![]() Lymphocyte apoptosis and caspase activity were analyzed by flow cytometry and fluorimetry, and residual 8-methoxypsoralen concentrations by liquid chromatography–mass spectrometry. Cells collected with a Spectra Optia apheresis system were suspended in plasma or physiological saline (NaCl) and incubated with 200 ng/ml versus 340 ng/ml photosensitizer before UVA irradiation (Macogenic G2 or UVA PIT system). Materials and MethodsĪll patients ( n = 13) included in this study received photopheresis as medically indicated. This study aimed to optimize treatment by varying the 8-MOP starting concentration and the cell suspension medium. Several factors influence the efficacy of ECP with the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet light A (UVA). IL-21 acute myeloid leukemia autologous feeder cells chimeric antigen receptor-modified NK cells closed GMP-compliant Prodigy system interleukin-3 receptor subunit alpha (CD123).Extracorporeal photopheresis (ECP), an apheresis-based therapy for various immunological diseases, works mainly by inducing apoptosis in lymphocytes. 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. 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. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123 pos AML cell line KG1a and primary AML blasts. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. 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. 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. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56 dimCD16 pos and CD56 brightCD16 dim&neg NK subsets on day 14 with cells cultivated in NK MACS ® media. 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. 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. Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). 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). Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10 6 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-VIVO ™10, CellGro ®, TexMACS ™, and NK MACS ®). Separation of primary human NK cells (CD56 +CD3 -) was carried out with the CliniMACS Prodigy ® in a single process, starting with approximately 1.2 × 10 9 leukocytes collected by small-scale lymphapheresis or from buffy coats. 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). However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. 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.
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