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Cancer Immunol Immunother. 2018 Jan;67(1):25-38. doi: 10.1007/s00262-017-2055-2. Epub 2017 Sep 6.

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

Author information

1
German Red Cross Blood Donation Service, Baden-Württemberg-Hessen, Frankfurt am Main, Germany.
2
Institute for Transfusion Medicine and Immunohematology, Goethe-University, Sandhofstrasse 1, 60528, Frankfurt am Main, Germany.
3
Institute for Transfusion Medicine, German Red Cross Blood Donation Service North-East, Blasewitzer Strasse 68/70, 01307, Dresden, Germany.
4
Department of Medicine, Division of Hematology, University of Washington, Seattle, WA, USA.
5
Georg-Speyer-Haus Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Straße 42-44, Frankfurt am Main, 60596, Germany.
6
German Cancer Consortium (DKTK) partner site Frankfurt/Mainz, Frankfurt am Main, Germany.
7
Institute for Transfusion Medicine, German Red Cross Blood Donation Service North-East, Blasewitzer Strasse 68/70, 01307, Dresden, Germany. t.tonn@blutspende.de.
8
Medical Faculty, Carl Gustav Carus Technical University Dresden, Dresden, Germany. t.tonn@blutspende.de.

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.

KEYWORDS:

CAR; Cancer immunotherapy; Glioblastoma; HER2; NK-92; Natural killer cells

PMID:
28879551
DOI:
10.1007/s00262-017-2055-2
[Indexed for MEDLINE]

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