Zoledronate-activated Vγ9γδ T cell-based immunotherapy is feasible and restores the impairment of γδ T cells in patients with solid tumors

Abstract

Gamma/delta (γδ) T cells play a role in innate immunity and exhibit cytotoxicity toward a large range of tumor types. Recent studies have shown that aminobisphosphonates may be applied to a culture in which a large number of γδ T cells are proliferated ex vivo. We carried out a clinical study of 25 patients with various solid tumors to determine further the safety, immunologic effect and feasibility of zoledronate-activated Vγ9γδ T cell-based immunotherapy. No severe toxicity was observed. In the cells used for the first treatment, the total cell number, frequency and number of CD3⁺ Vγ9⁺ γδ T cells were 409 ± 284 × 10⁷ cells, 56 ± 33% and 255 ± 242 × 10⁷ cells, respectively. Aminobisphosphonate therapy or chemotherapy resulted in the suppression of CD3⁺ Vγ9⁺ γδ T-cell proliferation. The numbers of CD3⁺ T cells, CD3⁺ Vγ9⁺ γδ T cells and CD27⁻ CD45RA⁻ Vγ9⁺ subsets in peripheral blood were significantly lower in patients than in healthy subjects (P <y 0.05). From such an impaired immunologic condition, the numbers and frequencies of CD3⁺ Vγ9⁺ γδ T cells and CD27⁻ CD45RA⁻ subsets significantly increased in patients treated with this immunotherapy. Zoledronate-activated Vγ9γδ T cell-based immunotherapy that restores the number of Vγ9γδ T cells in cancer patients may provide another mode of adoptive immunotherapy.

Introduction

Autologous activated lymphocyte therapy (ALT) for use in cancer treatment was first introduced by Rosenberg et al. (1) in the late 1980s as a form of adoptive immunotherapy, and to date has been developed further. We have developed and advanced ALT in collaboration with various university hospitals (2,3). With this therapy, peripheral blood mononuclear cells (PBMC) are activated and proliferated in culture by stimulation with an immobilized anti-CD3 antibody (OKT3) and interleukin(IL)-2, and these lymphokine-activated killer (LAK) cells are administered repeatedly to patients (2., 3., 4.). Flow cytometry analyzes of cultured cells from 678 patients at our institute showed that approximately 90% of the cultured cells were CD3⁺ alpha/beta (αβ) T cells; gamma/delta (γδ) T cells were 7 ± 8%, and natural killer (NK) cells 7 ± 10% (2).

The majority of mature T lymphocytes in peripheral blood (PB) are αβ T cells that express αβ T-cell receptor (TCR), whereas a small minority of γδ T cells expresses γδ TCR. While the role of human γδ T cells in tumor immunity has yet to be fully understood, it has been shown that they play a role in innate immunity and exhibit major histocompatibility complex (MHC) unrestricted cytotoxicity against a large range of tumor types (5). The majority of human γδ T cells in PB express a TCR comprising the variable segments γ9 and δ2 (Vγ9γδ T cells). Pyrophosphates, including isopentenyl pyrophosphate (IPP) generated by mammalian cells through the alternative mevalonate pathway, activate Vγ9γδ T cells (6). Recent studies have shown that aminobisphosphonates (aBP) can alter this pathway. aBP such as pamidronate and zoledronate are used clinically for osteolytic bone disease. Recently, some indirect mechanisms have been shown to exist. aBP could activate and induce the proliferation of γδ T cells in vivo, which may eradicate tumor cells, resulting in the induction of anti-tumor activity (7,8).

These previous observations suggested that aBP may be applied to a culture in which γδ T cells are predominantly activated and proliferated ex vivo (2,9,10). This form of adoptive immunotherapy using a large number of activated Vγ9γδ T cells may provide another mode of cancer immunotherapy. We have established previously an efficient large-scale ex vivo expansion of Vγ9γδ T cells from various cancer patients using zoledronate, and characterization of the expanded cells has shown that they have potent cytotoxic activity against various types of tumor cell lines (11). We have carried out a clinical phase I study of six patients with multiple myeloma (MM) to evaluate the zoledronate-activated Vγ9γδ T cell-based immunotherapy (9). This study showed the safety and some efficacy of the therapy, which resulted in a significant increase in the numbers of Vγ9γδ T cells in the PB and bone marrow of the patients. In the current study, we have determined further the safety, immunologic effect and feasibility of this therapy in patients with various solid tumors. This therapy should be applicable to patients who undergo other beneficial therapies such as chemotherapy. The effect of concurrent therapies on the proliferation of Vγ9γδ T cells was also determined.