We established an automated method to expand high numbers of clinical-grade NK cells with properties similar to their manually produced counterparts. This automated process represents a highly efficient tool to standardize NK cell processing for therapeutic applications.

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...

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4842 Obtaining pure and unaffected leukocyte populations is of utmost importance in diagnostic as well as research settings. So far, the isolation of functional leukocyte subpopulations from whole blood has been a time-consuming procedure, rendering the performance of downstream assays and analyses a challenging objective. We have developed a cell isolation technology that allows the purification of immune cells from human whole blood within 20 minutes. This novel technology requires a minimum of laboratory equipment. A cell isolation reagent is added to the anticoagulated blood sample and mixed briefly. While placed in a strong magnetic field, magnetically labeled non-target cells are depleted, while untouched target cells remain in the supernatant. Simultaneously, a reagent-assisted erythrocyte sedimentation phase occurs, which depletes ∼99.7 % of erythrocytes. Using this novel technology, Natural Killer cells, B cells, T cells, CD4+ T helper cells, CD8+ cytotoxic T cells and naïve B cells were isolated from 30mL of anticoagulated human whole blood. Target cells were recovered in a volume of 25–30 mL of supernatant (67% plasma, 33% Phosphate buffered saline) and average purities among white blood cells were 88.9% for NK cells, 88.2% for B cells, 97.8% for T cells, 93.0% for CD4+ cells, 78.9% for CD8+ T cells and 79.4% for naïve B cells, yields were 75.5%, 84.4%, 54.5%, 63.0%, 59.5% and 96.8% respectively (n >6 each). Red Blood cells were reduced by ∼99.7%, platelets by >99.9%. Cytotoxicity and proliferative capacity of isolated NK cells were measured in cytotoxicity assays with K562 target cells and proliferation assays with antibody loaded large magnetic beads respectively. Cytotoxicity and proliferation rate were comparable to those assessed using NK cells isolated by Ficoll density gradient separation or magnetic cell sorting (NK cell isolation kit). In vitro proliferation assays with total T cells, CD4+ T cells, CD8+ T cells or B cells revealed that proliferation rate was identical to that of target cells which were isolated by Ficoll density gradient separation and magnetic cell sorting. We furthermore compared the mRNA yields from cells isolated with either method (new technology vs. Miltenyi's isolation kits). The mRNA samples were subsequently subjected to gene expression analysis. Comparing the results obtained from samples isolated with the two different separation methods, we could not detect any significant differences in gene expression levels. These results demonstrate, that cells isolated with the novel whole blood cell isolation strategy, can be used for cell-based functional assays, as well as gene expression profiling. Additionally, overall processing time can be significantly reduced, which is highly desirable for sensitive downstream experiments. Disclosures: Kauling: Miltenyi Biotec GmbH: Employment. Huppert:Miltenyi Biotec GmbH: Employment. Soltenborn:Miltenyi Biotec GmbH: Employment. Hillenkötter:Miltenyi Biotec GmbH: Employment. Mohaupt:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment.