P0971 - In vivo validation of trogocytosis in neuroblastoma – a potential mechanism of resistance to anti-GD2 immunotherapy

Background
Anti-GD2 monoclonal antibody (dinutuximab) has improved survival for high-risk neuroblastoma, but many children recur with resistant disease. We discovered that neuroblastoma cells treated with natural killer (aNK) cells and dinutuximab lead to tumor cell death followed by acquisition of neuroblastoma GD2 receptor on the aNK cell surface ((GD2+)aNK), a phenomenon called trogocytosis. Following in vitro identification, we hypothesized that trogocytosis can be validated in vivo and that (GD2+)aNK cells would be targeted by aNK cells in the presence of dinutuximab in vitro.

Methods
Human NB cells (CHLA-255, KCNR) were treated with aNK cells and dinutuximab to create (GD2+)aNK cells, which were then incubated with fresh (GD2-)aNK cells +/- dinutuximab at varying time points. Cell death was analyzed by flow-based cytotoxicity assay. Mice bearing CHLA-255 and KCNR orthotopic xenografts were treated with a single dose of aNK cells and dinutuximab prior to sacrifice. Tumors were analyzed by flow for detection of (GD2+)aNK cells.

Results
Interestingly, we observed minimal killing in vitro of (GD2+)aNK cells when incubated with fresh (GD2-)aNK cells +/- dinutuximab. However, we observed significant (GD2+)aNK cell death by other (GD2+)aNK cells when compared to the combination groups (Figure 1A). In vivo, we verified the presence of (GD2+)aNK cells from treated xenograft tumors in two human NB cell lines (Figure 1B).

Conclusion
Our findings verify the mechanism of trogocytosis in an experimental animal model of neuroblastoma. Moreover, we found that (GD2+)aNK cells appear to kill each other, which may decrease the efficacy of this treatment strategy. The observed trogocytosis may represent a novel mechanism of resistance to immunotherapy in neuroblastoma.