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

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.

Optimizing NK-92 serial killers: gamma irradiation, CD95/Fas-ligation, and NK or LAK attack limit cytotoxic efficacy

“…In this report, we shed new light on potential limitations to NK-92 cell-mediated serial killing and therapeutic efficacy, specifically following irradiation, a current clinical practice preceding adoptive transfer. Prior studies have investigated the effects of irradiation on NK-92 cell-mediated killing; however, these studies used shorter cytotoxicity assays with different target cells and reported that NK-92 cytotoxic capacity is mostly retained for at least 24 h following irradiation [ 22 , 42 ]. Another study, using haNK cells 24-h post irradiation saw an increase in cytotoxicity toward multiple carcinoma cell lines compared to haNK cell killing immediately post irradiation [ 43 ].…”

“…The profound effects of irradiation on NK-92 cytotoxic capacity indicate that radiation effects extend beyond DNA damage and likely include direct damage to proteins [ 46 , 47 ]. Ionizing radiation produces radiolysis products, such as reactive oxygen species that inactivate proteome functions including those involved in killing and DNA repair [ 42 ]. Low energy electron irradiation, as an alternative to gamma irradiation, inhibits NK-92 cell proliferation while maintaining higher cytotoxic capacity and for longer periods of time and could therefore be considered for clinical applications [ 42 ].…”

“…Ionizing radiation produces radiolysis products, such as reactive oxygen species that inactivate proteome functions including those involved in killing and DNA repair [ 42 ]. Low energy electron irradiation, as an alternative to gamma irradiation, inhibits NK-92 cell proliferation while maintaining higher cytotoxic capacity and for longer periods of time and could therefore be considered for clinical applications [ 42 ]. This report also indicates that 2-h after 10 Gy gamma irradiation, there is lower expression of genes encoding multiple pathways that are critical to cell-mediated cytotoxicity [ 42 ].…”

“…Low energy electron irradiation, as an alternative to gamma irradiation, inhibits NK-92 cell proliferation while maintaining higher cytotoxic capacity and for longer periods of time and could therefore be considered for clinical applications [ 42 ]. This report also indicates that 2-h after 10 Gy gamma irradiation, there is lower expression of genes encoding multiple pathways that are critical to cell-mediated cytotoxicity [ 42 ]. Considering the additional impact of direct proteome damage by irradiation, alternative treatments such as induction of genetically introduced type II restriction enzymes and pretreatment of cells with certain topoisomerase inhibitors (Hudig et al, unpublished results) that only inflict damage to DNA could be used prior to adoptive transfer [ 47 ].…”

Two for one: targeting BCMA and CD19 in B-cell malignancies with off-the-shelf dual-CAR NK-92 cells

“…For clinical application, proliferation of NK-92 cells needs to be halted prior to infusion to avoid NK-92 cell engraftment in vivo, which could lead to the development of NK cell lymphoma. Although alternative methods are being examined [ 63 ], this “inactivation” step is most commonly performed by gamma-irradiation. We confirmed that 10 Gy gamma-irradiation effectively blocks NK-92 cell proliferation and leads to a gradual decrease in cell viability down to zero over the course of 1 week, paralleling the CAR expression kinetics after mRNA electroporation.…”