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Published Online: 1 October 2017

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells

Publication: Human Gene Therapy
Volume 28, Issue Number 10


The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56+CD3) was carried out with the CliniMACS Prodigy® in a single process, starting with approximately 1.2 × 109 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 106 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO10, CellGro®, TexMACS, and NK MACS®). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56+CD3- target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56dimCD16pos and CD56brightCD16dim&neg NK subsets on day 14 with cells cultivated in NK MACS® media. Moreover, effector cell expansion in manually performed experiments with NK MACS® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

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Information & Authors


Published In

cover image Human Gene Therapy
Human Gene Therapy
Volume 28Issue Number 10October 2017
Pages: 897 - 913
PubMed: 28810809


Published in print: October 2017
Published online: 1 October 2017
Published ahead of production: 16 August 2017
Published ahead of print: 15 August 2017
Accepted: 14 August 2017
Received: 13 August 2017


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Stephan Klöß* [email protected]
Institute for Cellular Therapeutics, GMPDU, IFB-Tx, Hannover Medical School, Hannover, Germany
Olaf Oberschmidt
Institute for Cellular Therapeutics, GMPDU, IFB-Tx, Hannover Medical School, Hannover, Germany
Michael Morgan
Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
Julia Dahlke
Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
Lubomir Arseniev
Institute for Cellular Therapeutics, GMPDU, IFB-Tx, Hannover Medical School, Hannover, Germany
Volker Huppert
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Markus Granzin
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Tanja Gardlowski
Institute for Cellular Therapeutics, GMPDU, IFB-Tx, Hannover Medical School, Hannover, Germany
Nadine Matthies
Institute for Cellular Therapeutics, GMPDU, IFB-Tx, Hannover Medical School, Hannover, Germany
Stephanie Soltenborn
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Axel Schambach
Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts.
Ulrike Koehl
Institute for Cellular Therapeutics, GMPDU, IFB-Tx, Hannover Medical School, Hannover, Germany


Correspondence: Dr. Stephan Klöß, Institute of Cellular Therapeutics, GMP Development Unit (OE 8885), IFB-Tx, Hannover Medical School (MHH), Feodor-Lynen-Str. 21, D-30625, Hannover, Germany. E-mail: [email protected]

Author Disclosure

The authors declare no financial or commercial conflicts of interest, except that A.S. is co-inventor on a patent application describing alpha-retroviral SIN vectors.

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