Abstract
BackgroundWith increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these “off-the-shelf” therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function.MethodsEffectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others.ResultsOur results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30.ConclusionsThe presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.
Highlights
The natural killer cell line NK-92 is derived from the peripheral blood of a lymphoma patient and is used as an anti-cancer advanced therapy medicinal product (ATMP) due to its high cytotoxic activity against tumor cells and its “off-the-shelf” availability [1]
An amperage of 0.05 mA was used for further experiments, as this dose showed consistent inhibition of cell growth and gave a safety margin to the parameter of 0.01 mA, which is important for implementing LEE irradiation into perspective clinical studies and uses
In contrast to nonirradiated control cells, which showed expected cell growth and stable viability over 13 days, irradiated NK-92 and CD123directed chimeric antigen receptor (CAR)-NK-92 cells showed a significant decrease in proliferation and viability
Summary
The natural killer cell line NK-92 is derived from the peripheral blood of a lymphoma patient and is used as an anti-cancer advanced therapy medicinal product (ATMP) due to its high cytotoxic activity against tumor cells and its “off-the-shelf” availability [1]. Due to the malignant origin of NK-92 cells, cell inactivation prior to their application is indispensable in order to stop cell proliferation and prevent possible tumorigenesis [2, 8, 9]. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function
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