Combination of immunodoulatory cyclophosphamide with anti-PD-1 monoclonal antibody therapy to improve survival in preclinical models of neuroblastoma

Abstract

Neuroblastoma (NB) is one of the most common childhood cancers, constituting 8% of paediatric cancers, and 15% of paediatric cancer deaths. The majority of patients have high risk disease, with poor outcome despite intensive treatment regimens. Current therapies, which include chemotherapy and radiotherapy, are highly toxic, with significant treatment related mortality, and there is little scope for further intensification. Alternatives strategies, such as immunotherapy are a priority for improving patient outcomes. A number of different immunotherapies, such as antiGD2 antibody therapy, have shown promise in NB, but combining these with conventional chemotherapy regimens is challenging. Classically, chemotherapy has been regarded as immunosuppressive but recent work has highlighted that this may not entirely be accurate for certain immunogenic chemotherapies. Furthermore, immunomodulatory antibodies such as checkpoint inhibitors have shown efficacy in adult cancers such as melanoma, with little investigation into their potency in paediatric tumours. Although NB tumours are not particularly inherently immunogenic, the application of an ‘immunogenic’ chemotherapy may render them susceptible to further immunomodulatory immunotherapy.

Firstly this study aimed to identify the immunomodulating effects of cyclophosphamide (CPM) treatment on immune cells, within murine NB models. To achieve this, CPM’s ability to induce ‘immunogenic cell death’ (ICD) markers both in vitro and in vivo was investigated. Induction of ectoCRT, Hsp-70 and HMGB1 expression was observed. Secondly, effects of different doses of CPM on the immune cell infiltrates in the tumour microenvironment were studied in vivo using syngeneic murine NB models. It was demonstrated that low dose CPM was able to selectively deplete and possibly reduce the suppressive activity tumour infiltrating Treg cells in both NXS2 and 9464D NB models, alongside maintaining and activating CD8+ and CD4+ T cells. Additionally, using an apoptosis resistant NB cell line and mouse strain, investigation into whether ICD induction or Treg depletion is most critical aspect of CPM immunomodulation was conducted. It was observed that overexpression of BCL-2 either in the tumour cell line in vitro or Treg cells in vivo, prevented apoptosis and CPM dependent depletion.

Finally, it was assessed whether immunogenic CPM could be efficiently combined with immunomodulatory antibody therapy to slow tumour growth, utilising a combination of syngeneic and spontaneous transgenic NB models. Experiments utilising the three in vivo models demonstrated that combination of CPM with anti-PD-1 antibody led to an increase in survival over monotherapies alone, which was further enhanced by a metronomic weekly dosing strategy. Ongoing work is exploring this approach, to refine and establish the most effective chemotherapy and antibody combination in NB preclinical models, along with defining the mechanisms behind combination efficacy.

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Identifiers

ORCID for Juliet Gray: orcid.org/0000-0002-5652-4722

ORCID for Stephen Beers: orcid.org/0000-0002-3765-3342

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