γδ T cell therapy for the treatment of non-small cell lung cancer

Abstract

γδ T cells are attractive effector cells for cancer immunotherapy as they can secrete cytokines abundantly and exert potent cytotoxicity against a wide range of cancer cells. They comprise 1-5% of peripheral blood T cells, the majority expressing the Vγ9Vδ2 T cell receptor that recognizes phosphoantigens. Direct in vivo activation of Vγ9Vδ2 T cells in cancer patients as well as adoptive transfer of ex vivo expanded Vγ9Vδ2 T cells has been investigated in several clinical trials. We previously established a large-scale in vitro expansion method for Vγ9Vδ2 T cells using zoledronate and interleukin-2 (IL-2). We found that Vγ9Vδ2 T cells from patients with advanced cancer as well as from healthy donors underwent extensive proliferation under these conditions. Such cultured Vγ9Vδ2 T cells retained cytokine secretion capacity and mediated cytotoxicity against a variety of cancer cell lines. Recently, we conducted a phase I clinical study to evaluate safety and potential anti-tumor effects of re-infusing ex vivo expanded γδ T cells in patients with advanced or recurrent non-small-cell lung cancer (NSCLC) refractory to or intolerant of current conventional treatments. There were no severe adverse events related to the therapy. All patients remained alive during the study period with a median survival of 589 days and median progression-free survival of 126 days. Six patients had stable disease (SD), whereas the remaining six evaluable patients experienced progressive disease (PD) four weeks after the sixth transfer. We conclude that adoptive transfer of zoledronate-expanded γδ T cells is safe and feasible in patients with NSCLC, refractory to other treatments.

Keywords: Non-small-cell lung cancer (NSCLC); adoptive transfer; cell therapy; γδ T cells.

Figure 1

Lymphoid stress surveillance (A) and Tissue resident γδ T cells (B). (A) Injury or stress that upregulate surface expression of ligands for γδ TCR or for NKG2D initiate the lymphoid stress-surveillance response; (B) The intrathymic developmental programming determines the tissue distribution of γδ T cells. The emerging γδ thymocyte subsets with distinct transcriptional modules bear particular TCR V regions, which control the acquisition of discrete sets of HRs and effector functions. Abbreviations: TCR, T cell receptor; HR, homing receptor.

Figure 2

Tumor cell ligands recognized by human γδ T cells. Left panel, IPP is an intermediate metabolite produced through the mevalonate/cholesterol production pathway in mammalian cells. Pharmacological agents that can block upstream (statins) or downstream (aminobisphosphonate) this pathway lead to decreased or increased intracellular IPP levels, respectively. Endogenous IPP accumulation is observed in diverse tumor cells; IPP metabolites can be converted into ApppI, which could then be presented at the cell surface with much higher affinity to γδ TCR than IPP. Right panel, in pathogen-infected cells (e.g., mycobacterial infection), bacterial HMB-PP, or HDMAPP produced through the DOXP pathway could be presented. Abbreviations: IPP, isopentenyl pyrophosphate; TCR, T cell receptor; HMB-PP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate; HDMAPP, 4-hydroxy-3-dimethylallyl pyrophosphate.

Figure 3

Strategies for γδ T cell based immunotherapy. Left panel, the adoptive cell transfer of in vitro expanded γδ T cells. Right panel, the in vivo activation of γδ T cells by phosphoantigens (e.g., BrHPP) or aminobisphosphonates and low-dose IL-2. The concomitant injection of aminobisphosphonate leads to intracellular accumulation of IPP/ApppI in tumor cells by blocking the mevalonate pathway, resulting in the sensitization of tumor cells to γδ T cells. Abbreviations: BrHPP, phosphorylated bromohydrin; IL-2, interleukin-2; IPP, isopentenyl pyrophosphate.

Figure 4

Typical phenotype of zoledronate-expanded γδ T cells by flow cytometric analysis. Fresh peripheral blood mononuclear cells or cells cultured for 14 days were stained with anti-CD3 and TCRVγ9 mAb. The CD3⁺Vγ9⁺ population was further analyzed with anti-CD27, CD45RA, NKG2D or CD69 mAbs.

Figure 5

γδ T cell cytotoxicity. γδ T cells (green staining with CFSE) recognized and killed the lung cancer cell line, NCI H460 (red staining with PKH-26), by direct contact. In tumor cells attacked by the γδ T cells, collapse of the cell membranes led to apoptosis. It took approximately 2 h to progress from A to C.