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Anti-disialoganglioside antibody internalization by neuroblastoma cells as a mechanism of immunotherapy resistance

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Abstract

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.

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Acknowledgements

The authors would like to acknowledge Drs. Yves DeClerck, Nora Heisterkamp, and Chintan Parekh for manuscript review, and Drs. John Li and Alan Wayne for their valuable input.

Funding

This work was supported, in part, by a T32 CA009659 training grant (RT) from the National Cancer Institute (PI Y.A. DeClerck) and by the V Foundation (SA); and in part by the CA170257P1 from the Department of Defense (SA), P01 CA217959 National Cancer Institute (co-I SA, PI R.C. Seeger, J Maris), T.J. Martell Foundation, and Nautica Malibu Triathlon (SA).

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Authors and Affiliations

  1. Children’s Hospital Los Angeles, The Saban Research Institute, 4650 Sunset Boulevard, MS 57, Los Angeles, CA, 90027, USA

    Rachelle Tibbetts, Kee Kiat Yeo, Sakunthala Muthugounder, Meng-Hua Lee, Cham Jung, Tania Porras-Corredor, Michael A. Sheard & Shahab Asgharzadeh

  2. Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Rachelle Tibbetts, Kee Kiat Yeo & Shahab Asgharzadeh

  3. Dana-Farber/Boston Childrens Cancer and Blood Disorders Center, Boston, MA, USA

    Kee Kiat Yeo

  4. Harvard Medical School, Harvard University, Boston, MA, USA

    Kee Kiat Yeo

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  1. Rachelle Tibbetts
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Contributions

RT and SA conceived and designed the study. RT, KKY, SM, ML performed the research and also analyzed the data. SM, CJ, TP, and MS contributed reagents and analysis tools. The manuscript was written by RT, SA, and MS. All authors edited and approved the submission of this manuscript.

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Correspondence to Shahab Asgharzadeh.

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Tibbetts, R., Yeo, K.K., Muthugounder, S. et al. Anti-disialoganglioside antibody internalization by neuroblastoma cells as a mechanism of immunotherapy resistance. Cancer Immunol Immunother 71, 153–164 (2022). https://doi.org/10.1007/s00262-021-02963-y

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