PO-413 Immune modulating properties of cyclophosphamide synergise with immunotherapy in preclinical models of neuroblastoma

Abstract

IntroductionNeuroblastoma (NB) is one of the most common childhood cancers accounting for 15% of paediatric cancer deaths. Current therapies, which include chemotherapy, are highly toxic with significant treatment related mortality. There is little scope for further intensification therefore alternatives strategies such as immunotherapy, are a priority for improving patient outcomes. Classically, chemotherapy is regarded as immunosuppressive but recent work has highlighted that some may illicit an immunogenic form of cell death. This work sets out to identify the immunomodulating effects of cyclophosphamide (CPM) on tumour infiltrating immune cells and whether these properties can synergise with immunomodulatory antibodies in preclinical models of NB.Material and methodsThe effects of CPM and doxorubicin on tumour cells were investigated in vitro by analyses of immunogenic cell death (ICD) markers. Two different in vivo subcutaneous murine NB models (NXS2 +NB9464D), were treated i.p with different doses of CPM. Tissues were harvested and detailed immunophenotyping performed. Combination of CPM and anti-PD-1 therapy was investigated in both models using tumour growth and survival as end points. Combination therapy was also assessed in TH-MYCN mice which develop spontaneous neuroblastoma.Results and discussionsChemotherapy application in vitro led to an increase in expression of the ICD markers, ecto-calreticulin and Hsp-70. CPM was found to have numerous immune modulating activities in vivo, including the reduction of intratumoral Treg cells, even at low doses (20 mg/kg), in both NB models. Combination therapy was shown to increase CD8+ and CD4+ percentages within tumours, along with an increase CD4+ effector and memory cell proportions. Anti-PD-1 therapy synergised with CPM to improve median survival and slow tumour growth. Metronomic dosing of CPM and anti-PD-1 antibody led to tumour regression in TH-MYCN tumour bearing mice.ConclusionThis work supports combining low dose CPM to enhance immunomodulatory antibody therapy, to generate therapeutic anti-neuroblastoma immunity. Ongoing work is focused on elucidating the mechanism of action of the CPM and anti-PD-1 combination therapy, along with identifying the optimal chemotherapy and antibody dosing strategies.