Results: 2

Figure 2

Figure 2. From: IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect.

Lenalidomide modulates MM expression of NK cell–activating ligands and in combination with IPH2101 enhances in vivo tumor-cell rejection. (A) Incubation of U266 MM cells in lenalidomide (5μM) for 5 days led to alteration of NK cell–ligand expression, representative results shown for each ligand (open curves are isotype and control, shaded curve is lenalidomide treated). The average Ab-binding capacity of MICA, DR4, ULBP2, and CD112 increased by 73.6% ± 26.6%, 95.6% ± 9.5%, 86.1% ± 16.1%, and 114% ± 6.9%, respectively, compared with control cells (P < .05 for all comparisons, left). The expression of ULBP-1, CD155, ULBP-3, and ULBP-4 was not affected (right). (B) A similar effect was observed in primary MM cells (n = 7) all of which expressed ULBP-1 at baseline; a representative result is shown. Lenalidomide (5nM) for 24 hours increased the expression of ULBP-1 by 153% ± 24% (P = .016), but did not reproducibly alter expression of any of the other ligands shown in all cases examined. (C) NK-cell lysis of U266 cells was augmented 1.22 ± 0.05-fold relative to control (P < .05) when targets were pretreated with lenalidomide to increase activating ligand expression as shown in panel A. However, this effect was lost when NK cells were preincubated in blocking Abs against NKG2D, DNAM-1, and TRAIL, suggesting functional relevance to lenalidomide's modulation of activating ligands on MM target cells. A similar effect was observed in patient-derived NK cells against autologous MM-cell targets (right; n = 2 independent experiments). (D) A single 5-μg dose of 5E6 led to a slight reduction of observed RMA cells, and pretreatment of animals with lenalidomide for 21 days enhanced this effect (control mean 16.5 ± 5.9 vs 5E6 plus lenalidomide, 7.8 ± 3.5, P < .01). Similar results were observed with 5E6 (10 μg) with lenalidomide at 14 or 28 days (E). The combination of 5E6 and lenalidomide significantly reduced tumor burden (11.8 ± 5.1 for control vs 3.4 ± 1.6 for 5E6 plus lenalidomide, P < .005).

Don M. Benson, et al. Blood. 2011 December 8;118(24):6387-6391.
Figure 1

Figure 1. From: IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect.

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).

Don M. Benson, et al. Blood. 2011 December 8;118(24):6387-6391.

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