Elotuzumab Enhances CD16-Independent NK Cell-Mediated Cytotoxicity against Myeloma Cells by Upregulating Several NK Cell-Enhancing Genes

Abstract

Multiple myeloma (MM) is an intractable hematological malignancy caused by abnormalities in plasma cells. Combination therapy using antibodies and natural killer (NK) effectors, which are innate immune cells with safe and potent antitumor activity, is a promising approach for cancer immunotherapy and can enhance antitumor effects. Elotuzumab (Elo) is an immune-stimulatory antibody that targets the signaling lymphocytic activation molecule family 7 (SLAMF7) expressed on the surface of MM and NK cells. We confirmed that Elo strongly promoted NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against SLAMF7-positive MM cells in a CD16-dependent NK cell line, and also activated expanded NK cells derived from peripheral blood mononuclear cells of healthy donors and patients with MM in the present study. However, the antitumor effects and genes involved in the direct promotion of NK cell-mediated activation using Elo in CD16-independent NK cells are not clearly known. In this study, we demonstrated that Elo pretreatment significantly enhanced CD16-independent NK cell-mediated cytotoxicity in both SLAMF7-positive MM.1S and SLAMF7-negative K562, U266, and RPMI 8226 tumor cells. Upon direct simulation of CD16-independent NK cells with Elo, increased levels of CD107a degranulation and IFN-γ secretion were observed along with the upregulation of granzyme B, TNF-α, and IL-1α gene expression. The enhanced NK cell function could also be attributed to the increased expression of the transcription factors T-BET and EOMES. Furthermore, the augmentation of the antitumor effects of CD16-independent NK cells upon pretreatment with Elo enhanced the expression of CRTAM, TNFRSF9, EAT-2, and FOXP3 genes and reduced the expression of HSPA6. Our results suggest that Elo directly promotes the cytotoxic function of CD16-independent NK cells against target cells, which is associated with the upregulation of the expression of several NK cell-enhancing genes.

Figure 1

Characterization of surface marker expressions on NK effector cells and target cell lines using FCM analysis: (a) different expression of CD16 (upper panel) and CD319 (SLAMF7) (lower panel) in the two human NK cell lines, NK-92MI and NK-92MI/CD16a. No significant difference in the MFI of CD319 between the two cell lines (n = 5, right panel), (b) representative results of CD16 (upper panel) and CD319 (lower panel) before (light gray, day 0) and after (dark gray, day 21) expansion of fresh PBMCs from a donor (left panel) and patient (right panel), (c) CD319 (upper panel) and CD279 (PD-1) (lower panel) in target K562, U266, RPMI 8226, and MM.1S cells. Histogram overlay showing representative results for each in comparison to the control. Light gray, isotype control; dark gray, stained with anti-CD16, anti-CD319, or anti-CD279 (a)–(c), and (d) representative result of CD56⁺CD3^– and CD56^–CD3⁺ before (day 0) and after (day 21; left panel) expansion of frozen PBMCs from a donor (upper panel) or patient (lower panel). Each data point represents mean ± SD of CD56⁺CD3^– (solid line) and CD56^–CD3⁺ (dotted line) on eNKs from donors and patients (right panel, day 0 to day 21, n = 3, respectively).

Figure 2

FCM-based assessment of the cytotoxicity of NK effector (E) cells against target (T) cells at different E : T ratios: (a) NK-92MI/CD16a (CD16-dependent) or (b) NK-92MI (CD16-independent) cells were incubated for 4 hr with the CFSE (2.5 µM)-prelabeled K562, U266, RPMI 8226, and MM.1S cells. The percentage of dead target cells (CFSE⁺FVD⁺) was analyzed among the total FVD dye-labeled cells (n = 5), and (c) freshly eNKs were collected and washed, and then were cocultured with CFSE-labeled K562. The percentage of eNK (day 21) was 75.4% ± 11.3% (D-eNK, left panel, n = 3) and 69.9% (Pt-eNK, right panel, n = 1). Results are presented as mean ± SD of independent experiments, respectively. The percentage of labeled control cells was subtracted in all assays.

Figure 3

NK-92MI/CD16a (E, CD16-dependent) cell-mediated cytotoxicity against K562, U266, RPMI 8226, and MM.1S target (T) cells pretreated without or with Elo (10 μg/mL and/or 20 μg/mL), and their IFN-γ production: (a) LDH assay showing the cytotoxicity of NK-92MI/CD16a against various target cells, (b) FCM analyses of dead target cells (CFSE⁺FVD⁺) at E : T ratios of 1 : 1 and 2.5 : 1. MM.1S cells were also pretreated with Elo and incubated with NK-92MI/CD16a after washing, (c) degranulation of NK-92MI/CD16a was assessed as CD107a⁺CD56⁺ expression on various target cells, when target cells were pretreated without (−) or with Elo (E : T = 1 : 1), using FCM assay. gray bars, and (d) ELISA-based detection of IFN-γ production after coculture with NK-92MI/CD16a and target cells, pretreated without (dotted line) or with (solid line) 10 µg/mL of Elo. Results for (a)–(d) are presented as mean ± SD of three to five independent experiments.  ^∗P < 0.05,  ^∗∗P < 0.01, and  ^∗∗∗P < 0.005.

Figure 4

LDH assay for the assessment of the eNK (E, frozen) cell-mediated cytotoxicity pretreated without or with Elo (10 μg/mL and/or 20 μg/mL) against K562, U266, RPMI 8226 (RPMI), and MM.1S target (T) cells: (a) results show the cytotoxicity when D-eNKs were cocultured with K562, U266, and RPMI pretreated without or with Elo (n = 3), (b) D-eNK-mediated cytotoxicity against MM.1S pretreated without or with Elo (E : T ratios of 1 : 1, n = 1; 2.5 : 1 to 10 : 1, n = 3). The percentages of eNKs were 55.4% ± 10.0%, 61.2% ± 7.2%, and 63.7% ± 5.9% for donor no. 1 (D#1), no. 2 (D#2), and no. 3 (D#3), respectively, (c) cytotoxicity of Pt-eNKs against K562, U266, and RPMI 8226. The percentages of eNKs were 44.6%, 68.7%, and 79.0% for patient no. 1 (Pt#1, ▪), no. 2 (Pt#2, ▴), and no. 3 (Pt#3, •), respectively, and (d) Pt-eNK cell-mediated cytotoxicity against MM.1S cells pretreated without or with Elo at different E : T ratios (n = 3). Results are presented as mean ± SD of independent experiments, respectively.  ^∗P < 0.05 and  ^∗∗∗P < 0.005.

Figure 5

NK-92MI (E, CD16-independent) cell-mediated cytotoxicity pretreated without or with Elo (10 μg/mL and/or 20 μg/mL) against K562, U266, RPMI 8226, and MM.1S target (T) cells, and their IFN-γ production: (a) LDH assay showing the cytotoxicity of NK-92MI pretreated without or with Elo (37°C, 30 min) against various target cells (E : T ratios of 1 : 1 and 2.5 : 1), (b) FCM analyses of dead target cells (CFSE⁺FVD⁺) at an E : T ratio of 2.5 : 1, when NK-92MI were pretreated without (−) or with Elo (37°C, 1 hr). All target cells were prelabeled with CFSE (2.5 μM) before being washed and recounted, and then incubated with the effector cells, (c) degranulation of NK-92MI was assessed as CD107a⁺CD56⁺ expression on various target cells, when NK-92MI were pretreated without (−) or with Elo (E : T = 1 : 1), using FCM assay, and (d) ELISA-based detection of IFN-γ production after coculture of NK-92MI with target cells without (dotted line) or with (solid line) 10 μg/mL of Elo. Results in (a)–(d) are presented as mean ± SD of four to five independent experiments for every assay.  ^∗P < 0.05,  ^∗∗P < 0.01, and  ^∗∗∗P < 0.005.

Figure 6

qPCR analysis of the relative mRNA expression of GZMB, PRF1, TNF-α (a), CRTAM, TNFRSF9, EAT-2, IL-1α, HSPA6, FOXP3, T-BET, and EOMES and (b) in NK-92MI (E, CD16-independent) cells pretreated without or with Elo (10 μg/mL, 37°C, 1 hr), and then cocultured with K562, U266, RPMI 8226 (RPMI), and MM.1S target (T) cells (E : T ratio of 10 : 1) for 1, 2, 4, and 24 hr. Gene expression levels are expressed as the ratio of the expression in the group pretreated with Elo to that in the group pretreated without Elo (black bar). The Elo-pretreated groups are shown at the time points of 1 hr (red), 2 hr (orange), 4 hr (blue), and 24 hr (green). Results are presented as mean ± SD of four independent experiments for every target cell.  ^∗P < 0.05,  ^∗∗P < 0.01, and  ^∗∗∗P < 0.005.