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Modulation of anti-tumour activity of ex vivo expanded NK cells against neuroblastoma via HDACi and PD1/PD-L1 blockade

  • S. Shen
    Affiliations
    Blood & Marrow Transplant Program, Kids Cancer Centre, Sydney Children's Hospital, Randwick, New South Wales, Australia

    Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia

    Children's Cancer Institute for Medical Research, Sydney, New South Wales, Australia
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  • T. O'Brien
    Affiliations
    Blood & Marrow Transplant Program, Kids Cancer Centre, Sydney Children's Hospital, Randwick, New South Wales, Australia

    Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia

    Children's Cancer Institute for Medical Research, Sydney, New South Wales, Australia
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      Neuroblastoma (NB) is an aggressive childhood malignancy with limited treatment options for patients with relapsed and refractory disease. Natural killer (NK) cells are highly cytotoxic immune cells that mediates cytotoxicity against NB, suggesting the possibility of NK cell infusions as a treatment. Human tumours however, develop multiple resistance mechanisms to evade immune destruction. Thus, combining NK therapy with immune-modulatory agents might be beneficial. In this study, we investigated the cytolytic activity of ex vivo expanded NK cells against NB, either alone or in combination with histone deacetylase inhibitor (HDACi) and/or Programmed death 1 (PD-1) blocker. NK cells were expanded from peripheral blood mononuclear cells by co-culture with irradiated K562-mbIL15-41BBL cell line (from Prof. D. Campana, National University of Singapore), and exhibited robust cytotoxicity against NB cell lines (SK-N-SH, SK-N-AS and SK-N-Be2), with killing ranging from 32.7 ± 10.8% to 65.8 ± 1.8% at E:T ratio of 1:1. Activation of NK cells with IL2 (1000 IU/ml) resulted in increased expression of activation receptors NKG2D and NKp30, NKp44, as well as surface expression of PD-1. PD-1 expression was also up-regulated on NK cells following co-culture with target NB cells. Treatment of NB cells with HDACi entinostat (ENT) up-regulated surface expression of NKG2D ligand MIC A/B as well as PD1 receptors PD-L1 & PD-L2. Blockade of PD1 via neutralizing mAb marginally increased NK cytolitic activity against NB, use of both anti-PD1 and ENT further increased killing by 1.5 ± 0.3 fold compared to NK alone. In subcutaneous NB model (5 × 105 SK-N-Be2 cells), immunodeficient NSG mice either received no cell therapy, or NK cells (1 × 107/mouse/treatment), or NK cells in combination with either ENT (3 mg/kg) or anti-PD-1 (10 mg/kg) or both. Treatments repeated 4 times on Days 4,7,10 & 13 post NB inoculation. NK cell treatment significantly delayed tumour progression 3 weeks post inoculation with overall significantly smaller tumour volume compared to no-NK group; and prolonged medium survival from 26 days to 29 days. While treatment with ENT or anti-PD-1 alone did not significantly improve survival, combined treatment of ENT & PD-1 blocker further increase medium survival to 33 days (p < 0.05). Taken together, our data suggest that NK cells are active anti-tumor cell therapy agents, the use of HDACi and PD-1/PD-L1 blockade acts to further increase the efficacy of NK cell therapy against NB.
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