神经母细胞瘤细胞对抗双唾液酸神经节苷脂抗体的内化作为免疫治疗耐药机制

Neuroblastoma (NBL) accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy that includes antibody targeting disialoganglioside GD2, a NBL antigen. Unfortunately, resistance to anti-GD2 immunotherapy is frequent and we aimed to investigate mechanisms of resistance in NBL. GD2 expression was quantified by flow cytometry and anti-GD2 antibody internalization was measured using real-time microscopy in 20 human NBL cell lines.

Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were performed on a subset of the cell lines (n = 12), and results were correlated with GD2 expression and antibody internalization. GD2 was expressed on 19 of 20 NBL cell lines at variable levels, and neutrophil-mediated ADCC was observed only in GD2-expressing cell lines.

We found no correlation between level of GD2 expression and sensitivity to neutrophil-mediated ADCC, suggesting that GD2 expression of many cell lines was above a threshold required for maximal ADCC, such that expression level could not be used to predict subsequent cytotoxicity.

Instead, anti-GD2 antibody internalization, a process that occurred universally but differentially across GD2-expressing NBL cell lines, was inversely correlated with ADCC. Treatment with endocytosis inhibitors EIPA, chlorpromazine, MBCD, and cytochalasin-D showed potential to inhibit antibody internalization; however, only MBCD resulted in significantly increased sensitivity to neutrophil-mediated ADCC in 4 of 4 cell lines in vitro.

Our data suggest that antibody internalization may represent a novel mechanism of immunotherapy escape by NBL and provide proof-of-principle that targeting pathways involved in antibody internalization may improve the efficacy of anti-GD2 immunotherapies.