Adoptive cellular therapy using T cells programmed with chimeric antigen receptor (CAR) to target tumor cells has demonstrated tremendous efficacy in numerous pre-clinical and clinical trials. However, current FDA approved therapies are autologous, T cells collected from a patient will be manufactured and used to treat that single patient, making this type of therapy costly, labor intensive, and difficult to efficiently deliver to all patients in need1. Allogeneic, or “off-the-shelf” CAR-Ts could offer a potential solution to some of these hurdles. In most studies CARs are transduced into conventional αβ T cells, while the potential of introducing CARs into other cell types has yet received relatively little attention.In human, 1-5% of circulating T cells express γδ TCR. γδ TCR-CD3 complex are responsible for antigen stimulation of γδ T cells and the downstream signaling cascade for T cell activation. Unlike αβ TCR, γδ TCR directly recognizes tumor in the non-MHC restricted fashion, which makes γδ T cells a better CAR carrier for allogeneic cellular therapy because they do not cause graft-versus-host-disease (GvHD). In chapter 2, we discussed the unique features of different subsets of human γδ T cells and their antitumor properties. We also summarized the past and the ongoing pre-clinical studies and clinical trials utilizing γδ T cell-based cancer immunotherapy. Human Vγ9Vδ2 T cells are the predominant γδ T cell population in peripheral blood. They bind to butyrophilin-3A1/2A1 (BTN3A1/2A1) complex on various tumors, and can be selectively expanded in vivo and ex vivo by Zoledronate (ZOL), a clinically approved aminobisphosphonate drug for treatment of osteoporosis and cancer. Despite some positive results from clinical trials for cancer treatments using this subset of T cells, many patients’ γδ T cells failed to respond to ZOL and did not show robust cytotoxicity against tumors. The more recent studies have suggested that not all Vγ9Vδ2 T cells are identical, and there is a huge variation between people. In chapter 3 of this dissertation, we present a new screening strategy to select and expand peripheral blood mononuclear cell (PBMC)-derived Vγ9Vδ2 T cells that are more powerful against tumors. In addition, engineering of these γδ T cells with CAR and interleukin 15 (IL-15) transgenes to enhance the efficacy and persistence of the cells is shown in this chapter. The current method of generating Vγ9Vδ2 T cells for adoptive cell therapy involves either in vitro or in vivo expansion of PBMC-derived γδ T cells using aminobisphosphonates, such as ZOL. However, this method generates highly variable yields of γδ T cells depending on PBMC donors; and most importantly, such a γδ T cell product will likely contain conventional αβ T cells and thereby incurring GvHD risk in patients receiving these allogeneic cell products. A novel method that can reliably generate a homogenous monoclonal population of γδ T cells at large quantities with a feeder-free differentiation system is thus pivotal to developing an off-the-shelf γδ T cell therapy. In chapter 4, we demonstrate a novel feeder free-serum free in vitro culture system to generate Vγ9Vδ2 T cells from hematopoietic stem cells (HSCs). This approach provides an alternative strategy to generate γδ T cells, and overcomes the challenges of using PBMC-derived γδ T cells for cancer treatments. In the last chapter, we summarize our works and point out important future directions that may advance the field of γδ T cell‐based cancer immunotherapy.

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