G-CSF as a suitable alternative to GM-CSF to boost dinutuximab-mediated neutrophil cytotoxicity in neuroblastoma treatment

Abstract

BackgroundCurrent immunotherapy for patients with high-risk neuroblastoma involves the therapeutic antibody dinutuximab that targets GD2, a ganglioside expressed on the majority of neuroblastoma tumors. Opsonized tumor cells are killed through antibody-dependent cellular cytotoxicity (ADCC), a process mediated by various immune cells, including neutrophils. The capacity of neutrophils to kill dinutuximab-opsonized tumor cells can be further enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which has been shown in the past to improve responses to anti-GD2 immunotherapy. However, access to GM-CSF (sargramostim) is limited outside of Northern America, creating a high clinical need for an alternative method to stimulate dinutuximab responsiveness in the treatment of neuroblastoma. In this in vitro study, we have investigated whether clinically well-established granulocyte colony-stimulating factor (G-CSF) can be a potentially suitable alternative for GM-CSF in the dinutuximab immunotherapy regimen of patients with neuroblastoma.MethodsWe compared the capacity of neutrophils stimulated either in vitro or in vivo with GM-CSF or G-CSF to kill dinutuximab-opsonized GD2-positive neuroblastoma cell lines and primary patient tumor material. Blocking experiments with antibodies inhibiting either respective Fc gamma receptors (FcγR) or neutrophil integrin CD11b/CD18 demonstrated the involvement of these receptors in the process of ADCC. Flow cytometry and live cell microscopy were used to quantify and visualize neutrophil-neuroblastoma interactions.ResultsWe found that G-CSF was as potent as GM-CSF in enhancing the killing capacity of neutrophils towards neuroblastoma cells. This was observed with in vitro stimulated neutrophils, and with in vivo stimulated neutrophils from both patients with neuroblastoma and healthy donors. Enhanced killing due to GM-CSF or G-CSF stimulation was consistent regardless of dinutuximab concentration, tumor-to-neutrophil ratio and concentration of the stimulating cytokine. Both GM-CSF and G-CSF stimulated neutrophils required FcγRIIa and CD11b/CD18 integrin to perform ADCC, and this was accompanied by trogocytosis of tumor material by neutrophils and tumor cell death in both stimulation conditions.ConclusionsOur preclinical data support the use of G-CSF as an alternative stimulating cytokine to GM-CSF in the treatment of high-risk neuroblastoma with dinutuximab, warranting further testing of G-CSF in a clinical setting.

CD47-SIRPα Checkpoint Inhibition Enhances Neutrophil-Mediated Killing of Dinutuximab-Opsonized Neuroblastoma Cells

“…Furthermore, the overall clinical response of neuroblastoma patients has been found to be further improved by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF), acting on myeloid cells including neutrophils, to the anti-GD2 treatment regime [ 14 , 21 , 22 , 23 , 24 ]. Overall, from the above-mentioned studies and a variety of others, it has further become apparent that neutrophils can be stimulated by cytokines such as GM-CSF or granulocyte colony-stimulating factor (G-CSF), given either alone or in combination with interferon-gamma (IFNγ), in order to improve their in vitro IgG-mediated cytotoxicity [ 25 , 26 , 27 , 28 , 29 ].…”

Correspondence on "G-CSF as a suitable alternative to GM-CSF to boost dinutuximab-mediated neutrophil cytotoxicity in neuroblastoma treatment" by Martinez Sanz et al

“…Previous work has shown that G-CSF may not be an appropriate agent for patients with high-risk neuroblastoma, as neuroblastoma cell lines express mRNA for the G-CSF receptor and are stimulated in response to exogenous G-CSF, leading to a more aggressive biologic behavior and increased invasiveness. Martinez Sanz et al 1 reported in vitro G-CSF did not show increased neuroblastoma cell growth or a change in the susceptibility of neuroblastoma to neutrophil-mediated cytotoxicity as compared with the control, and no evidence of effect on the neuroblastoma phenotype.…”

“…We read with great interest the work by Martinez Sanz et al 1 ‘G-CSF as a suitable alternative to GM-CSF to boost dinutuximab-mediated neutrophil cytotoxicity in neuroblastoma treatment,’ published in the Journal for ImmunoTherapy of Cancer on May 28, 2021. The authors note access to recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF; sargramostim (yeast-derived)) is limited outside of North America, potentially leading to suboptimal treatment of patients with neuroblastoma and therefore necessitating an alternative agent to stimulate dinutuximab immunotherapy-responsiveness in the treatment of neuroblastoma.…”

“…Martinez Sanz et al 1 highlight the critical need for access to sargramostim for all patients with high-risk neuroblastoma. The benefits of combining anti-GD2 antibody and sargramostim therapy are well known, but since worldwide access is limited and sometimes cost prohibitive, the focus of research outside of North America has shifted to identifying alternatives.…”

Response to: Correspondence on "G-CSF as a suitable alternative to GM-CSF to boost dinutuximab-mediated neutrophil cytotoxicity in neuroblastoma treatment" by Moraet al

“…We appreciate the interest of Dr Mora and Dr Chantada 1 in our recently published work proposing granulocyte colony-stimulating factor (G-CSF) as a suitable alternative to improve antibody treatment of patients with high-risk neuroblastoma. 2 The authors strongly advocate finding ways to increase accessibility of granulocyte-monocyte colony-stimulating factor (GM-CSF, sargramostim), used in combination with dinutuximab in North America, also in countries where this treatment is currently not available. We fully agree that all relevant stakeholders should participate in finding a permanent solution to prevent potentially suboptimal treatment of patients with high-risk neuroblastoma in the absence of sargramostim.…”