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

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Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

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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Abbreviations

APC:

Allophycocyanin

CRS:

Cytokine release syndrome

EGFRvIII:

Epidermal growth factor receptor variant III

EPCAM:

Epithelial cell adhesion molecule

Eu:

Europium

FDA:

Food and Drug Administration

FFP:

Fresh frozen plasma

GBM:

Glioblastoma

GD2:

Disialoganglioside

GZMB:

Granzyme B

HI:

Heat inactivated

hPL:

Human platelet lysate

HSA:

Human serum albumin

IMP:

Investigational medicinal product

NCR:

Natural cytotoxicity receptors

r:

Recombinant

SCGM:

Stem cell growth medium

TF:

Transferrin

TRF:

Time-resolved fluorometry

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Acknowledgements

This work was supported in part by grants from the German Federal Ministry of Education and Research (BMBF) (Cluster für individualisierte Immunintervention, Ci3; FKZ 131A009A, 131A009B, 131A009C) and the LOEWE Center for Cell and Gene Therapy Frankfurt (CGT). The LOEWE Center for Cell and Gene Therapy Frankfurt is funded by Hessisches Ministerium für Wissenschaft und Kunst, reference number: III L 5–518/17.004 (2013). We thank Barry J. Simon, Nantkwest Inc., for providing NK-92 cells from Nantkwest´s proprietary master cell bank. We thank Andrea Jochheim Richter, Kurt Schoenfeld and Hans Klingemann for helpful discussions and suggestions throughout the project.

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Authors and Affiliations

  1. German Red Cross Blood Donation Service, Baden-Württemberg-Hessen, Frankfurt am Main, Germany

    Paulina Nowakowska, Annette Romanski, Halvard Bonig & Erhard Seifried

  2. Institute for Transfusion Medicine and Immunohematology, Goethe-University, Sandhofstrasse 1, 60528, Frankfurt am Main, Germany

    Paulina Nowakowska, Annette Romanski, Halvard Bonig & Erhard Seifried

  3. Institute for Transfusion Medicine, German Red Cross Blood Donation Service North-East, Blasewitzer Strasse 68/70, 01307, Dresden, Germany

    Nicole Miller, Marcus Odendahl & Torsten Tonn

  4. Medical Faculty, Carl Gustav Carus Technical University Dresden, Dresden, Germany

    Torsten Tonn

  5. Department of Medicine, Division of Hematology, University of Washington, Seattle, WA, USA

    Halvard Bonig

  6. Georg-Speyer-Haus Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Straße 42-44, Frankfurt am Main, 60596, Germany

    Congcong Zhang & Winfried S. Wels

  7. German Cancer Consortium (DKTK) partner site Frankfurt/Mainz, Frankfurt am Main, Germany

    Congcong Zhang & Winfried S. Wels

Authors
  1. Paulina Nowakowska
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  2. Annette Romanski
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  3. Nicole Miller
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  4. Marcus Odendahl
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  6. Congcong Zhang
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  7. Erhard Seifried
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  8. Winfried S. Wels
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  9. Torsten Tonn
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Corresponding author

Correspondence to Torsten Tonn.

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Nowakowska, P., Romanski, A., Miller, N. et al. Clinical grade manufacturing of genetically modified, CAR-expressing NK-92 cells for the treatment of ErbB2-positive malignancies. Cancer Immunol Immunother 67, 25–38 (2018). https://doi.org/10.1007/s00262-017-2055-2

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  • DOI: https://doi.org/10.1007/s00262-017-2055-2

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