Mass Production of Highly Active NK Cells for Cancer Immunotherapy in a GMP Conform Perfusion Bioreactor
- NK cells have emerged as promising candidates for cancer immunotherapy, especially due to their ability to fight circulating tumor cells thereby preventing metastases formation. Hence several studies have been performed to generate and expand highly cytotoxic NK cells ex vivo, e.g., by using specific cytokines to upregulate both their proliferation and surface expression of distinct activating receptors. Apart from an enhanced activity, application of NK cells as immunotherapeutic agent further requires sufficient cell numbers and a high purity. All these parameters depend on a variety of different factors including the starting material, additives like cytokines as well as the culture system. Here we analyzed PBMC-derived NK cells of five anonymized healthy donors expanded under specific conditions in an innovative perfusion bioreactor system with respect to their phenotype, IFN gamma production, and cytotoxicity in vitro. Important features of the meander type bioreactors used here are a directed laminar flow of medium and controlNK cells have emerged as promising candidates for cancer immunotherapy, especially due to their ability to fight circulating tumor cells thereby preventing metastases formation. Hence several studies have been performed to generate and expand highly cytotoxic NK cells ex vivo, e.g., by using specific cytokines to upregulate both their proliferation and surface expression of distinct activating receptors. Apart from an enhanced activity, application of NK cells as immunotherapeutic agent further requires sufficient cell numbers and a high purity. All these parameters depend on a variety of different factors including the starting material, additives like cytokines as well as the culture system. Here we analyzed PBMC-derived NK cells of five anonymized healthy donors expanded under specific conditions in an innovative perfusion bioreactor system with respect to their phenotype, IFN gamma production, and cytotoxicity in vitro. Important features of the meander type bioreactors used here are a directed laminar flow of medium and control of relevant process parameters. Cells are cultivated under "steady state" conditions in perfusion mode. Our data demonstrate that expansion of CD3(+) T cell depleted PBMCs in our standardized system generates massive amounts of highly pure (>85%) and potent anticancer active NK cells. These cells express a variety of important receptors driving NK cell recruitment, adhesion as well as activation. More specifically, they express the chemokine receptors CXCR3, CXCR4, and CCR7, the adhesion molecules L-selectin, LFA-1, and VLA-4, the activating receptors NKp30, NKp44, NKp46, NKG2D, DNAM1, and CD16 as well as the death ligands TRAIL and Fas-L. Moreover, the generated NK cells show a strong IFN gamma expression upon cultivation with K562 tumor cells and demonstrate a high cytotoxicity toward leukemic as well as solid tumor cell lines in vitro. Altogether, these characteristics promise a high clinical potency of thus produced NK cells awaiting further evaluation.…
Author details: | Katharine Bröker, Evgeny Sinelnikov, Dirk Gustavus, Udo Schumacher, Ralf Pörtner, Hans Hoffmeister, Stefan LüthGND, Werner DammermannORCiDGND |
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DOI: | https://doi.org/10.3389/fbioe.2019.00194 |
ISSN: | 2296-4185 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/31457007 |
Title of parent work (English): | Frontiers in Bioengineering and Biotechnology |
Publisher: | Frontiers Research Foundation |
Place of publishing: | Lausanne |
Publication type: | Article |
Language: | English |
Date of first publication: | 2019/08/13 |
Publication year: | 2019 |
Release date: | 2020/12/01 |
Tag: | GMP; cytotoxicity; immunotherapy; mass production process; natural killer cells (NK cells); perfusion bioreactor; tumor immunity |
Volume: | 7 |
Number of pages: | 17 |
Funding institution: | Ministry for Science, Research and Cultural Affairs of Brandenburg |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
DDC classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Peer review: | Referiert |
Publishing method: | Open Access / Gold Open-Access |
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