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Disialoganglioside-specific human natural killer cells are effective against drug-resistant neuroblastoma

Abstract

The disialoganglioside GD2 is a well-established target antigen for passive immunotherapy in neuroblastoma (NB). Despite the recent success of passive immunotherapy with the anti-GD2 antibody ch14.18 and cytokines, treatment of high-risk NB remains challenging. We expanded the approach of GD2-specific, antibody-based immunotherapy to an application of a GD2-specific natural killer (NK) cell line, NK-92-scFv(ch14.18)-zeta. NK-92-scFv(ch14.18)-zeta is genetically engineered to express a GD2-specific chimeric antigen receptor generated from ch14.18. Here, we show that chimeric receptor expression enables NK-92-scFv(ch14.18)-zeta to effectively lyse GD2+ NB cells also including partially or multidrug-resistant lines. Our data suggest that recognition of GD2 by the chimeric receptor is the primary mechanism involved in NK-92-scFv(ch14.18)-zeta-mediated lysis and is independent of activating NK cell receptor/ligand interactions. Furthermore, we demonstrate that NK-92-scFv(ch14.18)-zeta is able to mediate a significant anti-tumor response in vivo in a drug-resistant GD2+ NB xenograft mouse model. NK-92-scFv(ch14.18)-zeta is an NB-specific NK cell line that has potential for future clinical development due to its high stability and activity toward GD2+ NB cell lines.

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Abbreviations

ADCC:

Antibody-dependent cellular cytotoxicity

ANOVA:

Analysis of variance

anti-IdAb:

Anti-idiotypic antibody

BSA:

Bovine serum albumin

CAR:

Chimeric antigen receptor

CD:

Cluster of differentiation

CHO:

Chinese hamster ovary

COG:

Children’s Oncology Group

DAPI:

4′,6-Diamino-2-phenylindole

EDTA:

Ethylenediaminetetraacetic acid

ELISA:

Enzyme-linked immunosorbent assay

E/T ratio:

Effector cell-to-target cell ratio

FACS:

Fluorescence-activated cell sorting

FasL:

Fas ligand

FBS:

Fetal bovine serum

FcγR:

Fc-gamma receptor

GCS:

Glucosylceramide synthase

GD2:

Disialoganglioside

GM2:

Monosialoganglioside

GM-CSF:

Granulocyte–macrophage colony-stimulating factor

GMP:

Good manufacturing practices

HLA:

Human leukocyte antigen

IFN-γ:

Interferon-γ

IgG:

Immunoglobulin G

IL-2:

Interleukin-2

IMDM:

Iscove’s modified Dulbecco’s medium

IU:

International unit

KIR:

Killer cell immunoglobulin-like receptor

LAK:

Lymphokine-activated killer cells

MFI:

Mean fluorescence intensity

MHC:

Major histocompatibility complex

MICA:

MHC class I-related protein A

NB:

Neuroblastoma

NK:

Natural killer

NSG:

NOD.Cg-Prkdc scid Il2rg tm1WjI/SzJ

PBS:

Phosphate-buffered saline

PE:

Phycoerythrin

PI:

Propidium iodide

PMA:

Phorbol-12-myristate-13-acetate

PPPP:

1-Phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol

RPMI:

Roswell Park Memorial Institute

scFv:

Single-chain fragment variable

SDS:

Sodium dodecyl sulfate

STR:

Short tandem repeat

TNF:

Tumor necrosis factor

TRAIL:

TNF-related apoptosis-inducing ligand

TRAIL-R:

TNF-related apoptosis-inducing ligand receptor

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Acknowledgments

We thank Dr. Barry J. Maurer for providing the PPPP; Tito Woodburn, Heather Kimmons, Heather Davidson, Malkanthi Mudannayake and Christin Eger for excellent technical assistance; Merck Serono and Merck KGaA for providing scFv(ch14.18) constructs; and the SIOPEN group for providing ch14.18/CHO. This work was financially supported by the German Cancer Foundation (Deutsche Krebshilfe, Holger N. Lode) and the South Plains Foundation (Nicole Huebener). Further support was provided by the University Medicine of Greifswald (Holger N. Lode), the Hector Stiftung (Nicole Huebener, Nikolai Siebert, Holger N. Lode), the Kind-Philipp-Stiftung für Leukämieforschung (Diana Seidel), Apeiron Biologics (Holger N. Lode) and National Cancer Institute grant CA82830 (C. Patrick Reynolds).

Conflict of interest

The authors have no conflict of interest to declare.

Ethical standard

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

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Correspondence to Holger N. Lode.

Additional information

Nicole Huebener and Holger N. Lode have share senior authorship.

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Seidel, D., Shibina, A., Siebert, N. et al. Disialoganglioside-specific human natural killer cells are effective against drug-resistant neuroblastoma. Cancer Immunol Immunother 64, 621–634 (2015). https://doi.org/10.1007/s00262-015-1669-5

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Keywords

  • Neuroblastoma GD2
  • Chimeric antigen receptor
  • Natural killer cell
  • Single-chain antibody