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Anti-CD20 antibodies such as rituximab are broadly used to treat B-cell malignancies. These antibodies can induce various effector functions, including immune cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Neutrophils can induce ADCC toward solid cancer cells by trogoptosis, a cytotoxic mechanism known to be dependent on trogocytosis. However, neutrophils seem to be incapable of killing rituximab-opsonized B-cell lymphoma cells. Nevertheless, neutrophils do trogocytose rituximab-opsonized B-cell lymphoma cells, but this only reduces CD20 surface expression and is thought to render tumor cells therapeutically resistant to further rituximab-dependent destruction. Here, we demonstrate that resistance of B-cell lymphoma cells toward neutrophil killing can be overcome by a combination of CD47-SIRPα checkpoint blockade and sodium stibogluconate (SSG), an anti-leishmaniasis drug and documented inhibitor of the tyrosine phosphatase SHP-1. SSG enhanced neutrophil-mediated ADCC of solid tumor cells but enabled trogoptotic killing of B-cell lymphoma cells by turning trogocytosis from a mechanism that contributes to resistance into a cytotoxic anti-cancer mechanism. Tumor cell killing in the presence of SSG required both antibody opsonization of the target cells and disruption of CD47-SIRPα interactions. These results provide a more detailed understanding of the role of neutrophil trogocytosis in antibody-mediated destruction of B cells and clues on how to further optimize antibody therapy of B-cell malignancies.

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.

Software modernization has been extensively researched, primarily focusing on observing the associated phenomena, and providing technical solutions to facilitate the modernization process. Software modernization is claimed to be successful when the modernization is completed using those technical solutions. Very limited research, if any, is reported with an aim at documenting the post-modernization impacts, i.e., whether any of the pre-modernization business goals are in fact achieved after modernization. In this research, we attempt to address this relative absence of empirical study through five retrospective software modernization case studies. We use an explanatory case study approach to document the pre-modernization business goals, and to decide whether those goals have been achieved. The intended benefits for each of the five cases we considered were all (partially) met, and in most cases fully. Moreover, many cases exhibited a number of unintended benefits, and some reported detrimental effects of modernization.