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.

Keywords: CAR-NK-92; NK-92; acute myeloid leukemia; chimeric antigen receptor; gamma irradiation; immune cell therapy; low energy electron irradiation; off-the-shelf therapy.

Figure 1

Cell proliferation of NK-92 and CD123-directed CAR-NK-92 is fully inhibited by gamma irradiation and LEEI. (A) Cell count of viable CD123-CAR-NK-92 cells (left) and viability (right) were determined for 4 days for non-irradiated cells (black circles, n = 3) and LEE-irradiated cells at 200 keV and 0.01 mA (grey squares, n = 1), 0.03 mA (grey triangles, n = 3) or 0.05 mA (grey circles, n = 3) on an automated cell counter (CASY). (B) Cell count of viable NK-92 (left, n = 5) and CD123-CAR-NK-92 cells (right, n = 9) was determined for 13 days after irradiation for non-irradiated cells (black), LEE-irradiated cells (grey) and gamma-irradiated cells (white) on an automated cell counter (Countess II). (C) Viability was measured by trypan blue staining on an automated cell counter (Countess II) for 13 days after irradiation of NK-92 (left, n = 5) and CD123-CAR-NK-92 cells (right, n = 9). Non-irradiated cells (black) were compared to LEE-irradiated cells (grey) and gamma-irradiated cells (white). (D) Flow cytometry analysis of LEE- or gamma-irradiated NK-92 (left, n = 4) and CD123-directed CAR-NK-92 (right, n = 7) cells after 7-AAD and Annexin V staining. 7-AAD^-/Annexin V⁺ (black) and 7-AAD⁺/Annexin V⁺ (white) subpopulations are shown. All values are indicated as means ± SEM, statistical significance is symbolized by asterisks (* for p ≤ 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001, and **** for p ≤ 0.0001, ANOVA or Kruskal-Wallis test adjusted for multiple comparisons by Dunn’s test).

Figure 2

LEE-irradiated cells show high in vitro functionality. (A+B): Cytotoxicity was measured via specific lysis of K562 cells by NK-92 cells (A, n = 5) or specific lysis of KG-1 cells by CD123-CAR-NK-92 cells (B, n = 9) by chromium-release-assay for 3 days after LEEI (grey) or gamma irradiation (white). Non-irradiated cells (black) were used as a control. Cells were coincubated at an effector to target (E:T)-ratio of 5:1. (C+D): Three days after LEEI or gamma irradiation, NK-92 (C, n = 4) or CD123-CAR-NK-92 (D, n = 7) cells were cocultivated with the target cell line K562 or KG-1 in an E:T ratio of 5:1 for 2 h, respectively. As controls, non-irradiated NK-92 or CD123-CAR-NK-92 cells with and without specific target cell stimulation, and K562 and KG-1 cells alone were used. Supernatant was harvested and analyzed with a LEGENDplex human CD8/NK panel. Dotted line represents detection limit of this assay. Values are indicated as means ± SEM, statistical significance is symbolized by asterisks (ns for p > 0.05, * for p ≤ 0.05, ** for p ≤ 0.01, Kruskal-Wallis test adjusted for multiple comparisons by Dunn’s test).

Figure 3

Irradiation decreases surface expression of CD56. (A+B): NK-92 (A, n = 4) and CD123-CAR-NK-92 cells (B, n = 7) were stained with anti-human CD56 antibody. Surface expression levels of CD56 are indicated as CD56^low (black) and CD56^high (grey). Representative contour plots of non-irradiated (control), LEE-irradiated, and gamma-irradiated cells on day 3 post-irradiation are shown. (C) Analysis of the CD56^low subpopulation of NK-92 (left) and CD123-CAR-NK-92 cells (right) regarding apoptosis: 7-AAD^-/Annexin V^- (lightest grey), 7-AAD⁺/Annexin V^- (dark grey), 7-AAD⁺/Annexin V⁺(light grey) and 7-AAD⁻/Annexin V⁺ (black). Representative contour plots of non-irradiated (control), LEE-irradiated, and gamma-irradiated cells on day 3 post-irradiation are shown. Values are indicated as means ± SEM, statistical significance is symbolized by asterisks (* for p ≤ 0.05 and ** for p ≤ 0.01, Kruskal-Wallis test adjusted for multiple comparisons by Dunn’s test).

Figure 4

Gamma irradiation and LEEI result in DNA damage and an accumulation in G2/M phases of CD123-directed CAR-NK-92 cells. (A) Alkaline comet assays were performed with LEE- and gamma-irradiated versus non-irradiated CD123-directed CAR-NK-92 cells 2 h (left) or 24 h (right) after treatment to determine irradiation-induced DNA strand breaks. Values are depicted as means ± SEM (n = 6 independent experiments, each carried out in technical triplicates, minimum number of 100 cell nuclei analyzed per sample). Statistical significance is symbolized by asterisks (ns for p > 0.05, * for p ≤ 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001, and **** for p ≤ 0.0001, Kruskal-Wallis test adjusted for multiple comparisons by Dunn’s test). (B) CD123-CAR-NK-92 cells (n = 8) were fixed and stained with propidium iodide (PI). Representative histograms of non-irradiated (control, left), LEE-irradiated (middle), and gamma-irradiated (right) cells are shown 2 h (d0, top), 2 days (d2, middle), and 6 days (d6, bottom) after treatment. Gating indicates cells defined as Sub-G1- (1), G1- (2), S- (3), G2/M phase (4) or polyploid (5). (C) Quantification of PI-staining of CD123-directed CAR-NK-92 (n = 8). Values are indicated as means ± SEM. (D) Light microscopy of non-irradiated (control, top), LEE-irradiated (center), and gamma-irradiated (bottom) CD123-directed CAR-NK-92 cells on day 10 post-irradiation. Arrows show enlarged cells. (E) Preliminary results of gene expression levels of gamma-irradiated (left) and LEE-irradiated (right) CD123-directed CAR-NK-92 cells both compared to non-irradiated cells 2 h after treatment. Heat maps of normalized, variance-stabilized and per gene standardized expression values of three technical replicates show up- (red) or down-regulated (blue) genes. The total number of significantly differentially regulated genes is indicated below (FDR < 0.01).