Figure 2.
Preserved NK cell reactivity and stability of a mAb/B-BiTE complex. (A) IFN-γ production by isolated human CD3+ T cells including CD8+ T cells (left) and CD4+ T cells (middle), and human NK cells (right) against K562/CD20 cells (top) or K562/CD38 cells (bottom), was measured in the presence of 7.5 μg/mL rituximab or daratumumab together with graded concentrations of B-BiTE. The concentration of mAb is indicated as a dotted line in each graph. The maximal IFN-γ production by responder cells against target cells was considered as 100%, and each reactivity was calculated relative to this value. (B) Human PBMCs were incubated with K562/CD20 cells (top) or K562/CD38 cells (bottom) in the presence of rituximab/B-BiTE or daratumumab/B-BiTE complex (mAb, 7.5 μg/mL; B-BiTE, 2.5 μg/mL) with different concentrations of human Poly-Ig. Competitive assays were performed, and the IFN-γ production by human CD8+ T cells (left), CD4+ T cells (middle), and NK cells (right) was measured by intracellular cytokine assay. IFN-γ production (%) in the presence of each concentration of Poly-Ig was calculated relative to that induced by rituximab/B-BiTE or daratumumab/B-BiTE for K562/CD20 cells or K562/CD38 cells in the absence of Poly-Ig and is plotted. (C) Poly-Ig (20 mg) was IV injected into irradiated NOD/Shi-scid IL-2rγ(null) mice (n = 3) twice with a 12-hour interval, on day 0 (Poly-Ig, 40 mg/mouse). Daratumumab/B-BiTE complex (daratumumab, 30 μg; B-BiTE, 10 μg) was similarly injected into the mice on day 1. Murine serum was collected at each time point (before treatment and 6, 12, 24, 48, and 72 hours after treatment) (top left). Then, human PBMCs were cocultured with K562 cells or K562/CD38 cells in wells containing each collected serum. A sample just after administration of daratumumab/B-BiTE (0 hour) was prepared by adding it to pretreated serum in vitro (40 μg/mL). IFN-γ production by CD8+ T cells (bottom left), CD4+ T cells (bottom middle), and NK cells (bottom right) against target cells was measured. The maximal IFN-γ production by responder cells was considered as 100%, and each reactivity was calculated relative to this value. The values obtained before treatment, at 0 hour, and at 72 hours after treatment were also compared directly (top right). Error bars show the SD. ∗P  < .05; ∗∗P  < .01; ∗∗∗P  < .001; n.s., not significant; Poly-Ig, polyclonal immunoglobulin.

Preserved NK cell reactivity and stability of a mAb/B-BiTE complex. (A) IFN-γ production by isolated human CD3+ T cells including CD8+ T cells (left) and CD4+ T cells (middle), and human NK cells (right) against K562/CD20 cells (top) or K562/CD38 cells (bottom), was measured in the presence of 7.5 μg/mL rituximab or daratumumab together with graded concentrations of B-BiTE. The concentration of mAb is indicated as a dotted line in each graph. The maximal IFN-γ production by responder cells against target cells was considered as 100%, and each reactivity was calculated relative to this value. (B) Human PBMCs were incubated with K562/CD20 cells (top) or K562/CD38 cells (bottom) in the presence of rituximab/B-BiTE or daratumumab/B-BiTE complex (mAb, 7.5 μg/mL; B-BiTE, 2.5 μg/mL) with different concentrations of human Poly-Ig. Competitive assays were performed, and the IFN-γ production by human CD8+ T cells (left), CD4+ T cells (middle), and NK cells (right) was measured by intracellular cytokine assay. IFN-γ production (%) in the presence of each concentration of Poly-Ig was calculated relative to that induced by rituximab/B-BiTE or daratumumab/B-BiTE for K562/CD20 cells or K562/CD38 cells in the absence of Poly-Ig and is plotted. (C) Poly-Ig (20 mg) was IV injected into irradiated NOD/Shi-scid IL-2rγ(null) mice (n = 3) twice with a 12-hour interval, on day 0 (Poly-Ig, 40 mg/mouse). Daratumumab/B-BiTE complex (daratumumab, 30 μg; B-BiTE, 10 μg) was similarly injected into the mice on day 1. Murine serum was collected at each time point (before treatment and 6, 12, 24, 48, and 72 hours after treatment) (top left). Then, human PBMCs were cocultured with K562 cells or K562/CD38 cells in wells containing each collected serum. A sample just after administration of daratumumab/B-BiTE (0 hour) was prepared by adding it to pretreated serum in vitro (40 μg/mL). IFN-γ production by CD8+ T cells (bottom left), CD4+ T cells (bottom middle), and NK cells (bottom right) against target cells was measured. The maximal IFN-γ production by responder cells was considered as 100%, and each reactivity was calculated relative to this value. The values obtained before treatment, at 0 hour, and at 72 hours after treatment were also compared directly (top right). Error bars show the SD. ∗P  < .05; ∗∗P  < .01; ∗∗∗P  < .001; n.s., not significant; Poly-Ig, polyclonal immunoglobulin.

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