Figure 1
Figure 1. IPH2101 increases NK-cell recognition of and cytotoxicity against MM targets. (A) IPH2101 (30 μg/mL) augmented NK cell–mediated killing of U266 MM cells measured by a target-based, europium-release cytotoxicity assay. At E:T ratios of 2.5:1, 5:1, and 10:1, IPH2101 increased cytotoxicity by an average of 1.76 ± 0.28-fold, 1.4 ± 0.09-fold, and 1.48 ± 0.08-fold relative to control, respectively (P < .05 for all comparisons, results represent n = 10 independent donor experiments). (B) In a complementary manner, NK-cell production of granzyme B (GrB) measured by ELISPOT assay served as an effector-cell–based killing assay. IPH2101 enhanced NK-cell GrB production against U266 targets (E:T 20:1, left, isotype control mean 139 ± 63 vs IPH2101 treated 348 ± 20, n = 3 independent experiments, P = .005). IPH2101 also enhanced patient-derived NK-cell GrB secretion against primary MM cell targets (right, E:T 10:1, control mean 59 ± 2.3 vs IPH2101 treated 103 ± 4.6). *P = .008, results represent n = 5 independent donor experiments. The black bar in each graph represents a positive control for the assay using effector cells versus the K562 cell line. (C) Pretreatment of NK cells with IPH2101 significantly increased the number of immune complexes observed between patient-derived effector cells and purified, autologous CD138+ MM cells (E:T 1:1, control mean 751 ± SD 181 vs IPH2101 treated 1225 ± 207, P = .041), but not against the residual CD138− normal BM cells (E:T 1:1, control mean 458 ± 208 vs IPH2101 treated 507 ± 218, P = nonsignificant, n = 3 independent donor experiments). (D) The increase in immune complexes was associated with enhanced NK-cell lysis of autologous CD138+ MM cell targets (control 42% ± 8% vs IPH2101 treated 63% ± 12%, *P = .0135), but no enhancement of cytotoxicity against CD138− autologous, normal BM elements (control 6% ± 5% vs IPH2101 treated 3% ± 2%, P = nonsignificant, E:T = 20:1, n = 3 independent donor experiments). (E) Left, freshly isolated, healthy donor NK cells were pretreated with lenalidomide (5μM), IPH2101 (30 μg/mL), or the combination, and IFN-γ production was measured by ELISPOT assay against primary MM cells in coculture for 4 hours. Lenalidomide (mean 609 spots/well ± 34) and IPH2101 (568 ± 20) each increased NK-cell IFN-γ production beyond control conditions (483 ± 15, *P < .05). NK cells pretreated with both lenalidomide and IPH2101 (807 ± 50) led to the greatest IFN-γ production of any condition, and the interaction was statistically significant, suggesting these agents induced a synergistic effect (**P < .0182, n = 3 independent donor experiments, similar results were obtained with NK cells vs U266 MM targets, data not shown). Right, patient-derived NK cells against autologous MM tumor cells. Lenalidomide (**mean 74 ± 9 spots/well), IPH2101 (***mean 44 ± 4 spots/well), and the combination (*mean 99 ± 7 spots/well) all enhanced IFN-γ production beyond control (20 ± 2) conditions, overall P < .0001, P < .05 for each pairwise comparison shown, representative findings of n = 3 independent experiments. (F) PBMCs incubated in control conditions or lenalidomide (10μM) and/or IPH2101 (30 μg/mL) served as effector cells against U266 MM cell targets. At an E:T ratio of 30:1, lenalidomide increased cytotoxicity as measured by specific release by 1.39 ± 0.10-fold relative to control (P < .01), IPH2101 increased the specific release by 1.48 ± 0.21-fold (P < .01), whereas the combination of lenalidomide and IPH2101 increased the specific release by 2.09 ± 0.21-fold relative to control (P < .001), which was also significantly greater than the cytotoxicity observed with either lenalidomide or IPH-2010 alone (P < .01 for each comparison). Similar results were obtained using at an E:T ratio of 60:1 (right). (G) Patient-derived NK-cell cytotoxicity against autologous MM target cells was enhanced by lenalidomide (mean 110 ± 8 spots/well), IPH2101 (106 ± 11), or the combination (128 ± 9) compared with control conditions (81 ± 7), overall ANOVA P = .0001, all pairwise, planned comparisons shown P < .05, representative data of n = 3 independent experiments. (H) Whole BM aspirates obtained from patients with MM (n = 5) were incubated with or without IPH2101 (30 μg/mL) and/or lenalidomide (5μM) for 24 hours. The percentage of NK cells expressing CD107a increased in response to lenalidomide and IPH2101 (20% ± 6%) compared with control (11.3% ± 2%, P < .05).

IPH2101 increases NK-cell recognition of and cytotoxicity against MM targets. (A) IPH2101 (30 μg/mL) augmented NK cell–mediated killing of U266 MM cells measured by a target-based, europium-release cytotoxicity assay. At E:T ratios of 2.5:1, 5:1, and 10:1, IPH2101 increased cytotoxicity by an average of 1.76 ± 0.28-fold, 1.4 ± 0.09-fold, and 1.48 ± 0.08-fold relative to control, respectively (P < .05 for all comparisons, results represent n = 10 independent donor experiments). (B) In a complementary manner, NK-cell production of granzyme B (GrB) measured by ELISPOT assay served as an effector-cell–based killing assay. IPH2101 enhanced NK-cell GrB production against U266 targets (E:T 20:1, left, isotype control mean 139 ± 63 vs IPH2101 treated 348 ± 20, n = 3 independent experiments, P = .005). IPH2101 also enhanced patient-derived NK-cell GrB secretion against primary MM cell targets (right, E:T 10:1, control mean 59 ± 2.3 vs IPH2101 treated 103 ± 4.6). *P = .008, results represent n = 5 independent donor experiments. The black bar in each graph represents a positive control for the assay using effector cells versus the K562 cell line. (C) Pretreatment of NK cells with IPH2101 significantly increased the number of immune complexes observed between patient-derived effector cells and purified, autologous CD138+ MM cells (E:T 1:1, control mean 751 ± SD 181 vs IPH2101 treated 1225 ± 207, P = .041), but not against the residual CD138− normal BM cells (E:T 1:1, control mean 458 ± 208 vs IPH2101 treated 507 ± 218, P = nonsignificant, n = 3 independent donor experiments). (D) The increase in immune complexes was associated with enhanced NK-cell lysis of autologous CD138+ MM cell targets (control 42% ± 8% vs IPH2101 treated 63% ± 12%, *P = .0135), but no enhancement of cytotoxicity against CD138− autologous, normal BM elements (control 6% ± 5% vs IPH2101 treated 3% ± 2%, P = nonsignificant, E:T = 20:1, n = 3 independent donor experiments). (E) Left, freshly isolated, healthy donor NK cells were pretreated with lenalidomide (5μM), IPH2101 (30 μg/mL), or the combination, and IFN-γ production was measured by ELISPOT assay against primary MM cells in coculture for 4 hours. Lenalidomide (mean 609 spots/well ± 34) and IPH2101 (568 ± 20) each increased NK-cell IFN-γ production beyond control conditions (483 ± 15, *P < .05). NK cells pretreated with both lenalidomide and IPH2101 (807 ± 50) led to the greatest IFN-γ production of any condition, and the interaction was statistically significant, suggesting these agents induced a synergistic effect (**P < .0182, n = 3 independent donor experiments, similar results were obtained with NK cells vs U266 MM targets, data not shown). Right, patient-derived NK cells against autologous MM tumor cells. Lenalidomide (**mean 74 ± 9 spots/well), IPH2101 (***mean 44 ± 4 spots/well), and the combination (*mean 99 ± 7 spots/well) all enhanced IFN-γ production beyond control (20 ± 2) conditions, overall P < .0001, P < .05 for each pairwise comparison shown, representative findings of n = 3 independent experiments. (F) PBMCs incubated in control conditions or lenalidomide (10μM) and/or IPH2101 (30 μg/mL) served as effector cells against U266 MM cell targets. At an E:T ratio of 30:1, lenalidomide increased cytotoxicity as measured by specific release by 1.39 ± 0.10-fold relative to control (P < .01), IPH2101 increased the specific release by 1.48 ± 0.21-fold (P < .01), whereas the combination of lenalidomide and IPH2101 increased the specific release by 2.09 ± 0.21-fold relative to control (P < .001), which was also significantly greater than the cytotoxicity observed with either lenalidomide or IPH-2010 alone (P < .01 for each comparison). Similar results were obtained using at an E:T ratio of 60:1 (right). (G) Patient-derived NK-cell cytotoxicity against autologous MM target cells was enhanced by lenalidomide (mean 110 ± 8 spots/well), IPH2101 (106 ± 11), or the combination (128 ± 9) compared with control conditions (81 ± 7), overall ANOVA P = .0001, all pairwise, planned comparisons shown P < .05, representative data of n = 3 independent experiments. (H) Whole BM aspirates obtained from patients with MM (n = 5) were incubated with or without IPH2101 (30 μg/mL) and/or lenalidomide (5μM) for 24 hours. The percentage of NK cells expressing CD107a increased in response to lenalidomide and IPH2101 (20% ± 6%) compared with control (11.3% ± 2%, P < .05).

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