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Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing.

ABSTRACT:

Background

With 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.

Methods

Effectiveness 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.

Results

Our 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.

Conclusions

The 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.

SUBMITTER: Walcher L

PROVIDER: S-EPMC8212864 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:NK-92 cells and their CAR-modified derivatives exhibit strong cytotoxic activity against tumors and are promising "off-the-shelf" therapeutics compared to CAR-T cells. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cells need to be inactivated before transfusion. With increasing clinical use, there is a growing demand for enhanced safety and an efficient method that stops cell proliferation, but better maintains the cytotoxic effector functions of the cells compared to commonly used gamma irradiation. Recently, low-energy electron irradiation (LEEI) was successfully applied for inactivation of bacteria and viruses for vaccine production, and was described as a method for NK-92 inactivation for the first time. In the present publication, data on extensive characterization of LEEI and its comparison with gamma irradiation for the inactivation of parental NK-92 cells as well as CD123-directed CAR-NK-92 cells are provided. Our results show that both irradiation methods cause a progressive decrease in cell viability and are therefore suitable for inhibition of proliferation. Notably, cytotoxicity of the NK cells was significantly reduced by gamma irradiation, but not by LEEI three days after irradiation, compared to non-irradiated cells. Both gamma irradiation as well as LEEI led to substantial DNA-damage and an accumulation of irradiated cells in the G2/M phase. In addition, transcriptomic analysis of cells irradiated with both methods revealed approximately 12-fold more differentially expressed genes after gamma irradiation compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in CD56 expression but no changes in the levels of the activating receptors NKp46, NKG2D, and NKp30. Conclusions: The present data show that LEEI efficiently inactivates (CAR )NK-92 cells and sustains their cytotoxic capacity better than gamma irradiation. Taking into account additional logistic advantages of LEEI proves a potent alternative in the manufacturing of (CAR-)NK-92 cells for clinical application.