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01-01-2018 | Original Article

Clinical grade manufacturing of genetically modified, CAR-expressing NK-92 cells for the treatment of ErbB2-positive malignancies

Authors: Paulina Nowakowska, Annette Romanski, Nicole Miller, Marcus Odendahl, Halvard Bonig, Congcong Zhang, Erhard Seifried, Winfried S. Wels, Torsten Tonn

Published in: Cancer Immunology, Immunotherapy | Issue 1/2018

Abstract

Background

The NK-92/5.28.z cell line (also referred to as HER2.taNK) represents a stable, lentiviral-transduced clone of ErbB2 (HER2)-specific, second-generation CAR-expressing derivative of clinically applicable NK-92 cells. This study addresses manufacturing-related issues and aimed to develop a GMP-compliant protocol for the generation of NK-92/5.28.z therapeutic doses starting from a well-characterized GMP-compliant master cell bank.

Materials and methods

Commercially available GMP-grade culture media and supplements (fresh frozen plasma, platelet lysate) were evaluated for their ability to support expansion of NK-92/5.28.z. Irradiation sensitivity and cytokine release were also investigated.

Results

NK-92/5.28.z cells can be grown to clinically applicable cell doses of 5 × 10⁸ cells/L in a 5-day batch culture without loss of viability and potency. X-Vivo 10 containing recombinant transferrin supplemented with 5% FFP and 500 IU/mL IL-2 in VueLife 750-C1 bags showed the best results. Platelet lysate was less suited to support NK-92/5.28.z proliferation. Irradiation with 10 Gy completely abrogated NK-92/5.28.z proliferation and preserved viability and potency for at least 24 h. NK-92/5.28.z showed higher baseline cytokine release compared to NK-92, which was significantly increased upon encountering ErbB2(+) targets [GZMB (twofold), IFN-γ (fourfold), IL-8 (24-fold) and IL-10 (fivefold)]. IL-6 was not released by NK cells, but was observed in some stimulated targets. Irradiation resulted in upregulation of IL-8 and downregulation of sFasL, while other cytokines were not impacted.

Conclusion

Our concept suggests NK-92/5.28.z maintenance culture from which therapeutic doses up to 5 × 10⁹ cells can be expanded in 10 L within 5 days. This established process is feasible to analyze NK-92/5.28.z in phase I/II trials.

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Title: Clinical grade manufacturing of genetically modified, CAR-expressing NK-92 cells for the treatment of ErbB2-positive malignancies
Authors: Paulina Nowakowska
Annette Romanski
Nicole Miller
Marcus Odendahl
Halvard Bonig
Congcong Zhang
Erhard Seifried
Winfried S. Wels
Torsten Tonn
Publication date: 01-01-2018
Publisher: Springer Berlin Heidelberg
Published in: Cancer Immunology, Immunotherapy / Issue 1/2018
Print ISSN: 0340-7004
Electronic ISSN: 1432-0851
DOI: https://doi.org/10.1007/s00262-017-2055-2

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