28 co-stimulatory endodomain and CD3z activation domain (4D5-28z CAR) via two-step retrovirus-based delivery. Both in vitro and in vivo cytotoxicity of 4D5-28z CB- and PB-gdT cells against selected solid tumor cell lines were assessed using bioluminescence-based methods.

Results We found that CB-gd cells were less amenable to CAR transduction, resulting in substantially lower frequencies of CAR-positive CB-gd T cells compared with PB-gd counterparts. Efforts are underway to optimize transduction to enhance CAR expression in CB-gd T cells. Despite fewer CAR-positive CB-gd T cells, we consistently observed that these cells exhibited increased in vitro cytotoxicity against SK-OV-3 ovarian tumor cells compared with non-CAR counterparts. However, our preliminary results demonstrated that both CAR- and non-CAR CB-gdT cells lacked persistence in vivo, alluding to the need for further genetic modifications of gd CAR-T cells to secrete cytokines which can help them overcome the immunosuppressive effects of TME and improve their in vivo functionality.

Conclusions Taken together, our findings highlight the potential of CB-gd T cells in allogeneic CAR immunotherapy by increasing the efficiency of their genetic engineering and in vivo anti-tumor efficacy.

Acknowledgements We thank staff of the Biological Resource Centre (BRC) for care and maintenance of mice and members of the laboratory for insightful discussions.

Ethics Approval Experiments with mice were approved by the Institutional Animal Care and Use Committee (IACUC) at BRC, A*STAR.