Paulina Nowakowska1,2, Annette Romanski1,2, Nicole Miller3, Marcus Odendahl3, Halvard Bonig1,2,4, Congcong Zhang5,6, Erhard Seifried1,2, Winfried S Wels5,6, Torsten Tonn7,8. 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.
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.
Entities:
Keywords:
CAR; Cancer immunotherapy; Glioblastoma; HER2; NK-92; Natural killer cells
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