Figure 6.
In vivo antimyeloma effects and on-target cytokine release mediated by a mAb/B-BiTE complex. (A) Target myeloma cells including MM1S/CD38high and MM1S/CD38low cells at different ratios were prepared. CD19– human PBMCs were cocultured with these target cells at an E:T ratio of 4:1 in the presence of daratumumab, or daratumumab/B-BiTE (daratumumab, 7.5 μg/mL; B-BiTE, 2.5 μg/mL), and the proportion of CD138+ MM1S cells among total cells was measured (left). The percentage of MM1S cells after treatment with antibodies was calculated relative to the frequency of MM1S cells without any antibodies and is plotted as residual cells (right). (B) Three million MM1S/CD38high cells were injected subcutaneously into the right flank of irradiated NOD/Shi-scid IL-2rγ(null) mice. After engraftment of the MM1S/CD38high cells, 1.0 × 107 freshly isolated human PBMCs were injected IV into the mice on day 4. On day 5, daratumumab (30 μg) or daratumumab/B-BiTE complex (daratumumab, 30 μg; B-BiTE, 10 μg) was similarly injected into each mouse, followed by measurement of tumor sizes (n = 6 mice per group). (C) Three million MM1S/CD38high cells were similarly inoculated into NOD/Shi-scid IL-2rγ (null) mice. After engraftment, 5.0 × 106 freshly isolated human T cells or 5.0 × 106 human T cells together with 5.0 × 106 CD3– PBMCs were injected IV into the mice on day 4. On day 5, daratumumab/B-BiTE complex was similarly injected into each mouse, followed by measurement of tumor sizes (n = 6 mice per group). (D) To assess the amounts of cytokines, the experiment in panel C was repeated again, and murine sera were collected at each time point. A series of human cytokines contained in each murine serum were measured using cytokine beads assays. Each dot represents an individual mouse. The Welch t test (2-sided) was performed for comparison, and error bars show the SD. Each of the groups as well as each of the time points in the group treated with both T cells and CD3– PBMCs were compared. Decreases in cytokines after treatment were not assessed statistically. ∗P  < .05; E:T, effector to target; n.s., not significant; SD, standard deviation.

In vivo antimyeloma effects and on-target cytokine release mediated by a mAb/B-BiTE complex. (A) Target myeloma cells including MM1S/CD38high and MM1S/CD38low cells at different ratios were prepared. CD19– human PBMCs were cocultured with these target cells at an E:T ratio of 4:1 in the presence of daratumumab, or daratumumab/B-BiTE (daratumumab, 7.5 μg/mL; B-BiTE, 2.5 μg/mL), and the proportion of CD138+ MM1S cells among total cells was measured (left). The percentage of MM1S cells after treatment with antibodies was calculated relative to the frequency of MM1S cells without any antibodies and is plotted as residual cells (right). (B) Three million MM1S/CD38high cells were injected subcutaneously into the right flank of irradiated NOD/Shi-scid IL-2rγ(null) mice. After engraftment of the MM1S/CD38high cells, 1.0 × 107 freshly isolated human PBMCs were injected IV into the mice on day 4. On day 5, daratumumab (30 μg) or daratumumab/B-BiTE complex (daratumumab, 30 μg; B-BiTE, 10 μg) was similarly injected into each mouse, followed by measurement of tumor sizes (n = 6 mice per group). (C) Three million MM1S/CD38high cells were similarly inoculated into NOD/Shi-scid IL-2rγ (null) mice. After engraftment, 5.0 × 106 freshly isolated human T cells or 5.0 × 106 human T cells together with 5.0 × 106 CD3– PBMCs were injected IV into the mice on day 4. On day 5, daratumumab/B-BiTE complex was similarly injected into each mouse, followed by measurement of tumor sizes (n = 6 mice per group). (D) To assess the amounts of cytokines, the experiment in panel C was repeated again, and murine sera were collected at each time point. A series of human cytokines contained in each murine serum were measured using cytokine beads assays. Each dot represents an individual mouse. The Welch t test (2-sided) was performed for comparison, and error bars show the SD. Each of the groups as well as each of the time points in the group treated with both T cells and CD3– PBMCs were compared. Decreases in cytokines after treatment were not assessed statistically. ∗P  < .05; E:T, effector to target; n.s., not significant; SD, standard deviation.

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