Abstract 6612: Anti-GD2 antibody internalization by neuroblastoma as a mechanism of immunotherapy resistance
Authors: Tibbetts Rachelle J. , Yeo Kee Kiat , Muthugounder Sakunthala , Asgharzadeh Shahab 
Published: September 2020

Abstract
Background: Neuroblastoma, a sympathetic nervous system cancer, accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy including the relatively recent introduction of antibody targeting disialoganglioside (GD2), a common neuroblastoma antigen. The mechanisms underlying therapeutic resistance, specifically to antibody-based therapy, in some patients remains poorly understood. Aims: We hypothesized that differential internalization of anti-GD2 antibody by tumor cells upon binding to surface GD2 may act as a mechanism of escape by decreasing tumor sensitivity to antibody-dependent cellular cytotoxicity (ADCC). We aimed to measure antibody internalization differences across 22 human neuroblastoma cell lines and investigate how it relates to GD2 expression and ADCC sensitivity.
Methods: Antibody internalization was measured using IncucyteS3 live cell imaging and anti-GD2 antibody (14G2a) conjugated to a pH-sensitive red fluorescent dye (pHrodo). GD2 surface expression was quantified by flow cytometry and BD QuantiBrite Beads. Neutrophil-mediated ADCC was measured in vitro via one of two methods - DIMSCAN and calcein-labeled tumor cells or IncucyteS3 and GFP-expressing tumor cells. Agents targeting endocytosis (chlorpromazine, cytochalasin-D, methyl-beta-cyclodextrin/MβCD, Pitstop2, and EIPA) were then used to assess inhibition of internalization and subsequently sensitization to ADCC.
Results: Internalization of 14G2a-pHrodo over 24 hours varied significantly across cell lines (AUC range: 50-529) with LAN1 demonstrating the highest internalization AUC (529 +/- 40). Internalization rate was not correlated with GD2 surface expression (r=0.15). However, a moderate inverse correlation was found between antibody internalization and sensitivity to neutrophil-mediated ADCC in the 10 cell lines tested (r=-0.57). Antibody internalization by LAN1 was significantly inhibited by treatment with EIPA, chlorpromazine, and MβCD (AUC 1.1, 3.9, 104, respectively), all without significant change in surface GD2 expression. Treatment with MβCD (2mM) additionally was able to sensitize LAN1 to neutrophil-mediated ADCC in a dose-dependent manner.
Conclusion: Anti-GD2 antibody internalization varies widely across human neuroblastoma cell lines, is inversely correlated with ADCC sensitivity, and, in a high internalizing cell line, can be effectively modulated by inhibitors of pinocytosis. The finding that membrane cholesterol depletion with MβCD sensitized LAN1 to neutrophil-ADCC suggests that membrane organization of GD2, specifically in relation to cholesterol-rich domains, may play a more significant role in antibody internalization than specific endocytosis processes. Elucidating further the mechanisms underlying differential internalization across cell lines could lead to the development of agents targeting internalization and ultimately augmenting tumor sensitivity to antibody-based immunotherapy.
Citation Format: Rachelle J. Tibbetts, Kee Kiat Yeo, Sakunthala Muthugounder, Shahab Asgharzadeh. Anti-GD2 antibody internalization by neuroblastoma as a mechanism of immunotherapy resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6612.