Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer

Abstract

The increasing evidence that gammadelta T cells have potent antitumor activity suggests their value in immunotherapy, particularly in areas of unmet need such as metastatic carcinoma. To this end, we initiated a phase I clinical trial in metastatic hormone-refractory prostate cancer to examine the feasibility and consequences of using the gammadelta T-cell agonist zoledronate, either alone or in combination with low-dose interleukin 2 (IL-2), to activate peripheral blood gammadelta cells. Nine patients were enlisted to each arm. Neither treatment showed appreciable toxicity. Most patients were treated with zoledronate + IL-2, but conversely only two treated with zoledronate displayed a significant long-term shift of peripheral gammadelta cells toward an activated effector-memory-like state (T(EM)), producing IFN-gamma and perforin. These patients also maintained serum levels of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), consistent with a parallel microarray analysis showing that TRAIL is produced by gammadelta cells activated via the T-cell receptor and IL-2. Moreover, the numbers of T(EM) gammadelta cells showed a statistically significant correlation with declining prostate-specific antigen levels and objective clinical outcomes that comprised three instances of partial remission and five of stable disease. By contrast, most patients treated only with zoledronate failed to sustain either gammadelta cell numbers or serum TRAIL, and showed progressive clinical deterioration. Thus, zoledronate + IL-2 represents a novel, safe, and feasible approach to induce immunologic and clinical responses in patients with metastatic carcinomas, potentially providing a substantially increased window for specific approaches to be administered. Moreover, gammadelta cell phenotypes and possibly serum TRAIL may constitute novel biomarkers of prognosis upon therapy with zoledronate + IL-2 in metastatic carcinoma.

FIGURE 1

γδ T-cell numbers and activation markers in patients treated with zoledronate or zoledronate + IL-2. PBMC were obtained from patients treated with zoledronate alone (A) or with zoledronate + IL-2 (B), before starting treatment (white columns) and 3 mo (gray columns), 9 mo (black columns), and 12 mo (hatched columns) after treatment. Where no data are shown, patients had already died. PBMC were double stained with anti-CD3 and anti-Vδ2 mAbs and the absolute number of Vγ9Vδ2 T cells was calculated. The X-axis labeling indicates individual patients and their clinical status (P, progression; SD, stable disease; PR, partial remission). PBMC were stained with anti-CD3, anti-Vδ2, anti-CD69, and anti–HLA-DR mAbs. Columns, mean percentage of γδ T cells expressing CD69 (C) and HLA-DR (D) molecules in patients treated with zoledronate alone (empty columns) or with zoledronate + IL-2 (filled columns); bars, SD.

FIGURE 2

Distribution of γδ T-cell subsets in patients treated with zoledronate + IL-2. PBMC were obtained from seven patients treated with zoledronate + IL-2, before starting treatment (month 0), and 3, 9, and 12 mo after treatment. After four-color staining of PBMC with anti-CD3, anti-Vδ2, anti-CD45RA, and anti-CD27 mAbs, the absolute number of Vγ9Vδ2 T_naive (CD45RA+CD27+), T_CM (CD45RA−CD27+), T_EM (CD45RA−CD27−), and T_EMRA (CD45RA+CD27−) cells was calculated. Top, bottom, and line through the middle of the boxes, 75th, 25th, and 50th percentiles. Lines that extend from the boxes, the highest and the lowest values from each subgroup. Lines within the boxes, median values.

FIGURE 3

Proliferation, IFN-γ production, and cytotoxic activity of γδ T cells in patients treated with zoledronate + IL-2. PBMC were obtained from seven patients treated with zoledronate + IL-2, before starting treatment (month 0), and 3, 9, and 12 mo after treatment. Cells were cultured in vitro with IPP and IL-2, as described in Materials and Methods. Proliferation was evaluated 7 d later, whereas IFN-γ production and BLT-esterase activities were assessed in the 48 and 24 h supernatants, respectively. O.D., absorbance. Points, mean; bars, SD.

FIGURE 4

A, Kaplan-Meier curves for 12-mo survival according to treatment with zoledronate (dotted lines) or zoledronate + IL-2 (black-filled lines). P < 0.05 by the log-rank, Breslow (generalized Wilcoxon) or Tarone-Ware tests. B, echo-color Doppler imaging of the prostate of patient 1B before (top) and 12 mo (bottom) after zoledronate + IL-2 therapy. White arrow (top), an area of hypervascularization at the level of a hypoechogenic nodule (the darkest area surrounding the white dotted line), which contains malignancy. Twelve months after therapy, the hypoechogenic area is reduced and only one normal vessel is found in its context, indicating tumor regression. Green box, the area in which the echo-color Doppler signal was registered.

FIGURE 5

Correlation between γδ T-cell numbers and clinical outcome. A, numbers of total γδ cells and (B) Vδ2+ γδ T_EM cells assessed at 9 mo after therapy are shown in three patients with partial remission, five patients with stable disease, and three patients with progression. •, patients treated with zoledronate + IL-2; △, patients treated with zoledronate alone. C, inverse correlation between numbers of total γδ cells and (D) of Vδ2+ γδ T_EM cells and PSA levels, as assessed at 9 mo after therapy, in three patients with partial remission, five patients with stable disease, and three patients with progression (P). •, patients treated with zoledronate+IL-2; △, patients treated with zoledronate alone.

FIGURE 6

Flow cytometry for intracellular TRAIL on cultured PBMC (A), influence of treatment (zoledronate ± IL-2) on serum TRAIL concentrations (B), and association between TRAIL concentrations and clinical outcome (C). A, percentage of Vγ9+CD3+ cells positive for TRAIL on day 3 (n = 5 healthy volunteers). Points, mean; bars, SE. B, TRAIL concentration in serum of patients treated with zoledronate only or zoledronate + IL-2, analyzed at indicated time points. See the legend to Fig. 2. C, TRAIL concentrations assessed at 9 mo after therapy are shown for three patients with partial remission, five patients with stable disease, and three patients with progression. Lines, median values.