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

Abstract

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 (CD56CD3) was carried out with the CliniMACS Prodigy in a single process, starting with approximately 1.2 × 10 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10 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 CD56CD3 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 CD56CD16 and CD56CD16 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 CD123 AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor nec...

Efficient and Robust NK-Cell Transduction With Baboon Envelope Pseudotyped Lentivector

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CAR-Expressing Natural Killer Cells for Cancer Retargeting

“…In the case of CD123, a CAR-NK cell-based concept would be a more practical approach. The GMP-compliant clinical-grade manufacturing of expanded and activated NK cells expressing CD123-directed CARs was proven feasible and the produced CAR-modified effector cells showed in vitro activity against both an AML cell line and patient samples [50]. …”

High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia

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