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Development of immunomonitoring of antibody-dependent cellular cytotoxicity against neuroblastoma cells using whole blood

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Abstract

Neuroblastoma, a childhood tumour of neuroectodermal origin, accounts for 15 % of paediatric cancer deaths, which is often metastatic at diagnosis and despite aggressive therapies, it has poor long-term prognosis with high risk of recurrence. Monoclonal antibody (mAb) therapy targeting GD2, a disialoganglioside expressed on neuroblastoma, has shown promise in recent trials with natural killer cell (NK)-mediated antibody-dependent cellular cytotoxicity (ADCC) thought to be central to efficacy, although other immune effectors may be important. To further enhance therapy, immunomonitoring of patients is essential to elucidate the in vivo mechanisms of action and provides surrogate end points of efficacy for future clinical trials. Our aim was to establish a ‘real-time’ ex vivo whole-blood (WB) immunomonitoring strategy to perform within the logistical constraints such as limited sample volumes, anticoagulant effects, sample stability and shipping time. A fluorescent dye release assay measuring target cell lysis was coupled with flow cytometry to monitor specific effector response. Significant target cell lysis with anti-GD2 antibody (p < 0.05) was abrogated following NK depletion. NK up-regulation of CD107a and CD69 positively correlated with target cell lysis (r > 0.6). The ADCC activity of WB correlated with peripheral blood mononuclear cells (r > 0.95), although WB showed overall greater target cell lysis attributed to the combination of NK-mediated ADCC, CD16+ granulocyte degranulation and complement-dependent cytotoxicity. Response was maintained in heparinised samples stored for 24 h at room temperature, but not 4 °C. Critically, the assay showed good reproducibility (mean % CV < 6.4) and was successfully applied to primary neuroblastoma samples. As such, WB provides a resourceful analysis of multiple mechanisms for efficient end point monitoring to correlate immune modulation with clinical outcome.

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Abbreviations

ADCC:

Antibody-dependent cellular cytotoxicity

AICC:

Antibody-independent cellular cytotoxicity

CDC:

Complement-dependent cytotoxicity

CTL:

CD8+ cytotoxic T lymphocytes

CV:

Coefficient of variance

EFS:

Event-free survival

FcγR:

Fc gamma receptor

LRS:

Leucocyte reduction system

mAb:

Monoclonal antibody

NK:

Natural killer cell

PBMC:

Peripheral blood mononuclear cells

RT:

Room temperature

WB:

Whole blood

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Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Cancer Sciences Unit, Antibody and Vaccine Group, Faculty of Medicine, Southampton General Hospital, University of Southampton, Tremona Road, Southampton, SO16 6YD, UK

    Ferdousi Chowdhury, Mark S. Cragg, Martin J. Glennie & Juliet C. Gray

  2. Department of Pediatric Oncology and Hematology, University of Greifswald, 17475, Greifswald, Germany

    Holger N. Lode

Authors
  1. Ferdousi Chowdhury
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  2. Holger N. Lode
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  3. Mark S. Cragg
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  4. Martin J. Glennie
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  5. Juliet C. Gray
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Correspondence to Ferdousi Chowdhury.

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Chowdhury, F., Lode, H.N., Cragg, M.S. et al. Development of immunomonitoring of antibody-dependent cellular cytotoxicity against neuroblastoma cells using whole blood. Cancer Immunol Immunother 63, 559–569 (2014). https://doi.org/10.1007/s00262-014-1534-y

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  • DOI: https://doi.org/10.1007/s00262-014-1534-y

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