Abstract
High-dose cytoreductive therapy and autologous transplantation have extended survival of patients with advanced neuroblastoma (NB), however, more targeted approaches are necessary to improve outcomes. Strategies that incorporate the cytolytic properties of γδ T cells represent a promising means of harnessing a broad-based innate recognition of stressed malignant cells. Cultured Vγ9Vδ2+ γδ T cells recognize non-peptide phosphoantigens and stress-associated NKG2D ligands expressed on malignant cells and exert a potent cytotoxic effect on NB cell lines and autologous NB in vitro. By blocking farnesyl pyrophosphate synthase in the mevalonate pathway of isoprenoid synthesis in monocytes, the aminobisphosphonate zoledronic acid (ZOL) promotes the accumulation of isopentenyl pyrophosphate, which is sensed by γδ T cells. Addition of IL-2 is also required for robust expansion γδ T cells. In this Phase 1 trial, we are examining a strategy to expand Vγ9Vδ2+ cells in vivo to determine if γδ T cells in sufficient number can be safely expanded. If successful, this strategy could be useful as a stand-alone therapy or in combination with infusion of autologous ex vivoexpanded γδ T cells.
The trial is a prospective, non-randomized trial that assesses two dose levels of recombinant IL-2 in combination with ZOL. Eligibility criteria include an age between 2 to 21 years and a diagnosis of refractory NB with no known curative therapeutic options. Treated patients (n= 3) received ZOL intravenously on day 1, and subcutaneous IL-2 on days 1 to 5 and 15 to 19 of every 28-day cycle. Cell counts and lymphocyte immunophenotyping were assessed weekly x 4 on ZOL/IL-2 treated patients. Bone marrow was examined for disease content and lymphocyte infiltration and tumor, when available, was examined for expression of NKG2D ligands. Cell counts and lymphocyte immunophenotyping were also performed at one time point on 16 healthy children between the age of 5 and 15 and nine Stage IV NB patients between the ages of 4 months and 18 years (disease controls).
Three patients were enrolled on the first cohort, each receiving an IL-2 dose of 3 x 106 IU/m2. The patient age ranged from 5 to 20 years at study entry. All patients were heavily pre-treated. Two patients received 1 cycle of therapy (B&C), and the other received 2 cycles (A). Expected grade 3 and 4 toxicities included electrolyte and blood count abnormalities. There were no unexpected grade 3 or 4 toxicities. There was one death attributed to tumor progression. Examination of the only available resected tumor revealed strong expression of the NKG2DL ULBP-4. Circulating CD3+, CD3+CD4, CD3+CD8+, CD16/56+, Treg and CD19+ did not differ significantly between healthy volunteers and NB patients. Interestingly, the CD3+γδ+ T cell count was significantly higher in healthy volunteers relative to NB disease controls and pre-treatment NB patients (212+93 vs. 89+42 p = 0.05). All three treated patients (A,B,C) showed a substantial increase in both γδ+ T cell percentage and count (3,14, and 10 fold) after the first week of ZOL/IL-2 therapy, however, treatment was only successful in raising γδ+ T cell counts into the range seen in healthy volunteers (125+37 p = 1.940). NK cells also increased (5, 2, and 5 fold). Interestingly, all ZOL/IL-2 treated patients also experienced a substantial increase in CD3+CD4+CD27hiCD127dim Tregcells that in patients B and C continued to increase weekly throughout the four weeks of observation (maximum 41% and 24% of total CD3+CD4+ T cells respectively).
The combination of ZOL and IL-2 at dose level 1 is well tolerated and restores γδ+ T cells to numbers characteristic of healthy volunteers. NK cells are also moderately expanded likely due to IL-2 effect. Progressive increases in circulating Treg cells may offset beneficial effects of this therapy and will require close observation in the second cohort.
No relevant conflicts of interest to declare.
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