Combined immunotherapy with anti-PDL-1/PD-1 and anti-CD4 antibodies cures syngeneic disseminated neuroblastoma

Valentina Rigo,Franco Locatelli,Silvano Ferrini,Michela Croce,Antonio Daga,Simonetta Astigiano,Maria Valeria Corrias,Concetta Quintarelli,Laura Emionite

doi:10.1038/s41598-017-14417-6

Abstract

Anti-PD-1 or anti-PD-L1 blocking monoclonal antibodies (mAbs) have shown potent anti-tumor effects in adult cancer patients and clinical studies have recently been started in pediatric cancers, including high-risk/relapsing neuroblastoma (NB). Therefore, we studied the effects of anti-PD-1/PD-L1 mAbs in two syngeneic models of disseminated NB generated by the injection of either Neuro2a or NXS2 cells, which express PD-L1. In addition, we tested the combination of these agents with the immune-enhancing cytokine IL-21, the Ecto-NTPDase inhibitor POM-1, an anti-CD25 mAb targeting Treg cells, or an anti-CD4 mAb. We previously showed that CD4-transient depletion removes CD4+CD25+ Treg cells and other CD4+CD25− regulatory subsets. Here we show that mono-therapy with anti-PD-1/PD-L1 mAbs had no effect on systemic NB progression in vivo, and also their combination with IL-21, POM-1 or anti-CD25 mAb was ineffective. The combined use of anti-PD-1 with an anti-CD4 mAb mediated a very potent, CD8-dependent, synergistic effect leading to significant elongation of tumor-free survival of mice, complete tumor regression and durable anti-NB immunity. Similar results were obtained by combining the anti-PD-L1 and anti-CD4 mAbs. These findings indicate that both PD-1/PD-L1 and CD4+ T cell-related immune-regulatory mechanisms must be simultaneously blocked to mediate therapeutic effects in these models.

Highlights

Immune checkpoints are fundamental for the physiological maintenance of tolerance and protection of tissues from the damage that an unregulated immune response may cause
The lack of effects of anti-PD-L1 or anti-Programed Death-1 (PD-1) monoclonal antibodies (mAbs) mono-therapy in our NB model does not seem related to differences in the type or dosage of mAb used, since the mAb clone and the doses are the same reported to be successful in other experimental models[31]
It is well known that the conversion of Treg cells from precursors and/or their expansion within the tumor microenvironment may represent an important mechanism of immune evasion[32]

Summary

Introduction

Immune checkpoints are fundamental for the physiological maintenance of tolerance and protection of tissues from the damage that an unregulated immune response may cause. We previously showed that immunotherapy with the immune-enhancing cytokine IL-2119 in combination with an anti-CD4 mAb was highly effective in a syngeneic model of disseminated NB, through the activation of a cytotoxic T-lymphocyte (CTL) response[20,21]. In this NB model, tumor progression leads to an increase of CD4+CD25+FoxP3+ Treg cells, CD4+CD25− T cells mediated immune regulatory functions, as the use of anti-CD25 mAb was ineffective[20]. We explored the possible cooperative effects of anti-PD-1 mAb combined with either IL-21, as immune enhancing agent, or with an anti-CD25 mAb or with the NTPDase-inhibitor POM-1, which target Treg cells, or with a cell-depleting anti-CD4 mAb, which transiently depletes the whole CD4+ T-cell population

Methods

Results

Conclusion